| Objective: The study of immunohistochemistry and immunocytochemistry have indicated that myocardial cells can synthesis and secret atrial natriuretic peptide (atrial natriuretic polypeptide, ANP); Moreover, the amount of the atrial natriuretic peptide granules (ANP granules) are the largest in myocardial cells of right auricle. Radioimmunoassay showed that the pericardial fluid in the pericardial cavity contains atrial natriuretic peptide, and which is to show physiological or pathological changes in the content of circumstances. It is lack of morphological evidence for the origin and destination of ANP in pericardial fluid, and not very clear for its releasing and normal forms of transport. In this study, we observed the right atrial appendage tissue in rats by light microscopy and electron microscopy to reveal the subtle and ultrastructural characteristics of the right auricle, investigate the relationship between the right atrial cell and the atrial cavity and pericardial cavity, for the morphological basis of the releasing and normal forms of transport of secretory granules.Methods: 1. Experimental animals and materials: Ten healthy adult male SD rats were choosed. The animals were sacrificed under deep anesthesia by intraperitoneal injection of pentobarbital sodium solution, cut the right atrial appendage for the samples preparation of the light microscopy and electron microscopy.2. Light microscopy sample preparation and observation: The right atrial appendage tissue in paraffin-embedded was stained by the hematoxylin-eosin (haematoxylin-eosin, HE) and observed by the light microscope (light microscope, LM); The frozen right atrial appendage tissue was stained by the 5'-nucleotide enzymes-alkaline phosphatase (5'-Nase-ALPase) Enzyme histochemical double-staining and observed by the light microscopy. It showed the minor organizational structure of the right atrial appendage, the array of endocardium, myocardium and epicardium, as well as the distribution characteristic of the vascular system inside the right atrial appendage.3. TEM sample preparation and observation: The ultra-thin right atrial appendage tissue sample was prepared, and observed by transmission electron microscope(TEM). Through the observasion,we revealed the ultrastructural characteristics of right atrial appendage, observed the distribution of atrial granules; to identify the types of blood capillary and lymphatic capillaries among the atrial myocytes and their structural characteristics.4. SEM sample preparation and observation: The conventional sample was prepared, we used scanning electron microscopy (scanning electron microscope, SEM) to observe the structural characteristics of the epicardial and endocardial surfaces; Moreover we could obverse the structural characteristics of the severing surface of the right atrial appendage by SEM, which was prepared by DMSO (dimethyl sulfoxide , DMSO) freeze-cracking technique.Results:1. LM observation:(1) HE staining of paraffin-embedded tissue: the epicardium is composed of a thin layer of connective tissue and a layer of epithelial (mesothelial) cells; the connective tissue in different parts is of various thickness,and we can see many vascular and lymphatic vessels. Beneath the epicardium is myocardium, we can see the ring and the longitudinal arrangement of myocardial (cell) fiber connected with each other as a net, capillaries are rich in the mesh. The endocardium is located in the innerest part, composed of the flat endothelial cells and endocardial connective tissue.(2) Enzyme histochemical double-staining of histological sections by LM observation showed that the structural characteristics of the right atrial appendage are similar to the HE staining sections. The lymphatic vessel is positive to 5'-Nase(brown), and with a thinner vessel wall, a larger and irregular lumen; the blood vessel showed positive to ALP(blue), with a thicker wall, a mostly smaller circular lumen.2.TEM observation: (1) The epicardial epithelium and endocardial epithelium of rat right atrial appendage are composed of flat cells; connections between epithelial cells are fluffy, and there is lots of finger-like tubby protrusions which is called microvilli that penetrate to the surface of pericardial cavity and basis. The connections between endothelial cells are more closely, and we can see short and sparse microvilli on the cells. Subepithelial and subendothelial parts are loose connective tissue, mainly composed of the collagen fiber bundles in regular pattern, fibroblasts, nerves, blood vessels, lymphatics, etc can also be found.(2) From the surface of the longitudinal section of atrial myocytes, we can see lots of sarcomeres and mitochondrias; Golgi complexes and many secretory granules can also be seen around the nucleus; the secretory granules were round or oval in shape, with a diameter of 100~300nm; they are with a high electron density, homogeneous, and with wrapped membranes; a large number of similar secretory granules are also distributed near the atrial muscle cell membrane; secretory granules were wrapped up with membrane and a small amount of cytoplasm to form papillary protrusions, similar to jagged structure, we also can see residual vacuoles after emptying. Emitted secretory granules wrapped with limiting membrane can be seen at the gaps among myocardial cells, or carried by phagocytes.(3) The blood capillaris among atrial myocytes of the right auricle are of continuous type, with non-fenestra and thick endothelium, red blood cells are common in the cavity. we can also observed the lymphatic capillaries among atrial myocytes, with loosely connected endothelial cells which belong to overlapping connections, chimeric connectivity, end-to-end connectivity and other forms of open connections; the lymphatic capillaries has thinner wall, large and irregular lumen, homogeneous substance is in the cavity. Secretory granules similar to that of myocardial cells in form and size often can be seen in the perivascular space, connective tissue spase and lymphatic perivascular space.3.SEM observation:(1) The rat epicardial surface is composed of squamous epithelium, whose shape is polygon, and the edge of each other is compacted in a wavy shape,the parts of the nuclear bulge slightly; microvilli exist in the epithelial cell surface, longer and more microvilli are seen at the junction of adjacent cells, trema -like structure also can be seen.(2) The endocardial surface is smoother, sparse short microvilli are on endothelial cells; we can see the gap of smallest cardiac veins, whose aperture is about 10 ~ 20μm.(3) Subepithelial and subendothelial collagen fibers were fascicular, intertwined to form grid-like structure at the frozen severing surface of the right atrial appendage. Conclusions: The subtle and ultrastructural characteristics of the rat right atrial appendage cue that: (1) atrial secretory granules of atrial natriuretic peptide release forms include the whole-releasing pattern together with membrane. (2) the secretory granules or atrial natriuretic peptide which were released into the tissue fluid can enter the capillaries and then into the the right atrium directly through the smallest vein; can also enter the lymphatic capillaries, transiting with the return of lymphatic channels; and also directly into the pericardial cavity with the heart tissue fluid through the epithelium trema of the outer membrane.In view of the relationship between body organ structure and function of mutual unity, it is worth further studying that the physiological and pathological significance of the pericardial cavity in atrial natriuretic peptide transit. |
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