Menzies Research Institute, University of Tasmania, Hobart, Tasmania, Australia.
PLoS One. 2011;6(7):e22040. doi: 10.1371/journal.pone.0022040. Epub 2011 Jul 15.
Organotypic brain slice culturing techniques are extensively used in a wide range of experimental procedures and are particularly useful in providing mechanistic insights into neurological disorders or injury. The cellular and morphological alterations associated with hippocampal brain slice cultures has been well established, however, the neuronal response of mouse cortical neurons to culture is not well documented.
In the current study, we compared the cell viability, as well as phenotypic and protein expression changes in cortical neurons, in whole brain slice cultures from mouse neonates (P4-6), adolescent animals (P25-28) and mature adults (P50+). Cultures were prepared using the membrane interface method.
Propidium iodide labeling of nuclei (due to compromised cell membrane) and AlamarBlue™ (cell respiration) analysis demonstrated that neonatal tissue was significantly less vulnerable to long-term culture in comparison to the more mature brain tissues. Cultures from P6 animals showed a significant increase in the expression of synaptic markers and a decrease in growth-associated proteins over the entire culture period. However, morphological analysis of organotypic brain slices cultured from neonatal tissue demonstrated that there were substantial changes to neuronal and glial organization within the neocortex, with a distinct loss of cytoarchitectural stratification and increased GFAP expression (p<0.05). Additionally, cultures from neonatal tissue had no glial limitans and, after 14 DIV, displayed substantial cellular protrusions from slice edges, including cells that expressed both glial and neuronal markers.
In summary, we present a substantial evaluation of the viability and morphological changes that occur in the neocortex of whole brain tissue cultures, from different ages, over an extended period of culture.
器官型脑片培养技术广泛应用于各种实验程序，特别有助于深入了解神经疾病或损伤的机制。海马脑片培养相关的细胞和形态变化已经得到很好的证实，然而，关于培养物中小鼠皮质神经元的神经元反应尚未得到很好的记录。
在本研究中，我们比较了来自新生（P4-6）、青少年（P25-28）和成年（P50+）小鼠全脑片培养物中的皮质神经元的细胞活力以及表型和蛋白表达变化。使用膜界面法制备培养物。
碘化丙啶标记细胞核（由于细胞膜受损）和 AlamarBlue™（细胞呼吸）分析表明，与更成熟的脑组织相比，新生组织在长期培养中明显更不易受损。来自 P6 动物的培养物显示突触标志物的表达显著增加，而整个培养期间生长相关蛋白的表达显著减少。然而，来自新生组织的器官型脑片培养物的形态分析表明，新皮层内神经元和神经胶质组织发生了实质性变化，明显丧失了细胞结构分层，并增加了 GFAP 表达（p<0.05）。此外，来自新生组织的培养物没有神经胶质界膜，并且在 14 DIV 后，从切片边缘显示出大量的细胞突起，包括表达神经胶质和神经元标志物的细胞。
总之，我们对来自不同年龄的全脑组织培养物在延长培养期间的新皮层中的活力和形态变化进行了全面评估。
