formazans and Brain-Ischemia

Topics: Animals; Brain; Brain Ischemia; Carotid Artery, Common; Cell Survival; Coronary Occlusion; Disease Models, Animal; Formazans; Mice; Neuroprotective Agents; Oxidoreductases; Taurine

A histo-enzymatic technique for visualizing and quantifying endogenous NAD(H) in brain tissue was developed, based on coupled enzymatic cycling reactions that reduce nitrotetrazolium blue chloride to produce formazan. Conditions were used where the endogenous level of nicotinamide adenine dinucleotides (NAD(H)) was the rate limiting factor for formazan production. Spontaneous degradation of NAD(+) that occurs during incubation of thawed tissue was minimized by the addition of nicotinamide mononucleotide, an inhibitor of NAD(+) glycohydrolases. Cryostat sections of brains obtained from rats immediately after decapitation and 30 min later were used to determine the effects of ischemia alone on brain NAD(H) levels and neuroanatomic distribution. The ischemic insult resulted in a greater than 50% decline in the rate of formazan generation in the CA1 pyramidal neuronal layer of the hippocampus and in the parietal cortex and striatum, but not in the CA3 and dentate gyrus (DG) subregions of the hippocampus. The ischemia-induced changes in NAD(H) levels were confirmed by utilizing spectrofluorimetric measurements of NAD(H) present in perchloric acid extracts of brain samples. This new histo-enzymatic technique is suitable for visualizing and quantifying relative NAD(H) levels in the brain. This assay could prove useful in identifying region-selective NAD(H) catabolism that may contribute to neurodegeneration.

Topics: Animals; Brain; Brain Ischemia; Cerebral Cortex; Corpus Striatum; Enzyme Inhibitors; Formazans; Hippocampus; Male; NAD; NAD+ Nucleosidase; Nicotinamide Mononucleotide; Nitroblue Tetrazolium; Perchlorates; Rats; Rats, Inbred F344; Spectrometry, Fluorescence; Staining and Labeling; Time Factors

1. Microglial cell activation occurs during brain injury, ischemia, and in several neurologic disorders. Recently, we isolated a transmissible cytotoxic activity (TCA) from the cerebrospinal fluid of a patient with brain ischemia. Such a TCA, associated with one or more protein(s) that supposedly had undergone in vivo misfolding, causes apoptosis in vitro in different cell lines, including microglial cells. The TCA producing cells and the potential in vivo role of such cytotoxic activity remains to be elucidated. Here, we investigated the in vitro effects of TCA on microglial cell immune functions.2. The murine microglial cell line RR4 was exposed to TCA, and then its response was evaluated as: (a) phagocytosis and antifungal activity against Candida albicans; (b) secretory pattern; and (c) levels of p38 phosphorylation.3. Unlike mock-treated controls, microglial cells exposed to TCA showed an increase in phagocytic activity. Unexpectedly, their capability to kill the ingested fungi significantly diminished. Moreover, TCA-treated cells produced amounts of macrophage inflammatory protein 1-alpha, tumor necrosis factor-alpha, and nitric oxide significantly higher than mock-treated cells. Finally, phosphorylation of p38 mitogen-activated protein kinase (MAPK) was detected in TCA-treated but not in mock-treated controls as early as 30 min after treatment.4. Overall, these results indicate that TCA causes a rapid molecular response in microglial cells, by the time, leading to an intriguing effector and secretory dysfunction.

Topics: Brain Ischemia; Cell Line; Cell Survival; Cytokines; Cytotoxicity, Immunologic; Formazans; Humans; Hypoxia, Brain; Microglia; Nitric Oxide; p38 Mitogen-Activated Protein Kinases; Phagocytosis; Tetrazolium Salts

To establish a simpler and more accurate method for evaluating in vitro ischemic injury and neuroprotective effects of drugs through improving experimental instrument and quantitative index in mouse brain slices.. An incubation instrument was developed and its application tested. 2,3,5-triphenyltetrazolium chloride (TTC) was used as a substrate to biosynthesize formazan standard in mouse brain slices, and formazan was isolated, purified and identified. Ischemic injury of mouse brain slices was induced by oxygen/glucose deprivation (OGD), the produced formazan from TTC in the cortex and striatum was measured at 490 nm spectrophotometrically. Edaravone and ONO-1078 were added into the incubation medium to observe their neuroprotective effects.. The incubation instrument worked well for incubating brain slices and obtaining stable results efficiently. Standard formazan was biosynthesized and purified with a purity of 99.3%, and showed a linear range of 0.05 - 1 mg/ml in absorbance at 490 nm (r=0.9997). OGD decreased formazan production in the cortex and striatum in a duration-dependent manner. Edaravone (0.01 to 1 micromol/L) recovered OGD-induced decrease of formazan production, but ONO-1078 showed no effect.. The incubation instrument and quantitative measurement of formazan developed in this study are efficient,accurate and simple for evaluating ischemic injury and neuroprotection,which can be used in screening of neuroprotective drugs in vitro.

Topics: Alprostadil; Animals; Antipyrine; Brain Ischemia; Edaravone; Formazans; Male; Mice; Mice, Inbred ICR; Neuroprotective Agents; Staining and Labeling; Tetrazolium Salts

Freshly sampled brain tissue exposed to 2,3,5-triphenyltetrazolium chloride (TTC) acquires a red color because mitochondrial enzymes reduce the colorless TTC to a red, water-insoluble formazan deposit. Pan-necrotic areas remain uncolored, which enables quantitation of experimental brain injury by optical scanning and image analysis of serial slices to determine the relative volume of red versus infarcted, non-stained, tissue. The accuracy of this method can be challenged, however, when infarction is accompanied by areas of partial, scattered injury where differences in coloration are difficult to see or quantify. We tested the feasibility of measuring scattered injury using a principle which underlies standard assays for in vitro cell survival, namely extracting deposited formazan with a solvent and measuring its level by spectrophotometry. Anesthetized, adult Sprague Dawley rats were subjected to 12 min of cerebral ischemia to produce selective, delayed neuronal death in hippocampus, striatum and cortex. Some rats also received 6 h of whole-body hypothermia treatment (31.5-32.5 degrees C) immediately after ischemia. Ischemia rats and non-operated controls were sacrificed 1 week later. Hippocampus and portions of cerebrum were incubated 90 min in a 2% TTC solution and then soaked in a measured volume of 50:50 ethanol and dimethylsulfoxide to extract the red formazan product. Spectrophotometric measurements of the extract showed a diminished formazan coloration (absorbance/g brain) in all samples from the untreated ischemia group compared to non-operated controls. This apparent brain injury was attenuated in the group of ischemia rats that received hypothermia treatment. We conclude that solvent extraction and spectrophotometric quantitation of formazan has potential utility as an objective way to index experimental brain injury even if this is diffuse in nature and not amenable to measurement by conventional image analysis techniques.

Topics: Animals; Brain; Brain Ischemia; Cell Death; Cerebral Cortex; Coloring Agents; Corpus Striatum; Formazans; Hippocampus; Hypothermia, Induced; Male; Neurons; Rats; Rats, Sprague-Dawley; Spectrophotometry; Tetrazolium Salts