You Si-Wei, Hellström Mats, Pollett Margaret A, LeVaillant Chrisna, Moses Colette, Rigby Paul J, Penrose Marissa, Rodger Jennifer, Harvey Alan R
Department of Ophthalmology, Xijing Hospital, The Fourth Military Medical University, Xi'an, China.
Laboratory for Transplantation and Regenerative Medicine, Dept. of Obstetrics and Gynecology, Sahlgrenska Academy, University of Gothenburg, Sweden.
Exp Neurol. 2016 May;279:197-211. doi: 10.1016/j.expneurol.2016.03.006. Epub 2016 Mar 10.
Peripheral nerve (PN) grafts can be used to bridge tissue defects in the CNS. Using a PN-to-optic nerve (ON) graft model, we combined gene therapy with pharmacotherapy to promote the long-distance regeneration of injured adult retinal ganglion cells (RGCs). Autologous sciatic nerve was sutured onto the transected ON and the distal end immediately inserted into contralateral superior colliculus (SC). Control rats received intraocular injections of saline or adeno-associated virus (AAV) encoding GFP. In experimental groups, three bi-cistronic AAV vectors encoding ciliary neurotrophic factor (CNTF) were injected into different regions of the grafted eye. Each vector encoded a different fluorescent reporter to assess retinotopic order in the regenerate projection. To encourage sprouting/synaptogenesis, after 6 weeks some AAV-CNTF injected rats received an intravitreal injection of recombinant brain-derived neurotrophic factor (rBDNF) or AAV-BDNF. Four months after surgery, cholera toxin B was used to visualize regenerate RGC axons. RGC viability and axonal regrowth into SC were significantly greater in AAV-CNTF groups. In some cases, near the insertion site, regenerate axonal density resembled retinal terminal densities seen in normal SC. Complex arbors were seen in superficial but not deep SC layers and many terminals were immunopositive for presynaptic proteins vGlut2 and SV2. There was improvement in visual function via the grafted eye with significantly greater pupillary constriction in both AAV-CNTF+BDNF groups. In both control and AAV-CNTF+rBDNF groups the extent of light avoidance correlated with the maximal distance of axonal penetration into superficial SC. Despite the robust regrowth of RGC axons back into the SC, axons originating from different parts of the retina were intermixed at the PN graft/host SC interface, indicating that there remained a lack of order in this extensive regenerate projection.
周围神经（PN）移植物可用于桥接中枢神经系统中的组织缺损。利用PN-视神经（ON）移植物模型，我们将基因治疗与药物治疗相结合，以促进成年受损视网膜神经节细胞（RGCs）的长距离再生。将自体坐骨神经缝合到横断的视神经上，并将远端立即插入对侧上丘（SC）。对照大鼠接受眼内注射生理盐水或编码绿色荧光蛋白（GFP）的腺相关病毒（AAV）。在实验组中，将三种编码睫状神经营养因子（CNTF）的双顺反子AAV载体注射到移植眼的不同区域。每个载体编码一种不同的荧光报告基因，以评估再生投射中的视网膜拓扑顺序。为了促进发芽/突触形成，6周后，一些注射AAV-CNTF的大鼠接受玻璃体内注射重组脑源性神经营养因子（rBDNF）或AAV-BDNF。手术后四个月，使用霍乱毒素B来可视化再生的RGC轴突。AAV-CNTF组的RGC活力和轴突向SC的再生明显更强。在某些情况下，在插入部位附近，再生轴突密度类似于正常SC中所见的视网膜终末密度。在SC浅层而非深层可见复杂的树突分支，许多终末对突触前蛋白vGlut2和SV2呈免疫阳性。通过移植眼的视觉功能有所改善，在两个AAV-CNTF+BDNF组中瞳孔收缩明显更大。在对照和AAV-CNTF+rBDNF组中，避光程度与轴突向浅层SC穿透的最大距离相关。尽管RGC轴突强劲地再生回到SC，但源自视网膜不同部位的轴突在PN移植物/宿主SC界面处相互混合，这表明在这个广泛的再生投射中仍然缺乏秩序。
