Neurotrophic factors and corneal nerve regeneration

Marta Sacchetti, Alessandro Lambiase

Department of Sense Organs, Sapienza University of Rome, Rome, Italy

Neurotrophic factors and corneal nerve regeneration

Marta Sacchetti, Alessandro Lambiase*

Department of Sense Organs, Sapienza University of Rome, Rome, Italy

How to cite this article:Sacchetti M, Lambiase A (2017) Neurotrophic factors and corneal nerve regeneration. Neural Regen Res 12(8):1220-1224.

neurotrophic keratitis; corneal sensitivity; nerve regeneration; nerve growth factor

Introduction

Several local and systemic conditions that affect the cornea such as diabetes, dry eye, herpes simplex keratitis and neurotrophic keratitis, are associated with impairment of corneal innervation leading to decrease in tear production and impairment of wound healing (Sacchetti and Lambiase, 2014; Mastropasqua et al., 2017). The cornea is characterized by peculiar properties that make it a unique tissue to be used as a model for regenerative medicine studies, in fact, the cornea can be directly visualized and corneal structures, including nerves may be easily evaluated for both function and morphology using standard instruments such as slit lamp andin vivoconfocal microscopy (IVCM) (Cruzat et al., 2016). Recently, the cornea model has been successfully used in phase I/II clinical trials to obtain regulatory approval for cell therapy in limbal stem cell def i ciency, a rare corneal disease, orphan of medical treatment. In fact, the possibility of a direct visualization of corneal structures, including the epithelium, associated with well standardized clinical safety and ef ficacy outcomes, allowed scholars to demonstrate the regenerative effects of epithelial stem cell transplantation (Rama et al., 2017).e cornea represents also a useful model to characterize neuroregeneration and to evaluate novel neuroregenerative therapeutic approaches in humans.

Instruments for the assessment of corneal sensitivity have been developed and some of them are available for clinical use. Among them, the most used is the Cochet-Bonnet aesthesiometer, that allows scholars to easily evaluate mechanical corneal sensitivity. More recently, other clinical devices able to assess more specific sensitivity patterns, including chemical, thermal and mechanical corneal sensitivity, such as the Belmonte’s no-contact esthesiometer, have been introduced for clinical and research purposes (Belmonte et al., 2004; Golebiowski et al., 2011).

In addition, morphology of corneal nerves can be easily evaluated and visualized by IVCM, that is a rapid and non-invasive imaging method (Cruzat et al., 2017).In vivoconfocal microscopy allows scholars to visualize corneal nerves in health and in several ocular and systemic pathologic conditions and to assess their morphology in terms of nerve density and length, nerve branching, ref l ectivity and tortuosity (Lambiase et al., 2013; Cruzat et al., 2017) (Figure 1). This technique has also been used to assess peripheral neuropathy in patients with diabetes, idiopathic small fibre neuropathy, Charcot-Marie-Tooth Neuropathy Type 1 (CMT1A), Fabry’s disease and paraneoplastic neurological syndromes (Cruzat et al., 2017). In addition, IVCM is used to evaluatein vivothe ef f ects of treatments on corneal nerveregeneration and to evaluate recovery of corneal innervation during pathological conditions or aer surgery (Tavakoli et al., 2013; Cruzat et al., 2017; Culver et al., 2017; Lewis et al., 2017).

Recently, a neurofluorescent mouse model has been developed to study nerve regenerationin vivo. Specif i cally, thethy1-YFP transgenic mouse allowsin vivovisualization of fl uorescent sensory nerves and axonal regeneration and degeneration can be visualized and quantif i edin vivoallowing the possibility to investigate the neurotrophic potential of molecules and drugs (Yu and Rosenblatt, 2007; Leckelt et al., 2016).

Corneal Innervation Morphology and Function

Corneal innervation is provided by trigeminal nerve bundles that lose their perineurium and myelin sheaths and penetrate into the corneal stroma at the corneoscleral limbus, branching into the stromal plexus and then into the subepithelial and subbasal nerve plexus that provide sensitivity to the corneal epithelium (Muller et al., 2003). Corneal primary afferent neurons are represented by polymodal receptors that respond to mechanical, thermal and chemical stimuli, mechanoreceptors activated by mechanical stimuli, and cold receptors that respond to corneal cooling (Muller et al., 2003). Corneal sensory nerves react to ocular surface damage by inducing symptoms of pain and irritation and by eliciting protective reflexes such as blinking and tearing. In addition, corneal nerves provide trophic support to the ocular surface tissues, stimulate wound healing and maintain anatomic integrity by releasing neuromediators and growth factors (Muller et al., 2003; Shaheen et al., 2014; Mastropasqua et al., 2017).

Production and release of neuromediators during neurodegenerative diseases of the ocular surface can be easily evaluated by non-invasive, simple techniques such as corneal and conjunctival impression cytology and tear sample collection, that allow scholars to perform biochemical and molecular evaluation (Tervo et al., 1995; Yamada et al., 2000; Lambiase et al., 2011; Sacchetti et al., 2011; Mantelli et al., 2015). Several studies using these techniques have extended results on pathogenic mechanisms and drug effects from animal models to human ocular conditions (Lee et al., 2006; Lee and Kim, 2015).

Clinical Evidence of Corneal Neuroregeneration

Total or partial loss of corneal innervation leads to the development of neurotrophic keratitis (NK).is is a rare, degenerative corneal disease characterized by corneal epithelium breakdown with scarce tendency to healing and development of epithelial defects that may progress to corneal ulcer, melting and eye perforation (Mastropasqua et al., 2017). Several ocular and systemic diseases can damage the fifth cranial nerve at various levels, from the trigeminal nucleus to the corneal nerve endings. The most common causes of NK are herpetic viral keratitis, followed by trigeminal ophthalmic branch damage due to intracranial neoplasia and/ or neurosurgical procedures. Other ocular causes of corneal nerve damage include chemical burns, physical injuries, ocular surgery, corneal dystrophies, and chronic topical medications use. Systemic diseases, such as diabetes, multiple sclerosis, congenital syndromes, neurotoxic therapies and leprosy, can also impair corneal sensitivity, leading to NK (Sacchetti and Lambiase, 2014; Hsu and Modi, 2015).

Patients with NK, having a decrease of ocular surface sensitivity, rarely complain symptoms of ocular surface discomfort, while they may refer impaired vision in most severe cases (Sacchetti and Lambiase, 2014).e lack of symptoms in NK patients should be considered in the management of patients with local or systemic conditions causing trigeminal damage, and these patients should be routinely evaluated by an ophthalmologist to identify signs of corneal involvement (Sacchetti and Lambiase, 2014). NK is classif i ed based on the severity of the corneal damage in 3 stages. Stage 1 is characterized by a cloudy appearance of the epithelium associated or not with corneal and conjunctival epithelial damage. Stage 2 is characterized by the presence of a persistent epithelialdefect that may progress to corneal ulcer, perforation and/or stromal melting in stage 3.

Figure 1 A cornea of a healthy eye observed by slit lamp exam with frontal and longitudinal (insert) view.

Figure 2 Neurotrophic corneal ulcer (A) associated with impaired corneal innervation as demonstrated by decreased nerve density and increased tortuosity (B) atin vivoconfocal microscopy imaging.

Diagnosis of NK requires a careful clinical history collection to identify risk factors for trigeminal impairment and a multidisciplinary approach including ophthalmologist, neurologist and neuro-radiologist.e ophthalmologist will perform a complete eye examination with the slit lamp to identify corneal and conjunctival signs of NK and to classify NK severity. The diagnosis of NK is confirmed by the presence of decreased corneal sensitivity. Corneal sensitivity can be qualitatively detected by touching the cornea with the tip of a cotton swab, while severity of corneal sensitivity impairment may be quantified by a corneal aesthesiometer (Sacchetti and Lambiase, 2014). The Cochet-Bonnet aesthesiometer, the most frequently used, assesses corneal sensitivity by touching the cornea with a nylon fi lament: the length of the fi lament required to induce a blink reaction or a patient response will quantify the corneal mechanical sensitivity threshold (Sacchetti and Lambiase, 2014).

Corneal nerve imaging by IVCM may also allow thein vivoevaluation of all corneal structures including epithelium, nerves, keratocytes and endothelium (Lambiase et al., 2013). In fact, an IVCM study demonstrated that all corneal structures showed changes in patients with impairment of corneal sensitivity supporting the evidence of the key role of corneal nerves in maintaining homeostasis, survival, metabolism, and renewal of corneal cells (Lambiase et al., 2013) (Figure 2).

Management of NK is a challenge for ophthalmologists because of the lack of specific treatments able to stimulate corneal nerve regeneration and to restore corneal nerve function. Actually, NK treatment options aim at improving corneal healing and preventing progression of corneal damage and treatments able to stimulate corneal nerve regeneration are highly sought-aer (Sacchetti and Lambiase, 2014; Mastropasqua et al., 2017). Currently, the use of preservative-free ocular lubricants is a fi rst-line therapy and can be associated with the use of therapeutic contact lens (Lambiase et al., 1999; Sacchetti and Lambiase, 2014). Recently, a new regenerating agent, Cacicol (Laboratoires Théa, France), has been proposed as therapeutic option for treatment of persistent epithelial defects (Aifa et al., 2012; Arvola et al., 2016).e results of a fi rst open-label study evaluating the ef f ects of Cacicol in 11 patients with refractory NK reported a complete corneal healing in 73% of cases (Aifa et al., 2012). These data were not confirmed in a second study that described a complete corneal healing in only 33% of 6 patients aer 6–10 weeks of treatment (Arvola et al., 2016). Currently, unresponsive cases of NK require a surgical approach such as tarsorraphy, amniotic membrane transplantation, or conjunctival fl ap.ese approaches are ef f ective in achieving corneal healing, however, they result in poor cosmetic outcome and impairment of visual function (Chen et al., 2000; Hick et al., 2005; Khokhar et al., 2005; Turkoglu et al., 2014; Rajak et al., 2015; Uhlig et al., 2015; Ilic et al., 2016).

In the last decades, novel therapeutic approaches aiming at stimulating epithelial healing and/or corneal nerve regeneration and at restoring corneal nerve function and sensitivity have been proposed. Among them, several studies reported an high success rate in patients with NK treated with autologous serum eye drops, topical non-gelified platelet-rich plasma (PRP) eye drops, and umbilical cord serum eye drops. All these agents provide growth factors, neuromediators, and nutrients to the ocular surface and different studies reported corneal healing in 70% to 97% of cases (Lugo and Arentsen, 1987; Alino et al., 1998; Jeng and Dupps, 2009; Geremicca et al., 2010; Guadilla et al., 2013; Giannaccare et al., 2015; Sanchez-Avila et al., 2017). Based on these encouraging results two randomized clinical trials (RCT) are ongoing to evaluate safety and ef fi cacy of topical plasma rich in growth factors and cord blood in patients with NK (NCT03084861, NCT02707120).

In the last decades, several growth factors and neuroptptides have been shown to exert a positive ef f ect on corneal regeneration, including nerve growth factor (NGF), epidermal growth factor (EGF), SP and insulin-like growth factor-1 (IGF-1), vascular endothelial growth factor (VEGF), sema-phorins, neurotrophins 3 and 4 (NT-3; NT-4), growth associated protein-43 (Daniele et al., 1992; Muller et al., 2003; Nishida et al., 2007; Yanai et al., 2015). Despite encouraging results showed by the dif f erent trophic factors in stimulating corneal healing in patients with NK, very few demonstrated the ef ficacy in restoring corneal sensitivity and nerve morphology as demonstrated byin vivoimaging.

Topical treatment with NGF also represents a promising strategy to induce a durable recovery of trigeminal nerve function. A fi rst open-label study evaluating the ef ficacy of NGF eye drops treatment in 12 patients with neurotrophic keratitis at stage 2 and 3 reported complete corneal healing in 100% of cases after 10 days to 6 weeks treatment associated with a recovery of corneal sensitivity in most of the patients (Lambiase et al., 1998). A subsequent open-label, non-comparative study confirmed the previous results showing a complete corneal healing in 45 out of 45 eyes with NK at stage 2 and 3 refractive to medical treatments within 6 weeks of treatment. In this study, patients also showed signif i cant improvement of corneal sensitivity, tear function and visual acuity (Bonini et al., 2000).

In addition, the potential ef f ect of NGF in inducing nerve regeneration has been clearly demonstrated in several human diseases, such as diabetic, traumatic and toxic neuropathy (Sacchetti and Lambiase, 2014). Recently, a novel recombinant human NGF has been developed and has successfully completed the clinical development phase and recently received marketing authorization in Europe (EU/1/17/1197) opening new prospective for its next clinical use for inducing corneal neuroregeneration (Ferrari et al., 2014).

An open study reported that treatment with oral nicergoline was ef f ective in reaching a complete corneal healing in 23 out of 27 eyes with NK and in improving corneal sensitivity.ese ef f ects were associated with a signif i cant increase in tear level of NGF (Lee and Kim, 2015).

Other neurotrophic factors are under investigation. Specifically, topical treatment with CNTF in mice with wounded corneas showed an increased nerve fi ber density 8 weeks aer wounding (Reichard et al., 2014).e administration of pigment epithelial-derived factor in association with docosahexaenoic acid aer experimental surgery in rabbits showed a 75% of recovery of corneal sensitivity after 7 weeks of treatment (He et al., 2015). Subconjunctival injection of neuropeptide FF in diabetic mice promoted corneal nerve injury recovery and epithelial wound healing (Dai et al., 2015).

Conclusions

The cornea represents a unique model to evaluate in vivo the morphology and function of sensory nerves, their role in maintaining tissue trophism, the crosstalk between nerves and cells and the ef f ect of neuroregenerative drugs.e “human model” of the corneal disease induced by sensory nerve impairment is called NK. In fact, all local and systemic conditions causing damage to the trigeminal nerve may decrease corneal sensitivity and lead to the development of NK that is characterized by the development of non-healing corneal defects associated with lack of ocular symptoms. A multidisciplinary approach to patients with NK will aid at early identifying patients with NK and at improving their clinical outcome.e management of NK is still a challenge, due to the lack of pathogenic treatments targeting nerve regeneration.e development of novel molecules stimulating corneal nerve regeneration will improve the clinical management and outcomes of this disease.

Author contributions:AL and MS substantially contributed to condecptuzlization, acquisition of data, writing the draand revising the article.

Conf l icts of interest:None declared.

Plagiarism check:Checked twice by ienticate.

Peer review:Externally peer reviewed.

Open access statement:

Open peer review reports:

Reviewer 1: Rayaz Ahmed Malik, Weill Cornell Medical College in Qatar, Qatar.

Reviewer 2: Steven Levy, MD Stem Cells, USA.

Comments to authors: This is an excellent review of the physiology of the corneal nerves and the etiologies and disease manifestations of Neurotrophic Keratitis (NK).e paper also provides a well researched review of the current and previously published interventions using various neurotrophic factors. Having additional information detailing the completed studies within the paper would be helpful.

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*< class="emphasis_italic">Correspondence to: Alessandro Lambiase, M.D., Ph.D., alessandro.lambiase@ uniroma1.it.

Alessandro Lambiase, M.D., Ph.D., alessandro.lambiase@ uniroma1.it.

orcid: 0000-0002-8974-991X (Alessandro Lambiase) 0000-0001-5569-778X (Marta Sacchetti)

10.4103/1673-5374.213534

Accepted: 2017-07-20