HHcy Animals Research Articles

Alzheimer's Disease-like Pathological Features in the Dorsal Hippocampus of Wild-Type Rats Subjected to Methionine-Diet-Evoked Mild Hyperhomocysteinaemia.

Multifactorial interactions, including nutritional state, likely participate in neurodegeneration's pathogenesis and evolution. Dysregulation in methionine (Met) metabolism could lead to the development of hyperhomocysteinaemia (hHcy), playing an important role in neuronal dysfunction, which could potentially lead to the development of Alzheimer's disease (AD)-like pathological features. This study combines proton magnetic resonance spectroscopy (1H MRS) with immunohistochemical analysis to examine changes in the metabolic ratio and histomorphological alterations in the dorsal rat hippocampus (dentate gyrus-DG) subjected to a high Met diet. Male Wistar rats (420-480 g) underwent hHcy evoked by a Met-enriched diet (2 g/kg of weight/day) lasting four weeks. Changes in the metabolic ratio profile and significant histomorphological alterations have been found in the DG of hHcy rats. We have detected increased morphologically changed neurons and glial cells with increased neurogenic markers and apolipoprotein E positivity parallel with a diminished immunosignal for the N-Methyl-D-Aspartate receptor 1 in hHcy animals. A Met diet induced hHcy, likely via direct Hcy neurotoxicity, an interference with one carbon unit metabolism, and/or epigenetic regulation. These conditions lead to the progression of neurodegeneration and the promotion of AD-like pathological features in the less vulnerable hippocampal DG, which presents a plausible therapeutic target.

Hydrogen sulfide attenuates hyperhomocysteinemia-induced blood-brain barrier permeability by inhibiting MMP-9

Backgroud: Hyperhomocysteinemia (HHcy) is implicated in various neurovascular disorders including vascular dementia, subarachnoid hemorrhage and stroke. Elevated homocysteine (Hcy) levels are associated with increased oxidative stress and compromised blood–brain barrier (BBB) integrity. Hydrogen sulfide (H2S) has recently emerged as potent neuroprotective molecule in various neurological conditions including those associated with HHcy. The present study evaluates the protective effect of sodium hydrogen sulfide (NaHS; a source of H2S) on HHcy-induced BBB dysfunction and underpin molecular mechanisms. Materials and methods: Supplementation of NaHS restored the increased BBB permeability in the cortex and hippocampus of HHcy animals assessed in terms of diffused sodium fluorescein and Evans blue tracer dyes in the brain. Activity of matrix metalloproteinases (MMPs) assessed by gelatinase activity and in situ gelatinase assay was restored to the normal in the cortex and hippocampus of HHcy animals supplemented with NaHS. Results: Application of gelatin zymography revealed that specifically MMP-9 activity was increased in the cortex and hippocampus of HHcy animals, which was inhibited by NaHS supplementation. Real-time RT-PCR analysis showed that NaHS administration also decreased mRNA expression of MMP-9 in the hippocampus of HHcy animals. NaHS supplementation was further observed to reduce water retention in the brain regions of Hcy treated animals. Conclusion: Taken together, these findings suggest that NaHS supplementation ameliorates HHcy-induced BBB permeability and brain edema by inhibiting the mRNA expression and activity of MMP-9. Therefore, H2S and H2S releasing drugs may be used as a novel therapeutic approach to treat HHcy-associated neurovascular disorders.

Effect of Methionine Diet on Time-Related Metabolic and Histopathological Changes of Rat Hippocampus in the Model of Global Brain Ischemia.

Hyperhomocysteinemia (hHcy) represents a strong risk factor for atherosclerosis-associated diseases, like stroke, dementia or Alzheimer’s disease. A methionine (Met)-rich diet leads to an elevated level of homocysteine in plasma and might cause pathological alterations across the brain. The hippocampus is being constantly studied for its selective vulnerability linked with neurodegeneration. This study explores metabolic and histo-morphological changes in the rat hippocampus after global ischemia in the hHcy conditions using a combination of proton magnetic resonance spectroscopy and magnetic resonance-volumetry as well as immunohistochemical analysis. After 4 weeks of a Met-enriched diet at a dose of 2 g/kg of animal weight/day, adult male Wistar rats underwent 4-vessel occlusion lasting for 15 min, followed by a reperfusion period varying from 3 to 7 days. Histo-morphological analyses showed that the subsequent ischemia-reperfusion insult (IRI) aggravates the extent of the sole hHcy-induced degeneration of the hippocampal neurons. Decreased volume in the grey matter, extensive changes in the metabolic ratio, deeper alterations in the number and morphology of neurons, astrocytes and their processes were demonstrated in the hippocampus 7 days post-ischemia in the hHcy animals. Our results suggest that the combination of the two risk factors (hHcy and IRI) endorses and exacerbates the rat hippocampal neurodegenerative processes.

Hydrogen sulfide attenuates hyperhomocysteinemia-induced mitochondrial dysfunctions in brain

Hyperhomocysteinemia (HHcy) has been implicated in the development of neurodegenerative conditions and mild cognitive disorders. Mitochondrial dysfunctions are the major mechanisms involved in homocysteine (Hcy)-induced neurotoxicity. Although, hydrogen sulfide has been reported as potent antioxidant, its effects on Hcy-induced mitochondrial dysfunctions have not been studied. Therefore, the present study has been designed to evaluate the protective effect of NaHS on Hcy-induced mitochondrial dysfunctions in brain. NaHS supplementation decreased reactive oxygen species and nitrite levels in the cortex and hippocampus of animals with HHcy. NaHS supplementation increased the activity of mitochondrial electron transport chain components; NADH dehydrogenase, cytochrome c oxidase and F0-F1 ATPase in the cortex and hippocampus of HHcy animals along with in-gel activity of complex I – complex V in the mitochondria isolated from the cortex and hippocampus of HHcy animals. Moreover, NaHS supplementation also increased the mitochondrial complex I, II and IV mediated oxygen consumption rate in Hcy treated mitochondria. NaHS administration prevented the Hcy-induced mitochondrial damage as suggested by the decreased mitochondrial swelling in the cortex and hippocampus of HHcy animals. NaHS supplementation decreased the activity of lactate dehydrogenase isozymes (1–5) in the brain regions of HHcy animals. The expression of protein kinase c δ was also decreased in the brain regions of HHcy animals following NaHS supplementation. This was accompanied by reduced activity of caspase-3 indicating anti-apoptotic effect of H2S. Taken together, the findings suggest that H2S supplementation ameliorates Hcy-induced oxidative stress and mitochondrial dysfunctions suggesting H2S releasing drugs may be a novel therapeutic approach to treat HHcy associated neurological disorders.

Hydrogen sulfide suppresses homocysteine-induced glial activation and inflammatory response

Neuro-inflammation plays a critical role in hyperhomocysteinemia (HHcy)-associated neurodegenerative disorders. Hydrogen sulfide (H2S) has been suggested as an endogenous neuromodulator and potent anti-inflammatory molecule. In present study, we have investigated the effect of NaHS supplementation (a H2S source) on inflammatory response in animals subjected to HHcy. NaHS adminstration restored the decreased levels of H2S and polysulfides with a concomitant increase in the activity of cystathionase (CSE) and cystathionine β‐synthase (CBS) in the brain regions of HHcy animals. NaHS supplementation reduced the expression of glial fibrillary acidic protein (GFAP) and ionized calcium binding adaptor molecule 1 (Iba1) suggesting attenuation of astrocyte and microglia activation in HHcy animals. Tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) were decreased in the cortex and hippocampus of HHcy animals following NaHS supplementation. Moreover, NaHS supplementation also decreased the TNF-α, IL-6 and MCP-1 in the serum of HHcy animals. NaHS supplementation reduced nitrite levels, 3-nitrotyrosine (3-NT) modified proteins and inducible nitric oxide synthase (iNOS) in the cortex and hippocampus of HHcy animals. However, NaHS administration increased endothelial nitric oxide synthase (eNOS) expression in brain regions of Hcy treated animals. Expression of platelet endothelial cell adhesion molecule (PECAM) was decreased in the microvessels from HHcy animals supplemented with NaHS. Furthermore, HHcy-induced memory deficits assessed by Morris water maze and novel object recognition test were reversed by NaHS administration. Taken together, the findings suggest that NaHS supplementation ameliorates Hcy-induced glia mediated inflammatory response and cognitive deficits. Therefore, H2S may be a novel therapeutic molecule to treat HHcy associated neurological disorders and neuro-inflammatory conditions.

Prenatal hyperhomocysteinemia induces oxidative stress and accelerates ‘aging’ of mammalian neuromuscular synapses

Enhanced levels of homocysteine during pregnancy induce oxidative stress and contribute to many age-related diseases. In this study, we analyzed age-dependent synaptic modifications in developing neuromuscular synapses of rats with prenatal hyperhomocysteinemia (hHCY). One of the main findings indicate that the intensity and the timing of transmitter release in synapses of neonatal (P6 and P10) hHCY rats acquired features of matured synaptic transmission of adult rats. The amplitude and frequency of miniature end-plate currents (MEPCs) and evoked transmitter release were higher in neonatal hHCY animals compared to the control group. Analysis of the kinetics of neurotransmitter release demonstrated more synchronized release in neonatal rats with hHCY. At the same time lower release probability was observed in adults with hHCY. Spontaneous transmitter release in neonates with hHCY was inhibited by hydrogen peroxide (H2O2) whereas in controls this oxidant was effective only in adult animals indicating a higher susceptibility of motor nerve terminals to oxidative stress. The morphology and the intensity of endocytosis of synaptic vesicles in motor nerve endings was assessed using the fluorescence dye FM 1-43. Adult-like synapses were found in neonates with hHCY which were characterized by a larger area of presynaptic terminals compared to controls. No difference in the intensity of FM 1-43 fluorescence was observed between two groups of animals. Prenatal hHCY resulted in reduced muscle strength assessed by the Paw Grip Endurance test. Using biochemical assays we found an increased level of H2O2 and lipid peroxidation products in the diaphragm muscles of hHCY rats. This was associated with a lowered activity of superoxide dismutase and glutathione peroxidase. Our data indicate that prenatal hHCY induces oxidative stress and apparent faster functional and morphological "maturation" of motor synapses. Our results uncover synaptic mechanisms of disrupted muscle function observed in hHCY conditions which may contribute to the pathogenesis of motor neuronal diseases associated with enhanced level of homocysteine.

Cerebroprotective effects of hydrogen sulfide in homocysteine-induced neurovascular permeability: Involvement of oxidative stress, arginase, and matrix metalloproteinase-9.

An elevated level of homocysteine (Hcy) leads to hyperhomocysteinemia (HHcy), which results in vascular dysfunction and pathological conditions identical to stroke symptoms. Hcy increases oxidative stress and leads to increase in blood-brain barrierpermeability and leakage. Hydrogen sulfide (H2 S) production during the metabolism of Hcy has a cerebroprotective effect, although its effectiveness in Hcy-induced neurodegeneration and neurovascular permeability is less explored. Therefore, the current study was designed to perceive the neuroprotective effect of exogenous H 2 S against HHcy, a cause of neurodegeneration. To test this hypothesis, we used four groups of mice: control, Hcy, control + sodium hydrosulfide hydrate (NaHS), and Hcy + NaHS, and an HHcy mice model in Swiss albino mice by giving a dose of 1.8 g of dl-Hcy/L in drinking for 8-10 weeks. Mice that have 30 µmol/L Hcy were taken for the study, and a H 2 S supplementation of 20 μmol/L was given for 8 weeks to all groups of mice. HHcy results in the rise of the levels of superoxide and nitrite, although a concomitant decrease in the level of superoxide dismutase, catalase, glutathione peroxidase, reduced glutathione, and arginase in oxidative stress and a concomitant decrease in the endogenous level of H 2 S. AlthoughH 2 S supplementation ameliorated, the effect of HHcy and the levels of H 2 S returned to the average level in HHcy animals supplemented with H 2 S. Interestingly, H 2 S supplementation ameliorated neurovascular remodeling and neurodegeneration. Thus, our study suggested that H 2 S could be a beneficial therapeutic candidate for the treatment of Hcy-associated neurodegeneration, such as stroke and neurovascular disorders.

Neuroprotective Effect of Hydrogen Sulfide in Hyperhomocysteinemia Is Mediated Through Antioxidant Action Involving Nrf2.

Homocysteine (Hcy) is a sulfur-containing amino acid derived from methionine metabolism. Elevated plasma Hcy levels (> 15µM) result in a condition called hyperhomocysteinemia (HHcy), which is an independent risk factor in the development of various neurodegenerative disorders. Reactive oxygen species (ROS) produced by auto-oxidation of Hcy have been implicated in HHcy-associated neurological conditions. Hydrogen sulfide (H2S) is emerging as a potent neuroprotective and neuromodulator molecule. The present study was aimed to evaluate the ability of NaHS (a source of H2S) to attenuate Hcy-induced oxidative stress and altered antioxidant status in animals subjected to HHcy. Impaired cognitive functions assessed by Y-maze and elevated plus maze in Hcy-treated animals were reversed on NaHS administration. Increased levels of ROS, lipid peroxidation, protein carbonyls, and 4-hydroxynonenal (4-HNE)-modified proteins were observed in the cortex and hippocampus of Hcy-treated animals suggesting accentuated oxidative stress. This increase in Hcy-induced oxidative stress was reversed following NaHS supplementation. GSH/GSSG ratio, activity of antioxidant enzymes viz; superoxide dismutase, glutathione peroxidase, glutathione reductase, and glutathione-S-transferase were decreased in Hcy-treated animals. NaHS supplementation, on the otherhand, restored redox ratio and activity of antioxidant enzymes in the brains of animals with HHcy. Further, NaHS administration normalized nuclear factor erythroid 2-related factor 2 expression and acetylcholinesterase (AChE) activity in the brain of Hcy-treated animals. Histopathological studies using cresyl violet indicated higher number of pyknotic neurons in thecortex and hippocampus of HHcy animals, which were reversed by NaHS administration. The results clearly demonstrate that NaHS treatment significantly ameliorates Hcy-induced cognitive impairment by attenuating oxidative stress, improving antioxidant status, and modulating AChE activity thereby suggesting potential of H2S as a therapeutic molecule.

Exercise intervention attenuates hyperhomocysteinemia-induced aortic endothelial oxidative injury by regulating SIRT1 through mitigating NADPH oxidase/LOX-1 signaling

Coronary artery disease (CAD) is a critical cardiovascular disease and a cause of high morbidity and mortality in this world. Hyperhomocysteinemia (HHcy) has been suggested as a risk factor for CAD. In addition, SIRT1 (sirtuin 1) has been reported to play a protective role in a variety of diseases, especially in the cardiovascular system. The main purpose of this study was to investigate the effects of exercise training on apoptosis and inflammation in HHcy animals. We also tested whether exercise protected against Hhcy-induced dysfunction of endothelium through modulation of SIRT1. C57BL mice (8 in each group) were fed with or without 1% L-methionine (w/w) in water for 4 months to induce HHcy. We found that Hhcy repressed SIRT1 and AMPK expression and increased NADPH oxidase activity. Plasma MDA, endothelium LOX-1 and p-p38 were up-regulated by Hhcy induction. NF-κB and it downstream molecules were activated under Hhcy situation, thereby promoting pro-inflammatory responses. Moreover, we also reported that Hhcy caused endothelium apoptosis involving Akt inhibition and mitochondria-dependent apoptotic pathways. Exercise training significantly protected against endothelium from Hhcy caused oxidative injuries. In addition, EX527 (SIRT1 inhibitor) reduced the therapeutic effects by exercise. Our results had indicated that exercise training prevent the development of atherosclerosis through SIRT1 activation and oxidative stress inhibition under Hhcy situation.

Hydrogen sulfide attenuates homocysteine-induced cognitive deficits and neurochemical alterations by improving endogenous hydrogen sulfide levels.

Hyperomocysteinemia (HHcy) has been associated with mild cognitive impairment and dementia. Hydrogen sulfide (H2 S) has been suggested to be an endogenous modulator of neuronal functions. However, the effect and mechanisms involved in beneficial effect of H2 S has not been investigated in homocysteine (Hcy)-induced cognitive deficits. This study has been designed to evaluate the effect of exogenous H2 S on behavioral deficits and neurochemical alterations in HHcy animals. Hcy levels were significantly elevated in plasma, cortex, and hippocampus of Hcy administered animals. A progressive decline in memory functions and increased anxiolytic behavior was observed in HHcy animals. This was accompanied by decrease in endogenous H2 S levels along with decreased activity of cystathionase (CSE) and cystathionine β-synthase (CBS). However, a significant increase in CSE and CBS mRNAs was observed. In addition, the catecholamine and serotonin levels were reduced and the activity of monoamine oxidase A and B were increased in brain regions of HHcy animals. Haematoxylin and eosin staining revealed higher number of pyknotic cells in brain regions of HHcy animals. H2 S administration was found to lower elevated plasma and brain Hcy levels. The activities of CBS, CSE, and levels of H2 S were restored in HHcy animals administered H2 S. Exogenous H2 S also ameliorated behavioral deficits accompanied by significant increase in catecholamines. Histological analysis revealed normal cell morphology in Hcy-treated animals supplemented with H2 S. These results clearly demonstrate that the protective effect of H2 S on Hcy-induced cognitive deficits is mediated through increased catecholamine and H2 S levels thereby suggesting its beneficial role in preventing HHcy-induced neurodegeneration. © 2016 BioFactors, 43(3):434-450, 2017.

Hyperhomocysteinemia Is Associated with Impaired Fracture Healing in Mice

Hyperhomocysteinemia (HHCY) has been shown to disturb bone metabolism and to increase the incidence of osteoporosis and osteoporotic fractures. However, there is a complete lack of information on whether these metabolic alterations affect bone repair. The aim of this study was to analyze the impact of HHCY on fracture healing. One group of mice was fed a homocystine-supplemented diet (n = 12), whereas another group received the accordant standard diet for control (n = 13). Four weeks after the stable fixation of a closed femoral fracture, animals were killed to prepare bones for histomorphometric and biomechanical analyses. In addition, blood samples were obtained to evaluate serum concentration of homocysteine (HCY). Quantitative analysis of blood samples revealed severe HHCY as indicated by significantly increased serum concentrations of HCY in animals fed the homocystine-supplemented diet (102.2 +/- 64.5 micromol/l) compared to controls (2.8 +/- 1.5 micromol/l). Biomechanical evaluation of bone repair revealed significantly decreased bending stiffness of the femora of homocystine-fed animals (45.5 +/- 18.2 N/mm) compared with controls (64.6 +/- 15.8 N/mm). Histomorphometric analysis demonstrated a slightly smaller callus diameter in HHCY animals but no significant differences in the tissue composition of the callus. In conclusion, the homocystine-supplemented diet leads to severe HHCY, which is associated with an impaired biomechanical quality of the healing bone.

Hyperhomocysteinemia causes cardiac rhythm disturbances due to a shift in atrial and ventricular gap junction protein distribution

Homocysteine (Hcy) is an amino acid byproduct in the metabolism of methionine. Hyperhomocyseinemia (HHcy) is associated with thromboembolic events. Hcy has been identified to correlate with atrial fibrillation and stroke in humans. The role of Hcy and its influence on atrial and ventricular gap junction proteins has been investigated. We hypothesize that changes in the Connexin (Cx) distribution and localization are responsible for cardiac arrhythmia. ECG was monitored in freely moving mice (Hcy‐diet for 12 weeks) with a subcutaneously implanted telemetric ECG probe. 2D transthoracic echocardiography was performed to assess regional wall motion abnormalities and ventricular diameter changes in M‐Mode. Immunoblotting was used for quantification, immunohistochemistry was used to visualize the localization of the gap junction proteins Cx 40, 43 and 45. A shift of Cx 43 to Cx 45 was seen in the ventricular myocardium, a loss of Cx 40 was detected in the atrial myocardium in HHcy animals. Arrhythmia, sudden cardiac death and left ventricular dysfunction along with right ventricular diameter changes were detected in the HHcy animal group.

Vascular dysfunction produced by hyperhomocysteinemia is more severe in the presence of low folate

Earlier we reported that dietary folate depletion causes hyperhomocysteinemia (HHcy) and arterial dysfunction in rats (Symons JD, Mullick AE, Ensunsa JL, Ma AA, and Rutledge JC. Arterioscler Thromb Vasc Biol 22: 772-780, 2002). Both HHcy and low folate (LF) are risk factors for cardiovascular disease. Therefore, the dysfunction we observed could have resulted from HHcy, LF, and/or their combination (HHcy + LF). We tested the hypothesis that HHcy-induced vascular dysfunction is more severe in the presence of LF. Four groups of rats consumed diets for approximately 10 wk that produced plasma homocysteine (microM) and liver folate (microg folate/g liver) concentrations, respectively, of 7 +/- 1 and 15 +/- 1 (Control; Con; n = 16), 17 +/- 2 and 15 +/- 2 (HHcy; n = 17), 10 +/- 1 and 8 +/- 1 (LF; n = 14), and 21 +/- 2 and 8 +/- 1 (HHcy + LF; n = 18). We observed that maximal ACh-evoked vasorelaxation was greatest in aortas and mesenteric arteries from Con rats vs. all groups. While the extent of dysfunction was similar between LF and HHcy animals, it was less severe compared with arteries from HHcy + LF rats. Maximal ACh-evoked vasorelaxation in coronary arteries was not different between Con and LF rats, but both were greater than HHcy + LF animals. In segments of aortas, 1) ACh-evoked vasorelaxation was similar among groups after incubation with the nonenzymatic intracellular O2(-) scavenger Tiron, 2) vascular O2(-) estimated using dihydroethidium staining was greatest in HHcy + LF vs. all groups, and 3) tension development in response to nitric oxide (NO) synthase inhibition was greatest in Con vs. all other groups. We conclude that HHcy + LF evokes greater dysfunction than either HHcy alone (aortas, mesentery) or LF alone (aortas, mesentery, coronary), likely by producing more O2(-) within the vasculature and thereby reducing NO bioavailability.