Imatinib inhibits pericyte-fibroblast transition and inflammation and promotes axon regeneration by blocking the PDGF-BB/PDGFRß pathway in spinal cord injury.

BACKGROUND: Fibrotic scar formation and inflammation are characteristic pathologies of spinal cord injury (SCI) in the injured core, which has been widely regarded as the main barrier to axonal regeneration resulting in permanent functional recovery failure. Pericytes were shown to be the main source of fibroblasts that form fibrotic scar. However, the mechanism of pericyte-fibroblast transition after SCI remains elusive. METHODS: Fibrotic scarring and microvessels were assessed using immunofluorescence staining after establishing a crush SCI model. To study the process of pericyte-fibroblast transition, we analyzed pericyte marker and fibroblast marker expression using immunofluorescence. The distribution and cellular origin of platelet-derived growth factor (PDGF)-BB were examined with immunofluorescence. Pericyte-fibroblast transition was detected with immunohistochemistry and Western blot assays after PDGF-BB knockdown and blocking PDGF-BB/PDGFRß signaling in vitro. Intrathecal injection of imatinib was used to selectively inhibit PDGF-BB/PDGFRß signaling. The Basso mouse scale score and footprint analysis were performed to assess functional recovery. Subsequently, axonal regeneration, fibrotic scarring, fibroblast population, proliferation and apoptosis of PDGFRß+ cells, microvessel leakage, and the inflammatory response were assessed with immunofluorescence. RESULTS: PDGFRß+ pericytes detached from the blood vessel wall and transitioned into fibroblasts to form fibrotic scar after SCI. PDGF-BB was mainly distributed in the periphery of the injured core, and microvascular endothelial cells were one of the sources of PDGF-BB in the acute phase. Microvascular endothelial cells induced pericyte-fibroblast transition through the PDGF-BB/PDGFRß signaling pathway in vitro. Pharmacologically blocking the PDGF-BB/PDGFRß pathway promoted motor function recovery and axonal regeneration and inhibited fibrotic scar formation. After fibrotic scar formation, blocking the PDGFRß receptor inhibited proliferation and promoted apoptosis of PDGFRß+ cells. Imatinib did not alter pericyte coverage on microvessels, while microvessel leakage and inflammation were significantly decreased after imatinib treatment. CONCLUSIONS: We reveal that the crosstalk between microvascular endothelial cells and pericytes promotes pericyte-fibroblast transition through the PDGF-BB/PDGFRß signaling pathway. Our finding suggests that blocking the PDGF-BB/PDGFRß signaling pathway with imatinib contributes to functional recovery, fibrotic scarring, and inflammatory attenuation after SCI and provides a potential target for the treatment of SCI.

M1-type microglia can induce astrocytes to deposit chondroitin sulfate proteoglycan after spinal cord injury.

After spinal cord injury (SCI), astrocytes gradually migrate to and surround the lesion, depositing chondroitin sulfate proteoglycan-rich extracellular matrix and forming astrocytic scar, which limits the spread of inflammation but hinders axon regeneration. Meanwhile, microglia gradually accumulate at the lesion border to form microglial scar and can polarize to generate a pro-inflammatory M1 phenotype or an anti-inflammatory M2 phenotype. However, the effect of microglia polarization on astrocytes is unclear. Here, we found that both microglia (CX3CR1+) and astrocytes (GFAP+) gathered at the lesion border at 14 days post-injury (dpi). The microglia accumulated along the inner border of and in direct contact with the astrocytes. M1-type microglia (iNOS+CX3CR1+) were primarily observed at 3 and 7 dpi, while M2-type microglia (Arg1+CX3CR1+) were present at larger numbers at 7 and 14 dpi. Transforming growth factor-ß1 (TGFß1) was highly expressed in M1 microglia in vitro, consistent with strong expression of TGFß1 by microglia in vivo at 3 and 7 dpi, when they primarily exhibited an M1 phenotype. Furthermore, conditioned media from M1-type microglia induced astrocytes to secrete chondroitin sulfate proteoglycan in vitro. This effect was eliminated by knocking down sex-determining region Y-box 9 (SOX9) in astrocytes and could not be reversed by treatment with TGFß1. Taken together, our results suggest that microglia undergo M1 polarization and express high levels of TGFß1 at 3 and 7 dpi, and that M1-type microglia induce astrocytes to deposit chondroitin sulfate proteoglycan via the TGFß1/SOX9 pathway. The study was approved by the Institutional Animal Care and Use Committee of Anhui Medical University, China (approval No. LLSC20160052) on March 1, 2016.

Transcranial direct current stimulation treated by multilead brain reflex instrument accelerates neural functional recovery in a rat model of stroke.

PURPOSE: Clinical research suggests that transcranial direct current stimulation (tDCS) at bilateral supraorbital foramen and inferior orbital rim and nose intersections may facilitate rehabilitation after stroke. However, the underlying neurobiological mechanisms of tDCS remain poorly understood, impeding its clinical application. Here, we investigated the effect of tDCS applied after stroke on neural cells. MATERIALS AND METHODS: Middle cerebral arterial occlusion (MCAO) reperfusion was induced in rats. Animals with comparable infarcts were randomly divided into MCAO group and MCAO + tDCS group. Recovery of neurological function was assessed behaviorally by modified neurological severity score (mNSS). Ischemic tissue damage verified histologically by TTC and HE staining. Immunohistochemical staining, real-time qPCR, and western blot were applied to determine the changes of neural cells in ischemic brains. RESULTS: The results reveal that tDCS treated by multilead brain reflex instrument can promote the recovery of neurological function, remarkably reduce cerebral infarct volume, promote brain tissue rehabilitation, and can effectively inhibit astrocytosis and enhance neuronal survival and synaptic function in ischemic brains. CONCULSIONS: Our study suggests that tDCS treated by multilead brain reflex instrument could be prospectively developed into a clinical treatment modality.

TrkA regulates the regenerative capacity of bone marrow stromal stem cells in nerve grafts.

We previously demonstrated that overexpression of tropomyosin receptor kinase A (TrkA) promotes the survival and Schwann cell-like differentiation of bone marrow stromal stem cells in nerve grafts, thereby enhancing the regeneration and functional recovery of the peripheral nerve. In the present study, we investigated the molecular mechanisms underlying the neuroprotective effects of TrkA in bone marrow stromal stem cells seeded into nerve grafts. Bone marrow stromal stem cells from Sprague-Dawley rats were infected with recombinant lentivirus vector expressing rat TrkA, TrkA-shRNA or the respective control. The cells were then seeded into allogeneic rat acellular nerve allografts for bridging a 1-cm right sciatic nerve defect. Then, 8 weeks after surgery, hematoxylin and eosin staining showed that compared with the control groups, the cells and fibers in the TrkA overexpressing group were more densely and uniformly arranged, whereas they were relatively sparse and arranged in a disordered manner in the TrkA-shRNA group. Western blot assay showed that compared with the control groups, the TrkA overexpressing group had higher expression of the myelin marker, myelin basic protein and the axonal marker neurofilament 200. The TrkA overexpressing group also had higher levels of various signaling molecules, including TrkA, pTrkA (Tyr490), extracellular signal-regulated kinases 1/2 (Erk1/2), pErk1/2 (Thr202/Tyr204), and the anti-apoptotic proteins Bcl-2 and Bcl-xL. In contrast, these proteins were downregulated, while the pro-apoptotic factors Bax and Bad were upregulated, in the TrkA-shRNA group. The levels of the TrkA effectors Akt and pAkt (Ser473) were not different among the groups. These results suggest that TrkA enhances the survival and regenerative capacity of bone marrow stromal stem cells through upregulation of the Erk/Bcl-2 pathway. All procedures were approved by the Animal Ethical and Welfare Committee of Shenzhen University, China in December 2014 (approval No. AEWC-2014-001219).