Ocular tolerance to a topical formulation of hyaluronic acid and chitosan-based nanoparticles.

PURPOSE: Hyaluronic acid-chitosan nanoparticles (HA-CS NPs) have the potential to serve as a reliable drug delivery system to topically treat ocular surface disorders. We evaluated the in vivo uptake by ocular structures, the acute tolerance, and possible alterations of tear film physiology in rabbits. METHODS: Fluorescent HA-CS NPs (fl-HA-CS NPs) were prepared by ionotropic gelation using fluoresceinamine-labeled hyaluronic acid and resuspended in buffer. fl-HA-CS NPs (30 microL, 0.5 mg/mL) and fluoresceinamine-HA conjugate (30 microL) were instilled into rabbit eyes every 30 minutes for 6 hours. In vivo uptake and acute tolerance were characterized 24 hours after the first instillation and compared with preinstillation measurements. Clinical signs, including tear production, lacrimal drainage system patency and reflux, and ocular surface pathology, were evaluated. RESULTS: The rabbits showed no signs of ocular discomfort or irritation after exposure to HA-CS NPs. No macroscopic alteration in ocular surface structures was observed. fl-HA-CS NPs were present inside conjunctival and corneal epithelial cells, although the distribution within the cells was different. The fl-HA-CS NPs had no significant effects on tissue morphology and functionality, tear production, or drainage. CONCLUSION: Taken together, these data demonstrate that HA-CS NPs are a safe drug carrier for ocular surface application.

Altered expression of neurotransmitter receptors and neuromediators in vernal keratoconjunctivitis.

BACKGROUND: There is growing evidence that autonomic innervation is involved in the pathogenesis of mucus hypersecretion, goblet cell hyperplasia, and conjunctival hyperreactivity. OBJECTIVE: To determine the expression of neurotransmitters and neurotransmitter receptors in vernal keratoconjunctivitis (VKC) tissues to evaluate whether neurogenic inflammation plays a role in this ocular atopic-related disorder. METHODS: Biopsy specimens of upper tarsal conjunctiva from 8 VKC patients with active inflammation and from 4 healthy subjects were processed for immunohistochemistry using anti-M1, anti-M2, and anti-M3 muscarinic receptors; beta1-adrenergic receptor; vasoactive intestinal peptide; nerve growth factor; and protein gene product 9.5, a marker of nerve fibers. RESULTS: In the conjunctival epithelium of VKC patients, M1 muscarinic receptor, nerve growth factor, and protein gene product 9.5 expression were decreased, whereas M2 and M3 muscarinic receptors and beta1-adrenergic receptor were irregularly distributed, compared with control subjects. Neurotransmitter receptors and vasoactive intestinal peptide expression were increased in the substantia propria-localized infiltrate of VKC compared with healthy tissue. Nerve growth factor and protein gene product 9.5 staining was also enhanced in the conjunctival stroma of VKC vs healthy conjunctiva. CONCLUSIONS: The inflamed conjunctiva of VKC patients demonstrated an obvious alteration in muscarinic and beta1-adrenergic receptor, vasoactive intestinal peptide, protein gene product 9.5, and nerve growth factor expression. These results substantiate the involvement of an autonomic dysfunction in the pathogenesis of VKC.

Alpha2-adrenergic receptors are present in normal human conjunctiva.

PURPOSE: Despite the passage of medications, including antiglaucoma drugs, through the ocular surface, and despite the increasing relevance of neurogenic inflammation in the ocular surface, the presence of some neuroreceptors in the conjunctiva has not been ascertained. This study describes the presence of alpha2-adrenergic receptors in normal human conjunctiva. METHODS: Immunofluorescence microscopy, electrophoresis, and Western blot analyses were done in human conjunctival biopsies and rat control tissues. Antibodies against alpha2-adrenergic receptor subtypes alpha2A, alpha2B, and alpha2C were used. RESULTS: Immunoreactivity for alpha2A- and alpha2B-, but not alpha2C-adrenergic receptors was evenly distributed in epithelial cells of human conjunctiva cryosections. Immunoreactive bands were detected for the three alpha2-adrenergic receptor subtypes: a major band of 48-50 kDa and fellow bands of 65-67 kDa. CONCLUSIONS: Normal human conjunctival epithelial cells express alpha2A-, alpha2B-, and alpha2C-adrenergic receptors. Further studies to determine the functional implications of these receptors in ocular surface homeostasis are warranted.

Impression cytology of the ocular surface: a review.

PURPOSE: To historically review the technique of impression cytology as a minimally invasive diagnostic tool for ocular surface pathology. METHODS: A comprehensive review of published literature cited in PubMed since the first description of impression cytology in 1977 up to date has been undertaken. RESULTS: A wide range of processing methods have been adapted to the technique of impression cytology (from conjunctiva, cornea or limbus): regular light microscopy with different stainings, transmission and scanning electron microscopy, immunofluorescence, immunocytochemistry, polymerase chain reaction analysis, immunoblotting analyses, or flow cytometry. At present, it is widely used as a non-invasive alternative to the full-thickness biopsy for the obtention of epithelial cells from the ocular surface. CONCLUSIONS: Impression cytology represents a non- or minimally invasive biopsy of the ocular surface epithelium with no side effects or contraindications. It has demonstrated to be a useful diagnostic aid for a wide variety of processes involving the ocular surface. In addition, and mainly during the last decade, its use as a research tool has experienced an enormous growth and has greatly contributed to the understanding of ocular surface pathology.

Characterization of a spontaneously immortalized cell line (IOBA-NHC) from normal human conjunctiva.

PURPOSE: To characterize a new nontransfected, spontaneously immortalized epithelial cell line from normal human conjunctiva (IOBA-NHC), both morphologically and functionally, to determine whether the differentiated phenotype of conjunctival epithelial cells is preserved. METHODS: Outgrowing cells from explanted conjunctival tissue were successively passaged and preliminarily characterized at passage 3 to assess epithelial origin. The cells were further characterized at passages 15 to 20, 40, 60, and 100 by analyzing (1) proliferation and in vitro behavior (viability, plating efficiency, colony forming efficiency and colony size, and Ki-67 protein expression), (2) karyotype and G-banding, (3) epithelial marker expression (cytokeratins, desmoplakins, EGF receptor), (4) absence of contaminating cell types, (5) expression of conjunctival differentiation markers (mucin gene expression), and (6) functional capability in response to proinflammatory stimuli. IOBA-NHC cells were analyzed by light and electron (transmission and scanning) microscopy, immunohistochemistry, electrophoresis and Western blot analysis, flow cytometry, and reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: IOBA-NHC cells showed high proliferative ability in vitro and typical epithelial morphology. Cytokeratins and GalNAc, GluNAc, mannose, and sialic acid residues were immunodetected in these cells. No contaminating cell types were found. MUC1, -2, and -4, but not -5AC or -7 mucin genes were expressed in every cell passage tested. Exposure of cells to inflammatory mediators (IFNgamma and/or TNFalpha) resulted in increased expression of intercellular adhesion molecule (ICAM)-1 and HLA-DR. CONCLUSIONS: Morphologic and functional characterization of the nontransfected, spontaneously immortalized IOBA-NHC cell line shows that this new cell line may be a useful experimental tool in the field of ocular surface cell biology.

Human epithelium from conjunctival impression cytology expresses MUC7 mucin gene.

PURPOSE: To prove that noninvasive methods of obtaining conjunctival tissue, such as conjunctival impression cytology (CIC), could be valid alternatives that are simpler, faster, and more convenient for patients than biopsy to analyze mRNA levels of mucin genes. METHODS: Using the semiquantitative reverse-transcriptase polymerase chain reaction, we studied the presence of the mucin genes described on the ocular surface thus far and attempted to detect the presence of MUC7 in CIC samples from 10 healthy donors. RESULTS: Conjunctival cells recovered by CIC expressed all the genes studied. There were no statistically significant differences between male and female subjects, and there was a significant correlation between the two eyes of the same donor only in the expression of MUC7. CONCLUSION: CIC is a valid, noninvasive technique to detect the mRNAs of ocular genes in healthy individuals. MUC1, MUC2, MUC4, MUC5AC, and MUC7 mucin genes could be all detected in each CIC sample. This technique may be a useful tool to study the expression of some genes in ocular diseases.