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Correlation of nitric oxide levels in the cerebellum and spinal cord of experimental autoimmune encephalomyelitis rats with clinical symptoms

100%

Ljubisavljevic S. , Stojanovic I. , Pavlovic D. , Milojkovic M. , Vojinovic S. , Sokolovic D. , Stevanovic I.

Acta Neurobiologiae Experimentalis

2012

tom 72

nr 1

Experimental autoimmune encephalomyelitis (EAE) is a well-established cell-mediated autoimmune inflammatory disease of the CNS, which has been used as a model of the human demyelinating disease. EAE is characterized by infiltration of the CNS by lymphocytes and mononuclear cells, microglial and astrocytic hypertrophy, and demyelination which cumulatively contribute to clinical expression of the disease. EAE was induced in female Sprague-Dawley rats, 3 months old (300 g ± 20 g), by immunization with myelin basic protein (MBP) in combination with Complete Freund's adjuvant (CFA). The animals were divided into 7 groups: control, EAE, CFA, EAE + aminoguanidine (AG), AG, EAE + N-acetyl-L-cysteine (NAC) and NAC. The animals were sacrificed 15 days after EAE induction, and the level of nitric oxide (NO') production was determined by measuring nitrite and nitrate concentrations in 10% homogenate of cerebellum and spinal cord. Obtained results showed that the level of NO' was significantly increased in all examined tissues of the EAE rats compared to the control and CFA groups. Also, AG and NAC treatment decreased the level of NO' in all tissues compared to the EAE group. The level of NO' is increased significantly in the spinal cord compared to the cerebellum. The clinical course of the EAE was significantly decreased in the EAE groups treated with AG and NAC during the development of the disease compared to EAE group and its correlates with the NO' level in cerebellum and spinal cord. The findings of our work suggest that NO' and its derivatives play an important role in multiple sclerosis (MS). It may be the best target for new therapies in human demyelinating disease and recommend the new therapeutic approaches based on a decreased level of NO' during the course of MS.

The simple isocratic HPLC-UV method for the simultaneous determination of reduced and oxidized glutathione in animal tissue

84%

Begic A. , Djuric A. , Gobeljic B. , Stevanovic I. , Lukic V. , Stanojevic I. , Ninkovic M. , Saso L. , Vojvodic D. , Djukic M.

Acta Chromatographica

2017

tom Vol. 29, no. 1

67--84

The aim of our work was to optimize and apply simple high-performance liquid chromatography method with ultraviolet detection (HPLC-UV) for simultaneous determination of reduced (GSH) and oxidized (GSSG) glutathione in biological matrix (specifically, the rat liver tissue was used herein), since the ratio between oxidized and reduced glutathione forms (GSSG-GSH) has been recognized as an important biological marker of oxidatively depleted GSH in oxidative stress (OS)-associated diseases and poisonings. An isocratic chromatographic separation of GSH and GSSG (2.8 min and 6.3 min, respectively) was performed with the mobile phase consisted of sodium perchlorate solution (pH adjusted to 2.8) at flow rate of 1 mL min−1, detection set at 215 nm, and column temperature of 40 °C. The method offers short run time, linearity in the range of 0.01-200 μM concentration for both compounds (R2 = 1), low limits of detection and quantification (GSH: 0.18 μM and 0.56 μM, GSSG: 0.52 μM and 1.58 μM, respectively), precision, accuracy (bias < 2%), and high reproducibility. Through suitable sample handling, an overestimation of GSSG was prevented. High recovery (>99%) was achieved. The method was successfully applied for the analysis of GSH and GSSG in liver homogenates of Wistar rats intraperitoneally exposed to cadmium (Cd) (1 mg kg−1 CdCl2/21 days). Regardless of other Cd-mediated hepatotoxicity mechanisms, herein, we have exclusively interpreted/emphasized oxidative GSH depletion. The presented method is acceptable for a routine analysis of GSH and GSSG in biological matrix, while the calculated ratio GSSG-GSH is considered as a valuable OS marker.