Eisuke Kume

Hepatic changes in the acute phase of streptozotocin (SZ)-induced diabetes in mice.

We have reported the streptozotocin (SZ)-induced hepatic lesions in the subacute phase (4 to 12 weeks after the treatment), which are characterized by appearance of oncocytic hepatocytes, cytomegalic hepatocytes and bile duct hyperplasia. In this study, we focused on the acute phase (6 to 48 hours after the treatment) of the SZ-induced hepatic lesions of mice to clarify the onset of the hepatic alterations, especially before the induction of hyperglycemia. Livers were taken from 8-week-old Crj:CD-1 (ICR) male mice at 6, 12, 24, 36 and 48 hours after the 200 mg/kg b.w. of SZ-injection. SZ-induced hyperglycemia was noted at 36 and 48 hours after the treatment, but the hepatic changes including lipid peroxidation, mitochondrial swelling, peroxisome proliferation and inhibition of hepatocyte proliferation occurred before the elevation of the serum glucose levels. The present findings indicate the direct effects of SZ on hepatocytes rather than the secondary effects of diabetes, and certain correlations between the hepatocytic changes in the acute phase and those in the subacute one. In addition, ulcer and submucosal edema of the gallbladder were observed at 36 or 48 hours after the SZ-treatment, which can be a novel finding in SZ-treated animal.

Hepatic changes of mice in the subacute phase of streptozotocin (SZ)-induced diabetes

Hepatic changes of mice in the subacute phase of streptozotocin (SZ)-induced diabetes were investigated biochemically and pathologically. Biochemically, the contents of serum glucose and of serum and liver lipids increased while the content of liver glycogen decreased in SZ-induced diabetic mice. Histopathologically, hypertrophy of hepatocytes due to an increase in number of intracytoplasmic acidophilic granules was common to SZ-induced diabetic mice. Electron microscopically, these hepatocytes were characterized by a prominent increase in number of mitochondria showing normal structure, a marked decrease of glycogen granules and poorly developed rough endoplasmic reticulum, which were common to so-called oncocytic cells. In some SZ-induced diabetic mice, bile duct hyperplasia with an appearance of cytomegalic hepatocytes was also observed.

Morphologic changes in hepatocyte nuclei of streptozotocin (SZ)-induced diabetic mice

Morphological examinations were carried out on hepatocyte nuclei of streptozotocin (SZ)-induced diabetic mice. The area of hepatocyte nuclei in diabetic mice was about two times larger than that in control mice, and the incidence of hepatocytes with intranuclear inclusions was 3.4 +/- 0.2% in diabetic mice and 0% in control mice, respectively. Although the incidence of binuclear hepatocytes was not significantly different between diabetic (14.5 +/- 4.6%) and control mice (16.4 +/- 4.4%), the morphology of the nuclei of binuclear hepatocytes was apparently different between diabetic and control mice. Namely, the nuclei of binuclear hepatocytes of control mice were round and identical in ultrastructural appearance, and they did not differ from those of mononuclear diploid hepatocytes. On the other hand, the nuclei of binuclear hepatocytes of diabetic mice were not identical in distribution pattern of chromatin granules, and they frequently varied in size and showed irregular contours.

Quality assessment of long-term stored formalin-fixed paraffin embedded tissues for histopathological evaluation

Histopathological examination of formalin-fixed paraffin-embedded (FFPE) tissues that had been stored for 30 years was conducted, and reconstructivity of the results was verified. These FFPE tissues, which were from all organs of male and female rats, were re-sectioned and histopathologically examined using hematoxylin and eosin (HE) staining. In particular, the stainability and morphology of HE sections and reproducibility of microscopic findings in the liver and kidney demonstrated in the original final reports were evaluated. Although the stainability of hematoxylin was slightly weaker and some morphological artifacts were observed in tissues in re-prepared slides, these deteriorations in the quality of HE sections were considered to be permissible for histopathological examination so long as control sections were also prepared. Most microscopic findings recorded in the original final reports were confirmed using re-prepared HE sections in the present study. While some focal findings, which were judged to be either incidental or spontaneous in nature, were not observed in the sections as expected, this was not considered to be a problem in reconstructing the results of the original histopathological examination because most findings related to the test articles were generally observed diffusely or multifocally in each organ. We concluded that results of the original histopathological examinations could be reconstructed using paraffin blocks that had been stored for up to 30 years.

Role of endotoxin in 6-sulfanilamidoindazole(6SAI)-induced arthritis in rats.

6-Sulfanilamidoindazole (6SAI) induces selflimiting arthritis in rats. Since close relationships exist between arthritis and endotoxin, four experiments were conducted to clarify the relationship between endotoxin and 6SAI-induced arthritis. Endotoxin levels in the plasma from the abdominal aorta and portal vein from rats that had 6SAI (500 mg/kg) administered orally for up to 7 days remained within the control values at day 1 and day 3, and were significantly elevated at day 7. Endotoxin levels in the synovial fluid from the same rats showed no significant change. Ankle swelling and redness in rats treated 11 consecutive days with 6SAI did not ameliorate when coadministered with an anti-endotoxin agent, polymyxin B sulfate. Histopathological examination on the ankles of rats treated orally with non-arthiritogenic sulfonamides including sulfonamide, sulfamethoxazole and sulfadimethoxin (250 and 500 mg/kg/day, each compound) for 2 weeks demonstrated no inflammatory changes, while hyperplasia/hypertrophy of thyroid epithelial cells were frequently observed. When histopathological changes in the ankles from rats coadministered with 6SAI and lipopolysaccharide (LPS, Escherihia coli O55:B5, 50 microg/kg, i.v.) were compared with those in rats treated with 6SAI or LPS alone, the ankles from the 6SAI+LPS treated animals had marked edematous inflammation in the synovium and surrounding connective tissues, whereas the LPS-group had only mild focal infiltration of polymorphonuclear leukocytes in the synovium and the 6SAI-group showed no apparent changes. These results suggest that endotoxin is not a direct cause but a possible acceralating factor of 6SAI-induced arthritis, and that the effects of 6SAI on gut bacteria is not related with the pathogenesis of this model.