Protein Expressions by Immunohistochemistry method:
- GFAP, Astroglial Marker protein: In this protocol, the
injured brain sections were incubated with anti-GFAP Antibody (1:100).
The injured brain significantly expressed up-regulated GFAP in the
injured hippocampus and cortex. It has been observed that the
treatment therapy applied i.e. FH, FFA, VPA, and VFA significantly
reduced the GFAP protein expression in astroglial cells in the
Cerebral Cortex and hippocampus. The standard drug treatment along
with the combination of a standard with FFA was more effective in the
hippocampus when compared to FH and FFA. Moreover, all the treatment
effect was noticeable in reducing the expression of GFAP in cortex
region (Figure 8).
- Iba-1, Microglial Marker protein: The injured brain sections
were incubated with anti-Iba-1 Antibody (1:100). Iba-1 is
predominantly expressed in the injured brain and is significantly
found to be up-regulated in brain-injured tissues especially the
hippocampus and cortex. It has been observed that the treatment
therapy applied i.e. FH, FFA, VPA, and VFA significantly reduced the
Iba-1 protein expression in microglial cells of the Cerebral Cortex
and hippocampus. VFA combination was found less effective when
compared to FH, FFA, and VPA in both the regions likewise FH in the
cortex (Figure 8).
- ROCK2, Axonal Marker protein: In this IHC procedure, the
injured brain sections were incubated with anti-ROCK2 Antibody
(1:200). ROCK2 is predominantly expressed in the injured brain i.e.
hippocampus and cortex and this also was found to be significantly
up-regulated. It has been observed that the treatment therapy applied
i.e. FH, FFA, VPA, and VFA significantly reduced the ROCK2 protein
expression in axon cells of the hippocampus and cortex. FH, FFA, and
VFA combinations were found less effective when compared to VPA in
Cerebral Cortex likewise FH and FFA combinations in the cortex (Figure
8).
- TRPM2, Neuronal Receptor protein: The s ections were
incubated with anti-TRPM2 Antibody (1:100). TRPM2 was predominantly
expressed in the injured brain and was found to be significantly
up-regulated in brain-injured tissues. It has been observed that the
treatment therapy applied i.e. FH, FFA, VPA, and VFA significantly
reduced the TRPM2 protein expression in the neurons of the hippocampus
and cortex. FH, FFA, and VFA combinations were found less effective
when compared to VPA in Cerebral Cortex likewise FH combination in the
cortex (Figure-8).
DISCUSSION AND CONCLUSION
Progression periods of head injury
studies showed a difference in brain physiology and a decrease in body
weight(Aadal et al., 2015). The neurological abnormalities confirmed the
disturbed brain pathophysiology in injury controls, as reported in
previous studies(Umschwief et al., 2010; Villasana et al., 2014). This
study also confirmed the weight loss, abnormal grip strength, poor motor
coordination, disturbed beam-walk, and abnormal routine functions in
injury groups due to abnormal brain conditions. PTZ challenge confirms
the progression of epileptogenesis and animals were reported for
behavioral arrests, complex partial dileptic, and tonic-clonic seizures.
Brain edema induces ICP due to excessive accumulation of fluid in
intra/extracellular spaces i.e. due to ruptured blood vessels and
damaged neurons(Shiozaki et al., 2005). In this study, the treatments
applied significantly restored the body weight, neurological
abnormalities, and no seizures were confirmed after 2 weeks for PTZ
challenge in treatment groups. FH and FFA significantly reduced the
brain edema volume but VPA and VFA combination were more effective to
restore the neurophysiological and behavioral abnormalities.
Post-injury BBB disruption abruptly increases Evan’s blue penetration to
parenchyma(Rákos et al., 2007) and VPA is reported to restore BBB in
transient focal brain ischemia rat model(Xuan et al., 2012). We detected
a very less amount of dye in the brain homogenates after treatment.
Especially in VPA and VFA has
significantly more impact to restore the BBB. TTC staining confirms
tissue viability i.e. less stained area means, more number of infarct
cells(Bederson et al., 1986). Infarction was significantly visible with
a high number of infarct cells in the striatum hippocampus and cerebral
cortex area in injury controls. But treatments reduced the apoptotic
neuronal count i.e. less hypoperfusion and a rich red stain. Secondary
insult of CNS exacerbates the free radical’s formation i.e. ROS/RNS in
the brain, resulting in oxidative stress(Lipton, 1999). These activities
alter neurotransmitters and enzyme levels in the brain. AChE activity
was reported acutely elevated in the brain of ischemic/blast injury but
lowered AChE activity was seen in the neo-cortex of TBI patients with
chronic cognitive symptoms(Östberg et al., 2011). There was a
significant decrease in the levels of AChE after a head injury but the
treatments significantly elevated the AChE levels in the hippocampus and
cerebral cortex. Increased catalase activity has also been restored with
treatment drugs. The injured brain degrades LPO into aldehydic
malondialdehyde(Ostergard et al., 2016). But the increased level
significantly came to normal with our treatments. When SOD enzyme
concentration was calculated, significant elevation due to injury was
estimated which has been reduced by the treatments applied. Antioxidants
such as GSH which acts as an intracellular buffer for ROS and get
increased in injury conditions were also significantly restored after
treatments.
CNS inflammation due to dopamine metabolism was altered at 28-Days
post-injury along with microglial activation(Van Bregt et al., 2012).
But altered levels of dopamine were significantly recorded stable after
the treatment applied confirmed lesser loss of neurons in the injured
brains. Mitochondrial dysfunction enhances ROS production, brain
apoptosis, and elevated levels of Mitochondrial complex-I in
injury(Sullivan et al., 2005; Y. Xiong et al., 1997). The present study
has found that the treatment groups significantly decrease the levels of
Mitochondrial complex-I when compared to injury controls. NSE plays a
crucial role in erythrocytes, neuronal cell glycolysis(Chabok et al.,
2012). We have found the increased NSE levels have been significantly
restored by treatment to maintain the normal functional amount of enzyme
in the blood and brain. It has been reported, hypoxia and brain insult
induce local up-regulation of NGF-β(Kossmann et al., 1996).
But a significant decrease in the
levels of NGF-β in the brain was recorded in this study. Previous
literature showed robust and significant elevation of UCHL-1 in the
acute phase which is the main marker for Parkinson’s Disease and over
the Day7 of the study period when compared the serum and CSF levels in
TBI patients(Mondello et al., 2012). But a significant decrease in the
levels of UCHL-1 in the brain and serum was assessed after 2 weeks in
injury controls which significantly elevated after the treatment. This
treatment may help in PD pathophysiology. Our findings have found the
overlapping insults with brain damage by pro-/inflammatory cytokines
upregulation. CSF and serum concentrations showed IL-10 elevation in
severe trauma patients up to Day22 to 6 months(Csuka et al., 1999). The
present study confirms blood serum levels for IL-10 significantly
increased after 2 weeks in injury controls which got a significant
decrease after treatment applied. TNF-α was reported elevated in
Marmarou’s model of hypoxic injury(Yan et al., 2011). But this study
significantly confirmed elevated TNF-α levels in injury controls and was
significant reduced after treatment. The functional deficits result in
disturbed COX-2 levels following diffuse TBI(Cernak et al., 2002). HO-2
has been found elevated in the adult rodent brain(Ewing and Maines,
1997). Nrf2 was found able to regulate HO-1 via the phosphorylated
PI3K/Akt/GSK3β pathway(Singh et al., 2017). But the overall fold change
in HO-2 gene expression was significantly less in injury groups which
get increased in treatment groups. But COX-2 and Nrf-2 expression was
found to be significantly high in injury groups and treatment restored
it to normal. NF-kβ activation may potentially involve long-term
inflammation following TBI(Nonaka et al., 1999). Increased IL-1β
expression after the primary insult exacerbates epileptogenicity(Semple
et al., 2017). The significantly elevated overall fold change in NF-ƙβ
and IL-1β expression in injury groups were decreased after treatment.
IL-6 was also reported elevated in mTBI(Goodman et al., 2011) and our
study and it was significantly decreased by the treatment applied.
Initial brain insult increased IL-10 overproduction by resident
microglia(D’Mello et al., 2009). But the overall fold change in IL-10
expression was found significantly low in injury controls which got
significantly elevated in disease treatment groups. TNF-α was found
elevated in our experiment and also reported elevated in the FPI injury
model(Knoblach et al., 1999) but the overall fold change in TNF-α
expression was found to be significantly decreased in treatment groups.
Glial cells and hippocampal neurons also involved in post-traumatic
dementia and neuroinflammation by releasing TNF-α and IL-1β via
PI3K/AKT/NF-κβ signaling pathway(Zhao et al., 2014). The phosphorylation
of Protein Kinase-B was found to increase in rat hippocampus at Day1
after the initial blast and last for at least 6 weeks(Wang et al.,
2017). Phosphatase and tensin homolog expression was found upregulated
after TBI(Ding et al., 2013). In the present study, we have found the
overall fold change in PK-B-Akt/ PTEN and PI-3k expression was found to
be significantly high in TBI and EPLT groups which were recorded
significantly decreasing in treatment groups. It confirmed the
neuroprotection abilities of these drugs in dementia comorbidities also.
H&E is the further insight into necrosis and apoptosis and our study
confirms the morphology of neurons in normal controls was normal size
and intact shape with prominent nucleus like the old literature
reported(Isaksson et al., 2001). But in the case of injury controls, DAI
was seen with neutrophil rarefaction, eosinophilic cytoplasm, shrunken
and pyknotic nuclei in the hippocampus and cerebral cortex. Our
treatment altered the apoptotic scoring by reducing the nuclear
pyknosis, karyolysis, and nuclear lacking cellular structures. We
haven’t found proper distinguished apoptotic neurons in the VPA group.
The present study investigated the widespread heterogeneous distribution
of different neuronal proteins. GFAP was found upregulated when CNS
insult was followed by reactive gliosis(Schiff et al., 2012). Iba-1
confirmed the activated microglial expression after TBI up to 2-3
weeks(Neri et al., 2018). The ROCK2 is an axonal protein expressed in
axonal retraction balls and intermittent swellings from 6hours to 1-week
post-injury(Zhang et al., 2016). Many studies reported elevated TRPM2
expression following experimental trauma in rats at Day3-5 post-trauma,
especially in DG neurons(Cook et al., 2010). In our injury controls,
GFAP and Iba-1 predominately over-expressed and significantly
up-regulated in the hippocampus and cortex region. ROCK2 and TRPM2 are
also predominantly over-expressed and significantly up-regulated. But
the treatment therapy significantly reduced the protein over-expression
in astroglial, microglial, and axon cells of the hippocampus and
cerebral cortex. VPA and VFA were found much effective treatments to
minimize the apoptotic neurons.
We also checked the toxicity profile in vital organs of all animals and
observed the location, cell size and boundary area of organ tissues were
intact and at the exact position in all the groups i.e. no major
significant changes were observed.
The present study demonstrated that the fasudil hydrochloride(10mg/kg,
i.p.) acts as a potent Rho-kinases inhibitor that stopped the
post-injury neuronal apoptosis and flufenamic acid(20mg/kg, oral) with
anti-inflammatory properties can modulate the inflammatory environment
of post-traumatic epileptogenesis. We confirmed the neuroprotective
nature of these drugs for holding and minimizing the epileptogenesis
progression (Figure-9). VPA alone was observed much efficient compared
to a combination of FFA in molecular and histopathological findings and
the FFA was observed potentially more neuroprotective compared to FH.
Our study was limited to cover few pathways of epileptogenesis but the
complexity of this condition needs more studies on regulatory mechanisms
of intracellular signaling molecules during epileptogenesis progression
for PTE.