Background and Purpose Due to the limitation in treatment window of the rtPA (recombinant tissue plasminogen activator), the development of delayed treatment for stroke is needed. In this study, we examined the efficacy of delayed post-stroke treatment (post 3–8 days) of the sonic hedgehog pathway agonist (SAG) on functional recovery and the underlying mechanisms. Methods We evaluated functional recovery at 1 month after stroke using locomotion analysis and Barnes maze test for cognitive function. We utilized a genetically inducible NSC-specific reporter mouse line (nestin-CreERT2-R26R-YFP) to label and track their proliferation, survival and differentiation in ischemic brain. Brain tissue damage, angiogenesis and cerebral blood flow recovery was evaluated using MRI techniques and immunostaining. Results Our results show that delayed treatment of SAG in stroke mice results in enhanced functional recovery both in locomotor function and cognitive function at one month after stroke. Further, utilizing the nestincreERT2-YFP mice, we showed that post-stroke SAG treatment increased surviving newly born cells derived from both SVZ and SGZ neural stem cells (NSCs), total surviving DCX+ (Doublecortin) neuroblast cells and neurons (NeuN+/YFP+) in the ischemic brain. SAG treatment also improved the brain tissue repair in ischemic region supported by our T2 weighted MRI, Cerebral Blood Flow (CBF) map by Arterial-Spin-Labeling (ASL) and immunohistochemistry (alpha-smooth muscle actin and CD31 immunostaining). Conclusions These data confirm an important role for the hedgehog pathway in post-stroke brain repair and functional recovery, suggesting a prolonged treatment window for potential treatment strategy to modulate shh pathway after stroke.

Recently the sonic hedgehog (shh) signaling pathway has been shown to play an important role in regulating repair and regenerative responses after brain injury, including ischemia. However, the precise cellular components that express and upregulate the shh gene and the cellular components that respond to shh signaling remain to be identified. In this study, using a distal MCA occlusion model, our data show that the shh signal is upregulated both at the cortical area near the injury site and in the adjacent striatum. Multiple cell types upregulate shh signaling in ischemic brain, including neurons, reactive astrocytes and nestin-expressing cells. The shh signaling pathway genes are also expressed in the neural stem cells (NSCs) niche in the subventricular zone (SVZ). Conditional deletion of the shh gene in nestin-expressing cells both at the SVZ niche and at the ischemic site lead to significantly more severe behavioral deficits in these shh iKO mice after cortical stroke, measured using an automated open field locomotion apparatus (Student’s t-test, p<0.05). In contrast, animals given post-stroke treatment with the shh signaling agonist (SAG) demonstrated less deficits in behavioral function, compared to vehicle-treated mice. At 7 days after stroke, SAG-treated mice showed higher values in multiple horizontal movement parameters compared to vehicle treated mice (Student’s t-test, p<0.05) whereas there were no differences in pre-stroke measurements, (Student’s t-test, p>0.05). In summary, our data demonstrate that shh signaling plays critical and ongoing roles in response to ischemic injury and modulation of shh signaling in vivo alters the functional outcome after cortical ischemic injury.

p53, a stress response gene, is involved in diverse cell death pathways and its activation has been implicated in the pathogenesis of Parkinson's disease (PD). However, whether the neuronal p53 protein plays a direct role in regulating dopaminergic (DA) neuronal cell death is unknown. In this study, in contrast to the global inhibition of p53 function by pharmacological inhibitors and in traditional p53 knock-out (KO) mice, we examined the effect of DA specific p53 gene deletion in DAT-p53KO mice. These DAT-p53KO mice did not exhibit apparent changes in the general structure and neuronal density of DA neurons during late development and in aging. However, in DA-p53KO mice treated with the neurotoxin MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), we found that the induction of Bax and PUMA mRNA and protein levels by MPTP were diminished in both striatum and substantia nigra (SN) of these mice. Notably, deletion of the p53 gene in DA neurons significantly reduced dopaminergic neuronal loss in SN, dopaminergic neuronal terminal loss at striatum and, additionally, decreased motor deficits in mice challenged with MPTP. In contrast, there was no difference in astrogliosis between WT and DAT-p53KO mice in response to MPTP treatment. These findings demonstrate a specific contribution of p53 activation in DA neuronal cell death by MPTP challenge. Our results further support the role of programmed cell death mediated by p53 in this animal model of PD and identify Bax, BAD and PUMA genes as downstream targets of p53 in modulating DA neuronal death in the in vivo MPTP-induced PD model.