Selective laser-activated lesioning of prelabeled fetal hippocampal grafts by intracellular photolytic chromophore.
Selective removal of grafted tissue is critical to assess the functional role of that tissue in the host, yet is technically difficult for well-dispersed neural grafts. We labeled fetal hippocampal cells with both a nuclear marker (5'-bromodeoxyuridine) and a cytoplasmic marker (latex microspheres) before grafting into normal adult hippocampus. A nontoxic chromophore, chlorin e6, was conjugated on to the surface of latex microspheres of the treatment group. Chlorin e6 produces cytotoxic singlet oxygen only during photoactivation. Grafted animals received transcranial exposure to various intensities of near-infrared laser light. Following laser exposure, grafts were observed in all groups except in transplants prelabeled with chlorin e6 latex microspheres. An optimal laser exposure of 50-100 J/cm2 (4-8 min) was found to selectively remove only the chlorin e6-containing grafted cells. With increasing doses of laser illumination, non-specific lesions of the host tissue surrounding the graft were induced. Quantitative graft analysis, in the absence of laser exposure, indicated that the survival of grafted cells was similar between control transplants labeled with latex microspheres alone and grafts labeled with latex microspheres plus chlorin e6. This is further evidence that chlorin e6 by itself was not toxic without laser exposure. The results clearly demonstrate that singlet oxygen-induced cell photolysis can result in selective, non-invasive removal of dispersed grafted cells located in adult hippocampus. This technique may facilitate defining specific mechanisms of action of grafted cells which mediate functional recovery in different host conditions.
Shetty, AK; Rapoza, D; Madison, RD; Turner, DA
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