Corneal Diabetic Neuropathy

Recently, a new approach to the detection of very early small fiber peripheral diabetic neuropathy has been proposed and validated. It involves the detection and quantification of the alteration of corneal nerves in diabetes, mainly the subbasal corneal nerve plexus [10]. This is a monolayer of nerve fibers located at the border between corneal epithelium and stroma, which may be detected in vivo even in a noninvasive way (see below) and probably represents the best model to have clinical information on diabetic peripheral neuropathy.

CORNEAL CONFOCAL MICROSCOPY

Corneal confocal microscopy (CCM) is a diagnostic test used to investigate at a microscopic level the different layers of the cornea. It is based on the same physical principle of any confocal microscope, allowing to have in focus just one layer of the examined tissue. Light reflected by any layer out of focus is eliminated allowing to have a high magnification, sharp image of the layer under investigation. Using corneal confocal microscope, the individual structures of any corneal layer may be easily documented: from the endothelium through the stroma (containing keratocytes, nerve fibers, and sometimes Langherans cells) up to the epithelium (with each layer) and tear film. The procedure may be a contact or noncontact one. The noncontact procedure allows to repeat CCM in a safe way, as much as necessary and with high reliability [10]. In our studies, CCM was performed by using Confoscan 4.0 (Nidek, Gamogori, Japan) equipped with an Achroplan nonapplanating ×40 immersion objective lens (Zeiss, Oberkochen, Germany) and with a Z-ring adapter system. Each examination is performed according to a standard procedure, as previously described [11]. Briefly, before the examination, a drop of topical anesthetic (0.4% oxybuprocaine chlorohydrate) is instilled in the lower conjunctival fornix of the eye. One drop of 0.2% polyacrylic gel is applied onto the objective tip to serve as an immersion fluid. The patient is positioned in the chin and forehead rest, and when an image of stroma appears on the monitor of the confocal microscope, the recording button is pressed and a micrometric motor-driven system automatically completes the alignment. The focal plane is automatically moved to reach the anterior chamber and begins recording several scans of the entire depth of the cornea. The Z-ring device is used for all examinations, and only the central cornea is analyzed. Illumination intensity is kept constant in all cases. The images collected using this procedure are analyzed in a qualitative and/or quantitative way. The endothelium is automatically analyzed using a dedicated software available with the machine. Both stromal and epithelial cells may be quantified in a semiautomatic way. The analysis of corneal sub-basal nerve plexus (CSNP) has been recently validated in a large group of normal and pathological eyes (Figs. 1 and 2) [10].

The assessment of CSNP was performed according to the following standardized procedure. The standard dimension of each image produced was 340 × 255 mm (area = 0.132 mm2) with an optical section thickness of 5.5 mm. For each examined cornea, the best sharp focus frame of CSNP was chosen. For each frame of the CSNP images, five parameters were analyzed: nerve fiber length (NFL), number of fibers (NF), number of branching (NBr), number of beadings (NBe), and fiber tortuosity (FT) (Fig. 3). NFL was calculated using an image processing computer tool, the Neuron J© program to

Fig. 1. Corneal subbasal nerve plexus (CSNP) in a normal subject, as shown by corneal confocal microscopy (CCM). It appears as a monolayer of straight nerve fibers with hyperreflective spots along the nerve (nerve beadings).

Fig. 2. CSNP in diabetes, examined with CCM. The most evident aspect is the reduction of nerve beadings (colored in red) along the nerve fibers.

outline nerve fibers from each CSNP frame. NFL for each image was calculated as the total length of the nerves (micrometers) divided by the area of the image (0.132 mm2) and expressed as micrometers per square millimeters (mm/mm2). NF was manually calculated and defined as the total number of principal nerve trunks and their branches per image. NBr was manually calculated and defined as the total NBr per image. NBe was defined as the number of hyperreflective points manually calculated considering 100 mm

Fig. 3. Normal nerve tortuosity in the corneal subbasal nerve layer.

of one fiber. The fiber was randomly chosen by the operator between all the best focused fibers. The same standard magnification was kept for all the images during the counting. The score system proposed by Oliveira-Soto and Efron [12] was used to analyze FT.

CORNEAL NERVES AND DIABETES

The cornea is the most densely innervated tissue in the body and is richly supplied by sensory and autonomic nerve fibers [13, 14]. Nerve bundles enter the cornea at the periphery in a radial manner parallel to the corneal surface. The nerve bundles lose their perineurium and myelin sheaths approximately 1 mm from the limbus and continue into the cornea surrounded by Schwann cell sheaths, and then subdivide several times into smaller branches. Stromal nerve trunks move from the periphery toward the corneal center and eventually turn 90°, proceeding toward the corneal surface and penetrating Bowman’s layer. After penetrating Bowman’s layer, the large nerve bundles divide into several smaller bundles, which turn another 90° and continue parallel to the corneal surface between Bowman’s layer and the basal epithelial cell layer, creating the subbasal corneal nerve plexus. The CSNP is characterized by local axon enlargements, or beading, which are accumulations of mitochondria and glycogen particles located at the periphery of the bundle. Corneal nerve fibers exert important trophic influences on the corneal epithelium and contribute to the maintenance of a healthy ocular surface [13]. Corneal abnormalities caused by diabetes include superficial punctuate keratopathy, recurrent epithelial defects, neurotrophic keratopathy, and corneal ulcer [15–19]. These abnormalities have been reported to occur in 50–74% of patients with diabetes who never underwent surgery, and many of these patients are asymptomatic [18, 19]. Corneal sensation is reduced in diabetic patients and progresses with the severity of neuropathy, suggesting that corneal nerve fiber damage accompanies diabetic somatic nerve fiber damage [20–22], one of the most important and invalidating diabetic chronic complica-

Fig. 4. Altered (increased) tortuosity of the subbasal nerve plexus in diabetes. This image is classified as stage 4 tortuosity.

tions [23]. A growing interest in corneal morphology in diabetic patients, especially in CSNP, is documented [21, 24–27]. Corneal nerve changes secondary to diabetes mellitus have been recently analyzed with CCM using a multiparametric approach and termed corneal diabetic neuropathy (CDN) [21].

CDN, as defined using CCM, is characterized by relevant modifications (vs. control subjects) of CSNP parameters which may be summarized as follows: decrease in the number of fibers, branching pattern and number of beadings, and increase in nerve tortuosity in diabetic patients (Fig. 4) [21]. Rosenberg et al. [22] found a reduction in long nerve fiber bundle in patients with mild to moderate neuropathy, and a reduction in corneal mechanical sensitivity only in patients with severe neuropathy, suggesting that decrease in nerve fiber bundle counts precede impairment of corneal sensitivity and that reduction in neurotrophic stimuli in severe neuropathy may induce a thin epithelium that may lead to recurrent erosions. Chang et al. [24] defined diabetic alterations in the corneal innervations using CCM, finding a decrease in nerve fiber density and nerve branch density and an increase of tortuosity, demonstrating that reduced density in basal epithelial cell is correlated with changes in innervations. Malik et al. [26] showed a progressive reduction in the number of corneal nerve fibers in diabetes, suggesting enhanced degeneration, and showed reduction in the number of corneal nerve branches, suggesting a reduction in regenerative capacity, with a progression of neuropathic severity. Quattrini et al. [27] quantified nerve fiber pathological changes using CCM and found a progressive reduction in corneal nerve fiber and branch density, but the latter was significantly reduced even in diabetic patients without neuropathy. Kallinikos et al. [25] demonstrated that tortuosity coefficient of nerve fibers was significantly greater in the severe diabetic neuropathic group than in control subjects and in the mild and moderate neuropathic groups, suggesting that this morphologic abnormality relates to the severity of somatic neuropathy and may reflect an alteration in the degree of degeneration in