| Objective: Stroke results in rapid brain damage as well as long-term functional deficits. Pharmacological and cell-based functional recovery for all patients with stroke, with treatment initiated greater than 24 hours after stroke, are therefore under intensive investigation. As a well-known Chinese herbal medicine, Salvia miltiorrhiza has been widely used for the treatment of ischemic disease, and the medicinal properties of this plant have been extensively studied. Salvianolic acids(Sals), a compound isolated from salvia miltiorrhiza, was demonstrated the neuroprotection on cerebral ischemia in the acute phase. However, the effect of delayed treatment with Sals after ischemic stroke and the molecular mechanisms responsible for the beneficial effects are fairly rare.Generation of new neuronal cells in the injured brain, correlated to neurological outcome, is a key component of post-stroke brain remodeling processes. Accumulating evidence revealed that neural progenitor cells(NPCs) are multipotent stem cells that are capable of self-renewal in specific brain regions such as the subventricular zone(SVZ) and dentate gyrus of the hippocampus, and neurogenesis persists throughout adulthood in these regions. Several stimuli such as stroke promotes neurogenesis in the SVZ and newly generated neuroblasts in the SVZ migrate to the ischemic penumbra to replace damaged neurons which contribute to functional recovery. Shh is involved in embryonic development and postnatal repair by regulating cell proliferation, migration and differentiation. The Sonic hedgehog(Shh) signaling pathway begins when the secreted Shh peptide binds to its membrane-bound receptor Ptch, thus relieving its inhibition of Smoothened(Smo). Next, a complex signaling cascade involving the transcription factors of the Gli family is triggered. Gli1 is the principal effector of shh signaling in NPCs and is classically used as a sensitive measure of pathway activation.Several growth and neurotrophic factors promote neurogenesis in adults, and even under ischemic condition, leading to functional recovery. Brain derived neurotrophic factor(BDNF) and Nerve Growth Facto(NGF) are physiologically and pathologically important in the control of survival, proliferation and migration of NPCs in the SVZ. However, little is known about the relationship between the shh signaling pathways and the expressions of BDNF and NGF under stroke.The present study investigated whether delayed treatment with Sals could enhance neurogenesis and promote long-term functional recovery after stroke, and whether the shh pathway is responsible for the beneficial effects.Methods:Unilateral neocortical ischemia was induced in adult male C57BL/6 mice by permanent occlusion of the distal middle cerebral artery(MCAO) and ipsilateral common carotid artery. Mice subjected to MCAO were delayed treated with Sals with or without a pharmacological smoothened inhibitor, cyclopamine(CYC). Sals, as well as CYC, was administered intraperitoneally once a day for 14 days initiated 24 hours after stroke. Neurologic function was evaluated by the rotarod-test and m NSS evaluation weekly after MCAO. Immunofluorescence staining were employed to measure the proliferation, migration and differentiation of NPCs. Protein and gene expressions of neurotrophic factors and shh signaling pathway components were measured at different time points after stroke by western blot and RT-PCR analyses respectively.Results:1 Neurological deficit was examined and scored by the rotarod test and m NSS evaluation weekly. In the ischemic mice, treatment with high dose of Sals starting 24 hours after stroke showed significant functional improvement from week 2 to week 4 after stroke onset compared with vehicle-treated mice(P<0.05). However mice with low dose did not show any functional difference any time points(P>0.05). Mice treated with CYC combined with Sals or CYC alone showed significantly reduced functional scores compared with Sals treated mice(P<0.05), but did not show any functional difference compared with control mice at any time points(P>0.05).2 Infarct volume was measured on hematoxylin and eosin(H&E) stained after 28 days of MCAO. No infarction was observed in the sham operated group. In Sals-treated group, infarct volume was not significantly reduced compared with MCAO group.(Sals vs. MCAO: 11.39%±1.73% vs. 11.60%± 2.07%, P>0.05).3 Sals promoted neuronal proliferation, migration and differentiation in the ipsilateral SVZ through the Shh signaling pathway.Treatment with Sals(30 mg/kg) significantly increased the number of Brd U+ cells in ipsilateral v SVZ and the DCX immunoreactive area in the ipsilateral v SVZ and d SVZ compared with vehicle-treated groups at 7, 14 and 28 days when the treatment was initiated at 24 hours after stroke onset(P<0.05), but no significant difference was observed in the contralateral SVZ among groups. Double immunostaining revealed that DCX positive cells in the SVZ were Brd U positive, indicating neurogenesis. Also Sals treatment caused a significant increase in Brd U/DCX co-labeled cells as compared to control mice at 7, 14 and 28 days in ipsilateral v SVZ(P<0.05), but not observed in the contralateral SVZ. CYC with Sals treatment of mice for 14 days led to an inhibition of proliferation as measured by Brd U incorporation in the v SVZ, the density of DCX positive cells in both v SVZ and d SVZ and Brd U/DCX co-labeled cells in ipsilateral v SVZ as compared with the Sals-treated group(P<0.05), showing that adult SVZ progenitors require shh signaling for cell proliferation.We observed that newly born, immature neurons are present in large numbers in peri-infarct cortex, showing that neural progenitor cells in the SVZ could migrate to the ischemic penumbra to replace damaged neurons. Moreover Sals significantly increased the number of Brd U/Neu N, Brd U/GFAP and Brd U/Iba1 co-labeled cells as compared to the vehicle control, which suggested that Sals induces long-term survival and influence the phenotypic fate of newborn neurons after stroke.Increased dendritic branching peri-infarct cortex was visualized with confocal techniques in mice treated with Sals 28 days after stroke. Our results showed that in all groups, most of the surviving cells were shown to express the immatureneuronal marker β-tubulin III(Tuj-1), and few expressing the mature neuronal markers such as MAP-2 and SMI312. Sals significantly enhenced the coexpression of DCX/MAP-2 as well as DCX/SMI312 compared to the vehicle control.To examine the effect of Sals and CYC on oligodendrogenesis in the ischemic brain, we measured oligodendrocyte progenitor cells(OPCs) and mature oligodendrocytes in the SVZ and white matter. Treated with Sals exhibited a significant increase in the number of NG2+ cells in ipsilateral SVZ compared to the vehicle control group after 28 days(P<0.05), but not observed in the contralateral SVZ. However, coadministration of CYC at some extent blocked the effects of Sals on oligodendrocytes(P<0.05). Double immunostaining revealed that NG2+ cells in the SVZ and corpus callosum were Brd U positive, indicating oligodendrogenesis. Double immunofluorescent staining revealed that Sals significantly increased the percentage of CNPase/Brd U positive cells along the peri-infarct corpus callosum compared with the vehicle group after 28 days(P<0.05).4 Shh signaling pathway mediated the expression of BDNF and NGF after ischemic stroke. Co-expression of Shh/DCX, Shh/Neu N, Shh/MAP-2 and Shh/SMI312 showed that no matter immature or mature neuron could secrete Shh. Wester blot and RT-q PCR revealed that treatment with Sals significantly upregulated the gene expression and the protein expression of Shh, Ptch and Gli1, as well as BDNF and NGF at 7 and 14 days after stroke in ipsilateral cortex rather than contralateral(P<0.05). Double immunostaining of BDNF or NGF with DCX, Tuj-1, Neu N, MAP-2 and SMI312 demonstrated that immature and mature neurons are likely the sources of Sals-induced neurotrophic factors upregulation. The co-treatment with CYC for 7 days abolished the ability of Sals to induce Ptch and Gli1 expression in ipsilateral cortex(P<0.05), but it did not suppress Sals-induced Shh expression(P>0.05). CYC also abolished the ability of Sals-increased protein expression of BDNF and NGF at 7 days(P<0.05).Conclusions:Delayed treatment for Sals markedly improved functional recovery after MCAO in mice. Concurrently, Sals treatment enhanced post-ischemic neurogenesis by facilitating neuronal proliferation, migration and differentiation as well as oligodendrogenesis in the ipsilateral SVZ, and activated Shh signaling pathway. In addition, Sals strongly potentiated both the protein and m RNA levels of BDNF and NGF in the ipsilateral cortex on days 7 and 14 post-MCAO. Furthermore, CYC reversed the enhanced post-ischemic neurogenesis and functional recovery observed after Sals treatment, suggesting that the effects of Sals likely involve activation of Shh signaling pathway and upregulation of trophic factors BDNF and NGF. |
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