Blood Perfusion Assessment and Mortality Analysis Following UCCAo in Male and Female Mice

The rationale for conducting LDI was to confirm that UCCAo resulted in cerebral hypoperfusion after the occlusion. Blood flux measurements were recorded at three time points: before occlusion, 1 h after occlusion and 14–16 weeks after occlusion (Fig. 3). LDI at the pre-occlusion stage provided baseline measurements of cerebral blood flow. After rUCCAo, notable reduction in blood perfusion was observed at 1-h post-occlusion in both male and female animals, confirming the successful induction of cerebral hypoperfusion. Importantly, this reduced blood flux persisted even at 14–16 weeks post-occlusion, further validating the development of CCH. In the BCCAo and PC group, mortality rates were higher in males. Notably, PC reduced mortality in both male and female animals compared to the respective day-specific BCCAo group. The higher mortality observed in the 7-day post-surgery cohorts reflects cumulative deaths recorded up to day 7, rather than mortality confined to a single time point.

Fig. 3

Blood perfusion and mortality rates following UCCAo in male and female mice. A Blood perfusion in the right common carotid artery before UCCAo as measured by LDI. B Blood perfusion 1 h post-UCCAo showing a marked decrease in perfusion in both male and female animals. C Blood perfusion at 14–16 weeks post-UCCAo indicating induction of CCH. D-i and D-ii Graphical representation of blood flux over the three time points (pre-occlusion, 1-h post-occlusion and 14–16 weeks post-occlusion) for males and females, respectively. E-i and E-ii Mortality rates in males and females, respectively. Mortality rates were higher in the BCCAo group compared to the PC in both sexes

Neurodeficit Score Analysis in Male and Female Mice Groups Following UCCAo and PC

On day 1, one-way ANOVA in males (Fig. 4A) revealed a significant difference among the groups [F(2, 21) = 35.61, p < 0.0001]. Bonferroni's post hoc comparisons demonstrated that the BCCAo had significantly higher NDS compared to both the sham (mean difference =  − 1.500, p < 0.0001) and PC group (p < 0.0001). No significant difference was observed between the sham and PC groups (p = 0.1666). On day 7 in males (Fig. 4C), Kruskal–Wallis test was performed which revealed significant differences in median NDS among the groups [H(2) = 14.52, p = 0.0007]. Dunn's post hoc test showed that the BCCAo group exhibited significantly higher NDS compared to the sham group (p = 0.0005). However, there were no significant differences between the sham and PC groups (p = 0.0773) or between the BCCAo and PC groups (p = 0.3558).

Fig. 4

Neurological-deficit score (NDS) across sham, BCCAo, and PC groups in male and female mice. A Day 1 NDS in male revealed significantly higher NDS in the BCCAo group compared to sham (p < 0.0001) and PC (p < 0.0001). B Day 1 NDS in female mice showed significantly higher NDS in the BCCAo group compared to sham (p < 0.0001) and PC (p = 0.0160). C Day 7 NDS in male mice revealed significantly higher NDS in the BCCAo group compared to sham (p = 0.0005). D Day 7 NDS in female mice showed significantly higher NDS in the BCCAo group compared to sham (p = 0.0040) and PC (p = 0.0123). (*) Indicates significant differences between the sham and the BCCAo or PC group; (#) Represents significant differences between the BCCAo and the PC group. Data represent the mean ± SEM, with n = 8 to 10 per group

On day 1 in females (Fig. 4B), one-way ANOVA revealed a significant difference among the groups [F(2, 24) = 13.59, p = 0.0001]. Post hoc Bonferroni analysis indicated that NDS was significantly higher in the BCCAo compared to the sham group (p < 0.0001) and the PC group (p = 0.0160). No significant difference was observed between the sham and PC groups (p = 0.1333). On day 7, in females (Fig. 4D), the Kruskal–Wallis test revealed significant differences in median NDS among the groups [H(2) = 12.00, p = 0.0025]. Dunn's post hoc test showed that the BCCAo group exhibited significantly higher NDS compared to the sham (p = 0.0040) and PC group (p = 0.0123). There was no significant difference between the sham and PC groups (p > 0.9999).

Assessment of Motor Coordination and Grip Strength Reveals Differential Recovery Following BCCAo and PC in Male and Female Mice

A two-way ANOVA was performed to analyze grip strength measurements in males (Fig. 5A) which revealed significant effects of group [F(1, 42) = 15.64, p = 0.0003], day [F(2, 42) = 22.17, p < 0.0001] and interaction between group and day [F(2, 42) = 4.510, p = 0.0168]. On day 1 in males, post hoc Bonferroni analysis showed that grip strength was significantly reduced in BCCAo compared to sham (p < 0.0001) and PC (p = 0.0019). Grip strength in PC was significantly reduced than in sham (p = 0.0096). On day 7 in males, grip strength remained significantly reduced in BCCAo compared to sham (p = 0.0416). No significant differences were observed between sham and PC (p > 0.9999) or BCCAo and PC (p = 0.2826). In females (Fig. 5B), a two-way ANOVA revealed significant effects of group [F(1, 50) = 12.75, p = 0.0008], day [F(2, 50) = 21.27, p < 0.0001], and interaction between group and day [F(2, 50) = 4.500, p = 0.0160). On day 1 females, post hoc Bonferroni analysis showed that grip strength was significantly reduced in BCCAo compared to sham (p < 0.0001) and PC (p = 0.0012). A significant reduction was also observed in sham vs. PC (p = 0.0223). On day 7 in females, grip strength measurements remained significantly reduced in BCCAo compared to sham (p = 0.0498); however, no significant differences were found between sham and PC (p > 0.9999) or BCCAo and PC (p = 0.1241).

Fig. 5

Grip strength and rotarod performance in male and female mice across sham, BCCAo, and preconditioning (PC) groups on days 1 and 7. A On day 1, grip strength in male mice showed significant reductions in BCCAo compared to sham (p < 0.0001) and PC (p = 0.0019), with PC lower than sham (p = 0.0096). On day 7, grip strength remained significantly lower in BCCAo compared to sham (p = 0.0416). B In females on day 1, grip strength was significantly reduced in BCCAo compared to sham (p < 0.0001) and PC (p = 0.0012). PC group showed lower grip strength measurement compared to sham (p = 0.0223). On day 7, grip strength in BCCAo remained lower than sham (p = 0.0498). C Rotarod performance in males on day 1 was significantly reduced in BCCAo compared to sham (p < 0.0001) and PC (p = 0.0014), with PC higher than BCCAo (p < 0.0001). On day 7, BCCAo had significantly lower latency to fall than sham (p = 0.0026). D In females, rotarod performance on day 1 showed reductions in BCCAo compared to sham (p = 0.0018), with PC higher than BCCAo (p = 0.0333). 7-day group showed a significant decrease in latency to fall in BCCAo vs. sham (p = 0.0183). Latency was significantly higher in PC compared to BCCAo (p = 0.0420). (*) Indicates significant differences between the sham and the BCCAo or PC group; (#) represents significant differences between the BCCAo and the PC group. Data represent the mean ± SEM, with n = 7 to 10 per group

Rotarod performance in male mice (Fig. 5C) was analyzed using two-way ANOVA which showed a significant interaction between group and day [F(2, 40) = 10.71, p = 0.0002], a significant group effect [F(1, 40) = 9.638, p = 0.0035] and a significant day effect [F(2, 40) = 44.37, p < 0.0001]. On day 1, post hoc Bonferroni tests indicated significant decrease in latency to fall in BCCAo (mean difference = 105.0, p < 0.0001) as well as in PC (p = 0.0014) when compared to sham. It was significantly higher in PC when compared to BCCAo (mean difference =  − 64.27, p < 0.0001). Post hoc Bonferroni tests on day 7 showed significantly decreased latency to fall in BCCAo when compared to sham (p = 0.0026), but no significant differences between sham and PC (p = 0.1188) or BCCAo and PC (p > 0.4443). Two-way ANOVA in female mice (Fig. 5D) revealed significant interaction [F(2, 46) = 0.1896, p = 0.8279], group effect [F(1, 46) = 0.3993, p = 0.5306], and day effect [F(2, 46) = 11.97, p < 0.0001]. Bonferroni’s post hoc tests on day 1 female showed significant decrease in BCCAo (p = 0.0018) compared to sham. Further, significant increase in PC (p = 0.0333) was observed compared to BCCAo. However, there were no significant differences between sham and PC (p > 0.9442). Post hoc Bonferroni tests in 7-day group indicated significant decrease in latency to fall in BCCAo compared to sham (p = 0.0183), but no significant differences were observed between Sham and PC (p > 0.9999). Significant increase was observed in PC as compared to BCCAo (p = 0.0420).

Sex-Specific Behavioral Alterations in Open Field, Novel Object Recognition, and Y-Maze Tests Following Preconditioning and BCCAo

OFT, NORT, and Y-maze were conducted on day 7. In OFT, differential effects were observed in both male (Fig. 6A, C) and female mice (Fig. 6B, D). The male mice did not show significant differences in distance traversed across the groups [F(2, 18) = 1.830, p = 0.1891]. Post hoc tests revealed no significant differences between sham, BCCAo, and PC groups (p > 0.05 for all comparisons). Female mice demonstrated significant differences in the distance traversed, as indicated by one-way ANOVA results [F(2, 19) = 9.095, p = 0.0017]. Post hoc Bonferroni analysis revealed a significant reduction in distance traveled in the BCCAo group compared to sham (p = 0.0015) and (PC) group (p = 0.0303). No significant difference was observed between the sham and PC groups (p = 0.6927). In terms of mean velocity, male mice did not show significant differences [F(2, 18) = 0.9502, p = 0.4052]. In contrast, female mice displayed significant differences between the groups [F(2, 25) = 6.633, p = 0.0049]. Post hoc Bonferroni analysis showed a significant reduction in mean velocity in the BCCAo group compared to sham (p = 0.0039), while no significant difference was observed between sham and PC (p = 0.1446) or BCCAo and PC (p = 0.4366).

Fig. 6

Motor and cognitive behavior assessments following BCCAo and preconditioning. A OFT male distance moved: no significant differences were observed between sham, BCCAo, and PC groups. B OFT female distance moved: the BCCAo group showed reduced distance compared to sham (p = 0.0015) and PC (p = 0.0303). C OFT male mean velocity: no significant differences were observed across groups. D OFT female mean velocity: the BCCAo group exhibited lower velocity compared to sham (p = 0.0039). E NORT male DI: the BCCAo group had reduced DI compared to sham (p = 0.0015) and PC (p = 0.0300). F NORT female DI: the BCCAo group showed reduced DI compared to sham (p = 0.0073). G Y-Maze male % alternation: no significant differences were observed. H Y-Maze female % alternation: no significant differences were observed. I Y-Maze male duration in novel arm: the BCCAo group spent less time in the novel arm compared to sham (p = 0.0045) and PC (p = 0.0478). J Y-Maze female duration in novel arm: the BCCAo group spent less time in the novel arm compared to sham (p = 0.0059). K Y-Maze male frequency to novel arm: the BCCAo group entered the novel arm less frequently than sham (p = 0.0051). PC showed fewer entries compared to sham (p = 0.0430). L Y-Maze female frequency to novel arm: the BCCAo group entered the novel arm less frequently than sham (p = 0.0125), while PC showed higher entries compared to BCCAo (p = 0.0028). (*) Indicates significant differences between the sham and the BCCAo or PC group; (#) represents significant differences between the BCCAo and the PC group. Data represent the mean ± SEM, with n = 7 to 10 per group

In NORT, one-way ANOVA revealed significant differences in the discriminative index (DI) in both male (Fig. 6E) and female mice (Fig. 6F). Male mice showed significant differences in DI [F(2, 19) = 9.137, p = 0.0017]. Post hoc Bonferroni's test revealed a significant decrease in the DI of the BCCAo group compared to sham (p = 0.0015) and PC (p = 0.0300). The PC group did not differ significantly from sham (p = 0.6864). Female mice also exhibited significant differences in DI [F(2, 23) = 5.807, p = 0.0091]. Bonferroni's post hoc analysis revealed a significant decrease in the DI of the BCCAo group compared to sham (p = 0.0073). However, there was no significant difference between sham and PC (p = 0.3891), nor between BCCAo and PC groups (p = 0.1573).

In Y-maze test in males (Fig. 6G), the one-way ANOVA showed no differences in % alternation [F(2, 20) = 1.583, p = 0.2301] among groups. Similarly in females, no significant differences were observed [Fig. 6H, F(2, 23) = 0.8857, p = 0.4260]. In Novel Arm test in males (Fig. 6I), the BCCAo mice spent significantly less time in the novel arm compared to sham group (p = 0.0045) as well as to the PC group (p = 0.0478). No change was observed between sham and PC (p = 0.9534). In females (Fig. 6J), the BCCAo group spent significantly less time in the novel arm compared to the sham group (p = 0.0059), while no differences were found between the sham and PC (p = 0.4238), or between the BCCAo and PC groups (p = 0.1731). In terms of the frequency of entries into the novel arm, male BCCAo mice (Fig. 6K) showed significantly fewer entries compared to the sham group (p = 0.0051). PC group showed significantly less frequency compared to sham group (p = 0.0430). No differences were observed between BCCAo and PC (p > 0.9999) while in female mice (Fig. 6L) in the BCCAo group significantly, fewer entries were observed compared to the sham group (p = 0.0125) as well as to PC (p = 0.0028). No differences were observed between sham and PC (p > 0.9999).

Differential Glial Activation in the Striatum Following Ischemic Preconditioning

To investigate the effects of IPC on astrocytic and microglial activation, we performed immunofluorescence analysis of GFAP (Fig. 7) and IBA1 (Fig. 8) expression in the striatum.

Fig. 7

GFAP immunostaining in the striatum. Representative images showing astrocytic activation (GFAP) in the striatum of male (A) and female (B) mice. Images were captured at × 60 magnification using an Olympus FV 10i confocal microscope. BCCAo group exhibits significantly increased astrocytic activation in both males and females. The preconditioned (PC) group shows moderately elevated GFAP expression compared to the sham group, with significantly lower number of GFAP-positive cells than the BCCAo group, indicating reduced astrocytic activation. Statistical analysis revealed significant differences between the sham and BCCAo groups (p = 0.0084, males; p = 0.0008, females) and between the BCCAo and PC groups (p = 0.0316, males; p = 0.0049, females). (*) Indicates significant differences between the sham and the BCCAo or PC group; (#) represents significant differences between the BCCAo and the PC group. Data represent the mean ± SEM, with n = 3 per group

Fig. 8

IBA1 immunostaining in the striatum. Representative images showing microglial activation (IBA1) in the striatum of male and female mice. Images were captured at × 60 magnification using an Olympus FV 10i confocal microscope. The sham group shows resting microglia while the BCCAo group exhibits significantly increased microglial activation, with amoeboid morphology in both males and females. The preconditioned (PC) group displays an intermediate morphology, transitioning toward a more ramified phenotype. Statistical analysis revealed significant differences between the sham and BCCAo groups (p = 0.0003, males; p = 0.0131, females) and between the BCCAo and PC groups (p = 0.0059, males; p = 0.0966, females). In males, PC showed significantly higher expression than sham (p = 0.0335). (*) Indicates significant differences between the sham and the BCCAo or PC group; (#) represents significant differences between the BCCAo and the PC group. Data represent the mean ± SEM, with n = 3 per group

Reactive Astrocytes Exhibit Reduced Hypertrophy in Preconditioned State

In males, a one-way ANOVA revealed a significant difference in number of GFAP-positive cells between groups [F(2, 6) = 12.85, p = 0.0068]. The BCCAo group exhibited significantly higher GFAP-positive cells compared to the sham group (p = 0.0084). The difference between PC and BCCAo was statistically significant (p = 0.0316) indicating reduced GFAP in PC. In females, one-way ANOVA revealed a significant difference in number of GFAP-positive cells between groups [F(2, 6) = 30.31, p = 0.0007]. The BCCAo group exhibited significantly higher number of GFAP-positive cells compared to the sham group (p = 0.0008). In the PC group, GFAP expression was moderately elevated compared to the sham group though not reaching significance, but significantly lower than in the BCCAo group suggesting reduced astrocytic activation (p = 0.0049).

Microglial Activation is Dampened in Preconditioned Male Mice

In males, a one-way ANOVA (F(2, 6) = 39.52, p = 0.0004) revealed that BCCAo exhibited significantly increased IBA1 expression compared to sham (p = 0.0003). PC exhibited significantly reduced IBA1 expression compared to BCCAo (p = 0.0059). However, when compared with sham, PC showed significantly higher (p = 0.0335) expression. In females (F(2, 6) = 10.06, p = 0.0121), a one-way ANOVA revealed significant differences in number of IBA1-positive cells between groups. The BCCAo group exhibited increased IBA1-positive cells compared to the sham group (p = 0.0131). While there was a trend toward reduced microglial activation in PC compared to the BCCAo group, it was, however, not statistically significant (p = 0.0966). Furthermore, no significant difference was observed between sham and PC (p = 0.4406).

Gene expression analysis reveals sex-divergent transcriptional responses to CCH-induced preconditioning

One-way ANOVA was performed followed by post-hoc Bonferroni corrections for statistical analysis of gene expression studies. In males, Hif1a levels (Fig. 9A) showed a significant increase in the BCCAo group compared to the sham group (adjusted p-value < 0.0001); however, no significant difference was observed between the BCCAo and PC groups (p = 0.9770). Significant increase was observed in PC compared to sham groups (p =  < 0.0001). In females, BCCAo also exhibited significantly elevated Hif1a levels (Fig. 9B) compared to sham (adjusted p value = 0.0378). PC did not significantly differ from either sham (p = 0.6615) or BCCAo (p = 0.4228) in females, suggesting potential sex-dependent differences in ischemic adaptation and preconditioning efficacy. In males, Becn1 levels (Fig. 9C) were significantly higher in the PC compared to sham (adjusted p-value = 0.0148), but no significant difference was observed between sham and BCCAo (p = 0.0988). This suggests that PC specifically enhances autophagic activity. No significant difference was observed between BCCAo and PC (p > 0.9999). In females Becn1 (Fig. 9D) expression was significantly elevated in both BCCAo and PC groups compared to sham (adjusted p values = 0.0141 and < 0.0001, respectively); however, this change was more pronounced in PC. The BCCAo vs. PC comparison could not approach significance (p = 0.0548), yet implying a trend toward increased autophagy in PC females.

Fig. 9

Sex-specific expression patterns of Hif1a, Becn1, Il1b, and Sox2 in sham, BCCAo, and PC. In males, Hif1a levels (A) were significantly elevated in the BCCAo group compared to sham (adjusted p-value < 0.0001), while PC showed significantly higher levels compared to sham (p < 0.0001). In females (B), BCCAo exhibited significantly elevated Hif1a levels compared to sham (p = 0.0378). For Becn1, males (C) showed significantly higher expression in PC versus sham (adjusted p-value = 0.0148), with no differences between other groups. In females (D), Becn1 expression was significantly elevated in both BCCAo and PC compared to sham (p = 0.0141 and < 0.0001, respectively). Male Il1b levels (E) were significantly elevated in BCCAo compared to sham (p = 0.0116). Similarly, in females (F), Il1b levels were significantly higher in BCCAo compared to sham (p = 0.0382). For Sox2, males (G) showed significantly reduced expression in BCCAo versus sham (p = 0.0147), significantly elevated expression in PC versus BCCAo (p = 0.0002). No significant differences in Sox2 expression were observed among female groups (H). Data represent the mean ± SEM, with n = 6–8 per group. In males, Il6 levels (I) were significantly elevated in the BCCAo group compared to sham (adjusted p-value = 0.0003), while PC remained significantly higher than sham (p = 0.0271). In females (J), BCCAo exhibited significantly elevated Il6 levels compared to sham (p < 0.0001), with PC also showing significantly higher levels than sham (p = 0.0004). (*) Indicates significant differences between the sham and the BCCAo or PC group; (#) represents significant differences between the BCCAo and the PC group. Data represent the mean ± SEM, with n = 6 to 8 per group

Male Il1b levels (Fig. 9E) were significantly elevated in the BCCAo group compared to sham (adjusted p value = 0.0116). PC exhibited no significant difference when compared to sham (p = 0.1739) or to BCCAo (p = 0.6706). In females, Il1b levels (Fig. 9F) were significantly higher in BCCAo compared to sham (adjusted p value = 0.0382) with PC showing no significant difference (p = 0.793). In males, Sox2 (Fig. 9G) expression was significantly lower in BCCAo compared to sham (adjusted p value = 0.0147). Sox2 expression was significantly elevated in PC compared to BCCAo (adjusted p value = 0.0002), while no significant differences were observed between sham and PC (p = 0.1848). No notable variations in Sox2 expression were observed in females (Fig. 9H) between the different groups. In males, Il6 levels (Fig. 9I) were significantly elevated in the BCCAo group compared to sham (adjusted p value = 0.0003). PC exhibited no significant difference when compared to BCCAo (p = 0.2285) but remained significantly higher than sham (p = 0.0271). In females, Il6 levels (Fig. 9J) were significantly higher in BCCAo compared to sham (adjusted p value < 0.0001), with PC also showing significantly higher levels than sham (p = 0.0004) but no significant difference from BCCAo (p > 0.9999).

Preconditioning Mediates Translational Regulation of Synaptic Strengthening and Neurotrophic Responses

PSD-95, synaptophysin and BDNF are critical markers of synaptic health, neuronal plasticity, and neuroprotection. By comparing the levels of striatal proteins between the BCCAo and PC groups at day 1 post-surgery, we aimed to assess the neuroprotective and synaptic preservation effects of preconditioning. For SYN, BDNF, and PSD-95 expression levels, statistical analyses revealed significant differences between the PC and BCCAo groups. Males exhibited a highly significant increase in BDNF levels (Fig. 10A) in the PC group compared to BCCAo (p < 0.0001). Females (Fig. 10B) also showed a significant increase (p = 0.0425) in BDNF levels. In males (Fig. 10C) a significant increase was observed (p = 0.0101) in SYN, whereas in females (Fig. 10D) the difference was not significant (p = 0.8178). Similarly, PSD-95 levels (Fig. 10E, F) were significantly elevated in both males (p = 0.0002) and females (p < 0.0001).

Fig. 10

Western blot analysis of striatal protein expression in males and females following PC and BCCAo. A In males significant increase in BDNF expression in the PC group was observed compared to BCCAo (p < 0.0001). B In females significant increase in BDNF expression in the PC group compared to BCCAo (p = 0.0425) was observed. C SYN in males significantly increased in the PC group compared to BCCAo (p = 0.0101). D SYN female: no significant difference in SYN expression between PC and BCCAo groups (p = 0.8178). E PSD-95 in males was increased significantly in the PC group compared to BCCAo (p = 0.0002) while in females (F) as well PSD-95 expression in the PC group increased compared to BCCAo (p < 0.0001). Data are presented as mean ± SEM. Data represent the mean ± SEM, with n = 6–7 per group

CCH Preconditioning Enhances VEGF Expression

The serum VEGF levels were significantly elevated in the PC group compared to the sham and BCCAo groups at day 1 post-surgery in both male and female cohorts, indicating an early response to preconditioning. In males (Fig. 11A) at day 1 VEGF levels in the PC group was significantly higher than both sham (p = 0.0003) and BCCAo (p = 0.0152). By day 7, VEGF levels in the BCCAo group were significantly elevated compared to sham (p = 0.0026) and the PC group also showed an increase relative to sham (p = 0.0122) indicating a sustained response. In females (Fig. 11B), VEGF levels in the PC group at day 1 were significantly higher than in both the sham (p < 0.0001) and BCCAo (p = 0.0035) groups, whereas no significant difference was observed between sham and BCCAo groups. By day 7, VEGF levels in the BCCAo group were significantly elevated compared to sham (p = 0.0184), while the PC group levels remained comparable to sham suggesting normalization in preconditioned females.

Fig. 11

Serum VEGF levels in males (A) and females (B) show significant time and group-specific changes. In males (A), on day 1, VEGF levels in the PC group were significantly higher than those in the sham (p = 0.0003) and BCCAo (p = 0.0152) groups. By day 7, VEGF levels in the BCCAo group showed a significant increase compared to sham (p = 0.0026), while the PC group also exhibited elevated levels relative to sham (p = 0.0122). In females (B), on day 1, the PC group displayed significantly elevated VEGF levels compared to both sham (p < 0.0001) and BCCAo (p = 0.0035). By day 7, VEGF levels in the BCCAo group were significantly higher than sham (p = 0.0184). (*) Indicates significant differences between the sham and the BCCAo or PC group; (#) represents significant differences between the BCCAo and the PC group. Data represent the mean ± SEM, with n = 7 per group