EPILEPTIC SEIZURE-INDUCED STRUCTURAL CHANGES IN GENETICALLY EPILEPTIC RAT BONE TISSUES

1. EPILEPTIC SEIZURE-INDUCED STRUCTURAL CHANGES IN GENETICALLY EPILEPTIC RAT BONE TISSUES Şebnem GARİP Istanbul Kemerburgaz University, Faculty of Medicine, MedicalBiochemistry

2. National Synchrotron Light Source Molecular Biophysics Laboratory

3. TERMINOLOGY Epilepsy; acommon chronic brain disordercharacterized 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

4. IS THERE A CURE FOR EPILEPSY? • There is no known cure for epilepsy as yet.

5. Epilepsy & Bone loss Open 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

6. MATERIALS & METHODS RESEARCH PLAN • Animal Studies • Control (Wistar rats) • Epileptic (WAG/Rij rats) Serum samples Kidney Liver Bone tissues; Femur Tibia Lumbar spine Bone tissues; Femur Tibia Lumbar spine Traditional Biochemical Studies • Imaging Studies • FTIR Microspectroscopy • Biomechanical Studies • Vickers Microhardness • Test Calcium 25(OH)D 1,25(OH)D PTH ALP Osteocalcin C-telopeptides • ELISA: • Westernblot Lumbar spine Lumbar spine SR-FTIR Imaging AFM Microscopy Nano-indentation Test

7. Interior Exterior • 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

8. Methacrylate Method (R.G Erben, The Journal of Histochemistry & Cytochemistry Volume 45(2): 307–313, 1997). 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 Fixation in EtOH, tissues kept in: 70% EtOH for 2 times 2 days each 95% EtOH 2 times 2 days each 100% 2-propanol 2 times 1 day each Xylene solution for 2 times 1 day each (Shenk et al., 1984) 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.

9. 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

10. FTIR Microspectroscopy ѵ1,ѵ3 phosphate (mineral) Amide I (protein) ѵ2CO32- carbonate (mineral) Garip et al., J Biomed Opt. 2013; Nov;18(11):111409. doi: 10.1117/1.JBO.18.11.111409.

11. FTIR Microspectroscopy SR-FTIR Microspectroscopy

12. RESULTS & DISCUSSION SR-FTIR Microspectroscopic Studies

13. IR parameters which give information about bone quality Carbonate Phosphate v4 Phosphate v4 Amide I Relative Carbonate Content: Mineral/matrix ratio: Phosphate band area (650-500 cm-1) /amide I (1588-1712 cm-1) band area Mineral content of bone[8] Carbonateband area (850-890 cm-1) /phosphate band area (650-500 cm-1) Carbonate substitution in hydroxyapatite crystals[8] Garip et al., J Biomed Opt. 2013; Nov;18(11):111409. doi: 10.1117/1.JBO.18.11.111409.

14. Mineral/Matrix Ratio Decreased mineral/matrix ratio immature bone and/or impaired mineralization

15. Carbonate/Mineral Ratio

16. IR parameters which give information about bone quality 563 603 1660 1690 Crystallinity Collagen Crosslinks Band intensity ratios of the sub-bands at 603 cm-1 and 563 cm-1 Crystal size in bone mineral[8] Band intensity ratios of the sub-bands at 1660 cm-1 and 1690 cm-1 Helical structure and function of collagen molecules [8] Garip et al., J Biomed Opt. 2013; Nov;18(11):111409. doi: 10.1117/1.JBO.18.11.111409.

17. Cross-links Ratio Decreased collagen crosslinks ratio Less mature collagen and/or Excessive bone turnover

18. Crystallinity Increased crystal size Less ordered orientation and rigidity

19. CONCLUSION epileptic seizures affected both bone mineral and matrix impaired mineralization due to vitamin D deficiency excessive bone turnover Garip et al., J Biomed Opt. 2013; Nov;18(11):111409. doi: 10.1117/1.JBO.18.11.111409.

20. Middle East Technical University Prof. Dr. Feride Severcan Molecular Biophysics (Lab 146) Group Brookhaven National Laboratory Prof. Dr. Lisa Miller Dr. Randy Smith

21. THANK YOU

22. Why Wistar Albino Glaxo rats from Rijswijk (WAG/Rij)? Animal studies were carried out in Kocaeli University • Genetically absence epileptic rats • Subgroup of WAG/Rij rats (mixed form of epilepsy) convulsive audiogenic seizures non-convulsive absence seizures • Characteristics of WAG/Rij rats are similar to those of outbred Wistar rats The advantage of investigating the effects of seizures alone on bone tissues

23. Increased B-type carbonate substitution impaired mineralization and/or excessive bone turnover Garip et al., 2012 (submitted to Epilepsia)

24. Why FTIR Microspectroscopy? • 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 identifiedwhich may have diagnostic value[18].

25. COLLAGEN CROSSLINKS Reducible crosslinks Non-reducible crosslinks

26. ELECTROMAGNETIC SPECTRUM

27. ELECTROMAGNETIC RADIATION * After interacting with an electromagnetic radiation, a matter will either absorb, emit, or scatter light particles. ABSORPTION ELECTROMAGNETIC RADIATION MATTER EMISSION SCATTERING

28. Region Wavenumber range (cm-1) Near IR 14000-4000 14000 4 cm-1 Middle IR 4000-400 Far IR 400-4 INFRARED REGION * Infrared spectrumis a map of the internal vibrational frequenciesversusenergy of interaction with infrared (IR) radiation.

29. ENERGY LEVEL DIAGRAM First excited state E N E R G Y Vibrational levels Ground state Distance between electrons and nucleus or between atoms in a molecule * Transitions between vibrational levels of theground state of a molecule result from the absorption of light in the infrared region of the electromagnetic spectrum.

30. Stretching Antisymmetric Stretching Bending Bending Stretching Antisymmetric Stretching Bending Bending TYPES OF MOLECULAR VIBRATIONS * Types of normal vibration in a linear and non-linear triatomic molecule.Atomic displacements arerepresented by arrows (in plane of page) and by + and – symbols (out of page plane).

31. Symmetric Stretching Antisymmetric Stretching Bending Animated representation of some of the molecular vibrations.

32. Instrumentation of an FT-IR Spectrometer

33. Stationary mirror Unmodulated incident beam Moving mirror Source Beam splitter He-Ne laser light Modulated exit beam Sample White light Reference interferometer Detector Detector Schematic representation of FT-IR spectrometer

34. Phosphate Proteins Phosphate Lipids ABSORBANCE Carbonate 3500 3000 2500 2000 1500 1000 500WAVENUMBER(cm-1)

35. 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.

36. BASIC MECHANISMS UNDERLYING SEIZURING AND EPILEPSY 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.