SS31 attenuates oxidative stress and neuronal apoptosis in early brain injury following subarachnoid hemorrhage possibly by the mitochondrial pathway

Ruiming Shen1*, Jian Zhou2*†, Ge Li3, Wuyan Chen4, Wangwang Zhong2, Zhenggang Chen2


• SS31 improved neurobehavioral performance, alleviated brain edema and and mitigated BBB permeability following SAH.
• SS31 attenuated the oxidative stress and suppressed SAH-induced neuronal apoptosis in a rat model of SAH.
• SS31 provided neuroprotective effects in early brain injury following subarachnoid hemorrhage possibly by inhibiting the mitochondrial pathway.


Background: SS31 has been shown to have neuroprotective effects in a number of neurological degenerative diseases. However, the mechanisms and its role of neuroprotection after subarachnoid hemorrhage (SAH) remain unexplored. The aim of the present study is to evaluate the neuroprotective effects of SS31 on early brain injury (EBI) induced by SAH in rats and the potential mechanisms of the protective effects of SS31.
Methods: Sprague-Dawley rats were randomly divided into four groups: Sham, SAH, SAH+vehicle, and SAH+SS31 groups. The SAH-induced prechiasmatic cistern rat model was established in this study. Neurological scores were evaluated at 24 h and 72 h after SAH. The brain edema, blood-brain barrier (BBB) permeability, neuronal apoptosis, malondialdehyde (MDA), glutathione peroxidase (GPx) and superoxide dismutase (SOD) activities, as well as the expression of mitochondrial and cytosolic cytochrome C (Cyt C), and Bax were analyzed at 24 h after SAH.
Results: When compared with the vehicle-treated group, treatment with SS31 significantly reduced MDA levels and restored the activities of GPx and SOD in the temporal cortex following SAH when compared with the vehicle-treated group. In addition, the levels of mitochondrial Cyt C and Bax respectively increased and decreased by SS31 treatment. Moreover, SS31 treatment ameliorated brain edema and Evans blue dye extravasation, improved neurological deficits, and decreased neuronal apoptosis at 24 h after SAH.
Conclusion: Our data provides initial evidence that SS31 could alleviate EBI after SAH through its antioxidant property and ability in inhibiting neuronal apoptosis, likely by modulating the mitochondrial apoptotic pathway.

Keywords: subarachnoid hemorrhage, early brain injury, SS31, oxidative stress, apoptosis, mitochondria


Subarachnoid hemorrhage (SAH) is a devastating cerebrovascular disease with high morbidity and mortality worldwide. Despite the fact that great progress has been made in the diagnosis and surgical treatment of SAH, effective therapeutic measures are still limited and clinical outcomes remain dismal. In the past few decades, delayed cerebral vasospasm has been considered to be the major cause of the poor clinical outcome after SAH. However, long-term neurological outcomes following SAH did not improve by reducing the incidence of cerebral vasospasm. The prevention of vasospasm does not appear to improve the prognosis of patients with SAH. Recently, a mounting number of studies have demonstrated that early brain injury (EBI), which describes the acute brain injury within the first 72 h after SAH, is the dominant cause of death in patients with SAH [1]. Thus, treatment for EBI is considered to be the main target of SAH patient management.
Although the exact pathological mechanism of EBI remains unknown, previous studies demonstrate that oxidative stress plays a critical role in the pathogenesis of EBI following SAH. Mitochondria act as the major cellular source of reactive oxygen species (ROS) and are particularly liable to oxidative insult [2]. Mitochondria impairment causes ROS over-production, damaging mitochondrial proteins, DNA, and lipids, ultimately triggering apoptosis, and leading to metabolic dysfunction. During this process, an increase of mitochondrial membrane permeability plays an important role in apoptotic cell death, causing the release of apoptosis mitochondrial proteins and promoting neuronal apoptotic death. Evidence has implicated a marked increase in apoptotic cell death after SAH, and the beneficial roles of anti-apoptotic therapeutics in SAH have also been established [3]. Therefore, apoptotic-related mechanisms must be rigorously regulated to inhibit neuronal death after SAH, particularly the regulation of the mitochondrial pathway of apoptosis.
SS31 is a cell-permeable, novel mitochondria-targeted peptide [4-5], which is capable of targeting and permeating mitochondria. SS31 then accumulates inside the inner membrane of mitochondria via mitochondrial membrane potential [6]. As a target anti-oxidant, SS31 can scavenge free radicals and excessive ROS, inhibiting lipid peroxidation, and mitigating mitochondrial swelling and oxidative injury [7]. Recently, SS31 has been shown to exert potential neuroprotective effects in models of neurodegenerative disorders, including Huntington’s disease [8], amyotrophic lateral sclerosis [5], diabetes-induced retinal degeneration [9-10], and Alzheimer’s disease [8, 11-13]. However, no data concerning the neuroprotective effect of SS31 after SAH are available. Therefore, there is an urgent need to investigate the potential effect of SS31 and possible mechanisms in EBI following SAH. The aim of the present study is to explore the neuroprotective role of SS31 in the modulation of mitochondrial function and apoptotic mechanisms in a rat model of SAH.

Materials and methods

Animal Ethical Approval

Adult male Sprague-Dawley rats weighing 250 to 300 g were used in the study. The rats were housed under standard conditions of temperature ((22±1 °C), relative humidity (30%) and lighting (a 12-h light/dark cycle). Drinking water and standard diet were given ad libitum. Protocols for experiments were approved by the Animal Care and Use Committee of Hainan Medical college (Haikou, China) and followed to the Guide for the Care and Use of Laboratory Animals set by the National Institutes of Health (NIH).

SAH Model

The prechiasmatic cistern SAH model used in this study was induced as described previously by Wang, et al [26][14]. Briefly, rats were anesthetized with 10% chloral hydrate (300 mg/kg) and subsequently placed in a stereotactic frame. Under the stereotactic guidance, fresh autologous non-heparinized arterial blood (0.3 mL) from the femoral artery was injected aseptically into the prechiasmatic cistern in 20 s with a syringe pump in the SAH group. The rats in the sham group were injected with 0.3 mL artificial cerebrospinal fluid. The animals were allowed to recover in a head-down prone position for 45 min post-SAH. At the end of the operation procedures, the rats were returned to their cages where water and food were supplied freely. And then, the amount of 20ml saline was injected subcutaneously. During the experiment, the heart rate and rectal temperature were measured, and the rectal temperature was maintained at (37 ± 0.5) °C with a warm pad.

Experimental design

A total of 192 rats were randomly divided into the following groups: Sham, SAH, SAH + vehicle, and SAH + SS-31 (3 subgroups: 2 mg/kg, 5 mg/kg, 10 mg/kg). SS-31 was purchased from AnaSpec, CA. and freshly prepared in phosphate buffer saline (PBS) just before injection. Rats in the SAH + SS-31 group were injected with 5 mg/kg SS-31 intraperitoneally 30 min post- SAH. SAH vehicle-treated group were given equal volumes of the vehicle (PBS) at the corresponding time point. The dosage used in the present study was chosen based on a previous report. All the rats of each group were killed at 24 h post-SAH.

Neurobehavioral Evaluation

The neurological scores were evaluated at 24 h and 72 h after SAH using the scoring methodology including appetite, activity, and neurologic deficits (Table 1) [14]. All neurobehavioral evaluations were performed by two veterinarians who were blinded to the experimental groups.

Brain water content

Brain water content was determined in light of a previous study [15]. In brief, the rat brain was quickly harvested 24 h after SAH. And the temporal cortical tissue was harvested and immediately weighed to obtain the wet weight (WW). The brain samples were dried for 72 h at 80 °C and weighted to obtain the dry weight (DW). Brain water content was calculated as [(WW – DW)/WW] × 100%.

BBB permeability

BBB permeability was measured by the extravasation of Evans Blue at 24 h after SAH according to a previous study [14]. Briefly, 2% EB dye (4 mL/kg) was administrated into the right femoral vein over 2 min and circulated for 1 h. Rats were reanesthetized and perfused transcardially using PBS to remove intravascular EB dye. After decapitation, the level of EB within brain tissue was assessed by the absorbance at 620 nm using a spectrophotometer.

Tissue processing

The tissue that contained the inferior basal temporal lobe was harvested for Western blot and biochemistry indexes. The whole brain was collected and then immersed in 4% buffered paraformaldehyde overnight for immunohistochemistry and TUNEL analysis.

Isolation of mitochondria

Mitochondria extract from the inferior basal temporal lobe were isolated following the instruction of the manufacturer with the use of Mitochondrial Isolation Kit for Tissue (Beyotime Institute of Biotechnology, Nantong, China). In brief, the temporal lobe was homogenized in ice-cold isolation buffer and centrifuged at 1200 g for 3 min. The supernatants were collected, centrifuging at 14 000 g for 14 min at 4°C. After removing the supernatants, the precipitate is mitochondria.

TUNEL Staining

The apoptotic cells were assessed by using a TUNEL detection kit (ISCDD, Boehringer Mannheim, Germany) according to the manufacturer’s instructions. Quantitative analysis of TUNEL-positive cells in the temporal cortex was conducted in six random non-overlapping fields (× 400) by two investigators blinded to the experiment.

Western blot analysis

In brief, after the fractionated proteins were transferred onto a polyvinylidene difluoride membrane, they were incubated with various primary antibodies: cytochrome c (1:1000, Abcam), Bax (1:200, Santa Cruz), Bcl-2(1:200, Santa Cruz), COX Ⅳ(1:1000, Cell Signaling Technology), β-actin(1:5000, Bioworld Technology), and cleaved caspase-3 (1:1000, Cell Signaling Technology). Subsequently, the membranes were incubated with the appropriate secondary antibodies. The ECL system (Millipore, Billerica) was used for autoradiograms. The band relative density was analyzed using the Image J software.

Immunohistochemical (IHC) staining

Dehydrated paraffin-embedded sections were prepared. The sections were incubated with caspase-3 antibody (1:200 dilution), and then incubated with secondary antibodies; the sections were finally prepared for DAB substrate solution. Neuronal cell apoptosis assessment was performed using IHC staining as previously described [16].

Mitochondrial malondialdehyde (MDA) level, superoxide dismutase (SOD), and glutathione peroxidase (GPx) activities

Mitochondrial MDA content, as well as SOD and GPx activities were determined by chemiluminescence methods as previously described [17].

Statistical analysis

Statistical analysis was conducted using the GraphPad Prism 7.0 (GraphPad Software Inc., La Jolla, CA, USA) software. Values are expressed as the mean ± SEM. Statistical comparisons between groups were analyzed with one-way ANOVA and Tukey’s test. A p-value less than 0.05 was considered statistically significant.


SS31 improves the neurobehavioral function and alleviates cerebral edema following SAH.

To explore the protective role of SS31 on neurological function, we set six groups as follows: Sham, SAH, SAH + vehicle, SAH + SS31 (three different dose groups: 2 mg/kg, 5mg/kg, 10 mg/kg). The neurological deficit at 24 h and 72 h after SAH were evaluated as previously reported. All experimental animals were prepared 24 hours before SAH. Within 3 days after insult, main effects were found for the neurological deficit at 24h (F[5,30] = 25.51, p<0.0001) and 72h (F[5,30] = 26.4, p<0.0001) after SAH. Post-hoc tests confirmed that there was no statistical difference between time points in the sham group. No obvious difference was detected between the SAH group and the SAH+vehicle group. The group of rats that received 2 mg/ kg SS31 exhibited a slightly improved motor performance following SAH, however, there was no statistically significant change compared to the vehicle-treated group of rats on 24 h and 72 h (p > 0.05, Fig. 1A). The scores of the rats treated with 5 mg/ kg SS31 were significantly improved compared to that of the vehicle-treated rats after SAH (p < 0.05, Fig. 1A), whereas a high dose of SS31 (10 mg/kg) did not perform a better neuroprotective effect. (p > 0.05; Fig. 1A).
Brain water content was determined to assess the neuroprotective role of SS31. There was significant main effect of group (F[5,30] =31.32; p < 0.0001). Post-hoc tests indicated that the brain water content was dramatically increased in the SAH group compared with the sham group. There was no significant difference between the vehicle-treated group and SAH + 2 mg/kg SS31 group (p > 0.05, Fig. 1B). However, the 5 and 10 mg/kg SS31 had significantly decreased brain water content compared to the vehicle-treated group (p < 0.001 and p < 0.01, Fig. 1B). Consistent with the neurological scores, the 5 mg/kg dose showed a markedly better effect in ameliorating the SAH-induced brain edema than the vehicle-treated group (p < 0.05; Fig. 1B). Based on these results, SS31 has a neuroprotective effect against SAH, and the dose of 5 mg/kg SS31 provided the best results and thus was used in the subsequent studies. SS31 treatment attenuated BBB permeability mediated by SAH. A notable increase in the extravasation of Evans blue dye in the temporal lobes was induced by SAH insult. There was a main effect of group (F[3,20] = 338.51, p<0.0001). Post-hoc analyses showed that no significant differences were found in BBB permeability between the SAH and SAH+vehicle groups as compared with that in the sham group at 24 h after surgery. BBB permeability was markedly ameliorated by SS31 treatment at 24 h after SAH. These results demonstrated that SS31 administration can attenuate BBB permeability mediated by SAH (p < 0.01, Fig. 1C). SS31 administration ameliorated neuronal apoptosis following SAH To explore whether SS31 administration could alleviate apoptosis in the inferior temporal cortex 24 h after SAH, we used NeuN/TUNEL double immunofluorescence analysis to assess neuronal apoptosis. There was a main effect of group (F[3,20] = 92.99, p<0.0001). Post-hoc tests confirmed that only a few TUNEL-positive cells were found in the sham group, whereas the apoptosis index was notably decreased following SS31 administration (Fig. 2). Collectively, these data suggest that SS31 possibly ameliorates neuronal apoptosis following SAH. To further investigate the effects of SS31 on SAH-induced apoptosis, we detected the expression of mitochondrial apoptosis-related proteins such as Bax and cytochrome c, after SAH. Main effects were found for Cyto C (cytosol) (F[3,20] = 19.69, p<0.0001), Cyto C (mit) (F[3,20] = 37, p<0.0001), Bax (cytosol) (F[3,20] = 19.62, p<0.0001), and Bax (mit) (F[3,20] = 27.31, p<0.0001). Post-hoc tests revealed that the levels of mitochondrial and cytosolic Bax protein were increased and decreased, respectively, after SAH, compared with the sham group, whereas mitochondrial and cytosolic cytochrome c levels were decreased and increased, respectively, after SAH, relative to the sham group (Fig. 3). These effects were reversed by SS31 administration, which suppressed mitochondrial translocation of Bax and subsequent cytosolic release of cytochrome c. These results demonstrated that SS31 can reduce neuronal apoptosis in the temporal cortex following SAH. SS31 administration downregulated the expression of caspase-3 The protective role of SS31 against neuronal apoptosis following SAH was also evaluated by immunohistochemistry. There was a main effect of SS31 on the caspase-3 expression after SAH (F[3,20] = 54.2, p<0.0001). Post-hoc tests showed that a few caspase-3-positive cells were found in the inferior temporal cortex of the sham group, while numerous caspase-3-positive cells dyed as brown were obvious in the SAH and vehicle-treated groups. Compared with the SAH + vehicle group, SS31 administration markedly mitigated the number of caspase-3-positive cells in the brain at 24 h after SAH (Fig. 4 A, B). These results demonstrated that SS31 treatment could inhibit caspase-3 expression following SAH. In addition, we also detected the cleaved caspase-3 expression after SAH (F[3,20] = 227, p<0.0001). Post-hoc tests confirmed that the cleaved caspase- 3 expression increased 24 h after SAH, while SS31 downregulated the cleaved caspase-3 expression (Fig 4 C, D). SS31 attenuates oxidative stress induced by SAH To elucidate the antioxidative effect of SS31 following SAH, we determined the production of MDA, GPx and SOD in the mitochondria. Main effects were found for MDA (F[3,20]=33.01, p<0.0001), GPx (F[3,20]=33.04, p<0.0001) and SOD (F[3,20]=25.77, p<0.0001). Post-hoc tests revealed that the activities of GPx and SOD were obviously decreased after SAH compared with the sham group (both p < 0.001). However, SS31 treatment increased the activities of GPx and SOD (both p < 0.01; Fig. 5B and C). Mitochondrial MDA levels in the SAH and vehicle-treated groups were increased compared with the sham group (p < 0.001), whereas SS31 administration restored the content of MDA. (p < 0.001, Fig. 5A). Discussion In the present study, we demonstrated the neuroprotective effects of SS31 in rats following SAH and explored its potential molecular mechanism. The main findings are summarized as follows: 1) SS31 administration exerted neuroprotection after SAH, specifically, it improved neurological function and reduced the secondary brain injury including BBB permeability and cerebral edema; 2) neuronal apoptosis was notably observed in this SAH model, and SS31 treatment suppressed SAH-induced neuronal apoptosis and oxidative stress following SAH. SS31 performs a variety of biological effects, such as promoting mitochondrial inner membrane electron transport, stimulating ATP synthesis, reducing ROS overproduction and inhibiting apoptosis [5, 7]. SS31 plays a critical role in multiple diseases, such as neurodegenerative diseases, brain ischemia-reperfusion [18], and ischemia-reperfusion injury. However, the role of SS31 in EBI after SAH is elusive. Previous studies demonstrated that SS31 penetrated the mitochondria after injection and exerted underling antioxidant properties. As for neurodegenerative diseases and ischemic brain injury, SS31 showed neuroprotective effects by the scavenging of mitochondrial ROS, ameliorating the release of Cyt C from mitochondria into the cytoplasm and mitigating oxidant insult. Furthermore, we found that 5 mg/kg of SS31 30 min post-SAH recognizably improved neurological function. It was also discovered that SS31 treatment was optimal at 24 h post-SAH, which is consistent with a previous study. Oxidative stress, which is a vital pathophysiological process resulting from secondary brain insult, is caused by overproduction of ROS after SAH [2, 19]. Mitochondria are the “powerhouses” of cells, playing a pivotal role in the brain lesion through mitochondria-related apoptosis or by ROS production. Mitochondria damage produces redundant ROS, hindering mitochondrial function and disrupting the normal balance of endogenous oxidant and antioxidant mechanisms [20-21]. MDA, a marker of lipid peroxidation, starts to elevate immediately after injury and keeps increasing even 24 h later. However, endogenous antioxidative enzymes such as SOD and GPx, convert peroxides into innocuous substances [22]. Mitochondrial metabolic dysfunction causes oxidative modification of proteins, lipids, and DNA, leading to an excessive increase in ROS. The results of our study showed that, compared with the vehicle-treated group after SAH, SS31 administration mitigated the mitochondrial MDA level and improved the activities of GPx and SOD, compared with the vehicle-treated group after SAH. These results indicated that SS31 remarkably attenuated the oxidative insult mediated by SAH. These findings are in clear consistency with prior studies. According to our findings, we can infer that SS31 performs antioxidant roles via protecting mitochondria following SAH. Growing data indicate that dysfunctional mitochondria can promote cell death through the mitochondrial apoptotic pathway [23-25]. Briefly, an increase in expression of pro-apoptotic factors relative to the expression of the BCL-2 family of proteins leads to the opening of the mitochondrial permeability transition pore (PTP) and increases the permeability of the mitochondrial membrane, resulting in mitochondrial cytochrome c release [26-27]. Cytochrome c is released from the mitochondrial intermembrane space into the cytoplasm, leading to activation of the mitochondrial-dependent apoptotic pathway and inducing the sequential activation of the caspase cascades [28], including caspase-3 activation, resulting in the degradation of DNA and essential proteins and ultimately terminating in apoptosis [29-30]. A similar phenomenon was observed in our work. Our data suggests that SS31 administration suppresses Bax translocation into the mitochondrial membrane, and mitigates the release of cytochrome c from the mitochondria to the cytoplasm after SAH. The findings indicated that the mitochondrial- dependent apoptotic pathway was activated. These changes further elicited the activation of the intrinsic apoptosis pathway, leading to neuronal apoptosis. The effects were reversed by SS31 treatment following SAH. The results described above reveal that SS31 provides neuroprotective effects in a rat model of SAH by inhibiting the mitochondrial-dependent apoptosis pathway following SAH. Our study suffers from certain limitations. First, an in vitro model of SAH should be used to investigate whether SS31 is involved in the protection against neuronal injury and oxidative insult. Second, mitochondrial morphology and mitochondrial membrane potential should be used to confirm the neuroprotective effects of SS31 after SAH. Therefore, further research should be conducted to address these issues. Conclusion The results of the present study demonstrated that SS31 ameliorated the oxidative stress by enhancing the activities of antioxidant enzymes, as well as inhibited the mitochondrial apoptotic pathway via suppressing Bax translocation and cytochrome c release from the mitochondria in a rat model of SAH. 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