Osteopontin attenuates early brain injury through regulating autophagy‐apoptosis interaction after subarachnoid hemorrhage in rats

Abstract Aim To determine the effect of osteopontin (OPN) on autophagy and autophagy‐apoptosis interactions after SAH. Methods The endovascular perforation model of SAH or sham surgery was performed in a total of 86 Sprague‐Dawley male rats. The temporal expressions of endogenous OPN and autophagy‐related proteins (Beclin 1, ATG5, LC3 II to I ratio) were measured in sham and SAH rats at different time points (3, 6, 12, 24, and 72 hours). Rats were randomly divided into three groups: Sham, SAH + Vehicle (PBS, phosphate‐buffered saline), and SAH + rOPN (5 μg/rat recombinant OPN). Neurobehavioral tests were performed 24 hours after SAH, followed by the collection of brain samples for assessment of autophagy and apoptosis proteins. These tests assessed whether an autophagy‐apoptosis relationship existed on the histological level in the brain. Results Endogenous OPN and autophagy‐related proteins all increased after SAH. rOPN administration improved neurological dysfunction, increased the expression of autophagy‐related proteins (Beclin 1, ATG5, LC3 II to I ratio) and antiapoptotic protein Bcl‐2, while decreasing the expression of proapoptotic proteins (cleaved Caspase‐3 and Bax). rOPN also regulated autophagy‐apoptosis interactions 24 hours after SAH. Conclusion rOPN attenuates early brain injury and inhibits neuronal apoptosis by activating autophagy and regulating autophagy‐apoptosis interactions.


| INTRODUC TI ON
Aneurysmal subarachnoid hemorrhage (SAH) remains to be one of the most devastating forms of stroke with high mortality and disability rates throughout the world. 1 In recent years, early brain injury (EBI) has been reported as a primary cause of mortality in SAH patients. 2,3 The initiation of many important pathological mechanisms that happens within minutes following aneurysm rupture, 4 requires ongoing research to improve the understanding of the pathophysiological events occurred. 5 Among the various pathological processes contributing to EBI, neuronal apoptosis has been reported to be an essential process that might explain the severe impact of SAH on short-term and long-term outcomes. 6 Meanwhile, as a highly conserved biological phenomenon to degrade intracellular components and help maintain cellular homeostasis, 7 autophagy activation after SAH has been shown to have neuroprotective effects. Several studies have reported that autophagy enhancing drugs can reduce apoptosis, while autophagy inhibitors aggregate cell apoptosis after SAH induction. [8][9][10] Autophagy is an important protective mechanism against apoptosis in ischemic cell injury. 11 Activation of autophagy protects neuronal apoptosis in EBI after SAH. 12 However, few studies are investigating the relationship between autophagy and apoptosis in EBI after SAH on the molecular and cellular level.
Osteopontin (OPN) is a secreted extracellular matrix glycoprotein with roles in a variety of physiological and pathological processes, including cerebral vascular remodeling, cell migration, and antiapoptotic processes. 13 In previous studies, administration of recombinant OPN (rOPN) has shown promising neuroprotective effects 14 such as stabilizing vascular smooth muscle cell phenotype after subarachnoid hemorrhage, 15 and attenuating inflammation after intracerebral hemorrhage. 16 Interestingly, recent studies showed that OPN was a potential enhancer of autophagy in an in-vitro model of abdominal aorta aneurysm 17 and human hepatocellular carcinoma. 18 This indicates that one of the important mechanisms underlying OPN's neuroprotective effects after SAH could be its antiapoptotic effect through autophagy activation. No previous research has investigated the influence of OPN on autophagy or autophagy-apoptosis interactions in SAH models. In the present study, we aimed to determine the regulatory role of OPN in autophagy modulation and autophagy-apoptosis interaction to understand the neuroprotective effects of OPN treatment in SAH better. The endovascular perforation model of SAH was conducted following previously described procedures. 19 All rats were randomly assigned to experimental groups. Briefly, rats were intubated in deep anesthesia and kept on a rodent ventilator (Harvard Apparatus) during surgery with 3% isoflurane in 65/35% medical air/oxygen to maintain anesthesia. The left external and internal carotid artery were exposed, and a 4-0 monofilament nylon suture was inserted into the left internal carotid artery through the dissociated external carotid artery until resistance was felt. The suture was advanced 3 mm further to perforate the bifurcation of the anterior and middle cerebral artery followed by immediate withdrawal. Sham rats underwent the exact same procedures except for the perforation. The incision sutured and rats were housed individually in heated cages following recovery from anesthesia. At 24 hours after sham or SAH surgery, SAH score was evaluated by an independent observer as previously described 20 : the total score (maximum SAH grade = 18) was calculated as the sum of six sub-scores based on six corresponding predetermined areas. Animals with mild SAH (SAH grade ≤ 8)

| Animals and SAH model
were excluded from the current study.
Western blot was performed to determine the expression of the four target proteins.

| Experiment 2: Localization study
To determine the distribution of endogenous OPN and autophagyinitiating protein Beclin1 in different types of cells after SAH, three rats were used for double immunohistochemistry staining, performed at the time point after SAH when the peak expression of the two target proteins occurs.

| Experiment 3: Outcome study
To evaluate the possible beneficial effects of rOPN after SAH, 27 rats were randomly divided into three groups (n = 9 per group): Sham, SAH + Vehicle (30 μL PBS), and SAH + rOPN (5 μg/rat recombinant Osteopontin in 30 μL PBS, 6359-OP-050, R&D Systems). rOPN dosage was chosen based on our previous research. 21 The nine rats per group included six rats for neurological tests and Western blot, and 3 for immunofluorescence staining. Vehicle or rOPN was delivered via the intranasal route 1 hour after SAH induction. Rats under isoflurane anesthesia were placed in a supine position and PBS or rOPN dissolved in PBS was administered alternately into the left and right nares, 5 μL each time with an interval of 2 minutes between each administration.
A total volume of 30 μL was administered intranasally to each animal.

| Neurobehavior assessment at 24 hours after SAH
Neurobehavior was evaluated at 24 hours after SAH induction using the previously described modified Garcia scoring system and beam balance test in a blinded fashion. 22 In modified Garcia test, 23 six parameters were tested, which allowed a total score of 18. Higher scores indicated a greater functional assessment of neurological outcome. These six tests included: spontaneous activity, symmetry in the movement of all four limbs, forepaw outstretching, climbing, body proprioception, and response to vibrissae touch. The beam balance test 24,25 consisted of rats walking on a 15-mm wide wooden beam for 1 minutes. The mean score was calculated based on three consecutive trials that were scored from 0 to 4 according to the rats' walking ability.

| Western blot analysis
Western blot tests were performed as previously reported. 26,27 Briefly, the whole left hemispheres were isolated and collected

| Double immunofluorescence staining and TUNEL staining
Rats were sacrificed at 24 hours after SAH induction. A series of 8 μmthick frozen brain tissue slices were prepared. Double immunofluorescence staining was performed as previously described. 28,29 The primary antibodies used were anti-GFAP (1:500, ab53554), anti-NeuN

| Statistical analysis
All data were expressed as mean ± SD (standard deviation). After the normality test, one-way ANOVA of the mean values followed by Tukey's post-hoc test was performed for multiple groups. A Kruskal-Wallis test was used for Garcia scores, beam balance test, and SAH grading scores. The analyses were performed using SPSS version 24.0 (IBM Corp.). Statistical significance was defined as P < .05.

| Mortality and SAH grades
A total of 86 rats were used: 15 rats were sham, 71 rats underwent SAH induction. Seven rats were excluded from the study due to mild SAH with grade ≤ 8 ( Table 1). The mortality (calculated after exclusion of low-grade rats) of SAH rats was 20.31% (13/64). No rats died in the sham group. Blood clots were mainly seen around the circle of Willis and ventral brain stem after SAH induction ( Figure 1A). The average SAH grades showed no significant differences between SAH + Vehicle group and SAH + rOPN group ( Figure 1B).

| Temporal expression of endogenous OPN and autophagy-related proteins after SAH
Western blot was performed to determine the expression of endogenous OPN and autophagy-related proteins (Beclin 1, ATG 5 and LC3) at 3, 6, 12, 24, and 72 hours on the left hemispheres of rats' brains after SAH. Results showed that endogenous OPN & LC3-II to I gradually increased from 3 hours after SAH induction, while Beclin 1 and ATG 5 got a sudden increase starting from 12 hours. All the autophagy-related proteins and OPN peaked at 24 hours. However, OPN and Beclin 1 level started to drop at 72 hours after SAH while the expression of ATG 5 and LC3 II/I remained stable till 72 hours after SAH ( Figure 1C).  Figure 2A) and Beclin 1 ( Figure 2B) were expressed in all three types of cells after SAH, and both were mainly expressed in neurons, indicating their potential participation in neuronal survival and function.

| Intranasal administration of rat rOPN ameliorated neurological deficits at 24 h after SAH
During the assessment of short-term neurobehavior, modified Garcia and beam balance scores were significantly lower in the SAH + Vehicle group than those in the sham group (P < .001, F I G U R E 2 Expression of endogenous osteopontin (OPN) and Beclin 1 in different cell types of rat brain at 24 h after SAH. Double immunofluorescence staining for endogenous OPN (A) or Beclin 1 (B) in neurons (NeuN, green), astrocytes (GFAP, green) and microglia (IBA-1, green) in the left basal cortex of rat brain at 24 h after SAH. Nuclei are stained with DAPI (blue). The red box in the brain slice image indicates the area observed. n = 3 per group. Scale bar = 50 μm Figure 3A). However, the intranasal administration of rOPN significantly improved neurological scores (SAH + rOPN group vs SAH + Vehicle group P < .05, Figure 3A).

| rOPN administration elevated the total amount of OPN protein in the brain while suppressing apoptosis at 24 h after SAH
At 24 hours after SAH induction or sham surgery, Western blot analysis was performed to detect protein expressions after SAH and rOPN administration. Our results indicated that intranasal administration of rat rOPN drastically increased the level of full-length OPN (62 kDa) and its cleavage products (20-50 kDa) in the left hemisphere (SAH + rOPN group vs. SAH + Vehicle group P < .01, Figure 3B).
Moreover, TUNEL staining demonstrated there was a significant increase in the number of TUNEL-positive cells at 24 hours after SAH when compared with the Sham group (P < .05, Figure 4). Also, rOPN administration remarkedly reduced the amount of TUNEL-positive cells in the brain as compared with SAH + Vehicle group (P < .05, Figure 4).

| rOPN administration influenced the interaction and balance between autophagy and apoptosis at 24 hours after SAH
Based on our results above, we further performed double immunofluorescence staining of apoptosis marker Caspase-3 with autophagy marker Beclin 1 to investigate the interaction of autophagy and apoptosis after SAH on the histological level ( Figure 5).
In the brain slices of sham rats, Beclin 1 and Caspase-3 were both on relatively low expression levels. In both SAH + Vehicle and  Caspase-3 staining while increasing the density of Beclin 1-positive cells near the "confrontation line" (Figure 6).

| DISCUSS IONS
Our Autophagy is a highly dynamic process of cellular component degradation, which is often reflected by the conversion of LC3-I to LC3-II to form autophagosomes. 33 Previous research demonstrated that autophagy has a neuroprotective role in EBI after SAH and that at least part of its protective role is through its anti-apoptotic effect. 6 Studies reported that specific therapeutic agents for SAH could activate cell autophagy while inhibiting apoptosis at the same time, 6,10,34 which was consistent with the results in the current study. Furthermore, in the study of Shao et al, it was shown that 3-MA inhibition of class III PI3K activity and autophagy activation 35 led to increased cell apoptosis after SAH. 34 In contrast, treatment with RAP, which induces autophagy by inhibiting mTOR, 36 significantly decreases apoptosis level in the EBI phase. 9 These previous results suggested that autophagy might be an upstream event to cell apoptosis after the onset of SAH.
However, these previous studies mainly focused on the quantification of autophagy and apoptosis-related proteins using Western blot and TUNEL-based cell count. 6,9,10,34 Few studies have investigated the interaction or balance between autophagy and apoptosis after SAH on the histological level. In a recent study by Guo et al, 12

| CON CLUS ION
In conclusion, rOPN administration could activate autophagy while attenuating apoptosis at 24 hours after SAH through regulating the interaction between Caspase-3 and Beclin 1. Further investigations of the relationship between autophagy and apoptosis on the histological level using the "autophagy-apoptosis costaining method" described in the current study are needed to better understand precise molecular mechanisms of the interactions between autophagy and apoptosis in the brain cells after SAH. Future studies should also consider how therapeutic agents such as OPN can influence this autophagy-apoptosis interaction to exert their neuroprotective effects.

ACK N OWLED G M ENTS
This study is supported by grants from NIH (NS081740 and NS082184) to Prof. John H. Zhang, and grants from Guangdong Province (2016B030230004) and Guangzhou City (201803040016), China to Prof. Xiaodan Jiang.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest. F I G U R E 6 rOPN administration influenced the interaction and balance between Beclin 1 and Caspase-3 at 24 h after SAH. Double immunofluorescence staining of Caspase-3 and Beclin 1 in Sham group, SAH + Vehicle group and SAH + rOPN group at 24 h after SAH induction. Sample size is 9, n = 3 per group. Localization of Caspase-3 can be cytoplasmic and nuclear. Staining in the nucleus is considered to be an indication of active Caspase-3. The dashed lines and the red box on brain slice images indicate the locations observed. Vehicle, phosphate-buffered saline; Scale bar = 50 μm