Corneal Nerve Aberrations in Bullous Keratopathy

7/27/2019 Corneal Nerve Aberrations in Bullous Keratopathy

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Corneal NerveAberrations in Bullous Keratopathy

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

PURPOSE: To study the corneal nerves in patients withchronic bullous keratopathy. DESIGN: Prospective observational case series with

histologic evaluation. METHODS: We studied 25 eyes of 25 bullous keratopa-

thy 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 pen-

etrating keratoplasty from 5 of the above patients and 6

normal control corneal buttons were stained as whole

mounts with acetylcholinesterase (AChE) method forcorneal nerve demonstration and scanned in multiple

layers with digital pathology scanning microscope. RESULTS: The density, branching pattern, and diame-

ter ofsub-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 1.12f.lm). Aberrations such as localized thickenings or excres-cences, 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 inner-

vation are present in corneas with bullous keratopathy

that are unrelated to any specific etiology of bullous

keratopathy. This study provides histologic confirma-

tion of novel in vivo confocal microscopy findings

related to corneal nerves in bullous keratopathy.

(Am J Ophthalmol 2011;151:840849. 2011 by

Elsevier Inc. All rights reserved.)

ULLOUS KERATOPATHY IS A COMMON CLINICAL

endpoint of corneal endothelial dysfunction or

damage resulting from a variety ofcauses. Commonconditions associated with bullous keratopathy include

cataract extraction with or without intraocular lens im-

plantation, primary or secondary (immune rejection) cor-

neal 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 MolecularMedical Sciences, Division of Pathology (J.L.), University ofNotting-ham, Nottingham, United Kingdom.

Inquiries to Harminder S. Dua, Division of Ophthalmology and VisualSciences, B Floor, Eye ENT Centre, Queens Medical Centre, Notting-ham University Hospitals NHS Trust, Derby Road, Nottingham. NG72UH, England, UK; e-mail:[email protected]

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 edem-

atous and thickened and eventually intraepithelial and sub-

epithelial 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 inadvanced cases.

6Painful episodes associated with photo-

phobia and tearing are attributed to nerve stretching and

irritation by epithelial and subepithelial bullae and to

rupture ofsurfacebullae with exposure of nerve endings.6,7

The histologic features of bullous keratopathy include

intracellular epithelial edema and bullous separation, stro-

mal 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 kera-

topathy has been enhanced by our ability to examine thetissue in vivo, thereby avoiding artifacts resulting from

tissue handling and processing.912

Although pain, attributed to nerve stretching and ex-

posure 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 vivoconfocal 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, in-

cluding some that were scheduled for penetrating kerato-

plasty (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 observa-

tions. We also intended to elucidate any nerve-related

anatomic basis for pain, which is a dominant feature of

advanced bullous keratopathy.

840 2011 BY ELSEVIER INC ALL RIGHTS RESERVED 0002 9394/$36 00
mailto:[email protected]:[email protected]:[email protected]:[email protected]


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VOL 151 NO 5 CORNEAL NERVES IN BULLOUSKERATOPATHY 841

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

Patient Sex AgePrimary

Diagnosis SurgeryMethod of

ExaminationDuration of

Disease (Months) CCT (j.Lm)Detection of the Sub-basal

Nerve by IVCMAbnormal Stromal

Nerves by IVCM

1 F 78 ABK Triple IVCM+histology 9 700 +2 F 81 PBK PKP IVCM+histology 34 628 +

3 F 88 PBK Triple IVCM+histology 9 6914 M 74 PBK Triple IVCM+histology 14 650 +5 M 74 FED Triple IVCM 31 598 +6 F 73 FED Triple IVCM 48 760 +7 F 78 FED PKP IVCM 7 750 +8 F 72 FED Triple IVCM 24 642 + +9 F 76 FED DSEK IVCM 11 776 +

10 F 78 FED PKP IVCM 4 760 +11 M 79 FED PKP IVCM 8 58812 F 58 FED PKP IVCM 14 730 +13 F 82 FED DSEK IVCM 8 760 +14 F 71 FED PKP IVCM 19 613 + +15 F 82 FED PKP IVCM 48 570

16 F 84 Idiopathic Triple IVCM 5 737 + +17 M 83 FED Triple IVCM 7 624 + +18 F 70 PBK PKP IVCM 10 68819 F 74 PBK PKP IVCM 12 670 +20 F 85 Glaucoma Triple IVCM 11 749 +21 M 71 PBK DSEK IVCM 6 766 +22 M 86 PBK PKP IVCM 28 633 +23 M 60 PBK PKP IVCM 18 663 +24 M 72 FED PKP IVCM+histology 39 695 +25 F 78 PBK DSEK IVCM 11 625

APB aphakic bullous keratopathy; CCT central corneal thickness; DSEK Descemet stripping endothelial keratoplasty; Ffemale;

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.

MATERIALSAND 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 clin-

ical 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 trans-

plant surgery. Twelve of 25 patients (48%) had penetrat-

ing keratoplasty alone, 9 of 25 (36%) had triple procedure

(penetrating keratoplasty and lens extraction with im-

plant) and 4 of 25 (16%) underwent Descemet stripping

endothelial keratoplasty (DSEK). Five host corneal but-

tons were available for histology. Whole mounts were

stained for corneal nerve demonstration using acetylcho-

linesterase technique and examined by light microscopy.

The diagnosis of bullous keratopathy was based on

history, slit-lamp examination, and central corneal thick-

ness (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, Ja-

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 Mod-

ule [RCM]; Heidelberg Engineering GmbH, Heidelberg,

Germany). The device uses a class I diode laser (670-nmwavelength) 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 resolutionand 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 undertopical anesthesia with MINIMS oxybuprocaine hydro-

chloride 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
http://www.accessdata.fda.gov/cdrh_docs/pdf4/K042742.pdfhttp://www.accessdata.fda.gov/cdrh_docs/pdf4/K042742.pdfhttp://www.accessdata.fda.gov/cdrh_docs/pdf4/K042742.pdfhttp://www.accessdata.fda.gov/cdrh_docs/pdf4/K042742.pdfhttp://www.accessdata.fda.gov/cdrh_docs/pdf4/K042742.pdfhttp://www.accessdata.fda.gov/cdrh_docs/pdf4/K042742.pdf


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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 ofsub-basal nerves is shown (Top left inset). Sub-basal nerveplexus appearance in bullous keratopathy (Top right, frame level = 32 f.lm). There is a reduction in the density and thickness ofthe nerves. Tortuous sub-basal nerves in bullous keratopathy (Middle left, frame level = 30 f.lm). Tortuous stromal nerves, somesurrounding darklacunae, observed at different depths within the stroma (Middle right, frame level = 189 f.lm; Bottom left, framelevel = 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 King-

dom) was used as coupling medium between the contact

cap and objective lens of the microscope.

Central and paracentral regions (approximately 7 X 7mm) 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


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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 ofearly sprouting (arrows). (Middle left) A relatively thickstromal 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 den-

sity, which is in mm/mm2; branching pattern which is

expressed as the percentage of nerve branches per total

number ofsub-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.

Qualitative morphologic evaluation of sub-basal and stro-

mal nerves was also carried out.

ACETYLCHOLINESTERASE TECHNIQUE FOR THE DEM-

ONSTRATION OF CORNEAL NERVES: In 5 patients where

in vivo confocal microscopy examination of their corneas

was performed preoperatively, their corneal buttons ob-

tained after penetrating keratoplasty for bullous keratopa-

thy were processed and stained as whole mounts for


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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 Nano-

Zoomer digital pathology (NDP) microscope system

(Hamamatsu, Hamamatsu City, Japan). The corneal but-

tons were examined at 40X magnification in multiplelayers 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.

CONTROL: Six normal eyes from 6 healthy subjectswith no previous ocular problems orsurgeries 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 cor-

neas 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 thelung, 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 Pvalue of


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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 micros-

copy findings. Mann-Whitney U test was used to test the

difference in the duration of bullous keratopathy in pa-

tients with and without demonstrable in vivo confocal

microscopy findings.

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). Branch-

ing 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).

Furthermore, these nerves appeared thinner and at-

tenuated compared to the normal sub-basal nerves

(Figure 1, Top right). The diameter of the main sub-

basal 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 orienta-

tion (Figure 1, Middle left).

Stromal nerves. On in vivo confocal microscopy exam-

ination, stromal nerves were seen in all patients studiedbut 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 sug-

gestive 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 hyper-


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TABLE 2. Histologic Features of Corneal Nerves in Bullous Keratopathy

Corneal

Button DiagnosisSub-basal

Nerve StatusNo. of

Perforation SitesLocalized Nerve

ThickeningsaNerve

SproutingaNo. ofNerves

Entering the Cornea

1 ABK Absent Absent +++ +++ 362 PBK Present 9 +++ +++ 33

3 PBK Absent 7 +++ +++ 304 PBK Present 10 ++ +++ 315 FED Absent 9 ++ +++ 34

ABK aphakic bullous keratopathy; FED Fuchs endothelial dystrophy; PBK pseudophakic

bullous keratopathy.aNerve 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 ofthe

abnormal in vivo confocal microscopy features observed in

TABLE 3. Central Corneal Thickness and Duration ofBullous Keratopathy in Patients With and Without Stromal

Nerve Changes by In Vivo Confocal Microscopyand Histology

Duration Between

corneas with bullous keratopathy were seen in any ofthe

controls.

HISTOLOGIC FINDINGS: Sub-basal nerves. Histologic

Patients (Number)

Without demonstrable in vivo

confocal microscopy

CCT (j.Lm)

Mean + SD

Diagnosis and

Scanning (Months)

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. Perfora-

tion 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 dem-

findings (15) 691.4+ 71.2 16.5+ 14.0

With demonstrable in vivo

confocal microscopy

findings (10) 669.5+ 51.0 18.7+ 12.5

With demonstrable histologic

findings (5) 672.8+ 31.9 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 pseudo-

phakic 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 kera-

topathy were seen in any of the controls.


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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) (Table3). CCT in patients with demonstrable in vivo confocal

microscopy stromal nerve changes was lower than that inpatients 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.

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 fewsub-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;

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 mor-

phology 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 combina-

tion 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 altera-

tion 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 to-

gether with subepithelial scarring. Scar tissue is hyper-

reflective 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

localized thickening or excrescences; and possible sprout-

ing of new nerves. On in vivo confocal microscopy the

loops and coils of stromal nerves were seen to surround

darkspaces. It ispossible 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 darkspaces seen on in

vivo confocal microscopy of bullous keratopathy correlate

to empty spaces reported on cross-sectional electron mi-

croscopy (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-regen-

eration ofnerves have been reported in a previous study

where 3 cases of bullous keratopathy secondary to glau-

coma 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 amplifica-

tion 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+ 87j.Lm.21,26The 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 cor-

neal 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


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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 associ-

ates,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 orpresence of sub-

basal nerves was commented on. Quantitative descriptorsof 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 sensi-

tivity returns back to near-preoperative levels by 3 months.31,32

In ourstudy, the abnormal nerve features were found in most of

the corneal quadrants and therefore it is unlikely that they wereattributable to previous cataract surgery.

Activated skin keratinocytes have been shown to syn-

thesize neuronal growth factors,3335 which in turn induces

nerve sprouting and hyperalgesia in experimental mod-

els.33,36

The cornea also contains a wide variety of neuro-

nal growth factors and epithelial cells and keratocytes

express growth factor receptors.37 It is therefore conceiv-

able that aberrant regeneration of nerves occurs in the

cornea and could play an important role in pain experi-

enced 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 corn-

eal 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 visualizedby in

vivo confocal microscopy. Hence, lack of stromal changeson in vivo confocal microscopy may be a limitation ofthe

ability of the technique to detect early changes rather than

absolute absence. Conversely, increased corneal clouding

with prolonged edema may obscure nerve changes (re-

duced 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 dura-

tion 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.); conductof 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 andapproval 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 isconsistent with the tenets of the Declaration of Helsinki. Informed, written consent was obtained from all patients.

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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/mm2

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


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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.


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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


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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 layaradalah 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


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16/25

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


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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


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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 sub-

basal 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).


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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


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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


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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 sub-

basal 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


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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 sepertifakoemulsifikasi diketahui menyebabkan gangguan yang lebih sedikit dari persarafan kornea dengan


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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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Corneal Nerve Aberrations in Bullous Keratopathy

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