Purpose: Ocular barriers employ drug transporters that play an important role in maintaining homeostasis in the eye. The solute carrier (SLC) and ATP-binding cassette (ABC) transporters present comprise proteins involved in the influx and efflux of drugs, hormones, and other biologically active compounds. However, ocular inflammations cause alterations in these ocular barriers but the detailed molecular mechanism underlying these inflammation-induced alterations is unknown. We hypothesize that the dysregulations in the gene expression levels of the transporters in response to inflammation have a role in the ocular barriers modulation. So, the aim of this study was to evaluate the status of various drug transporters in the experimental model of ocular inflammation.
Methods: Wistar rats of either sex (n=6) were used for this study. Endotoxin (LPS) was administered at the dose of 200 µg into the hind paw to establish an experimental model of ocular inflammation and saline was injected in control animals. The model was validated by analyzing the proinflammatory cytokine levels, functional assessment by electroretinogram (ERG) and morphological examinations done by slit lamp biomicroscope and fundoscopy using MICRON III imaging system. Then the model was checked for the suitability for drug transporter studies using a probe (Oat substrate). At 22nd hr both control and the rats challenged with LPS were injected with fluorescein sodium at the dose of 7.44µM. Aqueous humor was collected at 24 hrs for the quantitation of fluorescein using a spectrofluorometric method. Expression analysis of 15 transporters belonging to 5 subcategories viz., Organic anion transporter (Oat), Organic cation transporter (Oct), Nucleoside transporters (Nts), Peptide transporters (Pept), PGP efflux transporter (Abc1b) of SLC and ABC superfamily was done in the ocular barriers (Cornea, Iris Ciliary body, Retina-Choroid) by qRT-PCR at 24 hrs post LPS challenge. Fold change in the gene expression was calculated by a 2-ΔΔCt method.
Results: The aqueous humor levels of the proinflammatory cytokines were significantly higher in the inflammatory model as compared to the controls. The ‘b’ wave in ERG has shown a significant decrease in the amplitude and the image analysis revealed miosis with extensive vasodilatation in the inflammatory model as compared to the controls. Suitability assays revealed higher levels of sodium fluorescein in the inflammatory model. In the blood-ocular barriers, P-gp upregulated, Cnt1, Cnt3, and Ent1 were downregulated in the cornea as shown in figure 1; Cnt1, Ent1, P-gp upregulated, Cnt2, Cnt3, Oat1, Oat2, Oat3, Oct1, Oct3, and Pept2 were downregulated in the Iris ciliary body as shown in figure 2; Cnt3, Ent2, Oat3, Oct2, P-gp1, and P-gp2 were upregulated in the Retina-Choroid as shown in figure 3, significantly. All the values were found significantly different (p≤0.05) in comparison with respective control.
Conclusion: This study validated the LPS model as a suitable experimental model for ocular inflammation. Inflammation dysregulated the expression levels of drug transporters present on ocular barriers. So, the alterations in the ocular barriers in inflammatory conditions may be attributed to the dysregulation in the gene expression levels of these transporters. Further studies are warranted to elucidate the functional importance of the altered expression levels of the drug transporters in ocular inflammation.
Nabanita Halder– Associate Professor, Ocular Pharmacology and Pharmacy Division, Dr. R. P. Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi, India, New Delhi, Delhi
Atul Kumar– Chief and Professor of Ophthalmology, Department of Ophthalmology, Dr. Rajendra Prasad Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi, India, New Delhi, Delhi
Sundarajan Singh– Senior Scientist-IV, Department of Biophysics, All India Institute of Medical Sciences, New Delhi, India, New Delhi
Rohan Chawla– Assistant Professor, Department of Ophthalmology, Dr. R. P. Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi, New Delhi, Delhi
Velpandian Thirumurthy– Professor and In-Charge, Ocular Pharmacology and Pharmacy Division, Dr. R. P. Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi, India, New Delhi, Delhi