Protein Expressions by Immunohistochemistry method:
  1. 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).
  2. 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).
  3. 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).
  4. 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.