Corneal basal epithelial cell density was significantly decreased in neurotrophic keratitis (A) when compared with contralateral unaffected eyes (B and C). C, Error bars indicate standard deviation.aP = .04.
Corneal subbasal nerve length was significantly decreased in neurotrophic keratitis (A) when compared with contralateral unaffected eyes (B and C). C, Error bars indicate standard deviation.aP = .04.
The number of hyperreflective keratocytes was significantly increased in neurotrophic keratitis (A) when compared with contralateral unaffected eyes (B and C). C, Error bars indicate standard deviation.aP = .04.
Corneal endothelial cell density was significantly decreased in neurotrophic keratitis (A) when compared with contralateral unaffected eyes (B and C). C, Error bars indicate standard deviation. D, The lower endothelial cell density was related to the longer duration of neurotrophic keratitis (NK). R2 linear = 0.565.aP = .01.
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Lambiase A, Sacchetti M, Mastropasqua A, Bonini S. Corneal Changes in Neurosurgically Induced Neurotrophic Keratitis. JAMA Ophthalmol. 2013;131(12):1547–1553. doi:10.1001/jamaophthalmol.2013.5064
Neurotrophic keratitis (NK) represents a sight-threatening complication after trigeminal impairment. To our knowledge, the duration for which trigeminal injury may affect corneal structures and function has not been investigated previously.
To describe the long-term clinical, morphological, and functional outcomes of NK after neurosurgical trigeminal damage.
Design, Setting, and Participants
Observational case series performed at a corneal and ocular surface diseases referral center in 2010. Eight consecutive patients with monolateral NK from 1 to 19 years after neurosurgery and 20 age- and sex-matched healthy participants were included.
Main Outcomes and Measures
Complete eye examination, tear film function tests, corneal staining, and Cochet-Bonnet esthesiometry were performed. The number and density of corneal nerves, number of hyperreflective keratocytes, and corneal epithelial, endothelial, and keratocyte cell densities were evaluated by in vivo slit scanning confocal microscopy. Clinical and morphological data were compared with the contralateral unaffected eyes and with the eyes of healthy control participants.
All patients showed superficial punctate keratitis and dry eye in the NK eye and a healthy contralateral eye. Decreased corneal sensitivity was observed in all affected eyes (mean [SD], 2.0 [1.9] mm in the affected eyes vs 5.8 [0.3] mm in the contralateral unaffected eyes; P = .01) and was related to decreased subbasal nerve length (P = .04; R = 0.895). Corneal epithelial and endothelial cell densities were significantly decreased and the number of hyperreflective keratocytes was significantly increased in NK eyes compared with contralateral unaffected eyes and with the eyes of healthy participants. A longer duration of NK was associated with lower endothelial cell density (P = .046; R = −0.715).
Conclusions and Relevance
Corneal morphology and function were impaired even years after neurosurgical trigeminal damage, suggesting that assessment of tear film and corneal sensitivity as well as in vivo confocal microscopy examination should be performed in all patients with trigeminal impairment.
The cornea is the most densely innervated human tissue. Corneal sensory nerves provide protective and trophic support to the cornea by regulating corneal epithelium integrity, proliferation, and wound healing.1,2 Experimental and clinical data have clearly demonstrated that the impairment of corneal sensitive nerve function induces functional and morphological changes of the corneal epithelium, leading to epithelial defects with poor tendency to spontaneous healing.1-3 In humans, injury of the trigeminal nerve leads to a decrease or absence of corneal sensation and to development of neurotrophic keratitis (NK).1,2 This condition is characterized by impairment of corneal sensitivity, corneal epithelial changes ranging from superficial punctate keratopathy to corneal ulcer and perforation, stromal scarring, neovascularization, tear function impairment, and decreased blink reflex.
Currently, corneal changes observed in NK are considered a consequence of epithelial breakdown, but some evidence suggests that corneal nerve damage may also induce changes of keratocytes and corneal endothelium. In fact, the recent introduction of in vivo corneal confocal microscopy (IVCM) has allowed for investigation of the entire corneal structure including epithelium, subbasal nerve plexus, keratocytes, and endothelium in healthy and pathological human corneas.4,5 Specifically, decreased corneal sensation in diabetic patients was associated with decreased subbasal nerve and basal epithelium density as well as changes in corneal stromal keratocytes and endothelium.6-8 Patients with herpes simplex virus keratitis also showed a relationship between decreased corneal sensation and changes in subbasal nerve plexus and endothelium morphology.9-11 This evidence suggests that alteration of corneal sensitivity may affect all corneal structures; however, both diabetic keratitis and herpes simplex virus keratitis result from a combination of different mechanisms including decreased innervation and metabolic, immune, and cytopathic effects.
In this study, we evaluated corneal structures by IVCM in patients with monolateral NK after neurosurgical trigeminal damage to assess long-term changes of corneal sensitivity and morphology.
This study was performed in accordance with the Declaration of Helsinki. The study was approved by the institutional review board of the University of Rome, Campus Bio-Medico, and written informed consent was obtained from the patients and healthy volunteers before examinations were performed.
Inclusion criteria were the diagnosis of monolateral NK at stage 1 after neurosurgery1,2 and a corneal scarring grade of 0.5 or lower according to the Fantes scale.12 Exclusion criteria were the presence of diabetes mellitus, previous intraocular surgery, history of ocular trauma, herpetic keratitis, and/or other ocular-associated diseases, use of topical treatments with the exception of ocular lubricants, and use of contact lenses.
Eight eyes of 8 consecutive patients (mean [SD] age, 49  years; 6 female, 2 male) with monolateral NK caused by neurosurgical damage to the trigeminal nerve as well as their contralateral unaffected eyes were included in the study. Twenty eyes of 20 healthy subjects (mean [SD] age, 53  years; 15 female, 5 male) were also included as a control group.
The diagnosis of NK was based on the history of trigeminal damage after neurosurgical interventions for brain neoplasia associated with ipsilateral corneal hypoesthesia or anesthesia.
All patients underwent evaluation for best spectacle-corrected visual acuity as well as complete eye examination.
Mechanical corneal sensation was measured at the central cornea with the Cochet-Bonnet esthesiometer (Luneau Ophtalmologie). This uses nylon monofilaments that have a diameter of 0.027 mm (Toray Industries, Inc), range from 0 to 6 cm in length, and apply different pressures to the cornea, shortening in steps of 1.0 cm if a positive response is not obtained. If a positive response is obtained, the thread is advanced by 0.5 cm. The longest filament length resulting in a positive response was considered the corneal sensitivity threshold, which was verified twice.2
Tear function was evaluated by the Schirmer I and break-up time tests.13 Corneal fluorescein staining was graded from 0 to 5 according to the Oxford scale.13
In vivo slit scanning confocal microscopy examination (Confoscan 4; Nidek Technologies) was performed bilaterally in the central cornea of all subjects with a 40×/0.75 objective lens. All Confoscan 4 examinations were performed by the same operator (M.S.) using a Z-ring and an internal fixation target to stabilize images. Eyes were anesthetized with 1 drop of oxybuprocaine hydrochloride,0.4% (benoxinate hydrochloride). The objective lens of the microscope was disinfected with isopropyl alcohol (70% vol/vol, with swabs). Then, a large drop of Viscotears liquid gel (Carbomer 940; Novartis Pharma) was applied to the tip of the lens as an immersion substance. Full-thickness automated scanning mode with a 5-µm scan step was used during each examination. Each image represents a coronal section of approximately 425 × 320 µm with magnification of ×500 and a lateral resolution of 1 µm/pixel. Corneal thickness was assessed and a minimum of 3 representative images were evaluated for superficial and basal epithelium, subbasal nerve plexus, superficial and deep stromal layer, and endothelium.4,5
Two masked observers evaluated the confocal images (M.S. and A.M.). The epithelial, stromal, and endothelial cells were manually counted using Adobe Photoshop 6.0 software (Adobe Systems). All the cells were counted within a 0.05-mm2 frame to calculate the cell density, which was expressed as the number of cells per square millimeter. Nerve density was assessed by measuring the total length of the nerve fibers in micrometers per frame. Main nerve trunks were defined as the total number of main nerve trunks in 1 image after analyzing the images anterior and posterior to the analyzed image to confirm that these did not branch from other nerves. Nerve branching was defined as the total number of nerve branches in 1 image.14 Tortuosity and reflectivity were classified according to criteria described by Oliveira-Soto and Efron.15
Statistical analysis was performed using Wilcoxon rank test and Mann-Whitney U test to assess differences between groups. Spearman ρ test was used to correlate clinical, demographic, and morphological parameters. We used SPSS version 18 statistical software (SPSS Inc). P < .05 was considered statistically significant.
Clinical characteristics of the patients are summarized in Table 1. At the inclusion, all patients showed a stage 1 monolateral NK according to criteria described by Mackie1 with mild to severe superficial punctate keratitis and were being treated only with preservative-free artificial tears. All contralateral eyes showed absence of pathological changes and normal corneal sensitivity, Schirmer test results, and break-up times.
Patients underwent neurosurgery in the past 1 to 19 years (mean [SD], 7.5  years) and were diagnosed as having NK from 1 to 24 months after neurosurgery. All patients also had a history of corneal ulcer 1 to 18 years (mean [SD], 6.6  years) after the onset of NK. Four patients had damage of the seventh cranial nerve with mild facial hemipalsy (Table 1).
All affected eyes showed a significant decrease in mechanical corneal sensitivity evaluated by Cochet-Bonnet esthesiometry when compared with the contralateral unaffected eye (mean [SD], 2.0 [1.9] vs 5.8 [0.3] mm, respectively; P = .01). Fluorescein staining demonstrated mild to severe epitheliopathy in NK eyes compared with unaffected eyes (mean [SD] Oxford score, 1.6 [0.7] vs 0, respectively; P < .001). Eyes with NK also showed a significant decrease in break-up time compared with contralateral unaffected eyes (mean [SD], 5.0 [2.0] vs 7.5 [2.3] seconds, respectively; P = .004), while Schirmer test results were not significantly different between the contralateral and pathological eyes (P = .66).
No significant correlations were observed between corneal sensitivity and the tear function test and corneal staining results (P = .52 and P = .20, respectively), while the higher corneal sensitivity was significantly correlated with a longer history of NK (P = .048; R = 0.712).
Results of IVCM evaluations are showed in Table 2. Eyes with NK showed a significant decrease of superficial and basal epithelial cell densities when compared with both contralateral unaffected eyes (mean [SD] superficial epithelial cell density, 1068  vs 1877  cells/mm2, respectively; P = .047; mean [SD] basal epithelial cell density, 3241  vs 4366  cells/mm2, respectively; P = .04) (Figure 1) and with healthy eyes of control participants (mean [SD] superficial epithelial cell density, 2145  cells/mm2; P = .04; mean [SD] basal epithelial cell density, 5732  cells/mm2; P = .004).
Subbasal nerves were also significantly reduced as compared with both contralateral unaffected eyes (mean [SD] number of nerve trunks, 1.0 [1.2] vs 4.4 [1.1], respectively; P = .04; and mean [SD] total nerve length, 1814  vs 11 606  µm/mm2, respectively; P = .04) (Figure 2) and healthy eyes (mean [SD] number of nerve trunks, 4.5 [0.9]; P = .003; mean [SD] total nerve length, 15 250  µm/mm2; P = .001). In NK eyes, the lower subbasal nerve length directly correlated with lower corneal sensitivity values (P = .04; R = 0.895).
The number of hyperreflective keratocytes was significantly increased in NK eyes when compared with contralateral unaffected eyes (mean [SD], 4.0 [2.2] vs 1.5 [2.2] hyperreflective keratocytes/frame, respectively; P = .04) and with healthy eyes (mean [SD], 1.2 [1.2] hyperreflective keratocytes/frame; P = .02) (Figure 3).
Interestingly, corneal endothelial cell density was significantly reduced in NK eyes when compared with both contralateral unaffected eyes (mean [SD], 2187  vs 3059  cells/mm2, respectively; P = .01) and with healthy eyes (mean [SD], 2960  cells/mm2; P = .004) (Figure 4A-C). Eyes with NK showed a significant correlation between the longer duration of NK and the lower endothelial cell density (P = .046; R = −0.715) (Figure 4D).
No significant difference in all morphological parameters were observed in the subgroup of patients with both NK and lagophthalmos.
In this study, we demonstrated that trigeminal nerve damage induces long-term corneal functional and morphological changes. All patients with NK showed decreased corneal sensitivity, superficial punctate keratitis, and dry eye associated with corneal epithelium, nerve, keratocyte, and endothelium alterations as demonstrated by IVCM.
Our study confirmed that the impairment of corneal sensitivity in patients with NK is related to a significant impairment of subbasal nerve plexus. Similar associations between impairment of corneal sensitivity and nerve morphology changes have been previously demonstrated in patients with diabetes, dry eye, pseudoexfoliation syndrome, and herpes simplex virus keratitis and after refractive surgery.6,9,16-19
Other long-term changes in NK, such as decreased epithelial and increased hyperreflective keratocyte cell densities, were detected by IVCM. It is generally accepted that corneal sensory nerves play a key role in maintaining the vitality, metabolism, and replenishment of corneal epithelial cells.2 The lower epithelial cell density in NK eyes may result from the decreased subbasal nerve supply; however, a pathological effect on the epithelium from the associated dry eye cannot be excluded.20,21 In fact, loss of corneal sensation is known to lead to tear and blink reflex dysfunction and to the development of corneal epithelial damage due to a consequent loss of key trophic neural factors supplied in tears. In line with this hypothesis, our patients with NK had a decreased tear break-up time associated with punctate keratopathy.2,22,23
In patients with NK, the presence of increased hyperreflective keratocytes, a common finding following corneal injury, infection, or inflammation, demonstrates that the disease is still ongoing even years after the neurosurgical trigeminal injury.4
A surprising novel finding was a decrease in endothelial cell density in NK eyes. This gives a new perspective on the importance of ocular sensory innervation on survival of the human corneal endothelium. In fact, while in rabbits the innervation of the endothelium is well described, in humans no evidence of nerve to endothelium interaction has been reported.24 Sensory nerve impairment may result in changes in aqueous humor and in the corneal microenvironment that ultimately lead to a loss of trophic factors critical to endothelial cell function and survival.25 Previous studies have shown an impairment of the endothelium pump function in patients with NK, and decreased endothelial cell density was described in patients with herpes simplex virus keratitis, pseudoexfoliation syndrome, and diabetes—all conditions characterized by decreased corneal sensitivity and subbasal plexus changes.6,9,10,18,26-28 These data suggest that impairment of corneal sensory nerves induces a prompt alteration of endothelial function as demonstrated by previous studies but also loss of cells over time.26 Lower endothelial cell density was also related to a longer duration of NK, suggesting that impairment of corneal sensory nerves induces immediate functional and long-lasting morphological alterations of endothelial cells. Since there is no evidence that human endothelial cells divide under normal circumstances, endothelial cell density may be a useful marker for the evaluation of long-term alterations due to loss of trophic support. Although we included only patients with surgically induced trigeminal damage and monolateral NK in this study to avoid interindividual variability and comorbidities, the strength of this study is limited by the small number of patients. A larger trial with long-term follow-up should be performed to confirm this hypothesis.
In conclusion, this study demonstrated that impairment of corneal sensory nerves induces anatomical changes of all layers of the cornea and that NK never completely resolves. In vivo corneal confocal microscopy analysis confirmed the presence of active pathological changes involving all corneal structures, including endothelial cell loss, suggesting that IVCM should be performed for better long-term management of patients with NK.
Corresponding Author: Stefano Bonini, MD, Department of Ophthalmology, University of Rome, Campus Bio-Medico, Via Alvaro del Portillo, 200, 00128 Rome, Italy (firstname.lastname@example.org).
Submitted for Publication: March 5, 2013; final revision received May 2, 2013; accepted May 8, 2013.
Published Online: October 24, 2013. doi:10.1001/jamaophthalmol.2013.5064.
Author Contributions: Drs Lambiase and Sacchetti had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Drs Lambiase and Sacchetti contributed equally to this study.
Study concept and design: Lambiase, Sacchetti, Bonini.
Acquisition of data: Lambiase, Sacchetti, Mastropasqua.
Analysis and interpretation of data: Lambiase, Sacchetti.
Drafting of the manuscript: Lambiase, Sacchetti, Mastropasqua.
Critical revision of the manuscript for important intellectual content: Lambiase, Sacchetti, Bonini.
Statistical analysis: Lambiase, Sacchetti, Mastropasqua.
Administrative, technical, or material support: Mastropasqua.
Study supervision: Lambiase, Bonini.
Conflict of Interest Disclosures: None reported.