Corneal Sensitivity to Hyperosmolar Eye Drops: A Novel Behavioral Assay to Assess Diabetic Peripheral Neuropathy

Abstract

Purpose: Diagnosis of peripheral neuropathy (PN), which affects approximately 50% of the diabetic population, is subjective, with many patients seeking a diagnosis only after presenting with symptoms. Recently, in vivo confocal microscopy of subepithelial corneal nerve density has been promoted as a surrogate marker for early detection of PN, but imaging of corneal nerves requires sophisticated instrumentation, expertise in confocal imaging, cooperative patients, and automated analysis tools to derive corneal nerve density. As an alternative, we developed a simple screening method that is based on the sensitivity of corneal nerves to cause reflex eyelid squinting in response to hyperosmolar eye drops.

Methods: Eyes of control and type 2 diabetic rats were given an eye drop of a 290- to 900-mOsm solution, and the ocular response was video recorded. Other neuropathic end points including nerve conduction velocity and subepithelial cornea nerve density were determined.

Results: Motor and sensory nerve conduction velocity and total nerve fiber length of corneal nerves in the subepithelial layer were significantly decreased in diabetic rats. Applying the hyperosmotic solutions to the ocular surface caused an osmolarity-dependent increase in squinting of the treated eye in control rats. Squinting was almost totally blocked by preapplication of proparacaine or N-(4-tertiarybutylphenyl)-4-(3-chloropyridin-2-yl)tetrahydropyrazine-1(2H)-carbox-amide, a transient receptor potential melastatin-8 channel blocker. Squinting in response to the 900-mOsm solution was significantly reduced in diabetic rats.

Conclusions: Preclinical studies show that evaluation of corneal sensitivity may be an alternative method for the early detection of PN and has potential for translation to clinical studies.

Figures

Figure 1

Effect of application of isotonic and increasing hypertonic eye drop on eye closure (squinting) in normal Sprague-Dawley rats. Two protocols were used to access the effect of hyperosmolarity on squinting, which was examined as described in the Methods section. The graph on the right “Independent” used four untested rats for each different osmotic solution. The graph on the left “Repeated” used the same four rats with a 2-day rest between each application. For the latter group, the isotonic, 290-mOsm solution was applied first followed by increasing osmolarity with each application. Data are presented as the mean ± SEM for % visible surface area of the eye.

Figure 2

Effect of proparacaine or BCTC on eye closure (squinting) in response to an application of 900-mOsm solution in normal Sprague-Dawley rats. Squinting in response to an eye drop of a 290- or 900-mOsm solution was examined as described in the Methods section (A). For this study, we also examined the effect of proparacaine or BCTC on hyperosmotic effect. Five and 10 minutes prior to the addition of the hyperosmotic solution (900 mOsm), a drop of proparacaine hydrochloride ophthalmic solution, 0.5%, or 20 μL 50 μM BCTC was applied to the ocular surface. Data are presented as the mean ± SEM for % visible surface area of the eye. Four animals were examined. In B1–B3 and C1–C3 (maximum projection) and B4 and C4–C6 (axial projection), representative images are provided for immunostaining of corneal nerves with antibodies to neuronal class III tubulin (green [B1, C1]) and TRPM8 (red [B2, C2]). A merge imaged for each of the two examples are shown (B3, C3). In B, the fourth image is an axial projection of the epithelial layer of the cornea. B shows two single simple filaments penetrating the epithelial layers, whereas C demonstrates a bifurcating nerve with in the epithelium.

Figure 3

Effect of type 2 diabetes induced by high-fat diet and low-dose streptozotocin in Sprague-Dawley rats on innervation of the subbasal layer of the cornea and corneal sensitivity. Innervation of the subbasal layer of the cornea was determined by using corneal confocal microscopy as described in the Methods section. Corneal sensitivity was determined by using a Cochet-Bonnet filament esthesiometer as described in the Methods section. The number of rats in each group was the same as described in Table 1. Data are presented as the mean ± SEM for innervation of the cornea in mm/mm2 cm for corneal sensitivity. *P < 0.05 compared with control rats.

Figure 4

Effect of type 2 diabetes induced by high-fat diet and low-dose streptozotocin in Sprague-Dawley rats on eye closure (squinting) in response to an application of 290- and 900-mOsm solution in the treated and untreated eye. Squinting in response to an eye drop of a 290-and 900-mOsm solution was examined as described in the Methods section. The number of rats in each group was the same as described in Table 1. Data are presented as the mean ± SEM for % visible surface area of the eye. *P < 0.05 compared with control rats.

Figure 5

Effect of a 290-mOsm solution and 2% and 5% Muro 128 on eye closure (squinting) in normal Sprague-Dawley rats. Squinting in response to an eye drop of a 900-mOsm solution and 2% and 5% Muro 128 was examined as described in the Methods section. Data are presented as the mean ± SEM for % visible surface area of the eye.