Connexin40 correlates with oxidative stress in brains of traumatic brain injury rats

2.1 Animals

Male Wistar rats (4 months) were provided by SLAC (Shanghai). Animals were housed under controlled conditions on a 12 h light/dark cycle at 20°C and 60–#x2013;70% humidity. The Institutional Animal Care and Use Committee of The People’s Hospital of Pu Dong New Area approved all experimental protocols and animal handling procedures. A total of 300 rats were used in this study. Ten rats were employed for each group at each time point.

2.2 Induction of TBI and NAC treatment

The controlled cortical impact (CCI) model uses an impact system to deliver physical impact to the exposed dura of an animal, providing an easy and accurate method of investigating the effects and potential treatments for TBI. CCI models produce brain injury by using a pneumatic impactor to impact exposed brain after the dura was opened (Dixon, Clifton, Lighthall, Yaghmai, & Hayes, 1991; Romine, Gao, & Chen, 2014). We used CCI instrument (Hatteras Instruments, Cary, NC, USA) to produce TBI. Briefly, rats were anesthetized with the mix of 80 mg/kg ketamine and 10 mg/kg xylazine by intraperitoneal injection and placed in the stereotaxic frame on a thermostatically-controlled heating pad to maintain body temperature. A portable drill was used to create a 4 mm diameter craniotomy over the right parietal cortex between Bregma and Lambda, 1 mm away from the midline. Injury was produced using a pneumatic piston with a rounded metal tip (2.5 mm diameter) that was adjusted at an angle parallel to the surface of impact site and at the center of the craniotomy. A velocity of 4 m/s and a deformation depth 2 mm below the dura were used. Sham-operated rats underwent identical surgical procedures, but did not receive a CCI. The bone flap was immediately replaced and sealed, and the scalp was sutured closed.

The antioxidant N-acetylcysteine (NAC, 100 mg/kg, Sigma, USA) was administered by intraperitoneal injection right after TBI surgery. Therapies were assigned in a randomized and blinded fashion.

2.3 Neurological functional evaluation

Neurological deficits were evaluated at 6, 12, 24, 28 and 72 hours after CCI by an observer who was blinded to experimental group, using the Neurological severity score (NSS) on a 10-point scale according to Chen’s method (Chen, Constantini, Trembovler, Weinstock, & Shohami, 1996). On this scale, one point is awarded for failure of each of 10 tasks, such that the maximum score of 10 points represents severe neurological dysfunction, whereas 0 points indicates normal function.

2.4 Brain infarct volume measurement

Each rat was sacrificed right after the behavioral tests were completed. The brains were removed for infarct volume measurement using the triphenyltetrazolium chloride (TTC) staining method (Hu, Zhou, Hu, & Zeng, 2005). Briefly, five thin sections were selected to prepare slices at 2 mm intervals (from the anterior 5 mm to the anterior 13 mm) to determine the infarct areas. The slices were stained in 2% TTC (Sigma, USA) at 37°C for 20 min, and then fixed with 10% formaldehyde neutral buffer solution (pH 7.4) for 24 h. At that time, the infarct tissue was unstained, whereas the normal part was stained red. Using a computerized image analysis system (NIH Image-Pro plus 6.0), the infarct areas on each slice were summed and multiplied by slice thickness to give the infarct volume. The data were expressed as the percentage of infarct area, calculated using the following formula: infarct area = [volume of left hemisphere – (volume of right hemisphere – measured infarct volume)]/volume of left hemisphere.

2.5 Brain edema measurement

According to Hatashita’s method (Hatashita, Hoff, & Salamat, 1988), rats were sacrificed by cervical dislocation, and their brains were immediately removed and weighed to determine wet weight. Then, brains were dried in an oven at 110°C for 24 h and weighed again to determine dry weight. Percentage of brain water content was calculated using the following formula: brain water content (%) = (wet weight–#x2013;dry weight)/wet weight×100%. Ten rats were analyzed for each group.

2.6 Brain oxidative stress evaluation

Rats were anesthetized with isoflurane before sacrificed for brain tissue collection. Ten rats were analyzed for each group. Brain cortex from injured area was taken and stored at – 80°C till use. For biochemical determination of GSH, lipid peroxidation product malondialdehyde (MDA), nitric oxide (NO), and Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity, the tissue sample homogenized in 10 volumes of ice cold PBS (pH 7) until a uniform suspension was obtained. The homogenate was centrifuged at 20,000×g for 10 min at 4°C. The supernatant was collected for analysis.

Indicators of oxidative stress, including reduced GSH (an important antioxidant preventing damage to cellular components caused by ROS) and increased MDA (an end product of lipid peroxidation), NO and NADPH oxidase (sources of ROS) activity, were measured in brain cortex homogenate by using the commercial kits. GSH assay kit was purchased from Cayman Chemical Company (Ann Arbor, MI, USA). MDA levels were evaluated by the method of thiobarbituric acid (TBA) using a TBARS Assay Kit purchased from Cayman Chemical Company. NO (total) detection kit was purchased from Enzo life science (Shanghai, China). NADPH oxidase activity was measured by NAD/NADPH assay kit from Abcam (Cambridge, MA, USA).

2.7 Western blot analysis

The brain cortex protein from injured area was extracted using RIPA lysis buffer (Santa Cruz, Dallas, TX, USA) containing 1% protease inhibitor cocktail and the protein concentrations were determined by BCA method. After electrophoresis on 12% SDS-PAGE gel and transferred onto nitrocellulose membrane (PVDF, BIO-RAD, Hercules, CA, USA), the membranes were blocked, and then incubated with the primary antibodies (anti-Cx40 antibody, 1 : 200; Santa Cruz, sc-20466) at 4°C overnight. At the end of incubation period, the membranes were rinsed and incubated with donkey anti goat IgG-HRP secondary antibodies (1 : 5000; Santa Cruz, sc2020) for 1 hr at room temperature. Subsequently, membranes were washed and target protein was visualized by using an Amersham ECL Advance Western Blotting Detection Kit (GE Healthcare, USA). Images were captured and the densitometry of the bands was analyzed using Image-prosoftware.

2.8 Statistical analysis

Statistical analysis Statistical analysis was carried out using Graph Pad Prism software (version 6). The data are presented as mean±standard deviation (S.D). The significances of differences among testing groups were analyzed using one-way or two-way ANOVA followed by Turkey’s post hoc test. Values of P < 0.05 were regarded as significant.

3.1 TBI induced neurological deficits

The brain injuries were gradually increased from 6 h to 24 h post CCI, and severe damage continued till 72 h. As show in Fig. 1a, neurological severity score was significantly increased to 5 ∼ 7 (all P < 0.001) from 6 h ∼ 72 h in post TBI rats comparing with sham rats. Similar results were found in brain infarction volume and brain water content (Fig. 1b, c), consistently indicating severe neurological deficits after TBI.

3.2 TBI-induced oxidative stress

To evaluate the oxidative stress after TBI, we measured the levels of GSH, MDA, NO and the activity of NADPH oxidase at 6, 12, 24 and 48 hours post CCI. As show in Fig. 2, the brain GSH was gradually depleted after surgery (Fig. 2a), whereas the levels of MDA (Fig. 2b) and NO (Fig. 2c), and the activity of NADPH (Fig. 2d) were all elevated in time-dependent manners. The oxidative stress reached the peak at 24 h post TBI surgery, and remained till 72 h. The above results indicated that TBI was able to induce oxidative stress.

3.3 Cx40 expression

Cx40 expressions were measured by Western blot, as shown in Fig. 3. Cx40 was observed to be upregulated as early as 6 h (P < 0.01 vs sham) after TBI surgery (Fig. 3a, b), which from 12 h to 72 h post TBI gradually increased almost by 100% compared with sham rats (all P < 0.001). These results suggested potential involvement of Cx40 in the TBI-induced brain injuries.

3.4 Correlation between Cx40 expression and oxidative stress

To investigate the possible correlation between Cx40 expression and brain oxidative stress, Cx40 expression after TBI was compared to brain levels of GSH, MDA, NO and NADPH oxidase activity by using Spearman’s correlation coefficient analysis. The correlation coefficient (R²) was 0.5213 for GSH (Fig. 4a, P < 0.01), 0.5488 for MDA (Fig. 4b, P < 0.01), 0.4941 for NO (Fig. 4c, P < 0.01) levels and 0.4761 for NADPH oxidase activity (Fig. 4d, P < 0.01), respectively. The results indicated that the increased expression of Cx40 in cortex was positively correlated with oxidative stress after TBI surgery.

3.5 Effects of NAC on oxidative stress, TBI severity and Cx40 expression

To further elucidate the relationship between the upregulated Cx40 and oxidative stress after brain injury, we administered NAC, a commonly used antioxidant, to the TBI rats and assessed the neurological severity, oxidative stress and Cx40 expression at 24 h post surgery. Comparing with untreated TBI rats, a single dose of 100 mg/kg NAC greatly attenuated the neurological damage, as evidence by significantly decreased neurological severity score (P < 0.01), brain infarction volume (P < 0.01) and brain edema (all P < 0.05), accompanied with decreased oxidative stress characterized as higher GSH (P < 0.05), lower MDA (P < 0.01), lower NO (P < 0.01) and lower NADPH oxidase activity (P < 0.01) (Fig. 5a-d). Simultaneously, NAC greatly prevented the elevated Cx40 expression after TBI (P < 0.001vs TBI) (Fig. 5e, f).