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Takefumi Yamaguchi, Bernardo M. The density of DCs was significantly increased in affected Purchase this article with an account. Jump To

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Takefumi Yamaguchi, Bernardo M. The density of DCs was significantly increased in affected Purchase this article with an account. Jump To Takefumi Yamaguchi ; Bernardo M. Alerts User Alerts. You will receive an email whenever this article is corrected, updated, or cited in the literature.

You can manage this and all other alerts in My Account. This feature is available to authenticated users only. Get Citation Citation. Get Permissions. Bacterial keratitis BK is a potentially blinding ocular condition of the cornea, which can result in severe loss of vision due to corneal scarring, corneal perforation, or endothalmitis.

Recent studies have suggested that proinflammatory cytokines in tears may have a key role in the pathogenesis of several corneal diseases, including dry eye disease, 19 keratoconus, 20 , 21 graft-versus-host disease GVHD , 22 conjunctivitis, 23 as well as in the development of corneal neovascularization.

Furthermore, previous studies have assessed tear cytokine changes in either affected eyes 21 , 24 or in bilateral diseases, 22 — 25 but to our knowledge contralateral changes in tear cytokines in unilateral diseases have not been assessed before. Finally, although previous reports suggested that tear cytokine levels correlated with clinical findings, such as keratorefractive values in keratoconus 20 or with clinical severity score in dry eye patients, 25 , 26 it is unclear if intracorneal cellular changes in DCs and corneal nerves as shown by IVCM correspond with changes in tear cytokines.

We hypothesized that alterations in corneal DCs and corneal nerves by IVCM would correlate with changes in proinflammatory tear cytokines, and that unilateral BK would alter bilateral tear cytokine levels. Thus, in the current study, we aimed to quantify bilateral tear cytokines levels, and correlate them with alterations in corneal nerves and DCs as detected by IVCM in unilateral BK.

A prospective, single-center study was conducted in a masked fashion. A total of 10 patients with a diagnosis of acute unilateral BK was included in this study and compared to 14 normal age- and sex-matched healthy control subjects. We collected 54 tear samples from the affected and contralateral clinically unaffected eyes of BK patients as well as from both eyes of healthy control subjects.

In three BK patients, tear collection and IVCM were performed twice Patients 8—10 to analyze changes over a 1- to 2-week time period after the initial visit. In all 3 patients, the infiltration and corneal epithelial defect improved after the treatment was started. All patients and healthy control subjects underwent slit-lamp biomicroscopy. Patients with other causative organism, such as viral, fungal, and Acanthamaoeba keratitis, were excluded. None of the healthy controls had trauma, surgery, contact lens use, or other ocular surface diseases, such as dry eye disease or conjunctivitis.

None of the BK patients or healthy control subjects had any systemic immunological diseases or diabetes. All patients provided written informed consent after a detailed explanation of the nature of the study. Tear collection was conducted before the IVCM examination and instillation of any eye drops as described previously. Next, subjects were asked to look left, right, up, and down, four times without blinking, to mix the tear fluid content.

Then, the diluted tears with sterile saline were collected from the inferior fornix using the micropipette and transferred to an Eppendorf tube. This process was repeated for the contralateral eye and for both eyes of control subjects, always using a new micropipette sterile tip for each eye.

The technique used was the same as reported previously. After the instillation of topical anesthesia of 0. Further, hydroxypropyl gel was added to the outside tip of the TOMO-cap to improve optical coupling and the lens was advanced until the cap made contact with the surface of the cornea. Images were obtained from the epithelial layer to the endothelial layer using multiple scans in the sequence mode.

When a corneal ulcer was present with an epithelial defect or severe ulceration in eyes with BK, the ulcer and periphery of the ulcer were scanned. If the images of the ulcer were not suitable due to severe edema or opacity, the periphery of the ulcer Fig.

A minimum of 3 representative images of the sub-basal nerve plexus and epithelial DCs were selected for analysis for each eye. The images were selected from the layer immediately at or posterior to the basal epithelial layer and anterior to the Bowman's layer. The criteria to select the images were the best-focused and complete images, with the whole image in the same layer, without motion, without folds, and good contrast.

Sub-basal nerve fibers and DC density and morphology were analyzed as described previously. Nerve density was assessed by measuring the total length of the nerve fibers in micrometers per frame of 0. Number of nerves was defined as the number of total nerve fibers per frame of 0.

The DCs were defined morphologically as bright dendritiform, well-demarcated structures, predominantly found in the sub-basal area. The DC density was calculated by dividing the total number of DCs per each frame of 0. Figure 1. View Original Download Slide. Slit-lamp photographs and in vivo confocal microscopy images.

The representative slit-lamp photographs of a normal control eye A , a clinically affected eye with unilateral BK B , C , and its contralateral clinically unaffected eye D. Figure 1 Slit-lamp photographs and in vivo confocal microscopy images. Plates were washed with washing buffer by centrifugation, and Phycoerythrin-labeled streptavidin was added for 15 minutes of incubation in the dark at room temperature.

We used the STarStation v2. Data were analyzed using Prism for Windows version 6. To compare the sub-basal nerve density; DC density and morphology; and cytokine levels between control eyes, affected BK eyes, and contralateral unaffected eyes; 1-way ANOVA with Tukey's multiple comparisons post-test was performed.

Spearman's correlation coefficient analysis was used to evaluate the correlation among sub-basal nerve density, DC parameters, and cytokine levels. For each test, differences were considered statistically significant at a P value of less than 0. We collected 26 tear samples from 10 patients with BK and 28 tear samples from 14 healthy control subjects.

Figure 1 shows the slit-lamp photographs and IVCM images of the representative cases of each group. Demographic data of BK and the healthy control group are summarized in Table 1.

Clinical data of BK patients are presented in Table 2. The causative organisms were identified using culture examination in 5 of 10 patients. Five other patients were diagnosed with BK by their history and clinical findings, as well as their rapid response to the antibacterial therapy. Table 1 View Table. Demographics of Patients and Control Subjects. Table 1 Demographics of Patients and Control Subjects.

Table 2 View Table. Quantitative analysis of affected and unaffected eyes of BK patients and controls are shown in Figure 2. Patients with unilateral BK showed significant reduction in the sub-basal nerve plexus density and total number of nerve fibers in both eyes, when compared to controls. The total number of nerve fibers was reduced to 2. The DC density was increased to The DC area was increased to Figure 2.

Corneal sub-basal nerves and dendritiform cells. A P value of less than 0. Graph bars : represent average SE. Figure 2 Corneal sub-basal nerves and dendritiform cells.

The detailed tear cytokine concentrations measured by the multiplex assay are shown in Figure 3. Figure 3. Tear cytokine levels.

Error bars : represent the mean value for each group. Figure 3 Tear cytokine levels. Figure 4. Correlation between tear cytokine levels and DC density. Figure 4 Correlation between tear cytokine levels and DC density. Figure 5. Correlation between cytokine levels and DC morphology.

Figure 5 Correlation between cytokine levels and DC morphology. Figure 6. Correlation between nerve density and cytokine levels. Figure 6 Correlation between nerve density and cytokine levels. In all these 3 patients, the BK improved clinically after the antibiotic treatment started. The ILa level in the contralateral eyes were elevated at visit 1 and decreased as the BK improved.

Figure 7. Alterations of DC density and tear cytokines over time. In 3 cases case 8—10 in Table 2 , tear samples and IVCM were obtained on the same day at visits 1 and 2. In all of these 3 patients, the BK improved clinically after the antibiotic treatment started. Figure 7 Alterations of DC density and tear cytokines over time.

The current study evaluates the tear cytokine concentrations and alterations in corneal DCs and nerves by IVCM in healthy control subjects and patients with unilateral BK. Recent advances in the technique of cytokine protein measurements from small volumes of fluid have enabled us to evaluate the tear cytokine concentrations in ocular diseases, such as dry eye disease, keratoconus, GVHD, and corneal neovascularization.

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Try out PMC Labs and tell us what you think. Learn More. We investigated bilateral tear cytokine levels in patients with unilateral bacterial keratitis BK as associated with in vivo confocal microscopic IVCM alterations in corneal nerves and dendritiform immune cells DCs.