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EPILEPTIC SEIZURE-INDUCED
STRUCTURAL CHANGES IN GENETICALLY EPILEPTIC RAT
BONE TISSUES
Şebnem GARİP
Istanbul Kemerburgaz University, Faculty of Medicine, Medical Biochemistry
National Synchrotron Light SourceMolecular Biophysics Laboratory
TERMINOLOGY
Epilepsy; a common chronic brain disorder
characterized by recurrent seizures due to excessive discharge of cerebral neurons [1]
inability to control excitability of neurons
too many neurons firing at the same time
SUDDEN SEIZURE
IS THERE A CURE FOR EPILEPSY?
There is no known cure for epilepsy as yet.
Epilepsy & Bone lossOpen Problems?
bone disorders in epilepsy patients
The possible effects of epilepsy and seizures on bone:
• cannot be investigated in clinical studies
• no study with convenient animal model
The effects of anti-epileptic drugs and epileptic seizures on bone cannot be differentiated
Animal Studies
1. Control (Wistar rats) 2. Epileptic (WAG/Rij rats)
Bone tissues;FemurTibiaLumbar spine
Bone tissues;FemurTibiaLumbar spine
Serum samplesKidney Liver
Imaging Studies
• FTIR Microspectroscopy
Biomechanical Studies
• Vickers Microhardness Test
Traditional Biochemical Studies
• ELISA:
• Westernblot
Calcium25(OH)D1,25(OH)DPTHALPOsteocalcinC-telopeptides
MATERIALS & METHODSRESEARCH PLAN
SR-FTIR Imaging AFM Microscopy
Lumbar spine
Nano-indentation Test
Lumbar spine
• 50 to 70% mineral: [Ca10(PO4)6(OH)2]• 20 to 40% organic matrix: type I collagen, non-collagenous proteins• 5 to 10% water• <3% lipids
Exterior Interior
Fixation in EtOH, tissues kept in:70% EtOH for 2 times 2 days each95% EtOH 2 times 2 days each100% 2-propanol 2 times 1 day eachXylene solution for 2 times 1 day each(Shenk et al., 1984)
Infiltration of PMMA, tissues kept in:
Solution I (60 ml methylmethacrylate, 25 ml buthylmethacrylate, 5 ml methylbenzoate, 1.2 ml polyethylene glycol 200) for 4 days
Solution II (100 ml methylmethacrylate, 0.4 gr benzoyl peroxide) for 4 days
Solution III (100 ml methylmethacrylate, 0.8 gr benzoyl peroxide) for 4 days
Embedding in PMMA
Tissues embedded in Solution III added with 400 µl N,N-dimethyl-p-toluidine (accelerator of polymerization, for 100 ml of Sol III)
Solution III mixed on ice for 1 hour prior to usage,
Tissues kept at 4˚C for 3 days and put in oven at 60˚C for 1 day.
Methacrylate Method (R.G Erben, The Journal of Histochemistry & Cytochemistry Volume 45(2): 307–313, 1997).
• 2 µm sections for IR imaging• IR transparent BaF2 windows • 2000–500 cm-1 wavenumber region• 1.00 X 1.00 µm pixel size
Analysis;• Images were analysed by ISys.
Statistical Study;• Bonferroni t-test• *p ≤ 0,05; **p ≤ 0,01; ***p ≤ 0,001
FTIR Microspectroscopy
Amide I(protein)
ѵ1,ѵ3 phosphate(mineral)
ѵ2CO32-
carbonate(mineral)
Garip et al., J Biomed Opt. 2013; Nov;18(11):111409. doi: 10.1117/1.JBO.18.11.111409.
FTIR Microspectroscopy
SR-FTIR Microspectroscopy
RESULTS & DISCUSSION
SR-FTIR Microspectroscopic Studies
Phosphate v4Amide I
Phosphate band area (650-500 cm-1) /amide I (1588-1712 cm-1)
band area
Mineral content of bone [8]
Mineral/matrix ratio:
Carbonate band area (850-890 cm-1) /phosphate band area (650-500 cm-
1)
Carbonate substitution in hydroxyapatite crystals [8]
Relative Carbonate Content:
IR parameters which give information about bone quality
Car
bona
te
Phosphate v4
Garip et al., J Biomed Opt. 2013; Nov;18(11):111409. doi: 10.1117/1.JBO.18.11.111409.
immature bone and/or
impaired mineralization
RATIO CONTROL EPILEPTIC
Mineral/Matrix 7.7 ± 0.5 5.2 ± 0.3**
Decreased mineral/matrix ratio
Mineral/Matrix Ratio
RATIO CONTROL EPILEPTIC
Carbonate/Mineral 0.015 ± 0.002 0.010 ± 0.001*
Carbonate/Mineral Ratio
16601690
Band intensity ratios of the sub-bands at 1660 cm-1 and 1690 cm-1
Helical structure and function of collagen molecules [8]
Collagen Crosslinks
Band intensity ratios of the sub-bands at 603 cm-1 and 563 cm-1
Crystal size in bone mineral [8]
Crystallinity
603563
IR parameters which give information about bone quality
Garip et al., J Biomed Opt. 2013; Nov;18(11):111409. doi: 10.1117/1.JBO.18.11.111409.
RATIO CONTROL EPILEPTIC
Collagen Crosslinks
3.0 ± 0.3 2.2 ± 0.3*
Less mature collagenand/or
Excessive bone turnover
Decreased collagen crosslinks ratio
Cross-links Ratio
Increased crystal size
RATIO CONTROL EPILEPTIC
Crystallinity 0.50 ± 0.05 0.86 ± 0.05+++
Less ordered orientationand
rigidity
Crystallinity
epileptic seizures affected bothbone mineral and matrix
excessive bone turnover impaired mineralization due to vitamin D deficiency
CONCLUSION
Garip et al., J Biomed Opt. 2013; Nov;18(11):111409. doi: 10.1117/1.JBO.18.11.111409.
Middle East Technical University
Prof. Dr. Feride Severcan
Molecular Biophysics (Lab 146) Group
Brookhaven National Laboratory
Prof. Dr. Lisa Miller
Dr. Randy Smith
THANK YOU
Why Wistar Albino Glaxo rats from Rijswijk (WAG/Rij)?
• Genetically absence epileptic rats
• Subgroup of WAG/Rij rats (mixed form of epilepsy)
• Characteristics of WAG/Rij rats are similar to those of outbred Wistar rats
The advantage of investigating the effects of seizuresalone on bone tissues
non-convulsive absenceseizures
convulsive audiogenic seizures
Animal studies were carried out in Kocaeli University
CARBONATE TYPE CONTROL EPILEPTIC
A Type(878 cm-1)
3.13 ± 0.04 3.01 ± 0.02*
B Type(872 cm-1)
3.54 ± 0.03 3.88 ± 0.05**
L Type(866 cm-1)
3.22 ± 0.02 3.15 ± 0.1*
impaired mineralizationand/or
excessive bone turnover
Increased B-type carbonate substitution
Garip et al., 2012 (submitted to Epilepsia)
• non-disturbing technique which provides quantitative and structural information about biological samples [15].
• valuable technique due to its high sensitivity in detecting changes in the functional groups belonging to tissue components, such as lipids, proteins, carbohydrates and nucleic acids [16,17].
• By using FTIR Microspectroscopy, it is possible to study different regions of bone at molecular level without any dying processes [18].
• pathological regions and disease-induced changes can be identified which may have diagnostic value [18].
Why FTIR Microspectroscopy?
COLLAGEN CROSSLINKS
Reducible crosslinks
Non-reducible crosslinks
EELECTROMAGNETIC SPECTRUMLECTROMAGNETIC SPECTRUM
MATTER
ELECTROMAGNETIC RADIATION
ABSORPTION
EMISSION
SCATTERING
* After interacting with an electromagnetic
radiation, a matter will either absorb, emit,
or scatter light particles.
ELECTROMAGNETIC RADIATIONELECTROMAGNETIC RADIATION
14000 4cm-1
INFRARED REGIONINFRARED REGION
Region Wavenumber range (cm-1)
Near IR 14000-4000
Middle IR 4000-400
Far IR 400-4
* Infrared spectrum is a map of the internal vibrational frequencies versus energy of interaction with infrared (IR) radiation.
ENERGY LEVEL DIAGRAMENERGY LEVEL DIAGRAM
Distance between electrons and nucleus or between atoms in a molecule
ENERGY
First excited state
Ground state
Vibrational levels
* Transitions between vibrational levels of the ground state of a molecule result from the absorption of light in the infrared region of the electromagnetic spectrum.
Stretching
Antisymmetric
Stretching
Bending Bending
Stretching Antisymmetric
Stretching
Bending Bending
* Types of normal vibration in a linear and non-linear triatomic molecule. Atomic displacements are represented by arrows (in plane of page) and by + and – symbols (out of page plane).
TYPES OF MOLECULAR TYPES OF MOLECULAR VIBRATIONSVIBRATIONS
Symmetric Stretching
Antisymmetric Stretching
Bending
Animated representation of some of the molecular vibrations.
Instrumentation ofInstrumentation of an an FTFT--IRIR
SSpectrpectrometerometer
Sample
Detector
Modulated exit beam
Beam splitter
Source
Stationary mirror
Movingmirror
He-Ne laser light
White light
Detector
Referenceinterferometer
Unmodulatedincident beam
Schematic representation of FT-IR spectrometer
AB
SO
RB
AN
CE
3500 3000 2500 2000 1500 1000 500 WAVENUMBER(cm-1)
Lipids
Proteins
Phosphate
Carbonate
Phosphate
BASIC MECHANISMS UNDERLYING SEIZURING AND EPILEPSY
The basic mechanism of neuronal excitability is action
potential;
•an increase in excitatory synaptic neurotransmission,
•decrease of inhibitory neurotransmission
•alteration in voltage-gated ion channels
•alteration of intra- or extra-cellular ion
concentrations.
The major neurotransmitters in the brain are glutamate,
gamma-amino-butyric acid (GABA), acetylcholine (ACh),
norepinephrine, dopamine, serotonin, and histamine. Molecules, such
as neuropeptides and hormones, play modulatory roles that modify
neurotransmission over longer time periods.
BASIC MECHANISMS UNDERLYING SEIZURING AND EPILEPSY