In Vivo Confocal Microscopy Analysis Research Articles

Preoperative fluorometholone in glaucoma surgery: 1 Year clinical and anatomical outcomes with in vivo confocal microscopy (IVCM)

Aims/Purpose: To report preliminary data on the preoperative application of topical Fluorometholone in patients requiring glaucoma surgery due to primary open‐angle glaucoma (POAG), evaluating clinical and anatomical outcomes with in vivo confocal microscopy (IVCM) at 1 year follow‐up.Methods: We conducted a prospective study involving 54 eyes with POAG requiring glaucoma surgery: Trabeculectomy or Minimally Invasive Bleb Surgery (MIBS) (XEN® and PreserFlo®). The sample was randomized: the treatment group (25 eyes) received Fluorometholone 1mg/ml QID for 1 month pre‐surgery, while the control group (29 eyes) did not. IVCM of the conjunctiva was performed at baseline, after 1 month of treatment, and 1 year post‐surgery. Preoperative parameters included goblet cell density (GCD) and dendritic cell density (DCD) as inflammatory markers, while postoperative parameters were density and mean area of intraepithelial cysts (MMD and MMA) as indices of bleb function. Pre and post‐surgery stromal meshwork reflectivity (SMR) served as an indirect marker of stromal collagen.Results: Preoperative Fluorometholone led to significantly decreased in DCD (p < 0.001) compared to baseline and in DCD (p < 0.001) and SMR (p = 0.014) compared to the control group. At 1 year, there were no significant differences between the two groups in mean IOP, medications, needling procedures, and failure rates. IVCM analysis showed increased MMD in the treatment group compared to the control group (p = 0.028). In the separate analysis, the MIBS treatment subgroup group had lower IOP (p = 0.046) and less medication use (p = 0.032), but this was not observed in the trabeculectomy treatment subgroup. MMD increased in both the MIBS (p = 0.037) and trabeculectomy (p = 0.024) treatment subgroups.Conclusions: Preoperative Fluorometholone reduced ocular inflammation, improving bleb function at 1 year as detected by IVCM. However, its impact on overall bleb functionality remains unclear, with a clinically detectable improvement only in the MIBS group. Larger studies with longer follow‐up are necessary to fully understand the implications of preoperative Fluorometholone on long‐term surgical outcomes across different types of glaucoma surgeries.

Effects of full-thickness wedge resection on ocular surface and in vivo confocal microscopy findings in floppy eyelid syndrome patients.

To evaluate the effect of full-thickness wedge resection (FTWR) on ocular surface and in vivo confocal microscopy (IVCM) findings in patients with floppy eyelid syndrome (FES). The study included two groups: a surgical treatment (ST) group (26 eyes) consisting of patients who underwent FTWR surgery, and a conservative treatment (CT) group (30 eyes). Pre-treatment and post-treatment ocular surface disease index (OSDI), tear break-up time (TBUT), corneal fluorescein staining (CFS), IVCM findings along with the body mass index (BMI), FES grade, the presence and the treatment of obstructive sleep apnea syndrome (OSAS) were recorded and compared between the groups. The groups were comparable in terms of BMI, FES grade, and OSAS data. After six months, TBUT in the ST group significantly increased to 12.92 ± 1.15, compared to 8.10 ± 1.60 in the CT group (p = 0.000). The CFS and OSDI scores were significantly lower in the ST group (0.15 ± 0.37, 18.0 ± 8.3, respectively) compared to the CT group (0.90 ± 0.61, 27.3 ± 9.3, respectively) (p = 0.000). IVCM analysis revealed a significant decrease in dendritic cell count (ST: 22.0 ± 12.4, CT: 39.5 ± 15.1, p = 0.000) and nerve tortuosity (ST: 1.38 ± 0.64, CT: 2.00 ± 0.59, p = 0.000), with a significant increase in total nerve density (ST: 4.27 ± 0.83, CT: 3.57 ± 0.90, p = 0.002) in the ST group compared to the CT group after six months. In our retrospective cohort, FTWR surgery was shown to be an effective and reliable surgical treatment for FES, improving both ocular surface and IVCM findings. Patients with moderate to severe stages of FES not responding to conservative treatment may benefit from eyelid tightening.

Corneal complications of ocular GvHD

Corneal fluorescein staining is one of the tests recommended to diagnose and grade ocular graft versus host disease (GvHD) according to the International Chronic Ocular GvHD Consensus Group. Despite superficial punctate keratopathy is the most common corneal manifestation, patients with more severe stages may develop corneal neovascularization, sterile corneal ulceration and even perforation. Previous in vivo confocal microscopy (IVCM) studies reported significant microstructural changes in the cornea of patients with ocular GvHD, including a higher density of dendritic cells and globular immune cells, a network of hyper‐reflective activated keratocytes, and a lower density and higher tortuosity of sub‐basal corneal nerves. Dendritic cells act as antigen‐presenting cells, and play a key role in ocular surface immune homeostasis. Thus, their increased density may be indicative of immune activation and inflammation of the ocular surface in patients with ocular GvHD. Interestingly, Kheirkhah et al. reported higher dendritic cell density in patients with DED owing to systemic immune disease (i.e., Sjögren syndrome and ocular GvHD) compared to patients with DED of other origins. Recently, our group employed a fully automated IVCM analysis system to compare corneal sub‐basal nerve plexus in patients with DED owing to both ocular GvHD and Sjögren syndrome and in healthy control subjects. Although overall patients with DED showed lower density of nerve fibres and branches, shorter nerve fibres and higher fibre width compared to healthy controls, no significant difference was observed between patients with ocular GvHD and Sjögren syndrome for all IVCM metrics. In agreement with this observation, other studies reported no significant difference of IVCM parameters in DED patients with and without ocular GvHD after adjusting for clinical severity of dry eye. This suggests that the ocular surface changes observed by IVCM in ocular GvHD may be possibly reflective of the local disease severity rather than the underlying systemic process.

Structural Changes in Thin Keratoconic Corneas Following Crosslinking with Hypotonic Riboflavin: Findings on In Vivo Confocal Microscopy.

PurposeTo report structural changes observable in in vivo confocal microscopy (IVCM) in keratoconic corneas 400 μm treated with hypotonic riboflavin and collagen crosslinking (CXL).MethodsTen eyes of ten patients with progressive keratoconus and corneal thickness between 350 and 399 μm underwent CXL with hypotonic riboflavin. IVCM was performed preoperatively and at one month, three months, and six months after the procedure.ResultsIVCM analysis one month postoperatively showed complete absence of the subepithelial nerve plexus with gradual regeneration over six months in 8 of the 10 eyes, and poor regeneration in the remaining 2 eyes. The anterior stroma showed extracellular lacunae and hyper-reflective cytoplasm in a honeycomb appearance signifying edema at one month which gradually decreased over six months post CXL. Stromal keratocyte apoptosis was evident in the anterior stroma in all cases and extended to the posterior stroma in four eyes with gradual regeneration evident at three and six months. The specular endothelial count decreased by 8% (P = 0.005) post-CXL, but no corneas developed clinical signs of endothelial trauma.ConclusionIVCM analysis of thin corneas after hypotonic CXL showed posterior corneal structural changes. Posterior stromal changes were accompanied by a decrease in the endothelial cell count. This case series was a preliminary feasibility study that might necessitate conducting a well-designed controlled study.

Region of interest and directional analysis of subbasal nerves in wide-area corneal nerve plexus mosaics in type 2 diabetes mellitus

In vivo confocal microscopy (IVCM) imaging of the corneal subbasal nerve plexus (SBNP) is a clinical imaging modality gaining popularity for the diagnosis and follow-up of corneal neuropathy in various conditions such as diabetes mellitus. There remain, however, major limitations to the method, hindering its widespread clinical use. Finding the same exact area of the central cornea to standardize image acquisition is difficult without a reference point. Alternatively, creating wide-area mosaics of the SBNP is resource-intensive and has not yet been developed for routine clinical use. Here, we investigated whether IVCM analysis of the corneal SBNP in a predetermined, anatomically standardized region of interest (ROI) could be applied as an equivalent substitution for wide-area SBNP mosaic generation and analysis. Furthermore, we investigated nerve patterns outside the central corneal region for a possible relationship to type 2 diabetes mellitus status using a publicly available dataset. We found that corneal nerve fibre length density (CNFL) based on the ROI underestimated the mosaic-based CNFL by an average of 34% in 90% of cases (150 eyes), and did not exhibit a significant reduction with diabetes, as seen in the full SBNP. Outside the central cornea, nerve orientation differed depending on the anatomic region (left, central or right superior plexus, P < 0.001). Moreover, in long-term type 2 diabetes mellitus (≥ 10 years, 28 subjects), nerve density in the left superior sector of the SBNP was decreased (P < 0.001) while that in the central superior SBNP increased (P = 0.01) relative to 35 age-matched healthy subjects with normal glucose tolerance. These results indicate that subbasal nerve degeneration in type 2 diabetes mellitus can vary according to anatomic location, and regions with potential diagnostic value outside the central SBNP may warrant further investigation.

In Vivo Evaluation of Corneal Nerves and Epithelial Healing After Treatment With Recombinant Nerve Growth Factor for Neurotrophic Keratopathy

To evaluate the renewal of corneal nerve structure and function in patients with neurotrophic keratopathy (NK) treated with recombinant human nerve growth factor (rhNGF) eye drops. Prospective, interventional, before-and-after case series. This study included 18 patients with NK with a persistent epithelial defect or corneal ulcer, treated with topical rhNGF, and age-matched healthy controls. Patients underwent clinical examination with corneal fluorescein staining, Schirmer 1 tear test, assessment of corneal sensitivity with the Cochet-Bonnet esthesiometer, and morphologic examination of the nerves by invivo confocal microscopy (IVCM) at baseline and at 4 and 8weeks of treatment. IVCM analysis was used to assess corneal sub-basal nerve density, number of nerve branches, and the diameter of nerve fibers. A complete resolution of the epithelial defect was observed in all patients within 8weeks. Schirmer 1 test showed a significant improvement of tear film secretion. Change from baseline in corneal sensation was significant (P < .001) but did not approach that of healthy controls. After 8weeks of treatment, there was a significant increase in the mean nerve density in affected eyes as compared to baseline (P= .007) as well as in the number of nerve branches (P= .008) and nerve fiber diameter (P= .007). Topical treatment with rhNGF was effective in promoting complete corneal healing of persistent epithelial defects and corneal ulcers in patients with NK. This was associated with an improvement of corneal sensitivity and an increase of sub-basal nerve density, diameter, and number of nerve branches, indicating improvement in structure and function of corneal nerves.

Proinflammatory Markers, Chemokines, and Enkephalin in Patients Suffering from Dry Eye Disease.

Dry eye symptoms are among the leading complaints in ophthalmology. Dry eye disease (DED) is associated with significant pain affecting quality of life. Cellular and molecular mechanisms underlying ocular pain associated with DED are not fully understood. In this study, we investigated the ocular surface of patients with DED using in vivo confocal microscopy (IVCM) to quantify corneal nerve density and its relation with corneal inflammation. Gene expression of the proinflammatory markers HLA-DR, IL-6, CXCL12, and CCL2 and the receptors CXCR4 and CCR2, as well as PENK (enkephalin precursor), was therefore quantified in conjunctival impression cytology specimens. Thirty-two patients with DED and 15 age-matched controls were included. Subbasal nerve density was significantly lower in DED patients compared to controls. IVCM analysis revealed that DED patients had a significantly higher corneal dendritic cell density compared to controls. Conjunctival impression cytology analysis revealed that HLA-DR, IL-6, CXCR4, and CCL2/CCR2 mRNA levels were significantly increased in DED patients compared to controls, whereas PENK mRNA levels were significantly decreased. Similar results were obtained in vitro on immortalized human conjunctiva-derived epithelial cells challenged with osmotic stress that mimics the DED condition. These results demonstrate that proinflammatory molecules and endogenous enkephalin have opposite gene regulation during DED.

Photophobia and corneal crystal density in nephropathic cystinosis: an in vivo confocal microscopy and anterior-segment optical coherence tomography study.

To analyze the correlation between photophobia and corneal crystal density in nephropathic cystinosis using in vivo confocal microscopy (IVCM) and anterior-segment optical coherence tomography (AS-OCT). Forty eyes of 20 patients with nephropathic cystinosis aged 7 to 37 years were included in this study. Ophthalmologic investigations included clinician-assessed and self-assessed evaluations of photophobia, slit-lamp biomicroscopy analysis, the depth of crystal deposition (DCD) and the central corneal thickness (CCT) in the central cornea measured with AS-OCT, and IVCM analysis of the crystal density score (IVCM-CysS), inflammatory cell density (IVCM-inf), and nerve damage (IVCM-N). Age, sex, intraleukocyte cystine concentrations (ICC), and the need for renal transplantation were also recorded. The average subjective and objective photophobia scores were 2.10 ± 1.28 and 1.70 ± 1.41, respectively. Using AS-OCT, the average percentage of crystal infiltration (OCT-CysP) and was 49.56 ± 27.31% (range, 11.45%-95.81%). The mean IVCM-CysS was 8.84 ± 4.34, the mean density of inflammatory cells (IVCM-inf) was 178.28 ± 173.00 cells/mm2, and the mean IVCM-N score was 3.11 ± 2.11. Clinician- and self-assessed estimations of photophobia were correlated (R2 = 0.61). No significant correlation was observed between clinician- and self-assessed photophobia scores and ICC or sex. There were significant correlations between clinician- and self-assessed photophobia scores and age, OCT-CysP, IVCM-CysS, IVCM-inf, and IVCM-N. The IVCM-CysS was also correlated with OCT-CysP (R2 = 0.27), IVCM-inf (R2 = 0.37), and IVCM-N (R2 = 0.56). In vivo confocal microscopy and AS-OCT are reliable tools to quantify cystinosis corneal crystals. In patients with nephropathic cystinosis, the intensity of photophobia is associated with the density of crystals, infiltration of inflammatory cells, and nerve damage within the cornea.

Comparison of the anti‐inflammatory effects of artificial tears in a rat model of corneal scraping

AbstractPurpose The aim of the present study was to evaluate the safety and tolerance of cationic oil‐in‐water emulsion on debrided cornea, and to characterize its benefits on the corneal epithelium healing process.Methods A rat model of corneal scraping was used to characterize the effects of four commercially available artificial tears (Cationorm®, Vismed®, Optive® and Systane Balance®) on the recovery process of the debrided corneas. The upper part of the corneal epithelium was scraped mechanically prior to a 5‐day treatment with different artificial tears. At the end of the treatment, the ocular surface was evaluated clinically (corneal fluorescein staining, CFS) and by in vivo confocal microscopy (IVCM). Conjunctival function was assessed by goblet cell count and MUC5AC immunostaining.Results The four artificial tears were all well tolerated by the debrided cornea. By restoring an adequately hydrated ocular surface environment they promote corneal healing, as evidenced by CFS measurements of the scraped area. In contrast 0.02% BAK solution inhibits the healing process. IVCM analysis of the different layers of the cornea confirmed the benefits of the cationic emulsion (Cationorm®). Interestingly inflammatory cells infiltration in the stroma was at its lower following Cationorm® treatment, while 0.02% BAK treatment resulted in marked inflammation. The different treatments were all able to protect goblet cells function and MUC5AC expression.Conclusion BAK‐free cationic emulsion (Cationorm®) is well tolerated by debrided cornea and allow for a safe healing of the cornea. The findings suggest that Cationorm® have the potential to benefit patients with corneal epithelium disorder. Commercial interest

Reduced in vivo Ocular Surface Toxicity with Polyquad-Preserved Travoprost versus Benzalkonium-Preserved Travoprost or Latanoprost Ophthalmic Solutions

The study used a validated acute in vivo model to compare a new formulation of travoprost 0.004% ophthalmic solution (travoprost PQ), preserved with polyquaternium-1 (PQ), with commercially available formulations of benzalkonium-chloride (BAK)-preserved travoprost 0.004% ophthalmic solution (travoprost BAK) and BAK-preserved latanoprost 0.005% ophthalmic solution (latanoprost BAK). Adult male New Zealand albino rabbits (n = 36) were randomly divided into 6 groups. Phosphate-buffered saline (PBS), 0.001% PQ, 0.015% BAK, travoprost PQ, travoprost BAK or latanoprost BAK were applied onto rabbit eyes as 1 drop, for 15 times at 5-min intervals. The ocular surface reactions were investigated at hour 4 and day 1 using slitlamp examination; in vivo confocal microscopy (IVCM) for cornea, limbus and conjunctiva/conjunctiva-associated lymphoid tissue, conjunctival impression cytology and standard immunohistology in cryosections for detecting CD45+ infiltrating cells and MUC-5AC-labeled cells. PBS, PQ and travoprost PQ did not induce obvious irritation by clinical observation, changes in microstructures of the whole ocular surface as measured by IVCM analysis, inflammatory infiltration or cell damage as measured by impression cytology, altered levels of goblet cell counts or numerous CD45+ cells in the cornea. In contrast, all BAK-containing products induced diffuse conjunctival hyperemia and chemosis, abnormal changes in the ocular surface microstructure, significant total ocular surface toxicity scores, damaged epithelial cells, inflammatory cell infiltration and decreased goblet cell density. Travoprost PQ did not elicit ocular surface toxicity when administered to rabbit eyes. These results suggest a greater safety advantage for the ocular surface of patients receiving chronic glaucoma treatment with PQ-preserved drugs.

In vivo analysis of conjunctiva in gold micro shunt implantation for glaucoma

AimsTo describe the conjunctival epithelial features seen with in vivo confocal microscopy (IVCM) after gold micro shunt (GMS) implantation in the suprachoroidal space, in patients with uncontrolled glaucoma.MethodsThis was an...

An In Vivo Confocal Microscopy and Impression Cytology Evaluation of Pterygium Activity

To correlate pterygium activity with in vivo confocal microscopy (IVCM) and ex vivo impression cytology (IC) analyses. Twenty-eight pterygia from 16 patients were analyzed in this study. A pterygium activity score was obtained by summing four scores of ocular discomfort, pterygium hyperemia, keratitis, and the presence of Fuchs patches. Patients underwent pterygium IVCM analysis and collection of IC specimens on the pterygium. IC specimens were analyzed quantitatively by immunohistochemistry for goblet cell and dendritiform inflammatory cell density. IVCM images and IC results were compared to search for a possible correlation with the pterygium activity score. The presence of inflammatory cells, numerous blood vessels, and an irregular limit between the cornea and the pterygium with infiltration of hyperreflective cells in the adjacent corneal epithelium were the signs observed with IVCM associated with pterygium activity. Using IC, the density of goblet cells and dendritiform inflammatory cells was significantly correlated to the pterygium activity score (Spearman coefficient 0.765, P < 0.0001 and 0.799, P < 0.0001, respectively). In the same pterygium, dendritic inflammatory cell density and goblet cell density were also significantly correlated (Spearman coefficient 0.952; P < 0.0001). The correlation of pterygium cell changes with pterygium activity supports the role of inflammation and its relationship with goblet cells in the pathogenesis of pterygium.

Filtering Bleb Functionality: A Clinical, Anterior Segment Optical Coherence Tomography and In Vivo Confocal Microscopy Study

To provide a microscopic and macroscopic analysis by a clinical, in vivo confocal microscopy (IVCM) and anterior segment optical coherence tomography (AS-OCT) assessment of filtering blebs after glaucoma surgery. We retrospectively analyzed 60 eyes of 48 glaucomatous patients by slit-lamp, IVCM, and AS-OCT examinations. Patients were divided into 2 groups: successful blebs (group 1, 16 patients, 27 eyes) were defined as a one-third reduction in preoperative intraocular pressure without antiglaucoma medications and failed blebs (group 2, 32 patients, 33 eyes) as a less than one-third reduction in preoperative intraocular pressure without therapy. The examinations were performed from 1 to 96 months postoperatively. Diffuse or cystic clinical patterns were indicative for good functionality whereas flat or encapsulation was indicative for poor functionality. When comparing successful with failed blebs, the IVCM analysis showed a greater number (P=0.014), density (P=0.009), and total area of epithelial microcysts (P=0.017) and a lower density of connective tissue (P=0.006). The AS-OCT analysis showed a lower degree of bleb wall reflectivity (P<0.001). A significant correlation was found between the clinical and AS-OCT parameters, particularly for the cystic (100%) and diffuse (74%) patterns. All IVCM parameters did correlate well with the bleb functionality whereas, among the AS-OCT parameters, only the bleb wall reflectivity was significantly related to the filtering capability. Clinical and AS-OCT bleb classification showed a significant degree of concordance. As a consequence, simultaneous approach by clinical, microscopic, and tomographic assessment improves the clinician's ability in the postsurgery understanding and management of blebs.

An in Vivo Confocal Microscopy and Impression Cytology Analysis of Preserved and Unpreserved Levobunolol-Induced Conjunctival Changes

To provide an in vivo confocal microscopy (IVCM) and impression cytology analysis of preserved-and unpreserved levobunolol-induced changes of conjunctival epithelium. 27 eyes of 27 patients were consecutively randomized to receive preserved or unpreserved levobunolol; all patients had a recent diagnosis of primary open angle glaucoma (POAG) or ocular hypertension and were not previously treated with topical medications. IVCM and impression cytology were performed before and after six months of therapy. Goblet cells density and a conjunctival epithelium regularity index were considered in the IVCM analysis, whereas impression cytology specimens were graded and scored in accordance with Nelson's method. After six months of therapy, IVCM and impression cytology parameters showed significant differences with respect to baseline in both groups (p<0.001); significant differences were also found between the two groups (p<0.001). The IVCM analysis showed a goblet cells density reduction (61% and 17% from baseline, respectively in group 1 and 2) (p<0.001) and an higher index of epithelial regularity (p<0.001) in both groups; the impression cytology analysis showed an higher score in both groups (p<0.001). All the IVCM and impression cytology parameters correlated well with the conjunctival modifications induced by the topical therapy, suggesting the less toxicity of unpreserved drugs.