Minna Vesaluoma

In vivo confocal microscopy after herpes keratitis.

Purpose. To describe the confocal microscopic findings, with special reference to corneal subbasal nerves, after herpes simplex virus (HSV) keratitis. Methods In this study, 16 HSV eyes and 14 contralateral eyes of 16 patients, diagnosed with unilateral HSV keratitis 1–12 months earlier by the presence of dendritic corneal ulceration or microbiologic confirmation, were examined by in vivo confocal microscopy for evaluation of corneal morphology. Results. Herpes simplex virus eyes: In 2 eyes the surface epithelial cells appeared large, and no abnormalities were observed in the basal epithelial cells. In 2 eyes subbasal nerve fiber bundles were completely absent, in 3 eyes there was a reduced number of long nerve fiber bundles, and in 11 eyes the subbasal nerve plexus appeared normal. In 10 corneas, highly reflective dendritic structures were found at the level of the basal epithelial cells. Frequently these structures were found in the vicinity of stromal fibrosis. Areas with increased abnormal extracellular matrix were found in 11 eyes. Stromal nerves were not visualized in all corneas, but appeared normal when observed. Contralateral eyes: No abnormalities were observed in the epithelium. All corneas presented with a normal subbasal nerve plexus, but in 2 eyes dendritic particles were observed. Three corneas presented with activated keratocytes and increased amounts of abnormal extracellular matrix. Conclusions. When visualized by confocal microscopy, the subbasal nerve plexus appears relatively unaffected in cases with resolved HSV keratitis. Unidentified dendritic structures, presumably Langerhans cells, are frequently seen at the level of the basal epithelium in corneas with a history of herpetic disease.

In vivo confocal microscopy of patients with corneal recurrent erosion syndrome or epithelial basement membrane dystrophy

OBJECTIVE To characterize morphologic changes in corneas of patients with recurrent erosion syndrome or epithelial basement membrane dystrophy using in vivo confocal microscopy. DESIGN Observational case series PARTICIPANTS Fourteen eyes of eight patients with diagnosed epithelial basement membrane dystrophy and 13 eyes of seven patients with recurrent erosion syndrome were examined. METHODS Slit-lamp examination and in vivo confocal microscopy. The pathologic findings are presented as digitized images obtained from video tape recorded during the confocal microscopy. MAIN OUTCOME MEASURES The morphology of corneal surface epithelial cells, basal epithelial cells, subbasal nerve plexus, Bowmans layer, stromal keratocytes, and endothelium was analyzed. RESULTS The surface epithelium was intact in all but two eyes. One cornea (a basement membrane disorder with clinically visible dots) had multinucleate surface epithelial cells, and one eye with recurrent corneal erosions showed a freely floating surface epithelium sheet in the tear fluid. Patients in both groups showed islets of highly reflective cells with presumed intracellular deposits surrounded by normal cells in the basal epithelial cell layer. The basal epithelial cell area also showed other pathologic changes, including drop-shaped configurations, streaks, or ridges. Folding of the Bowmans layer was also observed in both groups. Anterior keratocytes showed signs of activation (highly reflective nuclei with visible processes) in some of the patients regardless of the clinical diagnosis, and in recurrent erosions even increased deposition of abnormal extracellular matrix in the anterior stroma was suspected. Posterior corneal keratocytes and endothelium appeared normal when examined. The subbasal nerve plexus showed various pathologic changes, such as short or strangely shaped nerve fiber bundles, decreased numbers of long nerve fiber bundles, only faintly visible long nerve fiber bundles (instead of the normally observed long parallel running interconnected bundles), or increased amounts of Langerhans cells, but only one patient (with recurrent erosion syndrome) lacked the subbasal nerve plexus. CONCLUSIONS In vivo confocal microscopy of corneas with recurrent erosions or epithelial basement membrane dystrophy showed deposits in basal epithelial cells, subbasal microfolds and streaks, damaged subbasal nerves, or altered morphology of the anterior stroma. Confocal microscopy cannot replace biomicroscopy in making a specific diagnosis, but it sometimes helps the diagnosis in corneas that appear normal under a biomicroscope.

Postoperative Inflammation, Microbial Complications, and Wound Healing Following Laser in situ Keratomileusis

Although the biology of corneal wound healing is only partly understood, healing after photorefractive keratectomy (PRK) and laser in situ keratomileusis (LASIK) differs in many respects, and the mechanisms appear to be differently controlled. There is less of an inflammatory and healing response after LASIK, but a longer period of sensory denervation. The cellular, molecular, and neural regulatory phenomena associated with postoperative inflammation and wound healing are likely to be involved in the adverse effects after LASIK, such as flap melt, epithelial ingrowth, and regression. Interface opacities in the early postoperative period include diffuse lamellar keratitis (DLK), microbial keratitis, epithelial cells, and interface opacities. Diffuse lamellar keratitis (sands of the Sahara syndrome) describes an apparently noninfectious diffuse interface inflammation after lamellar corneal surgery probably caused by an allergic or a toxic inflammatory reaction. Noninfectious keratitis must be distinguished from microbial keratitis to avoid aggressive management and treatment with antimicrobial drugs. Microbial keratitis is a serious complication after LASIK, but a good visual outcome can be achieved following prompt and appropriate treatment.

Laser in situ keratomileusis flap margin: wound healing and complications imaged by in vivo confocal microscopy

PURPOSE To examine the healing response of laser in situ keratomileusis flap margin in vivo. METHODS Forty-three eyes of 43 patients who had undergone myopic (n = 39) or hyperopic (n = 4) laser in situ keratomileusis were examined once after surgery. The flap margin was imaged by in vivo confocal microscopy at various depths, and the wound healing response, flap alignment, and complications were evaluated. Ten eyes were examined on day 3 postoperatively, 13 eyes at 1 to 2 weeks, 10 eyes at 1 to 2 months, five eyes at 3 months, and five eyes at 6 months or later. RESULTS At 3 days after laser in situ keratomileusis, the surface epithelium and basal epithelium appeared normal. Keratocyte activation was strongest at 1 to 2 weeks and 1 to 2 months, and an increased amount of haze was observed correspondingly. Intrastromal epithelial cells forming a plug could occasionally be perceived in the wound gape. Wound constriction was completed in most cases by 3 to 6 months or later. Good alignment was observed in 12 of 43 flaps (27.9%) and moderate and poor alignment in 17 of 43 flaps (39.5%) and 13 of 43 flaps (30.2%), respectively. Poor alignment was not associated with lamellar epithelial ingrowth. Epithelial ingrowth was associated with dense haze at the interface. Diffuse lamellar keratitis was imaged in two corneas after hyperopic laser in situ keratomileusis. CONCLUSIONS The laser in situ keratomileusis incision wound at the flap margin appears to heal after the sequence observed in incisional wounds in nonhuman primates. Complications, such as lamellar epithelial in growth and diffuse lamellar keratitis, were often observed, particularly after hyperopic laser in situ keratomileusis.

Corneal recovery after LASIK for high myopia A 2-year prospective confocal microscopic study

Aim: To quantify human corneal recovery after moderate to high myopic laser in situ keratomileusis (LASIK) in a 2-year prospective follow-up study. Methods: Fifteen eyes of 15 patients (mean refraction −10.1 (SD 2.4) D) were examined preoperatively and postoperatively at day 1, 5 days, 2 weeks, 1, 3 and 6 months and 2 years. Biomicroscopy, visual acuity and refraction were examined prior to imaging studies. An in vivo tandem scanning confocal microscope was used to obtain images from the central cornea. Subbasal nerve density was measured as the total length of nerve trunks in confocal image per mm2. Keratocyte density was calculated manually from stromal sublayers. The thickness of the altered keratocyte zone was measured on both sides of the LASIK interface. Results: At the end of the follow-up, all patients had a 20/20 BCVA, and nine of 15 patients were within ±0.5 D of the intended correction. The total corneal thickness remained unaltered, but epithelial hyperplasia was seen at 2 years. Keratocyte density in the anterior stroma and posterior to the flap interface showed a slight decrease during the follow-up. Subbasal nerve density decreased 82% in 5 days after LASIK. A gradual increase was observed from 2 weeks postoperatively, but even 2 years after the operation the nerve density was only 64% from the preoperative values. Conclusions: Subbasal nerve fibre density shows a gradual recovery throughout the follow-up. However, only three subjects showed totally regenerated subbasal nerve fibres at 2 years. This may correlate with the observed decrease in the density of the most anterior keratocytes. Corneal remodelling seemed to continue for at least 2 years.

Increased release of tumour necrosis factor-α in human tear fluid after excimer laser induced corneal wound

AIMS To measure the pre- and postoperative tear fluid tumour necrosis factor-α (TNF-α) concentration and release in patients undergoing excimer laser photorefractive keratectomy (PRK). METHODS Tear fluid samples from 18 PRK patients were collected with scaled microcapillary tubes preoperatively (day 0), on the second (day 2), and on the seventh (day 7) postoperative days. The TNF-α concentration was measured using a double antibody radioimmunoassay, and the TNF-α release was calculated by multiplying the concentration by the tear fluid flow in the collection capillary. RESULTS The mean tear fluid flow in the capillary was 22.5 μl/min (range 1.5–93.2) on day 0, 80.7 μl/min (3.0–219, p = 0.0002) on day 2, and 14.6 μl/min (1.8–41.7, NS) on day 7. The mean TNF-α concentration and release values were: day 0, 358 ng/l (110–680) and 9.5 pg/min (0.2–37.5, NS); day 2, 417 ng/l (< 5–750, NS) and 28.6 pg/min (0.6–81.5; p = 0.003); and day 7, 320 ng/l (< 5–735, NS) and 4.8 pg/min (0–25.4, NS), respectively. CONCLUSION TNF-α appears to be a component of normal tear fluid. In spite of hypersecretion caused by the corneal wound, TNF-α concentrations remain constant during wound healing. TNF-α release increases significantly during the 2 postoperative days following PRK, suggesting a role in corneal wound healing.

In vivo confocal microscopy of a family with Schnyder crystalline corneal dystrophy.

OBJECTIVE To analyze corneal morphology in Schnyder crystalline corneal dystrophy (SCCD) in vivo. DESIGN Observational case series. PARTICIPANTS Five eyes of four patients of various belonging to the same family were examined. METHODS The eyes were examined using in vivo confocal microscopy (CM). MAIN OUTCOME MEASURES The corneal morphology including keratocytes and stromal extracellular matrix, as well as basal epithelial/subepithelial nerves is, described. RESULTS The right eye of a 48-year-old male patient had been treated with anterior keratectomy and the left eye with phototherapeutic keratectomy (PTK). The right eye presented with increased stromal reflectivity owing to accumulation of extracellular matrix and large subepithelial crystalline deposits. Far fewer crystals could be observed in the left eye. The haze, however, was increased, either because of the dystrophy or the excimer laser treatment. The anterior keratocytes appeared irregular, and the subepithelial nerves were undetectable in both eyes. His 78-year-old mother showed more advanced changes with dense crystals, highly fibrotic stroma, and severely damaged corneal innervation. The partly irregular anterior keratocytes of the 9- and 7-year-old children contained intracellular deposits, although the corneas were clinically clear with only subtle subepithelial crystalline formation. Accumulation of similar reflective material was also observed in association with the prominent subepithelial nerves. CONCLUSIONS In the early stages of SCCD, highly reflective deposits accumulate intracellularly and around anterior keratocytes and along subepithelial nerves. With time, the normal corneal architecture becomes disturbed by large extracellular crystalline deposits and accumulation of highly reflective extracellular matrix resulting in central opacity and disruption of the subepithelial nerve plexus. Furthermore, neural regeneration after keratectomy appears delayed in SCCD.

The effects of experimental tear film removal on corneal surface regularity and barrier function

PURPOSE To evaluate corneal surface regularity and asymmetry, corneal thickness, barrier function, and contrast sensitivity after experimental removal of the precorneal tear layer. DESIGN Prospective, clinic-based, nonrandomized (self-controlled) comparative trial. PARTICIPANTS Six eyes of six healthy volunteers (three males, three females; age range, 29-40 years). METHODS A precorneal tear lesion was created by pressing a sterile Biopore (Millipore, Bedford, MA) Teflon membrane against the central cornea. Corneal topography with both the Topographic Modeling System (TMS-1; Computed Anatomy, Tomey Technology, Cambridge, MA) and the Orbscan (Orbscan Inc., Salt Lake City, UT) were performed before the lesion was created and 30 seconds, 1 hour, and 4 hours after the lesion was created. Surface regularity and surface asymmetry indices were evaluated by the TMS-1 topography system. Maximum and minimum keratometric readings, corneal fluorescein staining, contrast sensitivity, and corneal thickness were evaluated before and after the tear lesion. Cytologic membranes were stained for MUC4 mucin using an indirect immunofluorescent staining technique. Confocal microscopy was performed to evaluate the integrity of the corneal epithelium in two eyes. Analysis of variance with polynomial contrasts was used to examine time trends of the outcome variables. MAIN OUTCOME MEASURES The change in corneal surface regularity and asymmetry indices, corneal thickness, permeability to fluorescein dye, and contrast sensitivity before and after the lesion was made were compared. RESULTS The corneal epithelium in the area of the lesion showed intense fluorescein staining 30 seconds postlesion but appeared normal by 4 hours. Confluent, homogeneous staining for MUC4 mucin was observed on the membranes used to create the lesion in all cases. The surface regularity index measured with the TMS-1 increased after the lesion was created and decreased toward normal by 4 hours (P = 0.017). Corneal thickness measured by the Orbscan instrument significantly increased in the central (P = 0.001), superior (P = 0.006), inferotemporal (P < 0.001) and superotemporal (P = 0.001) cornea immediately following the lesion and returned to normal by 4 hours. The lesion caused a decrease in visual acuity at 6.30%, 4% and 2.5% contrast sensitivities 1 hour postlesion and these measurements returned to prelesion values by 4 hours (P = 0.085, P = 0.005, P = 0.043). CONCLUSIONS The precorneal tear layer serves as a permeability barrier and is essential for maintaining a smooth quality optical surface.

A novel non-invasive, in vivo technique for the quantification of leukocyte rolling and extravasation at sites of inflammation in human patients

A novel non-invasive, in vivo technique for the quantification of leukocyte rolling and extravasation at sites of inflammation in human patients

Tenascin and cytokines in tear fluid after photorefractive keratectomy.

BACKGROUND Basic knowledge of the substances involved in wound healing after photorefractive keratectomy (PRK) is essential for development of pharmacological intervention. We present preoperative and postoperative analysis of tear fluid extracellular matrix proteins and cytokines after PRK. METHODS Tear fluid samples from 70 patients (72 eyes) who had PRK (38 women and 32 men, mean age 31.5 yr) were studied. Samples from 18 patients (18 eyes) were analyzed in two different studies. RESULTS Mean preoperative tear fluid flow in the collection capillary (volume divided by tear collection time) varied from 4.5 to 22.5 microliters/min in five series of patients. It increased significantly during the first two postoperative days (range of means, 55.5 to 88.8 microliters/min, p < 0.01), and decreased to the preoperative level by day 7 (range of means, 9.7 to 18.2 microliters/min). The tenascin and cytokine release rates increased significantly during the first two days after PRK and returned to the preoperative level by day 7. Mean +/- standard error for tenascin: day 0 (5.2 +/- 1.9 ng/min); day 2 (22.7 +/- 6.1 ng/min; p = 0.02). Mean +/- standard error for HGF: day 0 (3.2 +/- 0.7 pg/min); day 1 (22.8 +/- 4.2 pg/min; p = 0.0003). Mean +/- standard error for TGF-beta 1: day 0 (63.3 +/- 19.6 pg/min); days 1-2 (826.2 +/- 253.7 pg/min; p = 0.001). Mean +/- standard error for VEGF: day 0 (166.0 +/- 29.6 pg/min); days 1-2 (824.4 +/- 165.1 pg/min; p = 0.0007). Mean +/- standard error for PDGF-BB: day 0 (0.42 +/- 0.19 pg/min); day 2 (27.6 +/- 5.8 pg/min; p = 0.0000). Mean +/- standard error for TNF-alpha: day 0 (9.5 +/- 2.6 pg/min); day 2 (28.6 +/- 5.9 pg/min; p = 0.003). Excluding PDGF-BB, all substances studied were present in normal human tear fluid. PDGF-BB was present in only 17% of the preoperative samples. CONCLUSION Corneal wounding induces an increased release of several growth modulating cytokines which may be involved in healing processes.