Background

Although eye drop applications are not considered innovative, they are typically used to generate effects in the anterior portion of the eye (Patel et al., 2013; Sung et al., 2015). For instance, eye drop medications used in glaucoma patients decrease the production of aqueous humor or affect drainage of fluid through the trabecular meshwork in the anterior chamber of the eye (Dikopf et al., 2017). However, others have begun to use agents that are dissolved in vehicles to exert their effects in the back of the eye at the retina. For instance, the neuropeptide pituitary adenylate cyclase activating polypeptide [PACAP] provides a well-established neurotrophic and neuroprotective effects in the eye against different retinopathies. Although the route of delivery is usually intravitreal, a recent study that used PACAP dissolved in benzalkonium-chloride was able to cross the ocular barriers and exert protection in ischemic conditions (Werling et al., 2017). Other studies showed that the alpha7 nicotinic acetylcholine receptor specific agonist, PNU-282987 (Bodnar et al., 2005; Hajos et al., 2005; Iwamoto et al., 2014), was able to cross ocular barriers and induce neurogenesis of adult mammalian neurons when delivered as eye drops, made by diluting a stock solution of PNU-282987 in lipophilic vehicle, DMSO, in PBS. (Webster et al., 2017). Previous dose and time-dependent studies showed that PNU-282987 was detectable in the retina by HPLC MS/MS after eye drop delivery. PNU-282987 levels were detected in the retina after topical application of the nAChR agonist to the bulbar conjunctiva (Mata et al., 2015).

BrdU is a synthetic nucleoside that is an analog of thymidine and is commonly used in the detection of proliferating cells in living tissues. To label mitotically active cells with BrdU, well-established methods for delivery in rodents is via intraperitoneal or intraocular injection (Karl et al., 2008; Lee et al., 2015; Xie et al., 2016). However, more robust labeling of mitotically active cells in the retina occurs if BrdU is added into the PNU-282987 eye drop solution (Webster et al., 2017) (Figure 1). Eye drop applications of agents that reach the retina are much less invasive than either intraperitoneal or intravitreal injections and have the added advantage that they can be introduced several times each day or week without the disadvantage of multiple injections. Here, the procedure for labeling mitotically active cells in the adult rodent retina with BrdU eye drops is provided after stimulation with the alpha7 nicotinic acetylcholine receptor specific agonist, PNU-282987. However, this method also has broader implications and can be used to deliver other proliferating agents in the retina.


Figure 1. Evidence of successful BrdU labeling. A. A confocal retinal section obtained from an adult SVJ 129 mouse treated only with PBS containing BrdU for 3 days. Cell nuclei are stained with DAPI (blue). No evidence of BrdU positive cells when the retina is processed with antibodies against BrdU. B. A confocal retinal section obtained from an eye treated with PNU-282987/BrdU eye drops for 3 days. BrdU positive cells are observed in all nuclear layers of the retina (green). ONL: outer nuclear layer, INL: inner nuclear layer, GCL: ganglion cell layer. Arrows point to BrdU positive retinal cells. Scale bars represent 50 µm.