Surgical management of infectious keratitis.

The successful management of infectious keratitis is usually achieved with a combination of tools for accurate diagnosis and targeted timely antimicrobial therapy. An armamentarium of surgical interventions is available in the acute stage which can be resorted to in a step wise manner or in combination guided by the response to treatment. Simple surgical modalities can facilitate accurate diagnosis e.g. corneal biopsy and alcohol delamination. Surgery to promote epithelial healing can vary from tarsorrhaphy, amniotic membrane transplantation or conjunctival flaps depending on the extent of infection, visual prognosis, availability of tissue and surgeon's experience. Collagen crosslinking has been increasingly utilized with successful results to strengthen the cornea and reduce the infective load consequently the need for further elaborate surgical interventions. It has shown encouraging results specially in superficial bacterial and fungal keratitis but for deeper infections, viral and acanthamoeba keratitis, its use remains questionable. When globe integrity is compromised, corneal gluing is the most commonly used procedure to seal small perforations. In larger perforations/fulminant infections a tectonic/therapeutic graft is advisable. Partial thickness grafts are increasingly popular to treat superficial infection or internally tamponade perforations. Peripheral therapeutic grafts face challenges with potential requirement for a manually fashioned graft, and increased risk of rejection due to proximity to the limbal vessels. Late stage visual rehabilitation is likely to require further surgical interventions after complete resolution of infection and inflammation. A preliminary assessment of corneal sensation and integrity of the ocular surface are key for any successful surgical intervention to restore vision.

Manifestation of Herpetic Eye Disease after COVID-19 Vaccine: A UK Case Series.

PURPOSE: To highlight the potential risk of herpetic eye disease (HED) reactivation following COVID-19 vaccine. METHODS: Retrospective analysis of all patients who presented with HED within 28 days post-first dose COVID-19 vaccination. RESULTS: Eleven eyes (n = 10 patients) were included. The mean interval between COVID-19 vaccination and ocular symptoms/signs was 12.3 ± 10.3 days. Four (40%) patients presented with HSV keratitis, and six (60%) patients presented with VZV keratitis (five had concurrent other signs of herpes zoster ophthalmicus). Common ocular signs included multiple scattered dendritic/pseudodendritic corneal epitheliopathy (90.9%), anterior uveitis (63.6%), and endothelitis (27.3%). All cases were successfully treated with topical and systemic antiviral treatment and/or topical corticosteroids (mean healing time = 3.9 ± 1.6 weeks). CONCLUSIONS: Our case series highlights the potential temporal association between HED and COVID-19 vaccine. Prophylactic antiviral treatment is recommended in patients with a history of HED prior to COVID-19 vaccination.

In vivo confocal microscopy features and clinicohistological correlation of limbal nerve corpuscles.

AIMS: To describe the in vivo confocal microscopy (IVCM) features of human limbal nerve corpuscles (LNCs) and correlate these with the histological features. METHODS: We examined 40 eyes of 29 healthy living subjects (17 female, 12 male; mean age=47.6) by IVCM. Four limbal quadrants were scanned through all epithelial layers and stroma to identify the LNCs and associated nerves. Ten fresh normal human corneoscleral discs from five deceased patients with a mean age of 67 years and 17 eye-bank corneoscleral rims with a mean age of 57.6 years were stained as whole mounts by the acetylcholinesterase (AChE) method to demonstrate LNCs and corneal nerves. Stained tissue was scanned in multiple layers with the NanoZoomer digital pathology microscope. The in vivo results were correlated to the histological findings. RESULTS: On IVCM, LNCs were identified in 65% of the eyes studied and were mainly (84%) located in the inferior or superior limbal regions. They appeared either as bright (hyper-reflective) round or oval single structures within the hyporeflective, relatively acellular fibrous core of the palisades or were clustered in groups, often located anterior to the palisades of Vogt. They measured 36 µm in largest diameter (range 20-56 µm). The in vivo features were consistent with the histology, which showed LNCs as strongly AChE positive round or oval structures. CONCLUSION: The strong correlation with histology will enable use of IVCM to study LNCs in normal and disease conditions.

12-year analysis of incidence, microbiological profiles and in vitro antimicrobial susceptibility of infectious keratitis: the Nottingham Infectious Keratitis Study.

BACKGROUND/AIMS: To examine the incidence, causative microorganisms and in vitro antimicrobial susceptibility and resistance profiles of infectious keratitis (IK) in Nottingham, UK. METHODS: A retrospective study of all patients who were diagnosed with IK and underwent corneal scraping between July 2007 and October 2019 (a 12-year period) at a UK tertiary referral centre. Relevant data, including demographic factors, microbiological profiles and in vitro antibiotic susceptibility of IK, were analysed. RESULTS: The estimated incidence of IK was 34.7 per 100 000 people/year. Of the 1333 corneal scrapes, 502 (37.7%) were culture-positive and 572 causative microorganisms were identified. Sixty (4.5%) cases were of polymicrobial origin (caused by ≥2 different microorganisms). Gram-positive bacteria (308, 53.8%) were most commonly isolated, followed by Gram-negative bacteria (223, 39.0%), acanthamoeba (24, 4.2%) and fungi (17, 3.0%). Pseudomonas aeruginosa (135, 23.6%) was the single most common organism isolated. There was a significant increase in Moraxella spp (p<0.001) and significant decrease in Klebsiella spp (p=0.004) over time. The in vitro susceptibilities of Gram-positive and Gram-negative bacteria to cephalosporin, fluoroquinolone and aminoglycoside were 100.0% and 81.3%, 91.9% and 98.1%, and 95.2% and 98.3%, respectively. An increase in resistance against penicillin was observed in Gram-positive (from 3.5% to 12.7%; p=0.005) and Gram-negative bacteria (from 52.6% to 65.4%; p=0.22). CONCLUSION: IK represents a relatively common and persistent burden in the UK and the reported incidence is likely underestimated. Current broad-spectrum antimicrobial treatment provides a good coverage for IK, although challenged by some level of antimicrobial resistance and polymicrobial infection.

Seasonal patterns of incidence, demographic factors and microbiological profiles of infectious keratitis: the Nottingham Infectious Keratitis Study.

PURPOSE: The purpose of this study is to examine the seasonal patterns of incidence, demographic factors and microbiological profiles of infectious keratitis (IK) in Nottingham, UK. METHODS: A retrospective study of all patients who were diagnosed with IK and underwent corneal scraping during 2008-2019 at a UK tertiary referral centre. Seasonal patterns of incidence (in per 100,000 population-year), demographic factors, culture positivity rate and microbiological profiles of IK were analysed. RESULTS: A total of 1272 IK cases were included. The overall incidence of IK was highest during summer (37.7, 95% confidence interval (CI): 31.3-44.1), followed by autumn (36.7, 95% CI: 31.0-42.4), winter (36.4, 95% CI: 32.1-40.8) and spring (30.6, 95% CI: 26.8-34.3), though not statistically significant (p = 0.14). The incidence of IK during summer increased significantly over the 12 years of study (r = 0.58, p = 0.049), but the incidence of IK in other seasons remained relatively stable throughout the study period. Significant seasonal variations were observed in patients' age (younger age in summer) and causative organisms, including Pseudomonas aeruginosa (32.9% in summer vs. 14.8% in winter; p < 0.001) and gram-positive bacilli (16.1% in summer vs. 4.7% in winter; p = 0.014). CONCLUSION: The incidence of IK in Nottingham was similar among four seasons. No temporal trend in the annual incidence of IK was observed, as reported previously, but there was a significant yearly increase in the incidence of IK during summer in Nottingham over the past decade. The association of younger age, P. aeruginosa and gram-positive bacilli infection with summer was likely attributed to contact lens wear, increased outdoor/water activity and warmer temperature conducive for microbial growth.

Clinical and in vivo confocal microscopic features of neuropathic corneal pain.

AIMS: To describe clinical and in vivo confocal microscopy (IVCM) features of neuropathic corneal pain (NCP) without clinically visible signs. METHODS: Prospective, observational study of 27 eyes of 14 patients who had continuous severe ocular pain for one or more years, with minimal or no ocular surface signs and were non-responsive to topical lubricants, steroids and/or ciclosporin. All patients were evaluated using Ocular Surface Disease Index, Oxford grading scale, Schirmer test 1, Cochet Bonnet esthesiometry and response to topical anaesthesia. Central and paracentral regions of the cornea of patients and seven healthy controls were studied by IVCM. Corneal epithelial thickness and sub-basal nerve density were measured in patients and controls. RESULTS: Four patients responded to topical anaesthesia (responsive group (RG)), indicating peripheral NCP while 10 patients did not show any improvement (non-responsive group (NRG)), indicating central NCP. Schirmer-1 test was within normal limits in the RG but significantly greater in the NRG (p<0.001). None of the other clinical parameters nor corneal epithelial thickness were statistically significantly different. The sub-basal nerve density was significantly reduced (p<0.008) in patients compared with controls. Stroma of all patients demonstrated activated keratocytes and spindle, lateral and stump microneuromas. There was a statistically significant greater number of microneuromas (p<0.0001) and activated keratocytes in RG compared with NRG. CONCLUSION: NCP without visible clinical signs does not represent typical dry eye disease. Distinct signs demonstrated on IVCM suggest that peripheral NCP, which responds to topical anaesthesia, and central NCP, which does not, are separate entities.

Corneal nerves in health and disease.

The cornea is the most sensitive structure in the human body. Corneal nerves adapt to maintain transparency and contribute to corneal health by mediating tear secretion and protective reflexes and provide trophic support to epithelial and stromal cells. The nerves destined for the cornea travel from the trigeminal ganglion in a complex and coordinated manner to terminate between and within corneal epithelial cells with which they are intricately integrated in a relationship of mutual support involving neurotrophins and neuromediators. The nerve terminals/receptors carry sensory impulses generated by mechanical, pain, cold and chemical stimuli. Modern imaging modalities have revealed a range of structural abnormalities such as attrition of nerves in neurotrophic keratopathy and post-penetrating keratoplasty; hyper-regeneration in keratoconus; decrease of sub-basal plexus with increased stromal nerves in bullous keratopathy and changes such as thickening, tortuosity, coiling and looping in a host of conditions including post corneal surgery. Functionally, symptoms of hyperaesthesia, pain, hypoaesthesia and anaesthesia dominate. Morphology and function do not always correlate. Symptoms can dominate in the absence of any visible nerve pathology and vice-versa. Sensory and trophic functions too can be dissociated with pre-ganglionic lesions causing sensory loss despite preservation of the sub-basal nerve plexus and minimal neurotrophic keratopathy. Structural and/or functional nerve anomalies can be induced by corneal pathology and conversely, nerve pathology can drive inflammation and corneal pathology. Improvements in accuracy of assessing sensory function and imaging nerves in vivo will reveal more information on the cause and effect relationship between corneal nerves and corneal diseases.

Survival of corneal nerve/sheath structures in organ-cultured donor corneas.

PURPOSE: To study the morphology of human corneal nerves in eye bank organ-cultured corneas and in corneal grafts post-transplantation. METHODS: Thirty-seven organ-cultured corneas were divided into: Group-A, anterior 300-400 µm of 20 corneas used for Descemets stripping endothelial keratoplasty, and Group-B, 17 full-thickness corneas unsuitable for transplantation. Corneas whole mounts were stained for nerves using acetylcholinesterase technique and examined by NanoZoomer digital pathology microscope. Central and sub-Bowman's stromal nerves and the sub-basal nerve plexus including perforation sites and terminal bulbs were studied. Ten eyes were imaged following penetrating keratoplasty using in-vivo confocal microscopy (IVCM) for the presence of sub-basal and stromal nerves at 1, 4-5 and 7-8 weeks postoperatively (five eyes) and in all the other five eyes, the final follow-up was at 12 weeks. RESULTS: Fifteen of twenty (75%) corneas had stromal nerves in Group-A and 15 of 17 (88.2%) in Group-B. Average number of stromal nerves entering peripherally were 9.1 (range: 1-36). 7.5 in Group-A and 10.8 in Group-B. Central stromal nerves were seen in eight samples in Group-A and nine in Group-B. Many stromal nerves terminated abruptly without demonstrable continuity through Bowman's membrane. No terminal bulbs or sub-basal nerves were detected. In-vivo confocal microscopy (IVCM) showed 4 of 5 in 9 of 10 (90%) donor corneas had stromal nerves 1 week postoperatively, which remained present in 8 of 10 (80%) corneas at 4-5 weeks and in 9 of 10 (90%) at 7-8 weeks postoperatively. All 5 corneas analysed at 12 weeks showed the same stromal nerves from 1 to 12 weeks postoperatively. Sub-basal nerves were absent in all corneas over the 12-week study period. CONCLUSION: This study provides further insight into the behaviour of corneal nerves in transplanted corneas. Corneal stromal nerves/nerve-sheaths are preserved in organ-cultured eye bank eyes and persist post-transplantation up to 3 months. These could provide directional guidance to regenerating nerves from host stroma.

Nerve terminals at the human corneoscleral limbus.

AIMS: To demonstrate and characterise distinct subepithelial compact nerve endings (CNE) at the human corneoscleral limbus. METHODS: Ten fresh human donor corneoscleral discs (mean age, 67 years) and 26 organ-cultured corneoscleral rims (mean age, 59 years) were studied. All samples were subjected to enzyme histochemical staining related to endogenous acetylcholinesterase present in nerve tissue and H&E staining. Whole-mount en face imaging with NanoZoomer digital pathology microscope and serial cross-section imaging with light microscope were undertaken. RESULTS: Nerves entering the corneoscleral limbus and peripheral cornea terminate under the epithelium as enlarged multiloculated and multinucleated ovoid structures within a 2 mm zone. They are closely associated with the rete pegs of the limbal palisades and the limbal epithelial crypts, often located within characteristic stromal invaginations of these structures. Their numbers ranged from 70 to 300 per corneoscleral rim. The size ranged from 20 to 100 µm. They had one or more nerve connections and were interconnected to other similar endings and to the limbal nerve plexus. CONCLUSION: Human corneoscleral limbus demonstrates a population of nerve terminals resembling CNE with distinct morphological features. They are closely associated with the limbal stem cell niches, suggesting a potential contribution to the niche environment.

Corneal hypoesthesia with normal sub-basal nerve density following surgery for trigeminal neuralgia.

PURPOSE: To evaluate the corneal sub-basal nerve plexus in patients presenting with hypoesthesia following surgery for trigeminal neuralgia. METHODS: Twenty-one patients who had unilateral medically uncontrolled trigeminal neuralgia and underwent ipsilateral surgery from 2006 to 2012 were included. Of these, 10 had microvascular decompression (MVD group) and 11 had balloon compression of the trigeminal ganglion (BC group). Slit lamp examination, Cochet-Bonnet aesthesiometery and in vivo confocal microscopy were carried out on both eyes of each patient. Nerve density data were statistically analysed. RESULTS: Corneal sensations and sub-basal nerve densities in MVD group were normal and equal in both the operated and unoperated sides, indicating that there was no intra-operative damage of the ophthalmic division of the trigeminal nerve (V1). However, those in BC group, despite having significantly reduced corneal sensations on the operated side (p = 0.007), did not demonstrate any significant difference in their sub-basal nerve densities (p = 0.477). No patient had any ocular symptoms. CONCLUSIONS: This study supports the hypothesis that complete ganglionic damage and/or postganglionic damage of V1 results in corneal hypoesthesia and neurotrophic keratitis, but partial ganglionic or preganglionic damage would preserve trophic function despite hypoesthesia and not result in clinically significant symptoms or signs of neurotrophic keratitis. The trophic and sensory functions of V1 are therefore independent and can be dissociated by disease or injury.

Calcified corneal nerves.

PURPOSE: To report a case of calcified corneal nerves in an eye with long-standing diabetic eye disease. METHODS: This is a case report where the clinical diagnosis was made using a slit-lamp biomicroscope and confirmed using in vivo confocal microscopy. Photoslit-lamp images were taken for both eyes, and nerve thickness measurements made using ImageJ software. RESULTS: A 70-year-old British white woman showed band keratopathy in the right eye and dense white lines and dots in her left cornea on slit-lamp examination. The left corneal images had an appearance exactly similar to stromal nerves and terminal bulbs reported before. In vivo confocal microscopy (IVCM) of the left cornea showed hyperreflective and thickened stromal and sub-Bowman nerves and their characteristic termination in bulbous thickenings. CONCLUSIONS: Clinically detected corneal nerve calcification has not been described before. This report presents the direct visualization of calcified corneal nerves and their terminal bulbs and IVCM characteristics.

Management of postkeratoplasty astigmatism by paired arcuate incisions with compression sutures.

PURPOSE: To analyse the efficacy of paired arcuate incisions and compression sutures technique in the management of post penetrating keratoplasty (PK) astigmatism. METHODS: A paired arcuate incision with compression sutures procedure was used to treat 26 eyes with post-PK astigmatism ranging from 6.00 to 16.50 dioptres (D). The incisions were placed at the 7.0 mm optical zone inside the graft-host junction at a depth of 80% of corneal thickness. A 45° paired arc length was planned for eyes with preoperative astigmatism between 6D and 9D, and a 60° paired arc length was planned for eyes with preoperative astigmatism of >9D. At 3 months, corneal topography and refraction was performed and suture(s) removed if indicated. Net and vector astigmatism changes were calculated to determine the efficacy of the procedure. The indications for PK included keratoconus, Fuch's endothelial dystrophy, pseudophakic bullous keratopathy and corneal scar. RESULTS: There was a statistically significant reduction in the mean magnitude of astigmatism from 9.66 ± 2.90D preoperatively to 4.37 ± 2.53D postoperatively in the whole group. The mean decrease in the astigmatism was 4.37 ± 2.05D (58.4%) and 6.23 ± 3.63D (52.6%) in patients with 6-9D and >9D, respectively. Vector power calculations also showed a significant astigmatism reduction in all groups. The safety and efficacy indices were 1.40 and 0.28, respectively. CONCLUSIONS: Manual astigmatic keratotomy is a viable technique with relatively good safety and efficacy outcomes. Based on the results we propose that increasing the arc length to a minimum of 60° for astigmatism of 6-9D, and to 75° for astigmatism >9D, is likely to have a greater beneficial effect.

Profile of sight-threatening infectious keratitis: a prospective study.

PURPOSE: To prospectively study patients presenting with sight-threatening corneal ulcers with a view to identify the predisposing factors, causative organisms, clinical signs and treatment outcomes. METHODS: Prospectively, over 3-year period, all cases with serious infective keratitis presenting to Queens Medical Hospital in Nottingham, UK, were recruited. Detailed information on the aetiology, culture results, signs & symptoms, the treatment given and the patient's response was collected and statistically analysed. RESULTS: One hundred and forty-three eyes of 129 patients were enrolled. Thirty-one patients were managed as out-patients, and 98 were treated as in-patients. The mean duration of admission was 9 ± 13 days but was significantly higher in older patients and in Acanthamoeba keratitis cases. The important risk factors were ocular surface disease (32%), contact lens wear (26%) and previous ocular surgery (20%). Old age, deep infiltration, steroid use and poor initial vision were risk factors for prolonged course of treatment in bacterial keratitis. Corneal scrapings were done in 89% of the cases, but positive results were obtained only in 41.7%. Staphylococcus aureus was the most common isolated bacteria (18.8%). Acanthamoeba and Pseudomonas aeruginosa were the second and third common causative organisms (16.6% and 15%, respectively). Overall, 8.3% needed corneal grafting, which survived in 83.3% and eradicated infection in 100%. CONCLUSION: Microbial keratitis is an important cause of ocular morbidity. Previous ocular disease is an important predisposing factor. Old age, steroid use and poor presenting visual acuity are important prognostic indicators. Corneal grafting is an effective option for managing recalcitrant corneal infections.

Wavefront-optimized excimer laser in situ keratomileusis for myopia and myopic astigmatism: refractive outcomes and corneal densitometry.

PURPOSE: To determine the refractive outcomes of wavefront-optimized laser in situ keratomileusis (LASIK) treatments, in particular to measure corneal densitometry after LASIK using the densitometry function of the Pentacam Scheimpflug system. SETTING: Division of Ophthalmology and Visual Sciences, University of Nottingham, United Kingdom. DESIGN: Cohort study. METHODS: Changes in postoperative visual acuity, refraction, and contrast sensitivity were evaluated after wavefront-optimized laser treatment. Corneal densitometry was evaluated with the Scheimpflug system before and after LASIK. RESULTS: One year postoperatively, the uncorrected distance visual acuity was 6/6 or better in 92% of eyes and 6/9 or better in all eyes. Eighty-six percent of eyes had no change in the corrected distance visual acuity (CDVA); 4% gained 1 or more lines. Wavefront-optimized LASIK was stable over 1 year postoperatively. Eighty-nine percent of eyes were within ±0.50 diopter (D) and 100% were within ±1.00 D of the intended correction 1 year postoperatively. Contrast sensitivity showed a nonsignificant improvement (1.55 ± 0.10 [SD] preoperatively to 1.57 ± 0.09 12 months postoperatively) (P > .05). There was a nonsignificant increase in corneal densitometry 1 year postoperatively (from 12.72 ± 2.43 to 13.04 ± 2.58) (P > .05). No correlation was found between corneal densitometry and contrast sensitivity or CDVA. CONCLUSIONS: Wavefront-optimized LASIK gave excellent refractive and visual outcomes and did not seem to affect corneal densitometry significantly 1 year postoperatively. However, larger studies may show a masked effect on corneal densitometry. FINANCIAL DISCLOSURE: No author has a financial or proprietary interest in any material or method mentioned.

Variable expression of human Beta defensins 3 and 9 at the human ocular surface in infectious keratitis.

PURPOSE: The authors have previously reported the presence of the antimicrobial peptides human beta defensin (hBD) 3 and hBD9 on the ocular surface (OS). These play an important role in infection and inflammation. In the present study, the authors studied the gene expression levels of hBD3 and hBD9 in healthy subjects and during and after healing of infectious keratitis. METHODS: Human OS specimens were obtained by impression cytology from healthy controls and patients with Acanthamoeba and Gram-negative and -positive bacterial keratitis (BK), both during active infection and after healing. The gene expression levels of hBD3 and hBD9 were determined using quantitative real-time polymerase chain reaction (RT-PCR). RESULTS: hBD3 and hBD9 were constitutively expressed in all healthy controls. During acute Acanthamoeba keratitis (AK), hBD3 levels were markedly increased and then returned close to normal levels after healing. In BK, hBD3 gene expression was moderately increased and then decreased after healing. In contrast to hBD3, hBD9 was significantly downregulated in both AK and Gram-positive BK, whereas it showed an insignificant decrease in Gram-negative BK. After healing, the expression showed upregulation except in Gram-positive BK, where it continued to decline. CONCLUSIONS: This is the first study that demonstrates the gene expression of hBD3 and hBD9 in response to infection. It illustrates that not all antimicrobial peptides (AMPs) behave in a similar manner. Some are upregulated and some are downregulated, suggesting a diverse role of AMP in infection and inflammation. The results point to a role of AMP-mediated host defense in Acanthamoeba keratitis as well.

Organization of the regenerated nerves in human corneal grafts.

PURPOSE: To examine by histopathology the degree of nerve regeneration in human corneal grafts and to determine the anatomic organization and morphology of the regenerated nerves. DESIGN: Experimental laboratory investigation. METHODS: Twelve corneal grafts from 12 patients (7 men and 5 women) aged 34-93 (mean, 66.9 years) were included. The most common indication for regrafting was late endothelial failure. The mean duration of graft survival was 6.41 years (range, 1-14 years). The freshly obtained specimens with a narrow rim of host tissue incorporating the graft-host junction were subjected to the acetylcholinesterase method for the demonstration of corneal nerves. RESULTS: Subbasal nerves were found in 75% and 25% of the grafts at the periphery and center, respectively. They were mostly originated from the host subbasal nerves. Regenerated stromal nerves were detected in 83% of the specimens; half of them showed extension into the center of the graft. A lack of the normal link between the subbasal and stromal nerves was observed and almost all of the regenerated stromal nerves were found to remain within the stroma and did not contribute to the epithelial innervation. CONCLUSIONS: A persistent anatomic disorganization of the corneal nerves in human grafts was found even 14 years after surgery. This could explain the significant reduction of corneal sensation reported in previous studies.

The effect of standard and transepithelial ultraviolet collagen cross-linking on human corneal nerves: an ex vivo study.

PURPOSE: To evaluate the early effect of standard and transepithelial collagen cross-linking on human corneal nerves in donor eyes by ex vivo confocal microscopy and acetylcholinesterase staining. DESIGN: Experimental laboratory investigation. METHODS: Eight human eye bank corneal buttons (mean age, 73.6 years) were included. Ultraviolet A collagen cross-linking was performed postmortem on 3 corneas with the standard protocol involving epithelial debridement and 4 corneas by the transepithelial approach. One cornea served as a control. Corneal nerves were evaluated using confocal microscopy and acetylcholinesterase histology. RESULTS: Confocal microscopy demonstrated the absence of subbasal nerves in corneas treated by the standard technique. These nerves were preserved in corneas treated by the transepithelial approach. Stromal nerves were visible in both groups. Histology of corneas treated by the standard technique revealed localized swellings of the stromal nerves with disruption of axonal membrane and loss of axonal continuity within the treatment zone. These changes were absent in corneas treated by the transepithelial approach. CONCLUSIONS: This study highlights the immediate effects of collagen cross-linking on the corneal nerves in an ex vivo model. The absence of subbasal nerves in the early phase of treatment appears to be attributable mainly to mechanical removal of epithelium, rather than ultraviolet light-induced damage. Localized swelling of the stromal nerves was the main difference between the 2 treatment protocols. Further research on laboratory animals would be necessary to verify these changes over a specified time course without the super-addition of postmortem changes.