Ahmad Muneer Otri

Corneal densitometry as an indicator of corneal health.

PURPOSE To establish prospectively the normal values of corneal density of healthy subjects using the Pentacam Scheimpflug system (Oculus, Inc., Wetzlar, Germany) and to investigate alteration in corneal density during active and healed stages of bacterial keratitis. DESIGN Prospective, comparative case series. PARTICIPANTS AND CONTROLS Sixty-four eyes of 40 healthy controls and 36 eyes of 35 patients with bacterial keratitis were studied. METHODS This study was conducted at the Queens Medical Centre, Nottingham, United Kingdom. A Pentacam system was used to study corneal density. Corneal densitometry readings in subjects with bacterial keratitis were recorded during the active stage and 4 to 6 weeks after complete healing. Densitometry was recorded at the site of infection and at a point in clear cornea furthest away from the infectious infiltrate. Corneal thickness also was measured. MAIN OUTCOME MEASURES Densitometry values of normal cornea, at the site of corneal ulcer or abscess, and at a distant point of clear cornea during active and healed keratitis. RESULTS The mean densitometry value of normal corneas was 12.3 ± 2.4. In infectious keratitis, the densitometry values were greatest at the site of the active infection and significantly more than in controls. The densitometry values at the points of clear cornea furthest away from the site of infection also were significantly higher than in controls during active disease, but failed to return to normal values, despite complete resolution of infection. The density of the infiltrates was much higher than that of residual scars after healing of ulcers. No correlation was found between the pachymetry and the densitometry values. CONCLUSIONS Densitometry of active infectious corneal infiltrates is more than that resulting from the corneal scarring after healing. Persistent increase in density of clear cornea furthest away from the focus of corneal infection suggests that the host response extends beyond the immediate area of infection and indeed may occur through the entire cornea. These changes persist beyond 4 weeks of healing, which was the duration of follow-up of this study. Densitometry can be used as an objective measure of the corneal response to infection and to monitor response to therapy.

In vivo confocal microscopic features of normal limbus

Aim To describe in vivo confocal microscopy (IVCM) features of the limbus in normal eyes as related to the palisades of Vogts. Methods 46 eyes of 29 consecutive volunteers were recruited in this observational study. A detailed examination by IVCM was performed in addition to a routine slit-lamp biomicroscopy. Size and density of corneal and limbal epithelial cells were measured and statistically analysed using SPSS version 8.0 software. Results Anatomical and morphological features were noted between corneal and limbal cells. Size and density differences reached to significant levels (p<0.05). Different shapes of palisades of Vogt have been described clearly by confocal microscope. Cell-like structures were observed in the peripheral end of the palisades which might represent limbal stem cell crypts. Conclusions Laser IVCM can be used to establish the features of the normal limbus. The identified features demonstrate quantitative changes in the basal epithelium between the limbus and the central cornea and morphological differences between pigmented or non-pigmented studied subjects. Further studies should be performed to correlate with histology the possible crypts which were observed in this study.

Ex vivo confocal microscopy of human corneal nerves.

Aims To evaluate the distribution, morphometry and the postmortem changes of the central and peripheral human corneal nerves by exvivo laser-scanning confocal microscopy (EVCM). Methods 24 eyes from 14 cadavers were retrieved at different time intervals after death and examined by EVCM. Five regions were examined in each eye: central, superior, inferior, temporal and nasal. In each region, corneal nerve images were categorised according to their anatomical location in the cornea into sub-basal, stromal and limbal nerves. Five nerve parameters were measured: density, orientation, diameter, numbers and branching pattern. Results Exvivo confocal scanning of a motionless eye allows high quality imaging and tracking of corneal and limbal nerves. Stromal nerves from the sub-Bowmans plexus perforate the Bowmans zone and terminate in bulb-like structures, from each of which a leash of sub-basal nerves arises. Following death, sub-basal nerve parameters showed significant changes. The density decreased from 9.23±4.48 to 0.45±0.07 mm/mm2, the diameter from 4.01±0.81 to 2.08±0.20 μm, the numbers from 8.3 to 1.0 and branching pattern from 39.38% to 0% (p<0.05) from day 1 to day 5 postmortem. Stromal and limbal nerves showed no significant changes in their density and diameter. Conclusions This study establishes a direct link between sub-basal nerves and the sub-Bowmans nerves via distinct terminal bulbs. Limbal nerves are the thickest, are seen in all quadrants and can be traced to the corneal centre. The sub-basal nerve plexus rapidly degenerates after death but stromal and limbal nerves survive during the first five days after death.

The effect of standard and transepithelial ultraviolet collagen cross-linking on human corneal nerves: an ex vivo study.

PURPOSE To evaluate the early effect of standard and transepithelial collagen cross-linking on human corneal nerves in donor eyes by ex vivo confocal microscopy and acetylcholinesterase staining. DESIGN Experimental laboratory investigation. METHODS Eight human eye bank corneal buttons (mean age, 73.6 years) were included. Ultraviolet A collagen cross-linking was performed postmortem on 3 corneas with the standard protocol involving epithelial debridement and 4 corneas by the transepithelial approach. One cornea served as a control. Corneal nerves were evaluated using confocal microscopy and acetylcholinesterase histology. RESULTS Confocal microscopy demonstrated the absence of subbasal nerves in corneas treated by the standard technique. These nerves were preserved in corneas treated by the transepithelial approach. Stromal nerves were visible in both groups. Histology of corneas treated by the standard technique revealed localized swellings of the stromal nerves with disruption of axonal membrane and loss of axonal continuity within the treatment zone. These changes were absent in corneas treated by the transepithelial approach. CONCLUSIONS This study highlights the immediate effects of collagen cross-linking on the corneal nerves in an ex vivo model. The absence of subbasal nerves in the early phase of treatment appears to be attributable mainly to mechanical removal of epithelium, rather than ultraviolet light-induced damage. Localized swelling of the stromal nerves was the main difference between the 2 treatment protocols. Further research on laboratory animals would be necessary to verify these changes over a specified time course without the super-addition of postmortem changes.

Correlation of central and peripheral corneal thickness in healthy corneas

PURPOSE To study the thickness profile of the normal cornea in order to establish any correlation between central and peripheral points. METHODS Sixty-seven eyes of 40 patients were subjected to central corneal thickness measurement (CCT) with an ultrasound pachymeter (UP) and corneal thickness mapping with the Oculus Pentacam. The corneal apex thickness (CAT), pupil centre thickness (recorded as CCT and corresponded to CCT of UP) and thickness at the thinnest location (CTL) were obtained and compared with each other. Corneal thickness data at 3 mm and 7 mm temporally, nasally, superiorly and inferiorly from the corneal apex were obtained. The mean corneal thickness values along the 2, 4, 6, 8 and 10 mm diameter concentric circles, with the CTL as the centre, were also obtained. The above data at different points were statistically correlated. RESULTS There was no significant difference between CCT readings measured by UP and Pentacam (P=0.721). There was high positive correlation between the CAT values and the thickness at 3 mm (R≥0.845, P<0.001) and at 7 mm points (R≥0.654, P<0.001). A gradual increase in thickness was noted from the centre to the periphery with a high positive correlation between the CTL values and the mean thickness at the circles of 2, 4, 6, 8 and 10 mm (R≥0.635, P<0.001). CONCLUSION The results suggest that central corneal thickness can serve as a good guide for predicting peripheral thickness. For surgical procedures specifically undertaken at mid-peripheral and peripheral zones, the actual measurements at the site of surgery may confer some advantage.

The morphologic characteristics of corneal nerves in advanced keratoconus as evaluated by acetylcholinesterase technique.

PURPOSE To study the morphologic characteristics of corneal nerves in patients with advanced keratoconus using the acetylcholinesterase technique in corneal whole mounts. DESIGN Prospective, observational case series. METHODS Fourteen corneal buttons from 14 keratoconic patients (9 males and 5 females; mean age, 34.3 years) who had undergone keratoplasty for advanced keratoconus and 6 corneal buttons from 6 normal corneas were included. Whole mounts were stained for acetylcholinesterase and were scanned with a novel digital pathology scanning microscope. RESULTS Seventy-one percent of keratoconic corneas demonstrated central stromal nerve changes, which included thickening, tortuosity, nerve spouting, and overgrowth. The nerve changes ranged from early to extensive and could be separated into 3 different grades. The central stromal nerves were abnormally thicker (18.9 ± 14.7 μm) than in controls (8.11 ± 3.31 μm; P < .001). The thickness of peripheral stromal nerves (12.6 ± 3.1 μm) was similar to that of controls (14.86 ± 5.60 μm; P = .072). Subbasal nerves showed changes in the form of loss of radial orientation and increased tortuosity, especially at the cone apex. At the cone base, a concentric arrangement of subbasal nerves was found in 43% of cases. Localized thickenings of subbasal nerves also were observed at their origin from the bulbous terminations of sub-Bowman nerves. The terminal bulbs, too, were enlarged. The mean diameter of the subbasal nerves in keratoconus (4.11 ± 0.60 μm) did not differ from that of the controls (4.0 ± 0.61 μm; P = .422). CONCLUSIONS This study provides additional histologic evidence of the involvement of corneal nerves in keratoconus and suggests further that they may play a role in the pathophysiologic factors and progression of the disease.

Localization and Gene Expression of Human β-Defensin 9 at the Human Ocular Surface Epithelium

PURPOSE Antimicrobial peptides (AMPs) are multifunctional host defense molecules. Human beta-defensin 9 (HBD9) has previously been shown to be downregulated during ocular surface (OS) infection or inflammation. Here, the authors aimed to study localization of HBD9 protein in different OS regions and to determine the role of Toll-like receptors (TLRs), nucleotide oligomerization domain (NOD)-like receptors, and proinflammatory cytokines in HBD9 expression. METHODS Immunolocalization of HBD9 protein was carried out on the normal human OS regions (cornea, limbus, and conjunctiva). Quantitative PCR analysis of HBD9 mRNA was performed in SV40-transformed human corneal epithelial cells (hCECs) treated for different durations with synthetic pathogen-associated molecular patterns (PAMPs) and recombinant cytokines. RESULTS HBD9 protein was constitutively expressed on OS epithelia. Corneal and limbal epithelia and corneal stroma demonstrated modest levels of HBD9, whereas conjunctival epithelium demonstrated high levels of HBD9 protein. TLR02, TLR03, TLR04, and TLR05 were shown to modulate HBD9 mRNA in hCECs. Similarly, NOD2 and IL-1beta were also shown to alter HBD9 in a time-dependent manner. In response to infection-related PAMPs and inflammatory cytokines, an initial increase in HBD9 mRNA levels was observed, followed by a significant downregulation. CONCLUSIONS This is the first demonstration of HBD9 protein expression at different OS regions. The authors also determined the role of various innate immune receptors in HBD9 mRNA modulation. Further understanding of the signaling mechanisms involved in the initial response of HBD9 to infection or inflammation is likely to indicate future therapeutic directions with this AMP.

Profile of sight-threatening infectious keratitis: a prospective study.

Purpose:  To prospectively study patients presenting with sight‐threatening corneal ulcers with a view to identify the predisposing factors, causative organisms, clinical signs and treatment outcomes.

Organization of the Regenerated Nerves in Human Corneal Grafts

PURPOSE To examine by histopathology the degree of nerve regeneration in human corneal grafts and to determine the anatomic organization and morphology of the regenerated nerves. DESIGN Experimental laboratory investigation. METHODS Twelve corneal grafts from 12 patients (7 men and 5 women) aged 34-93 (mean, 66.9 years) were included. The most common indication for regrafting was late endothelial failure. The mean duration of graft survival was 6.41 years (range, 1-14 years). The freshly obtained specimens with a narrow rim of host tissue incorporating the graft-host junction were subjected to the acetylcholinesterase method for the demonstration of corneal nerves. RESULTS Subbasal nerves were found in 75% and 25% of the grafts at the periphery and center, respectively. They were mostly originated from the host subbasal nerves. Regenerated stromal nerves were detected in 83% of the specimens; half of them showed extension into the center of the graft. A lack of the normal link between the subbasal and stromal nerves was observed and almost all of the regenerated stromal nerves were found to remain within the stroma and did not contribute to the epithelial innervation. CONCLUSIONS A persistent anatomic disorganization of the corneal nerves in human grafts was found even 14 years after surgery. This could explain the significant reduction of corneal sensation reported in previous studies.

Variable Expression of Human beta Defensins 3 and 9 at the Human Ocular Surface in Infectious Keratitis

PURPOSE The authors have previously reported the presence of the antimicrobial peptides human beta defensin (hBD) 3 and hBD9 on the ocular surface (OS). These play an important role in infection and inflammation. In the present study, the authors studied the gene expression levels of hBD3 and hBD9 in healthy subjects and during and after healing of infectious keratitis. METHODS Human OS specimens were obtained by impression cytology from healthy controls and patients with Acanthamoeba and Gram-negative and -positive bacterial keratitis (BK), both during active infection and after healing. The gene expression levels of hBD3 and hBD9 were determined using quantitative real-time polymerase chain reaction (RT-PCR). RESULTS hBD3 and hBD9 were constitutively expressed in all healthy controls. During acute Acanthamoeba keratitis (AK), hBD3 levels were markedly increased and then returned close to normal levels after healing. In BK, hBD3 gene expression was moderately increased and then decreased after healing. In contrast to hBD3, hBD9 was significantly downregulated in both AK and Gram-positive BK, whereas it showed an insignificant decrease in Gram-negative BK. After healing, the expression showed upregulation except in Gram-positive BK, where it continued to decline. CONCLUSIONS This is the first study that demonstrates the gene expression of hBD3 and hBD9 in response to infection. It illustrates that not all antimicrobial peptides (AMPs) behave in a similar manner. Some are upregulated and some are downregulated, suggesting a diverse role of AMP in infection and inflammation. The results point to a role of AMP-mediated host defense in Acanthamoeba keratitis as well.