Light and Electron Microscopic Study on the Mode of Cell Death Induced by Methotrexate Administration in the Crypt and Villus of Rat Small Intestine

Jung, Yang Ho; Nam, Kwang Il; Oh, Chang Seok; Ahn, Kyu Youn; Bae, Choon Sang; Kim, Baik Yoon; Park, Sung Sik

Abstract

Methotrexate (MTX), folic acid antagonist, is effective against the cancer, but is often accompanied by damage to the small intestine due to its inhibition of cell division. The nature of cell damage induced by MTX on the surface lining and crypt cells of the small intestine was investigated in the rat receiving MTX administration. MTX was injected once in every other day intraperitoneally to rats [0.03, 0.1, and 0.3 mg per 100 g body weight in small (SD), large (LD), and over dose (OD) groups, respectively] and the animals were sacrificed with various intervals after single, three-, five-times administrations of SD, single, three-, four-times administrations of LD, and single administration of OD. In case of three times injection of LD, intravenous injection was also made. Three portions, duodenum, jejunum, and ileum of the small intestine were studied by light and electron microscopy. Histologically, there were no appreciable morphological changes in villi and crypts after single and three times administrations of SD. The cells contained dense nucleus with clear cytoplasm, however, were rarely presented among the crypt cells. The duodenal villi were relatively normal in shape, but the number of crypts were somewhat decreased in five times administrations of SD. On the other hand, the villi of jejunum and ileum were atrophied and the epithelial cells became flattened. Crypts were atrophied and decreased in number. In single administration of LD, the shape of villi were normal in all of three portions. The cellular damages were noted in crypts of all of them, and the severity was most predominant at 6 hours than at 2, 4, or 24 hours after administration. In three times administrations of LD, more serious damages were noted in intravenous injection groups than intraperitoneal injection groups. Villi and crypt of the jejunum and ileum became atrophy, and epithelial cells were vacuolated. In four times administrations of LD, loss of villi and atrophied glands contained cellular debris within their lumen were noted in all three portions. In single administration of OD, there was no remarkable changes in histological structures of duodenum, jejunun, and ileum. With TUNEL staining, positive cells were present mainly in the crypt. The positive cells appeared among the glandular cells and were consistent with dense nucleus contained cells in hematoxylin-eosin stain. In small dose group the number of apoptotic cells in duodenum and ileum was increased in proportion to the times of administration, but that in jejunum was decreased after three times of administrations because of the destruction of crypts. Most prominent apoptotic changes of crypt cells were noted at 6 hours compare with 2, 4, and 24 hours after three times administrations of SD. Positive cells appeared most numerous at 6 hours after single administration of LD and OD. Destructive changes of crypt were found in jejunum and ileum after three times intravenous injection of LD, and were found in all portions after four times administrations of LD. Electron microscopic observation generally confirmed the findings of light microscopy. The apoptotic cells appeared in the crypt and was characterized by chromatin condensation and fragmentation, cytoplasmic condensation. Notable changes were observed in epithelial and crypt cells after five- and four-times administrations of SD and LD, respectively. The epithelial cells became flat and had many fat droplets in the cytoplasm. The crypts were atrophied and the glandular cells were very flat. These changes were more severe in jejunum and ileum than in duodenum. These results indicate that the damage of small intestine by MTX is more severe in jejunum and ileum than in duodenum, and also suggest that the cell death of the crypt is mediated by apoptosis, and that of the epithelial cell is due to fatty degeneration.