Purinergic 2Y 1 Receptor Stimulation Decreases Cerebral Edema and Reactive Gliosis in a Traumatic Brain Injury Model Lora Talley Watts, 1 Shane Sprague, 2 Wei Zheng, 1 R. Justin Garling, 2 David Jimenez, 2 Murat Digicaylioglu, 2 and James Lechleiter 1 Abstract Traumatic brain injury (TBI) is the leading cause of death and disability in children and young adults. Neuroprotective agents that may promote repair or counteract damage after injury do not currently exist. We recently reported that stimulation of the purinergic receptor subtype P2Y 1 R using 2-methylthioladenosine 5¢ diphosphate (2MeSADP) signifi- cantly reduced cytotoxic edema induced by photothrombosis. Here, we tested whether P2Y 1 R stimulation was neuro- protective after TBI. A controlled closed head injury model was established for mice using a pneumatic impact device. Brains were harvested at 1, 3, or 7 days post-injury and assayed for morphological changes by immunocytochemistry, Western blot analysis, and wet/dry weight. Cerebral edema and expression of both aquaporin type 4 and glial fibrillary acidic protein were increased at all time points examined. Immunocytochemical measurements in both cortical and hippocampal slices also revealed significant neuronal swelling and reactive gliosis. Treatment of mice with 2MeSADP (100 lM) or MRS2365 (100 lM) 30 min after trauma significantly reduced all post-injury symptoms of TBI including edema, neuronal swelling, reactive gliosis, and AQ4 expression. The neuroprotective effect was lost in IP 3 R2-/- mice treated with 2MeSADP. Immunocytochemical labeling of brain slices confirmed that P2Y 1 R expression was defined to cortical and hippocampal astrocytes, but not neurons. Taken together, the data show that stimulation of astrocytic P2Y 1 Rs significantly reduces brain injury after acute trauma and is mediated by the IP 3 -signaling pathway. We suggest that enhancing astrocyte mitochondrial metabolism offers a promising neuroprotective strategy for a broad range of brain injuries. Key words: aquaporin 4; edema; mitochondria; purinergic receptor; reactive gliosis; traumatic brain injury Introduction T raumatic brain injury (TBI) is a multi-faceted injury re- sulting in a range of symptoms and disabilities. It is considered the leading cause of death in both children and young adults. 1 Recent estimates by the Center for Disease Control and Prevention indicate that 1.7 million cases of TBI occur in the United States annually. 2 Damage associated with the initial physical trauma is called the primary injury phase. In the hours and days after TBI, there is a progression of molecular changes that lead to neuronal degeneration, inflammation, blood-brain barrier (BBB) dysfunc- tion and hyper-permeabilization. 3–6 Changes to the cellular mi- croenvironment contribute to the secondary injury phase when neuronal loss either permanently disables victims, decreasing their quality of life, or leads to their death. There are currently no neu- roprotective agents that are clinically available to counteract damage or promote repair after brain trauma. It has also been well established that TBI causes breakdown of the BBB, which is followed by vasogenic edema formation, 7,8 increased intracranial pressure, decreased cerebral blood flow, and subsequent ischemia. 9–11 Cytotoxic edema, because of the loss of adenosine triphosphate (ATP)-maintained ion homeostasis, generally precedes vasogenic edema. 12,13 TBI impairs oxygen de- livery to the brain, decreasing mitochondrial function and energy production. 14–17 Central to the regulation of water homeostasis are aquaporins, a family of membrane water channels. 18 Aquaporin 4 is the primary water channel expressed within astrocytic end feet surrounding the brain vasculature. It is presumed to play a key role in edema formation, especially during a state of inhibited energy production. Inhibition of glial mitochondrial metabolism increases astrocyte swelling and necrotic cell death, which has been impli- cated as a primary cause of cytotoxic brain edema. 19–22 Astrocytes, the most abundant cell type in the human brain, 23,24 play a vital role in supporting and protecting neuronal function and Departments of 1 Cellular and Structural Biology and 2 Neurosurgery, The University of Texas Health Science Center at San Antonio School of Medicine, San Antonio, Texas. JOURNAL OF NEUROTRAUMA 30:55–66 ( January 1, 2013) ª Mary Ann Liebert, Inc. DOI: 10.1089/neu.2012.2488 55
Transcript
Purinergic 2Y1 Receptor Stimulation DecreasesCerebral Edema and Reactive Gliosis
in a Traumatic Brain Injury Model
Lora Talley Watts,1 Shane Sprague,2 Wei Zheng,1 R. Justin Garling,2
David Jimenez,2 Murat Digicaylioglu,2 and James Lechleiter1
Abstract
Traumatic brain injury (TBI) is the leading cause of death and disability in children and young adults. Neuroprotective
agents that may promote repair or counteract damage after injury do not currently exist. We recently reported that
stimulation of the purinergic receptor subtype P2Y1R using 2-methylthioladenosine 5¢ diphosphate (2MeSADP) signifi-
cantly reduced cytotoxic edema induced by photothrombosis. Here, we tested whether P2Y1R stimulation was neuro-
protective after TBI. A controlled closed head injury model was established for mice using a pneumatic impact device.
Brains were harvested at 1, 3, or 7 days post-injury and assayed for morphological changes by immunocytochemistry,
Western blot analysis, and wet/dry weight. Cerebral edema and expression of both aquaporin type 4 and glial fibrillary
acidic protein were increased at all time points examined. Immunocytochemical measurements in both cortical and
hippocampal slices also revealed significant neuronal swelling and reactive gliosis. Treatment of mice with 2MeSADP
(100 lM) or MRS2365 (100 lM) 30 min after trauma significantly reduced all post-injury symptoms of TBI including
edema, neuronal swelling, reactive gliosis, and AQ4 expression. The neuroprotective effect was lost in IP3R2-/- mice
treated with 2MeSADP. Immunocytochemical labeling of brain slices confirmed that P2Y1R expression was defined to
cortical and hippocampal astrocytes, but not neurons. Taken together, the data show that stimulation of astrocytic P2Y1Rs
significantly reduces brain injury after acute trauma and is mediated by the IP3-signaling pathway. We suggest that
enhancing astrocyte mitochondrial metabolism offers a promising neuroprotective strategy for a broad range of brain
and subsequent ischemia.9–11 Cytotoxic edema, because of the loss
of adenosine triphosphate (ATP)-maintained ion homeostasis,
generally precedes vasogenic edema.12,13 TBI impairs oxygen de-
livery to the brain, decreasing mitochondrial function and energy
production.14–17 Central to the regulation of water homeostasis are
aquaporins, a family of membrane water channels.18 Aquaporin 4
is the primary water channel expressed within astrocytic end feet
surrounding the brain vasculature. It is presumed to play a key role
in edema formation, especially during a state of inhibited energy
production. Inhibition of glial mitochondrial metabolism increases
astrocyte swelling and necrotic cell death, which has been impli-
cated as a primary cause of cytotoxic brain edema.19–22
Astrocytes, the most abundant cell type in the human brain,23,24
play a vital role in supporting and protecting neuronal function and
Departments of 1Cellular and Structural Biology and 2Neurosurgery, The University of Texas Health Science Center at San Antonio School ofMedicine, San Antonio, Texas.
JOURNAL OF NEUROTRAUMA 30:55–66 (January 1, 2013)ª Mary Ann Liebert, Inc.DOI: 10.1089/neu.2012.2488
55
in modulating brain energy metabolism.25,26 P2Y1R activation in as-
trocytes provides a mechanism whereby local extracellular signals can
rapidly elevate release of Ca2 + through increased production of
and, consequently, increases respiration and ATP production.29–32
Our laboratory demonstrated that both in vitro and in vivo
neuroprotection could be enhanced by increasing astrocyte mito-
chondrial metabolism via P2Y1Rs.33,34 The goal of this study was
to investigate the therapeutic effects of stimulating astrocyte
mitochondrial metabolism using 2MeSADP and MRS2365, two
P2Y1R ligands, as a novel therapy to treat patients with TBI.
Methods
Controlled closed skull injury model
A pneumatic impact device was used to generate a moderate TBIleaving the skull and dura matter intact. To achieve this, C57BL/6mice were anesthetized with isoflurane (3% induction, 1% mainte-nance) in 100% oxygen. A body temperature of 37�C was maintainedusing a temperature-controlled heated surgical table. A small midlineincision was made on the scalp using aseptic surgical techniques. A5-mm stainless steel disc was positioned on the skull and fixed usingsuperglue on the right parietal bone between bregma and lamda overthe somatosensory cortex. The mouse was then positioned on a stagedirectly under the pneumatic impact tip. A calibrated impact wasdelivered at 4.5 m/sec at a depth of 2 mm, which generates a mod-erate injury in the mouse. Apneic episode after injury and rightingreflex after removal of the mouse from anesthesia were timed andrecorded (supplementary Fig. 3; see online supplementary material athttp://liebertonline.com). Scalp incisions were closed using 4-0 ny-lon braided suture and antibiotic ointment applied to the incision.
Mice were placed in a Thermo-Intensive Care Unit (BraintreeScientific model FV-1; 37�C; 27% O2) and monitored until fullyawake and moving freely. Thirty minutes after injury or sham(uninjured), mice were treated with either vehicle (saline) or100 lM 2-methylthioladenosine 5¢ diphosphate (2MeSADP) orMRS2365 [[(1R, 2R, 3S, 4R, 5S)-4-[6-Amino-2-methylthio)-9H-purin-9-yl]-2,3-dihydroxybicycl-[3.1.0]hex-l-yl]methyl] dipho-sphonic acid mono ester trisodium salt (100 lm) intravenously.Brains were harvested at 1, 3, and 7 days post-TBI. At selectedsurvival times, mice were anesthetized under isoflurane, sacrificed,and prepared accordingly for the assay to be performed. For im-munostaining, mice were perfused with 4% paraformaldehyde in5% sucrose and post-fixed overnight followed by placement in 30%sucrose for 48 h. Brains were then stored at - 80�C until sliced.
IP3R2 knockout mice
A breeding pair of IP3R type 2 knockout mice were provided toour laboratory by Dr. Ken McCarthy with Dr. Ju Chen’s permis-sion. The mice were made in Dr. Chen’s laboratory, the details ofwhich can be found in Li and associates.35
Edema formation
To quantify cerebral edema after TBI, brain water content wasmeasured. Briefly, mice were anesthetized using 3% isoflurane 1, 3,or 7 days post-TBI or sham procedure. Mice were decapitated andthe brain removed. Samples were placed in glass bottles andweighed (wet weight). The brains were dried at 105�C for 72 hand reweighed (dry weight). Brain water content was calculated asthe difference between wet and dry weights using the formula ([wetweight-dry weight]/wet weight) · 100%.
Nissl staining
Standard procedures were used for detection of Nissl body foundin the cytoplasm of neurons to identify the neuronal changes.
Briefly, brains were harvested as described above and sectionedat 25 lm and placed on gelatin-coated slides. The slides were driedat 37�C overnight. Slides were hydrated with graded alcohols todistilled water, 0.1% cresyl violet was applied for 7 min, followedby a wash in distilled water. The slides were then dehydrated,
FIG. 1. P2Y1R stimulation (2MeSADP) reduces edema forma-tion in a traumatically injured brain. (A) Wild-type C57 mice wereprepared for sham, traumatic brain injury (TBI), or TBI treatedwith 2MeSADP as described in the methods. Mice were sacrificedat 1, 3, or 7 days post-injury or sham surgery and their brains wereremoved and imaged with a Nikon D70 macro lens. Re-presentative images of brains from sham, TBI, and TBI + 2Me-SADP mice are shown in A. Note the midline shift in the 24-hmouse after TBI represented with dashed lines. (B) The samebrains from A were then immediately weighed and placed in anoven at 105�C to determine the percent water content by the wet/dry weight method. Histogram of the percent water weight cal-culated using the wet/dry weight method demonstrating an in-crease in water content after TBI. a = p < 0.05, b = p < 0.01, andc = p < 0.001 compared with sham; d = p < 0.05 and e = p < 0.01compared with TBI mice at each respective time point. n = 5(sham), n = 8 (day 1), n = 5 (day 3), and n = 5 (day 7) for each timepoint examined.
56 TALLEY WATTS ET AL.
cleared in xylene, and a cover-slip applied. Images were acquiredon a Zeiss AX10 microscope using a 40 · objective. To analyze thesoma area, three serial sections from each animal were stained asdescribed.
After staining, brightfield images were collected from the im-pacted cortex and CA3 region of the impacted side of the brain at40 · . Image J was then used as follows: The find edges function keywas used to determine the boundaries of each cell, the images werethen despeckled, and the measure function was used to determinethe area of each soma. The data were then averaged for each regionimaged, and this was done for each of the three sections per animal.The resulting data were then averaged among all the animals in thisexperimental series.
GFAP staining
Slides were washed in phosphate buffered saline (PBS), per-meabilized with 0.2% Triton-X 100 in PBS. Sections were thenblocked in 5% goat serum. Glial fibrillary acidic protein (GFAP)antibody was diluted (1:1000) in goat serum and applied to thesections for 1 h at 37�C. The sections were then washed and sec-ondary goat anti-rabbit IgG antibody was applied at 1:200 for30 min at 37�C. Nuclei were stained with Hoechst 33342 (100 lg/mL).Slides were washed with PBS, and Vectashield was used to mountthe cover-slip. Images were acquired on Nikon C1si microscope
using a 60 · 1.1 NA water immersion objective. The 10 · imageswere also collected and were used for the analysis of the number ofreactive astrocytes per field (1.26 mm · 1.26 mm).
Western blot analysis for GFAP and AQ4
Mice were anesthetized using isoflurane and subsequently de-capitated. The brain was removed and placed on ice for dissectioninto impacted and non-impacted hippocampus and cortex. Theisolated tissue was rapidly homogenized in chilled homogenizationbuffer (0.32M sucrose, 1mM ethylenediaminetetraacetic acid, 1MTris-HCL pH = 7.8) on ice using a Wheaton glass dounce (20strokes). The homogenate was transferred to a 2 mL tube andcentrifuged at 1000 g for 10 min at 4�C; the supernatant was col-lected and analyzed. Protein concentration was determined by thebicinchoninic acid assay using a 1:50 dilution. For each sample,100 lg of protein was aliquoted and Laemmli buffer containingb-mercaptoethanol added; the sample was placed in a heat block for3 min at 95�C. Samples were loaded on a 12% gel and ran at 80 Vfor 20 min followed by 40 min at 130 V.
Samples were transferred to nitrocellulose membrane at 100Vfor 1 h. The membrane was blocked with 5% milk in Tris-bufferedsaline with Tweet (TBS-T) for 30 min. GFAP (1:1000-ImgenexIMG-5083-A) or AQ4 (1:500; Santa Cruz SC-9888) was added andplaced at 4�C overnight. The membrane was washed with TBS-T
FIG. 2. P2Y1R stimulation (2MeSADP) reduces cytotoxic edema formation in neurons of the somatosensory cortex and CA3 regionof the hippocampus demonstrated by Nissl staining. Mice underwent sham or traumatic brain injury (TBI) surgery under isofluraneanesthetic (n = 6 for each group). A subset of TBI mice received 2MeSADP 30 min post-TBI. Mice were sacrificed (1, 3, or 7 days post-injury; n = 5 (day 1), n = 6 (day 3) and n = 6 (day 7)), perfused, sectioned at 25 lm, and Nissl staining was performed. Cytotoxic edemawas evaluated by measuring the soma area of 20 cells per field. (A, B, D, E) Representative images collected with a 20 · objective of thesomatosensory cortex (A, B) and the CA3 region of the hippocampus (D, E) for sham, TBI, and TBI + 2MeSADP treated mice. Thedashed box denotes the area enlarged and displayed below each image. (C) Line plots demonstrating an increase in neuronal soma sizeafter TBI injury in the cerebral cortex. (F) Line plots demonstrating an increase in neuronal soma size after TBI injury in the CA3 regionof the hippocampus. Mice treated with 2MeSADP demonstrated a slower progression of increased neuronal soma size. The soma areawas measured from 20 cells per field for each animal and were averaged. * = p < 0.05, ** = p < 0.01, and *** = p < 0.001 compared withsham; # = p < 0.05 and ## = p < 0.01 compared with TBI mice at each respective time point.
three times for 10 min. Secondary antibody for GFAP (Donkeyanti-rabbit horseradish peroxidase [HRP] conjugated (Immuno-Jackson Laboratories; 711-035-152; 1:20000) or AQ4 (donkeyanti-goat HRP conjugated; Santa Cruz; sc-2020; 1:5000) was ap-plied at room temperature for 1 h. The membranes were washedwith TBS-T for 15 min (3 times) and developed using the WesternLightning� Plus-ECL kit (PerkinElmer, Inc) following manufac-turer’s directions.
Statistical analysis
One-way analysis of variance (ANOVA) was used to comparethe differences among three or more groups. The Student t test wasused to compare the difference between two groups. The signifi-cance level was set at p < 0.05. Data are presented asmean – standard error of the mean (SEM). GraphPad Prism soft-ware (GraphPad Software Inc.) was used to perform statisticalanalyses.
Results
P2Y1R stimulation reduces whole brain vasogenicedema formation after TBI
Edema formation is a classic indicator of TBI that is thought to
occur in two stages. First, cytotoxic edema forms because of the
loss of intracellular ATP and the disruption of ion homeostasis.36,37
Within minutes to hours, the second stage of edema is initiated
when vascular fluid flows into the extracellular space (vasogenic
edema) and increases intracranial pressure, generally with neuro-
destructive consequences.38–41 To test the impact of P2Y1R stim-
ulation on both stages of edema formation, we first developed a
controlled closed head injury model for mice using a pneumatic
impact device. Whole brain vasogenic edema formation was
measured as an increase in brain tissue water content using the wet/
dry weight method as described previously.42
One-way ANOVA analysis of time post-injury and treatment
group revealed a significant increase in water content after TBI
compared with sham-treated mice at all time points measured
(Fig. 1). There was an 8.06% increase in water content after TBI at
24 h post-injury compared with sham mice (sham 72.41% – 0.75,
n = 5; 24 h TBI 80.47% – 1.56, n = 8). Three days post-injury, a
10.35% increase was observed in TBI mice (82.76% – 1.8; n = 5)
compared with sham mice, and the increase was maintained 7 days
post-injury (82.62% – 1.3; n = 5) with a 10.21% increase above
sham mice. When test mice were injected in the tail vein with the
purinergic agonist 2MeSADP (100 lM, 100 lL) 30 min post-
injury, there was a significant reduction in the percent of whole
brain water content compared with untreated TBI mice at day 1
(74.69 – 0.65, n = 5), day 3 (78.38 – 1.18, n = 5) and day 7 (79.08 –1.01, n = 5) (Fig. 1). In addition, measurement of hemisphere size
also demonstrated a significant increase following TBI and was
reversed with 2Me SADP treatment (Supp. Fig. 1).
FIG. 3. P2Y1R stimulation (MRS2365) reduces cytotoxic edema formation in neurons of the somatosensory cortex and CA3 region ofthe hippocampus demonstrated by Nissl staining. Mice underwent sham or traumatic brain injury (TBI) surgery under isofluraneanesthesia. A subset of TBI mice received MRS2365 30 min post-TBI by tail vein injection (n = 5 (day 1), n = 6 (day 3) and n = 5 (day7)). Mice were sacrificed (1, 3, or 7 days post-surgery), perfused, sectioned at 25 lm, and Nissl staining was performed. Cytotoxicedema was evaluated by measuring the soma area of 20 cells per field. (A, B) Representative images collected with a 20 · objective ofthe somatosensory cortex from sham, TBI, and MRS-treated TBI mice. The dashed box denotes the area enlarged and displayed beloweach image. (C) Line plot of the averaged soma area of 20 cells from each section averaged. (D, E) Representative images collected witha 20 · objective of the CA3 region of the hippocampus. (F) Demonstrates the averaged neuronal soma size in the CA3 region of thehippocampus for TBI and TBI + MRS treated mice compared with sham. * = p < 0.05, ** = p < 0.01 and *** = p < 0.001 compared withsham; # = p < 0.05 and ## = p < 0.01 compared with TBI mice at each respective time point.
58 TALLEY WATTS ET AL.
P2Y1R stimulation reduces neuronal cytotoxic edemaformation in the hippocampus and cortex after TBI
After TBI, Nissl staining of brain slices revealed significant
increases in the soma size of neurons in both the ipsilateral cortex
and hippocampus compared with sham mice, consistent with
cytotoxic edema formation (Figs. 2 and 3). Twenty-four h post-
TBI, the average soma size in the ipsilateral cortex increased to
64.2 – 3.3 (n = 6) lm2 compared with the sham-treated soma size
66.96 – 1.26 (n = 5) lm2, respectively (Fig. 3F). At day 7 post-
injury, there was no statistical difference in the cortex or CA3
region of the hippocampus between mice treated with MRS com-
pared with non-treated TBI mice.
IP3R type2 deficient (IP3R2-/-) mice were used to determine
whether the neuroprotective effects mediated by 2MeSADP and
MRS2365 were abolished when the IP3 receptor expressed in
FIG. 4. P2Y1R stimulation (2MeSADP) does not reduce cytotoxic edema formation in neurons of the somatosensory cortex or CA3region of the hippocampus in IP3R2 -/- mice demonstrated by Nissl staining. Mice underwent sham or traumatic brain injury (TBI)surgery under isoflurane anesthesia. A subset of TBI mice received 2MeSADP 30 min post-TBI by tail vein injection. Mice weresacrificed (1 day post-surgery), perfused, and sectioned at 25lm, and Nissl staining was performed. Cytotoxic edema was evaluated bymeasuring the area of 20 cells per field. (A, C) Representative images collected with a 20 · objective of the cerebral cortex and CA3region of the hippocampus. The dashed box denotes the area enlarged and displayed below each image. (B, D) Histograms of the somaarea of 20 cells from each section averaged for the somatosensory cortex (B) and the CA3 region of the hippocampus (D). n = 3 pergroup; * = p < 0.05 compared with sham mice at each respective time point.
AQ4 upregulation is reversed by purinergic stimulation
As stated earlier, AQ4 is the primary water channel in the brain,
and it is assumed to be intimately associated with the regulation of
water homeostasis.43–45 To determine the effects of TBI on AQ4
expression, Western blot analysis was used. We found significant
increases in AQ4 protein expression in both the cortex and hip-
pocampus of mice sacrificed at days 1 and 3 post-TBI (Figs. 5A, B).
Twenty-four h post-injury, there was a 7% – 6.08 (n = 6) increase
compared with sham-treated mice, which increased by 11.57% –4.89 (n = 5) and 19.47% – 8.93 (n = 5) at days 3 and 7 post-injury
within the ipsilateral cortex (Fig. 5A). There was a comparable
increase in AQ4 expression within the hippocampus at 1 and 3 days
post-injury with a 10.8% – 6.42 (n = 6) increase at 1 day and a
5% – 5.18 (n = 5) increase at 3 days compared with Sham mice
(Fig. 5B). To determine the effects of P2Y1R stimulation on AQ4
expression after TBI, 2MeSADP was tail-vein injected 30 min
post-injury. We found that AQ4 expression was not significantly
changed at any of the time points examined, consistent with a
reduction in brain edema post-trauma (Fig. 5).
Purinergic stimulation reduces GFAP expressionin the cortex and hippocampus after TBI
GFAP is an intermediate filament protein involved in the
structure and function of the cytoskeleton of astrocytes. Increased
GFAP expression is also frequently used as a marker of reactive
gliosis that occurs after brain injuries.46–48 To test our model of
FIG. 5. Increases expression of Aquaporin 4 is reversed with P2Y1R stimulation. Mice were prepared for sham or traumatic braininjury (TBI) under isoflurane anesthesia, with a subset of TBI mice receiving 2MeSADP 30 min post-TBI. The mice were sacrificed at 1,3, and 7 h, and the cortex and hippocampus were isolated from ipsilateral and contralateral hemispheres. Western blot analysis wasperformed against AQ4 (1:500) and normalized to actin. Representative samples from sham, TBI, and 2MeSADP treated TBI mice areshown for both the cortex (A) and hippocampus (B) from the impacted side of the brain. Histograms are shown as the mean of thepercent of control – standard error of the mean with an n = 5 for each group. * p < 0.05 compared with sham. For TBI mice n = 6 (day 1),n = 5 (day 3) and n = 5 (day 7) and for TBI + 2MeSADP n = 6 for each time point examined.
60 TALLEY WATTS ET AL.
TBI-induced reactive gliosis, we performed Western blot analysis
and immunofluorescent staining for GFAP expression in sham,
TBI, or TBI and 2MeSADP mice sacrificed at 1, 3, and 7 days post-
injury. First, Western blot analysis confirmed that TBI induced a
significant increase in GFAP expression in both the cortex and
hippocampus at 3 and 7 days post-injury (Fig. 6). Expression in-
creased to 116.92% – 6.19 (n = 6) and 145.64% – 12.15 (n = 6)
within the cortex and to 130.41% – 5.49 (n = 6) and 196.34% –18.28 (n = 7) within the hippocampus, compared with sham mice at
days 3 and 7, respectively. P2Y1R stimulation by 2MeSADP
maintained these levels close to control values at each of these time
points. (Figs. 6A, B).
Second, we also observed a significant increase in the number
of GFAP positive cells in both the cortex and hippocampus of TBI
mice at all times tested (Fig. 7). The mean number of GFAP posi-
tive astrocytes in the cortex was initially 39.4 – 7.2 (per 1.59 mm2),
which significantly increased to 82 – 5.8 (per 1.59 mm2), 65 – 5.7
(per 1.59 mm2) and 61.8 – 3.6 (per 1.59 mm2) at days 1, 3, and 7 post-
injury. In mice treated with 2MeSADP, the number of GFAP posi-
tive cells in the cortex were significantly reduced to 49 – 3.4 (per
1.59 mm2), 52.4 – 3.3 (per 1.59 mm2) and 41 – 2.77 (per 1.59 mm2) at
1, 3, and 7 days post-injury. The same pattern of reactive astrocytes
was observed in the hippocampus of mice after TBI (Fig. 7). Sham
with comparable values of 81.8 – 4.1 (per 1.59 mm2) and 83.2 – 3.9
(per 1.59 mm2) at 24 h, respectively (Fig. 8). In addition, the
number of GFAP positive astrocytes in the hippocampus of sham
IP3R2-/- mice was 447.9 – 36.3 per field (1.26 mm · 1.26 mm). TBI
increased these numbers to 589.3 – 66.2 (per 1.59 mm2) and
FIG. 6. Increased GFAP expression is reduced by 2MeSADP treatment. Mice underwent sham or traumatic brain injury (TBI) with agroup of TBI mice receiving 2MeSADP 30 min post-TBI. The mice were sacrificed at 1, 3, or 7 days post-injury, and the cortex andhippocampus were isolated from ipsilateral and contralateral hemispheres. Western blot analysis was performed against GFAP (1:1000)and normalized to actin. Representative samples from sham, TBI, and TBI + 2MeSADP (100 lM) are shown for the cortex (A) and thehippocampus (B). Histograms are shown as mean of the percent of control + / - standard error of the mean. * p < 0.05 and ** p < 0.01compared with sham. The sample size of cortex measurements for TBI mice was n = 6 per time point and for hippocampal measurementsfor TBI mice were n = 6 (day 1), n = 6 (day 3) and n = 7 (day 7). The sample size for TBI + 2MeSADP mice were n = 6 for both cortexand hippocampus measurements.
612.4 – 57.4 (per 1.59 mm2) at 24 h, respectively (Fig. 8). We
concluded that P2Y1R-stimulated reductions of GFAP positive cell
in TBI mice is dependent on IP3 signaling.
Discussion
Treatment options to minimize and/or reverse brain damage
after TBI are clearly needed. Our studies support a new treatment
paradigm that significantly reduces severe injury in the primary
injury phase of TBI, thereby also diminishing the elevated neuro-
toxic effects observed during the secondary injury phase.3–6 In
brief, we enhanced the endogenous neuroprotective effects of as-
trocytes by stimulating their mitochondrial metabolism. Our
working model is that stimulation of P2Y1Rs, which are primarily
expressed on astrocytes (Supplementary Fig. 2; see online sup-
plementary material at http://liebertonline.com), increases IP3-
mediated intracellular Ca2 + release, which, in turn, is sequestered
by mitochondria, activating Ca2 + sensitive dehydrogenases and
ultimately leading to increased ATP production.33,34 Higher ATP
levels can then be used for any energy-dependent function that is
needed of astrocytes during brain injury.
An early critical need for energy utilization after TBI is the
maintenance of ion homeostasis. TBI-induced ischemia decreases
oxygen dependent ATP production.17,49,50 This disrupts ion
homeostasis, which increases osmotic stress and leads to water
uptake, swelling, and, if left unchecked, cell lysis. The damaging
role of edema during the early phase of TBI is well recog-
nized.12,36,51 Edema increases intracranial pressure, which further
decreases cerebral blood flow.9–11 The precise mechanisms un-
derlying edema formation following TBI, however, have not been
fully elucidated.7,8 Edema formation is separated into cytotoxic
and vasogenic components.13 Cell swelling from loss of ATP-
maintained ion homeostasis is referred to as cytotoxic edema. We
demonstrated that neuronal cytotoxic edema formation was sig-
nificantly increased by our pneumatic model of TBI, in both the
ipsilateral cortex and hippocampus. The ability of 2MeSADP and
FIG. 7. Reactive gliosis increases with traumatic brain injury (TBI) and is reduced with P2Y1R stimulation in the somatosensorycortex and the CA3 region of the hippocampus. Mice were prepared for sham or TBI under isoflurane anesthesia. After the mice weresacrificed at 1, 3, or 7 days post-injury and perfused with 4% PFA in 5% sucrose, brains were sectioned on a cryostat at 25 lm, andGFAP (green) immunofluorescence was performed using an anti-GFAP antibody. Nuclei are stained with DAPI (blue). The number ofGFAP positive cells was evaluated using the Image J Cell Counter Pluggin. (A) Representative images collected with a 60 · objective ofsomatosensory cortex (upper image) with a 4 · magnified region (lower image) demonstrated by the dashed box of each upper image.(B) Histogram demonstrating an increase in GFAP positive cells, counted from a 10 · image with an area of 1.26 mm · 1.26 mm, withTBI that was reduced by administration of 2MeSADP. (C) Demonstrates representative images from the CA3 region of the hippo-campus obtained using a 60 · objective (upper image) with a 4 · magnified image demonstrated by the dashed box within each upperimage. (D) Histogram demonstrating an increase in reactive astrocytes after TBI. 2MeSADP treatment reduced the number of reactiveastrocytes. n = 6 per group; a = p < 0.05, b = p < 0.01, and c = p < 0.001 compared with sham; d = p < 0.05, e = p < 0.01, and f = p < 0.001compared with TBI mice at each respective time point.
62 TALLEY WATTS ET AL.
MRS2365 treatments to significantly reduce cell swelling is
consistent with our working hypothesis that P2Y1R stimulation
increases intracellular ATP levels, which in turn permits cells to
better maintain ion homeostasis post-injury.
The second component of edema formation is vasogenic, which
occurs when water moves from the vasculature into the extracel-
lular space of brain tissue. Vascular edema is maximum during the
height of TBI-induced BBB breakdown, which generally occurs
between 3 and 6 h post-injury. During the hours and days after the
initial insult, the processes associated with the secondary injury
continue to induce both cytotoxic and vasogenic edema.52–54 Our
pneumatic model of TBI clearly exhibited an expected progression
of vasogenic edema in the days post-injury, as indicated by
significant increases in whole brain water content and size. Tail-
vein injections of 2MeSADP were very effective at reducing
vasogenic edema, which presumably is because of decreased cy-
totoxic edema or potentially, energy-dependent regulation of the
BBB integrity by astrocytes.
AQ4 has been implicated as the primary water channel within
astrocytic end feet surrounding the brain vasculature. Both its
distribution and expression play a major role in the severity of
edema formation under normal and pathological conditions.55 Mice
deficient in AQ4 exhibit reduced water content and edema forma-
tion after acute water intoxication and ischemic stroke.56,57 Over-
expression of AQ4 accelerates the accumulation of water content
after acute water intoxication, which induces rapid swelling of the
brain.58 Sun and associates59 demonstrated that AQ4 mRNA is
upregulated in male rats after a focal cortical contusion in the areas
within the injured cortex. Ribeiro and colleagues60 reported an
increase in the expression of AQ4 on the endfeet of astrocytes
within 1 h of stroke induction. In addition, Tomura and coworkers61
demonstrated that AQ4 expression is increased after TBI using
lateral fluid percussion injury in rats, and Rao and colleagues62
demonstrated that in vitro trauma upregulates AQ4 expression after
fluid percussion injury. Our pneumatic model of TBI is consistent
with these observations, because we also found that AQ4 expres-
sion was significantly increased after injury.
For the mice treated with 2MeSADP, AQ4 expression was sig-
nificantly diminished post-injury, which again was likely because
of enhanced energization of astrocyte mitochondria.34 An energy
dependence of AQ4 expression is consistent with other reports in
the literature where injuries are associated with edema formation.
These injuries include intracerebral hemorrhage,63 stroke,64 and
acute water intoxication.44
Astrocytes are intimately involved in the maintenance of the
brain under normal conditions. Their functional role after brain
trauma, however, is less well understood. Astrocytes clearly play an
important role in the restoration of ion homeostasis, neurotrans-
mitter clearance, and they secrete a number of neurotrophic factors
and amino acids that are crucial to neuronal survival.65–67 Because
of the close association of astrocytes and the BBB as well as the
presence of crucial metabolic substrate transporters, astrocytes are
thought to play a prominent role in regulating the flow of essential
nutrients to neurons that promote survival post-injury.
GFAP, a well-known protein that is primarily expressed in as-
trocytes, increases its expression after brain injury, and its protein
levels are frequently used as a biological marker of TBI as well as a
prognosis indicator after an injury to the brain.68–70 Consistent with
a neuroprotective role, GFAP-null mice are more sensitive to both
TBI and cerebral ischemia.71,72 The production of neurotrophins,
which promote neuronal survival and function, has been associated
with reactive (high GFAP expression) astrocytes. GFAP is also
associated with increased inflammation, the production of glial
cytokines, and an increase in astrocyte numbers (astrogliosis).73–77
FIG. 8. P2Y1R stimulation is not protective against increased reactive astrocytes in IP3R2-/- mice. IP3R2 -/- mice were prepared forsham or TBI with a subset of mice receiving 2MeSADP 30 min post-TBI. The mice were sacrificed 24 h post-TBI and were perfusedwith 4% PFA. Brains were sectioned on a cryostat at 25 lm, and GFAP immunofluorescence was performed as described in Methods.The number of GFAP positive cells was evaluated using Image J Cell Counter Pluggin. (A) Demonstrates representative imagescollected with a 60 · objective of the somatosensory cortex. (B) Line plot demonstrating an increase in GFAP positive cells in thesomatosensory cortex after TBI, which was not reduced by administration of 2MeSADP. (C) Demonstrates representative imagescollected with a 60 · objective of the CA3 region of the hippocampus. (D) Line plot demonstrating an increase in GFAP positive cells inthe CA3 region of the hippocampus after TBI. Administration of 2MeSADP did not reduce the number of reactive astrocytes in IP3R2-/-mice. (C) * p < 0.01 compared with sham (n = 3 for each group).
Finally, GFAP is highly associated with the structural integrity of
the astrocyte membrane, which affords it greater control over the
extracellular environment, a role crucial to neuronal survival. In our
model of TBI, GFAP expression was significantly increased in as-
trocytes. We also found that treatment of injured mice with 2Me-
SADP significantly diminished GFAP expression, similar to its effect
on AQ4 levels. The reduction in both GFAP and AQ4 expression
could be a direct result on expression occurring during the secondary
injury phase. Because all indications of injury (swelling, GFAP
and AQ4 expression) were diminished by 2MeSADP treatment,
however, we cannot attribute enhanced neuroprotection to any one
specific process or injury phase, aside from the increased availability
of ATP for energy dependent functions. In fact, enhanced protection
during the primary phase of injury is beneficial simply because of
the consequential decrease in the second phase of injury. In this
light, the ability of P2Y1R stimulation to minimize edema may be
particularly important, because the maintenance of cell integrity is
clearly necessary to support other neuroprotective functions.
Because of the increasing prevalence of persons with TBI as well
as the lack of treatment options, it is imperative that new treatment
strategies are developed. We have established a new treatment
paradigm that enhances the natural neuroprotective functions of
astrocytes. Specifically, we demonstrated that enhanced astrocyte
mitochondrial metabolism via stimulation of P2Y1Rs, provides a
much needed energy boost that significantly decreases damage
from TBI. The strength of this approach is that specific underlying
neuroprotective mechanisms do not need to be identified and in-
dividually stimulated. Rather, stimulation of mitochondrial ATP
production benefits all of the energy-dependent neuroprotective
functions of astrocytes.
Acknowledgments
LTW, JDL, SS, and MD formulated the hypotheses, organized
and designed the studies. LTW, SS, WZ, and RJG performed ex-
periments and analyzed the data. DJ, MD, and JDL contributed
reagents and supplies necessary for the study. LTW, RJG, and JDL
wrote and edited the article and figures.
Author Disclosure Statement
No competing financial interests exist.
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Address correspondence to:
James Lechleiter, PhD
Department of Cellular and Structural Biology
STRF Neuroscience Program MC8253
The University of Texas Health Science Center at San Antonio
