Inflammatory Markers Estimation:
- Dopamine: Brain injury significantly increased the Dopamine
levels in brain tissue of disease control [i.e. TBI (25.91 ± 1.08),
EPLT (26.46 ± 0.87) groups as compared to normal controls [i.e.
Control (19.95 ± 1.99), Sham (20.29 ± 1.45). The treatment groups
showed a significant decrease in the levels of NSE when compared to
disease controls. The decrease in the concentration of NSE treatment
groups was calculated as [i.e. FH (20.95 ± 1.01), FFA (20.4 ± 0.93),
VPA (20.29 ± 1), VFA (20.2 ± 1.3)]. The levels of Dopamine were
significantly restored by the treatments applied.
- Mitochondrial complex-I: Brain injury damaged the ETC
machinery of mitochondria which results in apoptosis of the neurons.
Brain levels for Mitochondrial complex-I were markedly found to be
significantly increased in injury treated [i.e. TBI (6.77 ± 0.47),
EPLT (6.73 ± 0.45) groups as compared to normal controls [i.e.
Control (4.33 ± 0.69), Sham (4.46 ± 0.73). The decrease in the
concentration of treatment groups was calculated as [i.e. FH (5.40 ±
0.55), FFA (4.99 ± 0.72), VPA (4.78 ± 0.49), VFA (4.8 ± 0.48)] which
significantly restored the levels compared to injury groups.
- Neuron-Specific Enolase: Brain and serum levels for NSE were
markedly found to be significantly increased in injury treated [i.e.
brain tissue: TBI (22.33 ± 2.64), EPLT (22.44 ± 3.2) and blood serum:
TBI (10.4 ± 0.8) EPLT (10.8 ± 1.2)] groups as compared to normal
controls [i.e. brain tissue: Control (14.58 ± 1.04), Sham (15.25 ±
1.37) and blood serum: Control (7.5 ± 1.2), sham (7.7 ± 0.6)]. But
the treatment groups were shown a significant decrease in the levels
of NSE [i.e. brain tissue: FH (16.8 ± 2.3), FFA (16 ± 2.04), VPA
(15.8 ± 2.3), VFA (16.13 ± 1.98), and blood serum: FH (8.7 ± 1.01),
FFA (8.3 ± 1.01), VPA (7.8 ± 0.9), VFA (8.2± 1.1)] when compared to
disease controls.
- Nerve Growth Factor-Beta: Brain levels for Nerve Growth
Factor-Beta (NGF-b) were markedly found to be significantly increased
in injury treated [i.e. TBI (94.9 ± 6.1), EPLT (96.9 ± 6.8) groups
as compared to normal controls [i.e. Control (52.1 ± 5.1), Sham
(53.47 ± 6.2). But the decrease in the NGF-b concentration in
treatment groups was calculated as significant [i.e. FH (77.58 ±
8.2), FFA (68.56 ± 7.7), VPA (66.1 ± 7.6), VFA (60.43 ± 8.6)] when
compared to disease controls.
- Ubiquitin Carboxyl-terminal Hydrolase Isozyme L1: Brain and
serum levels for UCHL-1 were markedly found to be significantly
decreased in injury treated [i.e. brain tissue: TBI (7.58 ± 0.92),
EPLT (7.28 ± 1.13) and blood serum: TBI (4 ± 0.78) EPLT (3.96 ±
0.46)] groups as compared to normal controls [i.e. brain tissue:
Control (11.92 ± 1.29), Sham (12.18 ± 1.4) and blood serum: Control
(6.9 ± 0.71), sham (6.45 ± 0.7)]. The increase in the concentration
of UCHL-1 has been found significant in treatment groups [i.e. brain
tissue: FH (9.11 ± 0.75), FFA (9.46 ± 1.10) and blood serum: FH (4.69
± 0.36), FFA (5.19 ± 0.47)]. The concentration values to standard
drug therapy VPA, and its combination with FFA [i.e. brain tissue
VPA (10.91 ± 0.52), VFA (11.31 ± 1.18) and blood serum VPA (5.61 ±
0.5), VFA (5.79 ± 0.6)] was found more significantly increased when
compared to disease controls.
- Interleukin-1beta: Brain and serum levels for
IL-1β were markedly found to be
significantly increased in injury treated [i.e. brain tissue: TBI
(29.117 ± 2.34), EPLT (29.517 ± 3.69) and blood serum: TBI (11.23 ±
1.4) EPLT (11.83 ± 1.5)] groups as compared to normal controls
[i.e. brain tissue: Control (9.97 ± 1.9), Sham (10.47 ± 1.46) and
blood serum: Control (5.72 ± 1.11), sham (5.87 ± 1.36)]. The
decrease in the concentration of IL-1β treatment groups [i.e. brain
tissue: FH (14.95 ± 3.13), FFA (12.122 ± 3.08) and blood serum: FH
(7.4 ± 1.11), FFA (6.6 ± 0.97)] was found less when compared the
concentration values to standard drug therapy VPA, and its combination
with FFA [i.e. brain tissue VPA (12.017 ± 1.8), VFA (11.27± 1.61)
and blood serum VPA (6.33 ± 0.9), VFA (5.98 ± 0.68)]. The overall
treatment groups were shown a significant decrease in the levels of
IL-1β when compared to disease controls.
- Interleukin-10: Blood serum levels for IL-10 were markedly
found to be significantly increased in injury treated [i.e. TBI
(279.1 ± 4.3), EPLT (289.5 ± 9.6) groups as compared to normal
controls [i.e. Control (200.41 ± 9.1), Sham (210.83 ± 11.2)]. The
treatment groups were shown a significant decrease in the levels of
IL-10 [i.e. FH (232.08 ± 20.4), FFA (226.25 ± 16.1), VPA (221.6 ±
21.7), VFA (221.43 ± 15.9)] when compared to disease controls.
- Tumor necrosis factor-alpha: Brain and serum levels for TNF-α
were markedly found to be significantly increased in injury treated
groups [i.e. brain tissue: TBI (63.6 ± 5.8), EPLT (63.9 ± 4.8) and
blood serum: TBI (17.8 ± 1.87) EPLT (18.13 ± 2.6)] as compared to
normal controls [i.e. brain tissue: Control (26.2 ± 6), Sham (35.2 ±
6) and blood serum: Control (13.5 ± 3.71), sham (14 ± 3.22)]. The
decrease in the concentration of TNF-α treatment groups [i.e. brain
tissue: FH (38.13 ± 3.12), FFA (34.91 ± 6) and blood serum: FH (15.81
± 3.45), FFA (14.94 ± 1.6)] was found less when compared the
concentration values to standard drug therapy VPA, and its combination
with FFA [i.e. brain tissue VPA (36.22 ± 3.8), VFA (36.1± 4.4) and
blood serum VPA (15.43 ± 2.87), VFA (14.66 ± 2.6)]. But the overall
treatment groups were shown a significant decrease in the levels of
TNF-α when compared to disease controls.
Relative mRNA Expression: The relative mRNA expression of
pro-inflammatory/inflammatory genes has been assessed for overall change
(Figure-6).
- Cyclooxygenase-2: The
overall fold change in COX-2 expression was found to be significantly
high in TBI and EPLT groups when compared to Control and Sham. A
significant decrease in the expression of the COX-2 gene has been
found in injury treatment groups i.e. FH, FFA, VPA, and VFA. The
overall change in expression was similar in all treatment groups.
- Heme Oxygenase-2: The overall fold change in HO-2 expression
was found to be significantly less in TBI and EPLT groups when
compared to Control and Sham. But a significant increase in the
expression of the HO-2 gene has been found in disease treatment groups
i.e. FH, FFA, VPA, and VFA.
- Nuclear factor (erythroid-derived 2)-like 2: The overall fold
change in Nrf-2 expression was found to be significantly high in TBI
and EPLT groups when compared to Control and Sham groups. A
significant decrease in the expression of the Nrf-2 gene has been
found in disease treatment groups i.e. FH, FFA, VPA, and
VFA.
- Nuclear Factor-ƙβ: The overall fold change in NF-ƙβ
expression was found to be significantly high in TBI and EPLT groups
when compared to Control and Sham groups. And a significant decrease
in the expression of the NF-ƙβ gene has been found in disease
treatment groups i.e. FH, FFA, VPA, and VFA.
- Interleukin-1β: The overall fold change in IL-1β expression
was found to be significantly high in TBI and EPLT groups when
compared to Control and Sham. A significant decrease in the expression
of the IL-1β gene has been found in disease treatment groups i.e. FH,
FFA, VPA, and VFA. FH drug was found less effective than FFA, VPA, and
VFA in treatment groups while compared to disease controls.
- Interleukin-6: The overall fold change in IL-6 expression was
found to be significantly high in TBI and EPLT groups when compared to
Control and Sham groups. A significant decrease in the expression of
the IL-6 gene has been found in disease treatment groups i.e. FH, FFA,
VPA, and VFA. The overall change in expression was similar in all
treatment groups but FFA efficacy was found less compared to FH, VPA,
and VFA.
- Interleukin-10: The overall fold change in IL-10 expression
was found to be significantly increased in TBI and EPLT groups when
compared to Control and Sham groups. A significant decrease in the
expression of the IL-10 gene has been found in disease treatment
groups i.e. FH, FFA, VPA, and VFA. The VPA was found more effective to
reduce the change in expression in all treatment groups.
- Tumor necrosis factor-alpha: The overall fold change in TNF-α
expression was found to be significantly high in TBI and EPLT groups
when compared to Control and Sham groups. A significant decrease in
the expression of the TNF-α gene has been found in disease treatment
groups i.e. FH, FFA, VPA, and VFA. The overall change in expression
was similar in all treatment groups.
- Protein Kinase-B: The overall fold change in Akt expression
was found to be significantly high in TBI and EPLT groups when
compared to Control and Sham groups. A significant decrease in the
expression of the Akt gene has been found in disease treatment groups
i.e. FH, FFA, VPA, and VFA. The overall change in expression was
similar in all treatment groups but combination therapy VFA was less
effective than other treatments.
- Phosphatase and tensin homolog: The overall fold change in
PTEN expression was found to be significantly high in TBI and EPLT
groups when compared to Control and Sham groups. A significant
decrease in the expression of the PTEN gene has been found in disease
treatment groups i.e. FH, FFA, VPA, and VFA. The change in expression
was high in the combination VFA group in all treatment groups.
- Phosphatidylinositol 3-kinase: The overall fold change in
PI-3k expression was found to be significantly high in TBI and EPLT
groups when compared to Control and Sham groups. A significant
decrease in the expression of the PI-3k gene has been found in disease
treatment groups i.e. FH, FFA, VPA, and VFA. The overall change in
expression was similar in all treatment groups FH was found less
effective.
Hematoxylin-Eosin Staining for Neuronal Injury Scoring: It has
been found that the morphology of neurons was intact and good in the
case of the Control and Sham groups. They exhibited normal size, shape
and were arranged compactly with a prominent nucleus i.e. no apoptosis
was observed. The disease groups i.e. TBI and EPLT showed diffuse
neuronal injury with features such as rarefaction of neuropil,
eosinophilic cytoplasm, shrunken and pyknotic nuclei in hippocampus and
cortex region i.e. moderate and severe apoptosis was observed. In the
case of treatment groups, we have seen fewer apoptotic neurons as
compared to disease controls. The scoring system revealed mild apoptosis
in the case of FH, FFA, and VFA but no proper distinguished apoptotic
neurons were found in the VPA group (Figure-7).
Along with it, no significant histopathological changes have been found
in vital organs i.e. liver and kidneys of injury controls when compared
to treatment groups.