Irinotecan-induced alterations in intestinal cell kinetics and extracellular matrix component expression in the Dark Agouti rat

Abstract

Chemotherapy-induced mucositis is characterized by damage of mucous membranes throughout the alimentary tract (AT). Extracellular matrix (ECM) components play a vital role in maintaining mucosal barrier integrity by regulating cellular apoptosis, proliferation and differentiation of overlying epithelial cells. The aims of this study were to characterize the changes in epithelial cell kinetics and to investigate the expression of the ECM components in the gastrointestinal tract following irinotecan administration. Female dark agouti rats were treated with single 200 mg/kg dose irinotecan and killed at various time points (1, 6, 24, 48, 72, 96 and 14 h) after treatment. Ki67 immunostaining and TUNEL were used to assess proliferation and apoptosis, respectively, in the jejunum and colon. Masson trichrome staining and picro-sirius red staining were used to determine the level of collagen, and immunohistochemistry was used to further assess collagen IV, fibronectin and laminin 1 and 2 expression in these tissues. Irinotecan halved cellular proliferation in the jejunum and colon at 48 and 24 h, respectively, while apoptosis peaked at 6 h (P < 0.05). There was a substantial increase in total collagen deposits around crypts from 24 h in both regions. However, collagen IV expression decreased significantly in the crypt region in a delayed fashion (P < 0.05). Fibronectin expression decreased significantly in jejunum and colon from 6 to 24 h following treatment (P < 0.05). Irinotecan induced a significant alteration in epithelial cell kinetics in both the jejunum and colon, and this correlated with changes in ECM component expression. Changes in ECM expression may have a direct impact on the loss of mucosal layer integrity evident in chemotherapy-induced mucositis.

Figure 1

Changes in cell proliferation as indicated by Ki67 immunostaining in the (a) jejunum and (b) colon at 1, 6, 24, 48, 72, 96 and 144 h following the administration of 200 mg/kg irinotecan intraperitoneally. Cells positively stained for Ki67 were counted in 20 crypts and averaged. The data are mean number of stained cells/crypt + standard error; *P < 0.05 compared to untreated controls.

Figure 2

Changes in cell apoptosis as identified by TUNEL assay in the (a) jejunum and (b) colon at 1, 6, 24, 48, 72, 96 and 144 h following the administration of 200 mg/kg irinotecan intraperitoneally. The data are mean number of stained cells/field of view + standard error; *P < 0.05 compared to untreated controls.

Figure 3

Picro-sirius red staining demonstrating collagen in the (a) jejunum and (b) colon in a time course model of irinotecan-induced mucositis. Staining demonstrates collagen including the finest fibres and basement membranes, in red on a yellow background. Photomicrographs taken at 20× objective.

Figure 4

Masson trichrome staining demonstrating fibrous collagen in the (a) jejunum and (b) colon in a time course model of irinotecan-induced mucositis. Fibrous collagen is stained green on a purple background. Photomicrographs taken at 20× objective.

Figure 5

Extracellular matrix component staining in the jejunum. Top bars illustrate staining in villi while bottom bars illustrate staining in crypts at 1, 6, 24, 48, 72, 96 and 144 h following the administration of 200 mg/kg irinotecan intraperitoneally. All control and experimental tissue were graded by a qualitative scale. The data are means + standard error; significance indicated on the graph.

Figure 6

Extracellular matrix component staining in the colon. Top bars illustrate staining in the apical colon while bottom bars illustrate staining in the basal colon at 1, 6, 24, 48, 72, 96 and 144 h following the administration of 200 mg/kg irinotecan intraperitoneally. All control and experimental tissue were graded by a qualitative scale. The data are means + standard error; significance indicated on the graph.