establishing-porcine-ex-vivo-cornea-model-for-studying-drug

Establishing a Porcine Ex Vivo Cornea Model for Studying Drug Treatments against Bacterial Keratitis

When developing novel antimicrobials, the success of animal trials is dependent on accurate extrapolation of antimicrobial efficacy from in vitro tests to ...

L V Prasad Eye Institute

The aim of this review article is to present an overview of some of the seminal work published in the last 18 months (July 2014 to December 2015).

Update on fungal keratitis.

Corneal endothelial dystrophy is a progressive disease with gradual loss of vision and characterized by degeneration and dysfunction of corneal endothelial cells. Mutations in SLC4A11, a Na dependent OH transporter, cause congenital hereditary endothelial dystrophy (CHED) and Fuchs' endothelial corneal dystrophy (FECD), the two most common forms of endothelial degeneration. Along with genetic factors, oxidative stress plays a role in pathogenesis of several corneal diseases. In this study we looked into the role of SLC4A11 in antioxidant stress response in human corneal endothelial cells (HCEnC). We found increased expression of SLC4A11 in presence of oxidative stress. Depletion of SLC4A11 using targeted siRNA, caused an increase in reactive oxygen species, cytochrome c, lowered mitochondrial membrane potential, and reduced cell viability during oxidative stress. Moreover, SLC4A11 was found to be necessary for NRF2 mediated antioxidant gene expression in HCEnC. On the other hand, over expression of SLC4A11 reduces reactive oxygen species levels and increases cell viability. Lastly, CHED tissue specimens show evidence of oxidative stress and reduced expression of NRF2. In conclusion, our data suggests a possible role of SLC4A11 in regulating oxidative stress, and might be responsible for both the etiology and treatment of corneal endothelial dystrophy.

SLC4A11 depletion impairs NRF2 mediated antioxidant signaling and increases reactive oxygen species in human corneal endothelial cells during oxidative stress.

 is the leading cause of bacterial keratitis in the developing world with a growing trend of acquiring resistance against various antibiotics. In the current study, we determined the expression of different antimicrobial peptides (AMPs) in response to  in patients, as well as in primary and immortalized human corneal epithelial cells. We further focused on LL-37 and determined its expression in human cornea infected with  and studied the killing ability of LL-37 against  The expression of AMPs was determined by quantitative PCR and the phosphorylation of signaling proteins was evaluated by immunoblot analysis. LL-37 expression was also determined by immunofluorescence and Western blot method and the killing ability of LL-37 against  was determined by colony-forming units. Differential expression of antimicrobial peptides was observed in patients with  keratitis. Although  induced expression of the AMPs in human corneal epithelial cells (HCEC), it did not induce AMP expression in U937, a human monocyte cell line.  also caused activation of nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB)and mitogen activated protein kinase (MAPK) pathways in corneal epithelial cells. LL-37 was found to be effective against both laboratory and clinical strains of . LL-37 induction by  in human corneal epithelial cells was mediated by signal transducer and activator of transcription 3 (STAT3) activation, and inhibition of STAT3 activation significantly reduced LL-37 expression. Our study determines an extensive profile of AMPs expressed in the human cornea during  infection, and suggests the potential of LL-37 to be developed as an alternative therapeutic intervention to fight increasing antibiotic resistance among bacteria.

Differential Expression of Antimicrobial Peptides in Streptococcus pneumoniae Keratitis and STAT3-Dependent Expression of LL-37 by Streptococcus pneumoniae in Human Corneal Epithelial Cells.

Topical application of poorly water-soluble antibiotics cannot achieve the desired therapeutic concentration within cornea. The purpose of this study was to fabricate, characterize and evaluate in-vivo effectiveness of amphotericin B (AmB) containing microneedle ocular patch (MOP) against fungal keratitis. MOP containing free or liposomal AmB was fabricated using micromolding technique to mimic contact lens. MOPs were prepared using dissolvable polymeric matrix including polyvinyl alcohol and polyvinyl pyrrolidone. AmB loaded MOP were studied for their physical and mechanical properties, drug loading and dissolution rate, corneal insertion and drug permeability. MOP loaded with 100 µg AmB had a compression strength of 35.1 ± 6.7 N and required an insertional force of 1.07 ± 0.17 N in excised human cornea. Ex-vivo corneal permeation studies revealed significant enhancement in AmB corneal retention with the application of MOP compared with free AmB or liposomal AmB application. Furthermore, AmB loaded MOP application significantly (P < 0.05) reduced the Candida albicans load within cornea as evaluated in both ex-vivo model and in-vivo rabbit infection model. Histological examination showed that AmB MOP treatment improved the epithelial and stromal differentiation of corneal membrane. AmB containing MOPs can be developed as minimally invasive corneal delivery device for effective treatment of fungal keratitis.

Article	Year
Update on fungal keratitis. Current opinion in ophthalmology\| PubMed ID: 27093101	2016
SLC4A11 depletion impairs NRF2 mediated antioxidant signaling and increases reactive oxygen species in human corneal endothelial cells during oxidative stress. Scientific reports\| PubMed ID: 28642546	2017
Differential Expression of Antimicrobial Peptides in Streptococcus pneumoniae Keratitis and STAT3-Dependent Expression of LL-37 by Streptococcus pneumoniae in Human Corneal Epithelial Cells. Pathogens (Basel, Switzerland)\| PubMed ID: 30845777	2019
Amphotericin B containing microneedle ocular patch for effective treatment of fungal keratitis. International journal of pharmaceutics\| PubMed ID: 31678387	2019

Sanhita Roy

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