Person:

Cruzat, Andrea

The Boston keratoprosthesis (B-KPro), currently the most commonly used artificial cornea worldwide, can provide rapid visual rehabilitation for eyes with severe corneal opacities not suitable for standard corneal transplantation. However, the B-KPro presently needs a corneal graft as a tissue carrier. Although corneal allograft tissue is readily available in the United States and other developed countries with established eye banks, the worldwide need vastly exceeds supply. Therefore, a simple, safe, and inexpensive alternative to corneal allografts is desirable for the developing world. We are currently exploring reasonable alternative options such as corneal autografts, xenografts, noncorneal autologous tissues, and laboratory-made tissue constructs, as well as modifications to corneal allografts, such as deep-freezing, glycerol-dehydration, gamma irradiation, and cross-linking. These alternative tissue carriers for the B-KPro are discussed with special regard to safety, practicality, and cost for the developing world.

Purpose

To study and correlate corneal sensation in patients with herpes simplex keratitis (HSK) with density and morphology of subbasal corneal nerves by in vivo confocal microscopy (IVCM).

Design

Prospective, cross-sectional, controlled, single-center study.

Participants

Thirty-one eyes with the diagnosis of acute (n=7) or chronic (n=24) HSK and the contralateral clinically unaffected eyes were studied and compared to normal controls (n=15).

Methods

IVCM (Confoscan 4, Nidek) and corneal esthesiometry (Cochet-Bonnet) of the central cornea were performed bilaterally in all patients and controls. Patients were grouped into normal (>5.5 cm), mild (>2.5 to 5.5cm) and severe (≤2.5 cm) loss of sensation.

Main Outcome Measures

Changes in corneal nerve density, total nerve number, main nerve trunks, branching and tortuosity were evaluated after IVCM and correlated to corneal sensation, disease duration, and number of recurrences.

Results

HSK eyes, as compared to controls, demonstrated significant (p<0.001) decrease in mean nerve density (448.9±409.3 vs. 2,258.4±989.0 μm/frame), total nerve number (5.2±4.5 vs. 13.1±3.8), main nerve trunks (2.3±1.6 vs. 4.7±1.2) and nerve branches (3.2 ± 4.3 vs. 9.8±3.3). In contralateral unaffected eyes, mean nerve density (992.7±465.0 μm/frame), total nerve number (7.8±3.3), and branches (4.5±2.3) were significantly decreased as compared to controls (p<0.002). Reduced nerve density, total nerve count and main trunks in HSK eyes were significantly correlated with corneal sensation across all subgroups (p<0.001). Nerve density decreased within days of infection and was correlated to frequency of episodes in patients with HSK (p<0.02).

Conclusions

In vivo confocal microscopy reveals that the loss of corneal sensation in HSK correlates strongly with profound diminishment of the subbasal nerve plexus after herpes simplex virus (HSV) infection. Surprisingly, the contralateral clinically unaffected eyes also demonstrated a diminishment of the subbasal nerve plexus, as compared to normal subjects, revealing bilateral nerve alteration in an apparently unilateral disease.

Purpose.

To study the density and morphologic characteristics of epithelial dendritic cells, as correlated to subbasal corneal nerve alterations in acute infectious keratitis (IK) by in vivo confocal microscopy (IVCM).

Methods.

IVCM of the central cornea was performed prospectively in 53 eyes with acute bacterial (n = 23), fungal (n = 13), and Acanthamoeba (n = 17) keratitis, and in 20 normal eyes, by using laser in vivo confocal microscopy. Density and morphology of dendritic-shaped cells (DCs) of the central cornea, corneal nerve density, nerve numbers, branching, and tortuosity were assessed and correlated. It should be noted that due to the “in vivo” nature of the study, the exact identity of these DCs cannot be specified, as they could be monocytes or tissue macrophages, but most likely dendritic cells.

Results.

IVCM revealed the presence of central corneal DCs in all patients and controls. The mean DC density was significantly higher in patients with bacterial (441.1 ± 320.5 cells/mm2; P < 0.0001), fungal (608.9 ± 812.5 cells/mm2; P < 0.0001), and Acanthamoeba keratitis (1000.2 ± 1090.3 cells/mm2; P < 0.0001) compared with controls (49.3 ± 39.6 cells/mm2). DCs had an increased size and dendrites in patients with IK. Corneal nerves were significantly reduced in eyes with IK compared with controls across all subgroups, including nerve density (674.2 ± 976.1 vs. 3913.9 ± 507.4 μm/frame), total nerve numbers (2.7 ± 3.9 vs. 20.2 ± 3.3), main trunks (1.5 ± 2.2 vs. 6.9 ± 1.1), and branching (1.2 ± 2.0 vs. 13.5 ± 3.1; P < 0.0001). A strong association between the diminishment of corneal nerves and the increase of DC density was observed (r = −0.44; P < 0.0005).

Conclusions.

IVCM reveals an increased density and morphologic changes of central epithelial DCs in infectious keratitis. There is a strong and significant correlation between the increase in DC numbers and the decreased subbasal corneal nerves, suggesting a potential interaction between the immune and nervous system in the cornea.

Purpose.: To investigate the ability of bevacizumab to penetrate the cornea after topical application or subconjunctival injection.

Methods.: Bevacizumab 1% was topically applied three times a day to the corneas of mice (BALB/c) with intact corneas (n = 14), and with corneal neovascularization (n = 14). Animals were euthanized at 1, 6, 12, and 24 hours, and 2, 4, and 7 days for immunohistochemical analyses. Donkey anti-human IgG labeled with Cy3 was used for bevacizumab immunoreactivity detection. Additionally, one-time topical bevacizumab 1% was tested in corneas with denuded epithelium (n = 16). In another group (n = 16), a single dose of 0.5 mg bevacizumab was injected subconjunctivally. Animals were euthanized at 1, 6, and 24 hours, and 2, 4, 7, 14, and 21 days for immunohistochemical studies.

Results.: Bevacizumab was barely detected beyond the very superficial layer of the corneal epithelium in mice with intact corneas even after 7 days of topical administration. Application of bevacizumab in mice with corneal neovascularization; however, showed variable penetration into the corneal stroma. Experimentation with single application of topical bevacizumab in corneas with denuded epithelium or subconjunctivally injected bevacizumab showed intense staining for bevacizumab.

Conclusions.: Topically applied bevacizumab has limited capacity to penetrate the corneas with intact epithelium. However, bevacizumab can penetrate the neovascularized cornea after topical application. This study demonstrates that subconjunctivally injected bevacizumab in eyes with an intact cornea penetrates well into the corneal stroma.

Purpose

Herpes zoster ophthalmicus (HZO), thought to be a unilateral disease, results in loss of corneal sensation, leading to neurotrophic keratopathy. This study aimed to analyze bilateral corneal nerve changes in patients with HZO by in vivo confocal microscopy (IVCM) and their correlation with corneal sensation as a measure of nerve function.

Design

Prospective, cross-sectional, controlled, single-center study.

Participants

Twenty-seven eyes with the diagnosis of HZO and their contralateral clinically unaffected eyes were studied and compared with normal controls (n = 15).

Methods

In vivo confocal microscopy (Confoscan 4; Nidek Technologies, Gamagori, Japan) and corneal esthesiometry (Cochet-Bonnet; Luneau Ophthalmologie, Chartres, France) of the central cornea were performed bilaterally in all patients and controls. Patients were grouped into normal (>5.5 cm), mild (>2.5–5.5 cm), and severe (<2.5 cm) loss of sensation.

Main Outcome Measures

Changes in corneal nerve density, total nerve number, main nerve trunks, branching, and tortuosity were evaluated after IVCM and were correlated to corneal sensation, disease duration, and number of recurrences.

Results

Eyes with herpes zoster ophthalmicus had a significant (P<0.001) decrease in total nerve length (595.8±358.1 vs. 2258.4±989.0 μm/frame), total number of nerves (5.4±2.8 vs. 13.1±3.8), number of main nerve trunks (2.3±1.1 vs. 4.7±1.2), and number of nerve branches (3.2±2.3 vs. 8.4±3.7) as compared with controls. In the contralateral clinically unaffected eyes, total nerve length (1053.1±441.4 μm/frame), total number of nerves (8.3±2.9), and main nerve trunks (3.1±1.0) also were decreased significantly as compared with controls (P<0.01). Reduced nerve density, total nerve count, main trunks, and tortuosity was correlated significantly with corneal sensation across all subgroups (P<0.001).

Conclusions

Patients with unilateral HZO demonstrated a profound and significant bilateral loss of the corneal nerve plexus as compared with controls, demonstrating bilateral changes in a clinically unilateral disease. Loss of corneal sensation strongly correlated with subbasal nerve plexus alterations as shown by IVCM.

Purpose

To investigate the longitudinal alterations of subbasal corneal nerves in patients with infectious keratitis (IK) during acute phase, cessation of treatment and recovery phase by in vivo confocal microscopy (IVCM).

Design

Prospective, longitudinal, case-control, single-center study.

Subjects

Fifty-six eyes of 56 patients with the diagnosis of bacterial (n=28), fungal (n=15), and Acanthamoeba (n=13) keratitis were included in the study. Thirty eyes of 30 normal volunteers constituted the control group.

Methods

Corneal sensation and serial IVCM of the central cornea were performed prospectively, by using the Heidelberg Retina Tomograph 3/Rostock Cornea Module (Heidelberg Engineering, Germany). IVCM images were assessed at 3 time points: at the first visit of the patient to the cornea service, at cessation of antimicrobial treatment, and up to six months after the resolution of infection.

Main outcome measures

Total nerve number and length, main nerve trunks, branching and corneal sensation were assessed during the follow-up period.

Results

Corneal nerves were significantly reduced during the acute phase in eyes with IK compared with controls across all subgroups, with total nerve length of 5.47 ± 0.69 vs. 20.59 ± 1.06 mm/mm2; p<0.0001. At the cessation of treatment, corneal nerves in patients with IK had regenerated, including total nerve length (8.49 ± 0.94; p=0.02) and nerve branch length (4.80 ± 0.37; p=0.005). During the recovery phase, after resolution of infection, corneal nerves further regenerated, including total nerve length (12.13 ± 1.97; p=0.005), main nerve trunk length (5.80 ± 1.00; p=0.01) and nerve branch length (6.33 ± 0.76; p=0.003) as compared to the acute phase, but were still significantly lower when compared to controls (p<0.05 for all parameters). Corneal degeneration and regeneration correlated with corneal sensation (r=0.47, p=0.0009).

Conclusion

Patients with IK, suffering from profound loss of corneal nerves during the acute phase of infection, demonstrate an increase of corneal nerve density during the first six months after the resolution of infection. However, despite significant nerve regeneration, corneal nerve density does not fully recover and remains low as compared to controls. By providing an objective methodology to monitor corneal re-innervation, IVCM adds potentially important findings that may have implications for clinical management and surgical planning.

Purpose

To analyze the contralateral unaffected eyes of patients with microbial keratitis (MK) for any immune cell or nerve changes by laser in vivo confocal microscopy (IVCM).

Methods

A prospective study was performed on 28 patients with MK, including acute bacterial, fungal, and Acanthamoeba keratitis, as well as on their contralateral clinically unaffected eyes and on control groups, which consisted of 28 age-matched normal controls and 15 control contact lens (CL) wearers. Laser IVCM with the Heidelberg Retinal Tomograph 3/Rostock Cornea Module and Cochet-Bonnet esthesiometry of the central cornea were performed. Two masked observers assessed central corneal dendritiform cell density and subbasal corneal nerve parameters.

Results

The contralateral clinically unaffected eyes of patients with MK demonstrated significant diminishment in nerve density (15,603.8 ± 1265.2 vs. 24,102.1 ± 735.6 μm/mm2), total number of nerves (11.9 ± 1.0 vs. 24.9 ± 1.2/frame), number of branches (1.7 ± 0.2 vs. 19.9 ± 1.3/frame), and branch nerve length (5775.2 ± 757.1 vs. 12,715.4 ± 648.4 μm/mm2) (P < 0.001 for all parameters) compared to normal controls and CL wearers. Further, dendritiform cell density in the contralateral unaffected eyes was significantly increased as compared to that in controls (117.5 ± 19.9 vs. 24.2 ± 3.5 cells/mm2, P < 0.001).

Conclusions

We demonstrate a subclinical involvement in the contralateral clinically unaffected eyes in patients with unilateral acute MK. In vivo confocal microscopy reveals not only a diminishment of the subbasal corneal nerves and sensation, but also an increase in dendritiform cell density in the contralateral unaffected eyes of MK patients. These findings show bilateral immune alterations in a clinically unilateral disease.

Purpose

To evaluate density and morphology of corneal epithelial immune dendritic cells (DCs) in different subtypes of dry eye disease (DED) using in vivo confocal microscopy (IVCM).

Methods

This retrospective study included 59 eyes of 37 patients with DED and 40 eyes of 20 age-matched healthy controls. Based on clinical tests, eyes with DED were categorized into two subtypes: aqueous-deficient (n = 35) and evaporative (n = 24). For all subjects, images of laser scanning in vivo confocal microscopy (IVCM) of the central cornea were analyzed for DC density and DC morphology (DC size, number of dendrites, and DC field). These DC parameters were compared among all dry eye and control groups.

Results

Compared with the controls, patients with DED had significantly higher DC density, larger DC size, higher number of dendrites, and larger DC field (all P < 0.001). Comparison between aqueous-deficient and evaporative subtypes demonstrated that DC density was significantly higher in aqueous-deficient subtype (189.8 ± 36.9 vs. 58.9 ± 9.4 cells/mm2, P = 0.001). However, there were no significant differences in morphologic parameters between DED subtypes. When aqueous-deficient DED with underlying systemic immune disease (Sjögren's syndrome and graft versus host disease) were compared with nonimmune conditions, the immunologic subgroup showed significantly higher DC density, DC size, and number of dendrites (all P < 0.05).

Conclusions

Corneal IVCM demonstrated differential changes in DC density and morphologic DC parameters between subtypes of DED. These changes, which reflect the degree of immune activation and inflammation in DED, can be used for clinical practice and endpoints in clinical trials.

Purpose To compare the effects of post-penetrating keratoplasty (PK) and post-keratoprosthesis (KPro) surgery-related inflammation on the posterior segment of the eye and to assess inhibition of tumor necrosis factor alpha (TNFα) and interleukin-1 beta (IL-1β) on these effects. Methods: BALB/C (syngeneic) or C57BL/6 (allogeneic) corneas were transplanted onto BALB/C host beds as part of PK or miniature KPro (m-KPro) implantation. Intraocular pressure (IOP) was measured via an intracameral pressure sensor; tissues were harvested and analyzed 8 weeks after surgery. Expression of TNFα and IL-1β in the retina was analyzed using real-time quantitative (q)PCR. Optic nerve degeneration (axon count, circularity, and area) was assessed quantitatively using ImageJ software. After m-KPro implantation, mice were treated with saline, anti-TNFα, or anti-IL-1β antibody, and axonal loss was assessed after 10 weeks. Results: Mean IOP was within normal limits in the operated and fellow eyes in all groups. The mRNA expression of TNFα and IL-1β was highest in m-KPro groups with either syngeneic or an allogeneic carrier. We observed optic nerve degeneration in both allogeneic PK and m-KPro implanted eyes with an allogeneic carrier. However, TNFα blockade significantly reduced axonal loss by 35%. Conclusions: Allogeneic PK and m-KPro implants with an allogeneic carrier lead to chronic inflammation in the posterior segment of the eye, resulting in optic nerve degeneration. In addition, blockade of TNFα prevents axonal degeneration in this preclinical model of allogeneic m-KPro (alloKPro) implantation.

The repair of wounds through collective movement of epithelial cells is a fundamental process in multicellular organisms. In stratified epithelia such as the cornea and skin, healing occurs in three steps that include a latent, migratory, and reconstruction phases. Several simple and inexpensive assays have been developed to study the biology of cell migration in vitro. However, these assays are mostly based on monolayer systems that fail to reproduce the differentiation processes associated to multilayered systems. Here, we describe a straightforward in vitro wound assay to evaluate the healing and restoration of barrier function in stratified human corneal epithelial cells. In this assay, circular punch injuries lead to the collective migration of the epithelium as coherent sheets. The closure of the wound was associated with the restoration of the transcellular barrier and the re-establishment of apical intercellular junctions. Altogether, this new model of wound healing provides an important research tool to study the mechanisms leading to barrier function in stratified epithelia and may facilitate the development of future therapeutic applications.