"" Histological and Immunohistochemical Studies on the Cornea and Retina of Sofosbuvir Treated Rats

Research Article

Austin J Anat. 2017; 4(2): 1068.

Histological and Immunohistochemical Studies on the Cornea and Retina of Sofosbuvir Treated Rats

Issa NM and El-Sherif NM*

Department of Anatomy and Embryology, Faculty of Medicine, Menoufia University, Egypt

*Corresponding author: El-Sherif NM, Department of Anatomy and Embryology, Faculty of Medicine, Menoufia University, Egypt

Received: February 09, 2017; Accepted: March 06, 2017; Published: March 16, 2017

Abstract

Sofosbuvir (Sovaldi) is used in treatment of hepatitis C infection. The aim of this study is to demonstrate the effect of Sofosbuvir on the cornea and retina of adult male albino rat. Twenty adult male albino rats were used in this experiment. Animals were divided in two groups: Group I (Control) included ten rats were kept without any treatment throughout the experimental period and Group II (Sovaldi treated) included ten rats that received Sofosbuvir in the form of tablets with the trade name ‘Gratisovir’. Each tablet contained 400 mg of Sofosbuvir; these tablets were dissolved in distilled water and given for rats orally by gastric tube in a dose of 4mg/kg per day for 5 weeks. Sofosbuvir results in histological and immunohistochemical changes in cornea and retina of Sovaldi treated rats.

Keywords: Sofosbuvir; Cornea; Retina; E-cadherin; Fibronectin

Introduction

Sofosbuvir is a nucleotide analogue polymerase inhibitor with the same in vitro activity against all hepatitis c virus genotypes. Before the discovery of Sofosbuvir, different nucleoside analogs had been tested as antihepatitis C treatments, but these showed relatively low potency. This low potency comes in part because the enzymatic addition of the first of the three phosphate groups of the triphosphate is slow. The structure of Sofosbuvir, based on the protide approach, avoids this slow step by building the first phosphate group into the design of the drug during synthesis. Additional groups are attached to the phosphorus to temporarily mask the two negative charges of the phosphate group, thereby allowing entry of the drug into the infected cell [1]. Sofosbuvir and other nucleotide inhibitors of the HCV RNA polymerase show great difficulty for resistance development. This is an important advantage relative to other drugs of HCV that target other viral enzymes such as the protease, for which rapid development of resistance occurred, that lead to therapeutic failure [2]. It was reported that the Sovaldi treatment displays common side effects as chest pain, migraine, memory impairment, dyspnea, gastrointestinal reflux, alopecia, depression, muscle spasm and blurred vision [3]. Moreover vision loss seems to be a growing side effect of Sovaldi [4].

Methodology

Animals

Twenty adult male albino rats were used in this experiment, each of them is weighting 170-200 grams were used in this experiment. Food and water were provided ad libitum for one week before use in the anatomy department, faculty of medicine, Menoufia University. All aspects of animal care and treatment were carried out according to the local guidelines of the ethical committee for animal research.

Experimental design

The animals were divided in two groups: Group I (Control): included ten rats were kept without any treatment throughout the experimental period.

Group II (Sovaldi treated): included ten rats that received Sofosbuvir (Sofosbuvir, a product of Pharco Pharmaceuticals, Alexandria, Egypt, was available in the form of tablets with the trade name ‘Gratisovir’. Each tablet contained 400 mg of Sofosbuvir; these tablets were dissolved in distilled water and given for rats orally by gastric tube in a dose of 4 mg/kg per day for 5 weeks.

Histological study

Light microscope: The specimens of the two organs from the two groups were fixed in 10% neutral-buffered formalin, dehydrated in a graded ethanol series, cleared in xylene, and embedded in paraffin wax. Tissue sections of 5–7 μm thickness were cut using a rotating microtome, deparaffinized with xylene, and stained with H&E for LM examination of the corneas and retinas.

Immunohistochemical stains: Five micrometer sections were mounted, dried, and dewaxed in xylene. Rehydrated sections were then incubated with 3% hydrogen peroxide in humidified boxes to block the endogenous activity of peroxidase. Microwave-assisted antigen retrieval was performed for 20 min. Sections were then incubated overnight at 4C with:

1. Primary mouse monoclonal antirat E-cadherin antibody, (NeoMarkers/Lab Vision, Fremont, California, USA).

2. Primary rabbit antirat fibronectin antibody, (NeoMarkers/ Lab Vision, Fremont, California, USA) (Mouse monoclonal antibody).

After washing with phosphate-buffered solution, sections were incubated with biotinylated antirabbit secondary antibody for 30 min and then with streptavidin peroxidase conjugate for 30 min. Sections were then washed with phosphate buffer solution, and incubated with diaminobenzidine chromogen to detect immunoreactivity. Counterstaining was performed by Mayer’s hematoxylin. Positive reaction was visualized as brown coloration.

Morphometric and statistical analysis

A detailed morphometric analysis of the area percent of fibronectin and e cadherin expression was assessed in the ocular tissues of the different sections from each slide of different animals at a magnification of x400. This was performed using a Leica Qwin 500 MCO image analysis system (Wetzlar, Germany). Values of the measured parameters were presented as mean±standard deviation. The statistical analysis was processed according to the conventional procedures using the Statistical Program of Social Sciences (SPSS) software for windows (version 10.0) (IBM, USA).

Results

Cornea results

H&E light microscopy: Corneal examination of the control group showed normal histological structure formed of many layers: the outer stratified squamous nonkeratinized corneal epithelium, Bowman’s membrane, the stroma (substantia propria) and Descemet’s membrane (Figure 1). Corneal examination of SOV treated group showed focal areas of desquamation and separation of the epithelium. Some areas showed loss of corneal epithelium with apparent decrease in thickness of the epithelium. Corneal stromal collagen bundles were separated by multiple wide spaces, indicating corneal edema and showed cellular infiltration (Figures 2 and 3).