Abed Namavari

Corneal Neurotoxicity Due to Topical Benzalkonium Chloride

PURPOSE The aim of this study was to determine and characterize the effect of topical application of benzalkonium chloride (BAK) on corneal nerves in vivo and in vitro. METHODS Thy1-YFP+ neurofluorescent mouse eyes were treated topically with vehicle or BAK (0.01% or 0.1%). Wide-field stereofluorescence microscopy was performed to sequentially image the treated corneas in vivo every week for 4 weeks, and changes in stromal nerve fiber density (NFD) and aqueous tear production were determined. Whole-mount immunofluorescence staining of corneas was performed with antibodies to axonopathy marker SMI-32. Western immunoblot analyses were performed on trigeminal ganglion and corneal lysates to determine abundance of proteins associated with neurotoxicity and regeneration. Compartmental culture of trigeminal ganglion neurons was performed in Campenot devices to determine whether BAK affects neurite outgrowth. RESULTS BAK-treated corneas exhibited significantly reduced NFD and aqueous tear production, and increased inflammatory cell infiltration and fluorescein staining at 1 week (P < 0.05). These changes were most significant after 0.1% BAK treatment. The extent of inflammatory cell infiltration in the cornea showed a significant negative correlation with NFD. Sequential in vivo imaging of corneas showed two forms of BAK-induced neurotoxicity: reversible neurotoxicity characterized by axonopathy and recovery, and irreversible neurotoxicity characterized by nerve degeneration and regeneration. Increased abundance of beta III tubulin in corneal lysates confirmed regeneration. A dose-related significant reduction in neurites occurred after BAK addition to compartmental cultures of dissociated trigeminal ganglion cells. Although both BAK doses (0.0001% and 0.001%) reduced nerve fiber length, the reduction was significantly more with the higher dose (P < 0.001). CONCLUSION Topical application of BAK to the eye causes corneal neurotoxicity, inflammation, and reduced aqueous tear production.

Quality of Life Among Veterans With War-Related Unilateral Lower Extremity Amputation: A Long-Term Survey in a Prosthesis Center in Iran

Objective: To determine the factors that have an adverse effect on the long-term health-related quality of life (HRQOL) of veterans who have lost their extremities on the battlefield. Design: Cross-sectional study. Setting: Tertiary prosthesis center. Participants: One hundred forty-one male Iranian veterans who have sustained unilateral lower extremity amputation during the Iran-Iraq War (1980-1988) were evaluated after an average of 21.6 years (range, 20-27 years) after amputation. Intervention: No intervention. Main Outcome Measurements: Physical and mental HRQOL according to the Short Form-36 (SF-36) Health Survey. A cutoff point to define poor versus good HRQOL was calculated using the first quartile of SF-36 physical and mental component scores. Results: Poor physical HRQOL was positively associated with transfemoral amputation, phantom movement, low back pain, and a lower Barthel Index [odds ratios (ORs): 4.1, 7.8, 9.1, and 0.9, respectively). Poor mental HRQOL was associated with education level lower than high school diploma and the articular pain of the sound leg (OR = 2.9 and 6.5, respectively). Being employed or receiving disability was a factor that had a lower OR to associate with poor mental HRQOL (OR = 0.2). Conclusions: Alleviation of complaints such as low back pain and articular pain of the sound leg through appropriate medical management, granting facilities for continuing education, and employment are issues that should be considered by authorities and rehabilitative centers to increase HRQOL in amputee veterans.

Early results of Descemet-stripping and automated endothelial keratoplasty (DSAEK) in patients with glaucoma drainage devices.

Purpose: To evaluate the outcome of Descemet-stripping and automated endothelial keratoplasty (DSAEK) in patients with glaucoma tube shunts in the anterior chamber. Methods: Retrospective review of 4 patients with a history of tube shunt placement that experienced corneal decompensation and subsequently underwent DSAEK at 1 institution. Details of the surgical procedures as well as postoperative features including graft attachment, visual acuity, intraocular pressure (IOP), graft clarity, and central corneal thickness were recorded. Results: There were no graft detachments postoperatively. In all but 1 case, the corneal edema resolved with a corresponding decrease in corneal thickness. In 1 case, where there was excessive donor tissue manipulation intraoperatively, the edema failed to resolve. This patient underwent a repeat DSAEK with subsequent graft attachment and resolution of the corneal edema. In all 4 patients, the tube shunt was revised at the time of DSAEK. The tube was not tied or plugged in any of the patients. Except for 1 patient with fibrin reaction, there were no IOP spikes postoperatively. At the 6-month follow-up, the visual acuity had improved in all patients and the IOP had not changed significantly from preoperative levels. Conclusion: This small series suggests that DSAEK is a viable and effective option in patients with existing tube shunts. The presence of a tube did not affect the rate of graft dislocation; however, it appeared to increased the rate of complications postoperatively. These early outcomes support the use of DSAEK as an alternative to penetrating keratoplasty in this patient population.

Ocular Surface Extracellular DNA and Nuclease Activity Imbalance: A New Paradigm for Inflammation in Dry Eye Disease

PURPOSE We determined whether nucleases are deficient in the tear fluid of dry eye disease (DED) patients, and whether this causes extracellular DNA (eDNA) and neutrophil extracellular trap (NET) accumulation in the precorneal tear film, thus causing ocular surface inflammation. METHODS Exfoliated cells adhered to Schirmer test strips were collected on glass slides, and immunofluorescence confocal microscopy was used to evaluate neutrophils, eDNA, NETs, and their molecular components. Similar experiments were performed with mucoid films collected from the inferior conjunctival fornix or bulbar conjunctiva. We used quantitative PCR to evaluate eDNA signaling pathways and inflammatory cytokine expression. We also determined the amount of ocular surface eDNA and evaluated tear fluid nuclease activity. RESULTS eDNA, NETs, and neutrophils were present on the ocular surface in DED patients and abundant in mucoid films. NETs consisted of eDNA, histones, cathelicidin, and neutrophil elastase. Tear fluid nuclease activity was decreased significantly in DED patients, whereas the amount of eDNA on the ocular surface was increased significantly. Expression of genes downstream of eDNA signaling, such as TLR9, MyD88, and type I interferon, as well as the inflammatory cytokines interleukin-6 and tumor necrosis factor-α, was significantly increased in DED patients. CONCLUSIONS Extracellular DNA production and clearance mechanisms are dysregulated in DED. Nuclease deficiency in tear fluid allows eDNA and NETs to accumulate in precorneal tear film, and results in ocular surface inflammation. These findings point to novel therapeutic interventions in severe DED based on clearance of eDNA, NETs, and other molecular components from the ocular surface.

Rapamycin Inhibits the Production of Myofibroblasts and Reduces Corneal Scarring After Photorefractive Keratectomy

PURPOSE Corneal stromal scarring partly involves the production of corneal myofibroblasts. The purpose of this study was to examine the effects of rapamycin (an inhibitor of the mammalian target of rapamycin [mTOR] pathway) on myofibroblast formation in vitro and in-vivo. METHODS Human corneal fibroblasts were grown in culture and transformed into myofibroblasts using TGF-β (2 ng/mL). The phosphorylation (activation) of the mTOR pathway was examined by immunoblotting. Cell proliferation with and without rapamycin was examined by thiazolyl blue tetrazolium bromide (MTT) assay and Ki67 staining. The expression of the myofibroblast differentiation marker smooth muscle actin (SMA) was examined by immunostaining and immunoblotting. The functional effects of rapamycin were measured using a gel contraction assay. For in vivo studies, 140 μm laser ablation was performed on rabbit corneas followed by subconjunctival rapamycin or vehicle. Corneal haze development was graded at 4 weeks, while the expression of myofibroblast markers was examined by immunostaining and immunoblotting. RESULTS The TGF-β activated the mTOR pathway with peak phosphorylation at 2 to 4 hours. Treatment of corneal fibroblasts with rapamycin reduced their proliferation by 46% compared to control. Rapamycin significantly inhibited TGF-β-induced expression of myofibroblast markers (17.2% SMA positive cells with rapamycin compared to 69.0% in control). Rapamycin also significantly inhibited TGF-β-induced collagen gel contraction. In the rabbit eyes treated with rapamycin, corneal haze development was significantly less compared to controls (0.75 ± 0.4 vs. 2.17 ± 0.7). CONCLUSIONS Rapamycin appears to inhibit proliferation and differentiation of corneal myofibroblasts and, thus, may provide an effective therapeutic measure for preventing corneal scarring.

Cyclosporine Immunomodulation Retards Regeneration of Surgically Transected Corneal Nerves

PURPOSE To determine whether immunomodulation with cyclosporine (CsA) affects reinnervation after surgical transection of stromal nerves. METHODS Thy1-YFP+ neurofluorescent mice underwent lamellar corneal surgery and 3 days later, received artificial tears or CsA eye drops for 6 weeks. Serial in vivo wide-field stereofluorescent microscopy was performed to determine changes in nerve fiber density (NFD). Real-time quantitative PCR was performed to determine the expression of neurotrophins and cytokines (IL6 and TNF-α). Compartmental culture of trigeminal ganglion neurons was performed in Campenot devices to determine whether CsA directly affects neurite outgrowth. RESULTS Yellow fluorescent protein (YFP)-positive cells significantly increased at 3 and 7 days after surgery. The number of YFP-positive cells in the cornea was significantly lower in the CsA group than that in the control group. The percentage increase in NFD between 2 to 6 weeks was greater in the control group (80% ± 10%, P = 0.05) than that in the CsA group (39% ± 21%). The CsA group also exhibited lower expression of IL6 and TNF-α (P = 0.01). In compartmental culture experiments, neurite outgrowth toward side compartments containing CsA was significantly less (2.29 ± 0.4 mm, P = 0.01) than that toward side compartments containing vehicle (3.97 ± 0.71 mm). CONCLUSIONS Immunomodulation with CsA reduces the expression of cytokines (IL6) in the cornea and retards regenerative sprouting from transected corneal stromal nerve trunks. In addition, CsA has a direct growth inhibitory action on neurites as well.

Intravitreal bevacizumab (Avastin) for the treatment of cystoid macular edema in Behçet disease.

Purpose: To investigate the effect of bevacizumab on cystoid macular edema in Behçet disease. Methods: Twelve eyes of 11 patients with Behçet disease underwent optical coherence tomography, fluorescein angiography, and visual acuity measurement before and after the intravitreal injection of Avastin to evaluate its effect. Results: The visual acuity improved in 7 eyes. Foveal thickness and macular volume did not show statistically significant differences. Fluorescein angiography reduced in grading in 4 patients and was deteriorated in 2. Conclusions: The intravitreal injection of Avastin could be effective in the improvement of the vision in patients with uveitis secondary to Behçet disease.

Neurotrophins and nerve regeneration-associated genes are expressed in the cornea after lamellar flap surgery.

Purpose: To determine the in vivo expression of neurotrophins (NTs) and nerve regeneration-associated genes (RAGs) after surgically creating a hinged lamellar corneal flap in thy1-YFP mice. Methods: Lamellar corneal flaps with multiple hinges were created in thy1-YFP mice. Mice were killed at weeks 2, 4, and 8. Quantitative polymerase chain reaction was performed to determine the expression of NTs and RAGs in the corneas after lamellar transection. Nerve growth factor (Ngf), brain-derived neurotrophic factor (Bdnf), glial cell–derived neurotrophic factor (Gdnf), neurotrophin 3, neurotrophin 5, small proline–rich repeat protein 1A (Sprr1a), growth-associated protein 43 (Gap43), and beta III tubulin (Tubb3) gene expressions were analyzed. Whole-mount confocal immunofluorescence and Western analyses were performed for localization and abundance of robustly expressed genes. Results: Sprouts of fine YFP-positive fronds emanating from transected (injured) nerve bundles were seen in the flap area at 2 weeks onward. Bdnf and Sprr1a were robustly and significantly expressed at 2 weeks postoperatively (>2-fold increase in expression; P < 0.05). Bdnf localized to thy1-YFP+ cells in operated corneas. Sprr1a localized to corneal epithelial cell membranes. At 8 weeks, none of the NTs and RAGs had increased expression. Bdnf (&rgr; = 0.73, P = 0.001) and Sprr1a (&rgr; = 0.76, P = 0.001) showed a significant positive correlation with beta III tubulin. Conclusions: The neurotrophin Bdnf and RAG Sprr1a are robustly and significantly expressed during corneal nerve regeneration in vivo.

Semaphorin 7a Links Nerve Regeneration and Inflammation in the Cornea

PURPOSE We determined Semaphorin 7a (Sema7a) localization and abundance in naive corneas and in corneas after nerve-transecting lamellar flap surgery, and determined the effect of Sema7a supplementation on corneal nerve regeneration and inflammation. METHODS Immunolocalization and Western blot analyses were performed to evaluate the abundance of Sema7a in naive corneas and corneas undergoing nerve regeneration after lamellar corneal surgery in thy1-YFP+ neurofluorescent mice. We used compartmental cultures of dissociated trigeminal ganglion cells to determine the effect of Sema7a exposure on neurite outgrowth in vitro. Finally, a Sema7a pellet was implanted under the corneal flap after lamellar transection surgery to determine the neuronal and inflammatory effects of Sema7a supplementation in vivo. RESULTS Sema7a was expressed in the corneal epithelium and stromal keratocytes, but was more abundant in the epithelium (74.3%) compared to the stroma (25.7%, P = 0.02). Sema7a expression was increased significantly in the cornea after lamellar corneal surgery and was localized to stromal cells near the regenerating nerve fronds. Exposure of trigeminal neurites to Sema7a (20 nM) in the side compartment increased neurite length significantly. The implanted Sema7a pellet increased significantly YFP+ inflammatory cell influx into the cornea as well as increased corneal nerve length. CONCLUSIONS Sema7a is expressed constitutively in the cornea, and potently stimulates nerve regeneration and inflammatory cell influx. Therefore, this immune semaphorin links nerve regeneration and inflammatory processes in the cornea.

In Vivo Serial Imaging of Regenerating Corneal Nerves after Surgical Transection in Transgenic Thy1-YFP mice

PURPOSE To determine the effect of lamellar transection surgery on the nerve fiber density (NFD) and pattern of nerve regeneration in the cornea of thy1-YFP transgenic mice. METHODS Wide-field stereo fluorescence microscopy was used to obtain serial images of nerves in live thy1-YFP mice, which express a fluorescent protein in their axons. NFD (mm/mm(2)) was calculated from maximum intensity projection images as the total length of fibers within the area of the contour in which nerves were traced. Whole-mount confocal microscopy was performed to analyze the arrangement of nerves and the types of regenerating fibers. RESULTS NFD in normal corneas was 35.3 ± 1.8 mm/mm(2). Stereo fluorescence microscopy revealed the presence of a subbasal hairpin nerve layer and an intrastromal nerve trunk layer. After surgery, regenerative sprouting was observed from transected distal ends of intrastromal nerve trunks. NFD also increased, with this increase being maximal between 4 and 6 weeks after surgery. NFD approximated baseline values at 6 weeks and did not change any further at 8 weeks. Regenerated nerves did not readopt the normal corneal nerve arrangement. A dense interlacing network of regenerated nerves was present in the corneal bed. Branches from this network traversed the flap to innervate the epithelium. Immunofluorescence staining revealed that regenerating fronds contained peptidergic nociceptive fibers (positive for calcitonin gene-related peptide and substance P) and myelinated non-nociceptive fibers (positive for neurofilament 200). CONCLUSIONS Although corneal NFD recovers to normal levels by 8 weeks after nerve transection, the arrangement of regenerated nerves is abnormal.