In vivo confocal microscopy of equine fungal keratitis

Objective To describe in vivo corneal confocal microscopy of horses with fungal keratitis and correlate findings with clinical, histopathological, and microbiological evaluations of clinical cases and an ex vivo experimental equine fungal keratitis model. Animals studied A total of 12 horses with naturally‐acquired fungal keratitis and ex vivo equine corneas experimentally infected with clinical fungal isolates. Procedures Horses with naturally‐acquired fungal keratitis were examined with a modified Heidelberg Retina Tomograph II and Rostock Cornea Module. Confocal microscopy images of clinical isolates of Aspergillus fumigatus, Fusarium solani, and Candida albicans were obtained by examination of in vitro cultures and experimentally infected ex vivo equine corneas. Results Non‐specific in vivo corneal confocal microscopic findings in horses with fungal keratitis included leukocyte infiltrates, activated keratocytes, anterior stromal dendritic cell infiltrates, and vascularization. Linear, branching, hyper‐reflective structures that were 2–6 μm in width and 200 to >400 μm in length were detected in all horses with filamentous fungal keratitis. Round to oval hyper‐reflective structures that were 2–8 μm in diameter were detected in a horse with yeast fungal keratitis. The in vivo confocal microscopic appearance of the organisms was consistent with fungal morphologies observed during examination of in vitro cultures and infected ex vivo equine corneas. Conclusions In vivo corneal confocal microscopy is a rapid and non‐invasive method of diagnosing fungal keratitis in the horse. This imaging technique is useful for both ulcerative and non‐ulcerative fungal keratitis, and is particularly advantageous for confirming the presence of fungi in deep corneal stromal lesions.     

In vivo confocal microscopy in the normal corneas of cats, dogs and birds

OBJECTIVE: To evaluate the applicability of in vivo confocal microscopy (IVCM) in veterinary ophthalmology and analyze the morphology of living, healthy cornea. ANIMALS EXAMINED: Thirty-seven dogs, 34 cats and five birds. PROCEDURE: Various corneal sublayers were visualized in the central region using an in vivo confocal corneal microscope (HRTII/RCM). RESULTS: An investigation method was developed and adapted for use on animals with varying skull forms and eye positions. Real-time images of the epithelial cells, the corneal stroma and the endothelial layer were obtained. The corneal stromal nerve trunks and the subepithelial and basal epithelial nerve plexus were visualized. In dogs, full corneal thickness (FCT) was 585 +/- 79 microm (mean +/- SD) and endothelial cell density (ECD) 3175 +/- 776 cells/mm(2) (mean +/- SD). In cats, FCT was 592 +/- 80 microm and ECD 2846 +/- 403 cells/mm(2). There were no significant differences between canine and feline FCT and ECD and no morphologic differences could be seen between dogs and cats. The bird images revealed a number of structural differences. CONCLUSION: Noninvasive IVCM allows accurate detection of corneal sublayers, corneal pachymetry, endothelial cell density and corneal innervation in various animal species. For clinical usage, patients must be under general anesthesia. The confocal images provided anatomic reference images of various healthy corneal structures in dogs, cats and birds.     

In vivo confocal microscopy of the normal equine cornea and limbus

Objective  To describe morphologic features, pachymetry and endothelial cell density of the normal equine cornea and limbus by in vivo confocal microscopy.
Animals studied  Ten horses without ocular disease.
Procedure  The central and peripheral corneas were examined with a modified Heidelberg Retina Tomograph II and Rostock Cornea Module using a combination of automated and manual image acquisition modes. Thickness measurements of various corneal layers were performed and endothelial cell density determined.
Results  Images of the constituent cellular and noncellular elements of the corneal epithelium, stroma, endothelium, and limbus were acquired in all horses. Corneal stromal nerves, the subepithelial nerve plexus, and the sub-basal nerve plexus were visualized. Cells with an appearance characteristic of Langerhans cells and corneal stromal dendritic cells were consistently detected in the corneal basal epithelium and anterior stroma, respectively. Median central total corneal thickness was 835 μm (range 725–920 μm) and median central corneal epithelial thickness was 131 μm (range 115–141 μm). Median central endothelial cell density was 3002 cells per mm² (range 2473–3581 cells per mm²).
Conclusions  In vivo corneal confocal microscopy provides a noninvasive method of assessing normal equine corneal structure at the cellular level and is a precise technique for corneal sublayer pachymetry and cell density measurements. A resident population of presumed Langerhans cells and corneal stromal dendritic cells was detected in the normal equine cornea. The described techniques can be applied to diagnostic evaluation of corneal alternations associated with disease and have broad clinical and research applications in the horse.     

Corneal innervation in mesocephalic and brachycephalic dogs and cats: assessment using in vivo confocal microscopy

Objective To determine the density of the canine and feline corneal neural network in healthy dogs and cats using in vivo confocal microscopy (IVCM). Animals examined A total of 16 adult dogs (9 Mesocephalic breeds, 7 Brachycephalic breeds) and 15 cats (9 Domestic Short-haired cats (DSH), 6 Persian cats) underwent IVCM. Procedure Animals were examined with a confocal corneal microscope (HRTII/RCM; Heidelberg Retina Tomograph II/Rostock Cornea Module^®, Heidelberg Engineering, Dossenheim, Germany). The investigations focused on the distribution of the corneal nerves and quantification of central subepithelial and subbasal nerve plexus. Results The corneal stromal nerve trunks, subepithelial and subbasal nerve plexus were observed. The nerve fiber density (NFD) quantified in nerve fiber length in mesocephalic dogs were 12.39 ± 5.25 mm/mm² in the subepithelial nerve plexus and 14.87 ± 3.08 mm/mm² in the subbasal nerve plexus. The NFD of the subepithelial nerve plexus in DSH cats was 15.49 ± 2.7 and 18.4 ± 3.84 mm/mm² in the subbasal nerve plexus. The subbasal NFD of DSH cats was significantly higher than in mesocephalic dogs (P = 0.037). The subepithelial NFD in brachycephalic dogs, and Persian cats were 10.34 ± 4.71 and 9.50 ± 2.3 mm/mm², respectively. The subbasal NFD measured 11.80 ± 3.73 mm/mm² in brachycephalic dogs, and 12.28 ± 4.3 mm/mm² NFD in Persian cats, respectively. The subepithelial and subbasal NFD in Persian cats were significantly lower than in DSH cats (P = 0.028, respectively, P = 0.031), in contrast to brachycephalic vs. mesocephalic dogs. Conclusion The noninvasive IVCM accurately detects corneal innervation and provides a reliable quantification of central corneal nerves.     

Use of specular microscopy to determine corneal endothelial cell morphology and morphometry in enucleated cat eyes

The purpose of this study was to investigate the effect of age on endothelial morphology and morphometry in cats. The corneal endothelium was studied using a contact specular microscope. A total of 18 cats (Felis catus Linnaeus, 1758) were evaluated in this study. The subjects were divided into three groups of six cats each in function of age: G1 (1 to 3 months old), G2 (5 to 12 months old), and G3 (24 to 40 months old). The examination presented data as endothelial cell density (ECD), average cell area, corneal thickness, polymegathism, and pleomorphism. Results revealed ECD decrease in corneas of normal cats with age, as well as a corresponding increase in endothelial cell area and pleomorphism. The present work suggests that the endothelial parameters evaluated change with advancing age.     

In vivo measurement of central corneal thickness in normal chicks (Gallus gallus domesticus) with the optical low-coherence reflectometer

Objective To determine the practicability and accuracy of central corneal thickness (CCT) measurements in living chicks utilizing a noncontact, high‐speed optical low‐coherence reflectometer (OLCR) mounted on a slit lamp. Animals studied Twelve male chicks (Gallus gallus domesticus). Procedures Measurements of CCT were obtained in triplicate in 24 eyes of twelve 1‐day‐old anaesthetized chicks using OLCR. Every single measurement taken by OLCR consisted of the average result of 20 scans obtained within seconds. Additionally, corneal thickness was determined histologically after immersion fixation in Karnovsky’s solution alone (20 eyes) or with a previous injection of the fixative into the anterior chamber before enucleation (4 eyes). Results Central corneal thickness measurements using OLCR in 1‐day‐old living chicks provide a rapid and feasible examination technique. Mean CCT measured with OLCR (189.7 ± 3.34 μm) was significantly lower than histological measurements (242.1 ± 47.27 μm) in eyes with fixation in Karnovsky’s solution (P = 0.0005). In eyes with additional injection of Karnovsky’s fixative into the anterior chamber, mean histologically determined CCT was 195.2 ± 8.25 μm vs. 191.9 ± 8.90 μm with OLCR. A trend for a lower variance was found compared to the eyes that had only been immersion fixed. Conclusion Optical low‐coherence reflectometry is an accurate examination technique to measure in vivo CCT in the eye of newborn chicks. The knowledge of the thickness of the chick cornea and the ability to obtain noninvasive, noncontact measurements of CCT in the living animal may be of interest for research and development of eye diseases in chick models.     

Parachlamydia acanthamoebae in domestic cats with and without corneal disease

Corneal samples of cats with and without corneal diseases were screened with a pan‐Chlamydiales PCR and specific PCRs for Parachlamydia, Protochlamydia, Chlamydophila felis, Acanthamoeba and feline herpesviruses (FHV‐1). Several corneal samples tested positive for Parachlamydia and related Chlamydiales, indicating cat exposure to these intracellular bacteria.