Experimental Parameters Analysis:
Assessment of Animal’s Weight: Difference in weight was assessed for the abnormal metabolism after injury. And the comparison of the differences has been made to analyze the weight loss in injury controls and weight maintained in treatment groups.
Neurological Severity Scale Testing: A 28-point Neuroscore test was performed for each animal in this study, which ranging from simple observation to traversing a horizontal bar, involve in general condition, reflexes, behavior, motor tests, and grip strength [Stanford Behavioral and Functional Neuroscience Laboratory, Version 4.0]
Rotarod Test: Rotarod tests were employed for muscle coordination with a latency time of 120 sec.
Seizure confirmation of Epileptogenesis: Modified Racine scale was used for the assessment of neurobehavioral progression of seizures and monitored up to 2 hours following PTZ (35mg/kg). The maximum stage of seizure activity was analyzed in each 5-minute interval. Diazepam 4 mg/kg, i.p. was given an intraperitoneal route to terminate seizures if convulsions last for more than 2 hours.
2,3,5-Triphenyltetrazolium Chloride Staining: Whole-brain tissue viabilities and lesion size were estimated by 2,3,5- triphenyl tetrazolium chloride (TCC) staining. The excised brains were sliced into coronal sections of 2mm thickness and were incubated in a 2% TTC solution for 20-30min at 37°C.
Blood-Brain Barrier Permeability Estimation: A 2% (4 mL/kg i.p. of body weight) solution of Evans Blue in normal saline was injected and was measured by spectrophotometer (Shimadzu Japan) at 610nm. The brain tissue was quantified according to a standard curve and results were presented as ng/100mg(Manaenko et al., 2011).
Brain Edema Estimation: Whole-brain water content was measured with the wet-dry method for quantifying brain edema. The brains were weighed immediately after sacrifice the animals to yield wet weight. The tissues were placed for drying in a desiccating oven for 48 hours at 70oC and reweighed after 24 and 48 hours to calculate the weight loss. The %age of lost water content in the tissues was calculated by the average readings.
Biochemical Estimations : Biochemical imbalance alters the pathophysiology of TBI in the brain and such conditions open the doors for treatment check and development of antioxidant strategies to optimize brain insult i.e. tissue used was hippocampus and cortex.
Protein Assay: To estimate protein content in brain homogenates, the Biuret method was used in which bovine serum albumin was used as standard(GORNALL et al., 1949).
Acetylcholinesterase (AChE) activity: AChE activity has been determined by photometric method for cell suspensions, homogenates, and tissue extracts(Abass, 2014).
Catalase Activity: The activity of catalase was measured by the Luck, 1963 method.
Malondialdehyde (MDA): Thiobarbituric acid-reactive substances were measured to evaluated tissue LPO levels. MDA is a product of LPO that gives red light absorbance at 535nm after reacting with thiobarbituric acid.
Super Oxide Dismutase (SOD): The activity of SOD was estimated by the Kono et al. method which was developed in 1978.
Reduced Glutathione Assay (GSH): Jollow et al., in 1974 used DTNB as a substrate to developed yellow color and estimated reduced glutathione levels immediately at 412 nm as μmol GSH/g tissue.Enzyme-Linked Immunosorbent Assay Method ( ELISA) was proceeded in biological samples (i.e. brain tissue i.e. hippocampus, and cortex & blood serum) and analyzed over LISA Plus Microplate Reader in the form of concentration (pg/mL). The ELISA kits were procured from Diaclone and QAYEE-BIO.Gene Expression by Real-Time PCR: Brain tissues (especially hippocampus) were subjected to isolate total RNA from all experimental groups. The positive control of housekeeping gene β-actin was run. Different genes were standardized as per definite melting temperatures. The RT-PCR reaction conditions were set at 95°C for 15 min, 40 cycles of 95°C for 15 sec, and 61°C for 30sec. The termination step was set at 72°C for 30 sec. A sensitive and quantitative technique Real-time Polymerase chain reaction (i.e. TaqMan real-time QRT-PCR Applied Biosystem StepOnePlus, ThermoFisher) was used to simultaneously measure relative mRNA expression i.e. relative standard curve and comparative CT value of different biomarkers in the rat hippocampus.Histopathological Estimation: A 4% cold paraformaldehyde and PBS was perfused transcardially in experimental rats before their brains were removed and immersed in paraformaldehyde for 2–3 Days. Sagittal sections were selected after cut to study the hippocampus and cortex part in embedded paraffin blocks which were mounted and used for Hematoxylin and eosin staining H&E(10µm) and immunohistochemistry(5µm). Each group was observed under an optical microscope (OPUS) at magnifications of 20X & 40X and results have been shown @100µm scale. The scoring of apoptotic neurons in H&E staining was done based on Nuclear Pyknosis & Non-evident Nucleoli (NP&NN), Perineural Vacuolization (PV), Vascular Congestion (VC), and Brain Edema (BE)(Mena et al., 2004; Schmued et al., 1997; Schmued & Hopkins, 2000). All the primary antibodies were procured from Sigma-Merck and were diluted to the manufacturer’s protocol to optimized again for the perfect dilutions. The following primary Abs were used having sensitivity for Rat: a rabbit polyclonal anti-glial fibrillary acidic protein GFAP(Sigma-Aldrich Cat# G4546, RRID:AB_1840895) for astrocytes, a mouse monoclonal anti–ionized calcium-binding adaptor molecule-1, Iba1 (Sigma-Aldrich Cat# SAB2702364, RRID:AB_2820253) for microglia, a rabbit polyclonal anti-ROCK2(Sigma-Aldrich Cat# HPA007459, RRID:AB_1079828) for axonal marker protein, and a rabbit polyclonal anti-TRPM2(Sigma-Aldrich Cat# SAB2103193, RRID:AB_10665621) for neuronal receptor protein. For enzymatic detection (HRP or AP secondary conjugates) anti-goat anti-rat IgG H&L (Alexa Fluor® 488) was used. The scoring of neurons in IHC was done based on apoptotic and necrotic neurons. Examination Scale includes Negative (−), Weak (+), Moderate (++), and Strong (+++)(Moriyama et al., 1997; Seeger et al., 2003).
Statistical Analysis: Results of this study were expressed as mean ± SD. Intergroup difference was estimated by one-way analysis of variance, ANOVA test which was followed by Post hoc Bonferroni and Post hoc Dunnett test. The data for biochemical, histology and molecular studies were analyzed with appropriate or suitable parametric tests. The p<0.05 was considered significant. Graph-Pad Prism 9 software was used to analyze the records.