Protection by an Oral Disubstituted Hydroxylamine Derivative against Loss of Retinal Ganglion Cell Differentiation following Optic Nerve Crush

Abstract

Thy-1 is a cell surface protein that is expressed during the differentiation of retinal ganglion cells (RGCs). Optic nerve injury induces progressive loss in the number of RGCs expressing Thy-1. The rate of this loss is fastest during the first week after optic nerve injury and slower in subsequent weeks. This study was undertaken to determine whether oral treatment with a water-soluble N-hydroxy-2,2,6,6-tetramethylpiperidine derivative (OT-440) protects against loss of Thy-1 promoter activation following optic nerve crush and whether this effect targets the earlier quick phase or the later slow phase. The retina of mice expressing cyan fluorescent protein under control of the Thy-1 promoter (Thy1-CFP mice) was imaged using a blue-light confocal scanning laser ophthalmoscope (bCSLO). These mice then received oral OT-440 prepared in cream cheese or dissolved in water, or plain vehicle, for two weeks and were imaged again prior to unilateral optic nerve crush. Treatments and weekly imaging continued for four more weeks. Fluorescent neurons were counted in the same defined retinal areas imaged at each time point in a masked fashion. When the counts at each time point were directly compared, the numbers of fluorescent cells at each time point were greater in the animals that received OT-440 in cream cheese by 8%, 27%, 52% and 60% than in corresponding control animals at 1, 2, 3 and 4 weeks after optic nerve crush. Similar results were obtained when the vehicle was water. Rate analysis indicated the protective effect of OT-440 was greatest during the first two weeks and was maintained in the second two weeks after crush for both the cream cheese vehicle study and water vehicle study. Because most of the fluorescent cells detected by bCSLO are RGCs, these findings suggest that oral OT-440 can either protect against or delay early degenerative responses occurring in RGCs following optic nerve injury.

P16INK4a Upregulation Mediated by SIX6 Defines Retinal Ganglion Cell Pathogenesis in Glaucoma

“…Unilateral elevation of IOP in 1 month old C57BL/6J, p16 −/− , Thy1-CFP (Lindsey et al, 2013), Six6 +/− (Larder et al, 2011), p53 −/− (129-Trp53tm1Tyj/J, The Jackson Laboratory) mice was achieved through instilling the anterior chamber with saline solution and maintained at an elevated pressure of 90 mm Hg. The p16 −/− line was engineered to remove the common exon of E2 and E3, therefore the line is a null allele for both p16 and p19/Arf genes (Serrano et al, 1996).…”

Differential Protection of Injured Retinal Ganglion Cell Dendrites by Brimonidine

“…[11][12][13] These limitations also make it challenging to determine if neuroprotective treatments might preferentially target certain RGC types and whether there are different effects of neuroprotective treatments on dendritic remodeling among different RGC types.…”

“…This avoids a concern that some of the faintly fluorescent cells that were imaged could be amacrine cells; this concern arose in prior studies using Thy1-CFP23Jrs mice. 12,13,31 Second, assignment of each RGC to a group according to its dendritic arbor structure can be positively determined before injury-associated changes in dendrite structure might make it appear like an RGC of another group. Thus, changes in response to injury or neuroprotective treatment are positively associated with a particular RGC group.…”

“…A primary reason for the fewer groups in the present study is that, unlike the prior histological studies, the weeks after the severe optic nerve injury produced by ONC[11][12][13][14]41 , this model does have a days-long period between the onset of initial responses optic nerve injury and the elimination of Thy-1 expression during which degenerative events such as dendritic arbor remodeling and axonal beading occur 14. If the days-long period of dendritic pruning after ONC accurately models important aspects of what happens in RGCs of TON patients, then brimonidine may provide enhanced recovery in TON.…”

Complementary research models and methods to study axonal regeneration in the vertebrate retinofugal system

“…Alternatively, transgenic lines where RGCs are fluorescently labeled, such as expression of yellow fluorescent protein (YFP) under the Thy1 promoter (Thy1-YFP), could also prove valuable. Yet, Thy1 expression is known to decrease in injured RGCs (Lindsey et al 2013), and it remains unclear whether the YFP signal remains stable enough for in vitro studies.…”