Animal ethics statement. The protocols of all animal experiments were in accordance with the local and international guidelines on the ethical use of laboratory animals. All procedures adopted in this study were approved by the Henan Polytechnic University Animal Care and Use Committee (Approved number: HPU2021-006) and conducted under the control of the national guidelines in China in order to minimize the number and suffering of the laboratory animals used. This study is reported in accordance with ARRIVE guidelines where applicable 25.
Animal and TBI model. This research employed Sprague-Dawley rats (male) weighing between 250–300 g. The animal subjects were kept in a controlled environment with an alternating 12-hour light/12-hour dark cycle and unrestricted food and water supply.
To induce TBI for experimental purposes, Feeney’s weight-drop model was modified according to a method provided in previous research 5. Anesthesia was induced in the subjects with phenobarbital sodium (50 mg/kg) intraperitoneally, while ensuring spontaneous breathing. A midline incision of 20 mm was made on the skull. The skin and fascia were then retracted, and a bone window with a 5-mm diameter was meticulously created on the right parietal region by employing a dental drill. The center of the bone window was accurately set 1.5 mm behind and 2.5 mm laterally to the bregma. Throughout the process, the dura mater was kept intact. A steel weight of 40g, with a flat end, was released from a 25 cm height along a stainless-steel rod, which hit a small pillar (4 mm wide, 5 mm long) located on the dura mater. Consequently, the cortex was compressed to a depth of 5 mm by the pillar.
Sham-operated animals underwent the same anesthesia and surgical procedure but the contusion of the cortex. Core and cerebral temperatures were maintained between 36.8–37.2 ℃ with the aid of feedback warming lamps. The temperature was continuously monitored and tracked with rectal and temporalis muscle thermistors. Periodic testing of arterial blood samples was conducted to ensure all parameters stayed within normal physiological ranges. To keep their temperature around 37.0 ℃, the rats were placed on a warming pad until they regained mobility. Post-consciousness, the rats were returned to their cages with an unrestricted food and water supply.
Experimental groups and takinib treatment. Two experiments were performed. In the first, takinib purchased from Sigma (St. Louis, MO, USA) was dissolved in dimethylsulfoxide (DMSO) right before using it. Takinib solution (10 µg/rat and 20 µg/rat) or DMSO (5 µL) was intracerebroventricularly (ICV) administered 30 minutes after injury by employing a 1 µL Hamilton micro syringe. Two concentrations of takinib were used to evaluate its neuroprotective effects after TBI. Under the effects of phenobarbital sodium anesthesia, each subject was securely placed in a stereotactic frame in preparation for ICV injections.
The injection was administered at a position 1.5 mm left, 1 mm below, and 4.4 mm deep with respect to the bregma 26, with an injection duration of 10 minutes. Takinib or vehicle (DMSO) (n = 12 rats/group) was administered by an ICV injection 30 minutes after TBI. Neurological deficits, along with beam walking scores, were assessed. One day after TBI, the rats were deeply anesthetized, cerebral water levels were determined (n = 6), and brain tissue was collected (n = 6) to detect the levels of expression of p-TAK1 and TAK1 associated with different concentrations of takinib.
In the second stage of the experiment, rats were administered either takinib (10 µg/rat) or a vehicle half an hour post-TBI. After 24 hours, rats were euthanized, and brain tissue samples were collected. These samples were subjected to Western blot analysis, enzyme-linked immunosorbent assay (ELISA) (n = 8 rats/group), and TUNEL and Nissl staining (n = 6 rats/group).
Assessment of Neurological Behavior. The neurological condition of the mice was assessed at 24 hours post-TBI with the aid of the neurological severity score (NSS). This scoring system evaluates the ability of the mice to accomplish ten separate tasks related to motor skills, alertness, and balance. The score ranges from 0 (minimum deficit) to 10 (maximum deficit), where each task not completed earns one point (Table 1) 27. All these tests were performed by two researchers who were uninformed about the experimental groups.
Table 1
Neurological severity scoring.
Items | Description | Points |
Success/failure |
Exit circle | Ability and initiative to exit a circle of 30 cm diameter (time limit: 3 min) | 0 | 1 |
Mono-/hemiparesis | Paresis of upper and/or lower limb of contralateral side | 0 | 1 |
Straight walk | Alertness, initiative, and motor ability to walk straight, when placed on the floor | 0 | 1 |
Startle reflex | Innate reflex (flinching in response to a loud hand clap) | 0 | 1 |
Seeking behavior | Physiological behavior as a sign of “interest” in the environment | 0 | 1 |
Beam balancing | Ability to balance on a beam 7 mm in width for at least 10 s | 0 | 1 |
Round stick balancing | Ability to balance on a round stick 5 mm in diameter for at least 10 s | 0 | 1 |
Beam walk: 3 cm | Ability to cross a beam (length × width, 30 × 3 cm) | 0 | 1 |
Beam walk: 2 cm | Same task but with increased difficulty (beam width = 2 cm) | 0 | 1 |
Beam walk: 1 cm | Same task but with increased difficulty (beam width = 1 cm) | 0 | 1 |
Maximum score | | | 10 |
Beam walking score. The takinib-treated rats and those receiving a vehicle were subjected to behavioral and functional tests 24 hours post-injury. These tests were implemented by an observer unaware of the conditions of the experiment. Each rat was trained to walk the beam one day before the injury and once again right before anesthesia on the day of the injury.
The motor skills and coordination of animals are estimated by this score. The test was performed as previously described 28. The beam walking potential was graded from 1–7 as mentioned below:
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The rat couldn't place the damaged hind limb on the horizontal beam surface.
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The rat placed the damaged limb on the beam and remained in balance but failed to walk across.
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The rat dragged the damaged hind limb while walking the beam.
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The rat walked the beam, placing the injured limb on the beam surface once.
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The rat walked the beam, using the affected limb for support through less than half the steps.
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The injured limb was used by the rat for more than half the steps.
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A maximum of two-foot slips occurred while the rat walked across the beam. Seven was the highest possible score for non-operated rats.
Cerebral water content. As per a technique described in previous research, cerebral edema was assessed by employing the wet-dry method 6. In brief, cerebral tissue around the contusion cortex was quickly collected and wet weight measurement was carried out. These samples were then oven-dried at 110°C for 24 hours. and dry weight measurement was done. The cerebral water content was derived by the formula: percentage (%) of brain water = [(wet weight-dry weight)/wet weight] × 100.
Total/nuclear/cytosolic protein extraction.To obtain total protein from the cortex, tissues of appropriate size were mechanically lysed in a solution comprising 1 mM phenylmethylsulphonyl fluoride (PMSF), 20 mM Tris (pH 7.6), 0.2% sodium dodecyl sulfate, 1% Triton X-100, 0.11 IU/ml aprotinin, and 1% deoxycholate (all sourced from Sigma, China). Following centrifugation at 14,000 rpm and 4°C for 15 minutes, the resulting supernatant was gathered and preserved at -80°C for further analytical procedures.
For the extraction of cortex nuclear proteins, a previously established protocol was utilized 6. Homogenization of 50 mg of the fresh cortex was carried out in 0.4 mL ice-cold buffer A comprising 2 mM MgCl2, 0.1 mM ethylenediaminetetraacetic acid (EDTA), 10 mM KCl, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, pH 7.9), 1 mM dithiothreitol (DTT), and 0.5 mM PMSF (all supplied by Sigma, China). Fifteen microliters of Nonidet P-40 were introduced into this mixture.
Following centrifugation, the supernatant (cytoplasmic fraction) was collected, and the nuclear pellet was resuspended in 100 µL buffer B, comprising 1 mM DTT, 20 mM HEPES (pH 7.9), 420 mM NaCl, 1.5 mM MgCl2, 0.1 mM EDTA, 0.5 mM PMSF, and 25% (v/v) glycerol. After centrifugation at 14,000 g and 4°C for 15 minutes, the supernatant, comprising the nuclear proteins, was gathered and preserved at -80°C for further analytical processes.
Western blot analysis. To conduct this analysis, every lane of the sodium dodecyl-sulfate polyacrylamide gel electrophoresis was loaded with 40 µg of total or nuclear protein. The aforementioned proteins were subjected to an electrophoresis process and then transferred to a nitrocellulose membrane. The transfer protein-containing blot was blocked by a blocking buffer (1×Tris-buffered saline comprising Tween 20 with 5% w/v nonfat dry milk with no antibody) for an hour at 25 ℃. The blot was then subjected to overnight incubation at 4°C with primary TAK1 and p-TAK1 antibodies (1:200; Santa Cruz Biotech., CA), zonula occludens (ZO)-1, an inhibitor of NF-κB (IκB-α), cleaved caspase-3 (1:500; Cell Signaling Technology, MA), claudin-5, p65 (1:1,000, Abcam), ionized calcium-binding adaptor molecule 1 (Iba-1), β-actin and histone 3 (1:1,000; Proteintech, USA) and Bcl-2 and Bax (1:500; Santa Cruz Biotech., CA). Following incubation with the secondary horseradish peroxidase-conjugated immunoglobulin G (IgG), the generated signal was detected with the help of enhanced chemiluminescence detection reagents (Millipore Corporation, MA). Visualization of the blotted protein bands was achieved using the enhanced chemiluminescence detection reagents (Thermo Fisher Scientific). Employing ImageJ software, relative alterations in protein levels were calculated via the mean pixel density and normalized to histone 3 or β-actin.
ELISA. Quantification of IL-1β and TNF-α levels in the cerebral tissue was done with the aid of ELISA kits for rats as per the specific protocols (Beyotime Biotechnology, China). The amounts of inflammatory cytokines in the cerebral tissue were presented as pg/mg protein.
TUNEL staining. The rats were perfused sequentially in saline and 4% paraformaldehyde. The brains were extracted and kept in 4% paraformaldehyde for one night. To eliminate water, the brains were immersion in 15% sucrose for a day, then 30% sucrose for additional two days. Assessment of cerebral tissue apoptosis was done with the aid of the terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) assay (Roche, USA) as per the protocol. The cerebral sections (15 µm thick) were exposed to TUNEL reaction buffer (50 µL), followed by a one-hour incubation period at 37°C in a humid environment without light. This was followed by an overnight incubation period with an anti-NeuN antibody (1:200, Beyotime Biotechnology) at 4°C.
The sections were subjected to washing five times with phosphate-buffered saline (PBS) for 10 minutes each time and then subjected to incubation with secondary antibodies (AlexaFluor488 and AlexaFluor594, 1:200) for 2 hours at 25 ℃. After this, the PBS washing process was repeated, and the sections were stained for five minutes with the nuclear marker 4′,6-diamidino-2-phenylindole (DAPI, Beyotime Biotechnology). A ZEISS HB050 inverted microscope system (Zeiss, Germany) was used to image the basal temporal lobe using fluorescence microscopy after three further washes.
Nissl staining. The rats were put under anesthesia, and transcardial perfusion was carried out with saline and 4% paraformaldehyde. After extraction, the brains were post-fixed in paraformaldehyde for 24 hours. Following the fixation, the brains underwent dehydration and embedding in paraffin. The brain samples were then sectioned into pieces with a thickness of 4 µm. Nissl staining was done using Cresyl Violet, as per the previously mentioned methods 29,30. In the basal temporal lobe, the designated region for each subfield was marked, and cell counting was carried out for Cresyl Violet-stained neuronal cell bodies. Healthy neurons were observed to have large cell bodies, lightly stained cytoplasm, and spherical nuclei. Conversely, injured cells showed condensed nuclei, shrunken cell bodies, and dark-stained cytoplasm.
Statistical analysis. The obtained findings were presented as means ± standard deviation (SD). A comparative analysis of the means across various study groups (except the neurobehavioral scores) was done by means of a one-way analysis of variance (ANOVA) followed by Tukey’s test. Contrarily, the scores for the neural behavior of study subjects were assessed with the help of nonparametric tests (Kruskal–Wallis, and Dunn’s post-hoc test). The SPSS v 16.0 (SPSS, Inc., IL) was employed to conduct a statistical analysis of data. Variations in the data were deemed to be statistically significant at P < 0.05.