Intact Rat Brain Mitochondria from a Single Animal: Preparation and Properties

-The involvement of oxidative stress has been suggested as a mechanism for toxicity caused by methylmercury (MeHg). One of the major critical sites for oxidative stress is the mitochondria. In this research, to clarify the target site in mitochondria affected by MeHg, the individual activities of the mitochondrial electron transport chain (ETC) (I IV) were examined in the liver, cerebrum and cerebellum of MeHg-intoxicated rats. In addition, to elucidate the mechanism underlying MeHg toxicity, cytochrome c release, caspase 3 activity and histological study were examined in the cerebrum and cerebellum. The cerebellum was found to be an exclusive tissue in which significant MeHg-induced alterations were observed. The complex II activity in the cerebellum mitochondria significantly decreased after MeHg exposure. Cytochrome c release from mitochondria increased only in the cerebellum by MeHg exposure. However, no significant alterations in caspase 3 activity or histological structure were found in brain tissues. These results suggest that MeHg acts on the constituents of complex II in the cerebellum, and induces mitochondrial dysfunction, leading to a release of cytochrome c from mitochondria. These events were considered to occur at the early stage of MeHg intoxication.

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“…Therefore, we adopted the optimum isolation method for brain mitochondria according to Lee et al (1993). The cerebrum (approx.…”

Section: Preparation Of Mitochondria For Etc Activitiesmentioning

confidence: 99%

Methylmercury inhibits electron transport chain activity and induces cytochrome c release in cerebellum mitochondria

et al. 2011

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“…20 In brief, homogenate of hippocampal formation was prepared in cold (4°C) isolation medium, which consisted of 250 mmol/L sucrose, 10 mmol/L HEPES (pH 7.4), 1 mg/mL bovine serum albumin (fraction V), 0.5 mmol/L ethylenediaminetetraacetic acid, and 0.5 mmol/L ethylene glycol bis (␤-aminoethyl ether)-N,N,NЈ,NЈ-…”

Section: Isolation Of Mitochondriamentioning

confidence: 99%

“…Mitochondria were isolated according to the procedure described previously. 20 The resulting mitochondrial pellet was placed inside a nitrogen cell bomb and pressurized at 1200 psi for 7.5 minutes. 21 The samples obtained after nitrogen compression were centrifuged at 16 000 g for 15 minutes.…”

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confidence: 99%

Recurrent Hypoglycemia Exacerbates Cerebral Ischemic Damage in Streptozotocin-Induced Diabetic Rats

Dave

Tamariz

Desai

et al. 2011

Stroke

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Background and Purpose-Stroke and heart disease are the most serious complications of diabetes accounting for Ͼ65% of mortality among diabetics. Although intensive insulin therapy has significantly improved the prognosis of diabetes and its complications, it is associated with an elevated risk of recurrent hypoglycemia (RH). We tested the hypothesis that RH exacerbates cerebral ischemic damage in a rodent model of diabetes. Method-We determined the extent of neuronal death in CA1 hippocampus after global cerebral ischemia in control and streptozotocin-induced diabetic rats. Diabetic animals included an insulin-treated streptozotocin-diabetic (ITD) group and a group of ITD rats exposed also to 10 episodes of hypoglycemia (ITDϩrecurrent hypoglycemia: RH). Hypoglycemia (55 to 65 mg/dL blood glucose) was induced twice daily for 5 consecutive days. Results-As expected, uncontrolled diabetes (streptozotocin-diabetes, untreated animals) resulted in a 70% increase in ischemic damage as compared with the control group. Insulin treatment was able to lower ischemic damage by 64% as compared with the diabetic group. However, ITDϩRH rats had 44% more damage when compared with the ITD group. We also observed that free radical release from mitochondria is increased in ITDϩRH rats. Conclusions-This is the first report on the impact of RH in exacerbating cerebral ischemic damage in diabetic animals.Our results suggest that increased free radical release from mitochondria may be responsible for observed increased ischemic damage in ITDϩRH rats. RH thus may be an unexplored but important factor responsible for increased ischemic damage in diabetes. (Stroke. 2011;42:1404-1411.)Key Words: brain ischemia Ⅲ cardiac arrest Ⅲ diabetes Ⅲ free radicals Ⅲ glucose Ⅲ mitochondria Ⅲ stroke D iabetes is a devastating disease of epidemic proportions. It is estimated that 220 million patients are affected by diabetes worldwide. 1 Stroke and heart disease are the most serious complications of diabetes, because they account for approximately 65% of mortality among diabetics. 2 Epidemiological studies suggest that long-term diabetes increases the risk of cerebral ischemia as well as cardiovascular disease by 2 to 4 times as compared with the nondiabetic population. [3][4][5] The incidence of cerebral ischemia is greater in patients with Type 2 diabetes mellitus than Type 1 diabetes mellitus (T1DM). 3 Furthermore, cerebral infarction after ischemia is more extensive and common in diabetics, who also display slower recovery and worse survival rates than nondiabetic subjects. 6 Animal models corroborate these clinical observations and provide mechanistic insights into the pathophysiology of the effect of diabetes on cerebral ischemia. 7,8 It has been recognized that a high plasma glucose level is a key factor for the poor outcome observed after cerebral ischemia in diabetics. 9 Attaining tight glycemic control is a desirable goal for both patients with T1DM and Type 2 diabetes mellitus. Aggressive therapeutic interventions able to normalize glycohemog...

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“…All isolation procedures were carried out on ice. The procedure for the isolation of mitochondria from brain is as described in Lee et al (19) with minor changes. The brains from 15 fish were pooled for an experiment.…”

Section: Methodsmentioning

confidence: 99%

Comparison of the effects of ammonia on brain mitochondrial function in rats and gulf toadfish

Veauvy

Wang

Walsh

et al. 2002

American Journal of Physiology-Regulatory, Integrative and Comp

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We compared the effect of hyperammonemia on NADH levels in brain slices and on the rate of oxygen consumption from isolated nonsynaptic brain mitochondria in ammonia-sensitive Wistar rats with that in ammonia-tolerant gulf toadfish (Opsanus beta). The NADH content was significantly decreased (12% less than control after 45 min with 1 mM NH4Cl) in rat brain slices, but it was not affected in brain slices from toadfish (with both 1 and 6 mM NH 4Cl). The rates of oxygen consumption of different sets of enzymes of the electron transport chain (ETC; complexes I, II, III, and IV; II, III, and IV; and IV alone) were unaltered by hyperammonemic conditions in isolated nonsynaptic mitochondria from either rats or toadfish. These results lead us to conclude that the differing effects of ammonia on NADH levels in rat and toadfish brain slices must be due to aspects other than the direct effects of ammonia on enzymes of the ETC. Additionally, because these effects were seen in vitro, our studies enabled us to rule out the possibility that effects of ammonia on metabolism were via indirect systemic effects. These results are discussed in the context of current views on mechanisms of central nervous system damage in hyperammonemic states. hepatic encephalopathy; hyperammonemia; NADH; mitochondria; Opsanus beta; glutamine metabolism MAMMALS IN GENERAL are physiologically sensitive to increases in the concentration of ammonia in the body. In particular, excess brain ammonia 1 (i.e., as low as 500 to 1,000 mol/kg tissue wt) caused by liver failure (e.g., hepatic encephalopathy) or inborn errors in urea metabolism or by injection of ammonia in experimental animals leads to brain neuropathies. A general sequence of symptoms progresses from altered sleep patterns to muscular incoordination, stupor, coma, and death, at a rate that is dependent on the extent and rate of ammonia intoxication; a lethal dose of ammonia can cause symptoms within minutes [reviewed by Cooper and Plum (6) and more recently by Hazell and Butterworth (10)]. Much earlier literature focuses on the causes of these neurological symptoms being related to "cerebral energy failure." Indeed, in the early stages of hyperammonemia, brain metabolic rate decreases, with precipitous drops in whole brain creatine phosphate levels, whereas whole brain ATP content falls later in the onset of disease. Because the decline in whole brain ATP follows severe neurological impairment, many have argued that cerebral energy failure cannot be the cause of the impairment.Following this line of reasoning, many investigators have now focused attention on other changes associated with hyperammonemia and hepatic encephalopathy. One is the phenomenon of astrocyte swelling in hyperammonemia-induced brain pathologies. Astrocytes contain most of the brain's activity of the enzyme glutamine synthetase (GSase), which ostensibly detoxifies ammonia by combining it with glutamate to form glutamine (20). Thus, during hyperammonemia, glutamine accumulates in the brain and astrocytes then become swolle...

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Intact Rat Brain Mitochondria from a Single Animal: Preparation and Properties

Cited by 15 publications

References 28 publications

Methylmercury inhibits electron transport chain activity and induces cytochrome c release in cerebellum mitochondria

Methylmercury inhibits electron transport chain activity and induces cytochrome c release in cerebellum mitochondria

Recurrent Hypoglycemia Exacerbates Cerebral Ischemic Damage in Streptozotocin-Induced Diabetic Rats

Comparison of the effects of ammonia on brain mitochondrial function in rats and gulf toadfish

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