To establish a method for processing lymphoid organs suited to morphological, immunohistochemical and enzyme histochemical analyses for assessment of immunotoxicity, we examined a combination of fixation with periodate-lysine-paraformaldehyde (PLP) fixative and embedding in paraffin by the AMeX method (PLP-AMeX method). Spleen and thymus removed from monkeys and rats were fixed in PLP fixative for 6 hours at 4 degrees C. After fixation, specimens were processed and embedded in paraffin by the AMeX method. In hematoxylin and eosin-stained sections, tissue architecture was well preserved. In immunohistochemical staining, markers of T lymphocytes (CD3, CD4, CD8), B lymphocytes (monkey: CD20cy, rat: CD45RA) and macrophage (monkey; CD68, rat: ED-1) were well identified according to their specificities, although the staining intensity of CD8 in the monkey and CD4 in the rat were somewhat weaker in PLP-AMeX-prepared sections than in those frozen. In enzyme histochemical staining, alkaline phosphatase activity was well preserved in neutrophils. In toluidine blue- and Giemsa-stained sections, eosinophil granules and the metachromasia of granules in basophil/mast cells were clearly detectable. These findings suggest that the PLP-AMeX method is a powerful tool for assessment of immunotoxicity.

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Granulocyte colony-stimulating factor (G-CSF) is widely used to mobilize peripheral blood stem cells, and expected to restore cardiac function for patients with coronary artery diseases as a consequence of progression of atherosclerosis. Safety issues related to the administration of G-CSF to these patients, however, are still under study. The animal model for atherosclerosis was produced by feeding miniature swine a high-cholesterol diet for 3 months. G-CSF (5 or 10 microg/kg/day) was given to the animal model by daily subcutaneous injections for 10 days and 20 main arteries were evaluated pathologically. In addition, the general toxicological effects were studied on clinical signs, body weight, hematology, blood chemistry and pathology. In the G-CSF-treated groups, a variety of changes related to the major pharmacological activity of G-CSF including an increase in white blood cell (WBC) counts were observed. In many arteries, atherosclerotic lesions similar to Type I-V of the proposed classification by the American Heart Association were observed. No effects of the G-CSF treatment were seen on the histopathological findings, incidence, severity or distribution of atherosclerotic lesions. In addition, no infiltration of neutrophils to the lesions was observed. These findings suggest that the administration of G-CSF causes neither exacerbation or modification of atherosclerotic lesions nor adverse changes despite that a sufficient increase in WBC counts could be achieved in the peripheral blood.

Abstract:The authors have previously reported that the combination of fixation with periodate-lysineparaformaldehyde (PLP) fixative and embedding in paraffin by the AMeX method (the PLP-AMeX method) resulted in better preservation of immunoreactivities and endogenous enzyme activities. In the present study, we investigated whether PLP-AMeX-processed tissue would be useful for in situ hybridization (ISH). The pancreases were removed from rats treated with or without streptozotocin (STZ), and was fixed in PLP fixative. After fixation, the specimens were processed and embedded in paraffin by the AMeX method. Paraffin sections were made on which ISH and immunohistochemical staining were performed. ISH was performed according to the non-radioactive method using digoxigenin-labeled oligodeoxynucleotides probes and anti-digoxigenin antibody. For ISH, a 28S rRNA antisense probe was used for evaluation of the level of hybridizable RNA in the sections. 28S rRNA was clearly detected in the various cells of the pancreas. Using an insulin antisense probe, the presence of insulin mRNA was clearly identified in the pancreatic islet-cells. Furthermore, optimal results for ISH of insulin mRNA and immunohistochemical staining of insulin could be obtained in consecutive sections from a PLP-AMeX-processed block. In the rats treated with STZ, the histological changes of pancreatic islet-cells and the changes in staining intensity of immunohistochemical staining for insulin protein and ISH for mRNA could be observed in the PLP-AMeX-processed sections. These findings suggest that hybridizable RNAs are well preserved by the PLP-AMeX method, and that the method is useful for ISH as well as immunohistochemical and enzyme histochemical analysis. (J Toxicol Pathol 2004; 17: 171-176)