Horackova M, Croll R P, Hopkins D A, Losier A M, Armour J A
Department of Physiology and Biophysics, Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia, Canada.
Tissue Cell. 1996 Aug;28(4):411-25. doi: 10.1016/s0040-8166(96)80027-9.
Long-term (2-12 weeks) cultures of adult guinea-pig ventricular myocytes, cocultured with neurons derived from stellate or intrinsic cardiac ganglia, retain their functional properties (Horackova et al., 1993, 1994, 1995). The present study was designed to investigate the morphological and immunochemical properties of such neurons and their associated cardiomyocytes. Cultured myocytes studied by means of phalloidin-rhodamine (for F-actin) and an antibody raised against myomes revealed parallel myofibrils with striations typical of rod-shaped cardiomyocytes, even while myocytes changed from cylindrical to flattened form as they established intercellular contacts. Microtubular networks, identified by alpha-tubulin DM1A antibody, were arrayed longitudinally in myofibrils, being especially prominent during the formation of intercellular contacts between myocytes. Histochemically identified adult peripheral autonomic neurons cultured alone or with myocytes displayed a variety of shapes. alpha-Tubulin staining was associated with the somata and neurites of various-shaped neurons whether cultured alone or with myocytes. Cultured neurons derived from stellate and intrinsic cardiac ganglia also exhibited staining for the general neuronal marker PGP 9.5 (protein gene product 9.5), and for specific markers of the following neurochemicals: tyrosine hydroxylase, acetylcholinesterase, choline acetyltransferase, neuropeptide Y, vasoactive intestinal peptide, calcitonin gene-related peptide, bradykinin, oxytocin, and NADPH-diaphorase. These data indicate that: (a) adult ventricular myocytes cocultured with intrathoracic neurons retain the structural properties of adult myocytes found in vivo; (b) intrinsic cardiac and extrinsic intrathoracic neurons cultured alone or with cardiomyocytes display morphological characteristics similar to those of neurons studied in situ; (c) intrinsic cardiac and intrathoracic extracardiac neurons cultured alone or with cardiomyocytes display a variety of morphologies (unipolar, bipolar, and multipolar), larger and more multipolar neurons being present in cultures derived from stellate versus intrinsic cardiac ganglia; (d) such cultured neurons are associated with a number of neurochemicals, more than one chemical being associated with each neuron. This model presents an excellent opportunity to study the morphology of individual peripheral extracardiac and intracardiac neurons as well as their potential to produce various neurochemicals that are known to be involved in the neuromodulation of cardiomyocyte function.
成年豚鼠心室肌细胞与源自星状神经节或心脏固有神经节的神经元共培养2至12周，仍能保持其功能特性（霍拉科娃等人，1993年、1994年、1995年）。本研究旨在调查此类神经元及其相关心肌细胞的形态学和免疫化学特性。通过鬼笔环肽 - 罗丹明（用于F - 肌动蛋白）和一种针对肌球蛋白的抗体对培养的心肌细胞进行研究，结果显示，即使心肌细胞在建立细胞间连接时从圆柱形变为扁平形，仍存在具有杆状心肌细胞典型条纹的平行肌原纤维。通过α - 微管蛋白DM1A抗体鉴定的微管网络纵向排列在肌原纤维中，在心肌细胞间形成细胞间连接时尤为突出。单独培养或与心肌细胞一起培养的经组织化学鉴定的成年外周自主神经元呈现出多种形状。无论单独培养还是与心肌细胞一起培养，α - 微管蛋白染色都与各种形状神经元的胞体和神经突相关。源自星状神经节和心脏固有神经节的培养神经元还表现出对一般神经元标志物PGP 9.5（蛋白基因产物9.5）以及以下神经化学物质的特异性标志物的染色：酪氨酸羟化酶、乙酰胆碱酯酶、胆碱乙酰转移酶、神经肽Y、血管活性肠肽、降钙素基因相关肽、缓激肽、催产素和NADPH - 黄递酶。这些数据表明：（a）与胸内神经元共培养的成年心室肌细胞保留了体内成年心肌细胞的结构特性；（b）单独培养或与心肌细胞一起培养的心脏固有神经节和胸外神经元表现出与原位研究的神经元相似的形态特征；（c）单独培养或与心肌细胞一起培养的心脏固有神经节和胸外心脏神经元呈现出多种形态（单极、双极和多极），源自星状神经节的培养物中存在更大且更多极的神经元，而源自心脏固有神经节的培养物中则较少；（d）此类培养的神经元与多种神经化学物质相关，每个神经元与一种以上的化学物质相关。该模型为研究单个外周心脏外和心脏内神经元的形态以及它们产生已知参与心肌细胞功能神经调节的各种神经化学物质的潜力提供了绝佳机会。
