Hydrogen Sulfide Decreases Blood-Brain Barrier Damage via Regulating Protein Kinase C and Tight Junction After Cardiac Arrest in Rats

Cardiac arrest (CA) is a leading cause of death in America [1]. Even with effective primary cardiopulmonary resuscitation measures, only about 30% CA patients survive while most of them suffer from severe post-CA neurological defects due to cerebral injury [2]. Blood-brain barrier (BBB) plays an important role in the process of cerebral ischemia-reperfusion (I/R) injury induced by CA and resuscitation [3]. Therefore, efficacious therapies that target in decreasing cerebral injury will rely on methods that can preserve or recover the integrity of BBB.

Protein kinase C (PKC) represents a family of serine/threonine protein kinases which is integral to the structure and function of BBB [4-8]. The PKC family consists of 12 isozymes [9], among which 10 are expressed in brain microvascular endothelial cells and participate in a multitude of physiological functions including gene expression, cell proliferation and differentiation, and intracellular signal transduction pathways [10]. Previously, a study determined that I/R activated PKCs by enabling their translocation from the cytosol to cytomembrane [11]. In turn, the activated and translocated PKCs can phosphorylate the serine/threonine sites of relevant proteins, including tight junction (TJ) proteins which maintain the integrity of BBB [4]. PKCs, especially PKC-α, PKC-β, and PKC-δ, directly or indirectly act on TJ proteins such as ZO-1, Claudin-5, and Occludin, thereafter attenuating their expression [5-7]. However, another study had shown that PKC-η plays a vital role in the assembly and maintenance of TJ proteins [8].

Hydrogen sulfide (H₂S) has been reported as another gas, besides carbon monoxide and nitric oxide, with signal transduction modulatory capacity [12]. Many studies have shown that H₂S has the potential effects on decreasing oxidative stress damage, inhibiting cell apoptosis, regulating NMDA receptors, attenuating neurologic deficits, and promoting nerve functional recovery [13, 14], which were consistent with the results obtained in our previous studies [15]. Beyond these findings, inhalation of H₂S attenuates BBB permeability and brain edema in CA and resuscitation rats [16]. In addition, H₂S may potentially regulate the activity of PKC isoforms, especially PKC-α and PKC-ε [17]. However, the effects of H₂S on PKC isozymes and TJ proteins after CA and resuscitation remain unknown.

In this study, we investigated the effects of exogenous H₂S on the integrity of BBB; and further, we investigated whether these effects were associated with PKC and TJ in rats undergoing CA and resuscitation.

All experimental procedures were approved by the Institutional Animal Care and Use Committee of Harbin Medical University (Heilongjiang, China). Male Sprague-Dawley rats weighing 280 - 300 g (Beijing Vital River Laboratory Animal Technology Co., Ltd., Beijing, China) were housed under 12 h light/12 h dark cycles with food and water available ad libitum. Animals were used and treated in a non-stressful environment according to the guidelines of the Laboratory Animal Center of Harbin Medical University in this study. All studies involving animals are reported in accordance with the ARRIVE guidelines for reporting experiments involving animals [18, 19].

All rats were fasted except for free access to water before the experiment. Rats were anesthetized with 3-5% isoflurane mixed with 30% oxygen and 70% nitrogen as described previously with some minor modifications [20]. Rats were mechanically ventilated (InspiraASV, Harvard Apparatus, USA) with a frequency of 50 – 60 breaths/min and a tidal volume of 10 ml/kg after endotracheal intubation and the parameters were adjusted accordingly to maintain the arterial blood gas at pH 7.35–7.45, PaCO₂ 35–45 mmHg and PaO₂ ≥90 mmHg. A standard lead II electrocardiography was recorded continuously by subdermal electrodes and rectal temperature was monitored using a thermometer probe and maintained at 37 ± 0.5°C with the MP150 biological signals and data acquisition system (BIOPAC Systems, Inc., Goleta, CA, USA). Two 18-gauge catheters were inserted into the left femoral vein and the caudal ventral artery for drug and fluid administration and continuous arterial blood pressure monitoring respectively.

Cardiac arrest was induced by ventricular fibrillation as previously described [20]. Ventricular fibrillation was induced using an esophageal electrode (5F, Bard Peripheral Vascular, Tempe, AZ, USA) with a 1-minute pulse of a 12 V, 50 Hz alternating current. In case of spontaneous defibrillation, additional 20 seconds impulses were delivered [20]. CA was confirmed by an abrupt decrease in mean arterial pressure to < 15mmHg.

After 6 minutes of CA, cardiopulmonary resuscitation (CPR) was performed with sternal compression at a rate of 200/minute and mechanical ventilation was resumed with 100% oxygen. A bolus of epinephrine (0.02 mg/kg) and sodium bicarbonate (1 mEq/ kg) was administered. After 2 minutes of CPR, a biphasic shock of 2 J (HeartStart XL M4735A defibrillator, Philips Medical Systems, Andover, MA, USA) was performed for external defibrillation. Then the cycle of chest compressions for 1 minute followed by defibrillation were repeatedly performed until the return of spontaneous circulation (ROSC). Achievement of ROSC was defined by spontaneous cardiac rhythm in combination with a mean arterial pressure of 50 mmHg or greater. If CPR exceeded 10 minutes without ROSC, the CPR was stopped.

After that, all the resuscitated rats were randomly divided into three groups: CA / CPR group (CA group), 40 p.p.m. H₂S group (40H₂S group) and 80 p.p.m. H₂S group (80H₂S group). Rats in CA group inhaled 30% oxygen for 1 h, while rats in 40H₂S or 80H₂S group inhaled 30% oxygen with 40 p.p.m. or 80 p.p.m. H₂S, respectively, for 1 h. Blood gas analyses were performed and appropriate ventilator adjustments were made. Vascular catheters were removed, surgical wounds were sutured and infiltrated with 0.25% bupivacaine after 1 h of ROSC. Rats were then weaned from the ventilator, extubated, and placed in a chamber enriched in 50% oxygen with or without different concentrations of H₂S for another 1 h. Rats in Sham group were anesthetized and operated surgically as aforementioned without ventricular fibrillation.

The whole experiment consisted of two parts. In Part I, we examined brain water content, Evans blue leakage, and performed western blot analysis and immunofluorescence after 24 h of ROSC. In Part II, we recorded neuronal deficit score was detected at 24 h and 14 days after ROSC; we also evaluated the survival rate during the 14 days after ROSC. For experimental details, see Fig. 1.

Brain tissue was immediately dissected, weighed, and designated as the wet weight. The tissues were then placed in an oven (105°C) and dried for 72 h to obtain the dry weight. The brain water content was calculated by the formula: ([wet weight – dry weight]/wet weight) × 100% [21].

Two percent Evans blue (EB; Sigma, MO, USA) dissolved in saline was administered intravenously and circulated for 30 minutes. Then, rats were transcardially perfused with saline to remove residual EB from circulation. Brain tissues were rinsed with PBS followed by sectioning two 4 mm-wide slices from 1.8 mm anterior to the bregma to 6.2 mm posterior to the bregma. The cerebral cortex above the rhinal fissure was dissected from the two slices, respectively. Thereafter, cortex was homogenized in 50% trichloroacetic acid, and the samples were centrifuged at 21, 000 g for 30 min. The concentration of EB extracted in the supernatant was analyzed spectrophotometrically at 620 nm against a standard curve. The standard curve was constructed using dilution of Evans blue dye in a range from 3 to 400 ng/ml [15].

The brain microvessels were isolated as previously described with minor adjustments [22]. In brief, cerebral tissue of prefrontal cortex was homogenized in microvessel isolation buffer (pH = 7.4) containing 103 mM NaCl, 2.5 mM CaCl₂, 4.7 mM KCl, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄, 2.5 mM NaHCO₃, 15 mM HEPES, 10 mM glucose, 1 mM sodium pyruvate, and 10 g/L dextran (MW 64, 000) with protease inhibitor (PMSF, Beyotime Institute of Biotechnology, Jiangsu, China). Ice-cold dextran was added to the homogenate, oscillated, and centrifuged at 5, 600 g for 10 min at 4°C. The pellets were resuspended in microvessel isolation buffer, and passed through a 70 µm filter (Becton Dickinson, Franklin Lakes, NJ). The filtrate was centrifuged at 3, 000 g in isolation buffer at 4°C and the pellet consisting of microvessels was collected.

RIPA lysis buffer was added into the precipitate containing microvessels. After incubation on ice, the mixture was centrifuged at 16, 000 g for 20 min at 4°C and the supernatant was collected for detection of TJ proteins. In order to measure the translocation of PKC isozymes, proteins of cytoplasm and membrane were extracted respectively as previously described [20]. Another precipitate containing microvessels was resuspended in ice-cold lysis buffer containing 125 mM NaCl, 5 mM EDTA, 1% Nonidet P-40, 25 mM Tris (pH 7.5) and protease inhibitors and shaken on ice for 1 h. The lysate was centrifuged at 500 g for 10 min at 4°C to discard the pellet rich of nuclei. After that, supernatant was centrifuged again at 20, 000 g at 4°C for 20 min. The supernatant was collected as cytosolic fraction. The pellet was resuspended in lysis buffer containing 1% Triton X-100 and incubated on ice for another 1 h. Then, the hybrid was centrifuged at 20, 000 g at 4°C for 20 min and the supernatant was collected as membrane fraction.

Equal amounts of the protein samples were separated by 10% sodium dodecyl sulphate-polyacrylamide gel on Mini-PROTEAN Tetra Cell System (Bio-Rad, Hercules, CA) and then transferred onto polyvinylidene difluoride (PVDF) membranes. Then, membranes were blocked in 5% skim milk (Difco, Solarbio, Beijing, China) and then incubated with various primary antibodies against ZO-1 (1: 250; Invitrogen, Thermo Fisher Scientific, MA, USA), Occludin (1: 1000; Abcam, Cambridge, UK), PKC-β I (1: 1000; Abcam), PKC-β II (1: 1000; Abcam), Claudin-5 (1: 250; Bioss Biosynthesis Biotechnology, Beijing, China), PKC-α (1: 1250; Santa Cruz Biotechnology, CA, USA), PKC-δ (1: 500; SCBT) and PKC-ε (1: 500; SCBT) at 4°C overnight. The membranes were incubated with horseradish peroxidase-linked anti-rabbit IgG (1: 5000; ZSGB-BIO, Beijing, China) or anti-mouse IgG (1: 5000; ZSGB-BIO) at 25°C for 1 hour after three washes in TBST buffer and then again washed three times in TBST buffer. The ECL® kit (GE healthcare, UK) was used for image development and the density of the bands was quantified by ImageJ software (NIH).

The general neurological performance was assessed by one investigator who was blinded to the groups in all animals using the neurological deficit scale (NDS), which was established in rats undergoing CA previously [23] at 24 h after ROSC in each group. The score ranges from 80 points indicating a functionally normal to 0 representing brain or cardiac death. Survival rate was evaluated during the 14 days after ROSC.

The neuronal survival analysis in hippocampal CA1 region was investigated by an observer who was blinded to the experimental protocol after 14 days of ROSC. Rats were deeply anesthetized with isoflurane, perfused through the heart with 200 mL 0.1 mol/L phosphate-buffered saline and 4% paraformaldehyde. Hippocampi were removed quickly and fixed in 4% paraformaldehyde before paraffin embedding. Hematoxylin and eosin–stained sections of the hippocampus (corresponding to bregma -3.3 cm according to the atlas of Paxinos and Watson [24]) were examined. Viable neurons were defined as cells showing a distinct nucleus and nucleolus, while ischemic neurons were recognized by the pyknotic or karyorrhectic nuclei lacking a clear nucleolus. One observer, who was blinded to the experimental protocol, counted the numbers of normal-appearing pyramidal neurons per high-power field (×400). The number of neurons in the CA1 region of hippocampus was quantitated as the mean number from five sections per rat. In each section, the number of neurons was averaged from three random different vision fields in the CA1 region under microscope (×400) [25].

Data from the neurological deficit score was presented as the median, 25th, and 75th percentiles and statistical significance was assessed with the Kruskal–Wallis test, followed by post-hoc Mann–Whitney U tests, when the overall Kruskal-Wallis result was significant. Other data were presented as the mean ± SD and analyzed using ANOVA, followed by Bonferroni correction for the comparison between multiple groups. Kaplan–Meier survival curves were compared using log rank test. A P value less than 0.05 was considered statistically significant. Statistical analysis was performed using SPSS® software (SPSS Inc., Chicago, IL).

As shown in Fig. 2, the brain water content was 83.7 ± 1.0% at 24 h after ROSC in CA group, which was a significant increase compared with Sham group (78.1 ± 0.7%, P < 0.05). However, there was no significant difference in 40H₂S group and 80H₂S group when compared with the Sham group. In addition, The CA-mediated increase of brain water content was significantly inhibited in 40H₂S group (79.2 ± 0.8%, P < 0.05) and 80H₂S group (78.6 ± 0.7%, P < 0.05) indicating that inhalation of 40 p.p.m. H₂S or 80 p.p.m. H₂S decreased the brain water content after CA and resuscitation.

At 24 h after ROSC, a significant increase of EB leakage was observed in the whole brain tissue in CA group (1536 ± 171 ng/g) compared with Sham group (794 ± 98 ng/g, P < 0.05). Compared with the CA group, EB extravasation was significantly lower in 40H₂S group (1042 ± 121 ng/g) and in 80H₂S group (996 ± 149 ng/g) respectively (P < 0.05, Fig. 3). There was no significant difference between the 40H₂S group and the 80H₂S group.

As shown in Fig. 4, at 24 h after ROSC, an increased level of membrane-to-cytosol expression in PKC-α (Fig. 4A), β I (Fig. 4B), β II (Fig. 4C) and δ (Fig. 4D) was observed in CA group, but not PKC-ε (Fig. 4E). The membrane-to-cytosol ratios of the PKC-α (1.52 ± 0.09-fold of Sham group), β I (1.76 ± 0.25-fold of Sham group), β II (1.60 ± 0.31-fold of Sham group) and δ (2.04 ± 0.23-fold of Sham group) increased significantly, while decreased in PKC-ε (0.63 ± 0.11-fold of Sham group), in CA group compared with Sham group (P < 0.05). This data indicated that CA and resuscitation caused an overt activation of PKC-α, β I, β II and δ, and suppressed the activation of PKC-ε. Compared with CA group, the membrane-to-cytosol ratios of the PKC-α (1.13 ± 0.06-fold of Sham group in 40H₂S group, 1.08 ± 0.12-fold of Sham group in 80H₂S group), β I (1.22 ± 0.22-fold of Sham group in 40H₂S group, 1.15 ± 0.11-fold of Sham group in 80H₂S group), β II (0.95 ± 0.08-fold of Sham group in 40H₂S group, 1.01 ± 0.11-fold of Sham group in 80H₂S group) and δ (1.14 ± 0.09-fold of Sham group in 40H₂S group, 1.02 ± 0.10-fold of Sham group in 80H₂S group) decreased significantly, while increased in PKC-ε (0.91 ± 0.09-fold of Sham group in 40H₂S group, 0.96 ± 0.16-fold of Sham group in 80H₂S group) both in 40H₂S group and 80H₂S group (P < 0.05). These data indicated that inhalation of 40 p.p.m. or 80 p.p.m. H₂S inhibited the activation of PKC-α, β I, β II and δ, and promoted the activation of PKC-ε. However, no statistical differences were observed between the 40H₂S group and 80H₂S group.

The expressions of TJ proteins including Claudin-5, Occludin, and ZO-1 were also measured at 24 h after ROSC using western blot assays. As shown in Fig. 5A-C, a significant decrease in Claudin-5 (0.64 ± 0.17-fold of Sham group, P < 0.05), Occludin (0.66 ± 0.07-fold of Sham group, P < 0.05) or ZO-1 (0.58 ± 0.20-fold of Sham group, P < 0.05) was observed in CA group when compared with Sham group. However, H₂S treatment reverted the expression of these TJ proteins. Compared with the CA group, the levels of Claudin-5 (0.83 ± 0.18-fold of Sham group in 40H₂S group, 0.92 ± 0.13-fold of Sham group in 80H₂S group, respectively, P < 0.05), Occludin (0.85 ± 0.17-fold of Sham group in 40H₂S group, 0.97 ± 0.18-fold of Sham group in 80H₂S group, respectively, P < 0.05) and ZO-1 (0.89 ± 0.12-fold of Sham group in 40H₂S group, 0.88 ± 0.22-fold of Sham group in 80H₂S group, respectively, P < 0.05) were significantly increased at 24 h after ROSC. There was no significant difference between the 40H₂S group and the 80H₂S group.

As shown in Fig. 6A, at 24 h after ROSC, neurological deficit scale in CA group [47 (44, 51)], 40H₂S group [58 (50, 63)] and 80H₂S group [60 (53, 63)] were significantly lower than in the Sham group [79 (78, 80)] (all P < 0.05). Compared with CA group, the NDS in 40H₂S group and 80H₂S group were significantly increased (both P < 0.05). However, there was no significant difference between 40 H₂S group and 80H₂S group. Furthermore, at 14 d after ROSC (Fig. 6B), neurological deficit scale in CA group [50 (45, 54)], 40H₂S group [65 (58, 68)] and 80H₂S group [67 (64, 73)] were significantly lower than in the Sham group [80 (79, 80)] (all P < 0.05). Compared with CA group, the NDS in 40H₂S group and 80H₂S group were significantly increased (both P < 0.05).

At the end of the experiment (14 d after ROSC), all rats in Sham group had survived (100%, 5 of 5; Fig. 6C); while in the CA group, only 9 survived (45%, 9 of 20). The survival rate in 40H₂S groups and 80H₂S group were 75% (15 of 20) or 80% (16 of 20) respectively, both significantly higher than the CA group (P < 0.05).

Fourteen days following ROSC, the numbers of viable neurons in the CA1 region in the CA group (20 ± 6), 40H₂S group (33 ± 8) and 80H₂S group (36 ± 6) were significantly lower than those of the Sham group, where neuronal death was rarely seen (50 ± 10, both P < 0.05) (Fig. 6E). Nuclear pyknosis, karyorrhexis, and vacuolization were seen in the CA, 40H₂S and 80H₂S groups (Fig. 6D). However, neuronal density and cell morphologies were better preserved in the 40H₂S and 80H₂S group, where the neuron counts were significantly higher than that of the CA group (both P < 0.05). There was no significant difference between the 40H₂S group and the 80H₂S group.

Cerebral ischemic/reperfusion injury (CIRI) caused by CA/CPR is a key factor in the prognosis of patients greatly impacting their quality of life [2]. Therefore, identification of strategies that can help in reduction of damage caused by CIRI and its prevention as well as efficient treatment is a major focus of clinical research.

In addition to nitric oxide and carbon monoxide, H₂S has been shown to have signal transduction properties in recent years [12]. Abe et al [26].determined that endogenous H₂S may be a special kind of neuromodulator. In the central nervous system (CNS), endogenous H₂S is mainly generated through catalytic effects of 3-mercaptopyruvate sulfurtransferase and cystathionine-β-synthase on sulfur amino acids [13]. It is known that H₂S is not only involved in the neuroendocrine adjustment by the hypothalamus-pituitary-adrenal axis [27], but also affects cerebral blood flow by relaxing microvascular smooth muscles [28].

As an important endogenous gaseous mediator, H₂S plays an important role in CNS ischemic diseases. Various studies have confirmed that H₂S attenuates ischemic brain injury by mitigating oxidative stress, inhibiting cell apoptosis, opening K-ATP channels, relaxing blood vessels, and alleviating astrocytic activation [13, 14, 29]. Consistent with these previous findings, we found that H₂S improved neurological function and increased survival rate after CA and resuscitation in the present study. However, the therapeutic effect of H₂S on brain injury remains controversial. In an experimental model of middle cerebral artery occlusion in rats, Qu et al [30]. found that exogenous H₂S aggravated acute focal cerebral ischemic injury. This contradiction is attributed to the different dose of donor of H₂S, which lower rather than higher concentration of exogenous H₂S might offer a protection against the neuronal injury [31, 32].

Vasogenic edema is caused by increased permeability of brain capillaries and increased fluid leakage after CIRI; therefore, brain water content can directly represent the severity of cerebral edema [33]. Evans Blue assay is the most popular method for evaluation of BBB-disruption in experimental brain disease studies. Evans blue binds to serum albumin immediately after injection into the blood. The EB-albumin complex has a net molecular weight of ∼ 68 kDa which is impermeable across BBB in normal circumstances. In pathological condition when the integrity of the BBB is damaged and permeability is increased, EB-albumin complex leaks outward from blood microvascular endothelial cells (BMECs) gap into the brain tissue; therefore, Evans blue leakage can reflect the destruction of BBB [34]. The results of this study showed that brain water content and Evans blue leakage in brain tissue significantly increased at 24 h after ROSC, suggesting that CA caused significant damage to BBB and severe brain edema. After inhalation of 40 p.p.m. or 80 p.p.m. H₂S in the early stage after ROSC, brain water content was significantly reduced and Evans blue leakage was also significantly reduced. These data indicated that the protective effect of inhaled H₂S in rats after CA and resuscitation was associated with preservation of the integrity of BBB, reducing BBB permeability, and alleviating cerebral edema.

Previous studies have confirmed that the activation of PKC is the major factor in CIRI [5, 8, 35-38]. As previously reported, hypoxia-induced BBB permeability changes occur via a PKC-dependent mechanism. Activation of PKC modulates BBB endothelial cell permeability changes in vitro and in vivo BBB models [39]. A study of Shao et al [7]. showed that PKC-β and PKC-β II in human BMECs were activated after exposure to high glucose in an in vitro BBB model, and the activated PKC inhibited the expression of Occludin and undermined the integrity of BBB. Similar result was confirmed by transfecting BMECs with siRNA targeting PKC-β, in which the activation of PKC-β was impeded and the BBB damage was mitigated [35]. A recent study demonstrated that Go6983 decreased the phosphorylation levels of PKCδ and inhibited the release of MMP-9, which suggested that PKC-δ participated in PAR1-mediated MMP-9 release from pericytes, which lead to BBB dysfunction [40].

However, the role of PKC-ε is different from PKC isozymes mentioned above. Previous study have reported that IL-25 which is produced by BCECs up-regulates the expression of claudin-5 and protects against inflammatory cytokine-induced excessive BBB collapse through a PKC-ε-dependent pathway [41]. Similarly, Lucke-Wold and colleges [42] found that bryostatin-1, a specific PKC modulator, increased ameliorated BBB breakdown through reducing toxic PKC-α levels and increasing the neuroprotective isozyme PKC-ε. Consistent with these previous findings, our data showed that the expressions of PKC-α, PKC-β I, PKC-β II and PKC-δ in cerebral microvessels were significantly increased in the cell membrane which suggested the activation of these PKC isozymes after CA and resuscitation in this study. However, the activation of PKC-ε was significantly inhibited after ROSC.

As a third essential gas molecule, several studies have confirmed that H₂S could regulate the activation and expression of PKC [18, 43-45]. Tiong et al [17]. found that the protective effect of H₂S against 6-OHDA-induced cell apoptosis and death was PKC-α, ε-dependent. In another study, inhibition of PKC suppressed the H₂S-elevated Ca²⁺ influx in SH-SY5Y cells [43]. Furthermore, it was confirmed that H₂S reduced myocardium infarct size by activating PKC-ε and protecting the heart against ischemic/reperfusion injury [44]. In addition, Wei et al [45]. also found that exogenous H₂S activated PKC and down-regulated the expression of AQP4 in a local cerebral ischemia model. Similar to these investigations, the results in the present study indicated that the PKC-α, PKC-β I, PKC-β II and PKC-δ were inhibited while the PKC-ε was activated in cerebral microvessels after inhalation of 40 p.p.m. or 80 p.p.m. H₂S. The present findings, in conjunction with these previously published data [17, 43-45], suggest that PKC is involved in the protective effect of H₂S against CA and resuscitation.

The TJ protein is mainly responsible for the restriction of the paracellular transport of molecules across the BBB [46]. In an attempt to clarify the mechanisms underlying the improved BBB permeability observed after H₂S inhalation, we investigated the changes of TJ proteins. We found that the expression of Claudin-5, Occludin, and ZO-1 were significantly decreased after ROSC. However, inhalation of H₂S increased the expression of Claudin-5, Occludin, and ZO-1. In addition to this results, we also observed a decrease in Evans Blue extravasation and less BBB damage with improvement in neurological function after inhalation of H₂S. Furthermore, a significant improvement in the number of neurons in hippocampus and survival rate was also observed at 14 day after ROSC. These results showed that the increase in the TJ protein expression could be one of the mechanisms underlying the protective effect of H₂S against BBB disruption after CA and resuscitation.

Previous studies have indicated that activated PKC phosphorylates TJ proteins or regulates their expression [4-6, 47]. It has been shown that the activation of PKC-α disrupts the TJ and causes the disassembly of occluding junction [48]. Another study has shown that the damage of TJ following hypoxic/aglycemia was exacerbated by the over expression of PKC-β II which led to the increase of the hyperpermeability of BMECs [6]. Furthermore, Qi et al [5]. demonstrated that PKC-δ specifically resulted in the dysregulation of TJ components. Inhibition of PKC-δ significantly reduced the accumulation of PKC-δ in BMECs and prevented the hypertension-induced TJ disruption associated with BBB breakdown. In this study, we demonstrated that regulation of PKC isozymes by H₂S correlated with TJ proteins and BBB permeability changes induced by CA and resuscitation. Inhalation of H₂S improved the neurological function and survival rate, decreased the expression of PKC-α, PKC-β I, PKC-β II, PKC-δ, and increased the expression of PKC-ε. Taken together, these results suggest that it is likely that the protective effect of H₂S against CA and resuscitation was via a PKC/TJ-dependent pathway to alleviate BBB leakage. The inhalation of H₂S in the early CA setting could be used to mitigate BBB disruption and improve neurologic function.

However, the mechanism by which PKC modulates TJ proteins is still poorly understood. One hypothesis is that PKC may directly regulate the structure and function of TJ proteins, and the other is that PKC may be indirectly altering the TJ proteins by activating a downstream intracellular signaling pathway that in turn directly affects the TJ proteins [40]. Further investigations on the colocalization and distributions of TJ protein after CA and resuscitation with or without H₂S inhalation will be needed.

It remains controversial whether the effect of H₂S is dose-dependent or not. Johansen et al [49]. confirmed that the protective effect of NaHS on myocardium against ischemic/ reperfusion injury protection was not concentration-dependent. While the study of Gheibi et al [50]. in which a MCAO model was applied, the authors found that intraperitoneal injection of NaHS alleviated brain edema and the protective effect was dose-dependent. The contradiction may be due to the differences in experimental models, H₂S donors, concentration of drugs, as well as the models and timings of administering drugs. In our setting, H₂S gas was delivered via inhalation, and we did not observe a significant difference in almost all data between two concentrations, suggesting that therapeutic effect of inhalation of H₂S may have already reached the ceiling effect at 40 p.p.m. in this model. Further more in our next study, we will investigate whether H₂S inhalation has a dose-dependent effect at lower concentration or not and make an effort to find out the optimum concentration of H₂S needed for more beneficial effects and less side effects.

In conclusion, 40 p.p.m or 80 p.p.m H₂S inhalation significantly improved the integrity of BBB, mitigated brain edema, and improved neurological outcome and survival in rats after CA and resuscitation. The beneficial effect of H₂S could be associated with regulating PKCs. Potentially, the mechanism may be that inhalation of H₂S inhibits the activation of PKC-α, PKC-β I, PKC-β II and PKC-δ, and promotes the activation of PKC-ε, following which, the expression of Claudin-5, Occludin, and ZO-1 are increased to preserve the integrity of the BBB.

This research is supported by the National Natural Science Foundation of China (grant number 81372026, 81000822). The funding organizations had no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; and in the decision to submit the article for publication.

The authors declare that there is no conflict of interests.