Animals and Experimental Design
All of the procedures involving the animals used in this study were carried out in accordance with the guidelines of the Institutional Animal Care and Use Committee of the Nanjing University of Chinese Medicine. The present study used male (SD) rats (320–350 g) purchased from the Zhao Yan (Suzhou) New Drug Research Center. A 12-hour light/dark cycle was employed in an environment with both a controlled temperature and a controlled humidity, and the rats had constant free access to both water and food.
In the first experiment, 42 out of 44 rats were divided randomly into seven groups, including a sham group as well as a total of six SBI subgroups (these groups were 6 h, 12 h, 24 h, 48 h, 72 h, and 7 d after SBI). At the specific time points specified above, the rats were euthanized and their brain tissues from around the injured area were collected. The expression and localization of TFAM were determined via WB and IF, respectively.
In the second experiment, 48 rats (out of 49 total rats) were randomly divided into four groups, including a sham group, an SBI group, an SBI+TMP (40 mg/kg), and an SBI + Vehicle group. There were twelve rats in each group. The rats were sacrificed after neurological scoring at 48 h post-SBI. Brain tissue surrounding the injury was collected, and the expression levels of TFAM, bcl-2, and caspase-3 in their brain tissue were detected using WB. FJC and TUNEL staining were used to evaluate both necrosis and neuronal apoptosis, and the wet-dry method was employed to detect any brain edema. ROS and ATP levels were analyzed to assess mitochondrial function.
Surgical Brain Injury Rat Model
According to previous reports, the SBI rat model was established through an excision of a part of the right frontal lobe[27]. The SD rats were anesthetized through the administration of an intraperitoneal injection containing 40 mL/kg sodium pentobarbital. After successful anesthesia, the rats were placed on a stereotaxic apparatus in the prone position (SA-100, Shanghai Yuyan Instruments, China). The skin on the top of the head was disinfected with Aner iodine. The skin was cut, and the periosteum at the mid-sagittal line at the top of the skull was detached. A bone window with a diameter of about 5 mm was created at 2 mm before the right bregma and 2 mm beside the midline. The frontal lobe of the exposed area on the right side was removed. Once the bleeding stopped, the operation field was rinsed repeatedly with sterile normal saline until the flushing liquid was clear. The wound was disinfected and sutured. After the operation, the rats were closely monitored until they woke up after anesthesia.
Drug Injection
TMP (40 mg/kg, MCE, USA) dissolved in 10% DMSO + 90% corn oil was injected intraperitoneally 1 h after SBI. The SBI+Vehicle group was given 10% DMSO at the same time point and dose.
Western Blot
According to a previously published method[28], the rats’ brain tissues were completely disintegrated with RIPA lysis buffer and protease inhibitor (Beyotime, China). Then, the buffer mixture was centrifuged and the supernatant collected. Using a PierceTM BCA Protein Assay Kit (Thermo Fisher, USA), the supernatant was aliquoted to determine the protein concentration, as well as the loading amount. The remaining supernatant was mixed with 5X loading buffer (Beyotime, China) and boiled. The protein was isolated on SDS-polyacrylamide gels (Beyotime, China), and after that it was then transferred to a PVDF membrane (Millipore, USA). This membrane was sealed at 37°C for 1 h using A QuickBlockTM Western (Beyotime, China) and incubated with primary antibody overnight at 4°C. The antibodies used included rabbit anti-mtTFA (1:1000, Abcam, UK), rabbit anti-Bcl-2(1:1000, Abcam, UK), rabbit anti-caspase-3(1:2000, Abcam, UK), and mouse anti-β-actin (1:1000, Sigma, USA) as a control group. Then, the primary antibody was recovered, and a sample was incubated with the corresponding secondary antibody, including goat anti-rabbit IgG-HRP (Invitrogen, USA) and goat anti-mouse IgG-HRP (Invitrogen USA). Then, western chemiluminesent HRP substrate (Millipore, USA) and an imaging system (GE Healthcare Bio-Sciences AB, Sweden) were used to visualize the proteins, and the resulting bands were quantified with ImageJ software.
Immunofluorescent staining
Immunofluorescent (IF) staining was performed according to the methods in a previously[29]. Paraffin sections were first deparaffinized and then repaired by sodium citrate. Immunostaining permeable solution (Beyotime, China) was employed to penetrate the membrane. Tissue sections were then washed three times in PBS before being blocked with immunostaining blocking buffer (Beyotime, China) for 1 h. After that, the tissues were incubated with both primary and secondary antibodies. Finally, DAPI anti-fluorescence quenching solution (YEASEN, China) was then applied, and the tissues were observed using a fluorescence microscope after mounting (OLYMPUS, Japan). The antibodies used in the experiment included: rabbit anti-TFAM (Abcam, 1:500, UK), mouse anti-GFAP (Invitrogen, 1:1000, USA), goat anti-Iba1 (Abcam, 1:800, UK), mouse anti-NeuN (Abcam, 1:1000, UK), donkey anti-rabbit IgG, Alexa Fluor 488 (Invitrogen, 1:800, USA), donkey anti-mouse IgG, Alexa Fluor 555 (Invitrogen, 1:800, USA), and donkey anti-goat IgG.
Brain Water Content
The wet–dry method was employed to assess brain edema[30]. The rats were first anesthetized and then they were decapitated. Their brains were then removed immediately and then divided into ipsilateral and contralateral sections. To determine the wet weight (WW), each section was weighed using an electronic analytical balance, and each was then subsequently oven-dried at 100°C for 24 h to determine the dry weight (DW). The brain water content was calculated as [(WW − DW)/WW] × 100%.
Neurological Score
A modified Garcia score was used to assess and evaluate the rats’ neurological function, according to the methods used in a previous report[30]. This modified score is composed of seven parameters: locomotion, body proprioception, tactile response, symmetry of limb movement, lateral rotation, forelimb walking, and climbing ability. Each parameter has a score of between 0 and 3, and the maximum score is thus 21. A high score is associated with a small degree of nerve damage.
TUNEL Staining
TUNEL staining was used for the detection of neuronal apoptosis according to a previous method[31]. The paraffin sections were deparaffinized and then incubated in proteinase K (37°C for 20 min) and TUNEL detection liquid (Beyotime, China) (37°C for 60 min). TUNEL-positive neurons were then detected with a U-RFL-T fluorescence microscope (OLYMPUS, Japan).
Fluoro-Jade C Staining
Neuronal necrosis was detected by FJC staining with a Biosensis kit (Biosensis, USA)[32]. Paraffin sections were first deparaffinized, and then they were incubated in a potassium permanganate (at a concentration of 1:9 in distilled water) and FJC working solution (at a concentration of 1:10 in distilled water). The tissues were then rinsed with distilled water and dried at 60°C for at least 5 min. The dried tissues were soaked in xylene for 1 min and fixed on a coverslip with stilbene plasticizer xylene (DPX). The FJC-positive cells were then imaged using a U-RFL-T fluorescence microscope (OLYMPUS, Japan).
Measurement of ROS Levels
An ROS test kit (JianCheng, China) was used to measure the levels of ROS in brain tissues according to the method in a previously published study[33]. Briefly, the samples were decomposed with 0.01 mol/lPBS and centrifuged at 4°C and 500 × g for 10 min. Then, the supernatant (190 µL) and DCFH-DA (10 µL, 1 mol/l) were combined in micropores at room temperature for a total of 30 min. The mixture was then measured by fluorometry. A detergent-compatible protein assay kit (Bio-Rad, Hercules, CA, USA) was used to measure the concentration of protein; the ROS levels are expressed as fluorescent/mg protein.
Measurement of ATP Levels
To quantify the ATP levels in the rats’ brain tissue, an ATP assay kit (Beyotime, Shanghai, China) was used. Then, 20 mg of brain tissue was combined with 200 µL of lysis solution and homogenized. The fully lysed tissues were centrifuged at 12000 g for 5 min at 4ºC. ATP working reagent was added to the supernatant, and the absorbance was measured on a spectrophotometer (Thermo Fisher Scientific, USA). the sample’s ATP concentration was calculated according to the standard curve. A PierceTM BCA Protein Assay Kit (Thermo Fisher, USA) was also used to detect the protein concentration in the supernatant, and the ATP concentration was expressed as nmol/mg protein.
Statistical Analysis
GraphPad Prism 8.0 software was used to perform statistical analyses, and all data are presented as the mean ± standard deviation. One-way analysis of variance (ANOVA) for multiple comparisons and Student-Newman-Keuls post hoc test were used to determine the significant differences between the groups. P < 0.05 was considered statistically significant.