Snehal Sonawane

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.

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.

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.

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.

A Recurrence of Bilateral Diffuse Sclerosing Lobular Hyperplasia of Breast: A Case Report

Mammary sclerosing lobular hyperplasia is an uncommon benign fibroproliferative lesion of adolescent and young women, often of African American heritage with an incidence of ~3%. Patients generally complain of a palpable, painless, or slightly tender and well-defined lump in breast. Very rarely, this lesion may be bilateral and diffuse. The definitive diagnosis of sclerosing lobular hyperplasia requires histopathologic evaluation. Here, we describe a case of diffuse sclerosing lobular hyperplasia in a 29-year-old African American woman that required bilateral mastectomy and recurred bilaterally requiring second resections. This appears to be the first report of this phenomenon.

Endometrioid Endometrial Carcinoma With Spindle Cells–Aberrant p16 and p53 Expression

Endometrioid carcinoma is known for its diverse morphology and may pose a diagnostic dilemma when it presents with a spindle cell component. We present a case of a 65-year-old woman with postmenopausal bleeding. Physical examination showed a mass protruding from the external cervical os. The patient underwent biopsy followed by hysterectomy. Pathologic examination showed an endometrioid endometrial carcinoma with spindle cell differentiation arising in an endometrial polyp, which raised a variety of differential diagnoses. Prior reports of this tumor type showed nonaberrant immunohistochemical expression of p16 and p53. However, this case showed p16 and p53 overexpression indicating that there is a spectrum of these tumors.

Metastatic intestinal adenocarcinoma to a lymph node involved by follicular lymphoma: The importance of looking beyond the apparent

Composite tumors consisting of follicular lymphoma (FL) and colorectal or small intestinal adenocarcinoma are exceedingly rare, with only four cases published in the literature, to the best of our knowledge. While in most of these cases the clinical prognosis seems to be determined by the adenocarcinoma, at least one patient has shown rapid and aggressive progression of the FL. Here we report on a 62 year-old male with colonic adenocarcinoma metastatic to a retroperitoneal lymph node involved by FL, which illustrates the importance of carefully examining the histomorphology of lymphoid elements in surgical specimens.