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Increased Corneal Nerve Regeneration and Function with DHA Plus PEDF After Injury

This issue of the PUFA Newsletter reports research demonstrating that injured peripheral neurons are more resistant to damage from injury and recover more quickly and extensively when cellular concentrations of DHA and docosapentaenoic acid (DPA n-3) are elevated. In this article, Haydee Bazan and colleagues at Louisiana State University and the University of Illinois Medical Center describe improved corneal nerve regeneration and function in the presence of DHA and pigment epithelial-derived factor (PEDF). These investigators have previously reported that corneal nerve regeneration increases in the presence of DHA + PEDF following lamellar keratectomy. This surgical procedure is used to correct serious near-sightedness (myopia) and low to moderate far-sightedness.

The cornea is the transparent covering of the eye that together with the lens refracts light and provides about two-thirds of the eye’s optical power (Figure 1). It is highly enriched in sensory nerve fibers that respond to touch, temperature and chemicals. Corneal nerves modulate cell proliferation, differentiation and wound healing and have additional cellular functions. To maintain transparency, the cornea has no blood vessels. Instead, it obtains nutrients from the tear fluid on the outside and the aqueous humor on the inside. Neurotrophins supplied by nerve fibers also nourish it.

Damage to the corneal nerves reduces sensitivity, blinking and tear secretion. It can lead to such disorders as dry eye and eventually neurotrophic keratitis. Severe damage may impair epithelial wound healing and have more serious consequences. Mitigation of these pathologies in the corneas of mice has been associated with lipoxin A4 derived from arachidonic acid and neuroprotectin D1, derived from DHA, both of which have anti-inflammatory and wound-healing properties. It has been reported that DHA enhances the synthesis of neuroprotectin D1 in the presence of PEDF in retinal pigment epithelial cells and the cornea.

In these experiments, the investigators examined the function of regenerated corneal nerves treated with DHA and PEDF to determine if sensitivity and epithelial wound healing would be restored. Sensitivity was inferred from the presence of 2 neuropeptides that are markers of sensory neurons, calcitonin gene-related peptide and substance P. These peptides function in the ability to feel and modulate pain. Sensitivity was also measured using a pressure-sensitive esthesiometer (Figure 2).

Male New Zealand albino rabbits underwent lamellar keratectomy in the stroma of the right eye, with post-surgical topical application of moxifloxacin for prophylaxis. When performed in the midstroma, such surgery severs all corneal nerves. This is similar to the damage incurred after creating a LASIK flap. Immediately following surgery, animals were treated with a collagen shield impregnated with PEDF and DHA. The collagen shields were changed twice weekly for 6 weeks and the animals sacrificed 8 weeks after surgery.

Corneal sensitivity was measured weekly using a Cochet-Bonnet esthesiometer (Figure 2). The investigators assessed the touch threshold by varying the length of the monofilament in the esthesiometer until no blink response was observed. Blinking 50% or more of 4 test times was considered a positive response, i.e., sensitivity. If no blink response was elicited at a monofilament length of 0.5 cm, corneal sensitivity was considered 0.

The investigators evaluated epithelial wound healing by removing an 8-mm layer of corneal epithelial cells 4 weeks after the initial lamellar keratectomy and topical treatment with DHA + PEDF. This time period was selected because it reflects substantial but partial nerve regeneration. Treatment with DHA + PEDF continued for another 7 days and the corneas photographed every 24 hours until sacrifice at the end of the week’s treatment. To visualize the epithelial defect, the epithelium was stained with methylene blue.

Calcitonin gene-related peptide and substance P were assessed by immunohistochemical analysis of the stained and fixed whole corneas. Cell proliferation was measured by anti-Ki67 antibody staining sections of the corneal limbal area in animals receiving the epithelial wound and counting the number of positive cells in 10 images per animal.

Treatment with DHA + PEDF after surgical injury of the corneal nerves resulted in significant nerve regeneration (% corneal nerve area) in the treated animals compared with animals given only the treatment vehicle (12.2% ± 2.6% vs 5.6% ± 0.9%, respectively, P = 0.004) when assessed 8 weeks after surgery. Nerve regeneration did not differ significantly between the treated and non-injured control groups (12.2% ± 2.6% vs 13.6% ± 2.2%).

The nerves regenerated with DHA + PEDF treatment were shown to express calcitonin gene-related peptide to a similar extent as the control animals (45.4% ± 0.07% treated vs 44.2% ± 0.1%, control). Animals treated only with the treatment vehicle expressed 24.9% ± 0.04% peptide-positive neurons. These observations suggest that the regenerated neurons resulting from DHA + PEDF treatment resembled the functional control neurons. After injury, substance P was not found in the epithelial or subepithelial nerves of treated and control animals, but was present in the epithelial cells.

Measurements of corneal nerve sensitivity conducted weekly after surgery using the Cochet-Bonnet esthesiometer showed that central corneal sensitivity was low for the first 4 weeks (Table). By week 5, animals treated with DHA + PEDF gained significantly in sensitivity compared with the untreated animals, achieving measurement values approaching those of the control group. At the end of 8 weeks, sensitivity in the treated group remained significantly greater than in the vehicle-only group and did not differ significantly from the control group.

The investigators assessed wound healing in the epithelium of the cornea in animals with corneal nerve damage. The wound consisted of removing most of the epithelial cell layer. Treatment continued for another 7 days. As shown by the uptake of blue dye into the proliferating cells, control animals exhibited a gradual decrease in blue dye in the first 48 hours and by 72 hours the cornea showed almost no blue dye. The loss of blue dye indicated closure of the wound and increased growth of new epithelial cells. Similar results were observed in the DHA + PEDF group which had no blue dye remaining after 72 hours. The animals treated only with the treatment vehicle healed more slowly, exhibiting measurable blue dye after 72 hours.

These experiments confirmed previous findings from this laboratory showing that animals with severed corneal nerves undergo nerve regeneration when the cornea is treated with topical DHA + PEDF. In these studies, the investigators demonstrated that as the nerve fibers regenerate, sensitivity gradually returns, but that the total nerve area and level of corneal sensitivity 8 weeks after surgery remain significantly below pre-surgical values. The investigators showed that the regenerated nerves were functional and expressed calcitonin gene-related peptide in proportions similar to control animals and responded to touch at almost the same level as control animals 8 weeks after surgical injury. Further, corneas treated with DHA + PEDF after nerve damage and an epithelial cell wound exhibited rapid cellular repair that equaled or exceeded the measurements obtained in the control animals. The authors suggested that the nerve regeneration stimulated by DHA + PEDF was related to the production of neuroprotectin D1, which is derived from DHA and enhanced in the presence of PEDF.

A strong implication of these studies would be corneal treatment with DHA + PEDF after LASIK (laser-assisted in situ keratomileusis) surgery, which severely damages nerves. It has been reported that less than half the number of nerves remain in the sub-basal region one year after LASIK surgery compared with preoperative values. Immediate post-surgical treatment with DHA + PEDF might improve nerve regeneration and functionality and reduce the risk of dry eye in LASIK patients. It may be time for pilot studies in humans.

Cortina MS, He J, Li N, Bazan NG, Bazan HE. Recovery of corneal sensitivity, calcitonin gene-related peptide-positive nerves, and increased wound healing induced by pigment epithelial-derived factor plus docosahexaenoic acid after experimental surgery. Arch Ophthalmol 2012;130:76-83. PubMed

WORTH NOTING

Simon E, Bardet B, Grégoire S, Acar N, Bron AM, Creuzot-Garcher CP, Bretillon L. Decreasing dietary linoleic acid promotes long chain omega-3 fatty acid incorporation into rat retina and modifies gene expression. Exp Eye Res 2011;93:628-635. PubMed

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