P 95Cyclophosphamide-induced posterior coloboma formation in the rabbit
S. E. Coupland, G. Stoltenburg-didinger and E. A. Holtgrave
Purpose: Cyclophosphamide (CP) is well known for its teratogenic effects, particularly in the region of the head and neck, in both animal models and clinically. We investigated the teratogenic effect of cyclophosphamide on ocular development in the rabbit model.
Methods: White russian rabbit dams received CP by subcutaneous injection (12-15 mg/kg body weight) during pregnancy (day 11 or 12 of gestation). Foetuses were delivered by caesarian section at days 20, 22, 25, 28 and 29. Litter size varied between 6 and 9. Control dams were not treated with CP and foetuses were delivered on the corresponding days, as above. The foetuses were sacrificed, the heads fixed in 3% buffered formaldehyde and embedded in paraffin. Additional to conventional histological stainings, immunohistochemistry was applied using monoclonal antibodies against Ki-67 (MIB-1) and syndecan. Apoptosis was detected by labeling the 3OH-ends of fragmented DNA (Apoptag in situ apoptosis detection kit, Oncor).
Results: A posterior coloboma, with associated abnormalities of the hyaloid vasculature and "retinal dysplasia", was seen in 15 of 60 eyes (25%); these changes were bilateral in most cases. In four of eight fetuses, simultaneous abnormalities of the palate occurred. In the control eyes, normal development of the ocular structures was seen. Further, the anterior segments of all examined eyes were normal. Intense cellular proliferation was in the outer neuroblastic layer of the retina; here occasional apoptotic cells were was also observed. Syndecan expression was seen in a variety of tissues in both groups.
Conclusion: It could be demonstrated that CP lead to a higher incidence of posterior coloboma and of persistence of the hyaloid vasculature, through disruption of the embryogenesis of posterior ocular structures. Such morphological changes are to be seen in chromosomal abnormalities, such as trisomy 3. The investigations supports the hypothesis that CP leads to a disruption of apoptosis in embryonic tissues.
Universitätsklinikum Benjamin Franklin der Freien Universität Berlin, Hindenburgdamm 30, D-12200 Berlin