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Immunohistochemical staining was performed to further characterize 26 ENU-induced brain tumors (8 glioblastomas, 11 astrocytomas, and 7 oligodendrogliomas) from offspring of 20 Fischer 344 rats that had been treated with 20 mg/kg ENU intravenously 4 days before giving birth. Unstained slides from each tumor were stained with hematoxylin and eosin, as well as a panel of single-label immunohistochemical stains. These included α-glial fibrillary acidic protein (GFAP) antibody (recognizes a specific antigen on rat astrocytes), ionized calcium-binding adapter molecule 1 (Iba-1) antibody (recognizes a specific antigen on rat microglia and macrophages), and oligodendrocyte transcription factor 2 (Olig2) antibody (recognizes a specific antigen on rat oligodendrocytes). Introduction: N-ethyl-N-nitrosourea (ENU) is an alkylating agent that is used extensively in experimental neuro-oncogenesis. In rats, ENU causes neural neoplasms in up to 100% of offspring of dams given a single dose in the second half of gestation. Experimental Design: Immunohistochemical staining was performed to further characterize 26 ENU-induced brain tumors (8 glioblastomas, 11 astrocytomas, and 7 oligodendrogliomas) from a study in which 20 pregnant Fischer 344 rats were given 20 mg/kg ENU intravenously 4 days before giving birth. Materials and Methods: Hematoxylin & eosin slides were prepared from each animal. Immunohistochemical stains included glial fibrillary acidic protein (GFAP), ionized calcium-binding adapter molecule 1 (Iba-1), and oligodendrocyte transcription factor 2 (Olig2). Results: A total of 19 tumors were observed and diagnosed as oligodendrogliomas. Neoplastic cells stained most consistently with Olig2. There were 5 well-differentiated oligodendrogliomas. The remaining 14 tumors exhibited considerable cellular anaplasia and corresponding variability in Olig2 staining. These tumors also contained greater numbers of Iba-1+ microglial cells (diffuse) and GFAP+ astrocytic cells (largely distributed at the tumor edge and around blood vessels). The “ependymoma-like,” pseudorosette features of ENU-induced tumors stained positively for Iba-1, suggesting reactive microglia/macrophages. Conclusion: ENU-induced neoplasms consisted of variably differentiated oligodendrogliomas. While some tumors were well-differentiated, others were anaplastic with robust secondary infiltrates of reactive cells (microglial, astrocytic, endothelial). Impact Statement: A previous study indicated most spontaneous tumors in the rat were oligodendrogliomas, while chemically-induced neoplasms were malignant microglial tumors. Results for ENU are unique thus far for its induction of variably differentiated oligodendrogliomas. Abstract While brain tumors are rare in untreated rodents, rats are the most susceptible species of rodent to the development of brain tumors (Rice, 2000). N-ethyl-N-nitrosourea (ENU) and N-methyl-N-nitrosourea (MNU) are used extensively in experimental neuro-oncogenesis. ENU is an alkylating agent that is considered to be among the most carcinogenic chemicals. ENU causes neural neoplasms in up to 100% of offspring of rats given a single dose during the second half of pregnancy (Druckrey, 1966; Koestner, 1971). This investigation was undertaken to further characterize the cell of origin in ENU-induced brain neoplasms in Fischer 344 rats. Results for ENU are unique thus far for its induction of variably differentiated oligodendrogliomas. Neoplastic cells within ENU-induced tumors generally showed expression of Olig2. Olig2 staining was strong in the 5 well-differentiated tumors and variable in the 14 poorly differentiated tumors. Admixed with the Olig2+ cells in many of the tumors was a subpopulation of Iba-1+ cells, and the number of Iba-1+ cells was greater in poorly differentiated tumors. Iba-1+ cells were diffusely scattered with occasional cuffing around necrotic areas (reactive microglial cells). GFAP+ cells were present in small numbers within the neoplasms and were primarily concentrated along the periphery and surrounding blood vessels (reactive astrocytes). A previous study indicated most spontaneous tumors in the rat were oligodendrogliomas, while chemically-induced neoplasms were malignant microglial tumors (Kolenda-Roberts, 2013). Results for ENU are unique thus far for its induction of variably differentiated oligodendrogliomas. Druckrey H, Ivankovic S, Preussmann R. Teratogenic and carcinogenic effects in the offspring after a single injection of ethylnitrosourea to pregnant rats. Nature. 1966. 210: 1378-1379. Koestner A, Swenberg JA, Wechsler W. Transplacental production with ethylnitrosourea of neoplasms of the nervous system in Sprague- Dawley rats. Am J Pathol. 1971. 63: 37-56. Kolenda-Roberts H, Singletary E, Hardisty J. Immunohistochemical characterization of spontaneous and acrylonitrile-induced brain tumors in the rat. Toxicologic Pathology. 2013. 41(1): 98-108. Maekawa A, Mitsumori K. Spontaneous occurrence and chemical induction of neurogenic tumors in rats – influence of host factors and specificity of chemical structure. Critical Reviews in Toxicology. 1990. 20(4): 287-310. Rice JM, Wilbourn JD. Tumors of the nervous system in carcinogenic hazard identification. Toxicologic Pathology. 2000. 28(1): 202-214. Introduction References Methods Conclusion Discussion Results Immunohistochemical Characterization of ENU-induced Brain Tumors in F344 Rats Rebecca R. Moore 1 , Holly Kolenda-Roberts 2 , Nancy A. Harris 1 , Young-Man Cho 3 , Kumiko Ogawa 3 , Jerry F. Hardisty 1 , Rodney A. Miller 1 1 Experimental Pathology Laboratories, Inc., Research Triangle Park, NC, USA; 2 SNBL USA, Ltd, Everett, WA, USA; 3 National Institute of Health Sciences, Tokyo, Japan Well-differentiated Poorly differentiated/anaplastic H&E Olig2 Iba-1 GFAP IHC stain for Olig2 in poorly differentiated brain neoplasm showing anisokaryosis and binucleation of Olig2+ cells (neoplastic oligodendrocytes). IHC stain for Iba-1 in poorly differentiated brain neoplasm showing cuffing of Iba-1+ cells (reactive microglial cells) around area of necrosis.
