Kazuki Sakata

Pathophysiology of aganglionic colon and anorectum: An experimental study on aganglionosis produced by a new method in the rat

• Aganglionosis was produced in the rat by applying 0.1% benzalkonium solution for 30 min to the outer surface of the descending colon and the anorectum of a 1 cm long segment. This method was named the experimental anganglionosis-producing procedure or EAPP method. By this method, the aganglionic portion was found to be narrowed and the proximal segment become dilated as in megacolon. When the chemical was applied to a segment 3cm long or more, the animals succumbed. The portion treated with this method was confirmed to be dener-vated by Nissl stain, Bodian stain, supervital methylene blue stain, acetylcholinesterase stain and catecholamine fluorescence method. In the aganglionic colon produced by EAPP, the normal inhibitory reaction responding to different stimulations (intraluminal balloon inflation, electrical stimulation, or mechanical stimulation by pinching) to the proximal segment was found to be abolished. In the aganglionic anorectal segment produced by EAPP, the normal inhibitory reaction, recorded at the level of the internal sphincter muscle, to stimluations same as described above (i.e., the reaction similar to the recto-sphincteric reflex) was found to be abolished. The aganglionic segment produced by EAPP was confirmed to be functionally denervated and to have functionally intact smooth muscles by the fact that the segment examined in vitro was unresponsive to physostigmine but responsive to barium chloride showing normal contraction waves. The results of present study lend support to the assumption that aperistalsis observed in an aganglionic intestinal segment is caused by defect of intramural nerve plexi.

Selective destruction of intestinal nervous elements by local application of benzalkonium solution in the rat

Intestinal aganglionosis produced by serosal application of 0.1% benzalkonium solution to the colon of the rat was studied electronmicroscopically, and its was concluded that a higher susceptibility to the agent and a lower recovering ability of the nerve elements might be responsible for the phenomenon.

Late appearance of resistance to tumor rechallenge following cryosurgery: A study in an experimental mammary tumor of the rat

Abstract Resistance to tumor challenge following surgical and cryosurgical eradication of the tumor was studied, using an experimental mammary tumor of the rat, MRMT-1. It was revealed that rejection rate of the challenged tumor increased gradually following cryosurgery and reached its peak at 10 weeks after cryosurgery. No such phenomenon was observed after surgical excision of the tumor. Decreased incidence of lymph node metastases and decreased tumor weights in “take” cases also suggested an increased immunological activity against the tumor at 10 weeks after cryosurgery.

Immunological response of regional lymph nodes after tumor cryosurgery: Experimental study in rats

Seven days after inoculation of metastasizing rat mammary tumor No. 1 into the thigh of 5-week-old female Sprague-Dawley rats, the tumor was treated cryosurgically by two-cycle freezing and by contact methods at -170 degrees C. Weights of the thymus and the spleen, histological findings of the lumbar lymph nodes, phytohemagglutinin (PHA)-induced blastogenesis of lymphocytes obtained from the lumbar lymph nodes and peripheral blood, and resistance rate to tumor rechallenge were examined 1, 3, 6, 10, and 17 week(s) after cryosurgery, with the following results: Thymus weight gradually decreased by 3 weeks after cryosurgery, while spleen weight increased by 1 week, recovering the preoperative level at 6 weeks. Paracortical hyperplasia of the lumbar lymph nodes markedly increased in 1 week and sinus histiocytosis increased after 3 weeks, both remaining at high values until 10 weeks, while germinal center hyperplasia showed a high value at 3 weeks and thereafter decreased gradually. PHA-induced blastogenesis of the lumbar lymph nodes significantly increased 1 week after cryosurgery and remained at its high value until 10 weeks. PHA-induced blastogenesis of peripheral lymphocytes showed the lowest value at 3 weeks and then significantly increased at 6 weeks. Resistance rate to rechallenge showed the lowest value at 3 weeks, reaching the highest level 10 weeks after cryosurgery. From the above results, it was suggested that anti-tumor immunity (resistance to tumor rechallenge) induced by cryosurgery was at the lowest level at 3 weeks after cryosurgery, and gradually increased starting at 6 weeks.(ABSTRACT TRUNCATED AT 250 WORDS)

Antitumor immunologic reactivity in the relatively early period after cryosurgery: experimental studies in the rat.

Abstract An experimental investigation was performed on antitumor immunity in the relatively early postoperative period after cryosurgery, using a metastasizing rats mammary tumor, MRMT-1. Two weeks after its inoculation, surgical excision of the tumor, cryosurgery, surgical excision plus inoculation with freezing-thawing produced vaccine, or surgical excision plus fasting for 72 hr was performed, and postoperative follow-up was done on incidences of metastases, those of metastatic death, etc. Specific immunologic reactivity was examined in the surgical excision (SE) and cryosurgery (CR) groups. The FTV and fasting groups showed more metastatic deaths as compared with the SE group. The CR and SE groups did not differ significantly from each other in incidences of lung and lymph node metastases. Specific footpad reactivity at 2 and 3 weeks after treatment was lower in the CR group than in the SE group. Winns neutralization assay showed an inhibition of tumor growth at 1 and 3 week(s) after treatment both in the SE and in the CR groups, the inhibitory effect tending to be lower in the latter. Inactivated serum obtained at 1 week after treatment showed a facilitation of tumor growth in the SE group and a tendency of tumor suppression in the CR group, showing a significant difference between them. A mild reduction in antitumor immunity seen in the relatively early postoperative period following cryosurgery probably was not due to a blocking effect by superfluous antigens. Rather it was considered to be due to activation of suppressor cells, consequent on cryosurgical stress, and/or slow and steady absorption of antigens.

Experimental study on mechanism of absorption of cryonecrotized tumor antigens

On Day 7 of subcutaneous MRMT-1 tumor inoculation in the thigh of Sprague-Dawley rats, the tumor was treated cryosurgically by contact method. Postoperative changes in local blood circulation were observed by colloidal carbon perfusion and hydrogen-clearance methods, and the pathway and time course of tumor antigen absorption were observed by measuring uptake in serum and lymph nodes of [3H]thymidine injected intratumorally. Results were: At 0.5 to 1 hr after cryosurgery, vascular stasis, dilatation, and tortuosity were observed. There was no inflow of carbon and almost no uptake of [3H]thymidine. At 6 hr, vascular stasis and sludging become more marked. There was no inflow of carbon, while uptake of [3H]thymidine markedly increased. At 24 hr, some carbon inflow in fin vessels and partial recovery of blood circulation in the peritumoral region were observed. Rate of [3H]thymidine uptake was further increased. At 72 hr, carbon inflow and blood circulation increased further, while the cryonecrotized tumor showed hyaline degeneration. At 168 hr, increase of newly formed vessels and recovery of blood circulation were remarkable, but uptake of [3H]thymidine was decreased in parallel with increased demarcation of the cryonecrotized tumor. From these results, it was suggested that the absorption of cryonecrotized tumor antigens started through lymphatic channels in the early period and then took place also through newly formed capillaries surrounding the tumor at 24 hr after cryosurgery and continued until 72 hr after cryosurgery.

Effect of Intralipid-infusion on erythrocyte membrane in rabbits

Through Intralipid infusion in rabbits, the phospholipids derived from Intralipid were incorporated into erythrocytes, although Intralipid is mainly composed of triglycerides. This is supported by the increase in oleic acid and the compensatory decrease in linoleic acid of the phospholipids in the erythrocyte membrane, corresponding to the content of linoleic acid in the phospholipids from Intralipid. The excess phospholipid rendered the membrane more fluid, probably by overwhelming the rigidifying effect of the increased cholesterol content. Furthermore, the shape of erythrocytes was changed from biconcave to spur, dose dependently. The morphological alterations in erythrocyte membranes could not be completely elucidated by the changes in lipid. These results suggested that the alteration in lipid metabolism in Intralipid-infused rabbits caused various effects on the erythrocyte membrane, through the elevation of triglyceride, cholesterol, and phospholipid contents in plasma.

Stress ulcer accompanying subarachnoid hemorrhage—A new rat model

New rat model of stress ulcer, related to subarachnoid hemorrhage (SAH) was devised. A 0.2 ml of the arterial blood, obtained from another rat of the same litter, was injected into the cisterna magna and 4 days later, mild stress, consisting of restraint plus water-immersion for 3 hours to the injected rat, produced gastric ulcers. These lesions were far more marked than in usual rats undergoing the same stress (P<0.001). Using this “SAH-stress model”, effects of various counteracting measures were examined. Based on the results, we concluded that SAH produced a hypersensitivity or an abnormal exitability of the autonomic nervous centers, and the relatively mild stress effectively weakened the gastric defensive factors and strengthened the gastric aggressive factors, thus resulting in ulcer formation.

Neurogenic stress ulceration caused by laparatomy under anesthesia plus restraint. The device of a new rat model

We report herein, a new method devised of producing neurogenic stress ulceration in rats. An experimental subarachnoid hemorrhage (SAH) was produced in rats by injecting 0.2 ml of arterial blood from other rats into the cisterna magna. Three days later, the rats were laparotomized for 1 hour under ether anesthesia, followed by restraint for 3 hours in wakefulness. The SAH rats were found to develop stress ulcers (UL-I) in the glandular stomach, which were significantly (p<0.001) more marked than those in non-SAH rats. Measurements were performed on gastric acid secretion, an important aggressive factor. It was found that the SAH rats undergoing the laparotomy-restraint stress showed a more marked increase in gastric acid secretion and a more marked reduction in MBF, than the non-SAH rats. The effects of bilateral vagotomy, upper abdominal sympathectomy and bilateral adrenalectomy were examined, and it was revealed that the SAH rats were under the condition of hyperreactivity both in the sympathetic and parasympathetic systems and, on this basis, the laparotomy-restraint stress caused the stress gastric ulceration. In this rat model, the laparotomy stress was applied under anesthesia and any exposure to low temparatures which may have interfered with blood circulation was avoided.

Experimental studies on intestinal absorption following Martin's operation

Experimental studies were done on rats on compensatory absorptive capacity following Martins operation for extensive aganglionosis. Experimental aganglionosis was produced in the descending colon of rats by serosal application of 0.1% benzalkonium chloride solution. Wide side-toside anastomosis was performed between the aganglionic colon and the distal ileum, removing the remaining colon. As to absorptive capacity of water and electrolytes, this experimental intestine was compared, with other intestines, especially control intestine, in which similar side-to-side anastomosis was done between the normal colon and the normal ileum. “Experimental” as well as control intestine showed higher absorptive capacities of water, Na and Cl per unit length than did summed up values of the ileum and the colon per unit length. Postoperative body weight curves showed fairly good increases and appearance of feces showed a fair improvement in the experimental group. Autopsy of experimental intestine revealed marked dilatation of the anastomosed ileum and mucosal hypertrophy of the anastomosed colon. These results suggested a favorable compensatory absorptive capacity following Martins operation.