Envenomations by Bothrops and Crotalus Snakes Induce the Release of Mitochondrial Alarmins

Skeletal muscle necrosis is a common manifestation of viperid snakebite envenomations. Venoms from snakes of the genus Bothrops, such as that of B. asper, induce muscle tissue damage at the site of venom injection, provoking severe local pathology which often results in permanent sequelae. In contrast, the venom of the South American rattlesnake Crotalus durissus terrificus, induces a clinical picture of systemic myotoxicity, i.e., rhabdomyolysis, together with neurotoxicity. It is known that molecules released from damaged muscle might act as ‘danger’ signals. These are known as ‘alarmins’, and contribute to the inflammatory reaction by activating the innate immune system. Here we show that the venoms of B. asper and C. d. terrificus release the mitochondrial markers mtDNA (from the matrix) and cytochrome c (Cyt c) from the intermembrane space, from ex vivo mouse tibialis anterior muscles. Cyt c was released to a similar extent by the two venoms whereas B. asper venom induced the release of higher amounts of mtDNA, thus reflecting hitherto some differences in their pathological action on muscle mitochondria. At variance, injection of these venoms in mice resulted in a different time-course of mtDNA release, with B. asper venom inducing an early onset increment in plasma levels and C. d. terrificus venom provoking a delayed release. We suggest that the release of mitochondrial ‘alarmins’ might contribute to the local and systemic inflammatory events characteristic of snakebite envenomations

Poor Regenerative Outcome after Skeletal Muscle Necrosis Induced by Bothrops asper Venom: Alterations in Microvasculature and Nerves

artículo (arbitrado) -- Universidad de Costa Rica, Instituto de Investigaciones Clodomiro Picado. 2011Background: Viperid snakebite envenoming is characterized by prominent local tissue damage, including muscle necrosis. A frequent outcome of such local pathology is deficient skeletal muscle regeneration, which causes muscle dysfunction, muscle loss and fibrosis, thus provoking permanent sequelae that greatly affect the quality of life of patients. The causes of such poor regenerative outcome of skeletal muscle after viperid snakebites are not fully understood. Methodology/Principal Findings: A murine model of muscle necrosis and regeneration was adapted to study the effects of the venom and isolated toxins of Bothrops asper, the medically most important snake in Central America. Gastrocnemius muscle was injected with either B. asper venom, a myotoxic phospholipase A2 (Mtx), a hemorrhagic metalloproteinase (SVMP), or saline solution. At various time intervals, during one month, tissue samples were collected and analyzed by histology, and by immunocytochemical and immunohistochemical techniques aimed at detecting muscle fibers, collagen, endothelial cells, myoblasts, myotubes, macrophages, TUNEL-positive nuclei, and axons. A successful regenerative response was observed in muscle injected with Mtx, which induces myonecrosis but does not affect the microvasculature. In contrast, poor regeneration, with fibrosis and atrophic fibers, occurred when muscle was injected with venom or SVMP, both of which provoke necrosis, microvascular damage leading to hemorrhage, and poor axonal regeneration. Conclusions/Significance: The deficient skeletal muscle regeneration after injection of B. asper venom is likely to depend on the widespread damage to the microvasculature, which affects the removal of necrotic debris by phagocytes, and the provision of nutrients and oxygen required for regeneration. In addition, deficient axonal regeneration is likely to contribute to the poor regenerative outcome in this model.This study was supported by NeTropica (grant 2-N-2008), by Vicerrectoría de Investigación, Universidad de Costa Rica (project 741-A7-604). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP

Pharmacological characterization of mouse hind paw oedema induced by Bothrops insularis (jararaca ilhoa) snake venom

Bothrops snake venoms produce marked local effects, including oedema, haemorrhage and necrosis. The ability of Bothrops insularis venom to induce oedema in mice was investigated. Venom was injected into hind paws and the change in volume over time was measured by plethysmometry. B. insularis venom (0.01-2.5 mug/paw) induced paw oedema which, at high doses (greater than or equal to0.5 mug/paw), was accompanied by haemorrhage. The peak oedematogenic response occurred 3 h after venom injection with all doses and decreased gradually thereafter, but was still elevated with high doses after 24 h. Pretreating the mice with cyproheptadine (histamine H-1 and serotonin 5-HT2 receptor antagonist), mepyratnine (histamine H-1 receptor antagonist), L-NAME (inhibitor of nitric oxide synthase), indomethacin and rofecoxib (inhibitors of cyclooxygenases), and dexamethasone (indirect inhibitor of PLA(2)) significantly attenuated venom-induced oedema, whereas methysergide, a serotonin 5-HT1/5-HT2 receptor antagonist, had no effect. The administration of antivenom 30 min before or immediately after venom injection also significantly inhibited venom-induced oedema. These results show that B. insularis venom causes oedema in the mouse hind paw and that this response is mediated by histamine, nitric oxide, and arachidonic acid metabolites formed by cyclooxygenases 1 and 2. The neutralization by commercial antivenom indicates that the venom components responsible for oedema are recognized by the antivenom and share immunological identity with their counterparts in the venoms of mainland Bothrops species. (C) 2003 Elsevier Ltd. All rights reserved.42551552

Neutrophils do not contribute to local tissue damage, but play a key role in skeletal muscle regeneration, in mice injected with Bothrops asper snake venom

Local tissue damage induced by crotaline snake venoms includes edema, myonecrosis, hemorrhage, and an inflammatory response associated with a prominent cellular infiltrate. The role of neutrophils in the local tissue damage induced by Bothrops asper snake venom and by myotoxin 1, a phospholipase A(2) isolated from this venom, was investigated. Male Swiss mice were pretreated with either an antimouse granulocyte rat monoclonal immunoglobulin G (IgG) antibody or with isotype-matched control antibody. No significant differences in these local effects were observed between mice pretreated with antigranulocyte antibodies and those receiving control IgG. Moreover, myotoxicity induced by B. asper myotoxin I was similar in neutrophil-depleted and control mice. The role of neutrophils in the process of skeletal muscle regeneration was also assessed. Muscle regeneration was assessed by quantifying the muscle levels of creatine kinase and by morphometric histological analysis of the area comprised by regenerating cells in damaged regions of skeletal muscle. Mice depleted of neutrophils and then injected with B. asper venom showed a more deficient regenerative response than mice pretreated with control IgG. Moreover, a drastic difference in the regenerative response was observed in mice injected with myotoxin 1, because animals pretreated with control IgG showed a successful regeneration, whereas those depleted of neutrophils had abundant areas of necrotic tissue that had not been removed 7 days after injection, associated with reduced contents of creatine kinase. It is concluded that (1) neutrophils do not play a significant role in the acute local pathological alterations induced by the venom of B. asper, and (2) neutrophils play a prominent role in the process of skeletal muscle regeneration after injection of B. aspervenom and myotoxin 1, probably related to the phagocytosis of necrotic material and the recruitment of other inflammatory cells, two events directly associated with a successful muscle regenerative response.28444945

O ESTILO DE VIDA DE PACIENTES HIPERTENSOS DE UM PROGRAMA DE EXERCÍCIO AERÓBIO: ESTUDO NA CIDADE DE TOLUCA, MÉXICO

A hipertensão arterial, assintomática e desconhecida pela maioria dos seus por tadores, é importante fator de risco para complicações cardiovasculares e renais. Este é um estudo quantitativo e descritivo. Objetivou identificar os estilos de vida associados ao incremento do descontrole da pressão ar terial em pacientes hipertensos que acodem a um programa de exercício. A coleta dos dados ocorreu num centro de saúde do município do Toluca, México, durante agosto 2006 a março 2007. Foi utilizado um questionário aplicado a 200 pacientes hipertensos, através do referencial do "Modelo de Campo de Saúde da Lalonde". O estudo mostrou que o nível de pressão do grupo estudado possui relação estatisticamente significativa com o tabagismo, consumo de álcool e tratamento. A falta do controle da pressão arterial pode propiciar a evolução de doenças cardiovasculares e coronárias

Pollen analysis of Holocene sediments from the Poço das Antas National Biological Reserve, Silva Jardim, Rio de Janeiro, Brazil

The Poço das Antas National Biological Reserve is located in Rio de Janeiro State, southeast Brazil. This paper presents information on past environmental characteristics of the area through pollen analysis. Two sedimentary columns were collected and five samples were selected for radiocarbon dating. The following ages of the columns from bottom to top were detected: column 1 - 1.20-1.16 m: 6080 ±40 years BP, 0.775-0.735 m: 4090 ±40 years BP, 0.385-0.345 m: 1880 ±80 years BP; column 2 - 1.22-1.18 m: 3520 ±40 years BP, 0.23-0.19 m: 1810 ±40 years BP. Three samples from column 1 and two samples from column 2 were selected for pollen analysis: 1.20 m, 0.77 m and 0.37 m of column1 and 1.22 m and 0.21 m of column 2. Chemical treatment followed standard methodology. The palynological analysis shows that around 6080 years BP the study area was dominated by a rain forest and from around 4090 years BP the vegetation changed to a fragmented forest, restricted to low hills and surrounded by an open area of grassland and pioneer plants, swamps and peat areas. The pollen assemblage of the samples 3520, 1880 and 1810 years BP suggest the permanence of this kind of vegetation between 4000 years BP and the actual.<br>A Reserva Biológica de Poço das Antas localiza-se no Estado do Rio de Janeiro, na região sudeste do Brasil. O presente estudo teve por objetivo fornecer informações a respeito de características ambientais pretéritas da área por meio de análises palinológicas. Foram coletadas duas colunas de sedimentos e cinco amostras foram selecionadas para datação pelo método de radiocarbono, revelando as seguintes idades, da base ao topo dos testemunhos: coluna 1 - 1,20-1,16 m: 6080 ± 40 anos AP, 0,775-0,735 m: 4090 ± 40 anos AP e 0,38-0,34 m: 1880 ± 80 anos AP; coluna 2 - 1,22-1,18 m: 3520 ± 40 anos AP e 0,23-0,19 m, 1810 ± 40 anos AP. Foram retiradas três amostras da coluna 1 e duas da coluna 2 para análise palinológica: 1,20 m, 0,77 e 0,37 m da coluna 1 e 1,22 m e 0,21 m da coluna 2. O tratamento químico foi baseado em metodologia padrão de preparação de amostras palinológicas. A análise palinológica indica que em torno de 6080 anos AP a área de estudo era dominada pela Floresta Ombrófila e que, por volta de 4090 anos AP, a vegetação tinha mudado para uma floresta fragmentada, restrita às baixas colinas circundadas por áreas de campo aberto, pantanosas e turfosas. A permanência desse tipo de vegetação entre 4000 anos AP e o atual é sugerida pelas amostras datadas de 3520,1880 e 1810 anos AP

Evaluation of antivenoms in the neutralization of hyperalgesia and edema induced by Bothrops jararaca and Bothrops asper snake venoms

Neutralization of hyperalgesia induced by Bothrops jararaca and B. asper venoms was studied in rats using bothropic antivenom produced at Instituto Butantan (AVIB, 1 ml neutralizes 5 mg B. jararaca venom) and polyvalent antivenom produced at Instituto Clodomiro Picado (AVCP, 1 ml neutralizes 2.5 mg B. aspar venom). The intraplantar injection of B. jararaca and B. asper venoms caused hyperalgesia, which peaked 1 and 2 h after injection, respectively. Both venoms also induced edema with a similar time course. When neutralization assays involving the independent injection of venom and antivenom were performed, the hyperalgesia induced by B. jararaca venom was neutralized only when bothropic antivenom was administered iv 15 min before venom injection, whereas edema was neutralized when antivenom was injected 15 min or immediately before venom injection. On the other hand, polyvalent antivenom did not interfere with hyperalgesia or edema induced by B. asper venom, even when administered prior to envenomation. The lack of neutralization of hyperalgesia and edema induced by B. asper venom is not attributable to the absence of neutralizing antibodies in the antivenom, since neutralization was achieved in assays involving preincubation of venom and antivenom. Cross-neutralization of AVCP or AVIB against B. jararaca and B. asper venoms, respectively, was also evaluated. Only bothropic antivenom partially neutralized hyperalgesia induced by B. asper venom in preincubation experiments. The present data suggest that hyperalgesia and edema induced by Bothrops venoms are poorly neutralized by commercial antivenoms even when antibodies are administered immediately after envenomation.Universidad de Costa Rica/[741-A1-027]/UCR/Costa RicaFundação de Amparo à Pesquisa do Estado de São Paulo/[98/00257-0]/FAPESP/BrasilFundação Butantan///BrasilUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP