Person:

Baniasadi, Neda

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

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.