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(Investigative Ophthalmology and Visual Science. 2001;42:1969-1974.)
© 2001 by The Association for Research in Vision and Ophthalmology, Inc.

Improvement of HSV-1 Necrotizing Keratitis with Amniotic Membrane Transplantation

Arnd Heiligenhaus1,2, Dirk Bauer1,2, Daniel Meller1,3, Klaus-Peter Steuhl1 and Scheffer C. G. Tseng3

1 From the Department of Ophthalmology, University of Essen, Germany; 2 Department of Ophthalmology, St. Franziskus Hospital, Muenster, Germany; and the 3 Department of Ophthalmology, Ocular Surface and Tear Center, Bascom Palmer Eye Institute, University of Miami School of Medicine, Florida.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. Stromal herpes simplex virus keratitis (HSK) is an immune-mediated disease. Previous studies have indicated that T cells, neutrophils, and macrophages contribute to the tissue damage in HSK. It has been shown that human amniotic membrane promotes epithelial wound healing and has diverse anti-inflammatory effects. In this study, the effect of amniotic membrane transplantation (AMT) on corneal wound healing and on inflammation in mice with necrotizing HSK was examined.

METHODS. BALB/c mice were corneally infected with 105 plaque-forming units (PFU) of HSV-1 (KOS strain). In 16 mice that exhibited severe ulcerating HSK, the cornea was covered with a preserved human amniotic membrane as a patch. Corneas in 16 infected mice remained uncovered and served as a control. On days 2 and 7 after surgery, the amniotic membrane was removed (eight mice in each group), the HSV-1–infected cornea was evaluated clinically, and the eye was enucleated. Tissue sections were analyzed histologically for epithelialization and cellular infiltration and immunohistochemically with anti-CD3 mAb to T cells, anti-CD11b mAb to both macrophages and neutrophils, or anti-F4/80 mAb to macrophages.

RESULTS. Profound regression of corneal inflammation and rapid closure of epithelial defects were observed clinically within 2 days in the amniotic membrane-covered eyes, whereas HSV-1 keratitis and ulceration progressed in all mice in the control group (P < 0.001). Histologically, corneal edema and inflammatory infiltration, and immunohistochemically the number of CD3+, CD11b+, and F4/80+ cells in the cornea were markedly decreased at 2 and 7 days after amniotic membrane application, compared with the uncovered control corneas (P < 0.001).

CONCLUSIONS. AMT promotes rapid epithelialization and reduces stromal inflammation and ulceration in HSV-1 keratitis. AMT in mice with HSV necrotizing stromal keratitis appears to be a useful model for investigating the effect and the action mechanism of human amniotic membrane.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Herpes simplex virus-induced stromal keratitis (HSK) is a major cause of blindness worldwide. There is profound evidence that this is an immune-mediated disease.1 Induction and protection of HSK are not completely defined. After reactivation from latency in the trigeminal ganglion, herpes simplex virus (HSV) antigens are expressed and corneal antigens exposed. CD4+ T cells are the principal mediators orchestrating the inflammatory response. The corneal damage is caused by proinflammatory molecules and reactive radicals. Neutrophils and macrophages are well-known contributors to these complex processes.2 3 Besides neutrophils, T cells and macrophages contribute to tissue destruction in the cornea.4 5 6 Other studies have shown that HSV blepharitis and encephalitis are more severe after neutrophil depletion.7

Amniotic membrane, or amnion (i.e., the innermost layer of the placenta), consists of a thick basement membrane and an avascular stromal matrix. Clinical and experimental data indicate that amniotic membrane facilitates the proliferation and differentiation of epithelial cells, maintains the original epithelial phenotype, promotes goblet cell differentiation, and reduces scarring, vascularization, and inflammation.8 9 10 11 12 13 14 15 16 17 When used as a graft or a patch, amniotic membrane can promote healing of persistent corneal ulcers of different causes, including neurotrophic keratopathy of various underlying origins.18 19 Amniotic membrane transplantation (AMT) has been shown to be effective in the reconstruction of the conjunctival ocular surface.8 11 20 Rapid epithelialization has also been noted with AMT in patients with persistent sterile corneal ulceration.18 19 Typically, the wounds heal without inflammation after AMT.10 11 21

There is clinical support for the notion that the healing process in necrotizing herpes keratitis may be promoted by AMT.18 19 Although some in vitro observations indicate that amniotic membranes possess anti-inflammatory effects,22 23 24 there is no reproducible animal model available to investigate the biological effects of AMT on infectious HSK in vivo. This report demonstrates that use of human AMT in mice with necrotizing HSV-1 keratitis may be a useful model for studying these effects. Based on the clinical and morphologic parameters, we report that necrotizing HSV keratitis in mice is markedly improved by AMT.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Female BALB/c mice, 6 to 8 weeks of age, were used for all studies. All experimental procedures were conducted according to the ARVO Statement on the Use of Animals in Ophthalmic and Vision Research.

Virus
The HSV-1 KOS strain was kindly provided by Dave Knipe (Harvard Medical School, Boston, MA). The virus was propagated on Vero cells (CCL 81; American Type Culture Collection [ATCC], Rockville, MD). Infected Vero cell monolayers were harvested when confluent cytopathic effects were present. The infected cells were freeze thawed three times, homogenized, and centrifuged at 1500g. The collected supernatants were suspended in RPMI 1640 medium (Gibco, Eggenstein, Germany) and were used in all experiments. The virus-containing supernatants were aliquoted and stored at -80°C.

Corneal HSV-1 Infection
For the infection, mice were anesthetized intraperitoneally with ketamine hydrochloride (2 mg) and mepivacaine hydrochloride (400 ng) in 250 µl phosphate-buffered saline (PBS). The cornea of the right eye was scratched in a crisscross pattern (eight horizontal and eight vertical scratches) with a 27-gauge needle under a surgical microscope. Five microliters HSV-1 (KOS-strain) suspension containing 105 plaque-forming units (PFU) was placed on the cornea while the lids were held open for 10 seconds. The mice were treated daily with 0.5% gentamicin ophthalmic solution (Merck, Darmstadt, Germany).6 25 26

Preparation of Preserved Human Amniotic Membrane
Human amniotic membrane was prepared and preserved using our previously described method.8 13 18 Briefly, the human placenta was obtained shortly after elective cesarean delivery when human immunodeficiency virus, human hepatitis type B and C, and syphilis had been excluded by serologic tests. Under a laminar flow hood, the blood clots were removed by thorough washing with sterile saline solution containing 50 µg/ml penicillin, 50 µg/ml streptomycin, 100 µg/ml neomycin, and 2.5 µg/ml amphotericin B. The amnion was separated from the rest of the chorion by blunt dissection and was flattened onto nitrocellulose paper with a pore size of 0.45 µm, with the epithelium–basement membrane surface facing away from the paper. The paper with the adherent amniotic membrane was then stored at -80°C in sterile vials containing Dulbecco’s modified Eagle’s medium and glycerol at a ratio of 1:1 (vol/vol). The study was conducted in accordance with the tenets of the Declaration of Helsinki.

Experimental Design
On day 14 after corneal infection, 16 mice were chosen for the experiments after severe necrotizing ulcerating HSV keratitis had developed. The entire cornea and bulbar conjunctiva were covered with amniotic membrane with the epithelium facing up as a temporary patch and secured by tarsorrhaphy with three interrupted 10-0 nylon sutures.

For comparison, another 16 mice that underwent only tarsorrhaphy were used as a control group. A separate control group of three mice received AMT with the epithelial side facing down.

After the removal of the amniotic membranes on days 2 and 7 after transplantation, mice were clinically evaluated for signs of HSV keratitis. The eyes were then enucleated and were immediately frozen in liquid nitrogen or fixed in formalin. Tissue sections were stained according to a hematoxylin-eosin staining protocol and processed for immunohistochemistry.

Clinical Evaluation
After the corneal infection, the animals were observed daily with a surgical microscope (Carl Zeiss, Oberkochen, Germany) for the development of HSV-1 keratitis. The severity of clinical keratitis was graded as described previously.6 26 27 The scoring system was as follows: 0, clear cornea; +1, mild corneal haze; +2, moderate corneal opacity or scarring; +3, severe corneal opacity, iris visible; and +4, opaque cornea, iris not visible, necrotizing stromal keratitis with ulceration. Fluorescein dye solution was applied for the detection of corneal ulcers, and the presence of corneal ulcers was documented. The area of the epithelial defect was scored from grades 0 to 4: 0, no corneal defect; 1, defect in 25% of the corneal surface; 2, in 25% to 50% of the corneal surface; 3, in 50% to 75% of the corneal surface; and 4, in 75% to 100% of the corneal surface.

Histology
Specimens for light microscopy were fixed in McDowell solution (4% formaldehyde, 1% glutaraldehyde, 0.13% sucrose, 0.07 M sodium hydroxide, 0.08 M sodium phosphate [pH 7.2]), rinsed in a cacodylate buffer, dehydrated with ethanol, and embedded in paraffin. Five-micrometer sections were stained with hematoxylin-eosin.

Several eyes and amniotic membranes from each group were randomly chosen for analysis for the presence of neutrophils and mononuclear cells (lymphocytes, natural killer cells, plasma cells, and macrophages) in the central cornea, the limbus, and the conjunctiva. The number of total inflammatory cells and neutrophils was counted by means of bright-field microscopy independently by two investigators. Neutrophils were identified on the basis of their morphology under x250 magnification. Cell counts were performed in the central cornea under a 10 x 10-grid high-power field on eight eyes per group, two sections per eye. All counts were independently performed by two investigators in a masked fashion. Group means were compared with two-tailed Student’s t-test.

Presence and absence of epithelial defects was evaluated. Because ulcerations were present in all mice, corneal thickness (swelling) as another parameter for the severity of inflammation was also measured on a grid in micrometers in the midperipheral cornea.

Immunohistochemistry
After enucleation, the eye and the amniotic membranes were immediately snap frozen in liquid nitrogen and embedded in optimal-temperature cutting (OCT) compound (Miles Laboratory, Elkhart, IN) and stored at -80°C. Tissues were stained with the avidin-biotin-immunoperoxidase technique.6 26 Briefly, 5-µm cryostat sections were mounted on poly-L-lysine–coated slides (Sigma, Munich, Germany). The sections were fixed in cold acetone, incubated with bovine serum albumin (1:20 in PBS) for 30 minutes, and then stained with the primary antibodies in a moist chamber at 20°C for 20 minutes. The primary mAbs applied were as follows: rat anti-mouse CD3 (dilution 1:20 in PBS; PharMingen, San Diego, CA) for the detection of T cells; rat anti-mouse CD11b (dilution 1:20 in PBS; PharMingen) for the detection of neutrophils and macrophages; and rat anti-mouse F4/80 antigen (Serotec, Heidelberg, Germany) for the detection of macrophages. The tissues were then blocked for endogenous peroxidase using 3% hydrogen peroxide in PBS and rinsed with PBS. This was followed by an incubation for 30 minutes with a secondary biotinylated rabbit anti-rat antibody (dilution 1:10 in PBS; Dianova, Hamburg, Germany) that had been preabsorbed with 5% mouse serum protein. The sections were rinsed in PBS again. Avidin-biotin-peroxidase complexes (peroxidase-conjugated streptavidin, dilution 1:500 in PBS-BSA; Dako, Hamburg, Germany) were applied for 20 minutes. The reactions at sites of binding were developed in peroxidase substrate containing 3-amino-9-ethylcarbazole (Sigma) and hydrogen peroxide in 0.1 M acetate buffer. Specimens were then fixed in formalin (4%, in acetate buffer), counterstained with hematoxylin (Gills No. 3; Sigma), and coverslipped with Aquatex (Merck).

Eight eyes at each time point were analyzed in each group. All counts were independently performed by two investigators in a masked fashion. The number of positively stained cells was counted under a 10 x 10-grid high-power field (x250). Cell counts were performed on two sections per eye by scanning the central cornea at the site of ulceration. Group means were compared with Student’s t-test.

Statistical Analysis
We performed the entire experiment two times using the same experimental design. Fisher’s protected least-significant difference test was used to analyze the statistical significance of differences between mean values of clinical keratitis scores and the corneal thickness. Student’s t-test was used to determine the differences in cell numbers between the experimental groups in the histologic and immunohistochemical studies. P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Influence of the Amniotic Membrane on the Clinical Course of HSV-1 Keratitis
The control mice that were HSV infected but not treated with an amniotic membrane showed a profound progression of HSV-1 keratitis (Fig. 1) . In all corneas, the score for the area of ulceration progressed from 2.3 ± 0.5 at day 14, to 2.7 ± 0.4 at day 16, and 3.3 ± 0.6 at day 21 after infection. The inflammatory infiltration tended to progress (Fig. 1) , and 3 of the 16 control corneas were perforated.



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  		Figure 1. Influence of the amniotic membrane on the course of severe HSV-1 necrotizing keratitis in BALB/c mice. Fourteen days after corneal infection with HSV-1, all mice with +4 disease (opaque cornea, iris not visible, necrotizing stromal keratitis with ulceration) received an amniotic membrane patch or were left untreated. Severity of stromal keratitis was graded on days 2 and 7 after AMT. AMT significantly reduced the severity of keratitis (P < 0.001).

 
In contrast to the control eyes, corneal inflammation showed rapid regression in all the amniotic membrane–covered eyes within 2 days after AMT (P < 0.001). The corneal ulcers healed within the first 2 days after AMT in all mice (P < 0.001). The score for the area of the epithelial defect decreased from 2.5 ± 0.7 at day 14 to 0.5 ± 0.9 and 0.2 ± 0.3 at days 16 and 21 after infection, respectively—that is, days 2 and 7, respectively, after AMT (P < 0.05). Corneal perforation was not detected in any mouse of this group. Although no attempt was made to quantify corneal vascularization, the blood-filling corneal vessels appeared to be reduced 2 days after AMT. The inflammation, corneal edema, and ulceration also improved slightly from days 2 to 7 after AMT; however, these changes were not significant. Tarsorrhaphy did not reproduce the beneficial effect on the course of epithelial defects, the ulceration, the corneal vascularization, or stromal inflammatory infiltration, as noted by AMT. Two days after the use of murine conjunctiva as a patch (n = 3), the epithelial defect score remained 2.5 ± 0.9 and the corneal inflammation score still was in the range of 3.5 ± 0.7, both of which were not significantly different from scores in the control group.

Influence of the Amniotic Membrane on Histologic Appearance and Cellular Infiltration of the Cornea
Histologic Appearance.
At day 14 after corneal HSV-1 infection, the corneas of all mice had severe stromal edema, severe neutrophil infiltration, multifocal necrosis, severe ulceration, and intense vascularization. Although these histologic characteristics moderately progressed in the ensuing 7 days, the changes were not statistically significant (Fig. 2) .



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  		Figure 2. Influence of the amniotic membrane (AM) on the histology of severe HSV-1 necrotizing keratitis in BALB/c mice. Fourteen days after corneal infection with HSV-1, corneas with +4 disease were patched with an AM for 2 days (A) or left untreated (B). AM-patched corneas showed complete epithelialization and reduced stromal infiltration and thickness compared with the control. (C, D) Respective higher magnifications. Arrows: cells with abnormal morphology (C). Hematoxylin-eosin staining. Scale bars, 100 µm; magnification, (A, B) x120; (C, D) x250.

 
These findings were in sharp contrast to the histologic appearance of the mice that had been treated with an amniotic membrane as a temporary patch (Fig. 2) . There was a profound regression of epithelial and stromal HSV disease noted on day 2 after AMT. The epithelial defects had healed in all mice within 2 days. Compared with the mice in the control group, reduced corneal swelling was seen in all mice (386.6 ± 56.6 and 195.5 ± 45.5 µm, respectively; P < 0.01). Corneal vascularization, necrosis, and anterior chamber inflammation were also markedly improved in the AMT group. Between days 2 and 7 after AMT, no significant changes were noted in the histologic parameters examined, although there was a tendency to improve progressively (Fig. 1) .

Studies were performed to determine whether AMT influences the cellular infiltration in the cornea after HSV-1 infection. The numbers of neutrophils and other inflammatory cells were counted in the central cornea. The experiments showed that mice with +4 disease at 14 days after HSV infection had severe cellular infiltration with an extremely high number of inflammatory cells in the central cornea (Fig. 2 , Table 1 ). These control mice still maintained this high number of inflammatory cells in the central cornea at day 21 after infection. In contrast, the number markedly decreased at 48 hours after amniotic membrane application (P < 0.001; Fig. 2 , Table 1 ).


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  		Table 1. Influence of the Amniotic Membrane on the Number of Inflammatory Cells in BALB/c Mice with Severe HSV-1 Necrotizing Keratitis

 
Morphologic examination of corneas covered with amniotic membrane revealed that the number of neutrophils in the central cornea was increased in mice with HSV-1 necrotizing keratitis in the control group, but significantly decreased at days 2 and 7 after AMT (Table 1) . Nonviable neutrophils that showed cell shrinkage, condensation of the cytoplasm, and pyknotic nuclei were enumerated by a method that has been described previously.28 Cells with abnormal morphology are indicated by arrows in Figure 2C . The number of neutrophils that were not viable also increased in the central cornea of control mice during the ensuing course of necrotizing keratitis, whereas the number slightly decreased in the amniotic membrane–treated mice. The decrease, however, did not reach the level of significance. The percentage of total nonviable neutrophils in the central cornea is also presented in Table 1 . The majority of neutrophils in the central cornea of control mice showed normal PMN morphology. In contrast, after AMT, many of the neutrophils in the central cornea of amniotic membrane-covered corneas appeared to be nonviable.

Immunohistochemical Study of Inflammatory Cells in the Cornea.
Quantitative assessment of CD11b+ cells was performed in corneas of mice at day 14 after infection and 2 and 7 days afterwards. Groups of mice with and without AMT were compared (Table 2) . A dramatic reduction of the number of the diverse cell types was observed in the HSV-infected cornea after AMT. These differences were consistently noted in two different experiments. Two days after AMT, the number of CD11b+ cells in the cornea markedly decreased in the AMT-treated cornea, whereas CD11b+ cells continued to extravasate into the central cornea and onto the ulcerated surface in the control group (Fig. 3 , Table 2 , P < 0.001).


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  		Table 2. Influence of the Amniotic Membrane on Different Populations of Inflammatory Cells in BALB/c Mice with Severe HSV-1 Necrotizing Keratitis

 


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  		Figure 3. Influence of the amniotic membrane (AM) on CD11b cells in BALB/c mice with severe HSV-1 necrotizing keratitis. Fourteen days after corneal infection with HSV-1, corneas with +4 disease were patched with an AM for 2 days (A) or left untreated (B). Immunohistochemical staining with an antibody directed against CD11b showed numerous CD11b+ cells, predominantly in the anterior half of the corneal stroma of the control specimen, but the cell number was markedly reduced in the AM-treated cornea. Scale bar, 100 µm; magnification, x200.

 
AMT also caused a significant decrease of the CD3+ cell infiltration in the central cornea compared with the control group. Although the CD3+ cell number showed a tendency to increase during the subsequent course of HSV necrotizing keratitis in the control group, it was profoundly reduced at days 2 and 7 in amniotic membrane–covered corneas (Table 2 , P < 0.001). A similar result was observed in the F4/80 antigen–positive cells (Table 2 , P < 0.001). As early as 2 days after AMT, the number of F4/80+ cells was markedly reduced in the central cornea, compared with the control.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
AMT has been reported to be an effective surgical procedure for the reconstruction of the ocular surface and to reduce corneal inflammation.13 18 19 21 29 30 The clinical and morphologic data obtained in the current report support that AMT significantly modifies the course of necrotizing stromal keratitis induced by HSV-1. It was associated with a suppression of inflammation, rapid epithelialization, and a reduction of stromal necrosis. Although the anti-inflammatory effect of the amniotic membrane has been observed clinically, the underlying mechanism is not known. The data presented herein suggest that this mouse model of HSV-1 keratitis is ideal for investigating such an anti-inflammatory mechanism.

The mouse amniotic membrane is too small to investigate such an action in our mouse model. It has recently been shown that human amniotic membrane exhibits an anti-inflammatory action, as evidenced by clinical observation in human patients.8 10 11 13 20 29 30 31 In line with these observations, we also noted in the current study that human AMT as a temporary patch promoted rapid resolution of stromal inflammation and ulceration in experimental HSV-1 keratitis. This anti-inflammatory effect was mediated by a marked suppression of several populations of inflammatory cells, including CD3+ T cells, CD11b+ neutrophils, and F4/80+ macrophages.

This observation may be attributable to diverse factors. The membrane contains protease inhibitors, which exert an inhibitory effect on various proteinases,30 and as a result it may decrease inflammation and corneal destruction. It has been suggested that the amniotic membrane induces neutrophils to undergo programmed cell death and prevents their contributing to tissue destruction.32 33 Our preliminary unpublished study also indicated that the percentage of nonviable neutrophils was increased in the cornea of HSV-1–infected mice after AMT. Culturing of corneal keratocytes on the amniotic membrane reduces the expression of chemokines—that is, proteins that regulate leukocyte migration in response to inflammatory stimuli.24 Furthermore, expression of IL-1, a potent proinflammatory cytokine that can be produced by corneal epithelial cells,26 34 35 is markedly suppressed by the amniotic membrane.36 These data collectively indicate that the amniotic membrane may have a direct anti-inflammatory effect by suppressing inflammatory cells, but also may have an indirect effect through corneal epithelial cells by promoting rapid epithelial healing.

Kim et al.30 showed that AMT as a patch with the epithelial surface facing down was more effective than a patch with the stromal side down in treating acute alkali burns in rabbits. Our preliminary study did not reveal such a difference. The direct contact between the amniotic membrane and the cornea may not be of great importance for the improvement of HSK. In our experiments the membrane was secured in the cul du sac by tarsorrhaphy and was not firmly fixed to the cornea. This also suggests that the AMT may not serve primarily as a "mega-bandage contact lens" or barrier that keeps polymorphonuclear neutrophils (PMNs) and other inflammatory factors out of the cornea. It has recently been observed that PMNs were adherent to the stromal side of the amniotic membrane.32 Whether the matrix components or soluble factors released by the amniotic membrane are responsible for such an action deserves further investigation in this HSV-1 model in the future.

We did not note that the stromal inflammation completely resolved after AMT. There are several explanations. First, most membranes dissolved within a few days. Consequently, the membrane was not on the surface long enough to exert a continuous anti-inflammatory effect. Future studies are needed to determine whether multiple membranes, a technique that has been suggested recently for the reconstruction of deep corneal ulcers,19 will help. Second, significant suppression of inflammation was noted for at least 7 days. However, relapses were seen during the subsequent follow-up after removing the membrane. It may be helpful to repeat AMT to maintain the beneficial effect.37 Third, antiviral nonspecific immunity has been detected in the amniotic membrane.38 Whether this may be of relevance in the HSK model should be elucidated. Further studies are in progress to unravel the anti-inflammatory and antiviral effects of human amniotic membrane and to define their potential therapeutic role in treating refractory inflammatory diseases on the eye surface, including herpetic keratitis.


   Footnotes
 
Supported by Deutsche Forschungsgemeinschaft Grant He 1877/12-1.

Submitted for publication September 20, 2000; revised February 23, 2001; accepted March 21, 2001.

Commercial relationships policy: P (SCGT); N (all others).

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Arnd Heiligenhaus, Department of Ophthalmology, Ophtha-Lab, St. Franziskus Hospital, Hohenzollernring 74, 48145 Muenster, Germany. arnd.heiligenhaus{at}t-online.de


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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