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首页> 外文期刊>Brain research. Brain research protocols >Preservation and detection of lesion-induced collagenous scar in the CNS depend on the method of tissue processing.
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Preservation and detection of lesion-induced collagenous scar in the CNS depend on the method of tissue processing.

机译：在中枢神经系统中病变诱导的胶原性瘢痕的保存和检测取决于组织处理的方法。

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After traumatic injury, adult central nervous system (CNS) axons fail to regenerate. We have previously shown that one major impediment for axon regeneration is the basement membrane (BM) forming at the lesion center, by means of a wound healing collagenous scar, after lesioning the postcommisural fornix of the adult rat [Eur. J. Neurosci. 11 (1999) 632] [6]. This BM consists of a supramolecular network of collagen type IV, laminin (LN), nidogen, and associated proteoglycans [Crit. Rev. Biochem. Mol. Biol. 27 (1992) 93] [5]. Following axotomy, axons of the proximal stump of the transected postcommissural fornix fail to cross the lesion site. This regenerative failure is spatially and temporally highly correlated with the appearance of BM in the lesion site [Restor. Neurol. Neurosci. 15 (1999) 1] [7]. However, if the deposition of BM is prevented, the injured axons: (i) regenerate in their former pathway, (ii) are conductive across and behind the lesion site, and (iii) form chemical synapses in their target area, the mammillary body [Eur. J. Neurosci. 11 (1999) 632]. The developing BM is surrounded by neuropil and can easily be stained immunohistochemically using anti-collagen IV antibodies on fresh frozen sections (10 microm). To examine a clinically more relevant model of traumatic CNS injuries we developed a transection model of the thoracic dorsal corticospinal tract (CST) in the rat spinal cord. In contrast to fornix lesion this transection is performed in close proximity to the meninges. This involves the BM being completely washed out if fresh frozen tissue is used on slides. If the animals are sacrificed by perfusion with aldehydes the collagen IV and the LN antigen are masked by the fixative. To restore the correct immunohistochemical staining pattern (BM, blood vessels) a special protocol including an enzyme digestion is necessary. If thick sections are stained free floating, the tissue is destroyed due to the enzyme treatment. Here we present a method to prevent loss of the lesion-induced BM and to perform the correct immunohistochemical stainings of BM proteins in the traumatically injured spinal cord.
机译：创伤性损伤后，成人中枢神经系统（CNS）轴突无法再生。先前我们已经表明，轴突再生的一个主要障碍是在损伤成年大鼠的后室壁穹隆后，通过伤口愈合的胶原性瘢痕在损伤中心形成基底膜（BM）。 J.神经科学。 11（1999）632] [6]。这种BM由IV型胶原蛋白，层粘连蛋白（LN），胶原蛋白和相关蛋白聚糖的超分子网络组成[Crit。生物化学牧师。大声笑生物学27（1992）93] [5]。切开后，横切合后穹ni的近端残端的轴突无法穿过病变部位。这种再生衰竭在空间和时间上与病变部位BM的出现高度相关[Restor。神经元。神经科学。 15（1999）1] [7]。但是，如果防止了BM的沉积，则受伤的轴突：（i）在其先前的途径中再生，（ii）在病变部位及其后方传导，并且（iii）在其靶区域（乳突体）中形成化学突触。 [欧元。 J.神经科学。 11（1999）632]。发育中的BM被Neuropil包围，并且可以使用抗胶原IV抗体在新鲜的冷冻切片（10微米）上容易地进行免疫组织化学染色。为了检查临床上与中枢神经系统损伤相关的模型，我们开发了大鼠脊髓胸背皮质脊髓道（CST）的横断模型。与穹ni病变相反，该横切在靠近脑膜的地方进行。如果在载玻片上使用了新鲜的冷冻组织，这将导致BM被完全冲洗掉。如果通过灌注醛杀死动物，则胶原IV和LN抗原会被固定剂掩盖。为了恢复正确的免疫组织化学染色模式（BM，血管），需要特殊的方案，包括酶消化。如果厚的部分被染色成自由漂浮，则由于酶处理，组织被破坏。在这里，我们提出了一种方法，以防止损伤引起的BM丢失，并在受创伤的脊髓中进行BM蛋白的正确免疫组织化学染色。

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