Lisette Yco

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.

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.

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.

Keratocytes Derived from Spheroid Culture of Corneal Stromal Cells Resemble Tissue Resident Keratocytes

Purpose Corneal stromal cells transform to precursor cells in spheroid culture. We determined whether keratocytes derived from spheroid culture of murine corneal stromal cells resemble tissue resident keratocytes. Methods Spheroid culture was performed by seeding dissociated stromal cells onto ultra-low attachment plates containing serum-free mesenchymal stem cell culture medium. Spheroids were characterized with phenotype specific markers and stemness transcription factor genes. Spheroids and adherent cells in culture were induced to differentiate to keratocytes using keratocyte induction medium (KIM) and compared with tissue resident keratocytes. Results Stromal cells formed spheroids in ultra-low attachment plates, but not in polystyrene tissue culture dishes. Keratocan expression and abundance was significantly higher in spheroids as compared to adherent cells whereas alpha-smooth muscle actin (α-SMA) was significantly lower. As compared to adherent culture-derived cells, the expressions of keratocan, aldehyde dehydrogenase (ALDH3A1) and α-SMA in spheroid-derived cells approximated much more closely the levels of these genes in tissue resident keratocytes. Of the stemness genes, Nanog and Oct4 were upregulated in the spheroids. Conclusion Stemness transcription factor genes are upregulated in spheroids. Keratocytes derived from spheroids resemble tissue resident keratocytes, thus increasing manifolds the quantity of these cells for in-vitro experiments.