Corneal Nerve Aberrations in Bullous Keratopathy

MOUHAMED AL-AQABA, THAER ALOMAR, JAMES LOWE, AND HARMINDER S. DUA

PURPOSE:

To study the corneal nerves in patients with chronic bullous keratopathy. DESIGN: Prospective observational case series with histologic evaluation. METHODS: We studied 25 eyes of 25 bullous keratopathy patients of different etiologies (17 female, 8 male; mean age, 76.3 years) as well as 6 eyes of 6 normal control subjects (5 male, 1 female; mean age, 38 years). All subjects were scanned by laser scanning confocal microscope. Five corneal buttons obtained following penetrating keratoplasty from 5 of the above patients and 6 normal control corneal buttons were stained as whole mounts with acetylcholinesterase (AChE) method for corneal nerve demonstration and scanned in multiple layers with digital pathology scanning microscope. RESULTS: The density, branching pattern, and diameter of sub-basal nerves were significantly lower in corneas with bullous keratopathy compared with normal corneas (density: 4.42 1.91 mm/mm2 vs 20.05 4.24 mm/ mm2; branching pattern: 36.02% 26.57% vs 70.79% 10.53%; diameter: 3.07 0.64 f.lm vs 4.57 1.12 f.lm). Aberrations such as localized thickenings or excrescences, abnormal twisting, coiling, and looping of the (mid) stromal nerves were observed in the study group both by in vivo confocal microscopy and on histology. CONCLUSIONS: Striking alterations in corneal innervation are present in corneas with bullous keratopathy that are unrelated to any specific etiology of bullous keratopathy. This study provides histologic confirmation of novel in vivo confocal microscopy findings related to corneal nerves in bullous keratopathy. (Am J Ophthalmol 2011;151:840 849. 2011 by Elsevier Inc. All rights reserved.)

ULLOUS KERATOPATHY IS A COMMON CLINICAL

endpoint of corneal endothelial dysfunction or damage resulting from a variety of causes. Common conditions associated with bullous keratopathy include cataract extraction with or without intraocular lens implantation, primary or secondary (immune rejection) corneal graft failure, absolute glaucoma, and endothelial
Accepted for publication Nov 12, 2010. From the School of Clinical Sciences, Division of Ophthalmology and Visual Sciences (M.A., T.A., H.S.D.), and the School of Molecular Medical Sciences, Division of Pathology (J.L.), University of Nottingham, Nottingham, United Kingdom. Inquiries to Harminder S. Dua, Division of Ophthalmology and Visual Sciences, B Floor, Eye ENT Centre, Queens Medical Centre, Nottingham University Hospitals NHS Trust, Derby Road, Nottingham. NG7 2UH, England, UK; e-mail: Harminder.dua@nottingham.ac.uk

dystrophies such as Fuchs endothelial corneal dystrophy (Fuchs dystrophy). Unfortunately, bullous keratopathy of iatrogenic origin accounts for about 60% of cases.13 The 3 most common causes of bullous keratopathy are pseudophakic bullous keratopathy, aphakic bullous keratopathy, and Fuchs dystrophy. Currently, bullous keratopathy is the leading indication of penetrating keratoplasty/endothelial transplant and regraft.1,35 Initially, the corneal stroma becomes edematous and thickened and eventually intraepithelial and subepithelial fluid-filled vesicles, bullae, appear.1 Clinical features can vary from asymptomatic early disease, glare, and painless decrease in vision to profound loss of vision attributable to subepithelial scarring in advanced cases.6 Painful episodes associated with photophobia and tearing are attributed to nerve stretching and irritation by epithelial and subepithelial bullae and to rupture of surface bullae with exposure of nerve endings.6,7 The histologic features of bullous keratopathy include intracellular epithelial edema and bullous separation, stromal thickening, and endothelial loss. In addition, bullous keratopathy secondary to Fuchs dystrophy also shows Descemet membrane thickening with posterior guttata.8 With the advent of in vivo confocal microscopy our understanding of the pathologic findings in bullous keratopathy has been enhanced by our ability to examine the tissue in vivo, thereby avoiding artifacts resulting from tissue handling and processing.9 12 Although pain, attributed to nerve stretching and exposure as stated above, is a common manifestation and is often intractable, there is very little information on the state of the corneal nerves in bullous keratopathy. Routine histologic techniques and transverse sections of corneal tissue are not conducive to a proper evaluation of the nerves. Laser scanning confocal microscopy has provided new insights into the orientation and distribution of human corneal nerves in health and disease.13,14 However, it is not always clear what structures equate to the in vivo confocal findings. Studies on direct correlation of confocal microscopy findings with histology of the examined tissue are few. We used this mode of examination to assess the corneal nerves in corneas with bullous keratopathy, including some that were scheduled for penetrating keratoplasty (PKP). We were able to examine whole mounts of corneal buttons removed at PKP with a special nerve stain to delineate corneal nerves with a view to correlate the in vivo confocal microscopy findings with histologic observations. We also intended to elucidate any nerve-related anatomic basis for pain, which is a dominant feature of advanced bullous keratopathy.
RESERVED. 0002-9394/$36.00 doi:10.1016/j.ajo.2010.11.013

840

2011 BY

ELSEVIER INC. ALL RIGHTS

TABLE 1. Demographics and Clinical Data of Patients With Bullous Keratopathy

Primary Diagnosis Method of Examination Duration of Disease (Months) Detection of the Sub-basal Nerve by IVCM Abnormal Stromal Nerves by IVCM

Patient

Sex

Age

Surgery

CCT (j.Lm)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

F F F M M F F F F F M F F F F F M F F F M M M M F

78 81 88 74 74 73 78 72 76 78 79 58 82 71 82 84 83 70 74 85 71 86 60 72 78

ABK PBK PBK PBK FED FED FED FED FED FED FED FED FED FED FED Idiopathic FED PBK PBK Glaucoma PBK PBK PBK FED PBK

Triple PKP Triple Triple Triple Triple PKP Triple DSEK PKP PKP PKP DSEK PKP PKP Triple Triple PKP PKP Triple DSEK PKP PKP PKP DSEK

IVCM+histology IVCM+histology IVCM+histology IVCM+histology IVCM IVCM IVCM IVCM IVCM IVCM IVCM IVCM IVCM IVCM IVCM IVCM IVCM IVCM IVCM IVCM IVCM IVCM IVCM IVCM+histology IVCM

9 34 9 14 31 48 7 24 11 4 8 14 8 19 48 5 7 10 12 11 6 28 18 39 11

700 628 691 650 598 760 750 642 776 760 588 730 760 613 570 737 624 688 670 749 766 633 663 695 625

+ + + + + + + +

+ +

+ + + + + + + +

+ + +

+ + +

APB aphakic bullous keratopathy; CCT central corneal thickness; DSEK Descemet stripping endothelial keratoplasty; F female; FED Fuchs endothelial dystrophy; IVCM in vivo confocal microscopy; M male; PBK pseudophakic bullous keratopathy; PKP penetrating keratoplasty; Triple triple procedure: PKP + cataract extraction + intraocular lens implant.

MATERIALS AND METHODS

A PROSPECTIVE CONSECUTIVE CASE SERIES OF 25 EYES OF 25

patients with bullous keratopathy were examined by in vivo confocal microscopy. Patient demographics and clinical data are given in Table 1. Fuchs dystrophy and pseudophakic bullous keratopathy accounted for 88% of the cases. There were 17 female and 8 male patients with a mean age of 76.3 + 7.3 (58-88) years. All patients were white. All the patients were treated with corneal transplant surgery. Twelve of 25 patients (48%) had penetrating keratoplasty alone, 9 of 25 (36%) had triple procedure (penetrating keratoplasty and lens extraction with implant) and 4 of 25 (16%) underwent Descemet stripping endothelial keratoplasty (DSEK). Five host corneal buttons were available for histology. Whole mounts were stained for corneal nerve demonstration using acetylcholinesterase technique and examined by light microscopy. The diagnosis of bullous keratopathy was based on history, slit-lamp examination, and central corneal thickness (CCT) (mean + SD, 682.6 + 63.7 j.Lm; range 570-776 j.Lm). An ultrasonic pachymetry device (Tomey SP-3000, Pachymeter; Tomey Corporation, Nagoya, JaVOL. 151, NO.
5

pan) was used for the measurements of CCT. The average time between diagnosis of bullous keratopathy and in vivo confocal microscopy scan was 17.4 months (range 4-48 months). None of the patients had corneal vascularization.
CONFOCAL MICROSCOPY:

All 25 eyes were examined preoperatively by laser scanning confocal microscope (Heidelberg Retina Tomograph II Rostock Corneal Module [RCM]; Heidelberg Engineering GmbH, Heidelberg, Germany). The device uses a class I diode laser (670-nm wavelength) with a 63X water-immersion lens (Olympus, Tokyo, Japan). The images obtained using this lens are 400 X 400 j.Lm, and have 2- and 4-j.Lm lateral resolution and optical depth resolution, respectively (provided by the manufacturer at http://www.accessdata.fda.gov/cdrh_docs/ pdf4/K042742.pdf). Image magnification on screen was 300X. In vivo confocal microscopy was performed under topical anesthesia with MINIMS oxybuprocaine hydrochloride 0.4% (Bausch & Lomb Ltd, Surrey, United Kingdom). A digital camera mounted on a side arm furnished a lateral view of the eye and objective lens to monitor the position of the objective lens on the surface of 841

CORNEAL NERVES IN BULLOUS KERATOPATHY

FIGURE 1. In vivo confocal microscrographs of the corneal nerves. Normal appearance of the sub-basal nerve plexus seen in a healthy control (Top left, frame level = 59 f.lm). Bulbous termination of sub-basal nerves is shown (Top left inset). Sub-basal nerve plexus appearance in bullous keratopathy (Top right, frame level = 32 f.lm). There is a reduction in the density and thickness of the nerves. Tortuous sub-basal nerves in bullous keratopathy (Middle left, frame level = 30 f.lm). Tortuous stromal nerves, some surrounding dark lacunae, observed at different depths within the stroma (Middle right, frame level = 189 f.lm; Bottom left, frame level = 380 f.lm and Bottom right frame level = 331 f.lm). (Scale bar = 100 f.lm).

the eye. A drop of 0.2% polyacrylic gel (Viscotears liquid gel; Novartis Pharmaceuticals Ltd., Surrey, United Kingdom) was used as coupling medium between the contact cap and objective lens of the microscope. VOL. 151, NO.
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Central and paracentral regions (approximately 7 X 7 mm) of the cornea were scanned through all the layers. Frames from sub-basal (beneath basal cells of corneal epithelium) and stromal layers containing nerves were 842

CORNEAL NERVES IN BULLOUS KERATOPATHY

FIGURE 2. Correlation of confocal microscopy findings (Left column) with those observed on histology of whole mounts (Right column) in corneas with bullous keratopathy. (Top left and Top right) A mid-stromal nerve characterized by localized nerve excrescences or thickenings (arrowheads) suggestive of early sprouting (arrows). (Middle left) A relatively thick stromal nerve with ill-defined margins at its bifurcation seen on confocal microscopy at the level of 126 f.lm. This corresponds with (Middle right) extensive axonal sprouting seen at a stromal nerve bifurcation on histology. (Bottom left and Bottom right) Both confocal microscopy and histology images show looping, convolutions, and coiling of aberrant corneal nerves. (Scale bar = 100 f.lm.)

selected for analysis. Standard quantitative descriptors for nerve studies were examined.13,15 These were nerve density, which is in mm/mm2; branching pattern which is expressed as the percentage of nerve branches per total number of sub-basal nerve fibers within a single frame; and diameter of sub-basal nerves, in microns. The thickest region of each main sub-basal nerve within a single frame was selected for the thickness analysis and the average diameter of 3 measurements for each nerve was calculated. VOL. 151, NO.
5

Qualitative morphologic evaluation of sub-basal and stromal nerves was also carried out.
ACETYLCHOLINESTERASE TECHNIQUE FOR THE DEMONSTRATION OF CORNEAL NERVES: In 5 patients where

in vivo confocal microscopy examination of their corneas was performed preoperatively, their corneal buttons obtained after penetrating keratoplasty for bullous keratopathy were processed and stained as whole mounts for 843

CORNEAL NERVES IN BULLOUS KERATOPATHY

FIGURE 3. A photomicrograph of a corneal button with pseudophakic bullous keratopathy showing extensive aberrations in corneal nerves. Only a few fragmented sub-basal nerves are seen superiorly (Top right, inset) compared to similar area in a healthy control (Middle right, inset) where longer sub-basal nerves with bulbous terminations of sub-Bowman nerves are seen. Extensive axonal regeneration from stromal nerves is seen at the periphery (Top left and Bottom right, insets) and centrally (Bottom left, inset). The nerves are also thinner and convoluted compared to the healthy control (Middle left, inset). Scale bar of insets at Top left, Middle left, Bottom left and Bottom right = 200 f.lm, and of insets at Top right and Middle right = 100 f.lm.

cholinesterase enzyme using the acetylcholinesterase (AChE) technique.16 The protocol of the staining method has been described previously.17 Briefly, corneal buttons were fixed in cold 4% formaldehyde (pH 7) for 4 hours and then rinsed overnight in phosphate-buffered saline (PBS). Specimens were incubated in the stock solution containing acetylthiocholine iodide as a substrate for 24 hours at 37C. The acetylcholinesterase enzyme in the nerves reacted with acetylthiocholine iodide in the substrate to produce a brown coloration of the nerves. The color was intensified with a dilute solution of ammonium sulfide. Specimens were dehydrated by immersion in alcohol and cleared in xylene, as is standard for histologic preparation. The specimens were finally mounted between a slide and coverslip and scanned en face using a Hamamatsu NanoZoomer digital pathology (NDP) microscope system (Hamamatsu, Hamamatsu City, Japan). The corneal buttons were examined at 40X magnification in multiple layers from epithelium to endothelium at 10-j.Lm intervals. The images were then stacked and merged to give a single, holistic, detailed anatomic view of the stained corneal nerves. Image analysis was carried out using the software provided by the manufacturer and the areas of interest were then selected, automatically scaled, and exported to JPEG format. VOL. 151, NO.
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CONTROL:

Six normal eyes from 6 healthy subjects with no previous ocular problems or surgeries were selected as controls for in vivo confocal microscopy. There were 5 male and 1 female subjects with a mean age of 38 + 10.4 (range 31-49). Although mean age of the controls was less than that of the study group, it has been shown in the literature that no correlation exists between age and sub-basal nerve parameters.18 Six fresh postmortem corneas donated with family consent from 3 deceased patients (2 male, 1 female; mean age 57.3) with no previous ocular pathology or surgery were also stained with the AChE technique and used as controls. Causes of death were metastatic prostate carcinoma, adenocarcinoma of the lung, and post-renal transplant sepsis.
STATISTICAL TESTING:

Data were analyzed using an analysis tool pack for Microsoft Excel 2007 (Microsoft Corp., Redmond, Washington, USA) and SPSS 16.0 (SPSS Inc., Chicago, Illinois, USA). A P value of <.05 was taken as the threshold of statistical significance. The Student t test for independent samples was used to establish whether any differences in the sub-basal nerve density, branching pattern, and sub-basal and stromal nerve diameters between the normal subjects and patients with bullous keratopathy were significant. It was also used to 844

CORNEAL NERVES IN BULLOUS KERATOPATHY

FIGURE 4. A photomicrograph of a corneal button with aphakic bullous keratopathy, showing aberrant morphology and regeneration of the main stromal trunks, predominantly at mid-periphery. Nerve aberrations are demonstrated at a higher magnification (Top left, Top right, Bottom left, and Bottom right, insets). (Inset scale bar = 200 f.lm.)

test the difference in central corneal thickness in patients with and without demonstrable in vivo confocal microscopy findings. Mann-Whitney U test was used to test the difference in the duration of bullous keratopathy in patients with and without demonstrable in vivo confocal microscopy findings.

Furthermore, these nerves appeared thinner and attenuated compared to the normal sub-basal nerves (Figure 1, Top right). The diameter of the main subbasal nerves in patients with bullous keratopathy (3.07 + 0.64 j.Lm) was significantly lower than that of the controls (4.57 + 1.12 j.Lm) (P .001). Some nerves were abnormally tortuous and showed a bizarre orientation (Figure 1, Middle left). Stromal nerves. On in vivo confocal microscopy examination, stromal nerves were seen in all patients studied but changes were observed in 40% (10 out of 25) of bullous keratopathy cases. These consisted of relatively thin, tortuous, and convoluted nerves present mainly at the mid stroma (305.34 + 71.39 j.Lm depth). Their mean diameter was 5.35 + 1.3 j.Lm (range 3.12-8.86) (Figure 1, Middle right, Bottom left, and Bottom right). Some larger stromal nerves showed localized thickenings or excrescences suggestive of early sprouting (Figure 2, Top left). At the site of nerve bifurcations, hyper-reflective expansions with ill-defined blurry edges were noted in the central cornea (Figure 2, Middle left). At several places the stromal nerves formed distinct coils or loops appearing as hyper845

RESULTS
CONFOCAL MICROSCOPY FINDINGS:

Sub-basal nerves. Sub-basal nerves were found in 14 of 25 cases (56%) and were absent in 11 cases (44%). In cases where sub-basal nerves were detected, the mean sub-basal nerve density was 4.42 + 1.91 mm/mm2 (range 1.13-7.81 mm/mm2). This was significantly lower than the density in controls (20.05 + 4.24 mm/mm2) (Figure 1, Top left) (P .001). In addition, these nerves showed a decrease in number and in percentage of branching (branching pattern). Branching pattern of sub-basal nerve plexus in patients with bullous keratopathy (36.02% + 26.57%) was significantly lower than that of the controls (70.79% + 10.53%) (P .001). VOL. 151, NO.
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CORNEAL NERVES IN BULLOUS KERATOPATHY

TABLE 2. Histologic Features of Corneal Nerves in Bullous Keratopathy

Corneal Button Sub-basal Nerve Status No. of Perforation Sites Localized Nerve Thickeningsa Nerve Sprouting a No. of Nerves Entering the Cornea

Diagnosis

1 2 3 4 5

ABK PBK PBK PBK FED

Absent Present Absent Present Absent

Absent 9 7 10 9

+++ +++ +++ ++ ++

+++ +++ +++ +++ +++

36 33 30 31 34

ABK aphakic bullous keratopathy; FED Fuchs endothelial dystrophy; PBK pseudophakic bullous keratopathy. a Nerve changes are categorized as follows: (+) mild nerve changes involving 1 quadrant of the corneal button; (++) moderate nerve changes involving 2 or 3 quadrants; (+++) severe nerve changes involving all corneal quadrants.

reflective lines surrounding dark areas within the corneal stroma (Figure 1, Middle right, Bottom left, and Bottom right; Figure 2, Bottom left). All control eyes showed normal sub-basal and stromal nerves both quantitatively and qualitatively (Figure 1, Top left), as have been described previously.13,15 None of the abnormal in vivo confocal microscopy features observed in corneas with bullous keratopathy were seen in any of the controls.
HISTOLOGIC FINDINGS:

TABLE 3. Central Corneal Thickness and Duration of Bullous Keratopathy in Patients With and Without Stromal Nerve Changes by In Vivo Confocal Microscopy and Histology
Duration Between Patients (Number) CCT (j.Lm) Mean + SD Diagnosis and Scanning (Months)

Sub-basal nerves. Histologic features and correlation with in vivo confocal microscopy findings are illustrated in Figure 2 (Right column), Figure 3, and Figure 4. Sub-basal nerves were present in only 2 of 5 buttons examined (Figure 3, Top right inset). They were fragmented and seen only in the periphery of the buttons. There was a significant reduction in the number of perforation sites, as indicated by presence or absence of bulb-like structures just above the Bowman zone. Perforation sites were equal to or less than 10 in 4 buttons and absent in 1 button, while an average of 130 perforation sites were counted in the control buttons (Figure 3, Middle right inset). The details of the main histologic findings are shown in Table 2. Stromal nerves. The average number of stromal nerves entering the corneal buttons along their circumference was 32.8, which was less than the number in the control corneas (47). However, nerve bundles were distributed rather evenly around the circumference as in the controls. The mean stromal nerve diameter was 9.73 + 3.54 mm in the central cornea and 13.27 + 6.47 mm in the paracentral cornea. These figures were not statistically different from the mean stromal nerve diameter in the control corneas (8.11 + 3.31 mm in the center, P .081; 14.86 + 5.60 mm in the periphery, P .331). Aberrant nerves were observed within the corneal stroma in bullous keratopathy samples. Tortuous nerves originating from the main stromal nerve trunks and demVOL. 151, NO.
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Without demonstrable in vivo confocal microscopy findings (15) With demonstrable in vivo confocal microscopy findings (10) With demonstrable histologic findings (5)

691.4 + 71.2

16.5 + 14.0

669.5 + 51.0 672.8 + 31.9

18.7 + 12.5 21.0 + 14.4

CCT central corneal thickness.

onstrating bizarre twists and coils of irregular thickness were seen (Figure 2, Middle right and Bottom right; Figure 3, Top left, Bottom left, and Bottom right insets; Figure 4). This pattern was observed in all 5 corneal buttons, of which 1 was aphakic bullous keratopathy, 3 were pseudophakic bullous keratopathy, and 1 was Fuchs dystrophy. The control corneas showed normal straight nerves with dichotomous branching (Figure 3, Middle left inset). Additional novel findings included localized changes along the length of the nerve presenting as excrescences or thickenings (Figure 2, Top right) and sprouting of axons from such excrescences (Figure 2, Middle right). There was a strong morphologic correlation between histologic and in vivo confocal microscopy findings observed in the stromal nerves (Figure 2). All control eyes showed normal sub-basal and stromal nerves (Figure 3, Middle left and Middle right insets), as has been reported previously.17 None of the abnormal histologic features observed in corneas with bullous keratopathy were seen in any of the controls. 846

CORNEAL NERVES IN BULLOUS KERATOPATHY

 DURATION OF DISEASE AND CENTRAL CORNEAL THICKNESS: Patients with demonstrable stromal nerve

changes seen by in vivo confocal microscopy have had a longer mean duration of bullous keratopathy compared to those where such changes were not demonstrated, but the difference was not statistically significant (P .39) (Table 3). CCT in patients with demonstrable in vivo confocal microscopy stromal nerve changes was lower than that in patients where nerve changes were not observed. This difference in CCT was not statistically significant (P .76). The CCT of the 5 patients with demonstrable histologic changes was 672.8 j.Lm and the mean duration of bullous keratopathy in this group was 21.0 months.

DISCUSSION
IN VIVO CONFOCAL MICROCOPY HAS ENABLED DETAILED

wide-field examination of the cornea both grossly and at a cellular level. As the images generated by in vivo confocal microscopy are either bright or dark and lines or dots, interpretation of these images is often limited by lack of histologic correlation. However, a consensus on the morphology and patterns of sub-basal and stromal corneal nerves has emerged from a large body of data from in vivo confocal microscopy studies of the cornea.14 The AChE technique enables visualization of the corneal nerves. What makes it unique and ideal for confirmation of in vivo confocal microscopy findings is that, like in vivo confocal microscopy, it too generates en face images of the nerves examined, unlike cross-sectional histology. In combination with the NanoZoomer technology, it becomes a very powerful tool to study corneal nerves. Having reported the architecture and distribution of corneal nerves in normal eyes as revealed by the above methodology,17 we used this technique to study corneal nerves in bullous keratopathy and were able to correlate observed features with in vivo confocal microscopy images, thus providing histologic confirmation of the in vivo confocal microscopy findings. In bullous keratopathy significant reduction and alteration in the sub-basal nerve parameters were observed in vivo. These are likely to be related to damage caused by epithelial and subepithelial bullae and their rupture together with subepithelial scarring. Scar tissue is hyperreflective on in vivo confocal microscopy and can obscure the sub-basal nerves. However, histologic examination revealed a marked reduction or absence of sub-basal nerves, confirming the in vivo findings. In recent studies, both histologic and in vivo confocal microscopy,13,17 we demonstrated that sub-Bowman nerves perforate the Bowman zone and terminate as distinct bulb-like structures in the sub-basal plane from which a leash of sub-basal nerves arises. These were demonstrated in our control in vivo confocal microscopy and histology specimens but were strikingly reduced or absent in eyes with bullous keratopathy. This would suggest that the VOL. 151, NO.
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sub-basal nerves and the bulbous terminations of the sub-Bowman nerves undergo atrophy or degeneration. This is an interesting observation in the context of the pain experienced by patients with bullous keratopathy. Conventional wisdom is that stretching of nerves by epithelial bullae causes pain, and rupture of bullae is associated with exposure of nerve endings, resulting in severe pain. This study shows that there are very few sub-basal nerves remaining, raising the possibility that sub-Bowman nerves rather than sub-basal nerves may be contributing to the symptom of pain. In addition, this study also demonstrated a strong correlation between in vivo confocal microscopy and histologic features of the changes observed in stromal nerves such as thickening, twisting, looping, and coiling; localized thickening or excrescences; and possible sprouting of new nerves. On in vivo confocal microscopy the loops and coils of stromal nerves were seen to surround dark spaces. It is possible that the fluid accumulating in the corneal stroma displaces and stretches the surrounding collagen lamellae with the nerves, reducing them to thin septae between adjoining fluid lacunae. These lacunae tend to be roughly round or oval in shape. Nerves traversing these septae would therefore naturally assume the shape of the collagen matrix, partly explaining the tortuosity, looping, and coiling. The dark spaces seen on in vivo confocal microscopy of bullous keratopathy correlate to empty spaces reported on cross-sectional electron microscopy (EM) of these corneas.8,19 The overall length of the looped and coiled nerves cannot be explained only on the basis of stretching of the nerves and it is likely that aberrant regeneration is also taking place. Interestingly, similar stromal nerve changes described as hyper-regeneration of nerves have been reported in a previous study where 3 cases of bullous keratopathy secondary to glaucoma were examined using Hortega silver carbonate stain that is specific for Schwann cells.20 Mildly tortuous curvilinear structures seen on in vivo confocal microscopy in the anterior stroma of some normal corneas and in other conditions such as diabetes and Schnyders dystrophy and following laser (light amplification by stimulated emission of radiation) refractive surgery have been reported as tortuous corneal nerves.2125 The tortuous nerves reported by others in some normal corneas tended to be patchy, of small diameter (range 0.24-3.28 j.Lm), and often located in the anterior stroma at a mean depth of 140 + 87 j.Lm.21,26 The abnormal nerves that we observed in bullous keratopathy were more abundant, of thicker diameter (5.35 + 1.39 j.Lm), and located relatively deeper, in the mid stroma at a depth of 305.34 + 71.39 j.Lm. In recent years several in vivo confocal microscopy studies on corneas with Fuchs dystrophy6,11,27,28 and corneal edema of varied etiology have been conducted.29 None have described similar stromal nerve changes. The extent and duration of corneal edema in the eyes examined in these studies is unknown. However, the disappearance 847

CORNEAL NERVES IN BULLOUS KERATOPATHY

of sub-basal nerve fibers in Fuchs dystrophy has been reported in previous in vivo confocal microscopy and histologic studies.10,30 According to Mustonen and associates,10 the sub-basal nerves were detected in 7 out of 25 cases scanned by in vivo confocal microscopy. In their study they only evaluated cases of Fuchs dystrophy of varying severity and only absence or presence of subbasal nerves was commented on. Quantitative descriptors of sub-basal nerves were not studied and stromal nerves also were not examined. It is worth mentioning that limbal or corneal incisions from previous cataract surgery could have some effect on corneal innervation and sensitivity. Modern small-incision procedures such as phacoemulsification are known to cause less disruption of the corneal innervation with rapid recovery of corneal sensitivity. A recent study has shown that this effect is predominantly limited to the incision site and corneal sensitivity returns back to near-preoperative levels by 3 months.31,32 In our study, the abnormal nerve features were found in most of the corneal quadrants and therefore it is unlikely that they were attributable to previous cataract surgery. Activated skin keratinocytes have been shown to synthesize neuronal growth factors,3335 which in turn induces nerve sprouting and hyperalgesia in experimental models.33,36 The cornea also contains a wide variety of neuronal growth factors and epithelial cells and keratocytes express growth factor receptors.37 It is therefore conceivable that aberrant regeneration of nerves occurs in the cornea and could play an important role in pain experienced by these patients.

Form this study we are able to suggest that the various changes in sub-basal and stromal corneal nerves seen in bullous keratopathy are unlikely to be related to a specific etiology of bullous keratopathy. They were seen in Fuchs dystrophy, pseudophakic and aphakic bullous keratopathy. The limited number of cases does not enable us to make definite conclusions on any relationship between corneal nerve changes and the extent and duration of corneal edema. However, pachymetry as a measure of corneal edema did not correlate to the presence or absence of nerve changes, suggesting that the amount of edema may not be a factor. On the other hand, duration of edema may influence the type and extent of changes observed. Loss of sub-basal nerves would appear to be an early and universal event. Aberrant regeneration of stromal nerves appears to be related to duration but with certain caveatsif stromal nerve changes become more prominent with duration of edema then they are also more likely to be visualized by in vivo confocal microscopy. Hence, lack of stromal changes on in vivo confocal microscopy may be a limitation of the ability of the technique to detect early changes rather than absolute absence. Conversely, increased corneal clouding with prolonged edema may obscure nerve changes (reduced contrast and increased background noise) that may in fact be present. This is supported by the observation that 2 of our corneal buttons that were positive for aberrant nerves on histology were negative on in vivo confocal microscopy despite a longer duration of edema.

DR AL-AQABA IS FUNDED FOR HIS PHD STUDENTSHIP BY THE MINISTRY OF HIGHER EDUCATION AND SCIENTIFIC RESEARCH, Baghdad, Republic of Iraq. None of the authors has any proprietary/financial interest to disclose. Involved in design of study (M.A., H.S.D.); conduct of study (M.A., T.A., H.S.D.); data collection and analysis (M.A., T.A., J.L., H.S.D.); preparation of the manuscript (M.A., H.S.D.); and review and approval of the manuscript (M.A., J.L., H.S.D.).The study was approved by Nottingham Research Ethics Committee 2 (REC no. 06/Q2403/46) and is consistent with the tenets of the Declaration of Helsinki. Informed, written consent was obtained from all patients.

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13. Al-Aqaba MA, Alomar T, Miri A, Fares U, Otri AM, Dua HS. Ex vivo confocal microscopy of human corneal nerves. Br J Ophthalmol 2010;94(9):12511257. 14. Patel DV, McGhee CN. In vivo confocal microscopy of human corneal nerves in health, in ocular and systemic disease, and following corneal surgery: a review. Br J Ophthalmol 2009;93(7):853 860. 15. Oliveira-Soto L, Efron N. Morphology of corneal nerves using confocal microscopy. Cornea 2001;20(4):374 384. 16. Karnovsky MJ, Roots L. A direct-coloring thiocholine method for cholinesterases. J Histochem Cytochem 1964;12:219 221. 17. Al-Aqaba MA, Fares U, Suleman H, Lowe J, Dua HS. Architecture and distribution of human corneal nerves. Br J Ophthalmol 2010;94(6):784 789. 18. Erie JC, McLaren JW, Hodge DO, Bourne WM. The effect of age on the corneal subbasal nerve plexus. Cornea 2005;24(6):705709. 19. Akhtar S, Bron AJ, Hawksworth NR, Bonshek RE, Meek KM. Ultrastructural morphology and expression of proteoglycans, betaig-h3, tenascin-C, fibrillin-1, and fibronectin in bullous keratopathy. Br J Ophthalmol 2001;85(6):720 731. 20. Wolter JR. Hyper-regeneration of corneal nerves in bullous keratopathy. Am J Ophthalmol 1964;58:3138. 21. Visser N, McGhee CN, Patel DV. Laser-scanning in vivo confocal microscopy reveals two morphologically distinct populations of stromal nerves in normal human corneas. Br J Ophthalmol 2009;93(4):506 509. 22. Ciancaglini M, Carpineto P, Doronzo E, Nubile M, Zuppardi E, Mastropasqua L. Morphological evaluation of Schnyders central crystalline dystrophy by confocal microscopy before and after phototherapeutic keratectomy. J Cataract Refract Surg 2001;27(11):18921895. 23. Mocan MC, Durukan I, Irkec M, Orhan M. Morphologic alterations of both the stromal and subbasal nerves in the corneas of patients with diabetes. Cornea 2006;25(7):769 773. 24. Erie JC, Patel SV, Bourne WM. Aberrant corneal nerve regeneration after PRK. Cornea 2003;22(7):684 686. 25. Patel SV, Erie JC. Aberrant regeneration of corneal nerves after laser in situ keratomileusis. J Cataract Refract Surg 2003;29(2):387389.

26. Marfurt CF, Cox J, Deek S, Dvorscak L. Anatomy of the human corneal innervation. Exp Eye Res 2010;90(4):478 492. 27. Hara M, Morishige N, Chikama T, Nishida T. Comparison of confocal biomicroscopy and noncontact specular microscopy for evaluation of the corneal endothelium. Cornea 2003;22(6):512515. 28. Guthoff RF, Wienss H, Hahnel C, Wree A. Epithelial innervation of human cornea: a three-dimensional study using confocal laser scanning fluorescence microscopy. Cornea 2005;24(5):608 613. 29. Grupcheva CN, Craig JP, Sherwin T, McGhee CN. Differential diagnosis of corneal oedema assisted by in vivo confocal microscopy. Clin Experiment Ophthalmol 2001; 29(3):133137. 30. Adamis AP, Filatov V, Tripathi BJ, Tripathi RC. Fuchs endothelial dystrophy of the cornea. Surv Ophthalmol 1993; 38(2):149 168. 31. Kim JH, Chung JL, Kang SY, Kim SW, Seo KY. Change in corneal sensitivity and corneal nerve after cataract surgery. Cornea 2009;28(11):S20 25. 32. John T, Rao GN, Aquavella JV. Corneal sensitivity in aphakic and pseudophakic eyes. CLAO J 1988;14(2):101 104. 33. Shu XQ, Mendell LM. Neurotrophins and hyperalgesia. Proc Natl Acad Sci U S A 1999;96(14):76937696. 34. Hasan W, Zhang R, Liu M, Warn JD, Smith PG. Coordinate expression of NGF and alpha-smooth muscle actin mRNA and protein in cutaneous wound tissue of developing and adult rats. Cell Tissue Res 2000;300(1):97109. 35. Townley SL, Grimbaldeston MA, Ferguson I, et al. Nerve growth factor, neuropeptides, and mast cells in ultravioletB-induced systemic suppression of contact hypersensitivity responses in mice. J Invest Dermatol 2002;118(3):396 401. 36. Snider WD, McMahon SB. Tackling pain at the source: new ideas about nociceptors. Neuron 1998;20(4):629 632. 37. You L, Kruse FE, Volcker HE. Neurotrophic factors in the human cornea. Invest Ophthalmol Vis Sci 2000;41(3):692 702.

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Aberasi saraf kornea pada keratopati bulosa

MOUHAMED AL-AQABA, THAER ALOMAR, JAMES LOWE, AND HARMINDER S. DUA

Tujuan : untuk mempelajari saraf kornea pada pasien dengan keratopati bulosa kronik Design penelian : seri kasus prospektif observasional dengan evaluasi histologi Metode : kami mempelajari 25 mata pasien keratopati bulosa dengan beragam etiologi ( 17 perempuan, 8 laki-laki ; usia rata-rata 76,3 tahun) juga 6 mata dengan kondisi normal sebagai kontrol (5 laki-laki, 1 perempuan; usia rata-rata 38 tahun). Semua subjek diperiksa dengan menggunakan pemindaian laser mikroskop confocal. Ada Lima kornea dengan transplantasi kornea dari total keseluruhan pasien dan 6 kornea normal sebagai kontrol diwarnai dengan metode acetylcholinesterase (AChE) sebagai demonstrasi persarafan kornea lalu dipindai setiap lapisan dengan alat pemindai mikroskop patologi digital. Hasil : densitas, pola percabangan dan diameter saraf sub-basal secara signifikan lebih rendah pada kornea dengan keratopati bulosa dibandingkan kornea normal (densitas : 4,42 1,91 mm/mm 2 vs 20,05 4,24 mm/mm2 ; pola percabangan : 36,02% 26,57 vs 70,79% 10,53%; diameter 3,07 0,64 m vs 4,57 1,12 m). Aberasi seperti penipisan lokal atau eksresensi, twisting abnormal, coiling dan looping dari saraf stoma tengah diamati pada kedua grup baik dengan menggunakan mikroskop concofocal atau pun secara histologi. Kesimpulan : perubahan yang mencolok dalam persarafan kornea yang terdapat pada kornea dengan keratopati bulosa dalam penelitian ini tidak memiliki hubungan etiologi spesifik dari keratopati bulosa. Studi ini memberikan konfirmasi histologis novel in vivo dengan menggunakan mikroskop concofocal yang berkaitan dengan saraf kornea pada keratopathy bulosa. Keratopati bulosa adalah kondisi klinis umum yang merupakan titik akhir dari disfungsi endotel kornea atau merupakan hasil kerusakan yang diakibatkan penyebab yang beragam. Umumnya kondisi ini dihubungkan dengan keratopati bulosa termasuk ekstraksi katarak dengan atau tanpa implantasi lensa intraokular, kegagalan primer atau sekunder (penolakan secara imunologis) transplantasi kornea, glaukoma absolut, dan distrofi endotel seperti distrofi endotel kornea Fuchs (Fuchs distrofi). Sayangnya, keratopati bulosa yang iatrogenik terdapat pada kurang lebih 60% kasus. 3 penyebab yang paling umum dari keratopati bulosa adalah keratopati pseudophakik bulosa, keratopati aphakik bulosa, dan distrofi Fuchs. Saat ini, keratopati bulosa adalah indikasi utama dari transplantasi endotel kornea/penetrating

keratoplasti dan regraft. Awalnya, stroma kornea menjadi edematous dan menebal dan akhirnya intraepitel dan subepitel vesikel terisi cairan, "bula," muncul. Gejala klinis dapat sangat bervariasi mulai dari asimptomatik pada awal penyakit, silau, dan penurunan nyeri pada kehilangan visi mendalam disebabkan oleh jaringan parut subepitel dalam kasus-kasus lanjutan. Episode nyeri dikaitkan dengan fotofobia dan robekan dikaitkan dengan peregangan saraf dan iritasi bula pada epitel dan subepitel dan pecahnya bula permukaan dengan paparan ujung saraf. Gambaran histologis dari keratopati bulosa termasuk edema epitel intraselular dan pemisahan bula, penipisan stroma, dan hilangnya endotel. Sebagai tambahan keratopati sekunder pada distrofi Fuch juga terlihat. Penipisan membran desemen dengan gutata posterior. Dengan menggunakan mikroskop confocal secara in vivo pemahaman kami mengenai temuan patologi pada keratopati bulosa bertambah seiring dengan kemampuan dalam pemeriksaan jaringan secara in vivo. Meskipun sakit, disebabkan peregangan saraf dan paparan yang sudah dijelaskan sebelumnya di atas merupakan manifestasi umum dan sering sulit untuk diselesaikan hal ini disebabkan karena informasi tentang keadaan saraf kornea pada keratopati bulosa sangat sedikit. Teknik histologis rutin dan potongan melintang jaringan kornea untuk evaluasi yang tepat pada saraf sering kali tidak kondusif. Pemindaian laser mikroskop confocal telah tersedia dan memberikan wawasan baru ke dalam orientasi dan distribusi saraf kornea manusia di bidang kesehatan dan penyakit. Bagaimanapun tidaklah selalu jelas struktur apakah yang menyamakan dengan temuan confocal in vivo. Studi korelasi langsung temuan mikroskop confocal dengan histologi dari jaringan telah diperiksa. Kami menggunakan metode pemeriksaan ini untuk menilai persarafan kornea pada kornea dengan keratopati bulosa, termasuk beberapa yang dijadwalkan untuk dilakukan transplantasi kornea (penetrating keratoplasti / PKP). Kami dapat memeriksa keseluruhan dari area kornea yang diangkat pada PKP dengan pewarnaan saraf khusus untuk menggambarkan saraf kornea dengan maksud untuk mengkorelasikan in vivo temuan mikroskop confocal dengan pengamatan histologis. Kami juga menjelaskan setiap dasar anatomi saraf terkait untuk nyeri, yang merupakan fitur dominan keratopati bulosa lanjut.

Materi dan metode Studi seri kasus prospektif konsekutif dari 25 mata pasien dengan keratopati bulosa telah diperiksa dengan menggunakan mikroskop confocal in vivo. Sebaran demografi dan data klinis disajikan pada tabel 1. distrofi Fuchs dan keratopati pseudophakik bulosa berjumlah 88% dari keseluruhan kasus. Ada 17 pasien perempuan dan 8 pasien laki-laki dengan usia rata-rata 76,3 7,3 (58-88) tahun. Semua pasien adalah orang kulit putih. Semua pasien terlah di terapi dengan transplantasi kornea. Sebanyak 12 orang dari 25 pasien (48%) dilakukan translpantasi saja, 9 dari 25 (36%) menjalani triple procedure (transplantasi kornea dan ekstraksi lensa dengan implan) dan 4 dari 25 pasien (16%) menjalani descemet stripping endothelial keratoplasty (DSEK). Diagnosa keratopati bulosa berdasarkan dari riwayat, pemeriksaan slit-lamp dan ketebalan kornea sentral (Central corneal thickness , CCT) (rerata SD, 682,6 63,7 m; range 570-776 m). Sebuah alat ultrasonik pakimeter (Tomey SP-3000, pakimeter; tomey corporation, Nagoya , jepang) digunakan untuk menilai CCT. Rata-rata waktu antara diagnosis keratopati bulosa dan pemindaian mikroskop concofocal in vivo adalah 17,4 bulan (kisaran 4-48 bulan). Tak satu pun dari pasien memiliki vaskularisasi kornea.

Mikroskop confocal : dari 25 mata diperiksa preoperatif dengan menggunakan pemindaian laser mikroskop confocal. Alat ini mengginakan laser dioda kelas I (670-nm panjang gelombang) dengan 63x water0immersion lens. Gambar didapatkan menggunakan lensa ini 400 x 400 m, dan memiliki 2- dan 4- m resolusi lateral dan resolusi kedalaman optik, secara respektif. Magnifikasi gambar pada layar adalah 300x. Mikroskop confocal in vivo dilakukan dibawah anestesi topikal dengan menggunakan MINIMS oxybuprokain hydrochloride 0,4%. Digital kamera digunakan untuk menilai sisi pandang lateral dari mata dan lensa objektif digunakan untuk memonitor posisis dari lensa objektif pada permukaan mata. Tetes mata gel poliakrilik 0,2% digunakan sebagai medium coupling antara contak cap dan lensa objektif dari mikroskop.

Regio sentral dan parasentral (kurang lebih 7x 7 mm) dari kornea di pindai pada tiap lapis. Frames dari sub-basal (dibawah sel basal epitel kornea) dan tiap lapis stroma di dapatkan persarafan yang digunakan untuk dianalisis. Desktriptor kuantitatif standar untuk penelitian saraf di periksa. Termasuk densitas

saraf dalam mm/mm2 , pola percabangan dimana menggambarkan presentasi dari percabangan saraf per total serat nervus sub basal dalam sebuah frame tunggal dan diameter nervus sub-basal dalam mikron/ regio yang paling tipis pada tiap nervus sub-basal dalam single frame dipilih untuk dilakukan analisa ketebalannya. Teknik asetilkolinesterase untuk demonstrasi persarafan kornea. Pada 5 pasien dimana dilakukan pemeriksaan mikroskop confocal in vivo pada kornea mereka yang dilakukan saat preoperatif, pada kornea mereka di ambil setelah dilakukan transplantasi kornea untuk keratopati bulosa yang kemudian di proses dan diwarnai dengan menggunakan enzim kolinesterase dengan teknik asetilkolinesterase (AChE). Secara singkat, kornea difiksasi dalam formalin 4% (pH 7) selama 4 jam dan kemudian dibilas semalam di phosphate-buffered saline (PBS). Spesimen diinkubasi dalam larutan sediaan mengandung acetylthiocholine iodida sebagai substrat selama 24 jam pada 37 C. Enzin asetilkolinesterase pada saraf bereaksi dengan iodida asetilkolin dalam substrat untuk menghasilkan warna coklat pada saraf. Waarna ini lebih intense terlihat dengan tambahan dilute solution dari ammonium sulfida. Spesimen di dehidrasi dengan menggunakan immersi didalam alkohol dan dibersihkan dengan xylene sebagai standar preparat histologi. Sepesimen kemudian di pindai menggunakan Hamamatsu Nano Zoomer gigital pathologu (NDP) microscope system. Kornea diamati dengan pembesaran 40 x di tiap lapisan mulai dari epitel sampai dengan endotelium pada tiap interval 10 m. Gambar kemudian disatukan menjadi sebuah gambaran holistik dengan gambaran anatomis yang lebih detail dari persarafan yang sudah diwarnai. Analisis gambar dilakukan dengan menggunakan software.

Kontrol : enam mata normal dari enam subjek yang sehat dengan tanpa ada riwayat gangguan ocular sebelumnya atau bedah dipilih sebagai kontrol untuk mikroskop confocal in vivo. Ada 5 laki-laki dan 1 perempuan sebagai subjek dengan rata-rata usia 38 10,4 (range 31-49). Meskipun rata-rata usia dari kontrol kurang dari kelompok penelitian, telah dikatakan dalam literatur bahwa tidak ada korelasi antara usia dan parameter saraf sub-basal. Enam fresh postmortem kornea didonasikan oleh keluarga yang sudah dilakukan consent sebelumnya dari 3 pasien (2 laki-laki , 1 perempuan ; rata-rata usia 57,3) dengan tanpa riwayat gangguan ocular maupun bedah pada mata sebelumnya yang juga diwarnai dengan teknik AChE dan digunakan sebagai kontrol. Penyebab kematian dari pasien tersebut adalah karsinoma prostat dengan metastase, adenokarsinoma paru dan sepsis post transplantasi renal. Uji statistik : data di analisa dengan menggunakan perangkat analisis untuk mikrosoft exe 2007 dan SPSS 16.0. P valuse <.05 diambil sebagai signifikansi. Student t test untuk sample independen digunakan untuk memeriksa apakah ada perbedaan yang signifikan pada densitas nervus sub-basal, pola percabangan saraf, dan diameter sub-basal dan stroma antara subjek normal dan pasien dengan

keratopati bulosa. Student t test juga digunakan untuk memeriksa perbedaan ketebalan kornea sentral dengan atau tanpa penemuan in vivo mikroskop concofocal . Mann- Whitney U test digunakan untuk memeriksa perbedaan dari durasi keratopati bulosa pada pasien dengan atau tanpa penemuan in vivo mikroskop concofocal.

Hasil

Penemuan mikroskop confocal : nervus sub-basal, nervus sub-basal ditemukan pada 14 dari 25 kasus (56%) dan tidak ditemkan pada 11 kasus (44%). Pada kasus dimana nervus sub-basal berhasil diidetifiasi, rata-rata dari desitas nervus sub-basal adalah 4,42 1,91 mm/mm2 (range 1,13-7,81 mm/mm2). Ini secara signifikan lebih rendah daripada densitas pada kontrol (range 20,05 4,24 mm/mm2) (gambar 1, kiri atas) (P = .001). Sebagai tambahan, pada nervus ini menunjukan adanya penurunan jumlah dan presentase dari percabangan (pola percabangan). Pola percabangan dari pleksus nervus sub-basar pada pasien dengan keratopati bulosa (36,02% 26,57%) secara signifikan lebih rendah dari kontrol (70,79% 10,53%) (P=.001).

Lebih jauh lagi nervus ini tampak lebih tipis dan lebih lemah dibandingkan dengan nervus subbasal yang normal (gambar 1, atas kanan). Diameter dari nervus sub basal utama pada pasien dengan keratopati bulosa (3,07 0,64 m) secara signifikan lebih rendah daripada kontrol (4,57 1,12 m) (P= . 001). Beberapa nervus tampak berliku-liku secara abnormal dan menunjukan orientasi yang aneh (gambar 1, tengah kiri).

Nervus stroma. Pada pemeriksaan mikroskop confocal secara in vivo, nervus stroma terlihat pada semua pasien yang diteliti tetapi perubahan hanya terlihat pada 40% (10 dari 25) kasus keratopati bulosa. Ini terdiri darinervus yang relatif tipis, berliku-liku dan berbelit pada mid stroma (305,34 71,39 m kedalaman). Diameter rata-rata nya adalah 5,35 1,3 m (range 3.12-8.86) (gambar 1, tengah kanan, bawah kiri, dan bawah kanan). Beberapa nervus stroma yang lebih besar menunjukkan penipisan lokal atau excrescences (gamabr 2 atas kiri). Di lokasi pencabangan dua saraf, ekspansi hiper-reflektif dengan tepi kabur tidak jelas terlihat pada kornea pusat (Gambar 2, kiri Tengah). Pada beberapa tempat dari nervus stroma terbentuk coils atau loops sebagai garis hiper-reflektif yang dikelilingi area gelap didalam stroma kornea (gambar 1 , tengah kanan, bawah kiri dan bawah kanan; gambar 2 , bawah kiri) Semua mata kontrol menunjukkan nervus sub-basal dan stromal yang noral baik secara kuantitatif dan kualitatif (gambar 1, atas kiri) , yang sudah dijelaskan sebelumnya. Tidak ada satupun gambaran abnormal secara mikroskop confocal ditemukan pada kornea kontrol. Penemuan histologis : nervus sub-basal. Gambaran histologik dan hubungan dengan penemuan mikroskop confocal in vivo yang telah terilustrasi pada gambar 2 (kolom kanan), gambar 3, dan gambar 4. Nervus sub-basal terlihat pada 2 dari 5 kornea yang diperksa 9 gambar 3, atas kanan). Nervus sub-basal terlihat terfragmentasi dan terlihat hanya pada kornea perofer. Ada reduksi yang signifikan pada beberapa tempat perforasi sebagai indikasi dengan kehadiran atau ketidak hadiran dari struktur yang tampak seperti bulb tepat diatas zona Bowman. Tempat perforasi sebanding dengan atau kurang dari 10 pada 4 kornea dan tidak terlihat pada 1 kornea, sementara rata-rata perforasi terdapat pada 130 lokasi yang dibandingkan dengan kontrol (gambar 3, tengah kanan). Detail dari penemuan histologi utama ditunjukkan pada tabel 2.

Nervus stroma. Rata.rata nervus stroma yang memasuki kornea sepanjang lingkarannya adalah 32.8, dimana kurang dari jumlah yang ada pada kornea kontrol (47). Namun, berkas saraf didistribusikan lebih merata di sekitar lingkar seperti pada kontrol. Rata-rata ukuran diameter nervus stroma adalah 9.73 3.54 mm di dalam kornea central dan 13.27 6.47 mm di kornea paracentral. Ini menggambarkan tidak secara statistik berbeda dari rata-rata diameter nervus stroma pada kornea kontrol (8.11 3.31 mm di tengah, P = .081; 14.86 5,60 mm di perifer, P = .331) Saraf yang menyimpang diamati dalam stroma kornea pada sampel keratopati bulosa. Nervous yang berliku-liku berasal dari nervus stroma utama dan menunjukan adanya tikungan aneh dan kumparan dengan ketebalan yang tak beratudan (gambar 2, tengah kanan dan bawah kanan; gambar 3, atas kiri, dan bawah kanan: gambar 4). Pola ini diamati pada 5 kornea, dimana salah satunya merupakan keratopati aphakik bulosa, 3 keratopati pseudophakik bulosa dan 1 distrofi Fuchs. Kornea kontrol menunjukkan nervus normal dengan percabangan dikotom (gambar 3 , tengah kiri). Temuan baru tambahan termasuk perubahan lokal sepanjang panjang penyajian saraf sebagai penipisan (gambar 2, atas kanan) . ada korelasi kuat antara gamabaran histologi dan penemuan mikroskopi confocal in vivo yang diamati pada nervus stroma. Semua mata kontrol menunjukkan nervus sub-basal dan stromal yang normal (gambar 3 , tengah kiri dan tengah kanan) sebagaimana yang sudah dilaporkan sebelumnya. Tidak ada satu pun gambaran histologik abnormal yang ada pada kornea dengan keratopati bulosa ditemukan pada konea kontrol. Durasi dari penyakit dan ketebalan kornea sentral : pasien dengan perubahan saraf stroma yang dilihat dari mikroskop confocal in vivo rata-rata memiliki durasi yang lebih lama keratopathy bulosa nya dibandingkan dengan mereka di mana perubahan seperti itu tidak ditemukan, tetapi perbedaannya tidak signifikan secara statistik (P= .39) (tabel 3). CCT pada pasien yang ditunjukan pada mikroskop confocal in vivo menunjukkan adanya perubahan nervus stromal adalah lebih rendah dibandingkan pada pasien yang mana perubahan nervus tidak diamati. Perbedaan pada CCT ini tidak secara memuaskan signifikan (p=.76). CCT dari 5 pasien dengan perubahan histologik adalah 672.8 m dan rata-rata durasi dari keratopati bulosa pada kelompok ini adalah 21 bulan. Diskusi Mikroskop confocal in vivo detail. Pemeriksaan lebar lapangan kornea baik pada tingkat kasat mata dan pada tingkat sel . Sebagai gambar yang dihasilkan dalam mikroskop confocal in vivo yang baik

terang atau gelap dan garis atau titik , interpretasi citra ini sering dibatasi oleh kurangnya korelasi histologis . Namun, konsensus tentang morfologi dan pola sub - basal saraf stroma kornea telah muncul dari data dalam studi mikroskop confocal in vivo kornea . Teknik AChE memungkinkan visualisasi dari saraf kornea . Apa yang membuatnya unik dan ideal untuk konfirmasi temuan mikroskop confocal in vivo adalah bahwa, seperti dalam mikroskop confocal vivo , juga menghasilkan gambaran saraf yang diperiksa , tidak seperti histologi cross-sectional . Dalam kombinasi dengan teknologi NanoZoomer , menjadi alat yang sangat kuat untuk mempelajari saraf kornea . Setelah dilaporkan arsitektur dan distribusi saraf kornea pada mata normal seperti yang telah diungkapkan oleh metodologi di atas , kami menggunakan teknik ini untuk mempelajari saraf kornea pada keratopati bulosa dan mampu mengkorelasikan fitur yang diamati dengan mikroskop confocal in vivo sehingga memberikan konfirmasi histologis dari termuan tersbut. Pada keratopati bulosa terdapat reduksi yang signifikan dan terdapat perubahan pada parameter nervus sub-basal in vivo. Ini lebih dikaitkan pada kerusakan yang disebabkan oleh bula epitel dan subepitel dan ruptur dari bula tersebut dapat menyebabkan skar eubepitel. Jaringan skar tampak hiperreflektif pada mikroskop confocal in vivo dan dapat mengaburkan saraf sub basal. Namun, pemeriksaan histologi menunjukan reduksi dari tanda tersebut atau dengan tidak adanya nervus subbasal mengkonfirmasi penemuan in vivo. Pada penelitian baru-baru ini, baik histologik dan mikroskop confocal in vivo menunjukkan bahwa perforasi nervus sub-Bowman pada Bowman zone dan berakhir sebagai sturktur menyerupai bola pada bidang sub-basal. Ini dapat dilihat pada miskroskop confocal in vivo kontrol dan spesimen histologi namun pada keratopati bulosa terlihat berkurang atau tidak tampak sama sekali. Ini menunjukkan bahwa pada nervus sub basal dan akhir bulosa dari nervus sub-Bowman mengalami atrofi dan degenerasi. Ini merupakan observasi menarik dari konteks pengalaman nyeri pada pasien keratopati bulosa. secara konvensioal, pemanjangan dari nervus oleh bula epitel menyebabkan nyeri dan ruptur bula dikaitkan dengan pajanan dari ujung saraf yang menyebabkan nyeri. Penelitian ini menunjukan bahwa hanya ada beberapa nervus sub-basal yang tersisa yang meningkatkan kemungkinan bahwa nervus sub-Bowman dibandingkan dengann nervus sub-basal berkontribusi pada gejala nyeri. Dengan tambahan, penelitian ini menunjukkan korelasi yang kuat antara mikroskop confocal dan gambaran histologi dari perubahan yang diamati pada nervus stroma seperti penipisan, twisting, looping dan coiling; lokaslisasi penipisan atau excrescences; dan sebaran nervus baru yang mungkin. Mikroskop confocal in vivo dari loops dan coils dari nervus stroma terlihat mengelilingi daerah

gelap. Dan ini memungkinkan cairan terakumulasi pada stroma kornea dan pemanjangan kolagen disekitar lamell nervus, menurunkan mereka menjadi septa yang tipis yang berada diantara perbatasan cairan lakuna. Lakuna ini cenderung menjadi keras dengan bentuk bundar atau oval. Saraf yang melintasi septa ini secara natural diasumsikan memiliki bentuk matriks kolagen, sebagian dijelaskan berliku-liku, looping dan coiling. Ruang gelap yang terlihat pada mikroskop confocal in vivo dari keratopati bulosa berkaitan dengan ruang kosong yang dilaporkan pada mikroskop elektron cross-sectional (EM) pada kornea ini. Keseluruhan panjang dari loop dan coil nervus tersebut tidak dapat dijelaskan dengan hanya basik pemanjangan dari nervus. Menariknya, perubahan nervus stoma yang sama digambarkan sebagai hiper-regeneration of nerve telah dilaporkan pada penelitian sebelumnya dimana 3 kasus dari keratopati bulosa sekunder dari glaukoma diperiksa menggunakan Hortega silver dengan warna karbonat yang spesifik untuk sel schwann. Struktur lengkung agak berliku-liku terlihat di dalam mikroskop confocal in vivo dalam stroma anterior dari beberapa kornea normal dan dalam kondisi lain seperti diabetes dan distrofi Schnyder dan berikut Laser (amplifikasi cahaya menstimulasi emisi radiasi) bedah refraktif telah dilaporkan sebagai "tortuous corneal nerves". Saraf yang berliku-liku dilaporkan oleh orang lain di beberapa kornea yang normal cenderung tidak merata, dengan diameter kecil (kisaran 0,24-3,28 m), dan sering terletak di stroma anterior pada kedalaman rata-rata 140 87 M. Saraf abnormal yang kami amati dalam keratopati bulosa lebih berlimpah, diameter lebih tebal (5,35 1,39 M), dan terletak relatif lebih dalam, pada pertengahan stroma pada kedalaman 305,34 71.39 M. Dalam beberapa tahun terakhir beberapa studi dari mikroskop confocal in vivo pada kornea dengan distrofi Fuchs dan edema kornea dengan etiologi yang bervariasi telah dilakukan. Tidak ada yang menggambarkan perubahan saraf stroma serupa. Tingkat dan durasi edema kornea pada mata yang diperiksa dalam penelitian ini tidak diketahui. Namun, ketidak beradaannya serat nervus sub-basal ada distrofi Fuchs terlah dilaporkan pada penelitian mikroskop confocal in vivo sebelumnya. Berdasarkan Mustonen dan kawan0kawan, nervus sub-basal terdeteksi 7 dari 25 kasus yang dipindai dengan mikroskop confocal. Pada penelian mereka, mereka hanya mengevaluasi kasus dengan distrofi Fuchs dengan beragam derajat dan hanya mengomentari kehadiran atau keberadaan dari nervus basal. Desktripsi kuantitatif dari nervus sub-basal tidak diterliti dan nervus stroma juga tidak diperiksa. Perlu disebutkan bahwa sayatan limbal atau kornea dari operasi katarak sebelumnya bisa memiliki beberapa efek pada persarafan kornea dan sensitivitas. Prosedur kecil sayatan modern seperti fakoemulsifikasi diketahui menyebabkan gangguan yang lebih sedikit dari persarafan kornea dengan

pemulihan yang cepat pada sensitivitas kornea. Sebuah studi terbaru menunjukkan bahwa efek ini terutama terbatas pada situs sayatan dan sensitivitas kornea kembali kembali ke tingkat pra operasi dekat-dalam 3 bulan.pada penelitian kami, fitur saraf abnormal ditemukan di sebagian besar kuadran kornea dan karena itu tidak mungkin bahwa mereka disebabkan operasi katarak sebelumnya. Aktivasi dari keratinosit kulit telah terbukti mensintesis faktor pertumbuhan saraf, yang pada gilirannya menyebabkan saraf tumbuh dan hiperalgesia pada model eksperimental. Kornea juga mengandung berbagai faktor pertumbuhan saraf dan sel-sel epitel dan keratocytes mengekspresikan reseptor faktor pertumbuhan. Oleh karena itu dapat dibayangkan bahwa regenerasi menyimpang dari saraf dapat terjadi pada kornea dan dapat memainkan peran penting dalam rasa sakit yang dialami oleh pasien. Dari penelitian ini kami dapat menunjukkan perubahan yang beragam pada sub-basal dan nervus stroma kornea yang terlihat pada keratopati bulosa yang tidak biasanya berkaitan dengan etiologi spesifik keratopati bulosa. Perubahan ini biasanya terjadi pada distrofi Fuchs, keratopati pseudophakik dan aphakik bulosa. Dengan jumlah kasus yang terbatas tidak meumngkinkan untuk kita buat kesimpulan dari hubungan antara perubahan nervus kornea dengan tingkat dan durasi dari edema kornea. Namun, pakimetri sebagai penilaian edema kornea tidak berkorelasi dengan kehadiran atau ketidak hadiran dari perubahan nervus, menunjukan bahwa jumlah edema tidak menjadi faktor. Disisi lain , durasi dari edema dapat mempengaruhi jenis dan luasnya perubahan yang diamati. Kehilangan saraf sub-basal dapat dikaitkan dengan kejadian awal dan universal. Regenerasi yang menyimpang dari perubahan nervus stroma dapat dikaitkan dengan durasi tetapi dengan catatan- jika perubahan stoma menjadi lebih prominen dengan durasi edema maka mereka juga dapat divisualisasikan dengan mikroskop confocal in vivo. Oleh karena itu, kurangnya perubahan stroma pada mikroskop confocal in vivo dapat membatasi kemampuan dari teknik mendeteksi perunahan dini. Sebaliknya peningkatan kornea yang berkabut dengan edema berkepanjangan dapat mengaburkan perubahan tersebut. Hal ini didukung oleh pengamatan bahwa kornea yang positif terhadap penyimpangan histologi dan negatif pada mikroskop confocal in vivo meskipun memiliki edema yang lebih lama.

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