Estradiol therapy in adulthood reverses glial and neuronal alterations caused byperinatal asphyxia
Gustavo Ezequiel Saraceno a Mariacutea Laura Aoacuten Bertolino a Pablo Galeano a Juan Ignacio Romero aLuis Miguel Garcia-Segura b Francisco Capani aa Laboratorio de Citoarquitectura y Plasticidad Neuronal Instituto de Investigaciones Cardioloacutegicas ldquoProf Dr Alberto C Taquinirdquo (ININCA) UBA-CONICET Marcelo T de Alvear 2270C1122AAJ Buenos Aires Argentinab Instituto Cajal CSIC Avenida Doctor Arce 37 E-28002 Madrid Spain
The capacity of the ovarian hormone 17β-estradiol to prevent neurodegeneration has been characterized inseveral animal models of brain and spinal cord pathology However the potential reparative activity of thehormone under chronic neurodegenerative conditions has received less attention In this study we haveassessed the effect of estradiol therapy in adulthood on chronic glial and neuronal alterations caused byperinatal asphyxia (PA) in rats Four-month-old male SpraguendashDawley rats submitted to PA just afterdelivery and their control littermates were injected for 3 consecutive days with 17β estradiol or vehicleAnimals subjected to PA and treated with vehicle showed an increased astrogliosis focal swelling andfragmented appearance of MAP-2 immunoreactive dendrites decreased MAP-2 immunoreactivity anddecreased phosphorylation of high and medium molecular weight neurofilaments in the hippocampuscompared to control animals Estradiol therapy reversed these alterations These findings indicate thatestradiol is able to reduce in adult animals chronic reactive astrogliosis and neuronal alterations caused byan early developmental neurodegenerative event suggesting that the hormone might induce reparativeactions in the Central Nervous System (CNS)
lsevier Inc
copy 2010 Published by Elsevier Inc
Introduction
The neuroprotective effects of the ovarian hormone 17β-estradiolhave been widely described against a great variety of neuropatho-logical conditions in animal neuroprotective models These includeexperimental models of stroke Parkinsons disease Alzheimersdisease multiple sclerosis and excitotoxicity among others (Bourqueet al 2009 Garcia-Segura and Balthazart 2009 Kipp and Beyer2009 Kruse et al 2009 Lebesgue et al 2009 Pike et al 2009 Suzukiet al 2009) In most of these studies estradiol has been shown toprevent neuronal loss and gliosis when animals were treated with thehormone either before or shortly after the initiation of brain damageThere is little evidence that estradiol may repair brain tissue after achronic process of neurodegeneration in particular when this processhas been initiated at early life stages
Perinatal asphyxia is still an obstetric problem that might induceseveral alterations in the full-term births Of the affected newborns15ndash20die in thepostnatal period and25will remainwithpermanentneurobiological deficits like spasticity epilepsymental retardation (Hill
and Volpe 1981 Amiel-Tison and Ellison 1986 Vannucci and Perlman1997 Gunn 2000 Shankaran 2009) and visual alteration in adult life(Osborne et al 2004) Although the immature brain is relativelyprotected from hypoxia by adaptive mechanisms severe insults cantrigger self-sustaining damaging cascades lasting for days or weeks andresult in prominent injury (Ferriero 2004) The neurons that are mostsensitive and vulnerable to PA are located in the CA1 CA3 and CA4regions of the hippocampus in the cerebellum in layers III V and VI ofthe neocortex and in the neostriatum (Kirino 1982 Petito andPulsinelli 1984 Capani et al 2009) Some of the processes associatedwith asphyxia occur at synapses site including nitric oxide release(Capani et al 1997 2009 Loidl et al 1997) elevation of glutamate inthe extracellular space (Busto et al 1989 Choi and Rothman 1990)aberrant cell signaling (Kamme and Wieloch 1996) phospholipidicdegradation and lipooxidation (Farooqui et al 1994) and finallyexcessive release of free radicals (Capani et al 2001) In previousworkswe have shown long term biochemical cellular and subcellularalterations including reactive astrogliosis increase in the immunoreac-tivity for high weigh neurofilaments (200 kDa NF) (Cebral et al 2006)and high level of ubi-proteins and free ubiquitin at the post synapticdensity (PSD) in neostriatum (Capani et al 2009) Taken together thisdata suggests that PA could lead to neurodegenerative alterations(Capani et al 2009) These events are well correlated with behavioralalterations (Loidl et al 2000)
616 GE Saraceno et al Experimental Neurology 223 (2010) 615ndash622
We have extensively worked on experimental hypothermia astherapeutic tool to reduce thedamageproducebyhypoxia (Capani et al1997 2003 2009) Experimental hypothermia has shown to blockmostof the processes that trigger the mechanisms for the generation of thecell death (Capani et al 2009) Although hypothermia has been alreadyused in clinical trials it has been shown to be effective only in veryrestrictive neurological pathologies produced by PA (Gonzalez andFerriero 2008 Shankaran 2009)
According to our knowledge (Capani et al 2009) there is not aproper therapeutic tool to treat the neurological diseases produced byPA Therefore in this study we test if estrogens are effective to reducethe experimental chronic cytological alterations induced by PA in ratsOur main aims were to determine (i) whether alterations in the CA1area of hippocampus were still detectable in four-month-old animalssubmitted to perinatal asphyxia and (ii) whether these alterationscould be reverted in adult rats by the treatment with 17β-estradiol
Materials and methods
Animals
All procedures involving animals were approved by the Institu-tional Animal Care and Use Committee at the University of BuenosAires (School of Medicine) and conducted according to the principlesof the Guide for the Care and Use of Laboratory Animals (NIHPublications No 80-23 revised 1996) SpraguendashDawley female rats inthe fifteenth day of pregnancy were placed in individual cages andmaintained on a 1212 h lightdark cycle in a controlled temperature(21plusmn2 degC) and humidity (65plusmn5) environment The animals hadaccess to food (Purina chow) and tap water ad libitum One group ofanimals (n=4) were used as surrogate mothers another group(n=5) were assigned to perinatal asphyxia procedures and theremaining animals (n=5) were the mothers of the control pups
Induction of asphyxia
Five full-term pregnant rats on gestational day 22 were anesthe-tized (Dorfman et al 2006) rapidly decapitated and the uterus hornswere isolated through an abdominal incision and placed in a waterbath at 37 degC for 19 min (sub-severe perinatal asphyxia) (Bjelke et al1991 Van de Berg et al 2003 Capani et al 2009) We used 19 min asthe maximum time of PA because 21 or more minutes in thiscondition result in a survival rate lower than 3 (Capani et al 2009)Following asphyxia the uterus horns were rapidly opened the pupswere removed the amniotic fluid was cleaned and the pups werestimulated to breathe by performing tactile intermittent stimulationwith pieces of medical wipes for a fewminutes until regular breathingwas established The umbilical cord was ligated and the animals wereleft to recover for 1 h under a heating lamp When their physiologicalconditions improved they were given to surrogate mothers who haddelivered normally within the last 24 h The different groups of pupswere marked and mixed with the surrogate mothers normal litters(control animals that were left undisturbed) Wemaintained litters of10 pups with each surrogate mother
17β estradiol treatment
Sixteen adult male rats were injected ip 117 days after perinatalasphyxia with 17β estradiol (water soluble E4389 Sigma St Louis Mo250 microgkg) or vehicle (09 saline solution) Previous studies haveshown that doses of estradiol not very different from the one selected forthis study reduce gliosis in male rats after a penetrating brain injury orafter immune challenge (Tapia-Gonzalez et al 2008 Barreto et al2009) The injectionswere repeated daily for 3 days consecutively (up to119 days after perinatal asphyxia) Animals were distributed in 4experimental groups (i) control animals injected with vehicle (ii)
control animals injected with 17β estradiol (iii) animals submitted toperinatal asphyxia and injectedwith vehicle and (iv) animals submittedto perinatal asphyxia and treated with 17β estradiol
Tissue fixation and immunohistochemistry
At postnatal day 120 sixteen animals were anesthetized withchloral hydrate (28 wv 01 ml100 g body weight) and perfusedintracardially with 4 paraformaldehyde in 01 M phosphate bufferpH 74 Brains were removed and post-fixed in the same fixativesolution for 2 h at room temperature and then immersed overnight at4 degC in 01 M phosphate buffer pH 74 Coronal hippocampal sections(40 μm thickness) were obtained using a Vibratome (VT 1000 S LeicaMicrosystems Wetzlar Germany)
Immunohistochemistry was performed on free floating sectionsunder moderate shaking Endogenous peroxidase was quenched (3H2O2 30 methanol 70 PBS 01 M) and non-specific labeling wasblocked using 5 normal goat serum Sections were incubatedovernight at 4 degC with a mouse primary antibody that recognizes120 kDa neurofilaments (120 NF 1500 Chemicon InternationalTemecula CA USA) or a mouse primary antibody that recognizesphosphorylated high and medium molecular weight neurofilamentproteins (pHM NF 1250 Chemicon International Temecula CAUSA) overnight at 4 degC After several washes the sections wereincubated for 2 h at room temperature with secondary antibodies(Biotinylated anti mouse IgG diluted 1300 Vector Laboratories IncBurlingame CA USA) Labelingwas revealed using the ABC kit (VectorLaboratories Inc Burlingame CA USA) Peroxidase activity wasrevealed with 001 hydrogen peroxide using 33prime-diaminobenzidineas the chromogen (DAB Sigma) Immunostaining was absent whenthe first antibody was omitted
For immunofluorescence tissue sections were blocked for 30 minin phosphate-buffered saline (PBS) containing 03 Bovine SerumAlbumin (BSA Sigma St Louis MO USA) and 03 Triton X-100Sectionswere incubated overnight at 4 degCwith a rabbit polyclonal antiGFAP antibody (12000 Sigma St Louis MO USA) or a mousemonoclonal anti MAP-2 antibody (1500 Sigma St Louis MO USA)After washing in buffer tissue sections were incubated for 2 h at roomtemperature with Alexa 488 goat anti-rabbit IgG (1200 MolecularProbes) or Alexa 594 goat anti-mouse IgG (1200 Molecular Probes)Sections were counterstained with DAPI (Vector Laboratories IncBurlingame CA USA) to label cell nuclei and mounted withVectashield mounting medium Immunostaining was absent whenthe first antibody was omitted In order to minimize inter-assayvariations samples from all experimental groups were processed inparallel
Morphometric analysis
The volume fraction of immunoreactive material for MAP-2 120 NFand pHM NF was estimated using the point-counting method ofWeibel (1979) and a grid delimiting 5000 μm2 in the striatum radiatumof CA1 A total area of 75000 μm2 was evaluated in each animalPercentage of reactive areawasestimatedusing Image J Program(ImageJ 141o NIH USA) The number of GFAP immunoreactive astrocytes wasestimated in the striatum radiatum of CA1 by the optical dissectormethod (Howard and Reed 1998) using total section thickness fordissector height (Hatton and von Bartheld 1999) and a counting frameof 55times55 μm A total of 78 counting frames were assessed per animalSection thickness was measured using a digital length gauge device(Heidenhain-Metro MT 12ND221 Traunreut Germany) attached tothe stage of a Leitz microscope Cell nuclei from GFAP immunoreactivecells that came into focus while focusing down through the dissectorheightwere counted All counts were performed on coded sections Thevolume of the striatum radiatum of CA1 was estimated using the point-counting method of Weibel (1979) Since no significant differences in
617GE Saraceno et al Experimental Neurology 223 (2010) 615ndash622
this parameter were observed among the different experimentalgroups the changes in the number of GFAP immunoreactive cells perunit volume with the optical dissector method are assumed to reflectchanges in the total number of GFAP immunoreactive cells
Statistical analysis
Material from four rats was analyzed for each experimental groupand for each parameter studied (n=16) All statistical analyses wereperformed by two-way analysis of variances (ANOVAs) with birthcondition (CTL and PA) and treatment (Vhi and 17β) as the mainfactors When interaction effects were significant analyses of thesimple effects were carried out by post hoc comparisons usingStudents t-test (two-tailed) adjusted by Bonferroni correction In anycase neither the assumption of normal distribution (ShapirondashWilktest) nor equality of variances (Levenes test) was rejected Resultswere expressed as the meanplusmnSEM Differences with a probability of5 or less were considered to be significant (Pb005) All statisticalanalyses were done using the PASW Statistics 18 software (SPSS IncChicago IL USA)
Results
GFAP immunostaining in the CA1 hippocampal area Effects of PA and17β treatment
Hippocampal sections from PA animals treated with vehiclestained with GFAP and observed at confocal microscopy showed amarked astrogliosis in striatum radiatum of CA1 hippocampal area
Fig 1 Confocal microscope images of GFAP immunostaining from the striatum radiatum ofmarked reactive astrogliosis (panel C) respect to controls injected with vehicle and 17β estrastrogliosis in PA animals Scale bar 10 microm
which is a characteristic feature for chronic process of neurodegen-eration (Fig 1C) The morphometric analysis in the striatum radiatumof CA1 confirmed this data The two-way analysis of varianceindicated that the main factors of birth condition and treatmentwere both significant (F=2676 Pb0001 F=827 Pb001 respec-tively) and the interaction was also significant (F=2344 Pb001)Post hoc analysis of the simple effects revealed that treatment with17β estradiol in CTL rats had not significant effect on the number ofGFAP immunoreactive astrocytes in comparison to CTL rats injectedwith vehicle (P=ns see Figs 1-B 1-A and 2) PA rats injected withvehicle showed amarkedly significant increase of the number of GFAPimmunoreactive astrocytes in comparison to CTL rats treated withvehicle (Pb0005 see Figs 1-C 1-A and 2) Estradiol treatment in PArats significantly reduced the number of GFAP immunoreactiveastrocytes in comparison to PA rats treated with vehicle (Pb0005see Figs 1-D 1-C and 2) and moreover they did not show significantdifferences with CTL rats injected with vehicle (P=ns see Figs 1-D1-A and 2) suggesting a reversion of astrogliosis associated withhypoxia at birth by 17β estradiol treatment in the adulthood
Dendritic alterations induced by PA and reversion by 17β estradioltreatment
Confocal studies using MAP-2 were conducted to determine thedendritic changes after PA insult Animals subjected to PA and treatedwith vehicle showed focal swelling and markedly fragmentedappearance of MAP-2 immunoreactive apical dendrites in the CA1hippocampal area compared to control animals treated with vehicle(see Figs 3-A and 3-C) In addition the statistical analysis revealed
CA1 hippocampal area The hippocampus of PA animals treated with vehicle showed aadiol (panels A and B) See in panel D that 17β estradiol treatment reduce the reactive
Fig 2 Assessment of the number of GFAP immunoreactive astrocytes in the striatumradiatum of CA1 Estradiol by itself did not produce any statistical differences in thenumberof GFAP immunoreactive astrocytes since CTL rats treatedwith estradiol (CTL-17β) did notshowdifferences in thenumber ofGFAP+astrocytes in comparison to CTL rats treatedwithvehicle (CTL-Vhi) Animals that were submitted to PA and treated with vehicle (PA-Vhi)showeda significant increase ofGFAP immunoreactive astrocytes in comparison to CTL-Vhiand PA rats treated with 17β estradiol (PA-17β) Moreover number of GFAPimmunoreactive astrocytes in PA-17β rats did not differ from those seen in CTL-Vhi ratsdemonstrating that estradiol treatment in adult rats blocked the reactive astrogliosisassociated with a PA event Bars and error bars represent mean+SEM Pb0005 PA-Vhivs CTL-Vhi daggerdaggerPb0005 PA-Vhi vs PA-17β
618 GE Saraceno et al Experimental Neurology 223 (2010) 615ndash622
that neither the main factor of birth condition nor the main factor oftreatment was significant (F=166 P=ns F=299 P=ns respec-tively) but the interaction was (F=623 P=0016) Post hoc analysis
Fig 3 Confocal microscope images of MAP-2 immunostaining from the striatum radiatum of Cfragmented appearance of distal dendrites in the CA1 region of PA animals injected with vehand B) Asphyctic animals injected with 17β estradiol (panel D) shows similar morphologi
indicated that 17β estradiol treatment in CTL rats did not produce anysignificant changes of the percentage of reactive area of MAP-2positive dendrites with respect to CTL rats injected with vehicle(P=ns see Figs 3-B 3-A and 4) Rats subjected to PA and treatedwith vehicle showed a significant reduction of the percentage ofreactive area of MAP-2 positive dendrites in comparison to CTL ratsinjected with vehicle (Pb005 see Figs 3-C 3-A and 4) Estradioltreatment in adults rats that suffered from PA significantly improvedthe mean percentage of reactive area of MAP-2 positive dendrites incomparison to PA rats treated with vehicle (Pb005 see Figs 3-D 3-Cand 4) while no differences were detected between PA rats treatedwith 17β estradiol and CTL rats injected with vehicle (P=ns seeFigs 3-D 3-A and 4)
Effects of PA and 17β estradiol treatment on axonal neurofilaments
Axonal alterations were studied using immunocytochemistry formediummolecular weight neurofilaments (120 NF) and phosphorylat-edhighandmediummolecularweightneurofilaments (pHMNF) Fig 5shows a representative example of a hippocampal section immunos-tained for 120 NF No obvious differences in 120 NF immunostainingwere detected between animals subjected to PA and control animals Inagreement with the qualitative observations the statistical analysisshowed that neither the main factor of birth condition nor the mainfactor of treatment nor the interaction was significant (Fb1 F=23P=ns Pb1 respectively See Fig 6) In contrast noticeable changeswere seen in the volume fraction of immunoreactive material forphosphorylated high and medium molecular weight neurofilamentsafter PA and 17β treatment The two-way ANOVA revealed that the
A1 hippocampal area MAP-2 immunostaining revealed a focal swelling and amarkedlyicle (panel C) respect to control groups treated with vehicle and 17β estradiol (panels Acal characteristics to the control groups (panels A and B) Scale bar 10 microm
Fig 4 Assessment of the percentage of reactive area of MAP-2 positive dendrites in thestriatum radiatum of CA1 The statistical analysis revealed a significant decrease of thepercentage of reactive area of MAP-2 positive dendrites in PA rats treated with vehicle(PA-Vhi) in comparison to CTL rats treated with vehicle (CTL-Vhi) and PA rats treatedwith 17β estradiol (PA-17β) In addition PA-17β rats did not differ in the percentage ofreactive area of MAP-2 positive dendrites in comparison to CTL-Vhi rats suggesting areversion of dendritic alterations associated with perinatal asphyxia Estradiol by itselfdid not produce any differences in the percentage of reactive area of MAP-2 positivedendrites since CTL-17β rats did not differ in the percentage of reactive area of MAP-2positive dendrites from CTL-Vhi rats Bars and error bars represent mean+SEMPb005 PA-Vhi vs CTL-Vhi daggerPb005 PA-Vhi vs PA-17β
619GE Saraceno et al Experimental Neurology 223 (2010) 615ndash622
main effects of birth condition and treatment and the interaction wereall significant (F=4193 Pb0001 F=779 Pb001 F=158 Pb0001respectively) Post hoc analysis showed that treatment with 17βEstradiol in adults CTL rats had not any effect in the percentage of thereactive area of pHMNF (CTL-Vhi vs CTL-17β P=ns See Figs 7-A 7-B and 8) Rats subjected to PA and injected at adulthood with vehicleshowed a significantly reduced percentage of the reactive area of pHMNF than CTL rats treatedwith vehicle (Pb0005 see Figs 7-C 7-A and 8)
Fig 5 Optical microscope images show a representative example of a striatum radiatum of CAPA animals treated with vehicle (Panel C) respect to CTL injected with vehicle or 17β Estradishowed similar morphological characteristics to control groups (Panel A and B) Scale bar
The treatmentwith 17β Estradiol in PA rats enhanced the percentage ofthe reactive area of pHM NF in comparison to PA rats treated withvehicle (Pb0005 see Figs 7-D 7-C and 8) Furthermore 17β Estradioltreatment in PA rats increased the percentage of the reactive areaof pHM NF to a similar level to the one seen in CTL treated with vehicle(P=ns CTL-Vhi vs AP-17 β See Figs 7-A 7-D and 8)
Discussion
In this work we demonstrate that PA in rats results in permanentchanges in glial andneuronal cells in thehippocampus after fourmonthsof the induction of asphyctic insultWehave focus our studyonCA1 areaof hippocampus since it is one of the most affected areas by PA insult(Petito and Pulsinelli 1984) We have detected increased astrogliosisfocal swelling and fragmentation of CA1 apical dendrites decreasedMAP-2 immunoreactivity and decreased phosphorylated high andmediummolecular weight neurofilaments in the hippocampus of adultrats subjected to PA These alterations are well established markers ofneurodegenerative damage in the central nervous system Treatmentwith 17β estradiol has blocked these alterations in the hippocampalcytoskeleton Thus this data suggests that 17β estradiol could be apossible therapeutic tool for the neurological disorders produced by theasphyxia during birth
Cytoskeletal alterations are blocked by estradiol treatment
Estradiol treatment of adult rats that were subjected to perinatalasphyxia resulted in a decrease in the number of GFAP immunore-active astrocytes in the hippocampus to similar values to those seen incontrol animals These findings are in agreement with previousreports that indicate that estradiol reduces reactive astrogliosis afterdifferent forms of brain injury in adult animals (Arevalo et al in press
1 hippocampal area immunostained for 120 NF No obvious difference were observed inol (Panel A and B respectively) Asphyctic animals treated with 17β Estradiol (Panel D)10 μm
Fig 6 Assessment of the percentage of reactive area of medium molecular weightneurofilaments (120 NF) in the stratum radiatum of CA1 No differences among any ofthe groups were detected by the statistical analysis in agreement with the qualitativeobservation Bars and error bars represent mean+SEM
Fig 8 Assessment of the percentage of reactive area of phosphorylated high and mediummolecular weight neurofilament (pHM NF) in the stratum radiatum of CA1 Estradiol byitself did not produce any statistical differences in the percentage of reactive area of pHMNF since CTL rats treated with estradiol (CTL-17β) did not show differences in thepercentage of reactive area of pHM NF in comparison to CTL rats treated with vehicle(CTL-Vhi) Animals thatwere submitted to PA and treatedwith vehicle (PA-Vhi) showed asignificant decrease of the percentage of reactive area of pHM NF in comparison toCTL-Vhi and PA rats treated with 17β estradiol (PA-17β) Moreover the percentage ofreactive area of pHM NF in PA-17β rats did not differ from that seen in CTL-Vhi ratsdemonstrating that estradiol treatment in adult rats blocked the axonal alterationsassociated with a PA event Bars and error bars represent mean+SEM Pb0005 PA-Vhivs CTL-Vhi daggerdaggerPb0005 PA-Vhi vs PA-17β
620 GE Saraceno et al Experimental Neurology 223 (2010) 615ndash622
Barreto et al 2009) However most studies have tested the antiglioticproperties of estradiol when the hormone was administrated beforeor shortly after the induction of brain damage Our findings indicatethat the hormone is also able to reduce in adult animals chronicreactive astrogliosis caused by an early developmental neurodegen-erative event
Under neurodegenerative conditions astrocytes proliferate and theexpression of GFAP is increased (Eng and Ghirnikar 1994 Pekny andNilsson 2005)Alterations in dendrites are also a commonfindingunderneurodegenerative conditions (Ikonomidou et al 1989 Hsu andBuzsaacuteki 1993 Hori and Carpenter 1994 Matesic and Lin 1994Ramoacuten y Cajal 1995) and the aberrant phosphorylation of neurofila-ments is a hallmarkof axonal degeneration (Grant andPant 2000 Sihaget al 2007) Consistent with this data we have shown in previous workreactive astrogliosis in neostriatum of rat at six months after PA (Cebralet al 2006) In addition we have recently demonstrated that rats at six
Fig 7Optical microscope images show a representative example of a striatum radiatum of CApHMNF immunostaining were detected between PA treated with vehicle (panel C) and conasphyctic animals (panel D) Scale bar 10 microm
month after PA showed clear signs of synaptic neurodegeneration in theneostriatum and accumulation of ubi-proteins (Capani et al 2009)Taken together our findings indicate that the experimental model ofperinatal asphyxia used in this study causes neurodegenerativealterations that are still detectable in adult life The existence ofpermanent alterations in the hippocampus of rats subjected to perinatalasphyxia are in agreement with the permanent neurological deficitsassociated with this pathological condition in humans (Hill and Volpe1981 Amiel-Tison and Ellison 1986 Osborne et al 2004)
The alterations produced by perinatal asphyxia in the volumefraction of immunoreactivity material for phosphorylated high and
1 hippocampal area immunostained for phosphorylated HMNF Obvious differences introl groups (panels A and B) Estradiol treatment reverts the decrease of staining seen in
621GE Saraceno et al Experimental Neurology 223 (2010) 615ndash622
medium molecular weight neurofilaments are in agreement with ourprevious finding of NF alterations in the neostriatum of six month-oldrats (Cebral et al 2006) This data suggested that NF alterations are aconsistent modification induced by PA In addition abnormal NFaccumulations are found in several human neurological diseases suchas amyotrophic lateral sclerosis Parkinsons disease Alzheimersdisease progressive supranuclear palsy CharcotndashMariendashTooth diseasediabetic neuropathy giant axonal neuropathy and NF inclusion disease(Hirano 1994 Mori et al 1996 Bomont et al 2000 Shepherd et al2002) Since NF proteins are preferential targets for oxidative stress(Geacutelinas et al 2000 Hand and Rouleau 2002) estrogens can induce itsneuroprotective functionmodulating free radical productionMoreoverestrogen therapy was able to preserve the microtubule organization ofthedendrite severelymodifiedbyperinatal asphyxia A substantial bodyof evidences suggested that estrogens are related with the facilitationand preservation of neurite growth during the development of nervoussystem although the molecular mechanisms are not still fullyunderstood (Dominguez et al 2004) Recently it was proposed that17β-estradiol-mediated ERK activation is involved in the maintenanceof neuritic arborisation and neuronal morphology in pro-apoptoticconditions (Mintildeano et al 2008)
Conclusions
In conclusion ourfindings suggest that estradiol therapy in adult lifemay reverse neural deficits caused by early brain damage such as theones caused by perinatal asphyxia Therefore in addition to its wellcharacterized neuroprotective actions to prevent neuronal damageestradiol may also have neuro-reparative properties decreasing brainalterations caused by early life events Since estradiol may havenumerous undesirable peripheral effects further studies should addressthe effect of selective estrogen receptor modulators or non-feminizingestrogens whichmay represent amore adequate therapeutic approach
Acknowledgments
This research has been supported by Ministerio de Ciencia eInnovacioacuten Spain (BFU2008-02950-C03-01) IBRO Studentship 2008MAEC-AECID Fellowship 2008 UBACYTM407 and PIP5784 We alsothank to Dr Guelman who provided 17B Estradiol used in this studyGES MLA-B PG and JIR are fellowship holders from CONICET(Argentina)
References
Amiel-Tison C Ellison P 1986 Birth asphyxia in the fullterm newborn early assess-ment and outcome Dev Med Child Neurol 28 671ndash682
Arevalo MA Santos-Galindo M Bellini MJ Azcoitia I Garcia-Segura LM in pressActions of estrogens on glial cells implications for neuroprotection Biochim BiophysActa doi101016jbbagen200910002
Barreto G Santos-Galindo M Diz-Chaves Y Perniacutea O Carrero P Azcoitia I Garcia-Segura LM 2009 Selective estrogen receptor modulators decrease reactiveastrogliosis in the injured brain effects of aging and prolonged depletion of ovarianhormones Endocrinology 150 5010ndash5015
Bjelke B Andersson K Ogren SO Bolme P 1991 Asphyctic lesion proliferation oftyrosine hydroxylase-immunoreactive nerve cell bodies in the rat substantia nigraand functional changes in dopamine neurotransmission Brain Res 543 1ndash9
Bomont P Cavalier L Blondeau F Ben Hamida C Belal S Tazir M Demir ETopaloglu H Korinthenberg R Tuumlysuumlz B Landrieu P Hentati F Koenig M2000 The gene encoding gigaxonin a new member of the cytoskeletal BTBkelchrepeat family is mutated in giant axonal neuropathy Nat Genet 26 370ndash374
Bourque M Dluzen DE Di Paolo T 2009 Neuroprotective actions of sex steroids inParkinsons disease Front Neuroendocrinol 30 142ndash157
Busto R Globus MY Dietrich WD Martinez E Valdeacutes I Ginsberg MD 1989Effect of mild hypothermia on ischemia-induced release of neurotransmitters andfree fatty acids in rat brain Stroke 20 904ndash910
Capani F Loidl F Lopez-Costa JJ Selvin-Testa A Saavedra JP 1997 Ultrastructuralchanges in nitric oxide synthase immunoreactivity in the brain of rats subjected toperinatal asphyxia neuroprotective effects of cold treatment Brain Res 775 11ndash23
Capani F Loidl CF Aguirre F Piehl L Facorro G Hager A De Paoli T Farach HPecci-Saavedra J 2001 Changes in reactive oxygen species (ROS) production in ratbrain during global perinatal asphyxia an ESR study Brain Res 914 204ndash207
Capani F Loidl CF Piehl LL Facorro G De Paoli T Hager A 2003 Long termproduction of reactive oxygen species during perinatal asphyxia in the rat centralnervous system effects of hypothermia Int J Neurosci 113 641ndash654
Capani F Saraceno GE Botti V Aon-Bertolino L de Oliveira DM Barreto GGaleano P Giraldez-Alvarez LD Coirini H 2009 Protein ubiquitination inpostsynaptic densities after hypoxia in rat neostriatum is blocked by hypothermiaExp Neurol 219 404ndash413
Cebral E Capani F Selviacuten-Testa A Funes MR Coirini H Loidl CF 2006 Neostriatalcytoskeleton changes following perinatal asphyxia effect of hypothermia treatmentInt J Neurosci 116 697ndash714
Choi DW Rothman SM 1990 The role of glutamate neurotoxicity in hypoxicndashischemicneuronal death Annu Rev Neurosci 13 171ndash182
Dominguez R Jalali C de Lacalle S 2004 Morphological effects of estrogen oncholinergic neurons in vitro involves activation of extracellular signal-regulatedkinases J Neurosci 24 982ndash990
Dorfman VB Vega MC Coirini H 2006 Age-related changes of the GABA-B receptorin the lumbar spinal cord of male rats and penile erection Life Sci 78 1529ndash1534
Eng LF Ghirnikar RS 1994 GFAP and astrogliosis Brain Pathol 4 229ndash237Farooqui AA Haun SE Horrocks LA 1994 Ischemia and hypoxia In Siegel GJ
Hsu M Buzsaacuteki G 1993 Vulnerability of mossy fiber targets in the rat hippocampus toforebrain ischemia J Neurosci 13 3964ndash3979
Ikonomidou C PriceMTMosinger JL Frierdich G Labruyere J Salles KS Olney JW1989 Hypobaricndashischemic conditions produce glutamate-like cytopathology in infantrat brain J Neurosci 9 1693ndash1700
Kamme F Wieloch T 1996 Induction of junD mRNA after transient forebrainischemia in the rat Effect of hypothermia Brain Res Mol Brain Res 43 51ndash56
Kipp M Beyer C 2009 Impact of sex steroids on neuroinflammatory processes andexperimental multiple sclerosis Front Neuroendocrinol 30 188ndash200
Kirino T 1982 Delayed neuronal death in the gerbil hippocampus following ischemiaBrain Res 239 57ndash69
Kruse MS Rey M Barutta J Coirini H 2009 Allopregnanolone effects onastrogliosis induced by hypoxia in organotypic cultures of striatum hippocampusand neocortex Brain Res 1303 1ndash7
Lebesgue D Chevaleyre V Zukin RS Etgen AM 2009 Estradiol rescues neuronsfrom global ischemia-induced cell death multiple cellular pathways of neuropro-tection Steroids 74 555ndash561
Loidl CF Capani F Loacutepez-Costa JJ Selviacuten-Testa A Loacutepez EM Pecci-Saavedra J1997 Long term changes in NADPH-diaphorase reactivity in striatal and corticalneurons following experimental perinatal asphyxia neuroprotective effects ofhypothermia Int J Neurosci 89 1ndash14
Loidl CF Gavilanes AWVanDijk EH VreulsW Blokland A Vles JS SteinbuschHWBlanco CE 2000 Effects of hypothermia and gender on survival and behavior afterperinatal asphyxia in rats Physiol Behav 68 263ndash269
Matesic DF Lin RC 1994 Microtubule-associated protein 2 as an early indicator ofischemia-induced neurodegeneration in the gerbil forebrain J Neurochem 631012ndash1020
Mintildeano A Xifroacute X Peacuterez V Barneda-Zahonero B Saura CA Rodriacuteguez-Alvarez J2008 Estradiol facilitates neurite maintenance by a SrcRasERK signalling pathwayMol Cell Neurosci 39 143ndash151
Mori H Oda M Mizuno Y 1996 Cortical ballooned neurons in progressivesupranuclear palsy Neurosci Lett 209 109ndash112
Osborne NN Casson RJ Wood JP Chidlow G Graham M Melena J 2004 Retinalischemia mechanisms of damage and potential therapeutic strategies Prog RetinEye Res 23 91ndash147
Pekny M Nilsson M 2005 Astrocyte activation and reactive gliosis Glia 50 427ndash434Petito CK Pulsinelli WA 1984 Delayed neuronal recovery and neuronal death in rat
hippocampus following severe cerebral ischemiapossible relationship toabnormalitiesin neuronal processes J Cereb Blood Flow Metab 4 194ndash205
Pike CJ Carroll JC Rosario ER Barron AM 2009 Protective actions of sex steroidhormones in Alzheimers disease Front Neuroendocrinol 30 239ndash258
622 GE Saraceno et al Experimental Neurology 223 (2010) 615ndash622
Ramoacuten y Cajal S 1995 Histology of the Nervous System of Man and Vertebrates OxfordUniversity Press New York Translated by N Swanson and LW Swanson
Shankaran S 2009 Neonatal encephalopathy treatmentwith hypothermia J Neurotrauma26 437ndash443
Shepherd CE McCann H Thiel E Halliday GM 2002 Neurofilament-immunoreactiveneurons inAlzheimersdiseaseanddementiawithLewybodiesNeurobiolDis 9249ndash257
Sihag RK Inagaki M Yamaguchi T Shea TB Pant HC 2007 Role of phosphorylationon the structuraldynamics and functionof types III and IV intermediatefilaments ExpCell Res 313 2098ndash2109
Suzuki S Brown CM Wise PM 2009 Neuroprotective effects of estrogens followingischemic stroke Front Neuroendocrinol 30 201ndash211
Tapia-Gonzalez S Carrero P Pernia O Garcia-Segura LM Diz-Chaves Y 2008Selective oestrogen receptor (ER) modulators reduce microglia reactivity in vivoafter peripheral inflammation potential role of microglial ERs J Endocrinol 198219ndash230
Van de Berg WD Kwaijtaal M de Louw AJ Lissone NP Schmitz C Faull RLBlokland A Blanco CE Steinbusch HW 2003 Impact of perinatal asphyxia onthe GABAergic and locomotor system Neuroscience 117 83ndash96
Vannucci RC Perlman JM 1997 Interventions for perinatal hypoxicndashischemic ence-phalopathy Pediatrics 100 1004ndash1014
Weibel ER 1979 Stereological Methods Practical Methods for Biological MorphometryVol I Academic Press New York
616 GE Saraceno et al Experimental Neurology 223 (2010) 615ndash622
We have extensively worked on experimental hypothermia astherapeutic tool to reduce thedamageproducebyhypoxia (Capani et al1997 2003 2009) Experimental hypothermia has shown to blockmostof the processes that trigger the mechanisms for the generation of thecell death (Capani et al 2009) Although hypothermia has been alreadyused in clinical trials it has been shown to be effective only in veryrestrictive neurological pathologies produced by PA (Gonzalez andFerriero 2008 Shankaran 2009)
According to our knowledge (Capani et al 2009) there is not aproper therapeutic tool to treat the neurological diseases produced byPA Therefore in this study we test if estrogens are effective to reducethe experimental chronic cytological alterations induced by PA in ratsOur main aims were to determine (i) whether alterations in the CA1area of hippocampus were still detectable in four-month-old animalssubmitted to perinatal asphyxia and (ii) whether these alterationscould be reverted in adult rats by the treatment with 17β-estradiol
Materials and methods
Animals
All procedures involving animals were approved by the Institu-tional Animal Care and Use Committee at the University of BuenosAires (School of Medicine) and conducted according to the principlesof the Guide for the Care and Use of Laboratory Animals (NIHPublications No 80-23 revised 1996) SpraguendashDawley female rats inthe fifteenth day of pregnancy were placed in individual cages andmaintained on a 1212 h lightdark cycle in a controlled temperature(21plusmn2 degC) and humidity (65plusmn5) environment The animals hadaccess to food (Purina chow) and tap water ad libitum One group ofanimals (n=4) were used as surrogate mothers another group(n=5) were assigned to perinatal asphyxia procedures and theremaining animals (n=5) were the mothers of the control pups
Induction of asphyxia
Five full-term pregnant rats on gestational day 22 were anesthe-tized (Dorfman et al 2006) rapidly decapitated and the uterus hornswere isolated through an abdominal incision and placed in a waterbath at 37 degC for 19 min (sub-severe perinatal asphyxia) (Bjelke et al1991 Van de Berg et al 2003 Capani et al 2009) We used 19 min asthe maximum time of PA because 21 or more minutes in thiscondition result in a survival rate lower than 3 (Capani et al 2009)Following asphyxia the uterus horns were rapidly opened the pupswere removed the amniotic fluid was cleaned and the pups werestimulated to breathe by performing tactile intermittent stimulationwith pieces of medical wipes for a fewminutes until regular breathingwas established The umbilical cord was ligated and the animals wereleft to recover for 1 h under a heating lamp When their physiologicalconditions improved they were given to surrogate mothers who haddelivered normally within the last 24 h The different groups of pupswere marked and mixed with the surrogate mothers normal litters(control animals that were left undisturbed) Wemaintained litters of10 pups with each surrogate mother
17β estradiol treatment
Sixteen adult male rats were injected ip 117 days after perinatalasphyxia with 17β estradiol (water soluble E4389 Sigma St Louis Mo250 microgkg) or vehicle (09 saline solution) Previous studies haveshown that doses of estradiol not very different from the one selected forthis study reduce gliosis in male rats after a penetrating brain injury orafter immune challenge (Tapia-Gonzalez et al 2008 Barreto et al2009) The injectionswere repeated daily for 3 days consecutively (up to119 days after perinatal asphyxia) Animals were distributed in 4experimental groups (i) control animals injected with vehicle (ii)
control animals injected with 17β estradiol (iii) animals submitted toperinatal asphyxia and injectedwith vehicle and (iv) animals submittedto perinatal asphyxia and treated with 17β estradiol
Tissue fixation and immunohistochemistry
At postnatal day 120 sixteen animals were anesthetized withchloral hydrate (28 wv 01 ml100 g body weight) and perfusedintracardially with 4 paraformaldehyde in 01 M phosphate bufferpH 74 Brains were removed and post-fixed in the same fixativesolution for 2 h at room temperature and then immersed overnight at4 degC in 01 M phosphate buffer pH 74 Coronal hippocampal sections(40 μm thickness) were obtained using a Vibratome (VT 1000 S LeicaMicrosystems Wetzlar Germany)
Immunohistochemistry was performed on free floating sectionsunder moderate shaking Endogenous peroxidase was quenched (3H2O2 30 methanol 70 PBS 01 M) and non-specific labeling wasblocked using 5 normal goat serum Sections were incubatedovernight at 4 degC with a mouse primary antibody that recognizes120 kDa neurofilaments (120 NF 1500 Chemicon InternationalTemecula CA USA) or a mouse primary antibody that recognizesphosphorylated high and medium molecular weight neurofilamentproteins (pHM NF 1250 Chemicon International Temecula CAUSA) overnight at 4 degC After several washes the sections wereincubated for 2 h at room temperature with secondary antibodies(Biotinylated anti mouse IgG diluted 1300 Vector Laboratories IncBurlingame CA USA) Labelingwas revealed using the ABC kit (VectorLaboratories Inc Burlingame CA USA) Peroxidase activity wasrevealed with 001 hydrogen peroxide using 33prime-diaminobenzidineas the chromogen (DAB Sigma) Immunostaining was absent whenthe first antibody was omitted
For immunofluorescence tissue sections were blocked for 30 minin phosphate-buffered saline (PBS) containing 03 Bovine SerumAlbumin (BSA Sigma St Louis MO USA) and 03 Triton X-100Sectionswere incubated overnight at 4 degCwith a rabbit polyclonal antiGFAP antibody (12000 Sigma St Louis MO USA) or a mousemonoclonal anti MAP-2 antibody (1500 Sigma St Louis MO USA)After washing in buffer tissue sections were incubated for 2 h at roomtemperature with Alexa 488 goat anti-rabbit IgG (1200 MolecularProbes) or Alexa 594 goat anti-mouse IgG (1200 Molecular Probes)Sections were counterstained with DAPI (Vector Laboratories IncBurlingame CA USA) to label cell nuclei and mounted withVectashield mounting medium Immunostaining was absent whenthe first antibody was omitted In order to minimize inter-assayvariations samples from all experimental groups were processed inparallel
Morphometric analysis
The volume fraction of immunoreactive material for MAP-2 120 NFand pHM NF was estimated using the point-counting method ofWeibel (1979) and a grid delimiting 5000 μm2 in the striatum radiatumof CA1 A total area of 75000 μm2 was evaluated in each animalPercentage of reactive areawasestimatedusing Image J Program(ImageJ 141o NIH USA) The number of GFAP immunoreactive astrocytes wasestimated in the striatum radiatum of CA1 by the optical dissectormethod (Howard and Reed 1998) using total section thickness fordissector height (Hatton and von Bartheld 1999) and a counting frameof 55times55 μm A total of 78 counting frames were assessed per animalSection thickness was measured using a digital length gauge device(Heidenhain-Metro MT 12ND221 Traunreut Germany) attached tothe stage of a Leitz microscope Cell nuclei from GFAP immunoreactivecells that came into focus while focusing down through the dissectorheightwere counted All counts were performed on coded sections Thevolume of the striatum radiatum of CA1 was estimated using the point-counting method of Weibel (1979) Since no significant differences in
617GE Saraceno et al Experimental Neurology 223 (2010) 615ndash622
this parameter were observed among the different experimentalgroups the changes in the number of GFAP immunoreactive cells perunit volume with the optical dissector method are assumed to reflectchanges in the total number of GFAP immunoreactive cells
Statistical analysis
Material from four rats was analyzed for each experimental groupand for each parameter studied (n=16) All statistical analyses wereperformed by two-way analysis of variances (ANOVAs) with birthcondition (CTL and PA) and treatment (Vhi and 17β) as the mainfactors When interaction effects were significant analyses of thesimple effects were carried out by post hoc comparisons usingStudents t-test (two-tailed) adjusted by Bonferroni correction In anycase neither the assumption of normal distribution (ShapirondashWilktest) nor equality of variances (Levenes test) was rejected Resultswere expressed as the meanplusmnSEM Differences with a probability of5 or less were considered to be significant (Pb005) All statisticalanalyses were done using the PASW Statistics 18 software (SPSS IncChicago IL USA)
Results
GFAP immunostaining in the CA1 hippocampal area Effects of PA and17β treatment
Hippocampal sections from PA animals treated with vehiclestained with GFAP and observed at confocal microscopy showed amarked astrogliosis in striatum radiatum of CA1 hippocampal area
Fig 1 Confocal microscope images of GFAP immunostaining from the striatum radiatum ofmarked reactive astrogliosis (panel C) respect to controls injected with vehicle and 17β estrastrogliosis in PA animals Scale bar 10 microm
which is a characteristic feature for chronic process of neurodegen-eration (Fig 1C) The morphometric analysis in the striatum radiatumof CA1 confirmed this data The two-way analysis of varianceindicated that the main factors of birth condition and treatmentwere both significant (F=2676 Pb0001 F=827 Pb001 respec-tively) and the interaction was also significant (F=2344 Pb001)Post hoc analysis of the simple effects revealed that treatment with17β estradiol in CTL rats had not significant effect on the number ofGFAP immunoreactive astrocytes in comparison to CTL rats injectedwith vehicle (P=ns see Figs 1-B 1-A and 2) PA rats injected withvehicle showed amarkedly significant increase of the number of GFAPimmunoreactive astrocytes in comparison to CTL rats treated withvehicle (Pb0005 see Figs 1-C 1-A and 2) Estradiol treatment in PArats significantly reduced the number of GFAP immunoreactiveastrocytes in comparison to PA rats treated with vehicle (Pb0005see Figs 1-D 1-C and 2) and moreover they did not show significantdifferences with CTL rats injected with vehicle (P=ns see Figs 1-D1-A and 2) suggesting a reversion of astrogliosis associated withhypoxia at birth by 17β estradiol treatment in the adulthood
Dendritic alterations induced by PA and reversion by 17β estradioltreatment
Confocal studies using MAP-2 were conducted to determine thedendritic changes after PA insult Animals subjected to PA and treatedwith vehicle showed focal swelling and markedly fragmentedappearance of MAP-2 immunoreactive apical dendrites in the CA1hippocampal area compared to control animals treated with vehicle(see Figs 3-A and 3-C) In addition the statistical analysis revealed
CA1 hippocampal area The hippocampus of PA animals treated with vehicle showed aadiol (panels A and B) See in panel D that 17β estradiol treatment reduce the reactive
Fig 2 Assessment of the number of GFAP immunoreactive astrocytes in the striatumradiatum of CA1 Estradiol by itself did not produce any statistical differences in thenumberof GFAP immunoreactive astrocytes since CTL rats treatedwith estradiol (CTL-17β) did notshowdifferences in thenumber ofGFAP+astrocytes in comparison to CTL rats treatedwithvehicle (CTL-Vhi) Animals that were submitted to PA and treated with vehicle (PA-Vhi)showeda significant increase ofGFAP immunoreactive astrocytes in comparison to CTL-Vhiand PA rats treated with 17β estradiol (PA-17β) Moreover number of GFAPimmunoreactive astrocytes in PA-17β rats did not differ from those seen in CTL-Vhi ratsdemonstrating that estradiol treatment in adult rats blocked the reactive astrogliosisassociated with a PA event Bars and error bars represent mean+SEM Pb0005 PA-Vhivs CTL-Vhi daggerdaggerPb0005 PA-Vhi vs PA-17β
618 GE Saraceno et al Experimental Neurology 223 (2010) 615ndash622
that neither the main factor of birth condition nor the main factor oftreatment was significant (F=166 P=ns F=299 P=ns respec-tively) but the interaction was (F=623 P=0016) Post hoc analysis
Fig 3 Confocal microscope images of MAP-2 immunostaining from the striatum radiatum of Cfragmented appearance of distal dendrites in the CA1 region of PA animals injected with vehand B) Asphyctic animals injected with 17β estradiol (panel D) shows similar morphologi
indicated that 17β estradiol treatment in CTL rats did not produce anysignificant changes of the percentage of reactive area of MAP-2positive dendrites with respect to CTL rats injected with vehicle(P=ns see Figs 3-B 3-A and 4) Rats subjected to PA and treatedwith vehicle showed a significant reduction of the percentage ofreactive area of MAP-2 positive dendrites in comparison to CTL ratsinjected with vehicle (Pb005 see Figs 3-C 3-A and 4) Estradioltreatment in adults rats that suffered from PA significantly improvedthe mean percentage of reactive area of MAP-2 positive dendrites incomparison to PA rats treated with vehicle (Pb005 see Figs 3-D 3-Cand 4) while no differences were detected between PA rats treatedwith 17β estradiol and CTL rats injected with vehicle (P=ns seeFigs 3-D 3-A and 4)
Effects of PA and 17β estradiol treatment on axonal neurofilaments
Axonal alterations were studied using immunocytochemistry formediummolecular weight neurofilaments (120 NF) and phosphorylat-edhighandmediummolecularweightneurofilaments (pHMNF) Fig 5shows a representative example of a hippocampal section immunos-tained for 120 NF No obvious differences in 120 NF immunostainingwere detected between animals subjected to PA and control animals Inagreement with the qualitative observations the statistical analysisshowed that neither the main factor of birth condition nor the mainfactor of treatment nor the interaction was significant (Fb1 F=23P=ns Pb1 respectively See Fig 6) In contrast noticeable changeswere seen in the volume fraction of immunoreactive material forphosphorylated high and medium molecular weight neurofilamentsafter PA and 17β treatment The two-way ANOVA revealed that the
A1 hippocampal area MAP-2 immunostaining revealed a focal swelling and amarkedlyicle (panel C) respect to control groups treated with vehicle and 17β estradiol (panels Acal characteristics to the control groups (panels A and B) Scale bar 10 microm
Fig 4 Assessment of the percentage of reactive area of MAP-2 positive dendrites in thestriatum radiatum of CA1 The statistical analysis revealed a significant decrease of thepercentage of reactive area of MAP-2 positive dendrites in PA rats treated with vehicle(PA-Vhi) in comparison to CTL rats treated with vehicle (CTL-Vhi) and PA rats treatedwith 17β estradiol (PA-17β) In addition PA-17β rats did not differ in the percentage ofreactive area of MAP-2 positive dendrites in comparison to CTL-Vhi rats suggesting areversion of dendritic alterations associated with perinatal asphyxia Estradiol by itselfdid not produce any differences in the percentage of reactive area of MAP-2 positivedendrites since CTL-17β rats did not differ in the percentage of reactive area of MAP-2positive dendrites from CTL-Vhi rats Bars and error bars represent mean+SEMPb005 PA-Vhi vs CTL-Vhi daggerPb005 PA-Vhi vs PA-17β
619GE Saraceno et al Experimental Neurology 223 (2010) 615ndash622
main effects of birth condition and treatment and the interaction wereall significant (F=4193 Pb0001 F=779 Pb001 F=158 Pb0001respectively) Post hoc analysis showed that treatment with 17βEstradiol in adults CTL rats had not any effect in the percentage of thereactive area of pHMNF (CTL-Vhi vs CTL-17β P=ns See Figs 7-A 7-B and 8) Rats subjected to PA and injected at adulthood with vehicleshowed a significantly reduced percentage of the reactive area of pHMNF than CTL rats treatedwith vehicle (Pb0005 see Figs 7-C 7-A and 8)
Fig 5 Optical microscope images show a representative example of a striatum radiatum of CAPA animals treated with vehicle (Panel C) respect to CTL injected with vehicle or 17β Estradishowed similar morphological characteristics to control groups (Panel A and B) Scale bar
The treatmentwith 17β Estradiol in PA rats enhanced the percentage ofthe reactive area of pHM NF in comparison to PA rats treated withvehicle (Pb0005 see Figs 7-D 7-C and 8) Furthermore 17β Estradioltreatment in PA rats increased the percentage of the reactive areaof pHM NF to a similar level to the one seen in CTL treated with vehicle(P=ns CTL-Vhi vs AP-17 β See Figs 7-A 7-D and 8)
Discussion
In this work we demonstrate that PA in rats results in permanentchanges in glial andneuronal cells in thehippocampus after fourmonthsof the induction of asphyctic insultWehave focus our studyonCA1 areaof hippocampus since it is one of the most affected areas by PA insult(Petito and Pulsinelli 1984) We have detected increased astrogliosisfocal swelling and fragmentation of CA1 apical dendrites decreasedMAP-2 immunoreactivity and decreased phosphorylated high andmediummolecular weight neurofilaments in the hippocampus of adultrats subjected to PA These alterations are well established markers ofneurodegenerative damage in the central nervous system Treatmentwith 17β estradiol has blocked these alterations in the hippocampalcytoskeleton Thus this data suggests that 17β estradiol could be apossible therapeutic tool for the neurological disorders produced by theasphyxia during birth
Cytoskeletal alterations are blocked by estradiol treatment
Estradiol treatment of adult rats that were subjected to perinatalasphyxia resulted in a decrease in the number of GFAP immunore-active astrocytes in the hippocampus to similar values to those seen incontrol animals These findings are in agreement with previousreports that indicate that estradiol reduces reactive astrogliosis afterdifferent forms of brain injury in adult animals (Arevalo et al in press
1 hippocampal area immunostained for 120 NF No obvious difference were observed inol (Panel A and B respectively) Asphyctic animals treated with 17β Estradiol (Panel D)10 μm
Fig 6 Assessment of the percentage of reactive area of medium molecular weightneurofilaments (120 NF) in the stratum radiatum of CA1 No differences among any ofthe groups were detected by the statistical analysis in agreement with the qualitativeobservation Bars and error bars represent mean+SEM
Fig 8 Assessment of the percentage of reactive area of phosphorylated high and mediummolecular weight neurofilament (pHM NF) in the stratum radiatum of CA1 Estradiol byitself did not produce any statistical differences in the percentage of reactive area of pHMNF since CTL rats treated with estradiol (CTL-17β) did not show differences in thepercentage of reactive area of pHM NF in comparison to CTL rats treated with vehicle(CTL-Vhi) Animals thatwere submitted to PA and treatedwith vehicle (PA-Vhi) showed asignificant decrease of the percentage of reactive area of pHM NF in comparison toCTL-Vhi and PA rats treated with 17β estradiol (PA-17β) Moreover the percentage ofreactive area of pHM NF in PA-17β rats did not differ from that seen in CTL-Vhi ratsdemonstrating that estradiol treatment in adult rats blocked the axonal alterationsassociated with a PA event Bars and error bars represent mean+SEM Pb0005 PA-Vhivs CTL-Vhi daggerdaggerPb0005 PA-Vhi vs PA-17β
620 GE Saraceno et al Experimental Neurology 223 (2010) 615ndash622
Barreto et al 2009) However most studies have tested the antiglioticproperties of estradiol when the hormone was administrated beforeor shortly after the induction of brain damage Our findings indicatethat the hormone is also able to reduce in adult animals chronicreactive astrogliosis caused by an early developmental neurodegen-erative event
Under neurodegenerative conditions astrocytes proliferate and theexpression of GFAP is increased (Eng and Ghirnikar 1994 Pekny andNilsson 2005)Alterations in dendrites are also a commonfindingunderneurodegenerative conditions (Ikonomidou et al 1989 Hsu andBuzsaacuteki 1993 Hori and Carpenter 1994 Matesic and Lin 1994Ramoacuten y Cajal 1995) and the aberrant phosphorylation of neurofila-ments is a hallmarkof axonal degeneration (Grant andPant 2000 Sihaget al 2007) Consistent with this data we have shown in previous workreactive astrogliosis in neostriatum of rat at six months after PA (Cebralet al 2006) In addition we have recently demonstrated that rats at six
Fig 7Optical microscope images show a representative example of a striatum radiatum of CApHMNF immunostaining were detected between PA treated with vehicle (panel C) and conasphyctic animals (panel D) Scale bar 10 microm
month after PA showed clear signs of synaptic neurodegeneration in theneostriatum and accumulation of ubi-proteins (Capani et al 2009)Taken together our findings indicate that the experimental model ofperinatal asphyxia used in this study causes neurodegenerativealterations that are still detectable in adult life The existence ofpermanent alterations in the hippocampus of rats subjected to perinatalasphyxia are in agreement with the permanent neurological deficitsassociated with this pathological condition in humans (Hill and Volpe1981 Amiel-Tison and Ellison 1986 Osborne et al 2004)
The alterations produced by perinatal asphyxia in the volumefraction of immunoreactivity material for phosphorylated high and
1 hippocampal area immunostained for phosphorylated HMNF Obvious differences introl groups (panels A and B) Estradiol treatment reverts the decrease of staining seen in
621GE Saraceno et al Experimental Neurology 223 (2010) 615ndash622
medium molecular weight neurofilaments are in agreement with ourprevious finding of NF alterations in the neostriatum of six month-oldrats (Cebral et al 2006) This data suggested that NF alterations are aconsistent modification induced by PA In addition abnormal NFaccumulations are found in several human neurological diseases suchas amyotrophic lateral sclerosis Parkinsons disease Alzheimersdisease progressive supranuclear palsy CharcotndashMariendashTooth diseasediabetic neuropathy giant axonal neuropathy and NF inclusion disease(Hirano 1994 Mori et al 1996 Bomont et al 2000 Shepherd et al2002) Since NF proteins are preferential targets for oxidative stress(Geacutelinas et al 2000 Hand and Rouleau 2002) estrogens can induce itsneuroprotective functionmodulating free radical productionMoreoverestrogen therapy was able to preserve the microtubule organization ofthedendrite severelymodifiedbyperinatal asphyxia A substantial bodyof evidences suggested that estrogens are related with the facilitationand preservation of neurite growth during the development of nervoussystem although the molecular mechanisms are not still fullyunderstood (Dominguez et al 2004) Recently it was proposed that17β-estradiol-mediated ERK activation is involved in the maintenanceof neuritic arborisation and neuronal morphology in pro-apoptoticconditions (Mintildeano et al 2008)
Conclusions
In conclusion ourfindings suggest that estradiol therapy in adult lifemay reverse neural deficits caused by early brain damage such as theones caused by perinatal asphyxia Therefore in addition to its wellcharacterized neuroprotective actions to prevent neuronal damageestradiol may also have neuro-reparative properties decreasing brainalterations caused by early life events Since estradiol may havenumerous undesirable peripheral effects further studies should addressthe effect of selective estrogen receptor modulators or non-feminizingestrogens whichmay represent amore adequate therapeutic approach
Acknowledgments
This research has been supported by Ministerio de Ciencia eInnovacioacuten Spain (BFU2008-02950-C03-01) IBRO Studentship 2008MAEC-AECID Fellowship 2008 UBACYTM407 and PIP5784 We alsothank to Dr Guelman who provided 17B Estradiol used in this studyGES MLA-B PG and JIR are fellowship holders from CONICET(Argentina)
References
Amiel-Tison C Ellison P 1986 Birth asphyxia in the fullterm newborn early assess-ment and outcome Dev Med Child Neurol 28 671ndash682
Arevalo MA Santos-Galindo M Bellini MJ Azcoitia I Garcia-Segura LM in pressActions of estrogens on glial cells implications for neuroprotection Biochim BiophysActa doi101016jbbagen200910002
Barreto G Santos-Galindo M Diz-Chaves Y Perniacutea O Carrero P Azcoitia I Garcia-Segura LM 2009 Selective estrogen receptor modulators decrease reactiveastrogliosis in the injured brain effects of aging and prolonged depletion of ovarianhormones Endocrinology 150 5010ndash5015
Bjelke B Andersson K Ogren SO Bolme P 1991 Asphyctic lesion proliferation oftyrosine hydroxylase-immunoreactive nerve cell bodies in the rat substantia nigraand functional changes in dopamine neurotransmission Brain Res 543 1ndash9
Bomont P Cavalier L Blondeau F Ben Hamida C Belal S Tazir M Demir ETopaloglu H Korinthenberg R Tuumlysuumlz B Landrieu P Hentati F Koenig M2000 The gene encoding gigaxonin a new member of the cytoskeletal BTBkelchrepeat family is mutated in giant axonal neuropathy Nat Genet 26 370ndash374
Bourque M Dluzen DE Di Paolo T 2009 Neuroprotective actions of sex steroids inParkinsons disease Front Neuroendocrinol 30 142ndash157
Busto R Globus MY Dietrich WD Martinez E Valdeacutes I Ginsberg MD 1989Effect of mild hypothermia on ischemia-induced release of neurotransmitters andfree fatty acids in rat brain Stroke 20 904ndash910
Capani F Loidl F Lopez-Costa JJ Selvin-Testa A Saavedra JP 1997 Ultrastructuralchanges in nitric oxide synthase immunoreactivity in the brain of rats subjected toperinatal asphyxia neuroprotective effects of cold treatment Brain Res 775 11ndash23
Capani F Loidl CF Aguirre F Piehl L Facorro G Hager A De Paoli T Farach HPecci-Saavedra J 2001 Changes in reactive oxygen species (ROS) production in ratbrain during global perinatal asphyxia an ESR study Brain Res 914 204ndash207
Capani F Loidl CF Piehl LL Facorro G De Paoli T Hager A 2003 Long termproduction of reactive oxygen species during perinatal asphyxia in the rat centralnervous system effects of hypothermia Int J Neurosci 113 641ndash654
Capani F Saraceno GE Botti V Aon-Bertolino L de Oliveira DM Barreto GGaleano P Giraldez-Alvarez LD Coirini H 2009 Protein ubiquitination inpostsynaptic densities after hypoxia in rat neostriatum is blocked by hypothermiaExp Neurol 219 404ndash413
Cebral E Capani F Selviacuten-Testa A Funes MR Coirini H Loidl CF 2006 Neostriatalcytoskeleton changes following perinatal asphyxia effect of hypothermia treatmentInt J Neurosci 116 697ndash714
Choi DW Rothman SM 1990 The role of glutamate neurotoxicity in hypoxicndashischemicneuronal death Annu Rev Neurosci 13 171ndash182
Dominguez R Jalali C de Lacalle S 2004 Morphological effects of estrogen oncholinergic neurons in vitro involves activation of extracellular signal-regulatedkinases J Neurosci 24 982ndash990
Dorfman VB Vega MC Coirini H 2006 Age-related changes of the GABA-B receptorin the lumbar spinal cord of male rats and penile erection Life Sci 78 1529ndash1534
Eng LF Ghirnikar RS 1994 GFAP and astrogliosis Brain Pathol 4 229ndash237Farooqui AA Haun SE Horrocks LA 1994 Ischemia and hypoxia In Siegel GJ
Hsu M Buzsaacuteki G 1993 Vulnerability of mossy fiber targets in the rat hippocampus toforebrain ischemia J Neurosci 13 3964ndash3979
Ikonomidou C PriceMTMosinger JL Frierdich G Labruyere J Salles KS Olney JW1989 Hypobaricndashischemic conditions produce glutamate-like cytopathology in infantrat brain J Neurosci 9 1693ndash1700
Kamme F Wieloch T 1996 Induction of junD mRNA after transient forebrainischemia in the rat Effect of hypothermia Brain Res Mol Brain Res 43 51ndash56
Kipp M Beyer C 2009 Impact of sex steroids on neuroinflammatory processes andexperimental multiple sclerosis Front Neuroendocrinol 30 188ndash200
Kirino T 1982 Delayed neuronal death in the gerbil hippocampus following ischemiaBrain Res 239 57ndash69
Kruse MS Rey M Barutta J Coirini H 2009 Allopregnanolone effects onastrogliosis induced by hypoxia in organotypic cultures of striatum hippocampusand neocortex Brain Res 1303 1ndash7
Lebesgue D Chevaleyre V Zukin RS Etgen AM 2009 Estradiol rescues neuronsfrom global ischemia-induced cell death multiple cellular pathways of neuropro-tection Steroids 74 555ndash561
Loidl CF Capani F Loacutepez-Costa JJ Selviacuten-Testa A Loacutepez EM Pecci-Saavedra J1997 Long term changes in NADPH-diaphorase reactivity in striatal and corticalneurons following experimental perinatal asphyxia neuroprotective effects ofhypothermia Int J Neurosci 89 1ndash14
Loidl CF Gavilanes AWVanDijk EH VreulsW Blokland A Vles JS SteinbuschHWBlanco CE 2000 Effects of hypothermia and gender on survival and behavior afterperinatal asphyxia in rats Physiol Behav 68 263ndash269
Matesic DF Lin RC 1994 Microtubule-associated protein 2 as an early indicator ofischemia-induced neurodegeneration in the gerbil forebrain J Neurochem 631012ndash1020
Mintildeano A Xifroacute X Peacuterez V Barneda-Zahonero B Saura CA Rodriacuteguez-Alvarez J2008 Estradiol facilitates neurite maintenance by a SrcRasERK signalling pathwayMol Cell Neurosci 39 143ndash151
Mori H Oda M Mizuno Y 1996 Cortical ballooned neurons in progressivesupranuclear palsy Neurosci Lett 209 109ndash112
Osborne NN Casson RJ Wood JP Chidlow G Graham M Melena J 2004 Retinalischemia mechanisms of damage and potential therapeutic strategies Prog RetinEye Res 23 91ndash147
Pekny M Nilsson M 2005 Astrocyte activation and reactive gliosis Glia 50 427ndash434Petito CK Pulsinelli WA 1984 Delayed neuronal recovery and neuronal death in rat
hippocampus following severe cerebral ischemiapossible relationship toabnormalitiesin neuronal processes J Cereb Blood Flow Metab 4 194ndash205
Pike CJ Carroll JC Rosario ER Barron AM 2009 Protective actions of sex steroidhormones in Alzheimers disease Front Neuroendocrinol 30 239ndash258
622 GE Saraceno et al Experimental Neurology 223 (2010) 615ndash622
Ramoacuten y Cajal S 1995 Histology of the Nervous System of Man and Vertebrates OxfordUniversity Press New York Translated by N Swanson and LW Swanson
Shankaran S 2009 Neonatal encephalopathy treatmentwith hypothermia J Neurotrauma26 437ndash443
Shepherd CE McCann H Thiel E Halliday GM 2002 Neurofilament-immunoreactiveneurons inAlzheimersdiseaseanddementiawithLewybodiesNeurobiolDis 9249ndash257
Sihag RK Inagaki M Yamaguchi T Shea TB Pant HC 2007 Role of phosphorylationon the structuraldynamics and functionof types III and IV intermediatefilaments ExpCell Res 313 2098ndash2109
Suzuki S Brown CM Wise PM 2009 Neuroprotective effects of estrogens followingischemic stroke Front Neuroendocrinol 30 201ndash211
Tapia-Gonzalez S Carrero P Pernia O Garcia-Segura LM Diz-Chaves Y 2008Selective oestrogen receptor (ER) modulators reduce microglia reactivity in vivoafter peripheral inflammation potential role of microglial ERs J Endocrinol 198219ndash230
Van de Berg WD Kwaijtaal M de Louw AJ Lissone NP Schmitz C Faull RLBlokland A Blanco CE Steinbusch HW 2003 Impact of perinatal asphyxia onthe GABAergic and locomotor system Neuroscience 117 83ndash96
Vannucci RC Perlman JM 1997 Interventions for perinatal hypoxicndashischemic ence-phalopathy Pediatrics 100 1004ndash1014
Weibel ER 1979 Stereological Methods Practical Methods for Biological MorphometryVol I Academic Press New York
617GE Saraceno et al Experimental Neurology 223 (2010) 615ndash622
this parameter were observed among the different experimentalgroups the changes in the number of GFAP immunoreactive cells perunit volume with the optical dissector method are assumed to reflectchanges in the total number of GFAP immunoreactive cells
Statistical analysis
Material from four rats was analyzed for each experimental groupand for each parameter studied (n=16) All statistical analyses wereperformed by two-way analysis of variances (ANOVAs) with birthcondition (CTL and PA) and treatment (Vhi and 17β) as the mainfactors When interaction effects were significant analyses of thesimple effects were carried out by post hoc comparisons usingStudents t-test (two-tailed) adjusted by Bonferroni correction In anycase neither the assumption of normal distribution (ShapirondashWilktest) nor equality of variances (Levenes test) was rejected Resultswere expressed as the meanplusmnSEM Differences with a probability of5 or less were considered to be significant (Pb005) All statisticalanalyses were done using the PASW Statistics 18 software (SPSS IncChicago IL USA)
Results
GFAP immunostaining in the CA1 hippocampal area Effects of PA and17β treatment
Hippocampal sections from PA animals treated with vehiclestained with GFAP and observed at confocal microscopy showed amarked astrogliosis in striatum radiatum of CA1 hippocampal area
Fig 1 Confocal microscope images of GFAP immunostaining from the striatum radiatum ofmarked reactive astrogliosis (panel C) respect to controls injected with vehicle and 17β estrastrogliosis in PA animals Scale bar 10 microm
which is a characteristic feature for chronic process of neurodegen-eration (Fig 1C) The morphometric analysis in the striatum radiatumof CA1 confirmed this data The two-way analysis of varianceindicated that the main factors of birth condition and treatmentwere both significant (F=2676 Pb0001 F=827 Pb001 respec-tively) and the interaction was also significant (F=2344 Pb001)Post hoc analysis of the simple effects revealed that treatment with17β estradiol in CTL rats had not significant effect on the number ofGFAP immunoreactive astrocytes in comparison to CTL rats injectedwith vehicle (P=ns see Figs 1-B 1-A and 2) PA rats injected withvehicle showed amarkedly significant increase of the number of GFAPimmunoreactive astrocytes in comparison to CTL rats treated withvehicle (Pb0005 see Figs 1-C 1-A and 2) Estradiol treatment in PArats significantly reduced the number of GFAP immunoreactiveastrocytes in comparison to PA rats treated with vehicle (Pb0005see Figs 1-D 1-C and 2) and moreover they did not show significantdifferences with CTL rats injected with vehicle (P=ns see Figs 1-D1-A and 2) suggesting a reversion of astrogliosis associated withhypoxia at birth by 17β estradiol treatment in the adulthood
Dendritic alterations induced by PA and reversion by 17β estradioltreatment
Confocal studies using MAP-2 were conducted to determine thedendritic changes after PA insult Animals subjected to PA and treatedwith vehicle showed focal swelling and markedly fragmentedappearance of MAP-2 immunoreactive apical dendrites in the CA1hippocampal area compared to control animals treated with vehicle(see Figs 3-A and 3-C) In addition the statistical analysis revealed
CA1 hippocampal area The hippocampus of PA animals treated with vehicle showed aadiol (panels A and B) See in panel D that 17β estradiol treatment reduce the reactive
Fig 2 Assessment of the number of GFAP immunoreactive astrocytes in the striatumradiatum of CA1 Estradiol by itself did not produce any statistical differences in thenumberof GFAP immunoreactive astrocytes since CTL rats treatedwith estradiol (CTL-17β) did notshowdifferences in thenumber ofGFAP+astrocytes in comparison to CTL rats treatedwithvehicle (CTL-Vhi) Animals that were submitted to PA and treated with vehicle (PA-Vhi)showeda significant increase ofGFAP immunoreactive astrocytes in comparison to CTL-Vhiand PA rats treated with 17β estradiol (PA-17β) Moreover number of GFAPimmunoreactive astrocytes in PA-17β rats did not differ from those seen in CTL-Vhi ratsdemonstrating that estradiol treatment in adult rats blocked the reactive astrogliosisassociated with a PA event Bars and error bars represent mean+SEM Pb0005 PA-Vhivs CTL-Vhi daggerdaggerPb0005 PA-Vhi vs PA-17β
618 GE Saraceno et al Experimental Neurology 223 (2010) 615ndash622
that neither the main factor of birth condition nor the main factor oftreatment was significant (F=166 P=ns F=299 P=ns respec-tively) but the interaction was (F=623 P=0016) Post hoc analysis
Fig 3 Confocal microscope images of MAP-2 immunostaining from the striatum radiatum of Cfragmented appearance of distal dendrites in the CA1 region of PA animals injected with vehand B) Asphyctic animals injected with 17β estradiol (panel D) shows similar morphologi
indicated that 17β estradiol treatment in CTL rats did not produce anysignificant changes of the percentage of reactive area of MAP-2positive dendrites with respect to CTL rats injected with vehicle(P=ns see Figs 3-B 3-A and 4) Rats subjected to PA and treatedwith vehicle showed a significant reduction of the percentage ofreactive area of MAP-2 positive dendrites in comparison to CTL ratsinjected with vehicle (Pb005 see Figs 3-C 3-A and 4) Estradioltreatment in adults rats that suffered from PA significantly improvedthe mean percentage of reactive area of MAP-2 positive dendrites incomparison to PA rats treated with vehicle (Pb005 see Figs 3-D 3-Cand 4) while no differences were detected between PA rats treatedwith 17β estradiol and CTL rats injected with vehicle (P=ns seeFigs 3-D 3-A and 4)
Effects of PA and 17β estradiol treatment on axonal neurofilaments
Axonal alterations were studied using immunocytochemistry formediummolecular weight neurofilaments (120 NF) and phosphorylat-edhighandmediummolecularweightneurofilaments (pHMNF) Fig 5shows a representative example of a hippocampal section immunos-tained for 120 NF No obvious differences in 120 NF immunostainingwere detected between animals subjected to PA and control animals Inagreement with the qualitative observations the statistical analysisshowed that neither the main factor of birth condition nor the mainfactor of treatment nor the interaction was significant (Fb1 F=23P=ns Pb1 respectively See Fig 6) In contrast noticeable changeswere seen in the volume fraction of immunoreactive material forphosphorylated high and medium molecular weight neurofilamentsafter PA and 17β treatment The two-way ANOVA revealed that the
A1 hippocampal area MAP-2 immunostaining revealed a focal swelling and amarkedlyicle (panel C) respect to control groups treated with vehicle and 17β estradiol (panels Acal characteristics to the control groups (panels A and B) Scale bar 10 microm
Fig 4 Assessment of the percentage of reactive area of MAP-2 positive dendrites in thestriatum radiatum of CA1 The statistical analysis revealed a significant decrease of thepercentage of reactive area of MAP-2 positive dendrites in PA rats treated with vehicle(PA-Vhi) in comparison to CTL rats treated with vehicle (CTL-Vhi) and PA rats treatedwith 17β estradiol (PA-17β) In addition PA-17β rats did not differ in the percentage ofreactive area of MAP-2 positive dendrites in comparison to CTL-Vhi rats suggesting areversion of dendritic alterations associated with perinatal asphyxia Estradiol by itselfdid not produce any differences in the percentage of reactive area of MAP-2 positivedendrites since CTL-17β rats did not differ in the percentage of reactive area of MAP-2positive dendrites from CTL-Vhi rats Bars and error bars represent mean+SEMPb005 PA-Vhi vs CTL-Vhi daggerPb005 PA-Vhi vs PA-17β
619GE Saraceno et al Experimental Neurology 223 (2010) 615ndash622
main effects of birth condition and treatment and the interaction wereall significant (F=4193 Pb0001 F=779 Pb001 F=158 Pb0001respectively) Post hoc analysis showed that treatment with 17βEstradiol in adults CTL rats had not any effect in the percentage of thereactive area of pHMNF (CTL-Vhi vs CTL-17β P=ns See Figs 7-A 7-B and 8) Rats subjected to PA and injected at adulthood with vehicleshowed a significantly reduced percentage of the reactive area of pHMNF than CTL rats treatedwith vehicle (Pb0005 see Figs 7-C 7-A and 8)
Fig 5 Optical microscope images show a representative example of a striatum radiatum of CAPA animals treated with vehicle (Panel C) respect to CTL injected with vehicle or 17β Estradishowed similar morphological characteristics to control groups (Panel A and B) Scale bar
The treatmentwith 17β Estradiol in PA rats enhanced the percentage ofthe reactive area of pHM NF in comparison to PA rats treated withvehicle (Pb0005 see Figs 7-D 7-C and 8) Furthermore 17β Estradioltreatment in PA rats increased the percentage of the reactive areaof pHM NF to a similar level to the one seen in CTL treated with vehicle(P=ns CTL-Vhi vs AP-17 β See Figs 7-A 7-D and 8)
Discussion
In this work we demonstrate that PA in rats results in permanentchanges in glial andneuronal cells in thehippocampus after fourmonthsof the induction of asphyctic insultWehave focus our studyonCA1 areaof hippocampus since it is one of the most affected areas by PA insult(Petito and Pulsinelli 1984) We have detected increased astrogliosisfocal swelling and fragmentation of CA1 apical dendrites decreasedMAP-2 immunoreactivity and decreased phosphorylated high andmediummolecular weight neurofilaments in the hippocampus of adultrats subjected to PA These alterations are well established markers ofneurodegenerative damage in the central nervous system Treatmentwith 17β estradiol has blocked these alterations in the hippocampalcytoskeleton Thus this data suggests that 17β estradiol could be apossible therapeutic tool for the neurological disorders produced by theasphyxia during birth
Cytoskeletal alterations are blocked by estradiol treatment
Estradiol treatment of adult rats that were subjected to perinatalasphyxia resulted in a decrease in the number of GFAP immunore-active astrocytes in the hippocampus to similar values to those seen incontrol animals These findings are in agreement with previousreports that indicate that estradiol reduces reactive astrogliosis afterdifferent forms of brain injury in adult animals (Arevalo et al in press
1 hippocampal area immunostained for 120 NF No obvious difference were observed inol (Panel A and B respectively) Asphyctic animals treated with 17β Estradiol (Panel D)10 μm
Fig 6 Assessment of the percentage of reactive area of medium molecular weightneurofilaments (120 NF) in the stratum radiatum of CA1 No differences among any ofthe groups were detected by the statistical analysis in agreement with the qualitativeobservation Bars and error bars represent mean+SEM
Fig 8 Assessment of the percentage of reactive area of phosphorylated high and mediummolecular weight neurofilament (pHM NF) in the stratum radiatum of CA1 Estradiol byitself did not produce any statistical differences in the percentage of reactive area of pHMNF since CTL rats treated with estradiol (CTL-17β) did not show differences in thepercentage of reactive area of pHM NF in comparison to CTL rats treated with vehicle(CTL-Vhi) Animals thatwere submitted to PA and treatedwith vehicle (PA-Vhi) showed asignificant decrease of the percentage of reactive area of pHM NF in comparison toCTL-Vhi and PA rats treated with 17β estradiol (PA-17β) Moreover the percentage ofreactive area of pHM NF in PA-17β rats did not differ from that seen in CTL-Vhi ratsdemonstrating that estradiol treatment in adult rats blocked the axonal alterationsassociated with a PA event Bars and error bars represent mean+SEM Pb0005 PA-Vhivs CTL-Vhi daggerdaggerPb0005 PA-Vhi vs PA-17β
620 GE Saraceno et al Experimental Neurology 223 (2010) 615ndash622
Barreto et al 2009) However most studies have tested the antiglioticproperties of estradiol when the hormone was administrated beforeor shortly after the induction of brain damage Our findings indicatethat the hormone is also able to reduce in adult animals chronicreactive astrogliosis caused by an early developmental neurodegen-erative event
Under neurodegenerative conditions astrocytes proliferate and theexpression of GFAP is increased (Eng and Ghirnikar 1994 Pekny andNilsson 2005)Alterations in dendrites are also a commonfindingunderneurodegenerative conditions (Ikonomidou et al 1989 Hsu andBuzsaacuteki 1993 Hori and Carpenter 1994 Matesic and Lin 1994Ramoacuten y Cajal 1995) and the aberrant phosphorylation of neurofila-ments is a hallmarkof axonal degeneration (Grant andPant 2000 Sihaget al 2007) Consistent with this data we have shown in previous workreactive astrogliosis in neostriatum of rat at six months after PA (Cebralet al 2006) In addition we have recently demonstrated that rats at six
Fig 7Optical microscope images show a representative example of a striatum radiatum of CApHMNF immunostaining were detected between PA treated with vehicle (panel C) and conasphyctic animals (panel D) Scale bar 10 microm
month after PA showed clear signs of synaptic neurodegeneration in theneostriatum and accumulation of ubi-proteins (Capani et al 2009)Taken together our findings indicate that the experimental model ofperinatal asphyxia used in this study causes neurodegenerativealterations that are still detectable in adult life The existence ofpermanent alterations in the hippocampus of rats subjected to perinatalasphyxia are in agreement with the permanent neurological deficitsassociated with this pathological condition in humans (Hill and Volpe1981 Amiel-Tison and Ellison 1986 Osborne et al 2004)
The alterations produced by perinatal asphyxia in the volumefraction of immunoreactivity material for phosphorylated high and
1 hippocampal area immunostained for phosphorylated HMNF Obvious differences introl groups (panels A and B) Estradiol treatment reverts the decrease of staining seen in
621GE Saraceno et al Experimental Neurology 223 (2010) 615ndash622
medium molecular weight neurofilaments are in agreement with ourprevious finding of NF alterations in the neostriatum of six month-oldrats (Cebral et al 2006) This data suggested that NF alterations are aconsistent modification induced by PA In addition abnormal NFaccumulations are found in several human neurological diseases suchas amyotrophic lateral sclerosis Parkinsons disease Alzheimersdisease progressive supranuclear palsy CharcotndashMariendashTooth diseasediabetic neuropathy giant axonal neuropathy and NF inclusion disease(Hirano 1994 Mori et al 1996 Bomont et al 2000 Shepherd et al2002) Since NF proteins are preferential targets for oxidative stress(Geacutelinas et al 2000 Hand and Rouleau 2002) estrogens can induce itsneuroprotective functionmodulating free radical productionMoreoverestrogen therapy was able to preserve the microtubule organization ofthedendrite severelymodifiedbyperinatal asphyxia A substantial bodyof evidences suggested that estrogens are related with the facilitationand preservation of neurite growth during the development of nervoussystem although the molecular mechanisms are not still fullyunderstood (Dominguez et al 2004) Recently it was proposed that17β-estradiol-mediated ERK activation is involved in the maintenanceof neuritic arborisation and neuronal morphology in pro-apoptoticconditions (Mintildeano et al 2008)
Conclusions
In conclusion ourfindings suggest that estradiol therapy in adult lifemay reverse neural deficits caused by early brain damage such as theones caused by perinatal asphyxia Therefore in addition to its wellcharacterized neuroprotective actions to prevent neuronal damageestradiol may also have neuro-reparative properties decreasing brainalterations caused by early life events Since estradiol may havenumerous undesirable peripheral effects further studies should addressthe effect of selective estrogen receptor modulators or non-feminizingestrogens whichmay represent amore adequate therapeutic approach
Acknowledgments
This research has been supported by Ministerio de Ciencia eInnovacioacuten Spain (BFU2008-02950-C03-01) IBRO Studentship 2008MAEC-AECID Fellowship 2008 UBACYTM407 and PIP5784 We alsothank to Dr Guelman who provided 17B Estradiol used in this studyGES MLA-B PG and JIR are fellowship holders from CONICET(Argentina)
References
Amiel-Tison C Ellison P 1986 Birth asphyxia in the fullterm newborn early assess-ment and outcome Dev Med Child Neurol 28 671ndash682
Arevalo MA Santos-Galindo M Bellini MJ Azcoitia I Garcia-Segura LM in pressActions of estrogens on glial cells implications for neuroprotection Biochim BiophysActa doi101016jbbagen200910002
Barreto G Santos-Galindo M Diz-Chaves Y Perniacutea O Carrero P Azcoitia I Garcia-Segura LM 2009 Selective estrogen receptor modulators decrease reactiveastrogliosis in the injured brain effects of aging and prolonged depletion of ovarianhormones Endocrinology 150 5010ndash5015
Bjelke B Andersson K Ogren SO Bolme P 1991 Asphyctic lesion proliferation oftyrosine hydroxylase-immunoreactive nerve cell bodies in the rat substantia nigraand functional changes in dopamine neurotransmission Brain Res 543 1ndash9
Bomont P Cavalier L Blondeau F Ben Hamida C Belal S Tazir M Demir ETopaloglu H Korinthenberg R Tuumlysuumlz B Landrieu P Hentati F Koenig M2000 The gene encoding gigaxonin a new member of the cytoskeletal BTBkelchrepeat family is mutated in giant axonal neuropathy Nat Genet 26 370ndash374
Bourque M Dluzen DE Di Paolo T 2009 Neuroprotective actions of sex steroids inParkinsons disease Front Neuroendocrinol 30 142ndash157
Busto R Globus MY Dietrich WD Martinez E Valdeacutes I Ginsberg MD 1989Effect of mild hypothermia on ischemia-induced release of neurotransmitters andfree fatty acids in rat brain Stroke 20 904ndash910
Capani F Loidl F Lopez-Costa JJ Selvin-Testa A Saavedra JP 1997 Ultrastructuralchanges in nitric oxide synthase immunoreactivity in the brain of rats subjected toperinatal asphyxia neuroprotective effects of cold treatment Brain Res 775 11ndash23
Capani F Loidl CF Aguirre F Piehl L Facorro G Hager A De Paoli T Farach HPecci-Saavedra J 2001 Changes in reactive oxygen species (ROS) production in ratbrain during global perinatal asphyxia an ESR study Brain Res 914 204ndash207
Capani F Loidl CF Piehl LL Facorro G De Paoli T Hager A 2003 Long termproduction of reactive oxygen species during perinatal asphyxia in the rat centralnervous system effects of hypothermia Int J Neurosci 113 641ndash654
Capani F Saraceno GE Botti V Aon-Bertolino L de Oliveira DM Barreto GGaleano P Giraldez-Alvarez LD Coirini H 2009 Protein ubiquitination inpostsynaptic densities after hypoxia in rat neostriatum is blocked by hypothermiaExp Neurol 219 404ndash413
Cebral E Capani F Selviacuten-Testa A Funes MR Coirini H Loidl CF 2006 Neostriatalcytoskeleton changes following perinatal asphyxia effect of hypothermia treatmentInt J Neurosci 116 697ndash714
Choi DW Rothman SM 1990 The role of glutamate neurotoxicity in hypoxicndashischemicneuronal death Annu Rev Neurosci 13 171ndash182
Dominguez R Jalali C de Lacalle S 2004 Morphological effects of estrogen oncholinergic neurons in vitro involves activation of extracellular signal-regulatedkinases J Neurosci 24 982ndash990
Dorfman VB Vega MC Coirini H 2006 Age-related changes of the GABA-B receptorin the lumbar spinal cord of male rats and penile erection Life Sci 78 1529ndash1534
Eng LF Ghirnikar RS 1994 GFAP and astrogliosis Brain Pathol 4 229ndash237Farooqui AA Haun SE Horrocks LA 1994 Ischemia and hypoxia In Siegel GJ
Hsu M Buzsaacuteki G 1993 Vulnerability of mossy fiber targets in the rat hippocampus toforebrain ischemia J Neurosci 13 3964ndash3979
Ikonomidou C PriceMTMosinger JL Frierdich G Labruyere J Salles KS Olney JW1989 Hypobaricndashischemic conditions produce glutamate-like cytopathology in infantrat brain J Neurosci 9 1693ndash1700
Kamme F Wieloch T 1996 Induction of junD mRNA after transient forebrainischemia in the rat Effect of hypothermia Brain Res Mol Brain Res 43 51ndash56
Kipp M Beyer C 2009 Impact of sex steroids on neuroinflammatory processes andexperimental multiple sclerosis Front Neuroendocrinol 30 188ndash200
Kirino T 1982 Delayed neuronal death in the gerbil hippocampus following ischemiaBrain Res 239 57ndash69
Kruse MS Rey M Barutta J Coirini H 2009 Allopregnanolone effects onastrogliosis induced by hypoxia in organotypic cultures of striatum hippocampusand neocortex Brain Res 1303 1ndash7
Lebesgue D Chevaleyre V Zukin RS Etgen AM 2009 Estradiol rescues neuronsfrom global ischemia-induced cell death multiple cellular pathways of neuropro-tection Steroids 74 555ndash561
Loidl CF Capani F Loacutepez-Costa JJ Selviacuten-Testa A Loacutepez EM Pecci-Saavedra J1997 Long term changes in NADPH-diaphorase reactivity in striatal and corticalneurons following experimental perinatal asphyxia neuroprotective effects ofhypothermia Int J Neurosci 89 1ndash14
Loidl CF Gavilanes AWVanDijk EH VreulsW Blokland A Vles JS SteinbuschHWBlanco CE 2000 Effects of hypothermia and gender on survival and behavior afterperinatal asphyxia in rats Physiol Behav 68 263ndash269
Matesic DF Lin RC 1994 Microtubule-associated protein 2 as an early indicator ofischemia-induced neurodegeneration in the gerbil forebrain J Neurochem 631012ndash1020
Mintildeano A Xifroacute X Peacuterez V Barneda-Zahonero B Saura CA Rodriacuteguez-Alvarez J2008 Estradiol facilitates neurite maintenance by a SrcRasERK signalling pathwayMol Cell Neurosci 39 143ndash151
Mori H Oda M Mizuno Y 1996 Cortical ballooned neurons in progressivesupranuclear palsy Neurosci Lett 209 109ndash112
Osborne NN Casson RJ Wood JP Chidlow G Graham M Melena J 2004 Retinalischemia mechanisms of damage and potential therapeutic strategies Prog RetinEye Res 23 91ndash147
Pekny M Nilsson M 2005 Astrocyte activation and reactive gliosis Glia 50 427ndash434Petito CK Pulsinelli WA 1984 Delayed neuronal recovery and neuronal death in rat
hippocampus following severe cerebral ischemiapossible relationship toabnormalitiesin neuronal processes J Cereb Blood Flow Metab 4 194ndash205
Pike CJ Carroll JC Rosario ER Barron AM 2009 Protective actions of sex steroidhormones in Alzheimers disease Front Neuroendocrinol 30 239ndash258
622 GE Saraceno et al Experimental Neurology 223 (2010) 615ndash622
Ramoacuten y Cajal S 1995 Histology of the Nervous System of Man and Vertebrates OxfordUniversity Press New York Translated by N Swanson and LW Swanson
Shankaran S 2009 Neonatal encephalopathy treatmentwith hypothermia J Neurotrauma26 437ndash443
Shepherd CE McCann H Thiel E Halliday GM 2002 Neurofilament-immunoreactiveneurons inAlzheimersdiseaseanddementiawithLewybodiesNeurobiolDis 9249ndash257
Sihag RK Inagaki M Yamaguchi T Shea TB Pant HC 2007 Role of phosphorylationon the structuraldynamics and functionof types III and IV intermediatefilaments ExpCell Res 313 2098ndash2109
Suzuki S Brown CM Wise PM 2009 Neuroprotective effects of estrogens followingischemic stroke Front Neuroendocrinol 30 201ndash211
Tapia-Gonzalez S Carrero P Pernia O Garcia-Segura LM Diz-Chaves Y 2008Selective oestrogen receptor (ER) modulators reduce microglia reactivity in vivoafter peripheral inflammation potential role of microglial ERs J Endocrinol 198219ndash230
Van de Berg WD Kwaijtaal M de Louw AJ Lissone NP Schmitz C Faull RLBlokland A Blanco CE Steinbusch HW 2003 Impact of perinatal asphyxia onthe GABAergic and locomotor system Neuroscience 117 83ndash96
Vannucci RC Perlman JM 1997 Interventions for perinatal hypoxicndashischemic ence-phalopathy Pediatrics 100 1004ndash1014
Weibel ER 1979 Stereological Methods Practical Methods for Biological MorphometryVol I Academic Press New York
Fig 2 Assessment of the number of GFAP immunoreactive astrocytes in the striatumradiatum of CA1 Estradiol by itself did not produce any statistical differences in thenumberof GFAP immunoreactive astrocytes since CTL rats treatedwith estradiol (CTL-17β) did notshowdifferences in thenumber ofGFAP+astrocytes in comparison to CTL rats treatedwithvehicle (CTL-Vhi) Animals that were submitted to PA and treated with vehicle (PA-Vhi)showeda significant increase ofGFAP immunoreactive astrocytes in comparison to CTL-Vhiand PA rats treated with 17β estradiol (PA-17β) Moreover number of GFAPimmunoreactive astrocytes in PA-17β rats did not differ from those seen in CTL-Vhi ratsdemonstrating that estradiol treatment in adult rats blocked the reactive astrogliosisassociated with a PA event Bars and error bars represent mean+SEM Pb0005 PA-Vhivs CTL-Vhi daggerdaggerPb0005 PA-Vhi vs PA-17β
618 GE Saraceno et al Experimental Neurology 223 (2010) 615ndash622
that neither the main factor of birth condition nor the main factor oftreatment was significant (F=166 P=ns F=299 P=ns respec-tively) but the interaction was (F=623 P=0016) Post hoc analysis
Fig 3 Confocal microscope images of MAP-2 immunostaining from the striatum radiatum of Cfragmented appearance of distal dendrites in the CA1 region of PA animals injected with vehand B) Asphyctic animals injected with 17β estradiol (panel D) shows similar morphologi
indicated that 17β estradiol treatment in CTL rats did not produce anysignificant changes of the percentage of reactive area of MAP-2positive dendrites with respect to CTL rats injected with vehicle(P=ns see Figs 3-B 3-A and 4) Rats subjected to PA and treatedwith vehicle showed a significant reduction of the percentage ofreactive area of MAP-2 positive dendrites in comparison to CTL ratsinjected with vehicle (Pb005 see Figs 3-C 3-A and 4) Estradioltreatment in adults rats that suffered from PA significantly improvedthe mean percentage of reactive area of MAP-2 positive dendrites incomparison to PA rats treated with vehicle (Pb005 see Figs 3-D 3-Cand 4) while no differences were detected between PA rats treatedwith 17β estradiol and CTL rats injected with vehicle (P=ns seeFigs 3-D 3-A and 4)
Effects of PA and 17β estradiol treatment on axonal neurofilaments
Axonal alterations were studied using immunocytochemistry formediummolecular weight neurofilaments (120 NF) and phosphorylat-edhighandmediummolecularweightneurofilaments (pHMNF) Fig 5shows a representative example of a hippocampal section immunos-tained for 120 NF No obvious differences in 120 NF immunostainingwere detected between animals subjected to PA and control animals Inagreement with the qualitative observations the statistical analysisshowed that neither the main factor of birth condition nor the mainfactor of treatment nor the interaction was significant (Fb1 F=23P=ns Pb1 respectively See Fig 6) In contrast noticeable changeswere seen in the volume fraction of immunoreactive material forphosphorylated high and medium molecular weight neurofilamentsafter PA and 17β treatment The two-way ANOVA revealed that the
A1 hippocampal area MAP-2 immunostaining revealed a focal swelling and amarkedlyicle (panel C) respect to control groups treated with vehicle and 17β estradiol (panels Acal characteristics to the control groups (panels A and B) Scale bar 10 microm
Fig 4 Assessment of the percentage of reactive area of MAP-2 positive dendrites in thestriatum radiatum of CA1 The statistical analysis revealed a significant decrease of thepercentage of reactive area of MAP-2 positive dendrites in PA rats treated with vehicle(PA-Vhi) in comparison to CTL rats treated with vehicle (CTL-Vhi) and PA rats treatedwith 17β estradiol (PA-17β) In addition PA-17β rats did not differ in the percentage ofreactive area of MAP-2 positive dendrites in comparison to CTL-Vhi rats suggesting areversion of dendritic alterations associated with perinatal asphyxia Estradiol by itselfdid not produce any differences in the percentage of reactive area of MAP-2 positivedendrites since CTL-17β rats did not differ in the percentage of reactive area of MAP-2positive dendrites from CTL-Vhi rats Bars and error bars represent mean+SEMPb005 PA-Vhi vs CTL-Vhi daggerPb005 PA-Vhi vs PA-17β
619GE Saraceno et al Experimental Neurology 223 (2010) 615ndash622
main effects of birth condition and treatment and the interaction wereall significant (F=4193 Pb0001 F=779 Pb001 F=158 Pb0001respectively) Post hoc analysis showed that treatment with 17βEstradiol in adults CTL rats had not any effect in the percentage of thereactive area of pHMNF (CTL-Vhi vs CTL-17β P=ns See Figs 7-A 7-B and 8) Rats subjected to PA and injected at adulthood with vehicleshowed a significantly reduced percentage of the reactive area of pHMNF than CTL rats treatedwith vehicle (Pb0005 see Figs 7-C 7-A and 8)
Fig 5 Optical microscope images show a representative example of a striatum radiatum of CAPA animals treated with vehicle (Panel C) respect to CTL injected with vehicle or 17β Estradishowed similar morphological characteristics to control groups (Panel A and B) Scale bar
The treatmentwith 17β Estradiol in PA rats enhanced the percentage ofthe reactive area of pHM NF in comparison to PA rats treated withvehicle (Pb0005 see Figs 7-D 7-C and 8) Furthermore 17β Estradioltreatment in PA rats increased the percentage of the reactive areaof pHM NF to a similar level to the one seen in CTL treated with vehicle(P=ns CTL-Vhi vs AP-17 β See Figs 7-A 7-D and 8)
Discussion
In this work we demonstrate that PA in rats results in permanentchanges in glial andneuronal cells in thehippocampus after fourmonthsof the induction of asphyctic insultWehave focus our studyonCA1 areaof hippocampus since it is one of the most affected areas by PA insult(Petito and Pulsinelli 1984) We have detected increased astrogliosisfocal swelling and fragmentation of CA1 apical dendrites decreasedMAP-2 immunoreactivity and decreased phosphorylated high andmediummolecular weight neurofilaments in the hippocampus of adultrats subjected to PA These alterations are well established markers ofneurodegenerative damage in the central nervous system Treatmentwith 17β estradiol has blocked these alterations in the hippocampalcytoskeleton Thus this data suggests that 17β estradiol could be apossible therapeutic tool for the neurological disorders produced by theasphyxia during birth
Cytoskeletal alterations are blocked by estradiol treatment
Estradiol treatment of adult rats that were subjected to perinatalasphyxia resulted in a decrease in the number of GFAP immunore-active astrocytes in the hippocampus to similar values to those seen incontrol animals These findings are in agreement with previousreports that indicate that estradiol reduces reactive astrogliosis afterdifferent forms of brain injury in adult animals (Arevalo et al in press
1 hippocampal area immunostained for 120 NF No obvious difference were observed inol (Panel A and B respectively) Asphyctic animals treated with 17β Estradiol (Panel D)10 μm
Fig 6 Assessment of the percentage of reactive area of medium molecular weightneurofilaments (120 NF) in the stratum radiatum of CA1 No differences among any ofthe groups were detected by the statistical analysis in agreement with the qualitativeobservation Bars and error bars represent mean+SEM
Fig 8 Assessment of the percentage of reactive area of phosphorylated high and mediummolecular weight neurofilament (pHM NF) in the stratum radiatum of CA1 Estradiol byitself did not produce any statistical differences in the percentage of reactive area of pHMNF since CTL rats treated with estradiol (CTL-17β) did not show differences in thepercentage of reactive area of pHM NF in comparison to CTL rats treated with vehicle(CTL-Vhi) Animals thatwere submitted to PA and treatedwith vehicle (PA-Vhi) showed asignificant decrease of the percentage of reactive area of pHM NF in comparison toCTL-Vhi and PA rats treated with 17β estradiol (PA-17β) Moreover the percentage ofreactive area of pHM NF in PA-17β rats did not differ from that seen in CTL-Vhi ratsdemonstrating that estradiol treatment in adult rats blocked the axonal alterationsassociated with a PA event Bars and error bars represent mean+SEM Pb0005 PA-Vhivs CTL-Vhi daggerdaggerPb0005 PA-Vhi vs PA-17β
620 GE Saraceno et al Experimental Neurology 223 (2010) 615ndash622
Barreto et al 2009) However most studies have tested the antiglioticproperties of estradiol when the hormone was administrated beforeor shortly after the induction of brain damage Our findings indicatethat the hormone is also able to reduce in adult animals chronicreactive astrogliosis caused by an early developmental neurodegen-erative event
Under neurodegenerative conditions astrocytes proliferate and theexpression of GFAP is increased (Eng and Ghirnikar 1994 Pekny andNilsson 2005)Alterations in dendrites are also a commonfindingunderneurodegenerative conditions (Ikonomidou et al 1989 Hsu andBuzsaacuteki 1993 Hori and Carpenter 1994 Matesic and Lin 1994Ramoacuten y Cajal 1995) and the aberrant phosphorylation of neurofila-ments is a hallmarkof axonal degeneration (Grant andPant 2000 Sihaget al 2007) Consistent with this data we have shown in previous workreactive astrogliosis in neostriatum of rat at six months after PA (Cebralet al 2006) In addition we have recently demonstrated that rats at six
Fig 7Optical microscope images show a representative example of a striatum radiatum of CApHMNF immunostaining were detected between PA treated with vehicle (panel C) and conasphyctic animals (panel D) Scale bar 10 microm
month after PA showed clear signs of synaptic neurodegeneration in theneostriatum and accumulation of ubi-proteins (Capani et al 2009)Taken together our findings indicate that the experimental model ofperinatal asphyxia used in this study causes neurodegenerativealterations that are still detectable in adult life The existence ofpermanent alterations in the hippocampus of rats subjected to perinatalasphyxia are in agreement with the permanent neurological deficitsassociated with this pathological condition in humans (Hill and Volpe1981 Amiel-Tison and Ellison 1986 Osborne et al 2004)
The alterations produced by perinatal asphyxia in the volumefraction of immunoreactivity material for phosphorylated high and
1 hippocampal area immunostained for phosphorylated HMNF Obvious differences introl groups (panels A and B) Estradiol treatment reverts the decrease of staining seen in
621GE Saraceno et al Experimental Neurology 223 (2010) 615ndash622
medium molecular weight neurofilaments are in agreement with ourprevious finding of NF alterations in the neostriatum of six month-oldrats (Cebral et al 2006) This data suggested that NF alterations are aconsistent modification induced by PA In addition abnormal NFaccumulations are found in several human neurological diseases suchas amyotrophic lateral sclerosis Parkinsons disease Alzheimersdisease progressive supranuclear palsy CharcotndashMariendashTooth diseasediabetic neuropathy giant axonal neuropathy and NF inclusion disease(Hirano 1994 Mori et al 1996 Bomont et al 2000 Shepherd et al2002) Since NF proteins are preferential targets for oxidative stress(Geacutelinas et al 2000 Hand and Rouleau 2002) estrogens can induce itsneuroprotective functionmodulating free radical productionMoreoverestrogen therapy was able to preserve the microtubule organization ofthedendrite severelymodifiedbyperinatal asphyxia A substantial bodyof evidences suggested that estrogens are related with the facilitationand preservation of neurite growth during the development of nervoussystem although the molecular mechanisms are not still fullyunderstood (Dominguez et al 2004) Recently it was proposed that17β-estradiol-mediated ERK activation is involved in the maintenanceof neuritic arborisation and neuronal morphology in pro-apoptoticconditions (Mintildeano et al 2008)
Conclusions
In conclusion ourfindings suggest that estradiol therapy in adult lifemay reverse neural deficits caused by early brain damage such as theones caused by perinatal asphyxia Therefore in addition to its wellcharacterized neuroprotective actions to prevent neuronal damageestradiol may also have neuro-reparative properties decreasing brainalterations caused by early life events Since estradiol may havenumerous undesirable peripheral effects further studies should addressthe effect of selective estrogen receptor modulators or non-feminizingestrogens whichmay represent amore adequate therapeutic approach
Acknowledgments
This research has been supported by Ministerio de Ciencia eInnovacioacuten Spain (BFU2008-02950-C03-01) IBRO Studentship 2008MAEC-AECID Fellowship 2008 UBACYTM407 and PIP5784 We alsothank to Dr Guelman who provided 17B Estradiol used in this studyGES MLA-B PG and JIR are fellowship holders from CONICET(Argentina)
References
Amiel-Tison C Ellison P 1986 Birth asphyxia in the fullterm newborn early assess-ment and outcome Dev Med Child Neurol 28 671ndash682
Arevalo MA Santos-Galindo M Bellini MJ Azcoitia I Garcia-Segura LM in pressActions of estrogens on glial cells implications for neuroprotection Biochim BiophysActa doi101016jbbagen200910002
Barreto G Santos-Galindo M Diz-Chaves Y Perniacutea O Carrero P Azcoitia I Garcia-Segura LM 2009 Selective estrogen receptor modulators decrease reactiveastrogliosis in the injured brain effects of aging and prolonged depletion of ovarianhormones Endocrinology 150 5010ndash5015
Bjelke B Andersson K Ogren SO Bolme P 1991 Asphyctic lesion proliferation oftyrosine hydroxylase-immunoreactive nerve cell bodies in the rat substantia nigraand functional changes in dopamine neurotransmission Brain Res 543 1ndash9
Bomont P Cavalier L Blondeau F Ben Hamida C Belal S Tazir M Demir ETopaloglu H Korinthenberg R Tuumlysuumlz B Landrieu P Hentati F Koenig M2000 The gene encoding gigaxonin a new member of the cytoskeletal BTBkelchrepeat family is mutated in giant axonal neuropathy Nat Genet 26 370ndash374
Bourque M Dluzen DE Di Paolo T 2009 Neuroprotective actions of sex steroids inParkinsons disease Front Neuroendocrinol 30 142ndash157
Busto R Globus MY Dietrich WD Martinez E Valdeacutes I Ginsberg MD 1989Effect of mild hypothermia on ischemia-induced release of neurotransmitters andfree fatty acids in rat brain Stroke 20 904ndash910
Capani F Loidl F Lopez-Costa JJ Selvin-Testa A Saavedra JP 1997 Ultrastructuralchanges in nitric oxide synthase immunoreactivity in the brain of rats subjected toperinatal asphyxia neuroprotective effects of cold treatment Brain Res 775 11ndash23
Capani F Loidl CF Aguirre F Piehl L Facorro G Hager A De Paoli T Farach HPecci-Saavedra J 2001 Changes in reactive oxygen species (ROS) production in ratbrain during global perinatal asphyxia an ESR study Brain Res 914 204ndash207
Capani F Loidl CF Piehl LL Facorro G De Paoli T Hager A 2003 Long termproduction of reactive oxygen species during perinatal asphyxia in the rat centralnervous system effects of hypothermia Int J Neurosci 113 641ndash654
Capani F Saraceno GE Botti V Aon-Bertolino L de Oliveira DM Barreto GGaleano P Giraldez-Alvarez LD Coirini H 2009 Protein ubiquitination inpostsynaptic densities after hypoxia in rat neostriatum is blocked by hypothermiaExp Neurol 219 404ndash413
Cebral E Capani F Selviacuten-Testa A Funes MR Coirini H Loidl CF 2006 Neostriatalcytoskeleton changes following perinatal asphyxia effect of hypothermia treatmentInt J Neurosci 116 697ndash714
Choi DW Rothman SM 1990 The role of glutamate neurotoxicity in hypoxicndashischemicneuronal death Annu Rev Neurosci 13 171ndash182
Dominguez R Jalali C de Lacalle S 2004 Morphological effects of estrogen oncholinergic neurons in vitro involves activation of extracellular signal-regulatedkinases J Neurosci 24 982ndash990
Dorfman VB Vega MC Coirini H 2006 Age-related changes of the GABA-B receptorin the lumbar spinal cord of male rats and penile erection Life Sci 78 1529ndash1534
Eng LF Ghirnikar RS 1994 GFAP and astrogliosis Brain Pathol 4 229ndash237Farooqui AA Haun SE Horrocks LA 1994 Ischemia and hypoxia In Siegel GJ
Hsu M Buzsaacuteki G 1993 Vulnerability of mossy fiber targets in the rat hippocampus toforebrain ischemia J Neurosci 13 3964ndash3979
Ikonomidou C PriceMTMosinger JL Frierdich G Labruyere J Salles KS Olney JW1989 Hypobaricndashischemic conditions produce glutamate-like cytopathology in infantrat brain J Neurosci 9 1693ndash1700
Kamme F Wieloch T 1996 Induction of junD mRNA after transient forebrainischemia in the rat Effect of hypothermia Brain Res Mol Brain Res 43 51ndash56
Kipp M Beyer C 2009 Impact of sex steroids on neuroinflammatory processes andexperimental multiple sclerosis Front Neuroendocrinol 30 188ndash200
Kirino T 1982 Delayed neuronal death in the gerbil hippocampus following ischemiaBrain Res 239 57ndash69
Kruse MS Rey M Barutta J Coirini H 2009 Allopregnanolone effects onastrogliosis induced by hypoxia in organotypic cultures of striatum hippocampusand neocortex Brain Res 1303 1ndash7
Lebesgue D Chevaleyre V Zukin RS Etgen AM 2009 Estradiol rescues neuronsfrom global ischemia-induced cell death multiple cellular pathways of neuropro-tection Steroids 74 555ndash561
Loidl CF Capani F Loacutepez-Costa JJ Selviacuten-Testa A Loacutepez EM Pecci-Saavedra J1997 Long term changes in NADPH-diaphorase reactivity in striatal and corticalneurons following experimental perinatal asphyxia neuroprotective effects ofhypothermia Int J Neurosci 89 1ndash14
Loidl CF Gavilanes AWVanDijk EH VreulsW Blokland A Vles JS SteinbuschHWBlanco CE 2000 Effects of hypothermia and gender on survival and behavior afterperinatal asphyxia in rats Physiol Behav 68 263ndash269
Matesic DF Lin RC 1994 Microtubule-associated protein 2 as an early indicator ofischemia-induced neurodegeneration in the gerbil forebrain J Neurochem 631012ndash1020
Mintildeano A Xifroacute X Peacuterez V Barneda-Zahonero B Saura CA Rodriacuteguez-Alvarez J2008 Estradiol facilitates neurite maintenance by a SrcRasERK signalling pathwayMol Cell Neurosci 39 143ndash151
Mori H Oda M Mizuno Y 1996 Cortical ballooned neurons in progressivesupranuclear palsy Neurosci Lett 209 109ndash112
Osborne NN Casson RJ Wood JP Chidlow G Graham M Melena J 2004 Retinalischemia mechanisms of damage and potential therapeutic strategies Prog RetinEye Res 23 91ndash147
Pekny M Nilsson M 2005 Astrocyte activation and reactive gliosis Glia 50 427ndash434Petito CK Pulsinelli WA 1984 Delayed neuronal recovery and neuronal death in rat
hippocampus following severe cerebral ischemiapossible relationship toabnormalitiesin neuronal processes J Cereb Blood Flow Metab 4 194ndash205
Pike CJ Carroll JC Rosario ER Barron AM 2009 Protective actions of sex steroidhormones in Alzheimers disease Front Neuroendocrinol 30 239ndash258
622 GE Saraceno et al Experimental Neurology 223 (2010) 615ndash622
Ramoacuten y Cajal S 1995 Histology of the Nervous System of Man and Vertebrates OxfordUniversity Press New York Translated by N Swanson and LW Swanson
Shankaran S 2009 Neonatal encephalopathy treatmentwith hypothermia J Neurotrauma26 437ndash443
Shepherd CE McCann H Thiel E Halliday GM 2002 Neurofilament-immunoreactiveneurons inAlzheimersdiseaseanddementiawithLewybodiesNeurobiolDis 9249ndash257
Sihag RK Inagaki M Yamaguchi T Shea TB Pant HC 2007 Role of phosphorylationon the structuraldynamics and functionof types III and IV intermediatefilaments ExpCell Res 313 2098ndash2109
Suzuki S Brown CM Wise PM 2009 Neuroprotective effects of estrogens followingischemic stroke Front Neuroendocrinol 30 201ndash211
Tapia-Gonzalez S Carrero P Pernia O Garcia-Segura LM Diz-Chaves Y 2008Selective oestrogen receptor (ER) modulators reduce microglia reactivity in vivoafter peripheral inflammation potential role of microglial ERs J Endocrinol 198219ndash230
Van de Berg WD Kwaijtaal M de Louw AJ Lissone NP Schmitz C Faull RLBlokland A Blanco CE Steinbusch HW 2003 Impact of perinatal asphyxia onthe GABAergic and locomotor system Neuroscience 117 83ndash96
Vannucci RC Perlman JM 1997 Interventions for perinatal hypoxicndashischemic ence-phalopathy Pediatrics 100 1004ndash1014
Weibel ER 1979 Stereological Methods Practical Methods for Biological MorphometryVol I Academic Press New York
Fig 4 Assessment of the percentage of reactive area of MAP-2 positive dendrites in thestriatum radiatum of CA1 The statistical analysis revealed a significant decrease of thepercentage of reactive area of MAP-2 positive dendrites in PA rats treated with vehicle(PA-Vhi) in comparison to CTL rats treated with vehicle (CTL-Vhi) and PA rats treatedwith 17β estradiol (PA-17β) In addition PA-17β rats did not differ in the percentage ofreactive area of MAP-2 positive dendrites in comparison to CTL-Vhi rats suggesting areversion of dendritic alterations associated with perinatal asphyxia Estradiol by itselfdid not produce any differences in the percentage of reactive area of MAP-2 positivedendrites since CTL-17β rats did not differ in the percentage of reactive area of MAP-2positive dendrites from CTL-Vhi rats Bars and error bars represent mean+SEMPb005 PA-Vhi vs CTL-Vhi daggerPb005 PA-Vhi vs PA-17β
619GE Saraceno et al Experimental Neurology 223 (2010) 615ndash622
main effects of birth condition and treatment and the interaction wereall significant (F=4193 Pb0001 F=779 Pb001 F=158 Pb0001respectively) Post hoc analysis showed that treatment with 17βEstradiol in adults CTL rats had not any effect in the percentage of thereactive area of pHMNF (CTL-Vhi vs CTL-17β P=ns See Figs 7-A 7-B and 8) Rats subjected to PA and injected at adulthood with vehicleshowed a significantly reduced percentage of the reactive area of pHMNF than CTL rats treatedwith vehicle (Pb0005 see Figs 7-C 7-A and 8)
Fig 5 Optical microscope images show a representative example of a striatum radiatum of CAPA animals treated with vehicle (Panel C) respect to CTL injected with vehicle or 17β Estradishowed similar morphological characteristics to control groups (Panel A and B) Scale bar
The treatmentwith 17β Estradiol in PA rats enhanced the percentage ofthe reactive area of pHM NF in comparison to PA rats treated withvehicle (Pb0005 see Figs 7-D 7-C and 8) Furthermore 17β Estradioltreatment in PA rats increased the percentage of the reactive areaof pHM NF to a similar level to the one seen in CTL treated with vehicle(P=ns CTL-Vhi vs AP-17 β See Figs 7-A 7-D and 8)
Discussion
In this work we demonstrate that PA in rats results in permanentchanges in glial andneuronal cells in thehippocampus after fourmonthsof the induction of asphyctic insultWehave focus our studyonCA1 areaof hippocampus since it is one of the most affected areas by PA insult(Petito and Pulsinelli 1984) We have detected increased astrogliosisfocal swelling and fragmentation of CA1 apical dendrites decreasedMAP-2 immunoreactivity and decreased phosphorylated high andmediummolecular weight neurofilaments in the hippocampus of adultrats subjected to PA These alterations are well established markers ofneurodegenerative damage in the central nervous system Treatmentwith 17β estradiol has blocked these alterations in the hippocampalcytoskeleton Thus this data suggests that 17β estradiol could be apossible therapeutic tool for the neurological disorders produced by theasphyxia during birth
Cytoskeletal alterations are blocked by estradiol treatment
Estradiol treatment of adult rats that were subjected to perinatalasphyxia resulted in a decrease in the number of GFAP immunore-active astrocytes in the hippocampus to similar values to those seen incontrol animals These findings are in agreement with previousreports that indicate that estradiol reduces reactive astrogliosis afterdifferent forms of brain injury in adult animals (Arevalo et al in press
1 hippocampal area immunostained for 120 NF No obvious difference were observed inol (Panel A and B respectively) Asphyctic animals treated with 17β Estradiol (Panel D)10 μm
Fig 6 Assessment of the percentage of reactive area of medium molecular weightneurofilaments (120 NF) in the stratum radiatum of CA1 No differences among any ofthe groups were detected by the statistical analysis in agreement with the qualitativeobservation Bars and error bars represent mean+SEM
Fig 8 Assessment of the percentage of reactive area of phosphorylated high and mediummolecular weight neurofilament (pHM NF) in the stratum radiatum of CA1 Estradiol byitself did not produce any statistical differences in the percentage of reactive area of pHMNF since CTL rats treated with estradiol (CTL-17β) did not show differences in thepercentage of reactive area of pHM NF in comparison to CTL rats treated with vehicle(CTL-Vhi) Animals thatwere submitted to PA and treatedwith vehicle (PA-Vhi) showed asignificant decrease of the percentage of reactive area of pHM NF in comparison toCTL-Vhi and PA rats treated with 17β estradiol (PA-17β) Moreover the percentage ofreactive area of pHM NF in PA-17β rats did not differ from that seen in CTL-Vhi ratsdemonstrating that estradiol treatment in adult rats blocked the axonal alterationsassociated with a PA event Bars and error bars represent mean+SEM Pb0005 PA-Vhivs CTL-Vhi daggerdaggerPb0005 PA-Vhi vs PA-17β
620 GE Saraceno et al Experimental Neurology 223 (2010) 615ndash622
Barreto et al 2009) However most studies have tested the antiglioticproperties of estradiol when the hormone was administrated beforeor shortly after the induction of brain damage Our findings indicatethat the hormone is also able to reduce in adult animals chronicreactive astrogliosis caused by an early developmental neurodegen-erative event
Under neurodegenerative conditions astrocytes proliferate and theexpression of GFAP is increased (Eng and Ghirnikar 1994 Pekny andNilsson 2005)Alterations in dendrites are also a commonfindingunderneurodegenerative conditions (Ikonomidou et al 1989 Hsu andBuzsaacuteki 1993 Hori and Carpenter 1994 Matesic and Lin 1994Ramoacuten y Cajal 1995) and the aberrant phosphorylation of neurofila-ments is a hallmarkof axonal degeneration (Grant andPant 2000 Sihaget al 2007) Consistent with this data we have shown in previous workreactive astrogliosis in neostriatum of rat at six months after PA (Cebralet al 2006) In addition we have recently demonstrated that rats at six
Fig 7Optical microscope images show a representative example of a striatum radiatum of CApHMNF immunostaining were detected between PA treated with vehicle (panel C) and conasphyctic animals (panel D) Scale bar 10 microm
month after PA showed clear signs of synaptic neurodegeneration in theneostriatum and accumulation of ubi-proteins (Capani et al 2009)Taken together our findings indicate that the experimental model ofperinatal asphyxia used in this study causes neurodegenerativealterations that are still detectable in adult life The existence ofpermanent alterations in the hippocampus of rats subjected to perinatalasphyxia are in agreement with the permanent neurological deficitsassociated with this pathological condition in humans (Hill and Volpe1981 Amiel-Tison and Ellison 1986 Osborne et al 2004)
The alterations produced by perinatal asphyxia in the volumefraction of immunoreactivity material for phosphorylated high and
1 hippocampal area immunostained for phosphorylated HMNF Obvious differences introl groups (panels A and B) Estradiol treatment reverts the decrease of staining seen in
621GE Saraceno et al Experimental Neurology 223 (2010) 615ndash622
medium molecular weight neurofilaments are in agreement with ourprevious finding of NF alterations in the neostriatum of six month-oldrats (Cebral et al 2006) This data suggested that NF alterations are aconsistent modification induced by PA In addition abnormal NFaccumulations are found in several human neurological diseases suchas amyotrophic lateral sclerosis Parkinsons disease Alzheimersdisease progressive supranuclear palsy CharcotndashMariendashTooth diseasediabetic neuropathy giant axonal neuropathy and NF inclusion disease(Hirano 1994 Mori et al 1996 Bomont et al 2000 Shepherd et al2002) Since NF proteins are preferential targets for oxidative stress(Geacutelinas et al 2000 Hand and Rouleau 2002) estrogens can induce itsneuroprotective functionmodulating free radical productionMoreoverestrogen therapy was able to preserve the microtubule organization ofthedendrite severelymodifiedbyperinatal asphyxia A substantial bodyof evidences suggested that estrogens are related with the facilitationand preservation of neurite growth during the development of nervoussystem although the molecular mechanisms are not still fullyunderstood (Dominguez et al 2004) Recently it was proposed that17β-estradiol-mediated ERK activation is involved in the maintenanceof neuritic arborisation and neuronal morphology in pro-apoptoticconditions (Mintildeano et al 2008)
Conclusions
In conclusion ourfindings suggest that estradiol therapy in adult lifemay reverse neural deficits caused by early brain damage such as theones caused by perinatal asphyxia Therefore in addition to its wellcharacterized neuroprotective actions to prevent neuronal damageestradiol may also have neuro-reparative properties decreasing brainalterations caused by early life events Since estradiol may havenumerous undesirable peripheral effects further studies should addressthe effect of selective estrogen receptor modulators or non-feminizingestrogens whichmay represent amore adequate therapeutic approach
Acknowledgments
This research has been supported by Ministerio de Ciencia eInnovacioacuten Spain (BFU2008-02950-C03-01) IBRO Studentship 2008MAEC-AECID Fellowship 2008 UBACYTM407 and PIP5784 We alsothank to Dr Guelman who provided 17B Estradiol used in this studyGES MLA-B PG and JIR are fellowship holders from CONICET(Argentina)
References
Amiel-Tison C Ellison P 1986 Birth asphyxia in the fullterm newborn early assess-ment and outcome Dev Med Child Neurol 28 671ndash682
Arevalo MA Santos-Galindo M Bellini MJ Azcoitia I Garcia-Segura LM in pressActions of estrogens on glial cells implications for neuroprotection Biochim BiophysActa doi101016jbbagen200910002
Barreto G Santos-Galindo M Diz-Chaves Y Perniacutea O Carrero P Azcoitia I Garcia-Segura LM 2009 Selective estrogen receptor modulators decrease reactiveastrogliosis in the injured brain effects of aging and prolonged depletion of ovarianhormones Endocrinology 150 5010ndash5015
Bjelke B Andersson K Ogren SO Bolme P 1991 Asphyctic lesion proliferation oftyrosine hydroxylase-immunoreactive nerve cell bodies in the rat substantia nigraand functional changes in dopamine neurotransmission Brain Res 543 1ndash9
Bomont P Cavalier L Blondeau F Ben Hamida C Belal S Tazir M Demir ETopaloglu H Korinthenberg R Tuumlysuumlz B Landrieu P Hentati F Koenig M2000 The gene encoding gigaxonin a new member of the cytoskeletal BTBkelchrepeat family is mutated in giant axonal neuropathy Nat Genet 26 370ndash374
Bourque M Dluzen DE Di Paolo T 2009 Neuroprotective actions of sex steroids inParkinsons disease Front Neuroendocrinol 30 142ndash157
Busto R Globus MY Dietrich WD Martinez E Valdeacutes I Ginsberg MD 1989Effect of mild hypothermia on ischemia-induced release of neurotransmitters andfree fatty acids in rat brain Stroke 20 904ndash910
Capani F Loidl F Lopez-Costa JJ Selvin-Testa A Saavedra JP 1997 Ultrastructuralchanges in nitric oxide synthase immunoreactivity in the brain of rats subjected toperinatal asphyxia neuroprotective effects of cold treatment Brain Res 775 11ndash23
Capani F Loidl CF Aguirre F Piehl L Facorro G Hager A De Paoli T Farach HPecci-Saavedra J 2001 Changes in reactive oxygen species (ROS) production in ratbrain during global perinatal asphyxia an ESR study Brain Res 914 204ndash207
Capani F Loidl CF Piehl LL Facorro G De Paoli T Hager A 2003 Long termproduction of reactive oxygen species during perinatal asphyxia in the rat centralnervous system effects of hypothermia Int J Neurosci 113 641ndash654
Capani F Saraceno GE Botti V Aon-Bertolino L de Oliveira DM Barreto GGaleano P Giraldez-Alvarez LD Coirini H 2009 Protein ubiquitination inpostsynaptic densities after hypoxia in rat neostriatum is blocked by hypothermiaExp Neurol 219 404ndash413
Cebral E Capani F Selviacuten-Testa A Funes MR Coirini H Loidl CF 2006 Neostriatalcytoskeleton changes following perinatal asphyxia effect of hypothermia treatmentInt J Neurosci 116 697ndash714
Choi DW Rothman SM 1990 The role of glutamate neurotoxicity in hypoxicndashischemicneuronal death Annu Rev Neurosci 13 171ndash182
Dominguez R Jalali C de Lacalle S 2004 Morphological effects of estrogen oncholinergic neurons in vitro involves activation of extracellular signal-regulatedkinases J Neurosci 24 982ndash990
Dorfman VB Vega MC Coirini H 2006 Age-related changes of the GABA-B receptorin the lumbar spinal cord of male rats and penile erection Life Sci 78 1529ndash1534
Eng LF Ghirnikar RS 1994 GFAP and astrogliosis Brain Pathol 4 229ndash237Farooqui AA Haun SE Horrocks LA 1994 Ischemia and hypoxia In Siegel GJ
Hsu M Buzsaacuteki G 1993 Vulnerability of mossy fiber targets in the rat hippocampus toforebrain ischemia J Neurosci 13 3964ndash3979
Ikonomidou C PriceMTMosinger JL Frierdich G Labruyere J Salles KS Olney JW1989 Hypobaricndashischemic conditions produce glutamate-like cytopathology in infantrat brain J Neurosci 9 1693ndash1700
Kamme F Wieloch T 1996 Induction of junD mRNA after transient forebrainischemia in the rat Effect of hypothermia Brain Res Mol Brain Res 43 51ndash56
Kipp M Beyer C 2009 Impact of sex steroids on neuroinflammatory processes andexperimental multiple sclerosis Front Neuroendocrinol 30 188ndash200
Kirino T 1982 Delayed neuronal death in the gerbil hippocampus following ischemiaBrain Res 239 57ndash69
Kruse MS Rey M Barutta J Coirini H 2009 Allopregnanolone effects onastrogliosis induced by hypoxia in organotypic cultures of striatum hippocampusand neocortex Brain Res 1303 1ndash7
Lebesgue D Chevaleyre V Zukin RS Etgen AM 2009 Estradiol rescues neuronsfrom global ischemia-induced cell death multiple cellular pathways of neuropro-tection Steroids 74 555ndash561
Loidl CF Capani F Loacutepez-Costa JJ Selviacuten-Testa A Loacutepez EM Pecci-Saavedra J1997 Long term changes in NADPH-diaphorase reactivity in striatal and corticalneurons following experimental perinatal asphyxia neuroprotective effects ofhypothermia Int J Neurosci 89 1ndash14
Loidl CF Gavilanes AWVanDijk EH VreulsW Blokland A Vles JS SteinbuschHWBlanco CE 2000 Effects of hypothermia and gender on survival and behavior afterperinatal asphyxia in rats Physiol Behav 68 263ndash269
Matesic DF Lin RC 1994 Microtubule-associated protein 2 as an early indicator ofischemia-induced neurodegeneration in the gerbil forebrain J Neurochem 631012ndash1020
Mintildeano A Xifroacute X Peacuterez V Barneda-Zahonero B Saura CA Rodriacuteguez-Alvarez J2008 Estradiol facilitates neurite maintenance by a SrcRasERK signalling pathwayMol Cell Neurosci 39 143ndash151
Mori H Oda M Mizuno Y 1996 Cortical ballooned neurons in progressivesupranuclear palsy Neurosci Lett 209 109ndash112
Osborne NN Casson RJ Wood JP Chidlow G Graham M Melena J 2004 Retinalischemia mechanisms of damage and potential therapeutic strategies Prog RetinEye Res 23 91ndash147
Pekny M Nilsson M 2005 Astrocyte activation and reactive gliosis Glia 50 427ndash434Petito CK Pulsinelli WA 1984 Delayed neuronal recovery and neuronal death in rat
hippocampus following severe cerebral ischemiapossible relationship toabnormalitiesin neuronal processes J Cereb Blood Flow Metab 4 194ndash205
Pike CJ Carroll JC Rosario ER Barron AM 2009 Protective actions of sex steroidhormones in Alzheimers disease Front Neuroendocrinol 30 239ndash258
622 GE Saraceno et al Experimental Neurology 223 (2010) 615ndash622
Ramoacuten y Cajal S 1995 Histology of the Nervous System of Man and Vertebrates OxfordUniversity Press New York Translated by N Swanson and LW Swanson
Shankaran S 2009 Neonatal encephalopathy treatmentwith hypothermia J Neurotrauma26 437ndash443
Shepherd CE McCann H Thiel E Halliday GM 2002 Neurofilament-immunoreactiveneurons inAlzheimersdiseaseanddementiawithLewybodiesNeurobiolDis 9249ndash257
Sihag RK Inagaki M Yamaguchi T Shea TB Pant HC 2007 Role of phosphorylationon the structuraldynamics and functionof types III and IV intermediatefilaments ExpCell Res 313 2098ndash2109
Suzuki S Brown CM Wise PM 2009 Neuroprotective effects of estrogens followingischemic stroke Front Neuroendocrinol 30 201ndash211
Tapia-Gonzalez S Carrero P Pernia O Garcia-Segura LM Diz-Chaves Y 2008Selective oestrogen receptor (ER) modulators reduce microglia reactivity in vivoafter peripheral inflammation potential role of microglial ERs J Endocrinol 198219ndash230
Van de Berg WD Kwaijtaal M de Louw AJ Lissone NP Schmitz C Faull RLBlokland A Blanco CE Steinbusch HW 2003 Impact of perinatal asphyxia onthe GABAergic and locomotor system Neuroscience 117 83ndash96
Vannucci RC Perlman JM 1997 Interventions for perinatal hypoxicndashischemic ence-phalopathy Pediatrics 100 1004ndash1014
Weibel ER 1979 Stereological Methods Practical Methods for Biological MorphometryVol I Academic Press New York
Fig 6 Assessment of the percentage of reactive area of medium molecular weightneurofilaments (120 NF) in the stratum radiatum of CA1 No differences among any ofthe groups were detected by the statistical analysis in agreement with the qualitativeobservation Bars and error bars represent mean+SEM
Fig 8 Assessment of the percentage of reactive area of phosphorylated high and mediummolecular weight neurofilament (pHM NF) in the stratum radiatum of CA1 Estradiol byitself did not produce any statistical differences in the percentage of reactive area of pHMNF since CTL rats treated with estradiol (CTL-17β) did not show differences in thepercentage of reactive area of pHM NF in comparison to CTL rats treated with vehicle(CTL-Vhi) Animals thatwere submitted to PA and treatedwith vehicle (PA-Vhi) showed asignificant decrease of the percentage of reactive area of pHM NF in comparison toCTL-Vhi and PA rats treated with 17β estradiol (PA-17β) Moreover the percentage ofreactive area of pHM NF in PA-17β rats did not differ from that seen in CTL-Vhi ratsdemonstrating that estradiol treatment in adult rats blocked the axonal alterationsassociated with a PA event Bars and error bars represent mean+SEM Pb0005 PA-Vhivs CTL-Vhi daggerdaggerPb0005 PA-Vhi vs PA-17β
620 GE Saraceno et al Experimental Neurology 223 (2010) 615ndash622
Barreto et al 2009) However most studies have tested the antiglioticproperties of estradiol when the hormone was administrated beforeor shortly after the induction of brain damage Our findings indicatethat the hormone is also able to reduce in adult animals chronicreactive astrogliosis caused by an early developmental neurodegen-erative event
Under neurodegenerative conditions astrocytes proliferate and theexpression of GFAP is increased (Eng and Ghirnikar 1994 Pekny andNilsson 2005)Alterations in dendrites are also a commonfindingunderneurodegenerative conditions (Ikonomidou et al 1989 Hsu andBuzsaacuteki 1993 Hori and Carpenter 1994 Matesic and Lin 1994Ramoacuten y Cajal 1995) and the aberrant phosphorylation of neurofila-ments is a hallmarkof axonal degeneration (Grant andPant 2000 Sihaget al 2007) Consistent with this data we have shown in previous workreactive astrogliosis in neostriatum of rat at six months after PA (Cebralet al 2006) In addition we have recently demonstrated that rats at six
Fig 7Optical microscope images show a representative example of a striatum radiatum of CApHMNF immunostaining were detected between PA treated with vehicle (panel C) and conasphyctic animals (panel D) Scale bar 10 microm
month after PA showed clear signs of synaptic neurodegeneration in theneostriatum and accumulation of ubi-proteins (Capani et al 2009)Taken together our findings indicate that the experimental model ofperinatal asphyxia used in this study causes neurodegenerativealterations that are still detectable in adult life The existence ofpermanent alterations in the hippocampus of rats subjected to perinatalasphyxia are in agreement with the permanent neurological deficitsassociated with this pathological condition in humans (Hill and Volpe1981 Amiel-Tison and Ellison 1986 Osborne et al 2004)
The alterations produced by perinatal asphyxia in the volumefraction of immunoreactivity material for phosphorylated high and
1 hippocampal area immunostained for phosphorylated HMNF Obvious differences introl groups (panels A and B) Estradiol treatment reverts the decrease of staining seen in
621GE Saraceno et al Experimental Neurology 223 (2010) 615ndash622
medium molecular weight neurofilaments are in agreement with ourprevious finding of NF alterations in the neostriatum of six month-oldrats (Cebral et al 2006) This data suggested that NF alterations are aconsistent modification induced by PA In addition abnormal NFaccumulations are found in several human neurological diseases suchas amyotrophic lateral sclerosis Parkinsons disease Alzheimersdisease progressive supranuclear palsy CharcotndashMariendashTooth diseasediabetic neuropathy giant axonal neuropathy and NF inclusion disease(Hirano 1994 Mori et al 1996 Bomont et al 2000 Shepherd et al2002) Since NF proteins are preferential targets for oxidative stress(Geacutelinas et al 2000 Hand and Rouleau 2002) estrogens can induce itsneuroprotective functionmodulating free radical productionMoreoverestrogen therapy was able to preserve the microtubule organization ofthedendrite severelymodifiedbyperinatal asphyxia A substantial bodyof evidences suggested that estrogens are related with the facilitationand preservation of neurite growth during the development of nervoussystem although the molecular mechanisms are not still fullyunderstood (Dominguez et al 2004) Recently it was proposed that17β-estradiol-mediated ERK activation is involved in the maintenanceof neuritic arborisation and neuronal morphology in pro-apoptoticconditions (Mintildeano et al 2008)
Conclusions
In conclusion ourfindings suggest that estradiol therapy in adult lifemay reverse neural deficits caused by early brain damage such as theones caused by perinatal asphyxia Therefore in addition to its wellcharacterized neuroprotective actions to prevent neuronal damageestradiol may also have neuro-reparative properties decreasing brainalterations caused by early life events Since estradiol may havenumerous undesirable peripheral effects further studies should addressthe effect of selective estrogen receptor modulators or non-feminizingestrogens whichmay represent amore adequate therapeutic approach
Acknowledgments
This research has been supported by Ministerio de Ciencia eInnovacioacuten Spain (BFU2008-02950-C03-01) IBRO Studentship 2008MAEC-AECID Fellowship 2008 UBACYTM407 and PIP5784 We alsothank to Dr Guelman who provided 17B Estradiol used in this studyGES MLA-B PG and JIR are fellowship holders from CONICET(Argentina)
References
Amiel-Tison C Ellison P 1986 Birth asphyxia in the fullterm newborn early assess-ment and outcome Dev Med Child Neurol 28 671ndash682
Arevalo MA Santos-Galindo M Bellini MJ Azcoitia I Garcia-Segura LM in pressActions of estrogens on glial cells implications for neuroprotection Biochim BiophysActa doi101016jbbagen200910002
Barreto G Santos-Galindo M Diz-Chaves Y Perniacutea O Carrero P Azcoitia I Garcia-Segura LM 2009 Selective estrogen receptor modulators decrease reactiveastrogliosis in the injured brain effects of aging and prolonged depletion of ovarianhormones Endocrinology 150 5010ndash5015
Bjelke B Andersson K Ogren SO Bolme P 1991 Asphyctic lesion proliferation oftyrosine hydroxylase-immunoreactive nerve cell bodies in the rat substantia nigraand functional changes in dopamine neurotransmission Brain Res 543 1ndash9
Bomont P Cavalier L Blondeau F Ben Hamida C Belal S Tazir M Demir ETopaloglu H Korinthenberg R Tuumlysuumlz B Landrieu P Hentati F Koenig M2000 The gene encoding gigaxonin a new member of the cytoskeletal BTBkelchrepeat family is mutated in giant axonal neuropathy Nat Genet 26 370ndash374
Bourque M Dluzen DE Di Paolo T 2009 Neuroprotective actions of sex steroids inParkinsons disease Front Neuroendocrinol 30 142ndash157
Busto R Globus MY Dietrich WD Martinez E Valdeacutes I Ginsberg MD 1989Effect of mild hypothermia on ischemia-induced release of neurotransmitters andfree fatty acids in rat brain Stroke 20 904ndash910
Capani F Loidl F Lopez-Costa JJ Selvin-Testa A Saavedra JP 1997 Ultrastructuralchanges in nitric oxide synthase immunoreactivity in the brain of rats subjected toperinatal asphyxia neuroprotective effects of cold treatment Brain Res 775 11ndash23
Capani F Loidl CF Aguirre F Piehl L Facorro G Hager A De Paoli T Farach HPecci-Saavedra J 2001 Changes in reactive oxygen species (ROS) production in ratbrain during global perinatal asphyxia an ESR study Brain Res 914 204ndash207
Capani F Loidl CF Piehl LL Facorro G De Paoli T Hager A 2003 Long termproduction of reactive oxygen species during perinatal asphyxia in the rat centralnervous system effects of hypothermia Int J Neurosci 113 641ndash654
Capani F Saraceno GE Botti V Aon-Bertolino L de Oliveira DM Barreto GGaleano P Giraldez-Alvarez LD Coirini H 2009 Protein ubiquitination inpostsynaptic densities after hypoxia in rat neostriatum is blocked by hypothermiaExp Neurol 219 404ndash413
Cebral E Capani F Selviacuten-Testa A Funes MR Coirini H Loidl CF 2006 Neostriatalcytoskeleton changes following perinatal asphyxia effect of hypothermia treatmentInt J Neurosci 116 697ndash714
Choi DW Rothman SM 1990 The role of glutamate neurotoxicity in hypoxicndashischemicneuronal death Annu Rev Neurosci 13 171ndash182
Dominguez R Jalali C de Lacalle S 2004 Morphological effects of estrogen oncholinergic neurons in vitro involves activation of extracellular signal-regulatedkinases J Neurosci 24 982ndash990
Dorfman VB Vega MC Coirini H 2006 Age-related changes of the GABA-B receptorin the lumbar spinal cord of male rats and penile erection Life Sci 78 1529ndash1534
Eng LF Ghirnikar RS 1994 GFAP and astrogliosis Brain Pathol 4 229ndash237Farooqui AA Haun SE Horrocks LA 1994 Ischemia and hypoxia In Siegel GJ
Hsu M Buzsaacuteki G 1993 Vulnerability of mossy fiber targets in the rat hippocampus toforebrain ischemia J Neurosci 13 3964ndash3979
Ikonomidou C PriceMTMosinger JL Frierdich G Labruyere J Salles KS Olney JW1989 Hypobaricndashischemic conditions produce glutamate-like cytopathology in infantrat brain J Neurosci 9 1693ndash1700
Kamme F Wieloch T 1996 Induction of junD mRNA after transient forebrainischemia in the rat Effect of hypothermia Brain Res Mol Brain Res 43 51ndash56
Kipp M Beyer C 2009 Impact of sex steroids on neuroinflammatory processes andexperimental multiple sclerosis Front Neuroendocrinol 30 188ndash200
Kirino T 1982 Delayed neuronal death in the gerbil hippocampus following ischemiaBrain Res 239 57ndash69
Kruse MS Rey M Barutta J Coirini H 2009 Allopregnanolone effects onastrogliosis induced by hypoxia in organotypic cultures of striatum hippocampusand neocortex Brain Res 1303 1ndash7
Lebesgue D Chevaleyre V Zukin RS Etgen AM 2009 Estradiol rescues neuronsfrom global ischemia-induced cell death multiple cellular pathways of neuropro-tection Steroids 74 555ndash561
Loidl CF Capani F Loacutepez-Costa JJ Selviacuten-Testa A Loacutepez EM Pecci-Saavedra J1997 Long term changes in NADPH-diaphorase reactivity in striatal and corticalneurons following experimental perinatal asphyxia neuroprotective effects ofhypothermia Int J Neurosci 89 1ndash14
Loidl CF Gavilanes AWVanDijk EH VreulsW Blokland A Vles JS SteinbuschHWBlanco CE 2000 Effects of hypothermia and gender on survival and behavior afterperinatal asphyxia in rats Physiol Behav 68 263ndash269
Matesic DF Lin RC 1994 Microtubule-associated protein 2 as an early indicator ofischemia-induced neurodegeneration in the gerbil forebrain J Neurochem 631012ndash1020
Mintildeano A Xifroacute X Peacuterez V Barneda-Zahonero B Saura CA Rodriacuteguez-Alvarez J2008 Estradiol facilitates neurite maintenance by a SrcRasERK signalling pathwayMol Cell Neurosci 39 143ndash151
Mori H Oda M Mizuno Y 1996 Cortical ballooned neurons in progressivesupranuclear palsy Neurosci Lett 209 109ndash112
Osborne NN Casson RJ Wood JP Chidlow G Graham M Melena J 2004 Retinalischemia mechanisms of damage and potential therapeutic strategies Prog RetinEye Res 23 91ndash147
Pekny M Nilsson M 2005 Astrocyte activation and reactive gliosis Glia 50 427ndash434Petito CK Pulsinelli WA 1984 Delayed neuronal recovery and neuronal death in rat
hippocampus following severe cerebral ischemiapossible relationship toabnormalitiesin neuronal processes J Cereb Blood Flow Metab 4 194ndash205
Pike CJ Carroll JC Rosario ER Barron AM 2009 Protective actions of sex steroidhormones in Alzheimers disease Front Neuroendocrinol 30 239ndash258
622 GE Saraceno et al Experimental Neurology 223 (2010) 615ndash622
Ramoacuten y Cajal S 1995 Histology of the Nervous System of Man and Vertebrates OxfordUniversity Press New York Translated by N Swanson and LW Swanson
Shankaran S 2009 Neonatal encephalopathy treatmentwith hypothermia J Neurotrauma26 437ndash443
Shepherd CE McCann H Thiel E Halliday GM 2002 Neurofilament-immunoreactiveneurons inAlzheimersdiseaseanddementiawithLewybodiesNeurobiolDis 9249ndash257
Sihag RK Inagaki M Yamaguchi T Shea TB Pant HC 2007 Role of phosphorylationon the structuraldynamics and functionof types III and IV intermediatefilaments ExpCell Res 313 2098ndash2109
Suzuki S Brown CM Wise PM 2009 Neuroprotective effects of estrogens followingischemic stroke Front Neuroendocrinol 30 201ndash211
Tapia-Gonzalez S Carrero P Pernia O Garcia-Segura LM Diz-Chaves Y 2008Selective oestrogen receptor (ER) modulators reduce microglia reactivity in vivoafter peripheral inflammation potential role of microglial ERs J Endocrinol 198219ndash230
Van de Berg WD Kwaijtaal M de Louw AJ Lissone NP Schmitz C Faull RLBlokland A Blanco CE Steinbusch HW 2003 Impact of perinatal asphyxia onthe GABAergic and locomotor system Neuroscience 117 83ndash96
Vannucci RC Perlman JM 1997 Interventions for perinatal hypoxicndashischemic ence-phalopathy Pediatrics 100 1004ndash1014
Weibel ER 1979 Stereological Methods Practical Methods for Biological MorphometryVol I Academic Press New York
621GE Saraceno et al Experimental Neurology 223 (2010) 615ndash622
medium molecular weight neurofilaments are in agreement with ourprevious finding of NF alterations in the neostriatum of six month-oldrats (Cebral et al 2006) This data suggested that NF alterations are aconsistent modification induced by PA In addition abnormal NFaccumulations are found in several human neurological diseases suchas amyotrophic lateral sclerosis Parkinsons disease Alzheimersdisease progressive supranuclear palsy CharcotndashMariendashTooth diseasediabetic neuropathy giant axonal neuropathy and NF inclusion disease(Hirano 1994 Mori et al 1996 Bomont et al 2000 Shepherd et al2002) Since NF proteins are preferential targets for oxidative stress(Geacutelinas et al 2000 Hand and Rouleau 2002) estrogens can induce itsneuroprotective functionmodulating free radical productionMoreoverestrogen therapy was able to preserve the microtubule organization ofthedendrite severelymodifiedbyperinatal asphyxia A substantial bodyof evidences suggested that estrogens are related with the facilitationand preservation of neurite growth during the development of nervoussystem although the molecular mechanisms are not still fullyunderstood (Dominguez et al 2004) Recently it was proposed that17β-estradiol-mediated ERK activation is involved in the maintenanceof neuritic arborisation and neuronal morphology in pro-apoptoticconditions (Mintildeano et al 2008)
Conclusions
In conclusion ourfindings suggest that estradiol therapy in adult lifemay reverse neural deficits caused by early brain damage such as theones caused by perinatal asphyxia Therefore in addition to its wellcharacterized neuroprotective actions to prevent neuronal damageestradiol may also have neuro-reparative properties decreasing brainalterations caused by early life events Since estradiol may havenumerous undesirable peripheral effects further studies should addressthe effect of selective estrogen receptor modulators or non-feminizingestrogens whichmay represent amore adequate therapeutic approach
Acknowledgments
This research has been supported by Ministerio de Ciencia eInnovacioacuten Spain (BFU2008-02950-C03-01) IBRO Studentship 2008MAEC-AECID Fellowship 2008 UBACYTM407 and PIP5784 We alsothank to Dr Guelman who provided 17B Estradiol used in this studyGES MLA-B PG and JIR are fellowship holders from CONICET(Argentina)
References
Amiel-Tison C Ellison P 1986 Birth asphyxia in the fullterm newborn early assess-ment and outcome Dev Med Child Neurol 28 671ndash682
Arevalo MA Santos-Galindo M Bellini MJ Azcoitia I Garcia-Segura LM in pressActions of estrogens on glial cells implications for neuroprotection Biochim BiophysActa doi101016jbbagen200910002
Barreto G Santos-Galindo M Diz-Chaves Y Perniacutea O Carrero P Azcoitia I Garcia-Segura LM 2009 Selective estrogen receptor modulators decrease reactiveastrogliosis in the injured brain effects of aging and prolonged depletion of ovarianhormones Endocrinology 150 5010ndash5015
Bjelke B Andersson K Ogren SO Bolme P 1991 Asphyctic lesion proliferation oftyrosine hydroxylase-immunoreactive nerve cell bodies in the rat substantia nigraand functional changes in dopamine neurotransmission Brain Res 543 1ndash9
Bomont P Cavalier L Blondeau F Ben Hamida C Belal S Tazir M Demir ETopaloglu H Korinthenberg R Tuumlysuumlz B Landrieu P Hentati F Koenig M2000 The gene encoding gigaxonin a new member of the cytoskeletal BTBkelchrepeat family is mutated in giant axonal neuropathy Nat Genet 26 370ndash374
Bourque M Dluzen DE Di Paolo T 2009 Neuroprotective actions of sex steroids inParkinsons disease Front Neuroendocrinol 30 142ndash157
Busto R Globus MY Dietrich WD Martinez E Valdeacutes I Ginsberg MD 1989Effect of mild hypothermia on ischemia-induced release of neurotransmitters andfree fatty acids in rat brain Stroke 20 904ndash910
Capani F Loidl F Lopez-Costa JJ Selvin-Testa A Saavedra JP 1997 Ultrastructuralchanges in nitric oxide synthase immunoreactivity in the brain of rats subjected toperinatal asphyxia neuroprotective effects of cold treatment Brain Res 775 11ndash23
Capani F Loidl CF Aguirre F Piehl L Facorro G Hager A De Paoli T Farach HPecci-Saavedra J 2001 Changes in reactive oxygen species (ROS) production in ratbrain during global perinatal asphyxia an ESR study Brain Res 914 204ndash207
Capani F Loidl CF Piehl LL Facorro G De Paoli T Hager A 2003 Long termproduction of reactive oxygen species during perinatal asphyxia in the rat centralnervous system effects of hypothermia Int J Neurosci 113 641ndash654
Capani F Saraceno GE Botti V Aon-Bertolino L de Oliveira DM Barreto GGaleano P Giraldez-Alvarez LD Coirini H 2009 Protein ubiquitination inpostsynaptic densities after hypoxia in rat neostriatum is blocked by hypothermiaExp Neurol 219 404ndash413
Cebral E Capani F Selviacuten-Testa A Funes MR Coirini H Loidl CF 2006 Neostriatalcytoskeleton changes following perinatal asphyxia effect of hypothermia treatmentInt J Neurosci 116 697ndash714
Choi DW Rothman SM 1990 The role of glutamate neurotoxicity in hypoxicndashischemicneuronal death Annu Rev Neurosci 13 171ndash182
Dominguez R Jalali C de Lacalle S 2004 Morphological effects of estrogen oncholinergic neurons in vitro involves activation of extracellular signal-regulatedkinases J Neurosci 24 982ndash990
Dorfman VB Vega MC Coirini H 2006 Age-related changes of the GABA-B receptorin the lumbar spinal cord of male rats and penile erection Life Sci 78 1529ndash1534
Eng LF Ghirnikar RS 1994 GFAP and astrogliosis Brain Pathol 4 229ndash237Farooqui AA Haun SE Horrocks LA 1994 Ischemia and hypoxia In Siegel GJ
Hsu M Buzsaacuteki G 1993 Vulnerability of mossy fiber targets in the rat hippocampus toforebrain ischemia J Neurosci 13 3964ndash3979
Ikonomidou C PriceMTMosinger JL Frierdich G Labruyere J Salles KS Olney JW1989 Hypobaricndashischemic conditions produce glutamate-like cytopathology in infantrat brain J Neurosci 9 1693ndash1700
Kamme F Wieloch T 1996 Induction of junD mRNA after transient forebrainischemia in the rat Effect of hypothermia Brain Res Mol Brain Res 43 51ndash56
Kipp M Beyer C 2009 Impact of sex steroids on neuroinflammatory processes andexperimental multiple sclerosis Front Neuroendocrinol 30 188ndash200
Kirino T 1982 Delayed neuronal death in the gerbil hippocampus following ischemiaBrain Res 239 57ndash69
Kruse MS Rey M Barutta J Coirini H 2009 Allopregnanolone effects onastrogliosis induced by hypoxia in organotypic cultures of striatum hippocampusand neocortex Brain Res 1303 1ndash7
Lebesgue D Chevaleyre V Zukin RS Etgen AM 2009 Estradiol rescues neuronsfrom global ischemia-induced cell death multiple cellular pathways of neuropro-tection Steroids 74 555ndash561
Loidl CF Capani F Loacutepez-Costa JJ Selviacuten-Testa A Loacutepez EM Pecci-Saavedra J1997 Long term changes in NADPH-diaphorase reactivity in striatal and corticalneurons following experimental perinatal asphyxia neuroprotective effects ofhypothermia Int J Neurosci 89 1ndash14
Loidl CF Gavilanes AWVanDijk EH VreulsW Blokland A Vles JS SteinbuschHWBlanco CE 2000 Effects of hypothermia and gender on survival and behavior afterperinatal asphyxia in rats Physiol Behav 68 263ndash269
Matesic DF Lin RC 1994 Microtubule-associated protein 2 as an early indicator ofischemia-induced neurodegeneration in the gerbil forebrain J Neurochem 631012ndash1020
Mintildeano A Xifroacute X Peacuterez V Barneda-Zahonero B Saura CA Rodriacuteguez-Alvarez J2008 Estradiol facilitates neurite maintenance by a SrcRasERK signalling pathwayMol Cell Neurosci 39 143ndash151
Mori H Oda M Mizuno Y 1996 Cortical ballooned neurons in progressivesupranuclear palsy Neurosci Lett 209 109ndash112
Osborne NN Casson RJ Wood JP Chidlow G Graham M Melena J 2004 Retinalischemia mechanisms of damage and potential therapeutic strategies Prog RetinEye Res 23 91ndash147
Pekny M Nilsson M 2005 Astrocyte activation and reactive gliosis Glia 50 427ndash434Petito CK Pulsinelli WA 1984 Delayed neuronal recovery and neuronal death in rat
hippocampus following severe cerebral ischemiapossible relationship toabnormalitiesin neuronal processes J Cereb Blood Flow Metab 4 194ndash205
Pike CJ Carroll JC Rosario ER Barron AM 2009 Protective actions of sex steroidhormones in Alzheimers disease Front Neuroendocrinol 30 239ndash258
622 GE Saraceno et al Experimental Neurology 223 (2010) 615ndash622
Ramoacuten y Cajal S 1995 Histology of the Nervous System of Man and Vertebrates OxfordUniversity Press New York Translated by N Swanson and LW Swanson
Shankaran S 2009 Neonatal encephalopathy treatmentwith hypothermia J Neurotrauma26 437ndash443
Shepherd CE McCann H Thiel E Halliday GM 2002 Neurofilament-immunoreactiveneurons inAlzheimersdiseaseanddementiawithLewybodiesNeurobiolDis 9249ndash257
Sihag RK Inagaki M Yamaguchi T Shea TB Pant HC 2007 Role of phosphorylationon the structuraldynamics and functionof types III and IV intermediatefilaments ExpCell Res 313 2098ndash2109
Suzuki S Brown CM Wise PM 2009 Neuroprotective effects of estrogens followingischemic stroke Front Neuroendocrinol 30 201ndash211
Tapia-Gonzalez S Carrero P Pernia O Garcia-Segura LM Diz-Chaves Y 2008Selective oestrogen receptor (ER) modulators reduce microglia reactivity in vivoafter peripheral inflammation potential role of microglial ERs J Endocrinol 198219ndash230
Van de Berg WD Kwaijtaal M de Louw AJ Lissone NP Schmitz C Faull RLBlokland A Blanco CE Steinbusch HW 2003 Impact of perinatal asphyxia onthe GABAergic and locomotor system Neuroscience 117 83ndash96
Vannucci RC Perlman JM 1997 Interventions for perinatal hypoxicndashischemic ence-phalopathy Pediatrics 100 1004ndash1014
Weibel ER 1979 Stereological Methods Practical Methods for Biological MorphometryVol I Academic Press New York
622 GE Saraceno et al Experimental Neurology 223 (2010) 615ndash622
Ramoacuten y Cajal S 1995 Histology of the Nervous System of Man and Vertebrates OxfordUniversity Press New York Translated by N Swanson and LW Swanson
Shankaran S 2009 Neonatal encephalopathy treatmentwith hypothermia J Neurotrauma26 437ndash443
Shepherd CE McCann H Thiel E Halliday GM 2002 Neurofilament-immunoreactiveneurons inAlzheimersdiseaseanddementiawithLewybodiesNeurobiolDis 9249ndash257
Sihag RK Inagaki M Yamaguchi T Shea TB Pant HC 2007 Role of phosphorylationon the structuraldynamics and functionof types III and IV intermediatefilaments ExpCell Res 313 2098ndash2109
Suzuki S Brown CM Wise PM 2009 Neuroprotective effects of estrogens followingischemic stroke Front Neuroendocrinol 30 201ndash211
Tapia-Gonzalez S Carrero P Pernia O Garcia-Segura LM Diz-Chaves Y 2008Selective oestrogen receptor (ER) modulators reduce microglia reactivity in vivoafter peripheral inflammation potential role of microglial ERs J Endocrinol 198219ndash230
Van de Berg WD Kwaijtaal M de Louw AJ Lissone NP Schmitz C Faull RLBlokland A Blanco CE Steinbusch HW 2003 Impact of perinatal asphyxia onthe GABAergic and locomotor system Neuroscience 117 83ndash96
Vannucci RC Perlman JM 1997 Interventions for perinatal hypoxicndashischemic ence-phalopathy Pediatrics 100 1004ndash1014
Weibel ER 1979 Stereological Methods Practical Methods for Biological MorphometryVol I Academic Press New York
