Altered gastrointestinal motility in an animal model of Lesch-Nyhan disease

Abstract

Mutations in the HGPRT1 gene, which encodes hypoxanthine-guanine phosphoribosyltransferase (HGprt), housekeeping enzyme responsible for recycling purines, lead to Lesch-Nyhan disease (LND). Clinical expression of LND indicates that HGprt deficiency has adverse effects on gastrointestinal motility. Therefore, we aimed to evaluate intestinal motility in HGprt knockout mice (HGprt¯). Spontaneous and neurally evoked mechanical activity was recorded in vitro as changes in isometric tension in circular muscle strips of distal colon. HGprt¯ tissues showed a lower in amplitude spontaneous activity and atropine-sensitivity neural contraction compared to control mice. The responses to carbachol and to high KCl were reduced, demonstrating a widespread impairment of contractility. L-NAME was not able in the HGprt¯ tissues to restore the large amplitude contractile activity typical of control. In HGprt¯ colon, a reduced expression of dopaminergic D1 receptor was observed together with the loss of its tonic inhibitory activity present in control-mice. The analysis of inflammatory and oxidative stress in colonic tissue of HGprt¯ mice revealed a significant increase of lipid peroxidation associated with over production of oxygen free radicals. In conclusion, HGprt deficiency in mice is associated with a decrease in colon contractility, not dependent upon reduction of acetylcholine release from the myenteric plexus or hyperactivity of inhibitory signalling. By contrast the increased levels of oxidative stress could partially explain the reduced colon motility in HGprt¯ mice. Colonic dysmotility observed in HGprt¯ mice may mimic the gastrointestinal dysfunctions symptoms of human syndrome, providing a useful animal model to elucidate the pathophysiology of this problem in the LND.

Anti-inflammatory and antioxidant effects of muscarinic acetylcholine receptor (mAChR) activation in the rat hippocampus

“…Concentrations of IL-1β, IL-6, IL-10 and TGF-β1 were measured in the hippocampus homogenates (20 mg of tissue sample) using enzyme-linked immunosorbent assay ( ELISA) kits for rat (Cloud-Clone Corp, Wuhan, Hubei) according to the manufacturers’ protocols and as reported in Zizzo et al . 96 and in Mudò et al . 95 .…”

Anti-inflammatory and cognitive effects of interferon-β1a (IFNβ1a) in a rat model of Alzheimer’s disease

“…Concentrations of interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-10 (IL-10), and transforming growth factor-beta1 (TGF-β1) were measured in the hippocampus homogenates (20 mg of tissue sample) using enzyme-linked immunosorbent assay (ELISA) kits for rat (Cloud-Clone Corp, Wuhan, Hubei) according to the manufacturers’ protocols and as reported by Zizzo et al [34].…”

“…Samples were then incubated with 1 mM dichlorofluorescein diacetate (DCFH-DA) for 10 min at room temperature in the dark. The conversion of non-fluorescent DCFH-DA to the highly fluorescent compound 20,70-dichlorofluorescein (DCF) by esterase activity was used to monitor the presence of peroxides due to the oxidative burst in the brain [34]. The samples were analyzed by fluorimeter (Microplate reader WallacVictor 2 - 1420 Multilabel Counter; PerkinElmer, Inc.), using the excitation filter set at 485 nm and the emission filter set at 530 nm.…”

“…In the supernatant, total proteins were quantified by the Lowry method [32]. Volume corresponding to 50 μg of protein was used for total SOD enzymatic activity measurement, by using the SOD assay kit (Sigma–Aldrich) according to manufacturer’s instructions [34]. Absorbance was measured by using the iMark™ Microplate Absorbance Reader at 450 nm.…”

“…The lipid peroxidation assay kit (Sigma Aldrich) was used to detect the concentration of malondialdehyde (MDA), a final product of lipids peroxidation. Ten milligrams of hippocampal tissues was homogenized in 300 μl of MDA lysis buffer (supplied in the kit), and colorimetric reaction with thiobarbituric acid (TBA) was read on an iMark™ Microplate Absorbance Reader at 532 nm, according to manufacturer’s instructions [34].…”