Double immunofluorescence staining of Gas and Axl with neuro

Double immunofluorescence staining of Gas6 and Axl with neuronal specific nuclear protein (NeuN), glial fibrillary acidic protein (GFAP), and ionized calcium-binding adaptor molecule 1 (Iba-1), demonstrated that Gas6 and Axl are expressed by neurons, astrocytes and microglia/macrophages (Fig. 2). In both sham and MCAO animals, Gas6 and Axl were equally expressed in the neurons and astrocytes. In sham animals, Iba1 positive cells also localized with both Axl and Gas6, but after MCAO, Iba1 positive cells had increased expression of Gas6 and Axl compared to sham. rGas6 reduced alpha msh infarction and improved neurological function at 24h and 72h after MCAO. Cerebral ischemia/reperfusion leads to severe behavioral deficits and histological damage. MCAO resulted in infarction and neurological deficits at 24 and 72h (Fig. 3). Treatment with the median dose (MD) or high dose (HD) of rGas6 significantly decreased the infarct volume and improved the neurological function scores of rats 24h after MCAO (Fig. 3A, C–E p<0.05 vs. MCAO+Vehicle). MD rGas6 also decreased cerebral infarction and improved neurological function at 72h after injury (Fig. 3B, F–H p<0.05 vs. MCAO+Vehicle). Based on the short-term results indicating that the medium dose rGas6 was effective, we studied the long-term effects and mechanisms of rGas6 by using the MD. When the effects of stroke and treatment were investigated, it was found that MCAO+Vehicle has significantly higher infarct volumes than Sham+Vehicle, as well as greater functional deficits. On the other hand, no difference was observed between the Sham+Gas6 and MCAO+Gas6 groups in neurobehavior, although the MCAO+Gas6 had a significantly higher infarct volume than Sham+Gas6 (Fig. S2). Gas6 improved spatial learning, memory abilities and movement coordination ability 28days after MCAO. Untreated MCAO animals performed significantly worse on the modified Garcia scores, Morris water maze, and rotarod tests compared to sham animals. Both the single MD of rGas6 and the daily doses for three days of MD rGas6 group improved the modified Garcia neurological score compared with the MCAO+vehicle group at all time points (Figs. 4A, B p<0.05 vs. MCAO+Vehicle). No difference was observed between the single and multiple dose rGas6 treatment groups. In the visible platform trial for the Morris Water Maze, there was no difference among the four groups (Fig. 4C p>0.05 vs. Sham). For the hidden platform trials, all groups showed a gradual decrease in the escape latency to reach the hidden platform. The escape latency time was significantly reduced in all treatment groups compared to that of the vehicle group (Fig. 4D p<0.05 vs. MCAO+Vehicle). On the probe trial, the percentage of time spent in the target quadrant was higher in the treatment groups than in the MCAO+vehicle group (Fig. 4E p<0.05 vs. MCAO+Vehicle). Untreated MCAO rats performed worse in the rotarod test compared to sham rats (p<0.05). rGas6 significantly improved motor coordination for both the single and three days of MD rGas6 groups compared with the vehicle group in the 5RPM and 10RPM tests (Fig. 4F p<0.05 vs. MCAO+Vehicle). Western blot analysis showed that administration of rGas6 significantly increased the expression of Axl receptor and STAT1 24h after MCAO (Fig. 5A, B, D p<0.05 vs. MCAO+Vehicle), and enhanced the phosphorylation of Axl and STAT1 (Fig. 5A, C, E p<0.05 vs. MCAO+Vehicle). At the same time, the downstream proteins SOCS1 and SOCS3 increased after rGas6 (Fig. 5A, F, G p<0.05 vs. MCAO+Vehicle). R428, an inhibitor of Axl, effectively suppressed the expression of Axl and its phosphorylation, reducing the levels of STAT1, phosphorylated STAT1, SOCS1, and SOCS3 (Figs. 5A–G p<0.05 vs. MCAO+Vehicle). R428 also reversed the protective effects of Gas6 treatment on NF-κB activity (Fig. S3). R428 in MCAO animals did not have any significant effect on either infarction or behavior score compared to vehicle treated MCAO rats (Fig. S4, p>0.05 vs. MCAO+Vehicle, p<0.05 vs. Sham). Additionally, R428 given to MCAO rats treated with Gas6 did not have any significant change in infarction or behavior (Fig. 6 and S4, p>0.05 vs. MCAO+Vehicle, p<0.05 vs. Sham). Furthermore, knockdown of Axl with siRNA did not significantly reduce infarction or neurobehavior compared to MCAO+Vehicle (Fig. 6, p>0.05 vs. MCAO+Vehicle, p<0.05 vs. Sham).