Abstract
Purpose :
Dissociating the roles of Nestin(+)/Sox2(+) NPCs present in the optic nerve laminar region (ONLR) from those in the retina are difficult to achieve because: 1) Most current approaches to eliminate Sox2(+) cells result in depletion of these cells in both the retina and ONLR. 2) Most Nestin(+) transgenic animals do not express the nestin-associated transgene in the ONLR. 3) It is difficult to determine whether effective treatments are reaching the target cells. We wanted to identify an appropriate model and approach to selectively deplete ONLR-NPCs, using transgenic mice expressing nestin-promotor drivers.
Methods :
The C57BL/6-Tg(Nes-cre/ERT2)KEisc/J strain was crossed with ROSA 26-LoxP (DTA), and following Tamoxifen induction, ONLR's were assayed for elimination of nestin expression. The C57BL/6-Tg(Nes-TK*,-EGFP)145Sker/J) strain constitutively expresses GFP and viral thymidine kinase (Kernie, 2012). GFP(+) cells in the ONLR and retina were evaluated using immunohistochemistry. Nestin(+) cells were eliminated in the latter transgenic strain using either IP injection X 3 weeks of Ganciclovir (50mg/kg/d) or 3 week CNS adminstation of ganciclovir (GCV: Cytovene, 47.5mg/ml) using 2 or 4 week Alzet pumps attached to a intraventricular pump (Alzet pump 3). We evaluated ganciclovir distribution by doping the GCV fill with 0.5-2% fluorescein, and analyzing retinal and optic nerve fluorescence using a Heidelberg SD-OCT instrument with the blue peak fluorescent setting.
Results :
No elimination of ONLR-nestin(+) cells occurred in the the Eisch strain. The Kernie strain exhibited scattered GFP(+) cells in the retina and ONLR. Systemic GCV administration resulted in the loss of GFP immunopositivity in both tissues. Administration of GCV by intraventricular pump using both the 2- and 4 week (1/2 the rate of dosing) eliminated GFP(+) NPCs in the ONLR, but not the retina.Positive continuous GCV dosing in the intraventricular cannalized mice was demonstrable best with 1% fluorescein, but also easily detectable with 0.5% fluorescein.
Conclusions :
Selective elimination of ONLR-NPCs is possible via intraventricular dosing of GCV, and this can be reproducibly demonstrated at 2-, 3- and 4 weeks administration.The single 4 week Alzet pump used eliminated >90% of GFP(+) cells in the ONLR, while sparing retinal GFP(+) cells. Analysis of isolated ONLR-NPC functions in vivo are possible using this approach.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.