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Against the background of climate change, we studied the effects of a severe summer drought on buds of European beech (Fagus sylvatica L.) saplings and on leaves formed during the subsequent spring in trees attributed to different drought-damage classes. For the first time, we combined assessments of the vitality (assessed through histochemical staining), mass and stable carbon isotope ratios (δ13C) of buds from drought-stressed woody plants with morphological and physiological variables of leaves that have emerged from the same plants and crown parts. The number, individual mass and vitality of the buds decreased and δ13C increased with increasing drought-induced damage. Bud mass, vitality and δ13C were significantly intercorrelated. The δ13C of the buds was imprinted on the leaves formed in the subsequent spring, but individual leaf mass, leaf size and specific leaf area were not significantly different among damage classes. Vitality and δ13C of the buds are suitable indicators of the extent of preceding drought impact. Bud vitality may be used as a simple means of screening saplings for the flushing capability in the subsequent spring. European beech saplings are susceptible, but—due to interindividual differences—are resilient, to a certain extent, to a singular severe drought stress. [ABSTRACT FROM AUTHOR]

The aim of the study was to investigate the potential effect of ethanolic extract of Garcinia indica and its active principle (Garcinol) against isoprenaline hydrochloride (ISO) induced cardiotoxicity in rats. The cardiotoxicity was induced by myocardial infarction through subcutaneous administration of isoprenaline hydrochloride (25mg/kg b.w) for two consecutive days. The ethanolic extract of G.indica (250mg and 500mg/kg b.w) and garcinol (5mg/kg b.w) were given as pre-treatment for 28 days through oral gavage. A significant (P<0.05) increase in the activities of the serum marker enzymes (creatine phosphokinase, creatine kinase-MB, lactate dehydrogenease, aspartate transaminase and alanine transaminase), a prominent expression of LDH 1 and LDH 2 isoenzymes, increased levels of serum total cholesterol (TC), low density lipoprotein (LDL), very low density lipoprotein (VLDL), triglycerides (TG), and decreased level of high density lipoprotein (HDL) were observed in ISO-induced rats. The levels of TC, TG and free fatty acids (FFA) increased whereas phospholipids (PL) significantly (P<0.05) decreased in the heart tissue of ISO-induced rats. Further myocardium infarct size was observed by staining the heart tissue with triphenyltetrazolium chloride (TTC). Pretreatment with G.indica and garcinol significantly inhibited the effect of isoprenaline. Moreover, biochemical findings were supported by histopathological observations. The present study provide evidence for the first time, that G.indica and garcinol pre-treatment ameliorated myocardial injury in ISO-induced myocardial infarcted rats and exhibited significant cardioprotection. [ABSTRACT FROM AUTHOR]

To establish a dynamic and fine suspension culture, four different methods of tomato cell suspension culture were compared. Hypocotyl explants of the tomato cultivar Jina were used for callus induction on Murashige and Skoog (MS) Medium supplemented with three different phytohormone combinations. Then, one gram of each type of calli was transferred to 50 mL of liquid MS medium with four combinations of auxins and cytokinins to produce cell suspensions. The growth rate, judged by cell turbidity, cell fresh weight, and cell viability was evaluated. The best suspension culture was obtained by using friable calli formed on MS medium containing 1 mg L-1 NAA and 0.1 mg L-1 kinetin, transferred to the liquid MS supplemented with 2 mg L-1 NAA, 0.2 mg L-1 2, 4-D and 0.2 mg L-1 zeatin. [ABSTRACT FROM AUTHOR]

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Although used extensively in stroke research, there is limited knowledge of how 2, 3, 5-triphenyltetrazolium chloride (TTC)-treated rat brain sections are altered and if they can be used for immunohistochemical quantification after staining with TTC. In the present study, we hypothesized that TTC treatment (TTC+) would not interfere with collagen IV immunohistochemical staining compared with non-TTC-treated (TTC−) brain slices. We further hypothesized that there would be no difference in autofluorescence or nonspecific secondary antibody fluorescence between TTC+ and TTC− brain slices. Coronal brain sections of male Wistar rats (n = 5/group) were either treated with TTC or not after middle cerebral artery occlusion or sham surgery, and processed for immunohistochemical staining with mouse anti-collagen IV as the primary antibody, and goat anti-IgM as the secondary antibody. Four images were taken in the cerebral cortex of the contralateral side of infarction in each brain slice using an Olympus BX50 fluorescence microscope, and average intensity of the entire image was quantified using the Metamorph software. Compared with TTC− brain slices, TTC+ brain slices showed a significantly lower autofluorescence (P < 0.05), but was unchanged for nonspecific secondary antibody fluorescence. In addition, TTC+ brain slices had similar collagen IV staining intensity compared with TTC− brain slices. These results demonstrate that TTC+ brain slices are usable for immunohistochemical quantification. [ABSTRACT FROM AUTHOR]

Flaxseed is a herbaceous plant of the Linnaceae family that corresponds to the flax seed (Linum usitatissimum L.) it is traditionally used as oilseed. The interest for these seeds increases due to the recognition of some components with the health benefits. In this study, the effectiveness of the tetrazolium viability test in L. usitatissmum seeds was determined. Consequently, seeds of L. usitatissmum were presented to four treatments, which consisted of two (0.5% and 1%) and two exposure times (24h and 48h). Subsequently, the duration of the seeds was evaluated with the help of a stereoscope microscope, as the viable seeds of the red coloration in the embryo, in this way statistically significant differences can be obtained (P ≤ 0.05: Tukey HSD) for the T1 treatment (0.5%, 24h) with 65% viable seeds. Similarly, no differences were observed between treatments T2 (1%, 24h), T3 (0.5%, 48h) with viability of 86% and T4 (1%, 48h) with 89% of viability. In the germination test, an average of 86% was obtained in the five replicates with a standard deviation of 9.63. Concluding that the evaluation of viability in flax seed by means of the tetrazolium assay is efficient for the responses of 1% for 24 and 48 hours and for 0.5% at 48 hours of exposure. [ABSTRACT FROM AUTHOR]

Soybean (Glycine max L.) is a specie of the fabaceae family, of great importance for the high content of oils and proteins in its seed. The success of the production of this crop is mediated by factors such as the use of high quality seeds for planting. Therefore, the purpose of this study was to evaluate the tetrazolium test as a method to determine the viability of G. max seeds. Initially, the seeds were collected in the municipality of El Espinal- Tolima, Colombia; were treated with two concentrations (0.5 and 1.0%) and two exposure times (24 and 48 hours). Subsequently, they were studied in the stereoscope to determine its viability, taking as parameter the intensity of red coloration in the embryo. The highest percentages of viability were obtained with the treatments T2 (0.5%, 48 h) and T3 (1.0%, 24 h), these being statistically homogeneous with each other (p ≤ 0.05: Tukey HSD) with 97% and 95% viable seeds. In the germination test, an average of 96% was recorded for the five repetitions, and when both trials were compared, the T2 and T3 treatments did not show significant differences. Concluding that the viability test is effective at concentrations of 0.5% and 1.0% tetrazolium, during exposure times of 24 and 48 hours. [ABSTRACT FROM AUTHOR]

We developed a forced non-electric-shock running wheel (FNESRW) system that provides rats with high-intensity exercise training using automatic exercise training patterns that are controlled by a microcontroller. The proposed system successfully makes a breakthrough in the traditional motorized running wheel to allow rats to perform high-intensity training and to enable comparisons with the treadmill at the same exercise intensity without any electric shock. A polyvinyl chloride runway with a rough rubber surface was coated on the periphery of the wheel so as to permit automatic acceleration training, and which allowed the rats to run consistently at high speeds (30 m/min for 1 h). An animal ischemic stroke model was used to validate the proposed system. FNESRW, treadmill, control, and sham groups were studied. The FNESRW and treadmill groups underwent 3 weeks of endurance running training. After 3 weeks, the experiments of middle cerebral artery occlusion, the modified neurological severity score (mNSS), an inclined plane test, and triphenyltetrazolium chloride were performed to evaluate the effectiveness of the proposed platform. The proposed platform showed that enhancement of motor function, mNSS, and infarct volumes was significantly stronger in the FNESRW group than the control group (P

A potentiometric carbon paste sensor was fabricated for determination of sulfaquinoxaline (SQX) based on the use of ion-association complex of sulfaquinoxaline sodium with 2,3,5-triphenyltetrazolium chloride. The proposed sensor exhibited Nernstian slope of 58.4 ± 0.3 mV per decade for sulfaquinoxaline over a wide concentration range of 5.0 × 10 − 6 to 1.0 × 10 − 2 M, with a low detection limit of 3.0 × 10 − 6 M. The sensor manifested advantages of fast response time, satisfactory reproducibility, long life time, high thermal stability and, most importantly, excellent selectivities for sulfaquinoxaline relative to a wide variety of common foreign inorganic cations, anions, sugars and amino acids. The sensor was successfully used for determination of sulfaquinoxaline in pharmaceutical solution, blood serum, urine and milk samples. The isothermal coefficient of the electrode was calculated by the investigation of temperature effects on the electrode potential response. [ABSTRACT FROM AUTHOR]

Background: Traumatic brain injury (TBI), usually due to brain shaking or impact, affects the normal brain function and may lead to severe disability or even death. However, there is paucity of information regarding changes in the physiologic state of humans or animals after brain shaking.Methods: In this study, near-infrared spectroscopy (NIRS) was used to continuously monitor the concentration change of oxy-hemoglobin (HbO2) and deoxy-hemoglobin (HbR) to understand changes in the physiological state during and after brain shaking. Laser Doppler flowmetry was also used to monitor changes in cerebral blood flow under TBI to supplement the investigation. Triphenyltetrazolium chloride (TTC) staining was used to monitor changes of infarction volume corresponding to different impact strengths.Result: The experimental results indicated that concentration changes of HbO2 and total-hemoglobin (HbT) were significantly related to the impact strength. The infarction volume was also significantly related to the impact strength.Conclusion: Therefore, the non-invasive monitoring of concentration changes in HbO 2 , HbR, and HbT using NIRS may have a clinical application for the evaluation of TBI. [ABSTRACT FROM AUTHOR]

Three new fungal species of the genus Mortierella, Mortierella zychae, Mortierella ambigua, and Mortierella indohii, have been reported in Korea. The fungi were encountered during a study on the fungal community of soil samples collected from different locations in Korea. The species were identified based on molecular and morphological analyses. This study presents detailed descriptions of the morphological observations and molecular phylogenetic analysis of these three fungi. All three species were found to be sensitive to triphenyltetrazolium chloride staining. M. zychae demonstrated the highest intensity of mycelial staining, indicating that this species has the highest potential to produce arachidonic acid of the three species. The staining results indicated that the newly recorded species could potentially be useful for arachidonic acid production. [ABSTRACT FROM AUTHOR]

Plants are proven to be reliable sources of antimicrobial compounds in traditional medicinal systems. The leaf and stem extracts of Cestrum nocturnum were screened for antimicrobial activity. The extracts prepared in various solvents were examined against Escherichia coli, Klebsiella pneumonia, Proteus vulgaris, Pseudomonas aeruginosa, Salmonella paratyphi A, Salmonella paratyphi B, Shigella flexineri, Staphylococcus aureus. The ethyl acetate stem and leaf extracts were found to be most active. The results were compared with Penicillin and Streptomycin. [ABSTRACT FROM AUTHOR]

Abstract: Tetrazolium salts (TTZ) such as 2,3,5-triphenyltetrazolium chloride (TTC) are readily reduced by bacterial populations of various genus. The reduced form of these redox indicators is conspicuously colored allowing a quick and easy detection of growth. The studies are mainly confined to Gram negative bacteria because of an important toxic effect of tetrazolium salts on Gram positive bacteria. Indeed, we observed an important impact of different tetrazolium salts on Listeria monocytogenes growth, curiously limited to an increase in the duration of the lag phase. In this study, we demonstrate that increasing the iron concentration in a medium containing TTC leads to a significant decrease of the lag phase. L. monocytogenes growth was kinetically measured and growth parameters were estimated using the Baranyi model. While lag phase diminution was found to be iron concentration dependent, growth rate was not affected. Addition of iron enables growth of some strains totally inhibited by a 0.4 g/l of TTC and for the other a significant reduction of the latency is observed. The nature of the mechanism resulting in a decrease of the observed lag phase remains unclear. Then, the use of iron supplementation may be proposed to overcome the inhibitory effect of TTC on L. monocytogenes. [Copyright &y& Elsevier]

Abstract: For more than a century, the hippocampal sub-fields have been recognized as being differentially vulnerable to injury. While the cause remains unknown, the explanations generally considered have involved either vascular differences, or innate variability among cells. To examine the latter possibility, we prepared acute hippocampal slices from Sprague-Dawley rats, applied a brief period of oxygen–glucose deprivation (OGD; an in vitro model of ischemia), and assessed the viability of dissected sub-fields (CA1, CA3, dentate gyrus) by measuring mitochondrial 2,3,5-triphenyltetrazolium chloride (TTC) metabolism. In slices from young animals (15 weeks of age), post-OGD TTC metabolism was significantly reduced in the CA sub-fields relative to the dentate gyrus. Since previous studies found increasing age may worsen ischemic injury, we completed the same experiment using tissue from animals at 52 weeks of age, and found no differences in TTC metabolism across sub-fields. Given the established role of glutamate receptors in ischemic cell death, we examined two key subunit proteins (GluN1, found in all NMDA receptors, and GluA2, found in most AMPA receptors) across sub-fields and age to determine whether their expression complemented our viability data. We found that, relative to the CA1, the DG displayed greater GluN1 expression and lower GluA2 expression in both young and old animals. Our results confirm that regional vulnerability can be shown in a slice model, that the property is not intransigent, and that these features are likely not attributable to the expression pattern of key glutamate receptor subunits, but another molecular variable that changes over the lifespan. [Copyright &y& Elsevier]

The molybdenum content in the stems, leaves and fruit of tomatoes was determined by a new extraction method with Triphenyltetrazolium chloride. The method is rapid and simple with good reproducibility. It was established that fertilization has an effect upon the molybdenum content in the plant samples of tomatoes. Results show that the accumulation of molybdenum in tomatoes strongly depends on the chemical composition of the fertilizer, the content of molybdenum in it and the amount of fertilizer necessary for fertilization of the soil. [ABSTRACT FROM AUTHOR]

Abstract: Traumatic brain injury (TBI) is usually caused by brain shaking or impact. It can affect normal brain function and may even lead to disability or death. However, there are very few studies on the associated physiologic changes in humans or animals. In this study, a non-invasive, wireless multi-channel near-infrared spectroscopy (NIRS) was developed to continuously monitor the concentration change of oxyhemoglobin (HbO2), deoxyhemoglobin (HbR), and total hemoglobin (HbT) to elucidate changes in the physiological state of the brain during and after different strength impaction. The triphenyltetrazolium chloride (TTC) staining was also used to monitor changes of infarction volume after different strength impaction. The results indicated that the concentration changes of HbO2 and HbT, and the changes of infarction volumes were significantly related to the impact strength. In conclusion, the status of TBI can be clinically evaluated by detecting HbO2 and HbT changes. The system proposed here is stable, accurate, non-invasive, and mostly important wireless which can easily be used for TBI study. [Copyright &y& Elsevier]

Complex formation and liquid-liquid extraction were studied in systems containing Co(II), 4-(2-pyridylazo)resorcinol (PAR), tetrazolium salt (TZS) {2,3,5-triphenyl-2H-tetrazolium chloride (TTC) or 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride (INT)}, water and chloroform. The optimum conditions for the extraction of Co were found: pH, extraction time, concentration of the reagents PAR and TZS. The results show that the extracted species could be represented with the general formula (TZ+)[Co(PAR)2], where Co is in its +3 oxidation state and PAR is in a doubly deprotonated form. The following key constants and characteristics were established for both systems: constants of extraction (Kex), constants of association (β), constants of distribution (KD), recovery factors (R%), wavelengths of maximum absorption (λmax), molar absorptivities, Sandell's sensitivities, intervals of adherence to Beer's Law, limits of detection and limits of quantification. The ion-associate with INT is more stable and extractable (Log β = 5.0, Log Kex = 6.1, Log KD = 1.12, R = 92.8%). Beer's law for the system with this reagent is observed in a wider concentration interval (0.26-2.6 μg mL-1 Co) with a higher molar absorptivity coefficient (ε' = 5.2 x 104 L mol-1 cm-1 at λmax = 515 nm). [ABSTRACT FROM AUTHOR]

Abstract: This study investigates the enhancement effects of iodized oil and the features of hyperechoic focus changes in a target region of high-intensity focused ultrasound (HIFU) ablated uterine fibroids. Leiomyomas in the experimental group of 20 randomly assigned patients were ablated by HIFU under certain parameters 30 min after 1 mL of iodized oil was injected into the center of the myomas. The value of the gray scale and its area were observed by B-mode ultrasound in the target region and were carefully recorded at 0, 2, 4, and 5 min, respectively, after HIFU ablation. The samples were sectioned successively in a thickness of 1∼2 mm and stained by 2,3,5-triphenyltetrazolium chloride solution within 1 h after HIFU ablation. The TTC non-staining volumes were measured afterwards. All procedures in the control group of the other 20 randomly assigned patients were the same except that iodized oil was replaced by physiologic saline. The hyperechoic areas in the target region were observed in all fibroids of both groups. Compared with the control group, the gray scale values in the target region of the experimental group were higher 4 min after HIFU ablation. The enhanced gray scale area in the target region and the 2,3,5-triphenyltetrazolium chloride non-staining volumes in the experimental group were bigger. Based on our experience, HIFU sonication of leiomyomas injected with iodized oil produces hyperechoic foci that are greater than areas injected with saline. More studies are required to understand the clinical implications of these observations. [Copyright &y& Elsevier]

Solvent extraction of molybdenum(VI) ion associate with triphenyltetrazolium chloride ( TTC) has been studied. TTC was proposed as reagent for the spectrophotometric determination of micro amounts of molybdenum(VI) at λ 250 nm. The optimum conditions for extraction of molybdenum(VI) as an ionassociation complex with TTC has been determined. Beer's law is obeyed in the range of 0.5-10 μg/mL molybdenum(VI). The molar absorptivity of the ion-pair is 1 × 10 L/mol cm. The sensitivity of the method is 9.6 × 10 μg/cm. The characteristic values for the extraction equilibrium and the equilibrium in the aqueous phase are: distribution constant K = 32.64, extraction constant K = 2.19 × 10 association constant β = 6.71 × 10. The interferences of different cations, anions on molybdenum(VI) determination were also investigated. A sensitive and selective method for the determination of microquantities of molybdenum(VI) has been developed. The determination was carried out without preliminary separation of molybdenum. A novel procedure of molybdenum(VI) extraction and spectrophotometric determination in different plant samples was examined. [ABSTRACT FROM AUTHOR]

Abstract: When Zea mays callus cultures of two different genotypes were treated with the osmoticum mannitol (0.53M) for 24h their ability to reduce the tetrazolium derivative 2,3,5-triphenyltetrazolium chloride (TTC) to form the insoluble red compound formazan is stimulated. The formazan can be extracted with 95% ethanol for quantitation and this reaction has been used as a measure of viability since only live cells can carry out this reduction. In order to determine the cause of the increased TTC reduction caused by mannitol we carried out metabolic profiling analysis using GC–MS to identify 80 compounds. There were increases in sugar alcohols, hexoses except fructose and in total sugars. The total organic acid pools did not change and nitrogen containing compounds decreased slightly. Principle component analysis showed a large treatment effect due to changes in carbohydrate and nitrogen metabolism. These results indicate that the increased carbohydrate available for the citric acid cycle may be the cause of the increased TTC reduction observed after the mannitol treatment. [ABSTRACT FROM AUTHOR]

Abstract: Bacterial production of long chain polyunsaturated fatty acids (LC-PUFAs) is a promising biotechnological approach for the mass production of these valuable compounds, but extensive screening is currently needed to select a strain that meets industrial requirements. A method was developed for the rapid screening and isolation of eicosapentaenoic acid (EPA)-producing marine bacteria from mixed cultures using the dye 2,3,5-triphenyltetrazolium chloride (TTC). The method was first validated using two bacteria from the Shewanella genus, S. gelidimarina (known to contain EPA) and S. fidelis (known not to contain EPA), and subsequently applied to a range of bacterial samples collected from seven randomly selected New Zealand fish species. By incorporating TTC in both solid and liquid state fermentation treatments, a clear association between the reduction of TTC to the red-coloured triphenyl formazan (TF) and the presence of EPA within Gram-negative bacteria was confirmed. Incubation in 0.1% w/v TTC was optimal for colour response and cell growth in agar plates and liquid cultures. Bacteria that produce EPA reduced TTC to TF, but a number of non-EPA-producing bacteria also showed this capacity. By conducting a subsequent Gram staining, all EPA-producing strains were revealed to be G (−) rod bacteria while the non-producing ones were all G (+) cocci. The fatty acid methyl esters of the isolated bacteria that reduced TTC to TF were analysed using gas chromatography–mass spectrometry and the content of EPA was confirmed by gas chromatography. From a pool of 2.0×108 CFU/ml, this method allowed the rapid isolation of 16 bacteria capable of producing EPA. This new approach significantly reduces the number of samples submitted for GC analysis and therefore the time, effort and cost of screening and isolating strains of EPA-producing marine bacteria. [Copyright &y& Elsevier]

Abstract: Introduction: Infarct size (IS) is an important variable to estimate cardiac ischemia/reperfusion injury in animal models. Triphenyltetrazolium chloride (TTC) stains viable cells red while leaving infarcted cells unstained. To quantify IS, infarcted and non-infarcted tissue is often manually dissected and weighed (IS-DW). An alternative is to measure infarcted areas by cumulative planimetry (IS-CP). Methods: We prospectively compared these two methods in 141 Langendorff-prepared guinea pig hearts (1.44±0.02g) that were part of different studies on mechanisms of cardioprotection. Hearts were perfused with Krebs–Ringer''s and subjected to 30min global ischemia after various cardioprotective treatments. Two hours after reperfusion hearts were cut into 6–7 transverse sections (3mm) and stained for 5min in 1% TTC and 0.1M KH2PO4 buffer (pH 7.4, 38°C). Each slice was first scanned and its infarcted area measured with Image 1.62 software (NIH). Infarctions in individual slices of each heart were averaged (IS-CP) on the basis of their weight. After scanning, IS-DW was determined by careful manual dissection of infarcted from non-infarcted tissue and measuring their respective total weight. Results: We found limited tissue permeation of TTC in relation to the slice thickness leaving tissue in the center unstained, as well as significant cross-contamination of stained vs. unstained tissue after manual dissection. IS-CP and IS-DW ranged from 6.0 to 73.1% and 19.4 to 70.5%, respectively, and correlated as follows: IS-DW=(27.6±1.4)+(0.518±0.038) • IS-CP; r =0.75 (Pearson), p <0.001. In addition, IS-CP correlated better with return of function after reperfusion like developed left ventricular pressure, contractility and relaxation, and myocardial oxygen consumption. Discussion: Despite a good correlation between both methods, limited tissue permeation by TTC diffusion and limited precision in the ability to manually dissect stained from unstained tissue leads to an overestimation of infarct size by dissection and weighing compared to cumulative planimetry. [Copyright &y& Elsevier]

Background: Timing of leaf senescence is an important ecological trait of species in cool-temperate climates. In contrast to above-ground biomass, not much is known about timing and extent of root mortality of herbaceous perennials at the end of the growing season. Aims: We undertook to assess patterns of end-of-season root mortality in species of temperate wetlands, investigate if the interspecific variation in the pattern of leaf senescence in autumn is reflected in patterns of root mortality, and determine if coarse axile roots and fine lateral roots differ in their autumn mortality pattern in wetland graminoids. Methods: For six cool-temperate perennial wetland graminoids, we assessed whether their roots die back for the winter, if the species-specific timing of senescence corresponds to interspecific variation in leaf senescence, and if fine lateral roots and axile roots differ in their mortality pattern. Survival and mortality of lateral and axile roots, and above-ground senescence was assessed in Sudbury, Ontario, Canada, from 11 September to 11 December, 2007, a period during which most of the above-ground biomass died. Root mortality was assessed using 2,3,5-triphenyltetrazolium chloride. Results: In Carex oligosperma, Carex stricta, Phalaris arundinacea and Scirpus microcarpus the majority of lateral and axile roots showed little mortality until mid-December. Axile roots of Dulichium arundinaceum showed little mortality, but its lateral roots died in October. Both lateral and axile roots of Sparganium androcladum died in October. Conclusions: These results indicate that fine roots of many species do not senesce along with the leaves for winter in cool-temperate climates. In some species, however, all roots senesce in autumn. [ABSTRACT FROM AUTHOR]

Abstract: Using the controlled cortical impact (CCI) model, our laboratory compared edema in contralateral and ipsilateral regions to help clarify conflicting reports of contralateral edema and for enhanced assessment and interpretation of CCI injury pathophysiology. This investigation examined regional edema in response to graded injury severities over time with regards to tissue damage. Prior to injury rats were anesthetized with ketamine and xylazine (1:1). CCI injury parameters were set at 4.0m/s and 120 to 130ms. Rats were randomized to receive moderate or severe injuries set at 2.0 and 3.0mm depths, respectively. Cerebral edema and injury volume were examined separately following euthanasia with pentobarbital. Cerebral edema was measured using the wet–dry weight technique at 24 or 48h after injury. Sham animals underwent all surgical procedures except the impact injury. Injury volume was quantified using 2,3,5-triphenyltetrazolium chloride staining at 24h or 7 days after injury. The results of this investigation confirm that cerebral edema is absent in the uninjured, contralateral hemisphere after moderate and severe CCI injury. There were regional differences in cerebral edema formation in the hemisphere ipsilateral to injury that were dependent on injury severity and the length of time after injury. Tissue damage was reduced over 7 days following moderate CCI injury. Conclusions: (1) the absence of edema in the contralateral hemisphere allows it to serve as a valid control for edema formation, (2) misrepresenting injury volume because of edema continues to be a problem for evaluating CCI injury and treatment efficacy, and (3) reduced injury volume over 7 days following CCI injury suggests tissue recovery after initial dysfunction. [Copyright &y& Elsevier]

Abstract: The importance of the hippocampus to learning and memory has attracted significant attention to how the structure responds to damage. Although many studies have used either the acute hippocampal slice preparation or organotypic hippocampal slice cultures, little work has been done to determine if the choice of model is an important variable. The present study examined whether differences exist in how each model responds to a commonly studied ischemic-like paradigm, oxygen–glucose deprivation. Following the insult, synaptic activity was examined by recording orthodromically evoked CA1 subfield responses, while mitochondrial activity was assessed by spectrophotometric measurement of formazan produced by metabolism of 2,3,5-triphenyltetrazolium chloride. The insult significantly decreased both synaptic and mitochondrial activity within acutely prepared slices, but a disparity existed between these measures in cultured slices. While evoked activity was greatly reduced by an insult of moderate duration, a much longer period was required to cause a comparable decrease in formazan production. Quantitative immunoblotting revealed that one possible explanation for the discrepancy was an elevated expression of astrocytes, which display resistance to hypoxia–aglycemia. Our data indicate that acutely prepared and cultured slices respond differently to ischemic-like challenge; therefore, assays examining viability in these models must consider their innate differences. [Copyright &y& Elsevier]

m. zhu, l.j. yu, z. liu and h.b. xu. 2004To develop a fast isolation method for arachidonic acid-producing fungi of high yield.Relation between the staining degree of mycelia ofMortierella alpinastained with triphenyltetrazolium chloride (TTC) and arachidonic acid content in the fungal lipids was investigated. Results showed that staining degree of mycelia stained with TTC increased when arachidonic acid content in mycelia lipids increased. This finding was used to isolate strains of high arachidonic acid yield. Arachidonic acid producing fungi were selectively isolated from soil at a low temperature of 4°C and the mycelia of these isolates were stained with TTC.The strainM. alpinaM6 that had the highest staining degree had the highest arachidonic acid content (72·3%). The yield of arachidonic acid in this strain reached 4·82 g l−1.A fast and effective method to isolate strains of high arachidonic acid yield was established according to the finding that staining degree of mycelia ofM. alpinawas positively correlated with arachidonic acid content in mycelia lipid. [ABSTRACT FROM AUTHOR]

Mature embryo-derived calli obtained from three cultivars (cvs.) (Omrabi: drought-resistant; Belikh: salt-resistant; Cando: drought- and salt-sensitive) were exposed for one month to various iso-osmotic concentrations of NaCl, mannitol or polyethylene glycol (PEG) 6000. The lowest relative growth rate (RGR) for all cultivars were observed in the presence of PEG. Correlation between RGR and cell viability estimated by reduction of 2,3,5-triphenytetrazolium chloride (TTC) was significant in the case of salt stress only. NaCl increased the relative importance of the non-phosphorylating alternative respiratory pathway while osmotic stress in contrast increased the relative importance of the CN-sensitive pathway. Neither mineral concentration, nor proline, nor soluble sugar accumulation fully accounted for lowering the internal osmotic potential in PEG-treated calli. Calli exposed to PEG were able to recover while those treated with mannitol were not. After NaCl treatment, only calli obtained from the salt-resistant cultivar Belikh recovered. It is concluded that (i) salinity and water stress have contrasting effects on the relationships between growth and cell viability, (ii) these relationships are not the same during and after the stress treatment, and (iii) maintenance of growth potential after the stress relief is linked to the maintenance of ATP-producing respiratory pathway rather than to a stress-induced stimulation of the alternative pathway. [Copyright &y& Elsevier]

: ObjectivesWe examined whether delayed post-injection imaging of a new ultrasound contrast agent (BR-14) could produce prolonged opacification and hyperenhancement of myocardium subjected to coronary occlusion/reperfusion.: BackgroundWe hypothesized that ultrasound exposure destroyed BR-14 and eliminated visualization of sustained myocardial opacification from retained microbubbles.: MethodsWe studied eight open-chest dogs with 3 h of left anterior descending coronary artery (LAD) occlusion followed by 3 h of reperfusion. Myocardial contrast echocardiography (MCE) was performed before occlusion and 120 min after the onset of both occlusion and reperfusion. Ultrasound imaging was initiated 15 min after injection. Myocardial blood flow (MBF) was assessed by microspheres.: ResultsPre-occlusion images revealed uniform opacification of left ventricular myocardium greater than that of the cavity, with a mean intensity of the LAD bed of 8.66 ± 1.38 dB. During occlusion, MCE resulted in the appearance of a perfusion defect in the LAD risk area (intensity 2.08 ± 1.10 dB). After 120 min of reperfusion, the LAD risk-area myocardium manifested dense opacification of a higher intensity (“hot spot”) than baseline (13.7 vs. 8.7 dB), but with reduced MBF consistent with accumulation of a high concentration of microbubbles. Increased MCE intensity was associated with a greater myeloperoxidase score.: ConclusionsThese data establish that contrast opacification by BR-14 may be selectively retained within the perfusion bed of a coronary artery subjected to occlusion/reperfusion. Such opacification exhibits defects with occlusion, manifests hyperenhanced intensity (hot spot) with reperfusion, is associated with the level of myeloperoxidase activity, and conforms to the area of myocardium subjected to altered flow. [Copyright &y& Elsevier]

: ObjectivesWe sought to test the hypothesis of whether inflammatory cell infiltration in patients dying of an acute myocardial infarction (MI) is a multifocal event involving multiple coronary branches.: BackgroundCoronary instability is thought to reflect local disruption of a single vulnerable plaque. However, previous postmortem studies have not addressed the question of whether activation of inflammatory cells, particularly T lymphocytes, is limited to the culprit lesion only or rather diffuse in the coronary circulation.: MethodsWe performed a systematic flow cytometric study in three groups of autopsied patients (group 1 = acute MI; group 2 = old MI; group 3 = no ischemic heart disease). Cell suspensions of enzymatically digested coronary arteries were stained for flow cytometry with CD3, CD68, alpha-smooth muscle actin, and human leukocyte antigen (HLA)-DR antibodies.: ResultsThe coronary plaques showed: 1) a higher proportion of inflammatory cells in groups 1 and 2 than in group 3; 2) a higher percentage of T lymphocytes in group 1 than in group 2 (11.67 ± 0.70% vs. 5.67 ± 0.74%, p = 0.001) and in group 2 than in group 3 (p = 0.008); and 3) diffuse cell activation in the whole coronary tree of group 1, but not of group 2 subjects.: ConclusionsOur study suggests that lymphocytes may play a key role in coronary instability by determining activation of various cellular types throughout the coronary circulation. Activated T lymphocytes and their products may well represent a new target in both the treatment and prevention of acute coronary syndromes. [Copyright &y& Elsevier]

Cyclooxygenase (COX) is crucial in inflammation; COX-1 is constitutional, and COX-2 is inducible. In this study, neurological function and infarct volume were evaluated at 24 h after permanent endovascular middle cerebral artery occlusion (MCAO) in both COX-1-gene knockout (homozygous or heterozygous) and wide-type mice. Similar infarct volumes and neurological deficits were seen among mice of different genotypes. There was no difference among the groups in arterial blood pressure and regional cerebral blood flow during the first 30 min of ischemia. Our results failed to confirm the harmful effect of losing COX-1 activity due to gene knockout in a permanent endovascular MCAO mouse stroke model. [Copyright &y& Elsevier]

: ObjectivesThis study was designed to determine the enhancement profile of a necrosis-specific contrast agent (gadophrin III) in comparison to a standard extracellular agent on T1-weighted magnetic resonance (MR) images in acute and chronic myocardial infarctions (MIs).: BackgroundContrast-enhanced MR imaging demonstrated the ability to accurately quantify infarct size; however, some controversies persist about which contrast medium is best suited.: MethodsFifteen rabbits underwent thoracotomy and permanent occlusion of a branch of the left coronary artery. Two animals died before imaging, eight were examined 48 h after occlusion and five animals were imaged six weeks following induction of infarction. All animals received 50 μmol/kg of gadophrin-3 24 h before the MR examination. Continuous short-axis views were collected using an inversion recovery turbo fast low angle shot sequence. Imaging was repeated 5 to 10 min following additional injection of 100 μmol/kg of Magnevist. The area of hyperenhancement demarcated following gadophrin-3 injection was compared with the region of hyperenhancement seen on gadophrin-3 plus Magnevist enhanced image using triphenyltetrazolium chloride (TTC) staining as the standard of reference.: ResultsIn acute MI the mean difference in size of hyperenhancement seen on the two different in vivo MR scans was −1.8 ± 6.0 mm2 (p > 0.05). Both measurements showed excellent agreement with TTC staining. Chronic MIs showed no enhancement with gadophrin-3, whereas application of Magnevist resulted in hyperenhancement.: ConclusionsStandard extracellular contrast agents do not overestimate the size of acute MI. The combination of gadophrin-3 and Magnevist can distinguish acute and chronic myocardial injury because chronic MIs do not enhance with gadophrin-3. [Copyright &y& Elsevier]

We investigated whether pre-treatment with melatonin, a potent free radical scavenger and antioxidant, would protect against permanent focal cerebral ischemia without reperfusion in a rat middle cerebral artery occlusion (MCAO) model. A single dose of melatonin at 5, 15, or 50 mg/kg or the vehicle alone was given via an intraperitoneal injection at 0.5 h before permanent MCAO. Relative infarction volumes on day 3 were significantly reduced in the groups treated with melatonin at 5 (mean±SEM, 17.0±6.5%), 15 (18.1±5.8%), or 50 (20.6±5.0%) mg/kg when compared with the vehicle-treated group (37.1±2.8%) and so melatonin treatment achieved a relative reduction in infarct volume by 54.2, 51.2 and 44.5%, respectively. Melatonin did not affect the hemodynamic parameters. Thus, pre-treatment with melatonin at a dose between 5 and 50 mg/kg protects against focal cerebral ischemia without reperfusion. [Copyright &y& Elsevier]

The construction of a liquid triphenyltetrazolium cation (TT+) ion-selective electrode (ISE) based on [TT+]3[P(W3O10)4] salt dissolved in 2-nitrotoluene is described. The liquid membrane electrode exhibits a rapid and almost Nernstian response to triphenyltetrazolium cations in the concentration range from 2×10−4 to 1×10−2 mol l−1. The Nernstian slope is 56–58 mV decade−1 which remains constant for at least two months. The response is virtually unaffected by pH changes in the range 3–12. Major interferents are periodate and malate. Deviation from linearity is also observed in the presence of bromide, iodide and thiocyanate, due to their precipitation with triphenyltetrazolium cations. Analytical applications such as direct potentiometry for the determination of TT+ in aqueous solutions and indirect potentiometry for the assay of ascorbic acid (vitamin C) in pharmaceutical preparations are described. Ascorbic acid in the range of 150–500 mg l−1, under specified experimental conditions, can be determined with mean relative error of 1.9%. [ABSTRACT FROM AUTHOR]

Recent studies have repoarted that prolonged infusion of N-2-mercaptopropionyl glycine (MPG), a diffusible antioxidant, could limit infarct size in dogs. However, there are no comparable studies testing this agent in other species. We examined the efficacy of MPG in a rabbit model of infarction. Rabbit hearts were subjected to a 30-min coronary artery occlusion. Infarct size expressed as a percentage of risk zone was determined by either triphenyltetrazolium chloride (TTC) staining after 3 h of reperfusion (study 1) or by histology after 72 h of reperfusion (study 2). In study 1, 37 ± 2.6 % of the risk zone infarcted in the control group. Intravenous MPG at a rate of 100 mg/kg/h starting 15 min after the onset of ischemia and continuing until 1 h after reperfusion had no effect on infarct size (35.4 ± 3.4 % infarction). However, infusion of MPG until the end of reperfusion significantly reduced infarct size as measured with TTC to 17.2 ± 2.5 % (p < 0.01 vs. control group). In study 2, 48.6 ± 4.0 % of the risk zone infarcted in the control group. In the treatment group MPG was started as above and was continued for 4 h of reperfusion followed by an intramuscular injection at the termination of the intravenous infusion. No protection was seen after 72 h of reperfusion (43.8 ± 2.1 % infarction). These findings reveal that MPG at a dose and schedule that appeared to protect the dog heart could not effect sustained protection in the rabbit heart. TTC staining revealed that MPG appeared to have preserved viability for up to 3 h of reperfusion suggesting that failure may have been due to early withdrawal of the drug. Alternatively, early TTC staining may yield spurious results under conditions in which protection is dependent upon antioxidant or free radical scavenger treatment as has previously been suggested. It is concluded that MPG as administered in the previous canine studies does not limit infarct size in all species, thus raising a concern about MPG's potential efficacy in man. [ABSTRACT FROM AUTHOR]

Objectives: Recent studies suggest that nitric oxide (NO) is deleterious in models of shock and hypoxia-reoxygenation However, the role of endogenous NO in ischaemia-reperfusion injury in vivo remains controversial. We tested the hypothesis that blockade of endogenous NO produced during myocardial ischaemia-reperfusion or during reperfusion alone in vivo increases infarct size after coronary occlusion in the rabbit, and conversely, supplementation with L-arginine would reduce infarct size. Methods: Ketamine-xylazine anaesthetised New Zealand white rabbits were subjected to left coronary artery occlusion for 30 min and reperfusion for 120 min. The rabbits were divided into five groups: (1) saline (VEH); (2) L-nitro arginine (L-NA), a NO-synthase inhibitor, was infused intravenously (15 mg/kg bolus followed by 7.5 mg/kg h−1) before coronary occlusion to block NO synthase activity during ischaemia and reperfusion (IR); (3) L-NA was administered during reperfusion only (R) at the same dose as in the IR group; (4) D-arginine (D-ARG) (25 mg/kg bolus followed by 4 mg/kg min−1), the non-metabolised enantiomer of L-arginine was infused intravenously during reperfusion only; (5) L-arginine (L-ARG) (25 mg/kg bolus followed by 4 mg/kg min−1), the physiological precursor of NO, was infused intravenously during reperfusion only. Results: L-NA infusion in the IR and R groups caused an increase in mean arterial pressure and a decrease in heart rate; however, no significant change in pressure rate product (PRP) occurred immediately after drug infusion. PRP did not change significantly during the experiment across groups except at the end of reperfusion. The area at risk was comparable in all groups, averaging 29(1)%. The infarct size (triphenyltetrazolium chloride) expressed as a percent of area at risk was 27(2)% for the untreated vehicle group. In contrast, L-NA significantly (P < 0.05) increased infarct size in the IR group, 51(2)%; this augmented infarct size persisted when NO synthase activity was blocked during reperfusion only in the R group, 50(2)%. There was no significant (P < 0.05) difference in infarct size between the IR and the R groups. D-ARG-treated group showed a comparable increase in infarct size 48(2)% versus the IR and R groups. However, supplementation of NO with L-arginine (L-ARG) showed no reduction in infarct size, 24(3)%, over vehicle group (VEH). Conclusions: We conclude that (1) blockade of NO synthase activity with L-NA increases infarct size, (2) this effect was expressed primarily during reperfusion, (3) D-arginine mimicked the infarct augmentation of L-NA, while (4) L-arginine supplementation did not reduce infarct size. These data imply that endogenous NO production exerts a tonic cardioprotective effect on myocardial infarct following coronary reperfusion. [ABSTRACT FROM PUBLISHER]

Study objective – The aim of the study was to assess the value of triphenyltetrazolium chloride (TTC) staining as an indicator of non-viable myocardium after early reperfusion of ischaemic myocardium.Design – Left anterior descending artery occlusion was performed in pigs for various lengths of time and at two different sites (proximal and distal). After 120 min reperfusion, TTC was injected distal to the occlusion while the remainder of the myocardium was stained with Evans Blue. Myocardial enzymes were measured in non-ischaemic zone, regions of risk and in necrotic zones and related to staining characteristics.Subjects – 31 male Hampshire pigs, weight 34-39 kg, were studied. Twelve were excluded because of resistant ventricular fibrillation or poorly defined areas of infarction. In the remaining 19 pigs, proximal occlusion was carried out in 15 and distal in four. Occlusion lasted for 15 min in six animals, for 30 min in eight (four of which were the animals with distal occlusions), and for 45 min in five.Measurements and results – Biopsies from non-ischaemic zones, regions of risk and necrotic zones were analysed for creatine kinase and lactate dehydrogenase. In the 15 min group, myocardial creatine kinase in the region of risk (red stained) was similar to the non-ischaemic (blue) zone, but in the 30 min distal occlusion group it was reduced. After 30 and 45 min of proximal occlusion, creatine kinase activity in the necrotic (white) zone was reduced compared to the red zone in the same group, and in the red zone of both groups it was reduced compared to the non-ischaemic area.Conclusions – The red zone, as defined by TTC staining, may be associated with significant creatine kinase depletion after relatively brief periods of occlusion and subsequent reperfusion. This suggests that the red region may be a heterogeneous area of dead and viable cells. [ABSTRACT FROM PUBLISHER]