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.