Abstract
Aims
To evaluate the impact of herpes simplex virus (HSV)-induced scar location on bilateral corneal nerve alterations using laser in vivo confocal microscopy (IVCM).
Methods
Central and peripheral corneal subbasal nerve density (CSND) were assessed bilaterally in 39 patients with unilateral HSV-induced corneal scars (21 central scars (CS), 18 peripheral scars (PS)) using IVCM. Results were compared between patients and 24 age-matched controls. CSND was correlated to corneal sensation for all locations.
Results
Overall patients revealed significant decrease of CSND in the central and peripheral cornea (9.13±0.98 and 6.26±0.53 mm/mm2, p<0.001), compared with controls (22.60±0.77 and 9.88±0.49 mm/mm2). CS group showed a decrease in central (8.09±1.30 mm/mm2) and total peripheral nerves (5.15±0.62 mm/mm2) of the affected eyes, whereas PS group demonstrated a decrease in central (10.34±1.48 mm/mm2) and localised peripheral nerves only in the scar area (4.22±0.77 mm/mm2) (all p<0.001). In contralateral eyes, CSND decreased in the central cornea of the CS group (16.88±1.27, p=0.004), and in the peripheral area, mirroring the scar area in the affected eyes of the PS group (7.20±0.87, p=0.032). Corneal sensation significantly decreased in the whole cornea of the affected, but not in contralateral eyes (p<0.001). A positive correlation between CSND and corneal sensation was found in all locations (p<0.001).
Conclusions
Patients with HSV scar demonstrate bilateral CSND decrease as shown by IVCM. CSND and corneal sensation decrease in both central and peripheral cornea in affected eyes, although only in the scar area in PS group. Interestingly, diminishment of CSND was found locally in the contralateral eyes, corresponding and mirroring the scar location in the affected eyes.
INTRODUCTION
Herpes simplex virus (HSV)-1 keratitis is the most common infectious cause of unilateral blindness in developed countries.1 The incidence of HSV keratitis in the USA and Europe ranges from 4.1 to 31.5 cases per 100 000 per year, of which 42% comprise new cases and 58% recurrent cases.2 3 Due to the nature of the HSV, it can initiate the primary infection with obvious clinical symptoms or travel retrograde along sensory nerves to establish a latent infection in the trigeminal or other neurosensory ganglia.4 Viral reactivation and anterograde travel of the HSV along the nerves of infected ganglia can periodically occur and cause disease recurrence. The recurrence rate of HSV keratitis after an initial episode has been reported from 10% at 1 year up to over 60% at 20 years.5 Patients with an increased rate of recurrence, have demonstrated a higher risk for corneal scarring and neovascularisation.6
Recurrent HSV keratitis may induce persistent corneal inflammation or subclinical corneal infection, despite absence of clinical manifestations.7 The damage to sensory nerve fibres along the trigeminal nerve pathways, combined with chronic inflammation, results in corneal nerve loss and subsequent neurotrophic keratopathy (NK).8 9 The severity of NK varies greatly between patients with HSV keratitis. To date, the correlation of the location (central vs peripheral) of corneal scars with the severity of the NK and outcomes of this disease have not been studied.
Laser in vivo confocal microscopy (IVCM) is a high-resolution microscope that visualises the living cornea at a cellular level.8 This rapid and non-invasive technique has allowed for understanding of corneal microstructures in both health and disease.8–11 Currently, IVCM has been used for corneal nerve analysis in various ocular conditions, including HSV keratitis.9 11 12 Although the nature of HSV keratitis is typically unilateral, it can present in up to 12% of adult patients bilaterally.1 13 14 Clinically unilateral HSV keratitis was typically not thought to affect the unaffected eyes in patients.1 However, several recent articles have shown evidence of contralateral involvement in unilateral HSV cases, such as bilateral dry eye disease,15 and bilateral endothelial cell loss in patients with unilateral herpetic keratitis.16 17 Further, our group has previously reported bilateral decrease in corneal nerves in unilateral HSV keratitis.8 9
To date, known data merely revealed that unilateral HSV keratitis induces bilateral subbasal nerve loss and the regeneration of corneal nerves can occur very slowly.18 However, there have been no prognosticators that allow clinicians to predict the severity of corneal nerve damage and subsequent NK in patients with HSV keratitis. We hypothesised that the location of corneal scar can result in differential bilateral loss of subbasal corneal nerves in patients with unilateral HSV keratitis, hence affecting the severity of NK. To test this hypothesis, we performed a cross-sectional analysis to assess central and peripheral subbasal nerve density and corneal sensation in patients with central and peripheral scars (PS) from HSV keratitis.
SUBJECTS AND METHODS
Patients
This was a prospective, cross-sectional, single-centre study conducted in a controlled, single-blinded fashion. Thirty-nine patients who had unilateral HSV-induced corneal scarring treated at the Cornea Service of the Massachusetts Eye and Ear Infirmary, Boston, Massachusetts, between 2008 and 2014 were included in the study. All patients were diagnosed with HSV keratitis based on clinical presentation of at least one episode of characteristic epithelial or stromal HSV keratitis. Twenty-four age-matched and sex-matched healthy volunteers, who had no diabetes, history of ocular and systemic diseases, previous ocular surgery, current use of contact lens or ocular medication were selected from our reference control database for serving as normal controls. To prevent the statistical bias, only one eye of each control was used to compare with both affected and contralateral eyes of the patients. The primary outcomes aimed to assess the level of subbasal nerve loss in patients with HSV corneal scars.
All patients, who were in the remission stage with intact corneal epithelium, no signs of active ocular inflammation, no stromal infiltration, no keratouveitis and had clinically normal contralateral eyes, were enrolled during their regular follow-up visits. Patients and controls underwent a complete ophthalmological examination, including slit-lamp biomicroscopy, tonometry and fundus examination. The study group (39 HSV patients) was further divided into two subgroups based on the location of the corneal scar. If the location of the corneal scar was within the central 5 mm zone of the cornea, it was considered a central scar; otherwise, it was considered as a peripheral scar. Patients with multiple scars were excluded. Of the 39 patients, 21 patients had a central corneal scar (CS) and 18 patients had a peripheral corneal scar (PS).
Corneal esthesiometry
Corneal sensation was measured bilaterally by a Cochet-Bonnet esthesiometer (Luneau Ophthalmologie, Chartres, France) in all subjects as previously described,9 and the unaffected eye was measured first. Three different areas (one in the centre and two in the periphery) of each cornea were evaluated. For normal controls and the CS group, corneal sensation was measured in the central cornea, as well as the superior and inferior cornea. The average of superior and inferior corneal sensation represented peripheral corneal sensation. For the PS group, corneal sensation of the affected eyes was measured in the central cornea, the peripheral scar area (PA) and opposite area of the scar (peripheral cornea 180° away from the scar location: PO), and likewise in contralateral eyes (figure 1). The average of PA and PO represented peripheral corneal sensation of the PS group.
Figure 1.
The schematic of analysed areas of corneal sensation and laser in vivo confocal microscopy. Three analysed areas of the affected and contralateral eye of patient with herpes simplex virus (HSV) central corneal scar were assessed (A). Three analysed areas of the affected and contralateral eye of patient with HSV peripheral corneal scar was assessed (B). 
Represents the area of scar. 
Represents the analysed area. PA: the area of peripheral scar in affected eye; mirroring area in contralateral eye. PO: the area opposite to the scar (180° away from the scar); mirroring area in contralateral eye. In vivo confocal microscopy image of normal corneal subbasal nerve plexus in healthy controls (C). All corneal subbasal nerves are traced by the semiautomated tracing programme NeuronJ, a plug-in for ImageJ software: main nerve trunks are identified by yellow tracing and nerve branches are highlighted by pink tracing (D).
Laser IVCM
Laser IVCM (Heidelberg Retina Tomograph 3 with the Rostock Cornea Module (HRT3/RCM); Heidelberg Engineering GmbH, Heidelberg, Germany) was performed bilaterally for subbasal nerve assessment as previously described.19 Each eye was scanned in three different areas by obtaining images in the central and two peripheral corneal areas corresponding to the corneal esthesiometry. A random computer-generated number was assigned to each subject before IVCM scanning to provide masking to the observers (CC and RM).
Image analysis
An independent masked observer chose the three best-focused images with good contrast from each area of corneal subbasal layer.9 All images were analysed by two masked observers (CC and RM). Subbasal corneal nerves were traced and quantified using the semi-automated tracing programme NeuronJ,20 a plug-in for ImageJ. Subbasal nerves were categorised into main nerve trunks, nerve branches and total nerves.9
Statistical analysis
Statistical analysis was performed using SPSS V.20.0. The normality of data was investigated by Shapiro-Wilk’s test. Baseline characteristics between the patients and control group as well as among the patients with CS versus PS, but not between the eyes of same individual, were compared with analysis of variance (ANOVA), χ2 and Student’s t-test. Comparisons between nerve density and corneal sensation among the controls, affected eyes and contralateral eyes were performed with ANOVA with Scheffe post-hoc test. The analysis of covariance (ANCOVA) was used to evaluate the effect of variables such as sex, age, duration of the disease and number of recurrences on alterations of corneal nerve density and sensation. Pearson correlation coefficient was applied to assess the association between subbasal corneal nerve density and sensation. P values less than 0.05 were considered statistically significant.
RESULTS
Patient characteristics and study groups
Thirty-nine patients with unilateral HSV keratitis corneal scarring (21 CS and 18 PS) were enrolled in this study. Mean age of patients and healthy controls were 54.7±3.1 and 50.3±2.1 years, respectively. There was no statistically significant difference of baseline characteristics between patient groups and controls (all p>0.05) except there were more small scars (size <2 mm) in CS than PS (p=0.04) (table 1).
Table 1.
Demographic data of patients with herpes simplex virus keratitis (HSV) and controls
| Controls | All HSV patients | HSV central scar | HSV peripheral scar | P value | |
|---|---|---|---|---|---|
| Number of patients | 24 | 39 | 21 | 18 | – |
| Sex, male/female (n) | 9/15 | 19/20 | 9/12 | 10/8 | 0.55 |
| Age (years) | 50.3±2.1 | 54.7±3.1 | 53.0±4.4 | 56.6±4.3 | 0.41 |
| Time since first episode (years) | – | 11.1±2.0 | 11.6±2.8 | 10.5±3.1 | 0.79 |
| Time since last episode (years) | – | 2.0±0.5 | 1.3±0.5 | 2.8±0.8 | 0.12 |
| Number of recurrences | – | 6.7±1.2 | 6.7±1.6 | 6.7±1.9 | 0.99 |
| Corneal scar diameter <2 mm/>2 mm (n) |
– | 26/13 | 17/4 | 9/9 | 0.04* |
| Corneal sensation | 6.00±0.00 | ||||
| Affected eyes | |||||
| Central cornea | 3.35±0.36* | 3.62±0.50* | 3.03±0.53* | All <0.001* | |
| Peripheral cornea | 3.42±0.31* | 3.56±0.44* | 2.61±0.47* (PA) | All <0.001* | |
| 3.89±0.46* (PO) | <0.001* | ||||
| Contralateral eyes | |||||
| Central cornea | 5.67±0.14 | 5.82±0.12 | 5.50±0.27 | All >0.06 | |
| Peripheral cornea | 5.28±0.17 | 5.13±0.28 | 5.15±0.31* (PA) | 0.002* (PA) | |
| 5.76±0.10 (PO) | 0.006* (PO) | ||||
Values are presented as mean±SEM, unless otherwise noted.
PA represents peripheral cornea of the contralateral eye at the area corresponding to the area of scar in the affected eye.
PO represents peripheral cornea at 180° away from PA.
χ2 test was performed to analyse the difference of sex and size of corneal scars between the groups.
Analysis of covariance (ANCOVA) was performed to analyse the difference of age between the groups.
Independent t-test was performed to analyse the difference of duration of HSV episode between the groups.
Statistical significance when p<0.05 compared between study groups.
CS, central scar; PS, peripheral scar.
Corneal subbasal nerve alterations by IVCM
Subbasal corneal nerve density
The nerve density in the affected eyes of all HSV patients was significantly lower than controls for both central (9.13±0.98 vs 22.60±0.77 mm/mm2, p<0.001) and peripheral corneas (6.26±0.53 vs 9.88±0.49 mm/mm2, p<0.001). Similarly, the contralateral unaffected eyes showed significant nerve loss in the central (17.63±0.91 mm/mm2, p=0.002) and peripheral cornea (8.36±0.45 mm/mm2, p=0.016) as compared with controls. As expected, there was a significant difference in nerve density between the affected and contralateral unaffected eyes in both the central and peripheral cornea (p<0.00) (table 2).
Table 2.
Subbasal corneal nerve parameters in healthy controls, the affected eyes and contralateral eyes of patients with herpes simplex virus (HSV)-induced corneal scarrin
| Affected eyes | Central cornea |
Peripheral cornea |
|||||||
|---|---|---|---|---|---|---|---|---|---|
| Controls | All HSV patients | Central scar | Peripheral scar | Controls | All HSV patients | Central scar | Peripheral scar |
||
| Affected area | Opposite area | ||||||||
|
| |||||||||
| Total nerve density (mm/mm2) | 22.60±0.77 | 9.13±0.98* | 8.09±1.30* | 10.34±1.48* | 9.88±0.49 | 6.26±0.53* | 5.15±0.62* | 4.22±0.77* | 7.66±0.79 |
| Main nerve trunk density (mm/mm2) | 9.98±0.44 | 4.28±0.51* | 4.22±0.77* | 4.35±0.66* | 5.50±0.41 | 2.99±0.24* | 2.92±0.34* | 1.78±0.30* | 3.08±0.33* |
| Nerve branch density (mm/mm2) | 12.61±0.65 | 4.84±0.56* | 3.86±0.64* | 5.99±0.92* | 4.37±0.28 | 3.27±0.39* | 2.22±0.36* | 2.44±0.49 | 4.58±0.63 |
| Total nerve number (n/frame) | 20.9±1.6 | 8.5±1.0* | 7.2±1.2* | 10.0±1.5* | 8.7±0.7 | 5.4±0.5* | 4.1±0.5* | 4.0±0.8* | 7.0±0.8 |
| Main nerve trunk number (n/frame) | 4.0±0.2 | 1.8±0.2* | 1.7±0.3* | 1.8±0.3* | 2.2±0.2 | 1.3±0.1* | 1.3±0.2* | 0.8±0.1* | 1.3±0.2* |
| Nerve branch number (n/frame) | 16.9±1.6 | 6.7±0.8* | 5.4±1.0* | 8.2±1.2* | 6.5±0.6 | 4.0±0.5* | 2.8±0.5* | 3.2±0.7* | 5.6±0.7 |
| Contralateral eyes | Central cornea | Peripheral cornea | |||||||
|
| |||||||||
| Controls | All HSV patients | Central scar | Peripheral scar | Controls | All HSV patients | Central scar | Peripheral scar | ||
| PA | PO | ||||||||
| Total nerve density (mm/mm2) | 22.60±0.77 | 17.63±0.91* | 16.88±1.27* | 18.50±1.33 | 9.88±0.49 | 8.36±0.45* | 7.77±0.62 | 7.20±0.87* | 9.11±0.62 |
| Main nerve trunk density (mm/mm2) | 9.98±0.44 | 8.10±0.38* | 8.15±0.61 | 8.50±0.46 | 5.50±0.41 | 3.83±0.21* | 3.89±0.32 | 3.04±0.31* | 3.75±0.27 |
| Nerve branch density (mm/mm2) | 12.61±0.65 | 9.53±0.72* | 8.73±0.94* | 10.45±1.09 | 4.37±0.28 | 4.53±0.35 | 3.87±0.39 | 4.16±0.61 | 5.36±0.58 |
| Total nerve number (n/frame) | 20.9±1.6 | 17.0±1.2 | 15.4±1.5* | 18.8±1.8 | 8.7±0.7 | 7.7±0.6 | 6.5±0.6 | 6.6±0.9 | 9.1±1.0 |
| Main nerve trunk number (n/frame) | 4.0±0.2 | 3.3±0.2 | 3.4±0.3 | 3.3±0.2 | 2.2±0.2 | 1.7±0.1* | 1.7±0.1 | 1.4±0.1 | 1.6±0.1 |
| Nerve branch number (n/frame) | 16.9±1.6 | 13.6±1.1 | 12.1±1.4 | 15.5±1.7 | 6.5±0.6 | 6.0±0.6 | 4.9±0.5 | 5.2±0.8 | 7.5±1.1 |
Values are presented as mean±SEM, unless otherwise noted.
PA represents peripheral cornea of the contralateral eye at the area corresponding to the area of scar in the affected eye.
PO represents peripheral cornea at 180° away from PA.
Statistical significance when p<0.05 compared with controls.
CS, central scar; PS, peripheral scar.
Effect of scar location on corneal subbasal nerve density in the affected eyes
The eyes with CS revealed a significant diminishment of central nerves (8.09±1.30 mm/mm2) and globally for peripheral nerves (5.15±0.62 mm/mm2) as compared with controls (p<0.001), whereas the eyes with PS demonstrated similar diminishment of central nerves (10.34±1.48 mm/mm2, p<0.001) and localised peripheral nerves only at the scar area (PA) (4.22±0.77 mm/mm2, p<0.001), but not at the opposite site of the scar (PO) (7.66±0.79 mm/mm2, p=0.98). When we compared the nerve density between the eyes having CS and PS, there was no significant difference in nerve density (p>0.05) (figures 2 and 3).
Figure 2.
Comparison of total subbasal nerve density in the central and peripheral cornea between controls, affected eyes and contralateral eyes of patients with herpes simplex virus (HSV) corneal scarring. (A) All HSV patients versus controls; the bar graphs represent significant bilateral depletion of subbasal nerves in both the central and peripheral cornea when compared with controls. (B) The affected eyes of HSV patients versus controls; there is a significant decrease of total subbasal nerve density in both the central and peripheral cornea of patients with central scar as compared with controls. Meanwhile, a significant reduction of corneal nerve density of patients with peripheral scar is also revealed in the central and peripheral cornea but only at the area of scar. (C) Contralateral eyes of HSV patients versus controls; a significant decrease of total subbasal nerve density compared with controls is found in the central cornea of patient with central scar. CS: central scar, PS: peripheral scar, PA: the area of scar; PO: the area 180° away from the scar. Data presented as mean±SEM, unless otherwise noted. Error bars represents SE from the mean. Statistical significance when p<0.05 compared with controls; *P for total subbasal nerve density; ŧP for main nerve trunks; tP for nerve branches.
Figure 3.
(A) Slit lamp photograph shows central scar (CS) of patient with previous herpes simplex virus (HSV) keratitis. In vivo confocal microscopy (IVCM) images represent corneal subbasal nerve plexus in the central and peripheral cornea of healthy controls (A–C); affected eyes of patients (D–F); contralateral eyes of patients (G–I). Subbasal nerve density significantly decreased in both the central and overall peripheral cornea of the affected eyes and also the central cornea of the contralateral eyes (red arrows reflect statistically significant changes). (B) Slit lamp photograph shows peripheral scar (PS) of patient with previous HSV keratitis. IVCM images represent corneal subbasal nerve plexus in the central and peripheral cornea of healthy controls (J-L); affected eyes of patients (M-O); Contralateral eyes of patients (P-R). Significant corneal nerve depletion demonstrated in the central and peripheral cornea only at the area of scar (PA) in the affected eyes. Interestingly, there was a localised reduction of peripheral corneal nerves in the contralateral eyes detected at the area mirroring to the scar area in the affected eyes (red arrows reflect statistically significant changes). CS represents the patients with central scar and PS represents the patients with peripheral scar. PA: the area of scar; PO: the area 180° away from the scar.
Effect of scar location on corneal subbasal nerve density in the contralateral eyes
There was a differential loss of subbasal nerves between contralateral eyes of patients with CS and PS shown in figure 3. Patients with CS revealed a significant diminishment of the total number of nerves (15.4±1.5/frame, p=0.01) and density (16.88±1.27 mm/mm2, p=0.004) only in the central cornea compared with controls, whereas there was no significant nerve loss observed in the periphery (p>0.05). In contrast, patients with PS showed a decrease of total nerve density (7.20±0.87 mm/mm2, p=0.032) in the periphery, mirroring to the scar area in the affected eyes, while there was no significant subbasal nerve decrease in the central cornea.
Corneal sensation and correlation to subbasal nerve alterations
In the affected eyes of the patients with CS and PS, corneal sensation significantly decreased (p<0.001) in both central (3.67±0.38 and 3.13±0.40) and overall peripheral corneas (3.89±0.35 and 3.33±0.38 (PA), 3.53±0.41 (PO), respectively) as compared with normal controls (6.00±0.00). However, there was no significant difference of the corneal sensation between CS and PS groups in both central and peripheral corneas (table 1). Corneal sensation in the contralateral eyes of patients with CS and PS was slightly diminished in both central and peripheral corneas (p=0.096), when compared with controls. There was a significant decrease in sensation only in the peripheral area (5.15±0.31), mirroring the scar location in the affected eyes (p=0.014). A significant positive correlation between corneal sensation and the subbasal nerve density was found in both central (r=0.53, p<0.001) and peripheral corneas (r=0.27, p<0.01). We did not find an impact of sex, age, duration of the disease and number of recurrences on differential alterations of corneal nerve density and sensation in these patient groups (ANCOVA, all p>0.05).
DISCUSSION
This study compares bilateral subbasal corneal nerve alterations in HSV patients with regards to their central or peripheral corneal scar location. To the best of our knowledge, we are the first to assess the role of HSV-induced scar location on bilateral morphological and functional corneal nerve alterations. While both central and peripheral corneal HSV scars result in overall decrease of subbasal nerves and corneal sensation loss centrally, the peripheral cornea is only affected in patients with CS and in the specific area of PS. Interestingly, the degree of nerve damage observed is more severe in patients with CS as compared with patients with PS.
In addition, our study strikingly demonstrates that the location of the herpetic scar on the affected eye has a mirror effect on their respective contralateral eyes. These findings are in line with our previous findings, in which unilateral herpetic keratitis results in bilateral diminishment of corneal nerves.9 18 21 The presented correlation between location of corneal scarring and severity and pattern of corneal nerve damage may thus provide additional information that could be used to predict the prognosis of herpetic keratitis and its complications.
It could be speculated that patients with CS suffered from viral infection in the central cornea, and subsequently developed central corneal nerve loss and scarring, and that the peripheral nerve damage was due to peripheral dissemination of the virus. As a result, patients with CS not only show central corneal nerve loss, but also generalised peripheral corneal nerve decline. In contrast, patients with PS may have suffered from localised infection in the peripheral cornea and subsequent peripheral corneal nerve loss. Although the viral propagation may also occur from the peripheral infected area towards the central cornea, it is possible that viral dissemination across the cornea in a centripetal fashion has less effect than a centrifugal direction. Therefore, a significant decrease of corneal nerves is only detected in the central cornea and peripheral affected area, while the rest of the peripheral cornea is minimally affected.
The novel findings of nerve diminishment in the contralateral eyes mirroring the peripheral location in the affected eyes were presumed to be from bilateral symmetrical anatomy of the nervous system. The pattern of contralateral nerve loss occurring after the unilateral nerve damage have previously been discussed by several researchers.22–25 Moreover, previous animal studies demonstrated that after primary nerve injury, there were alterations occurring in sensory ganglia and the spinal cord, prior to contralateral nerve changes.26 27 Thus, with implicated circuits of the central nervous system, any peripheral nerve inputs from peripheral organs are transferred to higher centres in the spinal cord or brain, and then projected the outputs bilaterally along their corresponding nerve distributions.22 Likewise in the eyes, a transmission of neuronal signals bilaterally causing mirror-image alterations in unilateral eye disease is predicated.
The possible explanations for bilateral ocular involvement detected in patients with clinically unilateral eye disease have also been proposed.15–17 28 First, central regulation of the nervous system may initiate neural downregulation of the contralateral side.9 23 24 A previous study in mice also demonstrated congruous bilateral nerve changes after unilateral axotomy of the ciliary branches of ophthalmic nerve.29 These findings imply that unilateral peripheral nerve injury induce central sensitisation, subsequently affecting bilateral peripheral nerve changes.30 Second, the trigeminal nerve pathways cross the midline to reach the contralateral principal nucleus.24 31 32 This bilateral connection of trigeminal nerves and nuclei may allow HSV to spread directly from the primary affected eye to the contralateral eye.17 Clinically affected eyes exhibit a higher amount of viral replication and shedding, which may lead to more severe corneal nerve damage in the affected eyes as compared with the contralateral eyes.9 22
The current study demonstrates that the duration of the disease and number of recurrences did not have a significant effect on corneal nerve density and sensation. There are several rationales for this. First, there are various factors that affect corneal nerve alterations, in which complex neuro-immune mechanisms are simultaneously being orchestrated. Therefore, it is not just the number of recurrences, but the duration and severity of recurrences, the duration of a recurrence prior to a visit, the time between onset of a recurrence and initiation of anti- inflammatory therapy, which may all affect corneal nerve alterations. Second, duration is defined as time between first onset and enrollment visit, during which time the disease is in remission and can flare up. Third, the number of recurrences do not reflect the severity of each recurrence. Fourth, the number of recurrences do not include time of the last recurrence. Furthermore, we have previously shown that corneal sensation does not change until a threshold value of subbasal corneal nerve density is reached.18 Finally, Cochet-Bonnet esthesiometry assesses only mechanical nociceptors, and the result cannot be extrapolated to potential changes to polymodal and thermoceptors.9
One of the limitations of this study is that most of the patients with CS had relatively smaller scar sizes compared with PS. Therefore, bilateral effects of corneal nerve diminishment are not severe, resulting in significant decline of subbasal nerves appearing only in the central area of contralateral eyes, not including the periphery as it occurred in the affected eyes. Second, we did not differentiate our patients on the basis of types of HSV keratitis, which are different in their pathological mechanisms. We have previously demonstrated that decreased corneal nerves are evident within days of acute epithelial HSV keratitis and that comparison of acute HSV keratitis to chronic HSV keratitis did not demonstrate significant changes in corneal nerves.9 We have further shown that recovery of corneal nerves is minimal in HSV keratitis, and thus with each recurrence there is likely increased nerve loss,18 and thus a direct comparison will likely not show significant changes. Third, the evaluation of the peripheral corneal sensation and subbasal nerve density was confined in only two quadrants of the cornea to keep minimum manipulation. Nevertheless, we believe that sampling two areas, 180° away from each other were sufficient to represent the peripheral corneal changes without interspersing effect from one another. Finally, corneal sensation assessed by Cochet-Bonnet esthesiometer might not be optimal as it can assess only mechanical nociceptors,9 with limited stimulus step-range, resulting in suprathreshold of minimum stimulus detected.33 However, the data presented herein may provide evidence that could serve as a primary prognosticator for disease severity or risk, including the possibility of developing NK.
In conclusion, there is a differential effect of HSV-induced corneal scar location on bilateral corneal nerve alterations as detected by IVCM both in the central and peripheral cornea. Patients with central scars show more severe subbasal nerve damage, resulting in more profound decrease of sensation in the central and overall peripheral cornea in the affected eyes as compared with patients with peripheral corneal scars. Patients with both CS and PS also demonstrate decreased corneal nerve density in their contralateral clinically unaffected eyes, but interestingly only locally at the area corresponding to the scar location in their affected eyes. Nevertheless, corneal sensation of contralateral eyes is preserved. Once patient presents with HSV-induced corneal scars, particularly centrally, generalised subbasal nerve damage and subsequent NK is more likely in that eye and should be monitored more closely by clinicians.
Funding
NIH K08-EY020575 (PH), NIH R01-EY022695 (PH), Research to Prevent Blindness Career Development Award (PH, no grant number), MEEI Foundation (PH, no grant number), Falk Medial Research Trust (PH, no grant number), Johnstone Fund (DP-L, no grant number), Stevens Fund (DP-L, no grant number).
Footnotes
Correction notice This paper has been updated since it was published online. The second affiliation for author Andrea Cruzat has been updated.
Competing interests None declared.
Patient consent for publication Not required.
Ethics approval This study complied with the Health Insurance Portability and Accountability Act, adhered to the tenets of the Declaration of Helsinki, and was approved by the institutional review board/ethics committee of Massachusetts Eye and Ear Infirmary, Department of Ophthalmology, Harvard Medical School, Boston, MA, USA. All subjects gave a written informed consent after a detailed explanation of the nature of the study.
Data availability statement
Data are available upon reasonable request. The data that support the findings of this study are available from the corresponding author (PH), upon reasonable request.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
Data are available upon reasonable request. The data that support the findings of this study are available from the corresponding author (PH), upon reasonable request.