Effects of NaHS and hydroxylamine on the expressions of brain-derived neurotrophic factor and its receptors in rats after cardiac arrest and cardiopulmonary resuscitation

All animal experiments were performed according to the American experimental animal use guidelines (NIH Publications No. 80–23) and approved by the animal ethics committee of Xiamen University. Healthy male Sprague-Dawley rats (n = 240; age of 6–12 months; weight of 300–500 g) were provided by the experimental animal center of Xiamen University. The rats were divided into three groups: Intervention (n = 80), Inhibition (n = 80) and Control (n = 80). Twelve CA/CPR rats with successful ROSC were randomly and double-blindly selected from the surviving rats of these three groups on days 0, 1, 3, and 7, and served as subgroups for the result analysis. If there were more than 12 rats on the 7th day, only 12 rats were included in the 7-day subgroups. The general physiological characteristics of rats are shown in Table 1 and the rats’ survivals data are shown in Fig. 1.

CA rat model was established based on our previously reported method [18]. The ROSC rats received intervention immediately after ROSC as described in Section 2.3. ECG and hemodynamic monitoring were performed for 4 h. During the 4 h monitoring period, the rats with weak spontaneous breathing (PaCO2 < 60 mm mmHg) were continuously mechanically ventilated without any treatment. The breathing pattern was assessed every 15 min to decide whether to continue the mechanical ventilation [18]. If the rats were awake during the procedure, we intraperitoneally injected 3% sodium pentobarbital (30 mg/kg). After 4 h, mechanical ventilation was stopped, all tubes were removed and wounds were sutured. The rats were observed for 7 d. All the selected rats were intraperitoneally injected with 3% sodium pentobarbital (30 mg/kg). Cardiac puncture method was used to obtain 2–5 mL blood. All of the rats were killed by decapitation, and 3 mm brain tissues including cortex, hippocampus, and cerebellum were harvested, fixed in formaldehyde, and embedded in Paraffin.

The rats in Intervention group were intravenously injected with 1 mL sterile NaHS (a donor for H2S, CAS: 140650–84-6, Sigma, USA) at a dose of 14 μmoL/kg·d at 1 h after ROSC. The rats in Inhibition group were intravenously injected with 1 mL sterile diluted hydroxylamine (an inhibitor for H2S, CAS 7803-49-8, Sigma) at a dose of 40 μmoL/kg·d at 1 h after ROSC. The doses of NaHS and hydroxylamine were chosen based on our previous study and other references [19] [20] [21]. The rats in Control group received 1 mL sterile saline at 1 h after ROSC.

Before all the rats were euthanized, 1 mL of femoral vein blood sample was collected and added into 1 mL Eppendorf (EP) tubes with a coagulant agent. The blood samples were centrifuged at 4000 r/min for 15 min at 4 °C. The supernatant was collected, coded, and stored at − 80 °C for serum H₂S determination.

For each subgroup, six rats were perfused with saline through the heart, followed by 4% paraformaldehyde for 30 min. The brains were removed, fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned for immunohistochemistry assay. For the other six rats, hippocampus tissues were harvested, snap-freezing in liquid nitrogen, and stored at − 80 °C for quantitative real-time PCR and Western blot analyses.

A H₂S kit (BC2055, Solarbio, Beijing China) was used to detect the H₂S level. The following reagents were added in a 5 mL glass tube: 0.5 mL of 1% (W/W) zinc acetate, 2.5 mL of distilled water, 0.1 mL of serum sample or NaHS·H₂O (CAS: 140650–84-6, Sigma), 0.5 mL of 20 mM (7.2 M HCl) dimethyl-p-phenylenediamine sulfate, and 0.4 mL of 30 mM (1.2 M HCl) ferric chloride. The reagents were mixed and transferred to an incubator at 37 °C for 20 min to allow a complete color change. The mixtures were added with 1 mL 10% trichloroacetic acid, and then diluted with distilled water for a final volume of 5 mL. The mixtures (5 mL) were centrifuged at 12,000 rpm for 10 min. The supernatants were collected and measured at an absorbance of 620 nm. Linear regression equation was used to calculate the serum H₂S concentration. Each serum sample was measured 3 times, and the concentration of each sample was the mean of the three measurements. The final concentration (μmol/L) of H₂S in each group was averaged and presented as mean ± standard deviation.

The mRNA expressions of BDNF, TrkB, and p75NTR were measured by quantitative real-time PCR. Total RNA was isolated from the brain tissues using an RNeasy kit with Trizol (Invitrogen, USA), and reverse-transcribed into cDNA using a reverse transcription kit with M-MLV polymerase (Promega, USA). The sequence-specific primers used were as follows: BDNF-F, 5′-CGATTAGGTGGCTTCATAGGAG-3′ and BDNF-R, 5′-ACGAACAGAAACAGAGGAGAGATT-3′; TrkB-F, 5′-CAAGTTGGCGAGACATTCCA-3′ and TrkB-R, 5′-AGTCATCGTCGTTGCTGATGAC-3′; p75NTR-F, 5′-TTCCTTAGCCCCTCCCTTCT-3′ and p75NTR-R, 5′-CCTGCCTTTCTCTGGGTTTTAC-3′; ACTIN-F, 5′-GCTATGTTGCCCTAGACTTCGA-3′ and ACTIN-R, 5′-GATGCCACAGGATTCCATACC-3′. PCR was performed using an IQ5 PCR System (Bio-Rad, USA) with an initial denaturing step at 95 °C for 15 s, 45 cycles of denaturing at 95 °C for 5 s, and annealing at 60 °C for 30 s. The relative expressions of genes were determined using the ΔΔCt method [22] to normalize the target gene expression to that of the housekeeping gene (ACTIN).

The protein expressions of BDNF, TrkB, and p75NTR were measured by Western blot. Lysis buffer [400 μL; 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% nonidet P-40, 0.5% deoxycholic acid, 0.1% sodium dodecyl sulfate (SDS), 5 mM ethylenediaminetetraacetic acid, 2 mM phenylmethylsulfonyl fluoride, 20 μg/mL of aprotinin, 20 μg/mL of leupeptin, 10 μg/mL of pepstanin A, and 150 mM benzamidine] was added into 100 mg of brain tissues, and homogenized for 30 min on ice. The homogenate was transferred into 1.5 mL EP tubes and centrifuged at 12,000 rpm for 5 min at 4 °C. The supernatant was collected. Protein quantification was performed using the bicinchoninic acid method, and SDS-polyacrylamide gel electrophoresis was performed. The proteins were transferred onto polyvinylidene fluoride membranes, probed with appropriate primary and secondary antibodies, and then detected using a Pierce Fast Western Blot Kit ECL Substrate (Cat No. 35055, Thermo, USA). The primary antibodies (1:1000 dilution) used were as follows: rabbit anti-rat BDNF (H-117) (sc-20,981, Santa Cruz Biotechnologies, USA), rabbit anti-rat TrkB (H-181) (sc-8316, Santa Cruz Biotechnologies), rabbit anti-rat p75NTR (H-92) (sc-5634, Santa Cruz Biotechnologies), and rabbit anti-rat α-tubulin (sc-5546, Santa Cruz Biotechnologies). The secondary antibody (1:5000) used was goat anti-rabbit IgG (sc-2004; Santa Cruz Biotechnologies). Immunoreactivity was imaged using Perfection 3490 photo gel imaging systems (Epson, Japan) and analyzed using Image Pro PLUS (Media Cybernetics, USA). Protein expression was normalized to tubulin.

The protein expressions of BDNF, TrkB and p75NTR in the cortex, hippocampus, and cerebellum were detected by immunohistochemistry. Paraffin-embedded tissues were sectioned at a thickness of 10 μm and mounted onto a 1.35-μm thin polyethylene film (PALM GmbH; Wolfratshausen, Germany) overlaid on a glass slide. Sections were incubated with primary antibodies overnight at 4 °C. After wash, the sections were incubated with biotinylated goat anti-rabbit IgG (1:5000, sc-2004; Santa Cruz Biotechnologies) for 30 min. Positive staining was revealed using diaminobenzidine [SABC immunohistochemical staining kit (Cat. No. SA1025), Wuhan Boster Biological Engineering Co., Ltd., China)] according to the manufacturer’s instructions. The primary antibodies (1:1000 dilution) used were as follows: rabbit anti-rat BDNF (H-117) (sc-20,981, Santa Cruz Biotechnologies), rabbit anti-rat TrkB (H-181) (sc-8316, Santa Cruz Biotechnologies), and rabbit anti-rat p75NTR (H-92) (sc-5634, Santa Cruz Biotechnologies). The immunostained slides were imaged using Imaging-Pro Plus 6.0 [23]. Five fields were randomly selected in each section for integrated optical density (IOD) calculations. The IOD of each slice was the mean IOD of the five fields.

A total of 240 rats were used for the CA/CPR model. However, some rats died during ROSC or within the 7-d observation period. Thus, only 12 rats were selected on days 0, 1, 3, and 7 for the final analysis. The general physiological characteristics of the rats are shown in Table 1. After ROSC, no significant difference was found among the baseline characteristics of the rats in all the subgroups (p > 0.05).

The average serum H₂S concentrations of all the subgroups were calculated (Fig. 1). There was no significant difference in the average H₂S concentrations on day 0 (p > 0.05). After NaHS·H₂O stimulation, the serum H₂S concentrations in the Intervention group gradually increased over time (Fig. 1) and were significantly higher than the Control group (p < 0.05). Because the H₂S formation in the Inhibition group was inhibited by hydroxylamine (Fig. 1), the serum H₂S concentrations in Inhibition group was significantly lower than the Control group (p < 0.05), and were maintained at a low level during the 7-d observation period.

Survival rate was 82.5% (66/80) in Intervention group, 62.5% (50/80) in Inhibition group, and 71.3% (57/80) in Control group. The 95% CI of survival time was 6.015~6.753 days in Intervention group, 5.429~6.281 days in Inhibition group, and 5.728~6.520 days in Control group, which had a significant difference (χ² = 8.683, P = 0.013) (Fig. 2).

The relative mRNA expression levels of BDNF, TrkB and p75NTR in the brain tissues of CA/CPR rats after ROSC decreased over time during the 7-d observation period (Fig. 3). There was no significant difference in mRNA expression of BDNF, TrkB, and p75NTR among the three groups at 0-d after CPR (Fig. 3a-c). The mRNA levels of BDNF and TrkB were similar on days 0, 1, 3, and 7 in the Intervention group (Fig. 3a, b) (p > 0.05). However, the mRNA levels of BDNF and TrkB on days 1, 3, and 7 in the Intervention group were significantly higher than the Control group (p < 0.05). In contrast, the mRNA levels of BDNF and TrkB decreased over time in the Inhibition group and were significantly lower than the Control group (p < 0.05). The mRNA levels of p75NTR in the Inhibition group were significantly higher on days 1 and 3 than day 0, and also significantly higher than the Control group on days 1 and 3. In contrast, the mRNA level of p75NTR in the Intervention group decreased daily, and significantly decreased on days 1 and 3 compared with the Control group (p < 0.05).

There was no significant difference in the relative protein expressions of BDNF, TrkB, and p75NTR among the three groups on day 0 (Fig. 4). The expressions of BDNF and TrkB in the Intervention group decreased over time (Fig. 4a and b), but were significantly higher than the Control group on days 1, 3, and 7 (p < 0.05). The relative protein expressions of BDNF and TrkB in the Inhibition group decreased significantly than the Control group (p < 0.05) over time. In addition, the relative protein expression of p75NTR in the Intervention group significantly increased on days 1, 3, and 7, compared with the Control group (P < 0.05). In contrast, the relative protein expression of p75NTR in the Inhibition group reduced significantly compared with the Control group (p < 0.05).

The expressions of BDNF, TrkB and p75NTR in the cerebral cortex, hippocampal tissue, and cerebellum of CA/CPR rats after ROSC decreased over time during the 7-d observation period (Figs. 5 and 6). There was no significant difference in the IODs of BDNF, TrkB, and p75NTR in the cerebral cortex (Fig. 5A1-A3 and Fig. 6 A1-A3), hippocampal tissue (Fig. 5B1-B3 and Fig. 6B1-B3), and cerebellum (Fig. 5C1-C3 and Fig. 6C1-C3) among the three groups at 0-d after CPR. These findings are consistent with the imaging results (Fig. 6). On days 1, 3, and 7, the IODs of BDNF and TrkB significantly increased in the Intervention group (p < 0.05), but significantly decreased in the Inhibition group (p < 0.05), compared with the Control group. In contrast, the IOD of p75NTR significantly decreased in the Intervention group (p < 0.05), but significantly increased in the Inhibition group (p < 0.05) on days 1, 3, and 7.

There are some limitations in this study. Firstly, the age of the rats was variable (6–12 months). However, we did not find an association between the age and the survival time of the CA model. Secondly, we only demonstrate that H₂S can modulate the levels of BDNF and its receptors in ROSC rats, leading to an increased survival time; however, the underlying mechanisms need to be investigated in our future studies.