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