Efficient muscle regeneration after highly haemorrhagic Bothrops alternatus venom injection.

Bothrops alternatus snake venom is particularly characterized for inducing a prominent haemorrhage and affecting hemostasis as a consequence of 43.1% of metallo-proteinases and less than 10% of PLA2 (almost all non-myotoxic phospholipases) in its venomics. In addition, myonecrosis is the major local effect in viper envenoming which might lead to permanent sequela. Then, the rebuilding of the microvasculature at the local injured site acquires significance since represents one of the pivotal stages for subsequent skeletal muscle regeneration either at morphological or functional aspects. Due to the significance played by vasculature in this process, it is important to study by histology and immunohistochemical techniques, the muscular damage and the sequence of skeletal muscle reconstruction (degree of damage, reconstitution of muscle fibres and capillaries). In this work, we injected intramuscularly 50 or 100 µg per mouse of B. alternatus venom in gastrocnemius muscles. We provided a complete description and characterization of the different stages of myogenesis after mild (50 µg) and severe (100 µg) local injury induced by B. alternatus venom toxins. The regeneration was evaluated 24 h, 3, 7, 14 and 28 days after receiving venom injection. Finally, both doses induced an extended necrosis at the site of injection where, when critical steps in the regenerative process are taking place, an efficient tissue rebuilding is achieved. B. alternatus venom is characterized by the high percentage of exclusively class P-III metalloproteinases, and by the lack of class P-I metalloproteinases in its venom composition. This could explain the effectiveness of muscle regeneration after venom injection despite the severity of the initial phase of envenoming.

Haematologic alterations caused by Ipomoea carnea in experimental poisoning of guinea pig.

Ipomoea carnea subsp. fistulosa (Convolvulaceae) causes poisoning of goats, sheep and cattle in many tropical and subtropical countries. The pathophysiology of this poisoning mainly involves an abnormal glycoprotein metabolism. The aim of this study was to describe the potential toxicity of I. carnea in a guinea pig model through its effect on hematopoiesis in a time course study of 40 days. Experimental poisoning was achieved by feeding animals with "small balls" prepared with milled leaves of I. carnea mixed with commercial crushed pellets for rodents. Hematologic and biochemical parameters, bone marrow and spleencellularities, histopathologic evaluations and lectin-histochemistrywere performed during the scheduled time of the study.The treatment with "small balls" caused significant changes in the weight of spleen, a notable decrease in peripheral red blood cells, and concomitantwith morphological and histopathologicalalterationsin hematopoietic tissues. Overall, the present study suggested that 20 days ofthis treatmentcouldbe enough to develop bone marrow hypoplasia and vacuolation of white cells of spleen, blood and lymph nodes with a transient erythropoietic contribution of the splenic niche.Moreover, this work provides a cheap and simple method for detecting preclinical cases of intoxication by I. carnea in livestock.

Experimental intoxication of guinea pigs with Ipomoea carnea: behavioural and neuropathological alterations.

Ipomoea carnea is a toxic plant that affects goats, with symptoms being characterised by nervous disorders and death. Swainsonine and calystegines are the principal toxic components isolated from I. carnea, which also yields lysergic acid derivatives. The aim of this study was to improve the clinical characterisation of experimental intoxication by I. carnea in guinea pigs through the evaluation of behavioural changes and to perform a thorough histopathological analysis of the affected CNS. Leaves of I. carnea were administered to guinea pigs. Open-field gait analysis and monoamine levels were measured. The poisoned animals exhibited increased vocalisation, lethargy, and a reduction in the locomotion frequency after the fourth week of intoxication, as demonstrated in the open-field test. Significant differences were observed in hind-limb gait width by the last week of intoxication. After 65 days, the guinea pigs were euthanised, necropsied, and examined using light and electron microscopy. At the end of the experiment, plasma serotonin decreased. In contrast, dopamine decreased, and noradrenaline increased in urine. Brain sections were evaluated with conventional histological methods and immunohistochemistry (IHC), as well as by transmission electron microscopy (TEM). Vacuoles were observed throughout the brain, but they were particularly prominent in the brainstem. In addition, there were PAS-negative regions, and the Nissl substance was dispersed or absent, which was confirmed with the Kluver-Barreda stain. Moderate microgliosis was observed by immunohistochemistry. In the medulla oblongata, numerous ubiquitin-positive spheroids together with neuronal degeneration were observed in the nucleus gracilis/cuneatus. Furthermore, vacuoles were observed in astrocytes, oligodendrocytes, and endothelial cells by TEM. Our results showed that the behavioural effects may have been caused by alterations in the brain in conjunction with changes in monoamine levels. This research confirms the utility of this model for studying the pathogenesis of plant-induced lysosomal storage diseases.

Isolation and functional characterization of a new acidic PLA(2) Ba SpII RP4 of the Bothrops alternatus snake venom from Argentina.

An acidic protein with phospholipase A(2) activity was purified to homogeneity from the venom of the Northeast Argentinian viperid Bothrops alternatus by two chromatographic steps: a conventional gel filtration on Sephadex G-75 and reversed phase on C18 HPLC column. A molecular mass of 14185.48 Da was determined by mass spectrometry, displaying a homodimer conformation. The kinetic assay demonstrated a catalytically active phospholipase A(2) in correspondence with Asp49 PLA(2) group. The enzyme designated Ba SpII RP4 contains an amino acid composition of 121 residues and a calculated theoretical pI value of 4.88. Amino acid sequence alignments with other Bothrops PLA(2) revealed a high degree of homology sequence (90-56%). Ba SpII RP4 did not show myotoxic activity upon muscular fibers at doses up to 100 microg i.m. route injection or lethal response when it was i.p. injected at the hightest dose of 200 microg. This toxin generates slight biological activities like paw edema inflammation and a delay in the clotting time, although Ba SpII RP4 exhibited catalytic activity. The primary amino acid sequence, determined a quadruple-time of flight (Q-TOF) hybrid mass spectrometer Q-TOF Ultima from Micromass (Manchester, UK) equipped with a nano Zspray source operating in a positive ion mode and tandem mass spectrum, an ESI/MS mass spectrum (TOF MS mode) "de novo amino acid sequencing", also provides more database about the small group of the non-myotoxic PLA(2)s isolated up to the present.

Mice plasma fibrinogen consumption by thrombin-like enzyme present in rattlesnake venom from the north-east region of Argentina.

Due to variability of venom components from the same species of snakes that inhabit different regions, particular properties of the venom of Crotalus durissus terrificus that inhabits the North-East of Argentina were studied. Gyroxin, a thrombin-like enzyme, was isolated from this venom by gel filtration and affinity chromatography, it was found to be homogeneous according to SDS-PAGE, with a molecular weight of 33 kDa. "Gyroxin syndrome" in mice was tested and it showed changes in the animal behavior, confirming that the isolated thrombin-like enzyme is gyroxin. Effects of this enzyme and the crude venom on mice plasmatic fibrinogen levels were determined. The mice plasma fibrinogen decreased rapidly until incoagulability during the first hour after thrombin-like enzyme injection, then reaching its normal level 10 hours after injection; whereas crude venom resulted in a 60% decrease of the mice plasma fibrinogen, reaching its normal level after the same period of time. After 1 hour of gyroxin inoculation, intravascular coagulation was observed in histological cuttings of lung, cardiac muscle and liver. The isolated enzyme showed strong hydrolyzing activity on fibrinogen and fibrin in vitro, whereas the crude venom exhibited weak hydrolyzing activity on both substrates. It is probable that this very low activity is due to the low percentage of the enzyme in the crude venom. Decreasing of plasmatic fibrinogen levels may be due to either the coagulant or hydrolyzing actions of the enzyme.