EPILEPTIC
SEIZURE-INDUCED
STRUCTURAL CHANGES IN
GENETICALLY EPILEPTIC RAT
BONE TISSUES
Şebnem GARİP
Istanbul Kemerburgaz University, Faculty of Medicine,
Medical Biochemistry
Molecular Biophysics Laboratory
National Synchrotron Light Source
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 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
MATERIALS & METHODS
RESEARCH PLAN
Animal Studies
1. Control (Wistar rats)
2. Epileptic (WAG/Rij rats)
Bone tissues;
Femur
Tibia
Lumbar spine
Bone tissues;
Femur
Tibia
Lumbar spine
Imaging Studies
Biomechanical Studies
• FTIR Microspectroscopy
Lumbar spine
SR-FTIR Imaging
AFM Microscopy
• Vickers Microhardness
Test
Serum samples
Kidney
Liver
Traditional
Biochemical Studies
• ELISA:
Lumbar spine
Nano-indentation Test
• Westernblot
Calcium
25(OH)D
1,25(OH)D
PTH
ALP
Osteocalcin
C-telopeptides
Exterior
Interior
• 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
Methacrylate Method
(R.G Erben, The Journal of Histochemistry & Cytochemistry Volume 45(2): 307–313, 1997).
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)
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-ptoluidine (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.
• 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
ѵ1,ѵ3 phosphate
(mineral)
Amide I
(protein)
ѵ2CO32carbonate
(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
Carbonate
IR parameters which give information about bone quality
Phosphate v4
Amide I
Mineral/matrix ratio:
Phosphate v4
Relative Carbonate Content:
Phosphate band area (650-500
cm-1) /amide I (1588-1712 cm-1)
band area
Carbonate band area (850-890 cm1) /phosphate band area (650-500
cm-1)
Mineral content of bone [8]
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.
Mineral/Matrix Ratio
Decreased mineral/matrix ratio
immature bone
and/or
impaired mineralization
RATIO
CONTROL
EPILEPTIC
Mineral/Matrix
7.7 ± 0.5
5.2 ± 0.3**
Carbonate/Mineral Ratio
RATIO
Carbonate/Mineral
CONTROL
EPILEPTIC
0.015 ± 0.002
0.010 ± 0.001*
IR parameters which give information about bone quality
603
1690 1660
Collagen Crosslinks
563
Crystallinity
Band intensity ratios of the subbands at 1660 cm-1 and 1690 cm-1
Band intensity ratios of the subbands at 603 cm-1 and 563 cm-1
Helical structure and function of
collagen molecules [8]
Crystal size in bone mineral [8]
Garip et al., J Biomed Opt. 2013; Nov;18(11):111409. doi: 10.1117/1.JBO.18.11.111409.
Cross-links Ratio
Decreased collagen crosslinks
ratio
Less mature collagen
and/or
Excessive bone turnover
RATIO
Collagen
Crosslinks
CONTROL EPILEPTIC
3.0 ± 0.3
2.2 ± 0.3*
Crystallinity
Increased crystal size
Less ordered orientation
and
rigidity
RATIO
Crystallinity
CONTROL
EPILEPTIC
0.50 ± 0.05 0.86 ± 0.05+++
CONCLUSION
epileptic seizures affected both
bone mineral and matrix
excessive bone turnover
impaired mineralization
due to vitamin D deficiency
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)?
Animal studies were carried out in Kocaeli University
• Genetically absence epileptic rats
• Subgroup of WAG/Rij rats (mixed form of epilepsy)
non-convulsive absence
seizures
convulsive audiogenic
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
Increased B-type carbonate
substitution
impaired mineralization
and/or
excessive bone turnover
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*
Garip et al., 2012 (submitted to Epilepsia)
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
identified which may have diagnostic value [18].
COLLAGEN CROSSLINKS
Reducible crosslinks
Non-reducible crosslinks
ELECTROMAGNETIC SPECTRUM
ELECTROMAGNETIC RADIATION
*
After
interacting
with
an
electromagnetic
radiation, a matter will either absorb, emit, or
scatter light particles.
ABSORPTION
ELECTROMAGNETIC
RADIATION
MATTER
EMISSION
SCATTERING
14000
4
cm-1
INFRARED 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 DIAGRAM
E
N
E
R
G
Y
First excited state
Vibrational levels
Ground state
Distance between electrons and nucleus or between atoms in a molecule
* 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.
TYPES OF MOLECULAR VIBRATIONS
Stretching
Stretching
Antisymmetric
Stretching
Antisymmetric
Stretching
Bending
Bending
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).
Symmetric Stretching
Antisymmetric
Stretching
Bending
Animated representation of some of the
molecular vibrations.
Instrumentation of an
FT-IR Spectrometer
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
Phosphate
Proteins
ABSORBANCE
Phosphate
Lipids
Carbonate
3500
3000
2500
2000
1500
WAVENUMBER(cm-1)
1000
500
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.
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.