Comparison of Longitudinal In Vivo Measurements of Retinal Nerve Fiber Layer Thickness and Retinal Ganglion Cell Density after Optic Nerve Transection in Rat

Abstract

PurposeTo determine the relationship between longitudinal in vivo measurements of retinal nerve fiber layer thickness (RNFLT) and retinal ganglion cell (RGC) density after unilateral optic nerve transection (ONT).MethodsNineteen adult Brown-Norway rats were studied; N = 10 ONT plus RGC label, N = 3 ONT plus vehicle only (sans label), N = 6 sham ONT plus RGC label. RNFLT was measured by spectral domain optical coherence tomography (SD-OCT) at baseline then weekly for 1 month. RGCs were labeled by retrograde transport of fluorescently conjugated cholera toxin B (CTB) from the superior colliculus 48 hours prior to ONT or sham surgery. RGC density measurements were obtained by confocal scanning laser ophthalmoscopy (CSLO) at baseline and weekly for 1 month. RGC density and reactivity of microglia (anti-Iba1) and astrocytes (anti-GFAP) were determined from post mortem fluorescence microscopy of whole-mount retinae.ResultsRNFLT decreased after ONT by 17% (p<0.05), 30% (p<0.0001) and 36% (p<0.0001) at weeks 2, 3 and 4. RGC density decreased after ONT by 18%, 69%, 85% and 92% at weeks 1, 2, 3 and 4 (p<0.0001 each). RGC density measured in vivo at week 4 and post mortem by microscopy were strongly correlated (R = 0.91, p<0.0001). In vivo measures of RNFLT and RGC density were strongly correlated (R = 0.81, p<0.0001). In ONT- CTB labeled fellow eyes, RNFLT increased by 18%, 52% and 36% at weeks 2, 3 and 4 (p<0.0001), but did not change in fellow ONT-eyes sans CTB. Microgliosis was evident in the RNFL of the ONT-CTB fellow eyes, exceeding that observed in other fellow eyes.Conclusions In vivo measurements of RNFLT and RGC density are strongly correlated and can be used to monitor longitudinal changes after optic nerve injury. The strong fellow eye effect observed in eyes contralateral to ONT, only in the presence of CTB label, consisted of a dramatic increase in RNFLT associated with retinal microgliosis.

Bilateral retinal microglial response to unilateral optic nerve transection in rats

“…Especially for the first time, the long-term morphological alternations of microglial cells in the contralateral eye are shown to be sustained for up to 12 weeks. These results, together with the similar previous findings (Bodeutsch et al, 1999, Panagis et al, 2005, Sobrado-Calvo et al, 2007, Gallego et al, 2012, Choe et al, 2014, suggest that using the contralateral retina as a control in studies of retinal degeneration should be considered with caution. ANOVA with Dunnett's post hoc comparisons to the naive group were conducted in A, while paired-samples T test was applied to compare the ipsilateral versus contralateral data in B.…”

“…In addition, mechanism underlying the eccentric disparity in contralateral retina remains to be elucidated. Though it may be similar to that of the ipsilateral side described above as RGC reduction was also found in the contralateral retina (Choe et al, 2014), whether there is other unknown mechanism needs to be further investigated.…”

“…Though diverse descriptions of layer separation have been shown by other groups with different investigative methods (Santos et al, 2008, Bosco et al, 2011, Kezic et al, 2013, Choe et al, 2014, microglia were found, in the present study, to be mainly located in three separate layers by epifluorescence microscopy on retinal flat mounts: the RGC layer (GCL, including the nerve fiber layer), inner plexiform layer (IPL), and outer plexiform layer (OPL). Studies have shown that the most obvious microglial proliferation is detected in the GCL after ON transection, whereas microglia in the OPL remain undisturbed (Garcia-Valenzuela et al, 2005).…”

Topical Treatment With Bromfenac Reduces Retinal Gliosis and Inflammation After Optic Nerve Crush

“…This is of special importance in the central retina because in clinical practice, RFNL measurement is employed as an index of RGC loss. Our group and others 22,[58][59][60][61] have shown in animal models that following axotomy of the optic nerve or ocular hypertension there is a 9-day time lapse mismatch between the onset of RGC disappearance and the slower, more protracted, degeneration of the intraretinal axons, which form the RFNL. 22 It is tempting to suggest that such a time delay could be influenced by a RFNL swollen by the astrocyte and Müller cell hypertrophy that occurs upon lesion.…”

Assessing retinal ganglion cell damage

“…36,37 Cholera toxin subunit B (CTB) conjugated to a fluorophore has also been used as a retrograde tracer applied to the superior colliculus of rodents to reliably and specifically label RGCs. 38,39 Major limitations of retrograde tracers are that they do not persist in RGCs for extended periods of time. Furthermore, the tracer is taken up by microglia and macrophages when RGCs die after inducing injury, reducing the specificity of the label to RGCs.…”