The comprehensive analysis performed by Mirmoeini et al. provides important insight into the regulatory complexity of innervation-dependent corneal epithelial renewal. The breakthrough findings provide the missing linking factor by suggesting SCs as a new cellular component of the limbal niche that mediates corneal innervation-dependent epithelial renewal
[71][67].
4. Trophic Regulation of Sensory Innervation-Mediated Corneal Epithelial Renewal
Deficiencies in the signaling molecules, including neurotrophins and neurotransmitters, is a possible, if not a likely contributor to the demise of the epithelium after corneal denervation, because the deficiency results in a loss of regenerative capacity and progressive epithelial degeneration
[36,37,75,76][36][37][71][72]. Concentrations of acetylcholine and the neuropeptide substance P in the corneal epithelium and tears have been shown to decline after corneal denervation
[36,82,83][36][73][74]. In several in vitro experiments, pro-proliferative and cell migration-promoting effects of neuropeptides or neurotrophins, such as substance P in combination with IGF-1, have been observed in corneal epithelial cells and limbal stem cells
[84,85,86][75][76][77].
New insight to the mechanistic understanding of the regulation of LSCs during corneal epithelial renewal arrived from a recently reported comparative scRNA-seq analysis of dissociated corneal limbi that were harvested from healthy, de-epithelialized healing and denervated corneas
[71][67]. A complex regulatory trophic communication between limbal cell populations was proposed on the basis of the altered gene expression in the pathological conditions
[71][67]. This model of paracrine interactions suggests that NGF acts synergistically with other trophic factors, including CNTF, PDGF-α, and TGF-β, locally expressed by SCs and MSCs in the limbus, to regulate the activity of LSCs.
5. Currently Available Pharmacological Approaches to Treat NK
The current nonsurgical (i.e., conservative) treatments for NK include autologous serum topical applications
[93][78], topical nerve growth factor (see below), as well as lubricating agents, such as artificial tears. Less expensive growth factors, such as topical insulin (1 U/mL applied three times per day) have also been used to accelerate healing of epithelial defects in patients with NK
[94,95][79][80]. Others have used scleral lenses to slow the progression of NK. Surgical options have historically relied on tarsorrhaphy (suturing the eyelids together) to reduce the area of corneal surface exposed, and hence reducing evaporative losses and protecting the ocular surface. These methods help reduce the incidence of corneal ulceration in NK; however, many patients will eventually sustain a corneal ulcer despite these treatments.
Nerve Growth Factor (NGF)
Thus far, recombinant human (rhNGF; Cenegermin, Dompé, Milan, Italy) remains the only clinically approved topical treatment for NK
[66,97][81][82]. However, despite its low toxicity and evidence for NGF’s stimulation of LSC activity
[98][83] and corneal epithelial healing in human NK cases
[50[50][84][85],
99,100], NGF treatment of NK presents remaining concerns. Firstly, the treatment requires extremely high doses, prolonged treatment duration, and dosing every two hours. Secondly, NGF treatment fails in over 30% of patients, especially in those with more severe NK
[66,97][81][82]. These observations suggest that NGF alone may not be sufficient to fully compensate for a lack of innervation in NK patients.
In preclinical in vivo studies, the topical application of NGF significantly accelerated the healing of epithelial defects in physiologically innervated corneas, and conversely, topical application of NGF antibodies reduced the rate of healing
[49,102][49][86]. Placebo-controlled clinical studies that evaluated the therapeutic effect of topically applied NGF in patients with the corneal lesions of NK found significantly faster epithelial regeneration compared to the placebo group. There was a concomitant significant increase in corneal sensitivity, which suggested that the cornea had become reinnervated and that the reinnervation may be induced by the applied NGF
[48,103][48][87].
NGF significantly increases the expression, axonal transport, and secretion of certain neuropeptides such as substance P in sensory neurons, suggesting a possible pathophysiological connection between the observed effects of the trophic factor and neurotransmitters
[104][88]. The detection of NGF and its corresponding high-affinity receptor tropomyosin kinase A (TrkA) in corneal epithelial cells and LSCs in humans and murine species suggests partial autocrine or paracrine effects of NGF in the epithelium and LSCs
[48,49][48][49].
The expression of TrkA by both corneal sensory neurons and non-neuronal cells, and the local expression of other trophic factors rather than NGF suggested to be involved in corneal epithelial renewal, raise two possible explanations for the observed inconsistencies in the efficacy of NGF treatment in NK.
6. Strategies to Promote Corneal Reinnervation
6.1. Potential Treatment with Tacrolimus
Tacrolimus, a clinically approved immunosuppressant commonly used for various ophthalmic conditions
[121][89], including allergic keratoconjunctivitis
[122][90] and corneal transplantation
[123][91], has been shown to promote axon regrowth in vitro
[124,125,126,127][92][93][94][95] and axonal regeneration following nerve injury in vivo
[128,129,130,131,132][96][97][98][99][100]. The direct neurotrophic effect of tacrolimus is mediated by the chaperone-like FK506 binding protein (FKBP52)
[125[93][95][101][102],
127,133,134], which forms heterocomplexes with the 90 kDa heat-shock protein (Hsp90) and its co-chaperone p23
[134][102] in the neuronal nucleus. Injured neurons redistribute this complex to the growth cones of regenerating neurites upon cellular contact with tacrolimus, promoting their accelerated regeneration
[134][102]. FKBP52 also mediates neuronal growth cone guidance in response to attractive and repulsive chemotactic signals
[135][103]. The systemic delivery of tacrolimus accelerates axonal regeneration in vivo by 12% to 16%
[136,137][104][105]. Sustained local delivery of low-dose tacrolimus directly at the repair site of peripheral nerves increases the number of regenerating nerve fibers and accelerates their rate of regeneration
[138,139,140][106][107][108].
Given the well-documented clinical safety record of tacrolimus in ophthalmic applications
[141,142,143][109][110][111] as well as in other indications
[144,145,146[112][113][114][115][116],
147,148], this therapeutic approach could be seamlessly implemented in clinical trials. Furthermore, although a specialized drug delivery system designed for the sustained topical delivery of tacrolimus may not be necessary for preliminary clinical investigations, tacrolimus eye drops may be considered for self-administration during the day, albeit with a mean surface residence time of over 1.5 h
[149][117]. It remains to be seen whether this frequency of dosing is adequate for maintaining therapeutic levels of tacrolimus in the cornea, and future investigations will shed light on this topic.
6.2. Corneal Neurotization
Clinically, corneal “neurotization” surgery improves corneal wound healing and corneal innervation in patients with anesthetic corneas
[64][118]. Neurotization entails harvest and placement of healthy functional nerves into a previously denervated tissue to regain motor or sensory function. This approach was first reported in the peer-reviewed literature by Terzis et al., who used direct neurotization from the contralateral supratrochlear (STN) and supraorbital nerves (SON)
[150][119]. These nerves were transferred directly into the denervated cornea’s limbal area, which provided corneal sensation and enabled corneal healing in patients with anesthetic corneas. Later modifications of this procedure, which utilized nerve grafts to reduce invasiveness and allow for more distant sensory nerve sources, were similarly successful and made the procedure feasible for bilaterally affected patients and congenital patients with more widespread facial denervation
[151][120] (
Figure 4).
Figure 4. Neurotization of human cornea. The end of the sural nerve graft is coapted to the divided donor supraorbital nerve, permitting fibers to regenerate into the nerve graft. The nerve graft is passed subcutaneously from this incision into the contralateral upper lid counterincision. The graft is then passed into a subconjunctival plane and separated into its component fascicles, which are then tunneled and secured individually around the limbus. Axons of the donor nerve emerge from the graft, and grow into the corneal stroma and into the overlying epithelium. While most corneal neurotization patients gain sensation post-neurotization, many do not achieve the highest possible level of sensation as measured by standard Cochet–Bonnet esthesiometry (CBA, a variable length nylon monofilament). Those patients that achieved less than 50 mm of CBA (of maximum 60 mm) were not as protected against recurrent corneal ulceration post-operatively as those who achieved 60 mm on CBA
[152][121]. This observation suggests that corneal neurotization, while revolutionary, may not be sufficient to provide full corneal sensation in many cases, leaving room for assistance from topical therapeutics.