To observe the induction of ischemia, LDF recordings were performed and a decrease of LDF during intraluminal MCA thread occlusion and an increase in the LDF value during reperfusion were noted (Fig. 1A). In addition, cresyl violet stained sections were used to observe the ischemic hemisphere (Fig. 1A). According to the repeated measure of ANOVA, there was a significant day effect in the motor coordination (F = 11.617, p < 0.001) and grip strength test (F = 53.499, p < 0.001). Significant reductions in motor force and coordination skills were noticed in animals subjected to 30-min of MCAO. At the third day of ischemia, there was a decline in the grip strength test in both groups compared to pre-ischemic values while there was no significant difference between-groups. In the following days, melatonin treatment ameliorated grip strength of animals producing a significant increase at day 45 compared to the control group (p < 0.05) (Fig. 1B). Motor coordination of animals receiving melatonin increased in the post-ischemic period reaching statistically significant values at day 45 compared to vehicle-treated ischemic animals’ performance approaching to the baseline levels (p < 0.05) (Fig. 1C).
To assess how melatonin affects neuronal survival, immunohistochemistry against the neuronal marker NeuN was analyzed. Following 14 days after induction of ischemia, the percentage of surviving neurons in the ipsilateral hemisphere (IH) to the contralateral hemisphere (CH) continuously declined. After that period, a slow increase in neuronal survival was observed. Notably, this increase in neuronal survival was dramatically higher in mice receiving melatonin at day 55 (p < 0.05) (Fig. 2). The number of TUNEL-positive cells in the ischemic striatum, as revealed by TUNEL staining, was significantly reduced at the 14th day and at the 55th day in melatonin-treated animals compared with vehicle-treated controls according to the unpaired two-tailed t-test (Fig. 3).
The expression of Cdkn1a-p21, and IGF1 genes was significantly increased in the ipsilateral prefrontal cortex but significantly decreased in the contralateral striatum by melatonin treatment at the 3rd day of ischemia. In addition, an increase in the expression of IGF1 in the ipsilateral striatum and NGF in the contralateral striatum was observed that 55th day. At the 14th day, melatonin administration significantly reduced the expression of Nrp1 and IGF1 in ipsilateral striatum but increased IGF1expression levels in the contralateral prefrontal cortex. The observed change in the expression of the Cdkn1a gene at the 3rd day was also observed at the 14th day of ischemia. Indeed, a higher expression level was observed for the PTEN gene in both the contralateral and ipsilateral striatum at the 14th day of ischemia. At the 30th day of ischemia, melatonin treatment significantly reduced the expression of c-jun in both the cortex and striatum of the IH. In the CH, only the expression of IGF1 in the striatum was noted upon melatonin consumption at the 30th day of ischemia. Interestingly, the expression of the genes related to cell survival (Nrp1, NGF, c-jun, PTEN, and Casp3) significantly increased in the contralateral striatum, while their expression except that of Casp3, significantly decreased in the contralateral prefrontal cortex with melatonin administration at the 55th day of ischemia. In addition, at the 55th day, the expression of c-jun and PTEN significantly decreased in the ipsilateral striatum (Fig. 4).
The concentrations of proteins related to cell survival (AKT/ERK/JNK pathway) were significantly altered with melatonin therapy (Fig. 5A). There was a day (F = 670.24, p ≤ 0.001), treatment (F = 24.34, p ≤ 0.001), and brain hemisphere (IH vs CH) (F = 14.26, p = 0.001) effect in the phosphorylation of AKT. The post-hoc test showed that the ratio of phosphorylated AKT to total AKT in the CH was significantly downregulated at day 3 (p ≤ 0.001). However, at day 55, melatonin treatment significantly increased the pAKT/AKT ratio in the CH compared to control animals (p = 0.044) (Fig. 5B). In addition, there was a day (F = 47.47, p ≤ 0.001), and brain hemisphere (IH vs CH) (F = 185.47, p < 0.001) effect in the phosphorylation of ERK-1. However, the treatment effect was insignificant (F = 0.30, p = 0.590). ERK-1 phosphorylation was upregulated at day 30 and downregulated at day 3 in the CH upon melatonin administration (Fig. 5C). On the other hand, a significant day (F = 52.91, p < 0.001), brain region (F = 353.31, p < 0.001) and treatment effect (F = 60.49, p < 0.001) were observed on the phosphorylation of ERK-2. In the CH, a significant decrease in the ERK2 phosphorylation due to melatonin administration was observed at the 3rd and 30th days (Fig. 5D). In addition, JNK phosphorylation was also affected by recovery time (F = 108.08, p < 0.001 for JNK-1 and F = 26.59, p < 0.001 for JNK-2) and melatonin administration (F = 5.67, p = 0.023 for JNK-1 and F = 6.09, p = 0.019 for JNK-2). While there was no region effect in the JNK-1 expression, the region effect on the JNK-2 expression was significant (F = 41.46, p < 0.001). The expression of phosphorylated JNK-1 was significantly higher upon melatonin administration both in the IH and CH at the 14th day compared to control subjects (Fig. 5E). In the CH, the expression of phosphorylated JNK-1 was significantly decreased at both the 3rd and 55th days (Fig. 5E). Furthermore, the phosphorylation of JNK-2 was significantly upregulated at the 14th and 30th days in the IH and was significantly downregulated at the 3rd, 14th, and 55th days in the CH (Fig. 5F).
Melatonin treatment affected the cell cycle regulators to control cell proliferation and cellular transcription factors (Fig. 6A). Melatonin administration significantly reduced the expression of phosphorylated p38 at days 3, 14, and 30 in both IH and CH (Fig. 6B). Furthermore, in the IH, p53 expression was reduced at the 3rd and 14th days upon melatonin treatment. At days 30 and 55, p53 significantly increased in the melatonin-administered mice. In the CH, p53 expression was significantly lower at the 3rd day (Fig. 6C).
According to 3-way ANOVA, there was a significant day (F = 54.13, p < 0.001, F = 63.01, p < 0.001, respectively), region (F = 8.82, p = 0.006, F = 40.66, p < 0.001, respectively), and treatment (F = 11.07, p = 0.002, F = 73.81, p < 0.001, respectively) effects on the expression of CREB and Atf-1 proteins. These blots revealed that CREB protein was significantly decreased at the 3rd day and increased at the 30th and 55th days in the IH. In the CH, the concentration of CREB was significantly lower at days 3, 14 and 30 and significantly higher at the 55th day in melatonin-treated mice (Fig. 6D). Atf-1 expression in the melatonin group was significantly reduced at the 3rd day in the CH and significantly increased at the 30th and 55th days in the IH and at the 14th and 55th days in the CH (Fig. 6E).