3-5 months old Spraque Dawley female rats, weighing 250-350 g, were procured from Recep Tayyip Erdogan University Animal Care and Research Unit. All animals received humane care according to the criteria outlined in the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences and published by the National Institutes of Health. The study was approved by Recep Tayyip Erdogan University Animal Ethical Committee (ID: 2017/1).
2.1 Study Design
Animals’ care was performed in Recep Tayyip Erdogan University Animal Care and Research Unit. All animals were maintained and fed in aseptic conditions. Humidity and room temperature were kept at 55-60% and 22±2 °C, respectively. A 12 h light,12 h dark cycle was provided. Rats were allowed access to commercially available standard rat chow (Bayramoglu Feed and Flour Industry Trading Corporation Erzurum, Turkey) and tap water ad libitum along with the experiment.
After sufficient time to adaptto the laboratory conditions, eighteen experimental animals were randomized into three groups. The trauma group (n=6) received head trauma following anesthesia. Trauma+Dex group (n=6) received anesthesia and head trauma the same day; and 100µg/kg intraperitoneal dexmedetomidine daily for three days, beginning in the next day. The dose was determined according to the literature (Garrity et al. 2015). Control group (n=6) received anesthesia only.
Anesthesia was provided with 50 mg/kg intraperitoneal ketamine hydrochloride (Ketalar®, Eczacibası Parke-Davis, Istanbul, Turkey) and 10 mg/kg intraperitoneal xylazine HCl (Alfazyne®, Alfasan International BVWoerden, Holland).
Head trauma was performed with the Mild Traumatic Brain Injury Apparatus developed by Richelle Mychasiuk et al., by dropping a 150 mg weight onto the skull of sedated rats through a 100 cm long tube in free-fall motion (Mychasiuk et al. 2014). Each rat was prepositioned on a 38x27x27 cm3 box-shaped platform which housed a 38x25x15 cm3 sponge. There was a 10 cm gap between the 15 cm tall sponge and the upper edge of the box. An aluminum foil was placed on the upper surface of the box where the rats were laid during trauma. The distal end of the tube and the head of the rat were positioned at a distance of 4 cm.
On the fourth day, all rats were sacrificed by decapitation; the brain was immediately removed and divided into two parts for biochemical and histopathological analysis (Dixon et al. 2016). Biochemical, histopathological, and immunohistochemical analysis procedures are detailed below.
2.2 Tissue homogenization
Homogenization solution was prepared by mixing 20 mM sodium phosphate and 140 mM potassium chloride; the pH was adjusted to 7.4 (Rojas et al. 2012). One mL of this solution was mixed with 100 mg tissue and was centrifuged at 800 G for 10 mins at 4°C. The supernatant was used to measure the concentration of malondialdehyde produced due to degradation of unstable lipid peroxides via Thiobarbituric Acid Reactive Substances (TBARS), and concentration of intracellular glutathione.
2.3 TBARS measurement
TBARS level was measured by the protocol of Ohkawa (Ohkawa et al. 1979). The mixture of 200 µL of tissue supernatant, 50 µL of 8.1% sodium dodecyl sulfate, 375 µL of 20% acetic acid at pH of 3.5, and 375 µL of 0.8% thiobarbituric acid was vortexed. After incubation in boiling water bath for 1 h, it was cooled in ice water for 5 min and centrifuged at 750 G for 10 min. The resulting pink color was read on a spectrophotometer at 532 nm. The results were calculated as nmol/mg prt.
2.4 Total Sulfhydryl Content measurement
The sulfhydryl groups were measured with Ellman's reagent. 1000 µL of 3M Na2HPO4 and 250 µL of DTNB (4 mg DTNB prepared in 10 mL of 1% sodium citrate solution) were added to 250 µL of supernatant and vortexed. The absorbance was measured at 412 nm. The results were determined by the standard curve of reduced glutathione (1000 µM-62.5 µM) and given as nmol/mg prt.
2.5 Na/K ATPase measurement
Na/K ATPase activity was measured by the method of Yoshimura (YOSHIMURA 1973). 100 mg of brain tissue was homogenized after adding 1 mL of 10mM Tris-HCl at a pH of 7.4. Homogenates were centrifuged at 4000 rpm for 10 minutes at 4°C. One tube was prepared by adding 250 µL of KCl (0.8M), 250 µL of NaCl (4M), 250 MgL of MgCl (0.4) and 200 µL of Tris-HCl (1M) to 50 µL of the supernatant and incubated for 10 minutes. Another tube was prepared by adding 250 µL of ouabain (40mM), 250 µL of NaCl (4M), 250 MgL of MgCl (0.4) and 200 µL of Tris-HCl (1M) to 50 µL of the supernatant and incubated at 37°C for 10 minutes. After 10 min, 60 µL of ATP (25mM) was added to each tube and left at 37°C for 1 h. The reaction was stopped by adding 500 TCL of 10% TCA to each tube. Both tubes were centrifuged at 3000 G for 10 min at 4°C. Inorganic phosphate was measured by the Fiske and Subbarow method and the results were given as nmol/Pi/min/mg prt (Fiske and Subbarow 1925). A commercially available bicinchoninic acid kit was used for protein measurement.
2.6 Histopathological analysis procedure
The brain tissue samples were fixed in 10% neutral formaldehyde. After the fixation, specimens were dehydrated in an ascending series of alcohol, cleared in xylene and embedded in paraffin by routine laboratory methods. Hematoxylin&Eosin staining and evaluation with a light microscope (Leica DM6200, Germany; with Olympus DP20 camera attached) was performed by two histologists blinded to the study groups.
2.7 Immunohistochemical analysis
The following steps were performed for cleaved caspase-3 staining: the sections were deparaffinized and treated with 20 μg/mL proteinase-K solution in Phosphate Buffered Saline (PBS), rinsed in distilled water, immersed in 3% hydrogen peroxide. After several washes with PBS (pH 6.0), the sections were immersed in an equilibration buffer. Sections were incubated with anti-Cleaved Caspase-3 (1:200, ab2302, Rabbit polyclonal to active Caspase-3, Abcam, UK). After several washes, all sections were incubated in anti-digoxigenin-peroxidase. Cleaved caspase activity was revealed with 0.06% 3,3-diaminobenzidine tetrahydrochloride in PBS, and the sections were counterstained with Harris hematoxylin.
2.8 Semi-Quantitative analysis
Brain tissue sections were stained with Hematoxylin&Eosin. For each animal, three slides were selected randomly from eight slides containing the brain tissues. On each slide, the presence of ischemic neurons, edema and vasoconstriction were evaluated. All sections (3-4µm thick) prepared from brain tissue were stained with Caspase-3 staining. The evaluation was performed with a light microscope (Leica DM6200, Germany, x40 magnification). For each animal, six slides were selected randomly from 8 slides random areas. On each slide, the presence of Cleaved caspase-3 positive cells was determined. Two histologists blinded to the study groups evaluated and graded the slides in four categories as neuronal ischemia, brain edema, vascular congestion and cleaved caspase-3 staining positivity. The findings were graded as none (<5%), mild (6-25%), moderate (26-50%) or severe (>50%).
2.9 Statistical analysis
Statistical analysis was performed with SPSS ver. 12 (SPSS Statistical Program, IBM Corporation, USA). The normality of data was tested with the Kolmogorov-Smirnov test and QQ-plots. TBARS and total sulfhydryl values were presented as mean±standard deviation. Groups were compared with a one-way analysis of variance, followed by the Tamhane test. Na/K-ATPase activity, histological injury scores and caspase-3 positivity scores were presented as median (25%-75% interquartile range). Groups were compared with Kruskal-Wallis Test. In case of significant difference, two-group comparisons were performed with one-way analysis of variance, followed by the Tamhane test., a p-value < 0.05 was considered as statistical significance.