“Immunohistochemical Characterization of ENU-induced Brain Tumors in F344 Rats.” Moore RR (presenter), Kolenda-Roberts H, Harris NA, Cho YM, Ogawa K, Hardisty JF, Miller RA. The 33rd Society of Toxicologic Pathology Annual Meeting. Washington, DC. June 22-26, 2014. For full-resolution viewing, please open or save file as a PDF.

1. 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
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ethylnitrosourea of neoplasms of the nervous system in Sprague-
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Kolenda-Roberts H, Singletary E, Hardisty J. Immunohistochemical
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Introduction
References
Methods
Conclusion
Discussion
Results
Immunohistochemical Characterization of ENU-induced Brain Tumors in F344 Rats
Rebecca R. Moore1, Holly Kolenda-Roberts2, Nancy A. Harris1, Young-Man Cho3, Kumiko Ogawa3, Jerry F. Hardisty1, Rodney A. Miller1
1 Experimental Pathology Laboratories, Inc., Research Triangle Park, NC, USA; 2SNBL USA, Ltd, Everett, WA, USA; 3National 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.