Figure Legends
Figure 1: Histology and immunohistochemistry reveal features of
hippocampal sclerosis. Photomicrographs showing histology of the
MTLE-HS hippocampal samples and non-seizure control tissue sections.
Hematoxylin & Eosin stained MTLE-HS hippocampal specimens show (a) loss
of neurons, (b) highlighted by NeuN, (c) GFAP shows reactive
astrogliosis (arrow heads). Autopsy control specimens show (d) no loss
of neurons in H & E stained sections, (e) supported by NeuN IHC, (f) no
reactive gliosis as revealed in GFAP IHC.
Figure 2: Quantitative estimation of tryptophan-kynurenine
pathway metabolites. (a) concentration of tryptophan (autopsy control,
n=6, MTLE-HS, n=20; 1.068 ± 0.28 ng/mg of wet tissue in autopsy control
vs 0.086 ± 0.013 ng/mg of wet tissue in MTLE-HS), (b) concentration of
kynurenine (autopsy control, n=10, MTLE-HS, n=43; 2.895 ± 1.05 ng/mg of
wet tissue in autopsy control vs 1.74 ± 0.57 ng/mg of wet tissue in
MTLE-HS), (c) tryptophan kynurenine ratio (autopsy control, n=6,
MTLE-HS, n=20; 1.58 ± 0.80 in autopsy control vs 0.05 ± 0.009 in
MTLE-HS), (d) concentration of kynurenic acid (autopsy control, n=10,
MTLE-HS, n=43; 0.184 ± 0.046 ng/mg of wet tissue in autopsy control vs
0.0289 ± 0.0039 ng/mg of wet tissue in MTLE-HS), (e) concentration of
quinolinic acid (autopsy control, n=10, MTLE-HS, n=14; 11.67 ± 2.98
ng/mg of wet tissue in autopsy control vs 39.22 ± 9.23 ng/mg of wet
tissue in MTLE-HS), (f) quinolinic acid kynurenic acid ratio (autopsy
control, n=10, MTLE-HS, n=14; 90.34 ± 28.97 in autopsy control vs 242.0
± 20.25 in MTLE-HS), (g) concentration of glutamate (autopsy control,
n=10, MTLE-HS, n=12; 12.08 ± 3.01 ng/µg protein in autopsy control vs
10.57 ± 4.7 ng/µg protein in MTLE-HS), (h) concentration of GABA
(control, n=10, MTLE-HS, n=12; 1.18 ± 0.32 ng/µg protein in autopsy
control vs 1.15 ± 0.16 ng/µg protein in MTLE-HS), (i) glutamate GABA
ratio (autopsy control, n=10, MTLE-HS, n=12; 15.34 ± 4.98 in autopsy
control vs 8.46 ± 2.02 in MTLE-HS) in autopsy control samples and
hippocampal samples obtained patients with MTLE-HS. (j) Concentration ofde novo synthesis of kynurenic acid (non-seizure control, n=18,
MTLE-HS, n=26; 5.28 ± 1.13 ng/mg of wet tissue in non-seizure control vs
0.10 ± 0.03 ng/mg of wet tissue in MTLE-HS) and (k) concentration ofde novo synthesis of quinolinic acid (non-seizure control, n=12,
MTLE- MTLE-HS, n=12; 8.66 ± 2.28 ng/mg of wet tissue in non-seizure
control vs 22.33 ± 7.47 ng/mg of wet tissue in MTLE-HS) in non-seizure
control samples and hippocampal samples obtained patients with MTLE-HS.
Data are presented as a mean ± SEM. * denotes p<0.05, **
p<0.01, *** p<0.001. Mann Whitney Test.
Figure 3: Spontaneous excitatory postsynaptic currents were
enhanced in MTLE-HS hippocampal samples but spontaneous inhibitory
postsynaptic currents were unaffected. (a) Sample recordings of
spontaneous EPSCs recorded from pyramidal neurons in the cortical sample
obtained from non-seizure controls. Inset shows a single EPSC event at
an expanded time scale. The second trace shows absence of any
spontaneous EPSCs following perfusion of the slice with glutamate
receptor antagonists APV (50 µM) and CNQX (10 µM) for 10-min proving
that these events are glutamate receptor. The bottom trace shows sample
recordings of spontaneous EPSCs recorded from pyramidal neurons in the
hippocampal sample obtained from patients with MTLE-HS. Plots represent
data from eight neurons from eight patients for non-seizure control,
nine neurons from the hippocampal samples of nine MTLE-HS patients. (b)
In the hippocampal samples, cumulative distribution of inter-event
interval displaced toward lower intervals and (c) that of peak amplitude
displaced towards longer amplitude. (d) Sample recordings of spontaneous
IPSCs recorded from pyramidal neurons in the cortical sample obtained
from non-seizure controls and the hippocampal samples obtained from
patients with MTLE-HS. The bottom trace shows absence of any spontaneous
IPSCs following perfusion of the slice with GABAAreceptor antagonist bicuculline (10 µM) for 10-min proving that these
events are GABAA receptor mediated. Plots represent data
from five neurons from five patients for non-seizure control, five
neurons from the hippocampal samples of five MTLE-HS patients. (e)
Cumulative distribution of inter-event interval and (f) peak amplitude
of the IPSCs also did not alter. (g) Frequency and amplitude ratio of
EPSC/IPSC both were significantly higher in the hippocampal samples.
Data represented as mean ± SEM. *p<0.01, **p<0.01,
***p<0.001. Two tailed unpaired T-test/ Mann Whitney Test.
Figure 4: Spontaneous excitatory postsynaptic currents were not
suppressed after incubation with 200 µM kynurenine in the hippocampal
samples obtained from patients with MTLE-HS. (a) Sample recordings of
spontaneous EPSCs recorded from pyramidal neurons in cortical sample
obtained from non-seizure controls before and after incubation with200
µM kynurenine for 85 mins. Plots represent data from six neurons from
six patients for non-seizure control. (b) Cumulative distribution of
inter-event interval displaced towards longer intervals while that of
(c) peak amplitude displaced towards shorter amplitude. (d) Sample
recordings of spontaneous EPSCs recorded from pyramidal neurons in
hippocampus sample obtained from MTLE-HS patients without and with 200
µM kynurenine. Plots represent data from sixteen neurons from sixteen
patients for MTLE-HS. (e) Cumulative distribution of inter-event
interval and (f) peak amplitude did not displace. (g) Percentage
reduction of frequency was significantly reduced in the hippocampal
samples after incubation with 200 µM kynurenine. Data represented as
mean ± SEM. ***p<0.001. Two tailed unpaired T-test/ Mann
Whitney Test.
Figure 5: Spontaneous excitatory postsynaptic currents were
suppressed after 30 mins of perfusion with 10 µM kynurenic acid in the
hippocampal samples obtained from patients with MTLE-HS. (a) Sample
recordings of spontaneous EPSCs recorded from pyramidal neurons in
cortical sample obtained from non-seizure controls without and with 10
µM kynurenic acid. Plots represent data from six neurons from six
patients for non-seizure control. (b) Cumulative distribution of
inter-event interval displaced towards longer intervals while that of
(c) peak amplitude displaced towards shorter amplitude. (d) Sample
recordings of spontaneous EPSCs recorded from pyramidal neurons in the
hippocampus sample obtained from patients with MTLE-HS without and with
10 µM kynurenic acid. Plots represent data from ten neurons from ten
patients for MTLE-HS. (e) Cumulative distribution of inter-event
interval displaced towards longer intervals while that of (f) peak
amplitude displaced towards shorter amplitude. (g) Percentage reduction
of frequency was significantly increased in the hippocampal samples
after perfusion with 10 µM kynurenic acid. (h) Perfusion with 50 µM APV
significantly reduced frequency of spontaneous excitatory postsynaptic
currents in both non-seizure controls and hippocampal samples (n=6; 0.98
± 0.018 Hz without APV vs 0.41 ± 0.25 Hz with APV). (i) Percentage
reduction of frequency was significantly increased in the hippocampal
samples after perfusion with 50 µM APV respectively. (j) Within the
hippocampal samples, percentage reduction of frequency was significantly
increased after kynurenic acid treatment in comparison to kynurenine
incubation and after APV treatment in comparison to kynurenic acid. Data
represented as mean ± SEM. ***p<0.001, #p<0.05. Two
tailed unpaired T-test/ Mann Whitney Test/one way ANOVA with Dunn’s
posthoc test.
Figure 6: Altered enzyme expression levels of
tryptophan-kynurenine pathway contributes to reduced KYNA levels in the
hippocampal samples obtained from patients with MTLE-HS. (a) KAT II
mRNA expression did not alter significantly (autopsy control, n=6,
MTLE-HS, n=12; ΔCT 1.49 ± 0.25 in autopsy control vs
ΔCT 1.43 ± 0.44 in MTLE-HS) but (b) protein level
expression was significantly decreased in the hippocampal samples
(autopsy control, n=8, MTLE-HS, n=10; normalised expression 0.90 ± 0.03
in autopsy control vs normalised expression 0.76 ± 0.062 in MTLE-HS).
(c) IDO mRNA expression (autopsy control, n=6, MTLE-HS, n=10;
ΔCT 11.12 ± 1.0 in autopsy control vs
ΔCT 1.67 ± 0.89 in MTLE-HS) as well as (d) protein
expression (autopsy control, n=8, MTLE-HS, n=10; 246.4 ± 11.36 ng/ml in
autopsy control vs 294.7 ± 2.77 ng/ml in MTLE-HS) both significantly
increased in the hippocampal samples. (e) KMO mRNA (autopsy control,
n=6, MTLE-HS, n=12; ΔCT 7.11 ± 0.81 in autopsy control
vs ΔCT 5.95 ± 0.49 in MTLE-HS) as well as (f) protein
level expression (autopsy control, n=8, MTLE-HS, n=10; normalised
expression 0.79 ± 0.073 in autopsy control vs normalised expression 0.74
± 0.049 in MTLE-HS) did not alter. (g) PLP concentration was
significantly reduced in the hippocampal samples (autopsy control, n=6,
MTLE-HS, n=27; 5.03 ± 1.01 ng/mg of wet tissue in autopsy control vs
2.04 ± 0.75 ng/mg of wet tissue in MTLE-HS). (h) mRNA expression of PNPO
was significantly reduced in the hippocampal samples (autopsy control,
n=6, MTLE-HS, n=9; ΔCT 3.41 ± 0.43 in autopsy control vs
ΔCT 5.8 ± 0.29 in MTLE-HS). (i) Protein expression of
PNPO was also significantly reduced in the hippocampal samples (autopsy
control, n=8, MTLE-HS, n=10; normalised expression 0.58 ± 0.042 in
autopsy control vs normalised expression 0.39 ± 0.045 in MTLE-HS). The
Western blot images of KAT II, KMO, PNPO, and GAPDH represent single
band of KAT II at the predicted size of 50 KDa, KMO at 45 KDa, PNPO at
30 KDa and GAPDH at 37 KDa. Data represented as mean ± SEM. *
p<0.05, ** p<0.01, *** p<0.001. Mann
Whitney Test.
Figure 7: The concentration of metabolites in the hippocampal
samples was correlated with seizure duration of patients with MTLE-HS.(a) Scatterplot shows inverse correlation (sample size 40; Spearman’s
non-parametric correlation, r= -0.4712, p=0.0021; line of best fit Y=
-0.001385*X+0.06080, slope= -0.001385 ± 0.0004206,
R2=0.2220, F=10.84, p=0.0021) between concentration of
KYNA in the hippocampal samples obtained from patients with MTLE-HS and
seizure duration (years). (b) Scatterplot shows inverse correlation
(sample size 26; Spearman’s non-parametric correlation, r= -0.6173,
p=0.0008; line of best fit Y= -0.009452*X+0.2413, slope= -0.009452 ±
0.003495, R2=0.2336, F=7.313, p=0.0124) betweende novo synthesis of KYNA in the hippocampal samples obtained
from patients with MTLE-HS and seizure duration (years). (c) Scatterplot
shows correlation (sample size 14; Spearman’s non-parametric
correlation, r= 0.5423, p=0.0476; line of best fit Y= 3.683*X-10.84,
slope= 3.683 ± 1.135, R2=0.4675, F=10.53, p=0.0070)
between concentration of QUIN in the hippocampal samples obtained from
patients with MTLE-HS and seizure duration (years). (d) Scatterplot
shows no significant correlation (sample size 12; Spearman’s
non-parametric correlation, r= 0.5750, p=0.0542) between de novosynthesis of QUIN in the hippocampal samples obtained from patients with
MTLE-HS and seizure duration (years). (e) Scatterplot shows inverse
correlation (sample size 29; Spearman’s non-parametric correlation, r=
-0.4647, p=0.0111; line of best fit Y= -0.04474*X+1.880, slope= -0.04474
± 0.01921, R2=0.1673, F=5.425, p=0.0276) between
concentration of PLP in the hippocampal samples obtained from patients
with MTLE-HS and seizure duration (years).