Nicotine accelerates diabetes-induced retinal changes

Abstract

Purpose: To investigate the effects of nicotine on retinal alterations in early-stage diabetes in an established rodent model.

Materials and methods: Sprague-Dawley rats were examined using a combination of confocal scanning laser ophthalmoscopy and spectral domain optical coherence tomography to determine changes in retinal structure in response to nicotine exposure, diabetes and the combined effects of nicotine and diabetes. Diabetes was induced by a single injection of 65 mg/kg streptozotocin and nicotine injections were administered subcutaneously daily. Retinal thickness in the superior, inferior, nasal and temporal quadrants were determined based on the spectral domain optical coherence tomography (SD-OCT) volume scans (20° × 20°) centered on the optic disc. Segmentation of discrete retinal layers was performed on a subset of SD-OCT cross-sections to further examine changes in each treatment group. Survival of neurons within the ganglion cell layer (GCL) was assessed by confocal morphometric imaging.

Results: The control group did not experience any significant change throughout the study. The nicotine treatment group experienced an average decrease in total retinal thickness (TRT) of 9.4 µm with the majority of the loss localized within the outer nuclear layer (ONL) as determined by segmentation analysis (p < 0.05). The diabetic group exhibited a trend toward decreased TRT while segmentation analysis of the diabetic retinopathy (DR) group revealed significant thinning within the ONL (p < 0.05). The combination of nicotine and diabetes revealed a significant increase of 8.9 µm in the TRT (p < 0.05) accompanied by a decrease in the number of GCL neurons.

Conclusions: We demonstrated significant temporal changes in retinal morphology in response to nicotine exposure, diabetes and with the combined effects of nicotine and diabetes. These findings may have implications in determining treatment strategies for diabetic patients using products containing nicotine, such as cigarettes, smokeless tobacco, electronic cigarettes or smoking cessation products.

Keywords: Diabetes; SD-OCT; SLO; neurons; nicotine; retinal thickness.

Figure 1

Morphometric analysis of the rat retina non-invasively using SD-OCT. (A) SLO fundus image with corresponding overlay of the 20° × 20° field of view used for volumetric SD-OCT in the rat retina. (B) 3-D reconstruction of the retinal volume used to perform regional TRT measurements. (C) Example of the regional TRT thickness measurements performed for each animal utilizing the TruTrack^™ registration method and Heidelberg Engineering Eye Explorer version 5.1 analytical software (Baseline, 8 week follow up, differential retinal thickness map).

Figure 2

Multimodal imaging of the Sprague Dawley rat retina. (A) Fluorescein angiography highlighting the retinal vasculature (B) NIR reflectance SLO of the albino rat fundus. (C) Observed retinal layers using high resolution SD-OCT (D) Single 20° B-scan centered on the optic disc representing an average of 20 frames (E) Depiction of the retinal layers segmented and measured using OCT_Seg.

Figure 3

Segmented retinal thickness values were divided into 4 groups based on our ability to effectively delineate layer boundaries using the semi-automated technique. (A) The innermost layers of the RNFL, GCL and IPL did not demonstrate any significant changes in SD-OCT morphology associated with nicotine exposure or diabetes. (B) Likewise, the INL and OPL remained unchanged for all groups after 8 weeks. (C) The ONL was significantly thinner in the nicotine group and the diabetic group. (D)The outer retina measured from the RPE/BM interface to the IS/OS junction demonstrated a significant increase in thickness associated with the combination of nicotine exposure and diabetes.

Figure 4

Independent methods of analyzing TRT values yielded similar results in terms of significance with respect to retinal thinning in the nicotine exposed animals and retinal thickening in the DM group exposed to nicotine. Embedded Heidelberg Eye Explorer software (A) and OCT_Seg (B) yielded significant differences in both the nicotine exposed group and the combined nicotine and diabetes group after eight weeks. On average, the segmentation software measurements of TRT were 5.5 μm greater than the Heidelberg Eye Explorer software. For both measurement techniques, ANOVA among treatment groups at 8 weeks (P < 0.001). *Two-tailed t-test demonstrating statistically significant difference from baseline (P < 0.05)

Figure [5]

Confocal microscopy of labeled neurons in the ganglion cell layer (GCL) from flatmount preparations. Mean neuronal cell density was determined based on 12 confocal imaging fields (40X) in 3 representative animals from each treatment group. GCL was identified by the proximity to inner retinal vasculature. The data represent mean ± SEM for each image based on the experimental group (A) maximum intensity projection of an enhanced field of view is shown. (B) A 9.2% reduction in the density of stained GCL neurons was noted between the control group and the diabetic group treated with nicotine although no significant differences were observed at the conclusion of our study based on ANOVA (P > 0.05), Red: isolectin B4 staining of the vasculature, Green: GCL Neurons. Scale bar = 50 μm.