RESUMO
Gene therapy for neuropathic pain requires efficient gene delivery to both central and peripheral nervous systems. We previously showed that an adenoassociated virus serotype 9 (AAV9) vector expressing short-hairpin RNA (shRNA) could suppress target molecule expression in the dorsal root ganglia (DRG) and spinal cord upon intrathecal injection. To evaluate the therapeutic potential of this approach, we constructed an AAV9 vector encoding shRNA against vanilloid receptor 1 (TRPV1), which is an important target gene for acute pain, but its role in chronic neuropathic pain remains unclear. We intrathecally injected it into the subarachnoid space at the upper lumbar spine of mice 3 weeks after spared nerve injury (SNI). Delivered shTRPV1 effectively suppressed mRNA and protein expression of TRPV1 in the DRG and spinal cord, and it attenuated nerve injury-induced thermal allodynia 10-28 days after treatment. Our study provides important evidence for the contribution of TRPV1 to thermal hypersensitivity in neuropathic pain and thus establishes intrathecal AAV9-mediated gene delivery as an investigative and potentially therapeutic platform for the nervous system.
Assuntos
Dependovirus/genética , Vetores Genéticos/genética , Hiperalgesia/genética , RNA Interferente Pequeno/genética , Canais de Cátion TRPV/genética , Animais , Sequência de Bases , Dependovirus/imunologia , Modelos Animais de Doenças , Feminino , Gânglios Espinais/metabolismo , Expressão Gênica , Ordem dos Genes , Inativação Gênica , Técnicas de Transferência de Genes , Terapia Genética , Vetores Genéticos/administração & dosagem , Vetores Genéticos/imunologia , Hiperalgesia/terapia , Injeções Espinhais , Camundongos , Traumatismos dos Nervos Periféricos/genética , Traumatismos dos Nervos Periféricos/terapia , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/química , RNA Interferente Pequeno/metabolismo , Medula Espinal/metabolismo , Canais de Cátion TRPV/química , Canais de Cátion TRPV/metabolismoRESUMO
In the last decade, researchers and clinicians have reported that transplantation of bone marrow stromal cells (BMSCs) promotes functional recovery after brain or spinal cord injury (SCI). However, an appropriate scaffold designed for the injured spinal cord is needed to enhance the survival of transplanted BMSCs and to promote nerve regeneration. We previously tested a honeycomb collagen sponge (HC), which when applied to the transected spinal cord allowed bridging of the gap with nerve fibers. In this study, we examined whether the HC implant combined with rat BMSCs increases nerve regeneration in vitro and enhances functional recovery in vivo. We first evaluated the neurite outgrowth of rat dorsal root ganglion (DRG) explants cultured on HC with or without BMSCs in vitro. Regeneration of neurites from the DRGs was increased by BMSCs combined with HC scaffolds. In the in vivo study, 3-mm-long HC scaffolds with or without BMSCs were implanted into the hemisected rat thoracic spinal cord. Four weeks after the procedure, rats implanted with HC scaffolds containing BMSCs displayed better motor and sensory recovery than those implanted with HC scaffolds only. Histologically, more CGRP-positive sensory fibers at the implanted site and 5-HT-positive serotonergic fibers contralateral to the implanted site were observed in spinal cords receiving BMSCs. Furthermore, more rubrospinal neurons projected distally to the HC implant containing BMSCs. Our study indicates that the application of BMSCs in a HC scaffold in the injured spinal cord directly promoted sensory nerve and rubrospinal tract regeneration, thus resulting in functional recovery.
Assuntos
Células da Medula Óssea/metabolismo , Transplante de Medula Óssea/métodos , Traumatismos da Medula Espinal/metabolismo , Animais , Células da Medula Óssea/citologia , Colágeno , Feminino , Ratos , Ratos Endogâmicos F344 , Regeneração , Traumatismos da Medula Espinal/patologiaRESUMO
BACKGROUND: Flap necrosis remains a major complication of reconstructive surgery. To improve skin flap survival, various treatments with vasodilators, antiplatelet drugs, or the local administration of growth factors have been performed. However, the sufficient prevention of skin necrosis is not well established. Platelet-rich plasma has been used as an autologous factor and includes various growth factors. The authors evaluated whether or not platelet-rich plasma can improve skin flap survival in an experimental rat model. METHODS: Cranially based dorsal cutaneous flaps were elevated in 48 rats. The animals received subcutaneous injections of either platelet-rich plasma (100 µl) or platelet-poor plasma (100 µl). The rats were divided into three groups: the platelet-rich plasma group (n = 16), the platelet-poor plasma group (n = 16), and the nontreatment group (n = 16). Flap survival was measured and histologic specimens were collected on day 7. Real-time polymerase chain reaction specimens were collected after 8 hours, 24 hours, 3 days, and 7 days. RESULTS: Platelet-rich plasma significantly improved flap survival rates (61.2 percent) compared with the platelet-poor plasma treatment (35.8 percent) and nontreatment groups (28.0 percent). A histologic analysis showed that significantly fewer inflammatory cells and an increased blood vessel density were observed in the platelet-rich plasma rats versus the platelet-poor plasma or nontreatment rats. In addition, platelet-rich plasma treatment significantly increased the mRNA levels of vascular endothelial growth factor and platelet-derived growth factor. CONCLUSION: Platelet-rich plasma modulates the genes involved in angiogenesis and improves skin flap survival.