Factors Affecting Regeneration Through the Glial Scar

描述(申请人提供)：我们开发了一种新的胶质瘢痕体外模型，在该模型中，粗略梯度中的抑制CS-PG更接近于损伤后在体内发生的情况。典型的营养不良终球形成于成人感觉神经轴突的顶端，这些轴突被困在梯度中。现在，我们可以第一次提出关于营养不良生长锥体生物学的各种基本问题。我们希望在目标1中探讨的三个重要问题是：(A)营养不良状态是否是成年神经元特有的；(B)中枢神经系统神经元是否会对PG梯度环境做出类似的反应；(C)树突对抑制梯度的反应是否与轴突不同。最后，(D)我们想知道通过一种新的组合再生刺激策略，在潜在的营养不良轴突中发生了什么分子变化，这些轴突被诱导重新生长到有效的抑制梯度的边缘。我们的最终目标是在体外设计一种最佳的策略，用于克服生长锥体营养不良，并在体内刺激轴突再生，越过胶质瘢痕。在目标2中，基于我们的体外梯度模型的成功的再生促进策略的初步体内结果表明，有证据表明，在挤压根之前在DRG中诱导慢性无菌炎症，加上在挤压时将软骨素酶应用于根进入区，可以促进感觉神经轴突进入脊髓的强劲再生。我们假设，类似的策略可能会在更具临床相关性的损伤后模型中促进感觉纤维再生。我们建议研究这些纤维的功能功效。在目标3中，我们将利用一种新的扣带微损伤模型，在该模型中，人们可以使最小的损伤成为可能，但仍然清楚地识别那些已被切断并可能再生的轴突。在微损伤模型中，我们可以使用切割轴突的微吸管，在吸管从大脑中取出后注入桥梁构建细胞或其他因素。初步证据表明，注射软骨素酶联合未成熟星形胶质细胞可以明显刺激损伤后的再生。尽管如此，一旦穿过病变，纤维只会短距离生长。利用这个模型，我们假设，通过驱动细胞体附近再生神经元的内在生长潜力，再生将显著增强。

DESCRIPTION (provided by applicant): We have developed a new in vitro model of the glial scar in which inhibitory CS-PG in a crude gradient more closely resembles that which occurs in vivo after injury. Classic dystrophic endballs form at the tips of adult sensory axons that become trapped within the gradient. We can now ask, for the first time, a variety of basic questions concerning the biology of the dystrophic growth cone. Three important questions we wish to pursue in aim 1 are: (A) whether the dystrophic state is a peculiarity of adult neurons, (B) whether CNS neurons would respond similarly to the PG gradient environment and, (C) whether dendrites respond differently to the inhibitory gradient than do axons. Finally, (D) we would like to know what molecular changes occur in would- be dystrophic axons that are induced to regrow across the potently inhibitory rim of the gradient via a new combinatorial regeneration stimulating strategy. Our ultimate goal is to devise an optimal strategy in vitro that can be used to overcome growth cone dystrophy and stimulate axon regeneration past the glial scar in vivo. In aim 2, preliminary in vivo results based on a successful regeneration promoting strategy using our in vitro gradient model, have shown evidence that a combination of chronic sterile inflammation induced in the DRG prior to root crush, plus chondroitinase application to the root entry zone at the time of crush, can foster robust regeneration of sensory axons into the spinal cord. We hypothesize that a similar strategy may foster sensory fiber regeneration in a more clinically relevant post-injury model. We propose to study the functional efficacy of these fibers. In aim 3, we will utilize a novel microlesion model of the cingulum in which one can make the smallest lesion possible but still clearly identify only those axons that have been severed and have potentially regenerated. With the microlesion model, we can use the micropipette, that cuts the axons, to inject bridge building cells or other factors upon withdrawal of the pipette from the brain. Preliminary evidence shows that injection of chondroitinase combined with immature astroglia can stimulate regeneration clearly past the lesion. None-the-less, once past the lesion the fibers only grow short distances. Using this model we hypothesize that by also driving the intrinsic growth potential of the regenerating neurons at the vicinity of their cell body, regeneration will be significantly enhanced.