Activation of cerebral Ras-related C3 botulinum toxin substrate (Rac) 1 promotes post-ischemic stroke functional recovery in aged mice

Abstract

Brain functional impairment after stroke is common; however, the molecular mechanisms of post-stroke recovery remain unclear. It is well-recognized that age is the most important independent predictor of poor outcomes after stroke as older patients show poorer functional outcomes following stroke. Mounting evidence suggests that axonal regeneration and angiogenesis, the major forms of brain plasticity responsible for post-stroke recovery, diminished with advanced age. Previous studies suggest that Ras-related C3 botulinum toxin substrate (Rac) 1 enhances stroke recovery as activation of Rac1 improved behavior recovery in a young mice stroke model. Here, we investigated the role of Rac1 signaling in long-term functional recovery and brain plasticity in an aged (male, 18 to 22 months old C57BL/6J) brain after ischemic stroke. We found that as mice aged, Rac1 expression declined in the brain. Delayed overexpression of Rac1, using lentivirus encoding Rac1 injected day 1 after ischemic stroke, promoted cognitive (assessed using novel object recognition test) and sensorimotor (assessed using adhesive removal tests) recovery on days 14-28. This was accompanied by the increase of neurite and proliferative endothelial cells in the peri-infarct zone assessed by immunostaining. In a reverse approach, pharmacological inhibition of Rac1 by intraperitoneal injection of Rac1 inhibitor NSC23766 for 14 successive days after ischemic stroke worsened the outcome with the reduction of neurite and proliferative endothelial cells. Furthermore, Rac1 inhibition reduced the activation of p21-activated kinase 1, the protein level of brain-derived neurotrophic factor, and increased the protein level of glial fibrillary acidic protein in the ischemic brain on day 28 after stroke. Our work provided insight into the mechanisms behind the diminished plasticity after cerebral ischemia in aged brains and identified Rac1 as a potential therapeutic target for improving functional recovery in the older adults after stroke.

Keywords: Pak1; Rac1; aging; angiogenesis; brain-derived neurotrophic factor (BDNF); cerebral ischemia; cognitive recovery; neurite; sensorimotor recovery; stroke.

Figure 1

Schematic representation of the study design.

Figure 2

The protein level of Ras-related C3 botulinum toxin substrate (Rac) 1 in the cerebral hemisphere of aged mice. The protein level of Ras-related C3 botulinum toxin substrate (Rac) 1 was quantified by Western blotting. Relative protein expression was normalized to β-actin. n = 3/group. Data are presented as the mean ± SEM. *P < 0.05, vs. young mice (Mann-Whitney U test).

Figure 3

The effects of delayed overexpression of cerebral Ras-related C3 botulinum toxin substrate (Rac) 1 on functional recovery after ischemic stroke in aged mice. (A) Immunohistochemical assessment of Rac1 (Alexa Fluor 594 in red) on day 28 after stroke. Blue indicated cell nucleus staining using 4’,6-diamidino-2-phenylindole (DAPI). n = 4/group. *P < 0.05, vs. LV-blank group. (B, C) cognitive function assayed by novel object recognition test (*P < 0.05; B) and sensorimotor function assayed by adhesive removal test (C; ###P < 0.001, pre-stroke vs. 7 days after stroke in control vector group; *P < 0.05, vs. LV-Rac1 group). n = 9/group. Data are presented as the mean ± SEM. For immunohistochemistry, Mann-Whitney U test was used. For behavior outcome, two-way analysis of variance with post hoc Bonferonni test was used. LV-blank: Control vector; LV-Rac1: lentiviral vector encoding Rac1.

Figure 4

The effect of delayed inhibition of Ras-related C3 botulinum toxin substrate 1 on functional recovery after ischemic stroke in aged mice. (A) cognitive function assayed by novel object recognition test (*P < 0.05) and (B) sensorimotor function assayed by adhesive removal test (###P < 0.05, pre-stroke vs. 7 days after stroke in the vehicle control group; *P < 0.05, vs. vehicle control group). n = 10/group, Data are presented as the mean ± SEM. For behavior outcome, two-way analysis of variance with post hoc Bonferonni test was used.

Figure 5

The effect of delayed overexpression of cerebral Ras-related C3 botulinum toxin substrate (Rac) 1 on neurite density, endothelial proliferation, and tissue loss on day 28 after stroke in aged mice. (A) Neurite regrowth was quantified by the intensity of neurofilament (NFL) in immunohistochemistry (Alexa Fluor 594 in red), n = 4/group. (B) Angiogenesis was quantified by the colocalization of bromodeoxyuridine (BrdU) and cluster of differentiation (CD) 105 positive cells in immunohistochemistry (BrdU, Alexa Fluor 594 in red; CD105, Alexa Fluor 488 in green), n = 4/group. Blue indicated cell nucleus staining using 4’,6-diamidino-2-phenylindole (DAPI). (C) Quantification of brain tissue loss was assayed by cresyl violet staining. n = 4/group. Data are presented as the mean ± SEM. *P < 0.05, vs. lentivirus (LV)-blank. Mann-Whitney U test was used for comparison between the two groups. LV-blank: Control vector; LV-Rac1: lentiviral vector encoding Rac1.

Figure 6

The effect of delayed inhibition of Ras-related C3 botulinum toxin substrate 1 on neurite density, endothelial proliferation, and tissue loss on day 28 after stroke in aged mice. (A) Neurite regrowth was quantified by the intensity of neurofilament (NFL) in immunohistochemistry (Alexa Fluor 594 in red), n = 4/group. (B) Angiogenesis was quantified by the colocalization of bromodeoxyuridine (BrdU) and cluster of differentiation (CD) 105 positive cells in immunohistochemistry (BrdU, Alexa Fluor 594 in red; CD105, Alexa Fluor 488 in green), n = 4/group. Blue indicated cell nucleus staining using 4’,6-diamidino-2-phenylindole (DAPI). (C) Quantification of brain tissue loss was assayed by cresyl violet staining. n = 4/group. Data are presented as the mean ± SEM. *P < 0.05, vs. vehicle. Mann-Whitney U test was used for comparison between the two groups.

Figure 7

The effect of delayed inhibition of Ras-related C3 botulinum toxin substrate 1 on the expression of brain-derived neurotrophic factor (BDNF), phosphorylated p21-activated kinase (Pak) 1 (T212), and glial fibrillary acidic protein (GFAP) in the ipsilateral hemisphere on day 28 after stroke. Western blot was performed to quantitatively analyze the relative intensity of interest proteins by normalizing individuals to β-actin for BDNF and GFAP, and normalizing individuals to Pak1 for phosphorylated Pak1 (T212). n = 4/group. Data are presented as the mean ± SEM. *P < 0.05, vs. vehicle. Mann-Whitney U test was applied for comparison between two groups.