Time course studies on the functional evaluation of experimental chronic myocardial infarction in rats

In vivo models of myocardial infarction induced by coronary artery ligation (CAL) in rats usually suffer from high early mortality and a low rate of induction. This study investigated the time course initiation of chronic myocardial infarction (CMI) in albino rats and the possibility of reducing early mortality rate due to myocardial infarction by modification of the surgical technique. CAL was carried out by passing the suture through the epicardial layer around the midway of the left anterior descending coronary artery including a small area of the myocardium to avoid mechanical damage to the heart geometry. In addition, the role of endothelin-1 (ET-1) in rat heart with congestive heart failure was critically assessed. Time course initiation experiments were designed by sacrificing the animals at different time intervals and by carrying out physiological, biochemical, histopathological, electron microscopical and immunohistochemical studies. Specific markers of myocardial injury, viz. cardiac troponin-T (cTnT), high sensitivity C-reactive protein, lactate dehydrogenase and fibrinogen were measured at different time points. Serum marker enzymes and activities of lysosomal hydrolases were found to be elevated on the eighth day post-ligation. Histopathological studies demonstrated focal areas showing fibrovascular tissue containing fibroblasts, collagenous ground substance and numerous small capillaries replacing cardiac muscle fibers. Transmission electron micrographs exhibited mitochondrial changes of well-developed irreversible cardiac injury, viz. swelling, disorganization of cristae, appearance of mitochondrial amorphous matrix densities, significant distortion of muscle fibers and distinct disruption of the intercalated discs. Immunoblotting studies confirmed the presence of alpha 2-macroglobulin which supported the inflammatory response. The severity of the CMI was inferred by the measurement of the level of ET-1 in plasma and left ventricle which was significantly higher in the CMI rats than in the sham-operated rats. Immunohistochemical studies at different time intervals showed that there was a significant immunoexpression of ET-1 on the eighth day post-ligation. This study conclusively showed that ligation of left anterior descending artery minimized mortality and ET-1 was expressed during CMI.     

Low frequency and low intensity pulsed electromagnetic field exerts its antiinflammatory effect through restoration of plasma membrane calcium ATPase activity

Rheumatoid arthritis (RA) is a chronic inflammatory disorder affecting 1% of the population worldwide. Pulsed electromagnetic field (PEMF) has a number of well-documented physiological effects on cells and tissues including antiinflammatory effect. This study aims to explore the antiinflammatory effect of PEMF and its possible mechanism of action in amelioration of adjuvant induced arthritis (AIA). Arthritis was induced by a single intradermal injection of heat killed Mycobacterium tuberculosis at a concentration of 500 μg in 0.1 ml of paraffin oil into the right hind paw of rats. The arthritic animals showed a biphasic response regarding changes in the paw edema volume. During the chronic phase of the disease, arthritic animals showed an elevated level of lipid peroxides and depletion of antioxidant enzymes with significant radiological and histological changes. Besides, plasma membrane Ca²⁺ ATPase (PMCA) activity was inhibited while intracellular Ca²⁺ level as well as prostaglandin E₂ levels was noticed to be elevated in blood lymphocytes of arthritic rats. Exposure of arthritic rats to PEMF at 5 Hz × 4 μT × 90 min, produced significant antiexudative effect resulting in the restoration of the altered parameters. The antiinflammatory effect could be partially mediated through the stabilizing action of PEMF on membranes as reflected by the restoration of PMCA and intracellular Ca²⁺ levels in blood lymphocytes subsequently inhibiting PGE₂ biosynthesis. The results of this study indicated that PEMF could be developed as a potential therapy for RA in human beings.     

Effect of a derivatized tetrapeptide from lactoferrin on nitric oxide mediated matrix metalloproteinase-2 production by synovial fibroblasts in collagen-induced arthritis in rats

Matrix metalloproteinases (MMPs) constitute a family of zinc-dependent proteolytic enzymes, which degrade several components of extracellular matrix, in arthritic synovial cells. In cultured synovial fibroblasts, both nitric oxide (NO) and reactive oxygen species (ROS) are potent inducers of MMPs production. PEP1261, a tetrapeptide derivative used in this study, corresponds to residues of 39-42 human lactoferrin. The parent protein lactoferrin is able to inhibit the production of free radicals in rheumatoid joints and it regulates many aspects of inflammation. This study is aimed to examine the effects of PEP1261 on MMP-2 production in the presence of nitric oxide donor in cultured synovial fibroblasts from collagen-induced arthritic rats. PEP1261 affects a significant reduction in nitrite levels as well as in MMP-2 production in SNAP stimulated synovial fibroblasts and this is validated by gelatin zymography and immunoblot analysis. Furthermore, RTPCR analysis has demonstrated that PEP1261 inhibits MMP-2 mRNA expression in SNAP treated synovial fibroblasts. The results of this study suggest that PEP1261 possesses antiarthritic activity by inhibiting nitrite levels as well as MMP-2 expression better than control peptides viz., KRDS and RGDS.     

Inhibition of nitric oxide and caspase-3 mediated apoptosis by a tetrapeptide derivative (PEP1261) in cultured synovial fibroblasts from collagen-induced arthritis

In this study, the effect of (Boc-Lys (Boc)-Arg-Asp-Ser (tBu)-OtBu), a tetrapeptide derivative (PEP1261) was examined for antiproliferative potency and apoptotic induction. Synovial fibroblasts were isolated from collagen-induced arthritic (CIA) rats and exposed to peptides viz., PEP1261, and parental peptides (KRDS and RGDS). Viability of the cells decreased in the presence of PEP1261 at a lower concentration (0.1 mM) when compared to RGDS and KRDS (1 mM). The treatment of cells with peptides showed induction of apoptosis, resulting in the cleavage of caspase-3 as well as its substrate poly-(ADP-ribose) polymerase (PARP). Pretreatment of cells with caspase-3 inhibitor prevented inhibition of [³H] thymidine incorporation, DNA fragmentation, and cleavage of caspase-3 and PARP as confirmed by western blotting as well as annexin-V/PI-staining using flow cytometry. However, caspase-1 and caspase-2 inhibitors did not prevent the peptides from inducing apoptosis indicating that caspase-3 might have a role in the process of apoptosis induced by peptides. Treatment of synovial fibroblasts with nitric oxide donor, S-nitroso-N-acetyl-dl-penicillamine (SNAP) (500 μM) showed significant elevation of nitric oxide levels and resulted in absence of apoptosis by preventing the inhibition of [³H] thymidine incorporation. This was further evidenced by annexin V/propidium iodide (PI) staining and absence of DNA fragmentation, intra cellular caspase-3 activity and PARP cleavage. In contrast, SNAP followed by PEP1261 and parental peptides-induced apoptosis by lowering the levels of nitric oxide. These results suggested that PEP1261 suppressed the proliferation and induced apoptosis in cultured synovial fibroblasts from CIA rats. This study also confirmed that PEP1261 inhibited nitric oxide level in cultured synovial fibroblasts.     

Dexamethasone induced alterations in enzymatic and nonenzymatic antioxidant status in heart and kidney of rats

This study was designed to investigate the alterations in thiobarbituric acid reactants (TBA-reactants) and enzymatic and nonenzymatic antioxidant levels induced by dexamethasone (Dex) in heart and kidney and to find out whether these alterations induced by Dex and its hypertensive effect had any role in the maintenance of hypertension in this model. Administration of dexamethasone induced severe loss of body weight, significant increase in heart and kidney weights and also marked electrocardiographic changes. The protein content in heart and kidney increased significantly during Dex administration and returned to near normalcy after withdrawal. Total activity of lactate dehydrogenase showed a significant increase in heart till day 8 of treatment, whereas in serum, it exhibited a significant decrease. The activity of CK in heart showed an increase till day 8 of treatment and approached normalcy thereafter. In serum, CK exhibited a decrease till day 8, remaining insignificant thereafter. CKMB in heart showed an insignificant increase initially, reaching normal levels on Dex withdrawal, whereas in serum, it showed a significant decrease throughout the experimental period. Mean arterial pressure (MAP) and heart rate increased significantly, while a significant elevation in the ST segment was noticed during administration as well as after withdrawal of dex. The TBA-reactants levels were found to increase in heart and kidney during days 12 and 16 of administration with Dex and even after withdrawal of Dex, the levels were insignificantly elevated. The level of glutathione in heart and kidney increased from day 4 onwards and reached normalcy during the later stages of treatment and after withdrawal of Dex. The total sulfhydryl groups exhibited a significant increase in both heart and kidney throughout the experiment. The antioxidant enzymes such as catalase, superoxide dismutase, glutathione peroxidase and glutathione S-transferase exhibited a significant decrease in heart during Dex administration whereas, in kidney, they exhibited a significant increase during treatment and after withdrawal of Dex. Thus, Dex induced rise in mean arterial pressure, significant alterations in electrocardiographic parameters and also marked alterations in enzymatic and nonenzymatic antioxidant levels and in the TBA-reactants level in heart and kidney.     

Curcumin modulates free radical quenching in myocardial ischaemia in rats

This study was designed to investigate the protective effect of curcumin (CUR) against isoprenaline induced myocardial ischaemia in rat myocardium. The effect of single oral dose of curcumin (15 mg kg(-1)), administered 30 min before and/or after the onset of ischaemia, was investigated by assessing oxidative stress related biochemical parameters in rat myocardium. Curcumin pre and post-treatment (PPT) was shown to decrease the levels of xanthine oxidase, superoxide anion, lipid peroxides (LPs) and myeloperoxidase while the levels of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione-S-transferase (GST) activities were significantly increased after curcumin PPT. Histopathological and transmission electron microscopical studies also confirmed the severe myocardial damage occurring as a consequence of isoprenaline induced ischaemia and they also showed the significant improvement effected by curcumin PPT. These findings provided evidence that curcumin was found to protect rat myocardium against ischaemic insult and the protective effect could be attributed to its antioxidant properties as well as its inhibitory effects on xanthine dehydrogenase/xanthine oxidase (XD/XO) conversion and resultant superoxide anion production.     

Curcumin treatment modulates collagen metabolism in isoprot3erenol induced myoxardial necrosis in rats

This study was carried out to evaluate whether curcumin, a potent antioxidant, had any specific role in the synthesis and degradation of collagen in rat heart with mocardial necrosis, induced by isoproterenol.HCI (ISO). Myocardial necrosis was induced by administration of ISO (30 mg/100 g body weight subcutaneously twice at an interval of 24 h) and studies on collagen metabolism were carried out with curcumin (200 mg/kg) pre-and co-treatment with ISO. The incorporation of ₁₄C-proline into collagen was studied as an index of collagen synthesis. The heart weight /body weight ratio,heart RNA/DNA ratio and protein were found to increase significantly in ISO administered animals. Curcumin pre- and co-treatment with ISO reversed these changes and attenuated the development of cardiac hypertrophy two weeks after the second dose of ISO. Increased fractional synthesis rate and enhanced degradation of newly synthesized collagen were observed in ISO administered animals. Curcumin pre- and co-treatment with ISO was noticed to decrease the degree of degradation of the existing collagen matrix and collagen synthesis, two weeks after the second dose of ISO. The observed effects could be due to free radical scavenging capacity and inhibition of lysosomal enzyme release by curcumin.     

Protective role of curcumin against isoproterenol induced myocardial infarction in rats

The effect of curcumin on the biochemical changes induced by isoproterenol (ISO) administration in rats was examined. ISO (300 mg Kg^–1 administered subcutaneously twice at an interval of 24 h) caused a decrease in body weight and an increase in heart weight, water content as well as in the levels of serum marker enzymes viz creatine kinase (CK), lactate dehydrogenase (LDH) and LDH₁ isozyme. It also produced electrocardiographic changes such as increased heart rate, reduced R amplitude and ST elevation. Curcumin at a concentration of 200 mg.Kg^–1 when administered orally, showed a decrease in serum enzyme levels and the electrocardiographic changes got restored towards normalcy. Myocardial infarction was accompanied by the disintegration of membrane polyunsaturated fatty acids expressed by increase of thiobarbituric acid reactive substance (TBARS), a measure of lipid peroxides and by the impairment of natural scavenging, characterized by the decrease in the levels of superoxide dismutase, catalase, glutathione peroxidase, ceruloplasmin, alpha tocopherol, reduced glutathione (GSH) and ascorbic acid. The oral pretreatment with curcumin two days before and during ISO administration decreased the effect of lipid peroxidation. It was shown to have a membrane stabilizing action by inhibiting the release of -glucuronidase from nuclei, mitochondria, lysosome and microsome. Curcumin pre- and co-treatment decreased the severity of pathological changes and thus, could have a protective effect against the damage caused by myocardial infarction (MI).     

Studies on the immobilization of trypsin on sand

Trypsin was immobilized on sand using five different methods. Attempts were made to attach amino-functional groups onto sand using 3-aminopropyltriethoxysilane, hexamethylenetetramine, hexamethylenediamine, and melamine. Glutaraldehyde was used as a bifunctional agent in all the methods. Methods for the estimation of the proteolytic 1activity and protein content of immobilized trypsin were standardized. The maximum retained activity was observed for trypsin immobilized on sand via 3-aminopropytriethoxysilane and glutaraldehyde. Immobilized trypsin showed a shift in the pH optimum toward the acidic side over that of soluble trypsin in all five cases. The optimum temperature for both native and immobilized trypsin prepared by the silane-glutaraldehyde method was found to be 45°C. However, the pH and thermal stabilities of immobilized trypsin were observed to be better than that of the native enzyme.