Stargazin Interaction With Serine Racemase Mediates Cerebral Ischemia/Reperfusion Injury in Rats

D-Serine is thought to be involved in N-methyl-D-aspartate (NMDA)-type glutamate receptor-mediated neurotoxicity and plays a pathophysiologic role in stroke. D-Serine is synthesized by serine racemase (SR), which directly converts L-serine into D-serine. The deletion of SR has been reported to protect against cerebral ischemia damage. Additionally, SR catalytic activity is physiologically regulated by its binding to stargazin. However, whether the stargazin-SR interaction affects the level of stroke damage remains elusive. We showed that cerebral ischemia increased the interaction of stargazin and SR and decreased the levels of D-serine. Disrupting the stargazin-SR interaction by knocking down stargazin aggravated cerebral ischemic insults. We found that cerebral ischemia decreased the phosphorylation of stargazin at the Thr-321 residue, which was phosphorylated by cAMP-dependent protein kinase A (PKA). Treatment with the PKA inhibitor H89 blocked stargazin T321 phosphorylation, augmented the stargazin-SR interaction, decreased D-serine levels, and alleviated focal cerebral ischemic damage in rats subjected to middle cerebral artery occlusion and reperfusion (MCAO/R). Thus, the stargazin-SR interaction is a promising strategy in the treatment of stroke.


Introduction
Cerebral ischemia is a major cause of human neurological morbidity and mortality, and is associated with a poor prognosis worldwide. Cerebral ischemia is caused by an interrupted blood supply to the brain and can result in severe neurodegeneration and vascular dementia in patients [1].
Glutamate, the main excitatory neurotransmitter of the mammalian central nervous system (CNS), activates α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), N-methyl D-aspartate (NMDA), and kainate (KA) receptors [2]. NMDA receptor (NMDAR) is essentially a receptor-gated calcium ion channel with high permeability to calcium and sodium ions [3]. Immediately following ischemia, glutamate accumulates at synapses, leading to widespread stimulation of NMDARs, causing intracellular calcium overload and neurotoxicity [4][5][6][7]. It is recognized that activation of NMDARs requires the binding of both glutamate and glycine. D-serine is a physiologic coagonist with glutamate at NMDARs and is involved in NMDA receptor-induced neurotoxicity [8,9]. Serine racemase (SR) is an enzyme that converts L-serine to D-serine and its deletion has been reported to protect against cerebral ischemia and excitotoxicity [10]. The enzymatic activity of SR is enhanced by binding to PICK1 (protein interacting with C kinase 1) [11,12] and glutamate receptor-interacting protein [13], which are proteins associated with AMPA receptors. In contrast, SR activity is inhibited by binding to the phospholipid phosphatidylinositol 4,5-bisphosphate [14] and S-nitrosylation, which occurs following activation of NMDARs [15].
Stargazin (γ-2) is a member of the family of related transmembrane AMPA receptor regulatory proteins (TARPs), and it was initially recognized for the important role it plays in AMPA receptor tra cking [16]. SR binds to the C-terminal of stargazin, which promotes membrane localization of SR and inhibits SR catalytic activity. AMPA receptor activation leads to the association separation of SR and stargazin in the membrane, leading to enhanced catalytic activity of SR and increased generation of D-serine [17]. ImageJ 1.49 software (National Institutes of Health, Bethesda, MD, USA). The measurable analysis was carried out by observers who were blind to the experimental groups.

Immuno uorescence Analysis
During the in vivo experiments, the brain tissue samples were xed in 4% paraformaldehyde, embedded in para n, cut into 4-μm sections, and dewaxed immediately before staining. Finally, sections were observed using a uorescence microscope (OLYMPUS BX50/BX-FLA/DP70; Olympus Co., Tokyo, Japan) or a laser scanning confocal microscope (ZEISS LSM 880, Carl Zeiss AG, Oberkochen, Germany). A minimum of six random sections from each group were assessed, and representative results were displayed. We analyzed the relative uorescence intensity using ImageJ software, and the quantitative analysis was performed by observers who were blinded to the experimental groups.
Coimmunoprecipitation Immunoprecipitation was performed as described previously. Brie y, the brains of rats subjected to a sham or MCAO/R procedure were harvested, and the penumbral regions were chilled on ice and brie y washed with ice-cold arti cial cerebrospinal uid. Protein extracts were homogenized in RIPA lysis buffer (P0013; Beyotime, Shanghai, China). Supernatants (500 μg) were then incubated with 2-μg Stargazin antibody or normal rabbit IgG (negative control) for 3 h at 4°C with rotary agitation. The antibody-bound complexes were combined with 20-μL Protein A/G PLUS-Agarose and incubated overnight at 4°C with rotary agitation. Bound proteins were eluted by heating at 100°C in 20 μL 2× loading buffer and analyzed using immunoblotting.

Gas/Liquid Chromatography Analysis
The brain tissue homogenates collected from SD rats were cytolyzed in ultrapure water. The samples were centrifuged at 14,000 × g for 40 min at 4°C. Then, we acquired the supernatant liquid for gas/liquid chromatography analysis. For high-performance liquid chromatography (HPLC), a 2.1 mm*100 mm, 1.9μm C18 chromatographic column (Hypersil Gold; Thermo Fisher Scienti c, Shanghai, China) was used for chromatographic separation, with a column temperature of 25°C. Elution solvents consisted of 100% methanol (mobile phase B) and 5 mmol/L ammonium acetate (mobile phase C). Gradient elution (0-2 min, 90% C; 2-7 min, 90-10% C; 8-10 min, 90% C) was performed at a ow rate of 300 μL/min.

Transduction of Lentivirus
The titer of lentivirus (LV)-Cacng2-RNAi was 3× 10 9 TU/ml. In vivo, penumbra injection of the lentivirus was performed as described previously. We estimated the location of the penumbra before modeling based on previous modeling experiences and references because the infarcted area of MCAO/R modeling was relatively xed. We made a longitudinal cut (from top to bottom) approximately 2 mm from the sagittal suture through the right hemisphere, and then made a transverse diagonal cut at an approximate "2-o'clock" position to separate the core (striatum and overlying cortex) from the penumbra (adjacent cortex). Brie y, 2 weeks before MCAO/R operation, SD rats were anesthetized and xed in a stereotaxic apparatus (ZH-Lanxing B-type stereotaxic frame, AnhuiZhenghua Biological Equipment Co. Ltd., Anhui, China). The three injection sites were in the right hemisphere, and the stereotaxic coordinates were as follows: anteroposterior (AP) + 1.2 (site 1), 0.3 (site 2), −1.8 (site 3); mediolateral (ML) + 5.5; dorsoventral (DV) −3.5 mm from the skull. A 2-μL dose of LV was slowly infused into the penumbra at each injection site at a rate of approximately 0.5 μL/min. To avoid re ux, the needle was kept in place after injection for a further 5 min, withdrawn slightly, and then left in position for another 2 min before complete removal. Brain tissue within 2 mm of the injection site has a greater chance of being transduced with lentivirus, and the period of lentiviral expression in the brain is from 72 h to approximately 1 month after injection.

Modi ed Neurological Severity Score
We used the Modi ed Neurological Severity Score (mNSS) test, performed 24 h after reperfusion, to evaluate the MCAO/R model. The score, graded on a scale of 0-18, re ects motor, sensory, re ex, and balance functions, with higher scores indicating increased severity of neurological damage. The rats were trained and evaluated before surgery to ensure the normal baseline score of 0, and all scores were recorded by investigators who were blinded to experimental group assignment.

TTC Staining
Rats were euthanized 24 h after the MCAO/R procedure. The forebrain was extracted, frozen at −20°C for 10-20 min, and coronally dissected into 2-mm-thick slices beginning from the frontal pole. Then, the slices were immersed in TTC solution (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) at 37°C for 10-20 min. The infarct volume and total volume of each section were calculated using ImageJ software.

Rotarod Test
We used an accelerating rotarod test was used to evaluate the motor function of rats. The diameter of the rotarod spindle was 10 cm. The speed was increased from 10 to 30 rpm over 200 s, and maintained at 30 rpm for a maximum of 300 s. The time was recorded when the rat could not keep up with the speed and failed to stay on the rotarod spindle. Three rats could run simultaneously because there were two panels separating three runways, which prevented the rats from detecting each other. Three days before the test, each rat underwent training three times per day, with the nal three scores recorded as the baseline. After surgery, each rat performed the test trials three times per testing day (3,5,7,14), and the average of the scores was recorded as the nal score for the day.

Adhesive Removal Test
The adhesive removal test was chosen to assess the sensorimotor function of the rats at 3, 5, 7, and 14 days after MCAO/R. As described in previous studies, a rat was taken out and placed in a cage for 2-3 min to familiarize it with the testing environment. An adhesive tape (2 × 3 cm) was attached to the distal region of the left and right forelimbs as a tactile stimulus. The time to contact and remove both stimuli on each forelimb was recorded in three trials per day for 3 days. After the training, any rats that were unable to contact and remove the tapes in 10 s were excluded from the experimental group. Then, all the rats received a test trial on all testing days after MCAO/R or sham surgery. Each animal was tested three times at 2-min intervals, and the nal data are presented as the average time of the three tests.

Morris Water Maze
The Morris water maze test was used to examine spatial cognitive functional de cits. As previously described, a circular pool lled with opaque water was divided into four quadrants. A platform (10-cm diameter) was submerged in the pool (180-cm diameter) and maintained 2 cm under the water surface. The water temperature was maintained at 19-21°C, and the room temperature was maintained at 20-22°C. This test had two parts: a spatial learning test and a memory test. Before sham or MCAO/R surgery, the rats were trained for 3 consecutive days for the acquisition of spatial learning. Then, 22-26 days after MCAO/R, each rat was released from one of the three quadrants (except the one with the platform) and was allowed 60 s to look for the platform. Between swims, the animals were placed on the platform for 15-20 s to help them to gather the surrounding spatial cues. The average latency from three trials per day for 5 consecutive days was recorded as the nal data to re ect the learning abilities of the rats. At 27 days after MCAO/R, the underwater platform was removed, and each rat was allowed to swim in the pool for 60 s from the same starting position. Spatial memory was evaluated by recording the time at which each rat crossed the site where the platform had been previously located. The swimming speed of rat was recorded on each testing day to assess the general locomotor functions of rats.

Statistical Analysis
All data are presented as the mean ± standard error of the mean (SEM). GraphPad Prism 7 was used for all statistical analyses. Differences between groups were determined using unpaired t-tests. Statistical comparisons between groups were performed using one-or two-way analysis of variance (ANOVA), and post hoc least signi cant difference test was used for multiple comparisons. P-values < 0.05 were considered to indicate statistical signi cance.

Results
Decreased D-serine Level, Increased Stargazin-SR Interaction, and Decreased Stargazin pT 321 Level in the Penumbral Tissue after MCAO/R injury To identify SR catalytic activity after MCAO/R, we established an experimental model of MCAO/R in male SD rats and subsequently performed gas/liquid chromatography analysis of brain homogenate samples from penumbral tissue. Compared with the sham group, the D-serine level was decreased at 3 h after MCAO/R and rebounded at 6 h and 24 h ( Fig. 1a and  3.078, P < 0.05). Additionally, we used immuno uorescence labeling to determine the effects of MCAO/R on the stargazin-SR interaction. Our results indicated that the ratio of SR colocalized with stargazin was increased in the penumbral tissues of MCAO/R rats compared with that of sham rats ( Fig. 1c and 1d) (Sham: 1.00 ± 0.67, MCAO/R 3 h: 1.69 ± 0.34; Student's t test, t (2.25) = 10, P < 0.05). The results of the co-immunoprecipitation assays consistently showed that the stargazin-SR interaction was signi cantly higher 3 h after MCAO/R compared with the sham group, providing further evidence for an increase in the stargazin-SR binding in the penumbral tissues of MCAO/R rats ( Fig. 1e and 1f) (Sham: 1.00 ± 0.29, MCAO/R 3 h: 1.51 ± 0.38; Student's t test, t (2.58) = 10, P < 0.05).
It has been established that the binding between stargazin and SR is regulated by phosphorylation of stargazin at Thr-321 [17]. We therefore sought to investigate whether the stargazin pT 321 level changed as a result of stroke damage. Western blotting was subsequently performed to examine the protein levels of stargazin and stargazin pT 321 . Our results showed that MCAO/R 3 h increased the level of stargazin ( Fig. 1g and 1i We next sought to determine whether the increases in stargazin-SR binding regulates ischemic insult. To this end, we used lentivirus-mediated expression of speci c short hairpin RNAs (shRNAs) against stargazin. The transduction e ciency of stargazin lentivirus was examined by uorescence assays in vivo and in vitro ( Fig. 2a and 2b). Then, we veri ed the interfering effects of the stargazin lentivirus using western blotting. Our results showed that compared with the negative control group, the level of stargazin was signi cantly decreased in the LV-ShRNA-STG group ( Fig. 2c and 2d) (Sham: 1.00 ± 0.20, MCAO/R 3 h: 1.60 ± 0.14, MCAO/R 3 h + Ctr ShRNA: 1.64 ± 0.14, MCAO/R 3 h + ShRNA-STG: 1.06 ± 0.18, one-way ANOVA, F (3, 20) = 24.82, P < 0.01). Next, we examined whether the stargazin-SR interaction was affected by stargazin knockdown. The results of uorescent labeling showed a decreased proportion of SR colocalized with stargazin in penumbral-tissue neurons of the stargazin-downregulated rats compared with those of the control rats ( Fig. 2e and 2f) (MCAO/R 3 h + Ctr ShRNA: 1.00 ± 0.20, MCAO/R 3 h + ShRNA-STG: 0.57 ± 0.22, Student's t test, t (3.57) = 10, P < 0.05). Moreover, the results of the coimmunoprecipitation assay ( Fig. 2g and 2h) showed a reduction in the interaction between stargazin and SR in the stargazin-downregulated group compared with that in the negative control group (MCAO/R 3 h + Ctr ShRNA: 1.00 ± 0.06, MCAO/R 3 h + ShRNA-STG: 0.75 ± 0.13; Student's t test, t (4.26) = 10, P < 0.01). We next employed gas/liquid chromatography analysis to con rm the role of the stargazin-SR interaction downregulation in SR activity. Our results showed that the level of D-serine level was increased in the stargazing-downregulated group compared with that in the negative control group (Fig. 2i and 2j) (MCAO/R 3 h + Ctr ShRNA: 1.00 ± 0.08, MCAO/R 3 h + ShRNA-STG: 1.15 ± 0.11; Student's t test, t (2.73) = 10, P < 0.05). These ndings suggest that downregulation of stargazin increased the expression of Dserine, which may be associated with a reduction in the stargazin-SR interaction.

Genetic Knockdown of Stargazin Aggravates Ischemic Neuronal Death and Neurological Function after MCAO/R in Rats
Having determined that SR activity is affected by binding of stargazin to SR, we then explored whether stargazin downregulation affected neurological functions and brain damage in rats subjected to MCAO/R (Fig. 3a). Assessment of cerebral infarction 24 h after MCAO/R using TTC staining showed that total infarction volume in stargazin-knockdown rats was increased compared with the control rats ( Fig. 3b  0.36 ± 0.03; one-way ANOVA, F (2, 15) = 7.592, P < 0.05). Furthermore, mNSS was signi cantly higher in the stargazing-downregulated group compared with the negative control group (Fig. 3d) (3,34) = 80.72, P < 0.0001). To further evaluate the functional-recovery effect of stargazin downregulation in rats subjected to MCAO/R, we used the rotarod test to assess the post-stroke locomotor activity, the adhesive-removal test to evaluate forelimb sensorimotor asymmetries, and the Morris water maze test to examine the spatial learning and memory function. Untreated MCAO/R rats performed signi cantly worse in all three tests compared with the sham-treated rats (Fig. 3e-3k). Moreover, rats treated with shRNA-stargazin showed a signi cant increase in the time spent on the accelerating rotarod compared with the MCAO/R +LV-shRNA-control rats (Fig. 3e) (ShRNA-STG [F (3,196) = 204.00, P < 0.0001], test day [F (4,196) = 124.20, P < 0.0001], with an interaction observed between these two factors [F (12,196) = 16.63, P < 0.0001]). The sensorimotor function was aggravated in MCAO/R + LV-shRNA-stargazin rats compared with that of the MCAO/R+LV-shRNA-control rats, as demonstrated by the signi cantly shortened contact time (Fig. 3f) (ShRNA-STG [F (3,196) = 101.00, P < 0.0001], test day [F (4,196) = 91.86, P < 0.0001], with an interaction observed between these two factors [F (12,196) = 11.12, P < 0.0001]) and removal time (Fig. 3g) (ShRNA-STG (F (3,196) = 140.10, P < 0.0001), test day (F (4,196) = 128.90, P < 0.0001), with an interaction observed between these two factors (F (12,196) = 15.70, P < 0.0001]). The Morris water maze test, performed 22 to 27 days post-MCAO/R (Fig. 3a), showed that stargazin downregulation aggravated spatial learning, as evidenced by the decreased time spent seeking out the underwater hidden platform compared with the time taken by the lentivirus-negative control rats ( Fig. 3h and 3i) (ShRNA-STG [F (3,190) = 29.64, P < 0.0001], test day [F (4,190) = 21.37, P < 0.0001], with an interaction observed between these two factors (F (12,190) = 0.21, P = 1.00]). Unlike the other tests used in our study, the memory test indicated no difference in memory function between the MCAO/R +LV-shRNAstargazin group and the MCAO/R + LV-shRNA-control group. Additionally, the swimming speed was similar among the rats used in our present study (Fig. 3j)  These results indicated that downregulation of stargazin aggravated stroke outcomes, which may be associated with increased expression of D-serine regulated by stargazin-SR interaction.

Discussion
Our study has provided two key ndings. First, MCAO/R increased the stargazin-SR interaction and reduced the D-serine expression in penumbral tissue, which is associated with a low phosphorylation level of stargazin-Thr-321. Second, inhibition or further promotion of stargazin-SR interaction enhanced or attenuated D-serine expression, ischemic neuronal death, and neurobehavioral de cits. Collectively, these results suggested that enhanced stargazin-SR interaction in penumbral tissue in MCAO/R rats served as a protective mechanism in the context of cerebral ischemia/reperfusion injury.
In the past 20 years, the NMDA receptor has become the major focus of studies for treating stroke [20][21][22][23][24]. Activation of NMDA receptors is considered to be a primary cause of excitotoxicity in ischemic insult as a result of its high permeability to Ca 2+ [25]. Opening of the NMDA receptor channels requires the activation of two distinct compulsory co-agonist binding sites, one that binds glutamate and the other that binds glycine or D-serine [26,9]. During brain ischemia, D-serine release could potentiate excitotoxic actions of glutamate acting at NMDA receptors [26]. Degrading endogenous D-serine by D-amino acid oxidase (DAAOX), an enzyme that e ciently degrades extracellular D-serine, protects neurons from damage induced by simulated ischemia [27]. SR deletion mice, in which D-serine levels are reduced by 90%, have diminished neurotoxicity following MCAO [10]. Nevertheless, these results suggest only that endogenous D-serine is required for NMDA receptor-mediated excitotoxicity, but they do not demonstrate that increased D-serine is released during ischemia. In this study, we used gas/liquid chromatography analysis to detect endogenous D-serine levels at multiple time points after MCAO/R. Our data showed that the D-serine levels signi cantly reduced in the 3 h after MCAO/R, and subsequently returned to normal levels 6 and 24 h after MCAO/R. D-serine has a half-life of nearly 17 h, indicating slow metabolism in the brain [28]. The D-serine levels decreased only 3 h after stroke onset in the present study, suggesting the inhibition of SR activity. One of the main questions arising from this study is how SR activity becomes inhibited in response to cerebral ischemic insult. SR catalytic activity is thought to be controlled by multiple mechanisms. For instance, NMDAR activation triggers the production of nitric oxide (NO), which leads to the S-nitrosylation of SR and inhibition of SR activity [15]. SR is physiologically inhibited by PIP2 [14] and binds to stargazin, which also inhibits its activity [17]. In this study, we showed increased expression levels of stargazin and its interaction with SR in the penumbra. We also demonstrated that stargazin becomes dephosphorylated at pT321 in cortical neurons following ischemic insult. Some studies have indicated that the stargazin C terminus contains a sequence for phosphorylation by cAMP-dependent protein kinase A (PKA). Meanwhile, it has been shown that the stargazin C terminus is phosphorylated at the Thr-321 residue [19], and the binding between stargazin and SR is inhibited by phosphorylation of stargazin at Thr-321 [17]. Our results showed that the application of the PKA inhibitor H89 promoted stargazin dephosphorylation at pT321, augmented the stargazin-SR interaction, and inactivated SR. Additionally, H89 protected against ischemic neuronal death and improved neurological function. Indeed, our data suggested that the PKA inhibitor H89 reduced brain infarction and improved neurological functions as a result of dephosphorylation of pT 321 , which allowed stargazin to bind and inactivate SR. However, some studies have found the protective effects of H89 on cerebral ischemia, which might be not related to the inhibition of PKA. It has been reported that H89 may contribute to brain recovery after ischemic stroke by regulating proteins related to neuronal apoptosis and synaptic plasticity [29]. Thus, H89 confers protection from brain damage in many aspects of nervous system function. Conversely, several reports have shown that the effects of some neuroprotective agents are eliminated by H89 intervention [30][31][32]. One explanation for these ndings is that these neuroprotective agents protect neurons against ischemia through the cAMP/PKA/CREB signaling pathways, promote the phosphorylation of p-CREB, and play important anti-apoptotic roles. The mutation of stargazin-Thr-321 to alanine, which mimics dephosphorylation and augments stargazin-SR binding, are needed in the future research because pharmacological methods are nonspeci c.
We next silenced the stargazin gene to determine the effects of separating the stargazin-SR interaction on cerebral ischemic insult. Our data showed that downregulating the expression of stargazin increased MCAO/R insult to cortical neuronal cells and aggravated neurological function; the mechanism driving these events may be associated with the decreased stargazin-SR interaction and the increased expression of D-serine. Stargazin is a 40-kDa neuronal transmembrane protein that controls the surface and synaptic expression of AMPARs [33][34][35][36][37][38]. S-nitrosylation of stargazin increases interaction with the AMPAR subunit GluA1, resulting in increased surface expression of AMPARs [39]. Although we directly demonstrated that stargazin knockdown increased SR activity and reduced stargazin-SR binding, we did not examine the in uence of stargazin knockdown on AMPAR surface expression because the regulation of stargazin to AMPAR surface expression was nNOS dependent [39] and cerebral ischemia reduced nNOS expression [40]. Thus, it would not be possible that increased AMPAR surface expression is mediated by stargazin following cerebral ischemic insult. In addition, stargazin has been reported to bind to PSD95, AP-2, and AP-3A to regulate NMDA-induced AMPA receptor tra cking [19,41]. Thus, directly silencing stargazin can cause severe side effects. Although previous studies have identi ed the rst 66 N-terminal amino acids of SR as critical fragments for stargazin binding, the key binding sites between stargazin and SR remain to be determined [17]. It is possible to develop a small-molecular inhibitor or generate a cell membrane permeable peptide to speci cally disturb the stargazin-SR interaction.
Therefore, further studies are warranted to clarify whether stargazin-SR binding is involved in the various forms of AMPA receptor tra cking.
In summary, our results showed that under ischemic insult, stargazin was dephosphorylated at pT 321 and could bind to SR to inhibit SR activity, resulting in a decrease in D-serine release (Fig 6). Thus, dephosphorylated stargazin at pT 321 can be considered to be a protective mechanism against brain damage in stroke.

Data Availability
Data that support the ndings of this study are available from the corresponding author upon request. D-serine (D-ser) level, stargazin (STG)-serine racemase (SR) interaction, and phosphorylation level of stargazin-Thr-321 (STG-pT321) in the ischemic penumbra of MCAO/R rats. a-b. Gas/liquid chromatography analysis and quanti cation of D-serine in the ischemic penumbra. (*P < 0.05 vs. sham, n = 6 per group). c-d. Double immuno uorescence analysis was performed using antibodies against stargazin (green) and SR (red) in brain sections. Nuclei were uorescently labeled with DAPI (blue).
Western blotting of stargazin and phosphorylation of stargazin-Thr-321 in the ischemic penumbra of sham and MCAO/R rats (**P < 0.01 vs. sham, n = 6 per group). All data are presented as the mean ± SEM.  Effects of H89 on the phosphorylation level of stargazin-Thr-321 (STG-pT321), stargazin (STG) -serine racemase (SR) interaction and D-serine (D-ser) level. a-e. Western blot analysis of phosphorylation level of stargazin-Thr-321 in the ischemic penumbra of MCAO/R + DMSO/H89 rats (*P < 0.05 vs. MCAO/R + DMSO, n = 6 per group). f. Double immuno uorescence analysis was performed with antibodies against stargazin (green) and SR (red) in brain sections. Nuclei were uorescently labeled with DAPI (blue).  Schematic representations of potential mechanisms of stargazin interaction with serine racemase mediated cerebral ischemia/reperfusion injury in the MCAO/R model