Alterations of epithelial barrier properties of rat ileum following X irradiation
I. Dublineau, F. Lebrun, S. Grison, C. Strup and N.M. Griffiths
Meeting of the Physiological Society, 8-10 July 2002, Liverpool (GB)
Exposure of the digestive tract to ionising radiation results in both morphological and functional alterations of the small intestine with the terminal ileum being a particular site of injury. The aim of this work was to characterize, in parallel, epithelial barrier properties of rat ileum as determined by the passage of different sized molecules and spatial localization junctional proteins following increasing radiation dose.
Animals were exposed to a single hemi-body irradiation (9 Gy) under gaseous anaesthesia (isoflurane 2.5%, 0.4 L /min) and studied 3 days after exposure. After euthanasia (overdose of sodium pentobarbitone), samples of terminal ileum were placed in Ussing chambers for measurements of 14C-mannitol, FITC-Dextran-4kDa and 3H-Dextran-70kDa fluxes and short-circuit current (Isc) and transepithelial resistance (RT). Ileal samples were treated either for immunohistochemical analyses of junctional proteins (ZO-l, beta-catenin) and pan-cytokeratin using confocal microscopy or for standard histological analyses with haematoxylin-eosinsaffron staining. . All experiments were conducted according to the French regulations for animal experimentation (Ministry of the Agriculture Act No. 87848, October 19, 1987).
Following a 6Gy irradiation Isc was increased from 26±5 microA/cm2 (controls N=22) to 64±16 microA/cm2 (irradiated N=5) (mean±SEM, p<0.01 one-way ANOVA). RT was increased from 35±3 ohms.cm2 (controls N=22) to 50±3 ohms.cm2 (irradiated N=5, p<0.05) but permeability to mannitol and dextran was unchanged. Analysis of pan-cytokeratin and beta-catenin staining showed a slight increase of cell size with nevertheless a well-structured network in both villi and crypts. Standard histology confirmed these results.
At three days after 8 Gy RT was decreased by 50% concomitant with increased permeability to mannitol (x3), Dextran-4kDa (x3) and Dextran-70kDa (x2). A reduced and modified pattern of beta-catenin staining was observed suggesting a relocalization of this protein. The effect of irradiation on ZO-1 was more marked with very little staining being detected in either villi or crypts. An amplification of these effects occurred with increasing radiation dose where for > or = 10Gy RT was quasi-null and mannitol and dextran permeability was markedly increased (x7). Major histological alterations with epithelial discontinuity were also observed.
In conclusion, these results demonstrate radiation dose-dependent differences in the response of rat small intestinal epithelium: at a lower dose (6Gy) increased RT was observed whereas an opposite effect (decreased RT, enhanced permeability) was obtained at higher doses (> or =8Gy). The latter were associated with a disorganization of the small intestinal epithelium.