Morphological Studies Of The Chemical Nature Of Rat Pineal Hormones And The Pathway Of Hormone Granules From Pineal Body To Cerebrospinal Fluid

The pinealocytes have processes and secretory granules. The nerve fibres innervating the pineal gland could also release neuropeptides. So it is not clear that many neuropeptides detected in the mammalian pineal gland might be store in the processes of pinealocytes, or nerve fibres innervating the gland. Regulated exocytosis of peptide hormone secretory granules or dense-core granules has been well examined. But there may be another releasing pattern that involves a whole-releasing together with granular membrane. Because the mammalian pineal body is remarkable for its vascularization lacked an endothelial blood-brain barrier, the route used by pineal secretions to reach their different target areas is currently believed to be the systemic circulation. But there is another fact that the concentration of melatonin in CSF of subarachnoid space was significantly higher than the serum level in healthy male volunteers. So target sites located in the brain might be reached by means of the cerebrospinal fluid (CSF). The aim of our study was to identify the characteristics of neuropeptides in the pineal body of rats, and the transport pathway of rat pineal secretions from pineal body to cerebrospinal fluid of subarachnoid space, so that we can go further to interpret the function of pineal body, and provide structural foundation for the diagnosis and treatment of the related diseases. The contents and results of our study were as follows:一A primary culture of rat pineal gland cellsObjective: Because pineal gland cells in culture can get rid of the influence of nerve fibres and body fluid, the culture of pineal gland cells is a good method to study the synthesis and secretion of neuropeptides in pinealocytes. Thus, we undertook the development of such a primary culture technique of rat pineal gland cells, and in addition studied morphology and biochemistry, to find the best period of growth and function for the further study of neuropeptide in pinealocytes.Methods: Sprague-Dawley rats born 3 days previously were cleaned with 75% ethanol and decapitated between 9:00 and 10:00 in spring. When the top of the skull is turned up as a flap from the rear, the superficial pineal gland adheres to the flap. After pineal glands were rinsed by cold D-Hank's balanced salt solution, the meninges and vessels were removed carefully from the pineal gland. Enzymatic dissociation of the tissue was performed by incubation with 0.125% trypsin and 0.02%EDTA for 15 min at 37℃. Enzyme activity was stopped with serum containing medium (DMEM/F12, containing 10% newborn bovine serum and 100IU/ml penicillin and 100IU/ml streptomycin) followed by mechanical dissociation of the tissue with pipettes. Dissociated cells were plated onto glass coverslips in 6 well cell culture cluster flat bottom with lid. Cells were incubated under 5% CO2 at 37℃with medium as above. Half of the medium was replaced at day 3,day 7,day 10 and day 13. The growth conditions of cells were observed with phrase contrast microscope everyday. Cells grown on glass coverslips at day 12 in vitro were fixed for 60 min with 4% paraformaldehyde in 0.1M phosphate buffer(PB, pH7.4) for immunocytochemical staining , followed by an incubation in 1%TritonX-100 in PBS for 30 min at room temperature. Coverslips were then incubated overnight at 4℃with primary rabbit polyclonal antibody against 5-HT. Binding of the 5-HT antibody was visualized with a biotin-conjugated goat anti-rabbit IgG as the second antibody, a horseradish peroxidase-conjugated streptavidin complex and DAB as the chromogen. Cells at day 15 in vitro were fixed for transmission electron microscopy with 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1M PB(pH7.4)at 4℃for 3 hour, and then the specimens were performed as routine for TEM. Cells grown on glass coverslips at day 15 in vitro were fixed for scanning electron microscopy with 1% glutaraldehyde in 0.1M PB(pH7.4)at 4℃for 1 hour, followed dehydrated with graded ethanol series, spurted with gold and observed under scanning electron microscope. Results: At 6 hour after plating, some cells became attached to the cover glass. During the first 4 day, the proliferation was very slowly. From 5 day to 6 day, the proliferation increased. From 7 day to 8 day, some cells aggregated and formed typical nest that some small dark round, oval-shape, triangle or polygon pinealocytes assembled like pebble paved a road on the top of flat fibroblast-like cells. From 8 day to 12 day, the proliferation is high and the cells aggregate into many nests. From 12 day to 15 day, the scattered slices of fibroblast-like cells meets, the road pebble-liked cells on it became dispersed and the nest became untypical. At same time, small amounts big irregular glia-like cells that have many thick and long processes could be observed on the holes in the nests or beside the nests. 5-HT-immunoreactive cells appeared bipolar neuron in vitro after cultured for 12 days. The transmission electron microscopic investigation of cultured pinealocytes after 15 days in vitro showed more prominent rough endoplasmic reticulum, densed mitochondria, many spherical clear vesicles and few 300nm in diameter large dense core secretory granules. The scanning electron microscopic investigation of cultured pinealocytes after 15 days in vitro showed oval-shape cells.Conclusion: we have tried to set up a rat pineal gland cells primary culture system. The method is simple, operative and cheap. From 7 day to 8 day, some cells aggregate into typical nest that some small dark round, oval-shape, triangle or polygon pinealocytes assembled like pebble paved a road on the top of flat fibroblast-like cells. From 10 days to 15 days is the best period for the cultured pineal gland cells to grow. This period is also the best time to study by immunocytochemical and transmission electron microscopy.二An observation of somatostatin immunoreactive cells and fibers in the rat pineal bodyObjective: Because pinealocytes have processes and nerve fibres innervating the pineal body could also release neuropeptides, it is not clear whether many neuropeptides detected in the mammalian pineal gland might be stored in the processes of pinealocytes, or in the nerve fibres innervating the gland. So we explored whether somatostatin could be synthesized and secreted by pineal body itself or not.Methods:1.In vivo immunocytochemistry observation of somatostation in the pineal bodyThe experiments were performed during daytime (9:00AM to 11:00 AM) in spring. After anaesthesised by intraperitoneal injection of ethylcarbamate (0.4ml/100g), 15-day old Sprague-Dawlay rats were perfused with 0.9% physiological saline rapidly and 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 1 hour at +4℃. Then the superficial pineal gland was excised and immersed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 72 hours at +4℃for immunocytochemical staining. Frozen section is 14μm thick. The sections were incubated in 1%TritonX-100 in PBS for 30 min at room temperature, and then incubated overnight at 4℃with primary rabbit polyclonal antibody against somatostatin. Binding of the somatostatin antibody was visualized with a biotin-conjugated goat anti-rabbit IgG as the second antibody, a horseradish peroxidase-conjugated streptavidin complex and DAB as the chromogen.2.Expression of somatostatin in rat pineal cells in primary culturePineal glands were obtained from newborn Sprague-Dawlay rats of both sexes for pineal cell culture. Cells grown on glass coverslips at day 12 in vitro were fixed for 60 min with 4% paraformaldehyde in 0.1M PB(pH7.4), for immunocytochemical staining, followed by an incubation in 1%TritonX-100 in PBS for 30 min at room temperature. Coverslips were then incubated at 4℃with primary rabbit polyclonal antibody against somatostatin for 36 hours. Binding of the somatostatin antibody was visualized with a biotin-conjugated goat anti-rabbit IgG as the second antibody, a horseradish peroxidase-conjugated streptavidin complex and DAB as the chromogen.3.Pre-embedding staining TEM observation with immunogold technique of somatostation in the pineal bodyThe experiments were performed during daytime (9:00AM to 11:00 AM) in spring. Newborn Sprague-Dawlay rats were killed by decapitation after anaesthesised by intraperitoneal injection of ethylcarbamate (0.4ml/100g). Then the superficial pineal gland was rapidly excised and immersed in a fixative solution with 3% paraformaldehyde and 1% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for 1 hour at +4℃, and 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 2 hours at +4℃. Then the specimens were performed as routine for TEM. Following polymerization, the tissues were cut and thin sections were mounted on nickel grids previously coated with Formvar films. The grid-mounted tissue sections were then incubated at 4℃with primary rabbit polyclonal antibody against somatostatin for 70 hours. Binding of the somatostatin antibody was visualized with a colloidal gold(15nm)labled goat anti-rabbit IgG (1:10 dilution) for 60min at 37℃. Following immunolabeling, the tissue sections were stained with uranyl acetate and lead citrate and examined in the transmission electron microscope. Results:1.In vivo immunocytochemistry observation of somatostation in the pineal bodyThe 15-day old rat pineal parenchyma contains a small number of oval-shape or triangle somatostatin-immunoreactive positive cell, whose diameter is bigger than that of background cells. Some of the immunoreactive positive cells have one or two processes emerging from the cell body. Some varicose somatostatin-immunoreactive positive fibers were occasionally observed in pineal parenchyma.2.Expression of somatostatin in rat pineal cells in primary culture Somatostatin-immunoreactive positive cells appeared round, oval-shape, triangle or fusiform with two or three processes emerging from the cell body in vitro after cultured for 12 days.3.Pre-embedding staining TEM observation with immunogold technique of somatostation in the pineal bodyThe pineal parenchyma of 1-day old rat is mainly composed of pinealocytes. Some large dense core secretory granules, scattered in a small number of pinealocytes.In the pre-embedding staining TEM tissue sections applying immunogold technique of somatostation, some gold particles were scattered or clustered over the cytoplasm of a small number of pinealocytes.Conclusion: The rat pineal contains a small number of somatostatin- immunoreactive positive cell and fibers. A small number of pinealocytes have large dense core secretory granules. In the cytoplasm of a small number of pinealocytes exist somatostatin-immunoreactive positive substance. Some primary cultured pineal cells could synthesize and secrete somatostatin.三Structural foundation of the transport pathway of rat pineal secretions from pineal body to cerebrospinal fluid of subarachnoid spaceObjective: The mammalian pineal body is remarkable for its vascularization lacked an endothelial blood-brain barrier. The route used by pineal secretions to reach their different target areas is currently believed to be the systemic circulation. Because the concentration of melatonin in CSF of subarachnoid space was significantly higher than the serum level in healthy male volunteers, an alternative hypothesis is that target sites located in the brain might be reached by means of the cerebrospinal fluid (CSF). Regulated exocytosis of peptide hormone secretory granules or dense-core granules has been well examined. But there may be another releasing pattern that involves a whole-releasing together with granular membrane. In order to explore the releasing pattern of rat pineal secretory granules, and the structural foundation of a direct extracellular transport pathway of pineal secretions from pineal body to cerebrospinal fluid of subarachnoid space, Sprague-Dawlay rats were observed using TEM, SEM and SEM with freeze-fractured technique.Methods: The experiments were performed during spring, and the healthy Sprague-Dawlay rats were killed by decapitation, after anaesthesised by intraperitoneal injection of ethylcarbamate (0.4ml/100g) during daytime (9:00AM to 11:00 AM). Then the superficial pineal gland was rapidly excised. Four specimens of 12-month old rat were immersed in a fixative solution with 3% paraformaldehyde and 1% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for 48 hours at +4℃, and then the specimens were performed as routine for TEM. Another 9 specimens of 1.5-month old rat, and 9 specimens of 12-month old rat were immersed in 0.1 M phosphate buffer (pH 7.4) containing 3% paraformaldehyde and 1% glutaraldehyde for 48 hours at +4℃, and then the specimens were performed as routine for SEM, and the pineal capsule of the superficial pineal body were observed. At the same period, after anaesthesised as above, 4 rats of 12-month old were perfused with 0.9% physiological saline. Then the rats were killed by decapitation and the superficial pineal gland was rapidly excised. After immersion 1%OsO4 for 1 hour at 4℃, the specimens were performed as routine for SEM with freeze-fractured technique.Results:1.Observation with transmission electron microscopeThe blood capillaries in the rat pineal body were endowed with fenestrated endothelium (50nm), separated from the broad perivascular space by basement membrane; the pinealocytes were also separated from the connective tissue space by basement membrane. Between pinealocytes exist intercellular canaliculus formed by the exact opposition of two shallow gutters of the surface of adjacent pinealocytes. The canaliculi varied in diameter and are lined smoothly. Some membrane-like structures could be observed in the canaliculi. But the junctional complexes have not been observed. These canaliculi linked up with connective tissue space of pineal capsule and perivascular space; the pineal capsule was composed of squamous epithelial cells and subepithelial connective tissue. In subepithelial connective tissue space existed pinealocytic body and processes, et al. Two arrangement styles of epithelial cells could be observed. One is that parallel squamous epithelial leaflets piled up and formed overlapped junctions(1μm between parallel epithelial leaflets). The other is that squamous epithelium cells form a single layer and some epithelial openings(2.5μm)between epithelial cells were observed.2.Observation with scanning electron microscope with freeze-fractured techniqueOn the fractured surface of the pineal body, between pinealocytes exist intercellular canaliculus formed by the exact opposition of two shallow gutters of the surface of adjacent pinealocytes. Much broad perivascular space could be observed. The intercellular canaliculi linked up with perivascular space.3.Observation with scanning electron microscope of rat pineal surfaceCribriform and trumpet-shaped epithelial stomata are scattered on the pineal capsule. Cribriform epithelial stomata are seen most commonly in 1.5-month old rats. Cribriform epithelial stomata are composed of many round pores, which pierced through the full-thickness of the periphery of the capsule endothelium. The pores are dense and ranges from 200~500nm in diameter;Trumpet-shaped epithelial stomata are seen in 1.5-month and 12-month old rats. Trumpet-shaped epithelial stomata are located among epithelial cells of pineal capsule. They are round or elliptic in shape and ranges from 1~4μm in diameter. On the surface of pineal capsule, many spherule and 500~1000nm in diameter secretory granules-like structures and membrane fragment were observed. Some round or protrudent macrophages were observed occasionally.Conclusion: The pinealcyte has three types of functional surfaces: perivascular, intercellular and canalicular. Between pinealocytes exist intercellular canaliculus formed by the exact opposition of two shallow gutters of the surface of adjacent pinealocytes. The releasing pattern of secretory granules of pineal body includes a whole releasing together with granular membrane. The pineal secretions may be released into perivascular space by perivascular surfaces or into intercellular canaliculus by canalicular surfaces. The pineal secretory granules in CSF of subarachnoid space would rather derive from pineal body by pineal perivascular space--intercellular canaliculus-- overlapped junctions/epithelial openings among epithelial cells, than by CSF flow from pineal recess or by systemic circulation. Because of the alternation of the physico-chemical property, such as osmotic pressure, secretory granules expanded. Some granules membrane ruptured and pineal hormone were released into CSF.