In vivo microscopy of internal organs.
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Biomedical subjects
Publications and source records attributed to R S McCuskey.
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The nature of the intensely fluorescent cells (IFC) in the livers of male Sprague-Dawley rats was evaluated using histochemical and pharmacological techniques. The number and distribution of IFC in portal areas were quantified using some of these techniques. Cells which were metachromatic as well as fluorescent for serotonin, histamine, and heparin were observed in the connective tissue of the portal space, hilus, and capsule of the liver. Cells with the characteristics of chromaffin, enterochromaffin, or enterochromaffin-like cells were not seen in these locations. Intravenous administration of compound 48/80 a known mast cell degranulator, caused a significant decrease in the number of fluorescent and metachromatic cells in the portal areas of the liver. However, no significant difference was found in the number of cells counted in either the 48/80 or control groups when comparing the data from several histochemical methods. These results provided evidence that: (1) IFC demonstrate both fluorescence and metachromasia and, therefore, are mast cells, (2) compound 48/80 causes a comparable decrease in the number of serotonin and histamine fluorescent as well as metachromatic cells, indicating concomitant rather than differential release of serotonin, histamine, heparin, and/or other metachromatic substances, and (3) each of the three histochemical methods appears of equivalent sensitivity when used to study the effect of various factors (i.e., 48/80) on the release of endogenous substances from IFC. The results of this study indicate that the effects of 48/80 in vivo might be mediated through the release of various vasoactive substances from these IFC (mast cells).
Control, polycythemic, and anemic spleens of Sprague-Dawley rats surgically removed under pentobarbital sodium anesthesia (0.06 mg/g ip) were perfused in vitro by methods similar to those reported by Song and Groom (Am. J. Physiol. 220: 779-784, 1971). Red Blood cell (RBC) washout curves obtained for each group could be described adequately by a series of three exponentials--fast, intermediate, and slow--representing functional compartments in the spleens. Best-fit estimates of the parameters were used to characterize the flow distributions, cellular capacities, and t1/2 for washout of these components. It is thought that the fast compartment represents flow through intrasplenic vessels (including shunts) that bypass the red pulp, the intermediate compartment represents RBCs that flow through less impeded passageways in the red pulp, and the slow compartment consists of more tortuous, convoluted pathways through the red pulp. During polycythemia and anemia, inflow to the intermediate component was decreased, which suggested decreased filtration. In the slow compartment, the shutdown during polycythemia suggested RBC storage whereas during anemia control levels of inflow but increased t1/2 for washout suggested sequestration or erythropoiesis.
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During the course of a neurohistochemical and two independent electron microscopic studies of the mouse spleen, unmyelinated adrenergic nerves containing numerous dense core and lucent vesicles and devoid of neurolemma were observed adjacent to reticular cells and lymphocytes in the white pulp. Some of these nerves formed an intimate relationship with these cells. Since adrenergic substances have been reported to modulate the cell cycle of lymphocytes in vitro, these findings are suggestive of a neural influence on the cell cycle of lymphocytes in vivo.
In vivo microscopic study of the responses of the various segments of the hepatic microvascular system to adrenergic, cholinergic, and other aminergic substances and their antagonists have revealed the relative distribution of receptor sites in these various vessels. The results also suggest a possible interaction of cholinergic substances with periportal mast cells. The release of serotonin, histamine and possibly other products from these cells results in dramatic alterations in blood flow through the hepatic sinusoids due to the adhesion of leukocytes and platelets to the endothelium.
The intrahepatic distribution of nerves in the rat was studied using neurohistochemical and electron microscopic methods. Innervation was restricted primarily to vessels in the portal space and hilus. Both adrenergic and cholinergic fibers were observed in the adventitia of hepatic arteries, and to a lesser extent adjacent to portal veins. Some of the cholinergic fibers, however, were not contiguous with the vasculature. Near the hilus many of these fibers were associated with ganglia while peripherally some coursed into the immediately adjacent parenchyma where end bulbs abutted on hepatocytes. Ultrastructurally, scattered small nerves, devoid of neurolemma, were found contiguous with the portal lamina of hepatocytes. Nerve fibers deeper within the lobule were not seen but numerous gap junctions were observed between contiguous hepatocytes. Central and sublobular hepatic veins lacked innervation but adrenergic nerves were demonstrated in the walls of larger hepatic veins. Innervation of the biliary system was sparse. While nerves were interposed between vessels and bile ducts, such nerves tended to be associated more closely with the vasculature.
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The use of improved in vivo microscopic methods has permitted a reevaluation of the "open" vs. "closed" theory of blood flow through the splenic red pulp. The microscopic methods used for studying the spleen in vivo are described as are the results, both of which were presented in motion picture form at this symposium. The results strongly suggest that, in general, there is little or no continuity of endothelium through the splenic red pulp. Instead, blood flows from "arterial" capillaries lined with endothelium into channels within the red pulp formed by the cytoplasmic processes of reticular cells. In vivo these processes appear similar to endothelium. Blood leaves the red pulp by passing through apertures in the endothelium of sinuses and venules.
Metallic chambers were implanted into the proximal tibiae of rabbits to permit microscopic examination of living bone in situ. The bone repair process secondary to the injury produced during installation of the chamber, was visualized. Six to 8 weeks after implantation, osteoid and/or bone could be seen. The effects of various doses of disodium ethane-1-hydroxy-1, 1-diphosphonate (EHDP) on the repair and regeneration processes following chamber implantation were studied. Data from various techniques indicated that: (1) following low dose EHDP (0.25 mg/kg/day) chambers contained bone tissue morphologically and ultrastructurally indistinguishable from controls; and (2) with higher doses of EHDP (2.5 or 10 mg/kg/day) chamber contained spicules of normal osteoid, osteoblasts and osteocytes, but were devoid of osteoclasts. The effects of the various regimes of EHDP also were assessed on regenerated, trabecular bone contained within the tibia chambers three months after implantation of the chambers. Data from various methods of analysis supported the following conclusions: (1) the low dose of EHDP (0.25 mg/kg/day) had no toxic effects on the trabecular bone within the chambers but there appeared to be an increase in bone formation as compared to saline control; (2) higher doses of EHDP (2.5 or 10mg/kg/day) were not toxic to bone cells but thick osteoid seams formed on the trabecular bone within the chambers. No osteoclasts were found associated with the bone apparently due to the coverage of bone surfaces by osteoid seams; and (3) osteoid which accumulated after EHDP treatment of 2.5 mg/kg/day for 2 months remained uncalcified for as long as 2 months following withdrawal of EHDP administration. The results showed the value of tibial chamber for examining microscopically living bone in situ and demonstrated the inhibitory effect of EHDP on mineralization of newly formed osteoid and a lack of effect on bone cells.
Neurohistochemical techniques were used to confirm morphologically the distribution of adrenergic and cholinergic nerves to the splenic microvasculature. The results form the basis of this report. Using these methods, adrenergic innervation was observed only in the adventitia of arteries and arterioles. No cholinergic innervation was found in this site. No adrenergic or cholinergic innervation could be demonstrated to the channels of the red pulp, venules or veins. These data provided morphological evidence that in the murine spleen only splenic arteries and arterioles are innervated; and these have only an adrenergic innervation.
Extruded filaments of unmodified polypropylene (PP) with and without antioxidant were implanted subcutaneously in hamsters in order to determine their rate of degradation. Specimens were removed periodically during a 5 month test period and analyzed by infrared spectroscopy and dynamic mechanical testing. The analyses show that degradation beigns to occur after only a few days. Although the reaction sequence is not known, several factors suggest that the in vivo degradation process is similar to autoxidation which occurs in air or oxygen. The infrared data indicate that the hydroxyl content of the implants increases at a rate of 0.061 mg/g polypropylene per day during the initiation phase of the reaction. An induction time of 108 days was extablished. Carbonyl bonds appear after an implantation time of 50--90 days and increase therafter. Mechanical tests indicate a decrease in the dynamic loss tangent, tan delta, during the first month of implantation for unmodified polypropylene. No change in the infrared spectra or tan delta was observed, however, for implants containing an antioxidant. Thus, it is apparent that polypropylene filaments implanted subcutaneously in hamsters degrade by an oxidation process which is retarded effectively by using an antioxidant. While the findings reported are specific to subcutaneous polypropylene implants, they suggest that degradation of other systems may involve similar processes. This notion suggests directions for further research on increasing the in vivo stability of synthetic polymers. Long-term effects of polymer implantation upon tissue were not studied in this work.
Within a transparent chamber enclosing subcutaneous tissue of the hamster cheek pouch, allogeneic femoral marrow was grafted. This permitted in vivo microscopic study of the grafts for 14 days. Five to seven days after grafting, blood flow was established within sinusoids arranged in interconnecting polygonal networks, a pattern characteristic of marrow. All vessels appeared to have a complete and continuous endothelial lining. The graft contained foci of erythropoiesis, granulopoiesis, and megakaryocytopoiesis, but no osteogenic activity. Degeneration of the grafts began to occur between days 9 and 13 after implantation. Generally, this was preceded by infection within the chamber, fibroblastic proliferation, and an accumulation of acid mucopolysaccharide in the stroma. The concomitant loss of hemopoiesis and accumulation of acid mucopolysaccharides was consistent with our hypothesis reported previously that excessive concentrations of acid mucopolysaccharide in the hemopoietic microenvironment are not conducive to supporting hemopoiesis, especially erythropoiesis. Degeneration of the grafts probably was the result of infection since there was no evidence of an immunologic response to the graft, and in chambers not containing grafts, similar infections and changes in the connective tissue were observed. The technique of grafting marrow into the hamster cheek pouch chamber provides a model for long-term, in vivo microscopic study of bone marrow. However, methodologic improvements, especially in the control of infections, are needed.
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