Jurzak M, Müller A R, Schmid H A, Gerstberger R
Max-Planck-Institut für physiologische und klinische Forschung, W.G. Kerckhoff-Institut, Bad Nauheim, Germany.
Brain Res. 1994 Oct 31;662(1-2):198-208. doi: 10.1016/0006-8993(94)90813-3.
A primary culture system of cells derived from two circumventricular organs (CVO) of the rat brain was established. The subfornical organ (SFO) and the organum vasculosum of the lamina terminalis (OVLT) were dissected from the rostral wall of the third ventricle and its cells taken into culture after mechanical dissociation. The cells were cultured in a modified microculture chamber system ensuring relatively high cell density despite their low absolute number. When animals were injected with Evans blue prior to cell preparation, the macroscopically visible penetration of the dye into the parenchyma of the CVOs could be used as guidance during tissue isolation and labelled cells could be identified in culture. Cultured CVO neurones and astrocytes were identified using antibodies against cell type specific marker proteins. The histochemical NADPH-diaphorase staining was used for the detection of nitric oxide synthase in tissue sections of both CVOs and in their cultured neurones. In addition, angiotensin II (ANG II)-evoked elevations of the intracellular Ca2+ concentration ([Ca2+]i) in single cultured OVLT neurones were measured. The described methods will be useful for further characterization of CVO neurones and astrocytes.
建立了一种源自大鼠脑两个室周器官（CVO）的细胞原代培养系统。从第三脑室的前壁分离出穹窿下器官（SFO）和终板血管器（OVLT），其细胞经机械解离后进行培养。细胞在改良的微量培养室系统中培养，尽管其绝对数量较少，但能确保相对较高的细胞密度。在制备细胞前给动物注射伊文思蓝，染料在宏观上可见地渗入CVO实质，这可在组织分离过程中用作指导，并且在培养物中可识别标记细胞。使用针对细胞类型特异性标记蛋白的抗体鉴定培养的CVO神经元和星形胶质细胞。组织化学NADPH - 黄递酶染色用于检测两个CVO的组织切片及其培养神经元中的一氧化氮合酶。此外，还测量了血管紧张素II（ANG II）引起的单个培养的OVLT神经元细胞内Ca2 +浓度（[Ca2 +] i）的升高。所描述的方法将有助于进一步表征CVO神经元和星形胶质细胞。
