Difficulty in detecting low levels of polymyxin resistance in clinical Klebsiella pneumoniae isolates: evaluation of Rapid Polymyxin NP test, Colispot Test and SuperPolymyxin medium.

Polymyxins are important therapeutic options for treating infections, mainly those caused by carbapenem-resistant Klebsiella pneumoniae. Specific chemical characteristics of polymyxins make it difficult to perform antimicrobial susceptibility testing, especially within the clinical laboratory. Here we aimed to evaluate the performance of three phenotypic methods: Rapid NP Polymyxin Test, ColiSpot test and the SuperPolymyxin medium. To accomplish this, 170 non-duplicate clinical K. pneumoniae isolates were analysed (123 colistin-resistant and 47 susceptible). The sensitivity and specificity obtained for Rapid Polymyxin NP Test, Colispot and SuperPolymyxin medium were, respectively, 90% and 94%, 74% and 100%, and 82% and 85%. Very major errors occurred more frequently in low-level colistin-resistant isolates (MICs 4 and 8 µg/mL). Rapid Polymyxin NP proved to be a method capable of identifying colistin-resistant strains in acceptable categorical agreement. However, major errors and very major errors of this method were considered unacceptable for colistin-resistance screening. Although the Colispot test is promising and easy to perform and interpret, the results did not reproduce well in the isolates tested. The colistin-containing selective medium (SuperPolymyxin) showed limitations, including quantification of mucoid colonies and poor stability. Nevertheless, Colispot and SuperPolymyxin medium methods did not present acceptable sensitivity, specificity and categorical agreement. It is essential to use analytical tools that faithfully reproduce bacterial resistance in vitro, especially in last-line drugs, such as polymyxins, when misinterpretation of a test can result in therapeutic ineffectiveness.

Células-tronco mononucleares associadas ao plasma rico em plaquetas na consolidação de falha óssea no cão. Estudo piloto / Mononuclear stem cells associated with platelet-rich plasma in the consolidation of bone defects in dogs. A pilot study

A padronização de técnicas e o teste da associação do Plasma Rico em Plaquetas (PRP) e das Células-Tronco Mononucleares (CTMs) na consolidação de falhas ósseas corticais, por meio de avaliação clínica, biomecânica, radiológica e histológica, é avaliada em um estudo piloto. Foram utilizados seis cães adultos, fêmeas, sem raça definida, pesando entre 5 e 10kg, separados por sorteio aleatório em seis tratamentos. Foi confeccionada uma falha elíptica de 1,0x0,4cm na cortical medial diafisária da tíbia direita de cada animal, sendo preenchida de acordo com o tratamento proposto. No cão I, a falha foi preenchida com solução fisiológica (SF); no II, com o PRP; no III, com a fração total das células mononucleares (FTCM); no IV, com a fração vascular estromal (FVE); no V, com o PRP associado à FTCM; no VI, com a associação PRP e FVE. Foram realizadas avaliações: clínicas, diariamente; dos graus de claudicação, semanalmente; radiológica e perimetria da coxa, antes, no pós-operatório imediato, aos 7, 14, 21 e 30 dias; biomecânica, antes do procedimento, aos 10, 20 e 30 dias; e biópsias, aos 15 e 30 dias. A FTCM obteve uma contagem e viabilidade média de 2,0x108cél. e 90%, respectivamente, enquanto a FVE obteve 3x106cél. e 50%. O PRP concentrou, em média, sete vezes o número inicial de plaquetas do sangue total, de 250.000 µl-1 plaquetas no sangue total para 1.750.000 µl-1 plaquetas no PRP. Obteve-se padronização adequada de técnicas, possibilitando o teste da associação entre as células-tronco mononucleares (CTMs) e o plasma rico em plaquetas (PRP), assim como seu uso isolado, no reparo de falhas ósseas corticais, indicando a possibilidade de a associação FTCM e PRP ser o melhor tratamento...

The standardization of techniques and tests of the association of Platelet Rich Plasma (PRP) and Mononuclear Stem Cells (MSCs) in the consolidation of cortical bone defects by clinical, biomechanical, radiological, and histological analysis is evaluated in a pilot study. Six adult female dogs of mixed breed, weighing between 5 and 10kg, separated by random draw in six treatments were used. An elliptical failure of 1.0 x0.4cm was done in the medial diaphyseal cortical of the right tibia of each animal, that was filled according with the proposed treatment. In dog I, the failure was filled with saline (S), in dog II with PRP, in dog III with total mononuclear cell fraction (TMCF), in dog IV with stromal vascular fraction (SVF), in dog V with association of PRP and TMCF, and in dog VI with an association of PRP and SVF. Daily clinical evaluation, weekly degrees of lameness, radiological and girth before, immediate postoperative, 7, 14, 21 and 30 days, biomechanics before the procedure, at 10, 20 and 30 days, and biopsies at 15 and 30 days were performed. The TMCF got a count and viability of 2,0x108cells and 90% respectively, while for SVF it was 3x106cells and 50%, respectively. The PRP concentrated on average seven times the original number of platelets from whole blood, platelets from whole blood 250.000 μl-1 to 1.750.000 μl-1 platelets in PRP. This afforded adequate standardization of techniques, enabling the test of association between mononuclear stem cells (MSCs) and platelet-rich plasma(PRP), as well as their separate use to repair cortical bone defects, indicating the possibility of the association between FTCM and PRP to be the best treatment...

Efficiency and economic feasibility of pest control systems in watermelon cropping.

The aim of this work was to determine the efficiency and feasibility of two different watermelon pest control systems on pest infestations, natural enemies, and on the productivity and sustainability of watermelon cropping. Two independent experiments were carried out during the dry season of 2011. Both experiments were carried out using a randomized block experimental design, with three treatments; weekly application of pesticide (WAP), integrated pest management (IPM), and nonpesticide application (control); and four replicates. Arthropods sampling was performed every 2 d by direct counting at five randomly selected points in each plot. Samples were taken by beating the leaves from the apical portion of the plant against a white plastic tray. Arthropods that moved along the soil surface were sampled weekly using pitfall traps. Both WAP and IPM treatments negatively affected the arthropod population. We conclude that IPM is an attractive strategy for watermelon cropping both economically and environmentally because it provides the grower with an option to lower production cost, achieves the same production, and there is less need for pesticide application when compared with the prophylactic control treatment when pesticides are applied on a weekly basis. This has not been reported for watermelon before.

Differential expression of Fos protein in the rat brain induced by performance of avoidance or escape in the elevated T-maze.

Analysis of Fos protein expression was used to map brain areas activated by exposure of male Wistar rats to the elevated T-maze, an animal model of anxiety where tasks of inhibitory avoidance or one-way escape can be separately performed. The apparatus consists of three elevated arms--one enclosed and two open. In the inhibitory avoidance task--considered to represent learned fear--the time taken by rats to leave from the enclosed arm in three consecutive trials is measured. One-way escape task is measured by recording the time taken by animals to withdraw from the open arm and is thought to reflect innate fear. Control animals were placed three times at the end of the transversal arm of a T-maze composed of three enclosed arms and withdrawal latencies from this arm was similarly measured. Performance of avoidance task increased Fos-like immunoreactivity in the medial amygdaloid nucleus, in the anterior hypothalamic nucleus and in the median raphe nucleus. In contrast, performance of escape task enhanced Fos-like immunoreactivity in the basolateral amygdaloid nucleus and in the dorsal periaqueductal gray matter of the mesencephalon. Both behavioural tasks promoted an increase in Fos-like immunoreactivity in the paraventricular nucleus of the thalamus and in the dorsomedial hypothalamic nucleus. Therefore, the obtained results indicate that different sets of brain structures were, respectively, activated by inhibitory avoidance and one-way escape. This evidence supports the original hypothesis that two types of fear/anxiety are generated in the elevated T-maze.

Effects of acute and chronic fluoxetine treatments on restraint stress-induced Fos expression.

Chronic treatment with antidepressants has been shown to attenuate behavioral changes induced by uncontrollable stress. The mechanisms and brain sites of this effect, however, remain controversial. The objective of the present work was to investigate the effects of chronic and acute treatment with fluoxetine (FLX), a selective serotonin reuptake blocker, on Fos expression in animals submitted to restraint stress. Male Wistar rats (n = 3-9/group) received, during 1 or 21 days, intraperitoneal. Injections of vehicle (saline + 0.2% Tween-80, 1 ml/kg) or FLX (10 mg/kg). One hour after the last injection they were forced restrained for 2 h and sacrificed immediately after. Non-stressed animals were sacrificed 2 h after the last injection. The brains were removed and processed for immunohistochemistry. Fos-like immunoreactivity (FLI) was quantified by a computer system. In acutely treated animals FLX decreased stress-induced FLI in the medial amygdala (MeA), bed nucleus of the stria terminalis (BNST), ventrolateral part, and dorsolateral periaqueductal gray (PAG). After chronic treatment, however, the drug induced a significant increase in FLI in the BNST (ventrolateral and medial parts), lateral septal nucleus (LSN, dorsal part), dorsal raphe nucleus (DRN), and locus coeruleus in restrained group. In non-restrained animals chronic treatment with FLX increased FLI in the MeA, BNST (ventrolateral and dorsolateral parts), LSN (dorsal and intermediate parts), dorsolateral and dorsomedial PAG and in the DRN. The results suggest that chronic fluoxetine treatment induce plastic changes that result in a different regional pattern of Fos expression.

Nitrogen regulation of Saccharomyces cerevisiae invertase. Role of the URE2 gene.

The regulation of extracellular enzymes is of great biotechnological interest. We studied the regulatory role of the URE2 gene on the periplasmic invertase of Saccharomyces cerevisiae, because its periplasmic asparaginase is regulated by the URE2/GLN3 system. Enzymatic activity was measured in the isogenic strains P40-1B, the ure2 mutant P40-3C, and the P40-3C strain transformed with the pIC-CS plasmid carrying the URE2 gene. The assays were performed using midlog and stationary phase cells and nitrogen-starved cells from these growth phases. During exponential growth, the level of invertase in both wild-type and ure2 mutant cells was comparable. However, the invertase activity in ure2 mutant cells from stationary phase was sixfold lower than in the wild-type cells. When P40-3C cells were transformed with the pIC-CS plasmid, the wild-type phenotype was restored. On nitrogen starvation in the presence of sucrose, the invertase activity in wild-type cells from midlog phase decreased three times, whereas in stationary cells, the activity decreased eight times. However, invertase activity doubled in ure2 mutant cells from both phases. When these cells were transformed with the aforementioned plasmid, the wild-type phenotype was restored, although a significant invertase decrease in stationary cell was not observed. These results suggested that the URE2 protein plays a role in invertase activity.

Differential expression of c-fos mRNA and Fos protein in the rat brain after restraint stress or pentylenetetrazol-induced seizures.

1. c-fos mRNA expression and Fos protein expression were investigated by in situ hybridization and immunohistochemistry after 30 min of forced restraint stress or pentylenetetrazol (PTZ; 64 mg/kg, i.p.)-induced seizures. 2. Forced restraint stress and PTZ-induced seizures generated c-fos mRNA expression of distinct intensities, but in similar brain regions, including the hippocampus, the amygdala, the piriform cortex, the paraventricular hypothalamic nucleus, the habenula, and parts of the cerebral cortex. 3. The distribution of Fos-like immunoreactivity induced by stress or seizures only partially overlap. No Fos-like expression was found in the hippocampus or the habenula after restraint stress. Nevertheless, both areas presented Fos-like expression after PTZ-induced seizures. 4. Our results support the suggestion that immediate early gene expression in vivo may exhibit both region- and stimulus-specific expression.

Assay for in vivo yeast invertase activity using NaF.

The methods used for invertase activity determination are based on the measurement of glucose or reducing sugars produced by the enzymatic hydrolysis of sucrose into glucose and fructose. When whole yeast cells are used in these assays, the monosaccharides formed by the action of the periplasmic enzyme can be taken up and metabolized, leading to errors on the enzyme activity determination. This study reports a method for a more accurate invertase activity measurement by blocking the glycolytic pathway. In this method the cells were preincubated with 50 mM sodium fluoride, and inhibitor of enolase. This in vivo measurement of the enzyme activity, under initial rate conditions, was performed using cell concentrations up to 64 mg cell/ml. The results obtained showed that this method is particularly useful for cells with low invertase activity.

c-fos immunoreactivity in the brain following electrical or chemical stimulation of the medial hypothalamus of freely moving rats.

c-fos immunoreactivity was used to map brain areas in which neurons reacted either to electrical stimulation or to microinjection of the excitatory amino acid kainate and of the GABAA antagonist, SR-95531, applied to the medial hypothalamus of freely moving rats. All these stimulations induced flight behavior of moderate intensity. Immunoreactive cells were found within a radius of 0.5 mm around the stimulated area. Distally, clusters of labeled cells were found ipsilaterally in the piriform and entorhinal cortices, in several amygdaloid nuclei, in the bed nucleus of the stria terminalis, in the septo-hypothalamic nucleus, in the paraventricular, anterior and dorsomedial hypothalamic nuclei, the the paraventricular thalamic nucleus, in the dorsal periaqueductal gray extending to the cuneiform nucleus, and bilaterally in the supramammillary decussation and the locus coeruleus. The specificity of the brain areas thus labeled was indicated by the unilateral pattern of activation as well as by the different pattern obtained after control microinjection of saline. Therefore, these results are likely to provide sound information about the brain structures involved in defensive-aversive behavior evoked from the medial hypothalamus.

Regional distribution of Fos-like immunoreactivity in the rat brain after exposure to fear-inducing stimuli.

Fos protein immunohistochemistry was used to identify the neural substrate of fear/anxiety. The structures activated by exposure of Long Evans male rats (280-300 g) to the elevated plus-maze, a widely used animal model of anxiety, were compared with those activated by chemical stimulation of two aversive areas of the brain, the dorsal periaqueductal gray matter and the medial hypothalamus. Three different patterns of activation were obtained: Pattern 1 resulted from microinjection of the excitatory amino acid kainate (60 pmol; N = 5) or of the GABA(A) receptor antagonist SR-95531 (16 pmol; N = 3) into the dorsal periaqueductal gray matter and consisted mainly of caudal structures; Pattern 2 was observed after kainate injection (60 pmol; N = 4) into the medial hypothalamus and had a predominantly prosencephalic distribution; Pattern 3 extended from rostral to caudal brain regions and was induced by microinjection of either SR-95531 (16 pmol; N = 1) or kainate (120 pmol; N = 3) into the medial hypothalamus, as well as by 15-min exposure to the plus-maze (N = 3). Control animals were either injected with saline into the MH (N = 3) or the PAG (N = 3) or were exposed for 15 s to the elevated plus maze (N = 3) and exhibited no significant labeling. These results further support the participation of periventricular structures in the regulation of fear and aversion.

Regional distribution of Fos-like immunoreactivity in the rat brain after exposure to fear-inducing stimuli

Fos protein immunohistochemistry was used to identify the neural substrate of fear/anxiety. The structures activated by exposure of Long Evans male rats (280-300 g) to the elevated plus-maze, a widely used animal model of anxiety, were compared with those activated by chemical stimulation of two aversive areas of the brain, the dorsal periaqueductal gray matter and the medial hypothalamus. Three different patterns of activation were obtained: Pattern 1 resulted from microinjection of the excitatory amino acid kainate (60 pmol; N = 5) or of the GABA(A) receptor antagonist SR-95531 (16 pmol; N = 3) into the dorsal periaqueductal gray matter and consisted mainly of caudal structures; Pattern 2 was observed after kainate injection (60 pmol; N = 4) into the medial hypothalamus and had a predominantly prosencephalic distribution; Pattern 3 extended from rostral to caudal brain regions and was induced by microinjection of either SR-95531 (16 pmol; N = 1) or kainate (120 pmol; N = 3) into the medial hypothalamus, as well as by 15-min exposure to the plus-maze (N = 3). Control animals were either injected with saline into the MH (N = 3) or the PAG (N = 3) or were exposed for 15 s to the elevated plus maze (N = 3) and exhibited no significant labeling. These results further support the participation of periventricular structures in the regulation of fear and aversion.

Role of the amygdala and periaqueductal gray in anxiety and panic.

The amygdala (AM) and the periaqueductal gray (PAG) represent the rostral and the caudal pole, respectively, of a longitudinally organized neural system, that is responsible for the integration of behavioral and physiological manifestations of defensive reactions against innate and learned threats. Microinjection of benzodiazepine (BZD) anxiolytics, GABAA receptor agonists or 5-HT receptor antagonists into the AM has anxiolytic effects in conflict tests and other models of conditioned fear, while similar administration of 5-HT or of a 5-HT1A receptor agonist has anxiogenic effects. On the other hand, in the test of electrical stimulation of the PAG, microinjection of 5-HT, 5-HT mimetics, or of drugs that enhance the action of endogenous 5-HT into the same brain area has an antiaversive effect, like BZD and GABAA agonists. Furthermore, microinjection of midazolam, of the NMDA receptor antagonist AP-7, or of the 5-HT1A/1B receptor blocker propranolol increased the exploration of the open arms of the elevated plus-maze, having therefore an anxiolytic effect. These results point to an inhibitory role of the GABA-BZD system in both the AM and the PAG. In contrast, 5-HT seemingly enhances conditioned fear in the AM, while inhibiting unconditioned fear in the PAG. Thus, 5-HT2/1C antagonists reportedly release punished behavior when injected into the AM, whereas they antagonized the antiaversive effect of 5-HT, zimelidine and 5-HT1A/1B receptor blockers in the PAG. Since reported clinical studies revealed that one of such compounds, ritanserin, relieves generalized anxiety but tends to aggravate panic disorder, a relationship may be established between the AM and anxiety and the PAG and panic.

What brain structures are active during emotions? Effects of brain stimulation elicited aversion on c-fos immunoreactivity and behavior.

Aversive behavior is produced by stimulating some brain structures, such as the dorsal periaqueductal gray and the medial hypothalamus. We have used c-fos immunoreactivity to map brain areas which are influenced by stimulation of these two structures. Stimulation was produced in freely moving rats by electrical stimulation or by microinjections of either excitatory amino acids or GABA blocking drugs. Behavior was monitored to detect emotional changes. The effects on labeling induced by the stimulation of either structure were then compared. Structures labeled include the amygdala, the stria terminalis, the supramamillary area, the hypothalamus, the periaqueductal gray, the superior colliculus, the nucleus cuneiformis, and the locus coeruleus. Regardless whether chemical or electrical stimulation was used or the structure stimulated, there was a large overlap among the brain areas labeled. We then compared our results with data from the literature where other methods of inducing aversion have been used, including pain and stress. There was remarkable similarity in the patterning of labeling irrespective of the type of stimulation (central-peripheral, chemical-electrical). There was, however, one interesting difference produced by central vs. peripheral stimulation. Labeling was unilateral in the former case and bilateral in the latter case. Our results suggest that there is a neural substrate that mediates aversive behavior, no matter how it is produced. Nevertheless, that peripheral stimulation produces mainly bilateral activation of this substrate whereas central stimulation produces mainly unilateral activation suggests that natural peripheral stimuli are also integrated at a higher functional level. Future work could be directed toward explicit comparisons of central versus peripheral stimulation to identify the structures involved in higher level integration of aversive behavior.

Induction of Fos immunoreactivity in the brain by exposure to the elevated plus-maze.

The Fos protein immunohistochemistry technique was used to map the brain areas activated by a 15-min exposure of rats to the elevated plus maze, an ethologically based animal model of anxiety. Two hours after the test, labeling was found mainly in the piriform and entorhinal cortices, amygdala, midline thalamic nuclei, several medial hypothalamic nuclei, periaqueductal gray matter, superior and inferior colliculus, cuneiform nucleus, dorsal raphe nucleus and locus coeruleus. These results support a participation of these structures in anxiety.

Defense reaction elicited by microinjection of kainic acid into the medial hypothalamus of the rat: antagonism by a GABAA receptor agonist.

Electrical stimulation of either the midbrain central gray or the medial hypothalamus induces a defense reaction in the rat, characterized mainly by increased locomotion, rearing, and leaping. However, microinjection of the excitatory amino acid glutamate was effective only in the former region. Because excitatory amino acids do not depolarize axons of passage, it was suggested that the hypothalamus is devoid of soma/dendrites of neurons commanding the defense reaction. In the present study, we show that a subtoxic dose (60 pmol) of another excitatory amino acid, kainic acid, injected into the medial hypothalamus significantly enhanced locomotion and rearing of Wistar rats systematically observed in an open field. Similar behavioral changes have been reported following microinjection of drugs impairing GABAergic neurotransmission. Local pretreatment with the GABAA receptor agonist THIP (2 nmol) blocked the effect of kainic acid. Therefore, the medial hypothalamus of the rat seems to contain a population of neuronal cell bodies commanding the defense reaction, which is activated by excitatory amino acids and tonically inhibited by GABAergic fibers.

Myocardial protection by verapamil and reperfusion following coronary occlusion.

1. The hypothesis that early verapamil (VP) treatment in acute myocardial ischemia can enhance the effects of subsequent reperfusion was tested in open-chest dogs submitted to 3 h of left anterior descending artery occlusion and 2 h of reperfusion. 2. Arterial pressure and heart rate were monitored continuously. The area at risk (AR) was determined by left atrial injection of 99technetium-labeled microspheres soon after occlusion. The area of necrosis (AN) was identified histologically with triphenyl tetrazolium chloride and calculated as percent of AR. Myocardial preservation is reported as percent of AR spared from necrosis (AR-AN) x 100/AR. 3. Fourteen dogs received 0.2 mg VP, iv, 15 min after occlusion and 9 untreated dogs served as controls. Verapamil significantly reduced heart rate but did not affect blood pressure or the pressure-heart rate product. 4. Myocardial preservation was significantly greater in verapamil-treated dogs than in control animals (51 +/- 20 vs 31 +/- 19%, mean +/- SD). However, area at risk (%) in the left ventricle was not significantly different in treated and control animals (31 +/- 12 vs 32 +/- 4%). 5. These data indicate that verapamil protects the ischemic myocardium in this occlusion/reperfusion model and that the mechanism of protection is probably related to a non-hemodynamic, metabolic activity of verapamil.

Mycrocardial protection by verapamil and reperfusion following coronary occlusion

The hypothesis that early verapamil (VP) treatment in acute myocardial ischemia can enhance the effects of subsequent reperfusion was tested in open-chest dogs submitted to 3h of left anterior descending artery occlusion and 2 h of reperfusion. 2. Arterial pressure and heart rate were monitored continuosusly. The area at risk (AR) was deteminede by left injection of99 technetium-labeled microspheres soon after occlusion. The area of necrossis (AN) was indentified histologically with triphenyl tetrazolium chloride and calculated as percent of Ar. Myocardial preservation is reported as percent of AR spared from necrosis (AR-An) x 100/AR). 3. Fouteen dogs received 0.2 mg VP, iv, 15 min after occlusion and 9 untreated dogs served as controls. Verapamil signficantly reduced heart rate but did not affect blood pressure or the pressure or the pressure-heart rate product. 4. Myocardial preservation was significantly greater in verapamil-treated dogs than in control animals (51ñ20 vs 31 ñ 19%, mean ñ SD). However, area at risk (%) in the left ventricle was not significantly different in treated and control animals (31 ñ 12 vs 32 ñ 4%). 5. These data indicate that verapamil protects the ischemic myocardium in this occlusion/reperfusion model and that the mecanism of protection is probably related to a non-hemodynamic, metabolic activity of verapamil .

Defense reaction elicited by microinjection of Kainic acid in the medial hypothalamus of the rat

In order to localize groups of neurons commanding the defense reaction, a subtoxic dose (66 pmol) of kainic acid was microinjected into the medial hypothalamus of the rat. After drug treatment, the animals were placed inside a shuttle-box for 15 min and the number of midline crossings, rearings and forward leaps was recorded. Autonomic changes such as occurrence of micturition and defectation were also measured. Injection of kainic acid significantly increased locomotion, rearing and micturition, indicating that the medial hypothalamus of the rat contains perikarya/dendrites of neurons integrating the defense reaction

Defense reaction elicited by microinjection of kainic acid in the medial hypothalamus of the rat.

In order to localize groups of neurons commanding the defense reaction, a subtoxic dose (66 pmol) of kainic acid was microinjected into the medial hypothalamus of the rat. After drug treatment, the animals were placed inside a shuttle-box for 15 min and the number of midline crossings, rearings and forward leaps were recorded. Autonomic changes such as occurrence of micturition and defecation were also measured. Injection of kainic acid significantly increased locomotion, rearing and micturition, indicating that the medial hypothalamus of the rat contains perikarya/dendrites of neurons integrating the defense reaction.

Effects of calcium blockers on ventricular fibrillation during acute myocardial infarction.

1. The effects of the calcium blockers bepridil and verapamil on latency time to ventricular fibrillation (VF) and VF incidence were assessed in 109 anesthetized dogs, submitted to coronary occlusion and reperfusion. 2. In 19 dogs (Group I) submitted to global left ventricular ischemia, both bepridil and verapamil significantly (P less than 0.05) prolonged latency time as compared to 14 untreated controls. However, VF was not prevented by any of these drugs. Both drugs were given 15 min before coronary ligation. 3. In 76 dogs (Group II) submitted to regional myocardial ischemia (left anterior descending coronary artery (LAD) occlusion for 2 hours followed by 30 min reperfusion), VF incidence during occlusion was significantly reduced by verapamil as compared to controls (0/21 vs 10/25; P less than 0.05) but not by bepridil (10/25 vs 12/30; P = ns). During reperfusion, however, neither drug affected fibrillation incidence or latency time. 4. No correlation was observed between anti-arrhythmic drug effects and infarct size as measured by triphenyl tetrazolium chloride. 5. We conclude that both bepridil and verapamil significantly delayed the occurrence of fibrillation in acute ischemia due to coronary ligation. However, only verapamil prevented fibrillation and this effect was restricted to the occlusion phase. In contrast, during reperfusion, neither drug prevented fibrillation. Thus, VF during occlusion and reperfusion is likely to be mediated by different mechanisms.