Camundongos como modelos de doenças hemostáticas

Mice initiated to be used for experimental purposes in laboratories in laboratories in the early 20 th century, simultaneously with the rediscover of Mendel’s Laws. Ever since, they are considered excellent models for genetic studies, as they have high biological similarity with humans -between 70 and 90%-, and have a genome easily handled by means of molecular techniques and breeding procedures. Furthermore, mice have a short gestation period, shorter life cycle in comparison to other animals and easy handling in laboratory facilities. Around the 1990s, genetically modified animals were produced as models for mimicking hemostatic diseases. These genetically modified animals showed hemostatic alterations characteristic of human diseases, as hemophilia A -caused by mutations in coagulation factor VIII-, hemophilia B -caused by mutations in coagulation factor IX- and von Willebrand disease -vWD- caused by mutations in von Willebrand factor-. In this context, the animal models of hemostatic disorders have become important tools for evaluating new treatments and studying rare disease conditions.

Os camundongos foram introduzidos nos laboratórios com fins experimentais no início do século XX juntamente com a redescoberta da Lei de Mendel. Desde então, são considerados excelentes modelos para o estudo de genética, por apresentarem alta similaridade biológica com o ser humano, compreendida entre 70 e 90% e um genoma facilmente manipulado por meio de técnicas moleculares e sistemas de acasalamentos. Além disso, os camundongos apresentam um período de gestação curto, seu ciclo de vida é menor se comparado a outros animais e são de fácil manuseio em laboratório. Por volta dos anos 1990, foi introduzida a utilização de animais geneticamente modificados como modelos para reprodução de doenças hemostáticas. Esses animais geneticamente modificados apresentavam alterações hemostáticas características de doenças humanas, como as hemofilias do tipo A -causada por mutações no fator VIII da coagulação-, hemofilia do tipo B -causada por mutações no fator IX da coagulação- e doença de von Willebrand -vWD- causada por mutações no fator de von Willebrand -vWF-. Os modelos de animais com distúrbios hemostáticos se tornaram importantes ferramentas para testar novas formas de tratamento e estudar condições raras das doenças.

Involvement of von Willebrand factor and botrocetin in the thrombocytopenia induced by Bothrops jararaca snake venom

Patients bitten by snakes consistently manifest a bleeding tendency, in which thrombocytopenia, consumption coagulopathy, mucous bleeding, and, more rarely, thrombotic microangiopathy, are observed. Von Willebrand factor (VWF) is required for primary hemostasis, and some venom proteins, such as botrocetin (a C-type lectin-like protein) and snake venom metalloproteinases (SVMP), disturb the normal interaction between platelets and VWF, possibly contributing to snakebite-induced bleedings. To understand the relationship among plasma VWF, platelets, botrocetin and SVMP from Bothrops jararaca snake venom (BjV) in the development of thrombocytopenia, we used (a) Wistar rats injected s.c. with BjV preincubated with anti-botrocetin antibodies (ABA) and/or Na2-EDTA (a SVMP inhibitor), and (b) VWF knockout mice (Vwf-/-) injected with BjV. Under all conditions, BjV induced a rapid and intense thrombocytopenia. In rats, BjV alone reduced the levels of VWF:Ag, VWF:CB, high molecular weight multimers of VWF, ADAMTS13 activity, and factor VIII. Moreover, VWF:Ag levels in rats that received BjV preincubated with Na2-EDTA and/or ABA tended to recover faster. In mice, BjV caused thrombocytopenia in both Vwf-/- and C57BL/6 (background control) strains, and VWF:Ag levels tended to decrease in C57BL/6, demonstrating that thrombocytopenia was independent of the presence of plasma VWF. These findings showed that botrocetin present in BjV failed to affect the extent or the time course of thrombocytopenia induced by envenomation, but it contributed to decrease the levels and function of plasma VWF. Thus, VWF alterations during B. jararaca envenomation are an ancillary event, and not the main mechanism leading to decreased platelet counts.

Parental behavior and anxiety in isogenic and outbred mice given access to two types of nesting materials

The objective was to investigate if providing two types of nesting materials could modulate parental behavior and anxiety in laboratory mice. For that, 54 full-sib BALB/cJ and 50 randomly mated Swiss Webster mouse pairs were employed in a completely randomized designwith a 2×2 factorial arrangement (two genetic groups and with/ without access to nesting materials). Eight pieces of disposable polypropylene caps/ hairnets and 3g of cotton were provided as nesting materials to half the cages from each genetic group. Maternal and paternal behaviors were recorded on the third reproductive cycle, twice a week, for three weeks, using scan sampling. The behaviors were recorded every 10s for a period of 10min (totaling 60 records) each day. Dams (N=40) were tested in the elevated plus maze (EPM) on the fifth reproductive cycle, 7–10 days postpartum. Their 21-days-old weanlings (N=208) were tested in the same apparatus. Total number of closed arm entries was used as measurement of motor activity, the percentage of time spent on open arms as measurement of anxiety and head dipping time as measurement of exploratory behavior. The provision of nesting materials increased the frequency of dams licking pups (1.72±0.20 vs. 1.10±0.21, P=0.0342) and of sires resting in contact with pups (25.0±1.5 vs. 18.9±1.5, P=0.0050), while simultaneously decreased the frequency of sires in non-contact rest with pups (6.4±1.1 vs. 10.8±1.2, P=0.0074). Swiss Webster dams were recorded nursing their pups in the arched-back posture more frequently than BALB/cJ dams (9.63±0.89 vs. 7.13±0.86, P=0.0187) and Swiss Webster sires showed a higher frequency of nest building than BALB/cJ sires (0.80±0.16 vs. 0.31±0.15, P=0.0281). Motor activity was higher (8.00±0.43 vs. 2.83±0.41, P<0.0001) and anxiety was lower (3.2±28.4 vs. 2.4±3.1%, P<0.0001) in Swiss Webster than in BALB/cJ dams. Anxiety (26.6±3.2 vs. 37.9±3.5%, P=0.0168) was also lower in Swiss Webster than in BALB/cJ weanlings. The availability of nesting materials increased the time dams spent in head dipping (10.9±2.0 vs. 4.6±1.9, P=0.0087), but weanling behaviors in the EPM were unaffected. Slight differences in parental behavior and contrasting patterns of anxiety and motor activity were found between genetic groups. The provision of nesting materials promoted an intensification of favorable parent-offspring interactions and enhanced exploratory behavior of dams.

Comparative study of platelet aggregation and secretion induced by Bothrops jararaca snake venom and thrombin

Victims of Bothrops jararaca snakebites manifest bleedings, blood incoagulability, platelet dysfunction, and thrombocytopenia, and the latter has been directly implicated in the genesis of hemorrhagic diathesis. We addressed herein the direct effects of B. jararaca venom (BjV) on ex vivo platelet aggregation and granule secretion in washed human and mouse platelets. BjV directly aggregated platelets, but the extent of platelet aggregation was lower in human than mouse platelets. On the other hand, BjV (24.4 mu g/mL) and thrombin (0.1 U/mL) induced a similar extent of ATP and platelet factor 4 (PF4) secretion in both species. BjV-induced platelet aggregation was independent of the platelet dense body content, as in pearl mouse (Ap3b1(-/-))platelets, whose dense bodies are deficient in adenine nucleotides and serotonin, the extent of platelet aggregation was superior to that induced in BALB/c or C57BL/6 mice. BjV-induced beta-hexosaminidase secretion in human platelets was less intense than that evoked by thrombin, and the contrary was observed in mouse platelets. Irreversible inactivation of platelet cyclooxygenase 1 by acetylsalicylic acid did not reduce BjV-induced platelet aggregation. BjV exerted no cytotoxic activity in human and mouse platelets, as evaluated by lactate dehydrogenase loss. Eptifibatide, which inhibits the binding of fibrinogen to platelet glycoprotein complex GPIIb-IIIa, differently blocked BjV-induced platelet aggregation in mice and humans. BjV-induced platelet aggregation did not depend on snake venom serine proteinases nor metalloproteinases in mice, whilst serine proteinases were rather important for platelet aggregation in humans. Our results show that BjV induces direct activation, aggregation, and secretion in human and mouse platelets, but it exerts diverse responses in them, which should be considered in comparative studies to understand pathophysiological events during Bothrops envenomation.

Parental behavior and anxiety in isogenic and outbred mice given access to two types of nesting materials

The objective was to investigate if providing two types of nesting materials could modulate parental behavior and anxiety in laboratory mice. For that, 54 full-sib BALB/cJ and 50 randomly mated Swiss Webster mouse pairs were employed in a completely randomized designwith a 2×2 factorial arrangement (two genetic groups and with/ without access to nesting materials). Eight pieces of disposable polypropylene caps/ hairnets and 3g of cotton were provided as nesting materials to half the cages from each genetic group. Maternal and paternal behaviors were recorded on the third reproductive cycle, twice a week, for three weeks, using scan sampling. The behaviors were recorded every 10s for a period of 10min (totaling 60 records) each day. Dams (N=40) were tested in the elevated plus maze (EPM) on the fifth reproductive cycle, 7–10 days postpartum. Their 21-days-old weanlings (N=208) were tested in the same apparatus. Total number of closed arm entries was used as measurement of motor activity, the percentage of time spent on open arms as measurement of anxiety and head dipping time as measurement of exploratory behavior. The provision of nesting materials increased the frequency of dams licking pups (1.72±0.20 vs. 1.10±0.21, P=0.0342) and of sires resting in contact with pups (25.0±1.5 vs. 18.9±1.5, P=0.0050), while simultaneously decreased the frequency of sires in non-contact rest with pups (6.4±1.1 vs. 10.8±1.2, P=0.0074). Swiss Webster dams were recorded nursing their pups in the arched-back posture more frequently than BALB/cJ dams (9.63±0.89 vs. 7.13±0.86, P=0.0187) and Swiss Webster sires showed a higher frequency of nest building than BALB/cJ sires (0.80±0.16 vs. 0.31±0.15, P=0.0281). Motor activity was higher (8.00±0.43 vs. 2.83±0.41, P<0.0001) and anxiety was lower (3.2±28.4 vs. 2.4±3.1%, P<0.0001) in Swiss Webster than in BALB/cJ dams. Anxiety (26.6±3.2 vs. 37.9±3.5%, P=0.0168) was also lower in Swiss Webster than in BALB/cJ weanlings. The availability of nesting materials increased the time dams spent in head dipping (10.9±2.0 vs. 4.6±1.9, P=0.0087), but weanling behaviors in the EPM were unaffected. Slight differences in parental behavior and contrasting patterns of anxiety and motor activity were found between genetic groups. The provision of nesting materials promoted an intensification of favorable parent-offspring interactions and enhanced exploratory behavior of dams.

Comparative study of platelet aggregation and secretion induced by Bothrops jararaca snake venom and thrombin

Victims of Bothrops jararaca snakebites manifest bleedings, blood incoagulability, platelet dysfunction, and thrombocytopenia, and the latter has been directly implicated in the genesis of hemorrhagic diathesis. We addressed herein the direct effects of B. jararaca venom (BjV) on ex vivo platelet aggregation and granule secretion in washed human and mouse platelets. BjV directly aggregated platelets, but the extent of platelet aggregation was lower in human than mouse platelets. On the other hand, BjV (24.4 mu g/mL) and thrombin (0.1 U/mL) induced a similar extent of ATP and platelet factor 4 (PF4) secretion in both species. BjV-induced platelet aggregation was independent of the platelet dense body content, as in pearl mouse (Ap3b1(-/-))platelets, whose dense bodies are deficient in adenine nucleotides and serotonin, the extent of platelet aggregation was superior to that induced in BALB/c or C57BL/6 mice. BjV-induced beta-hexosaminidase secretion in human platelets was less intense than that evoked by thrombin, and the contrary was observed in mouse platelets. Irreversible inactivation of platelet cyclooxygenase 1 by acetylsalicylic acid did not reduce BjV-induced platelet aggregation. BjV exerted no cytotoxic activity in human and mouse platelets, as evaluated by lactate dehydrogenase loss. Eptifibatide, which inhibits the binding of fibrinogen to platelet glycoprotein complex GPIIb-IIIa, differently blocked BjV-induced platelet aggregation in mice and humans. BjV-induced platelet aggregation did not depend on snake venom serine proteinases nor metalloproteinases in mice, whilst serine proteinases were rather important for platelet aggregation in humans. Our results show that BjV induces direct activation, aggregation, and secretion in human and mouse platelets, but it exerts diverse responses in them, which should be considered in comparative studies to understand pathophysiological events during Bothrops envenomation.

The gingival vein as a minimally traumatic site for multiple blood sampling in guinea pigs and hamsters

Laboratory animals are still necessary in scientific investigation and vaccine testing, but while novel methodological approaches are not available for their replacement, the search for new, humane, easy, and painless methods is necessary to diminish their stress and pain. When multiple blood samples are to be collected from hamsters and guinea pigs, the number of available venipuncture sites-which are greatly diminished in these species in comparison with other rodents due to the absence of a long tail-, harasses animal caregivers and researchers. Thus, this study aimed to evaluate if gingival vein puncture could be used as an additional route to obtain multiple blood samples from anesthetized hamsters and guinea pigs in such a way that animal behavior, well-being or hematological parameters would not be altered. Thus, twelve anesthetized Syrian golden hamsters and English guinea pigs were randomly allocated in two groups: a control group, whose blood samples were not collected, and an experimental group in which blood samples (200 microliters) were collected by gingival vein puncture at weekly intervals over six weeks. Clinical assessment, body weight gain and complete blood cell count were evaluated weekly, and control and experimental animals were euthanized at week seven, when the mentolabial region was processed to histological analyses. Multiple blood sampling from the gingival vein evoked no clinical manifestations or alteration in the behavioral repertoire, nor a statistically significant difference in weight gain in both species. Guinea pigs showed no alteration in red blood cell, leukocyte or platelet parameters over time. Hamsters developed a characteristic pattern of age-related physiological changes, which were considered normal. Histological analyses showed no difference in morphological structures in the interdental gingiva, confirming that multiple blood sampling is barely traumatic. Thus, these results evidence that blood collection from multiple gingival vein puncture is minimally invasive and traumatic to hamsters and guinea pigs, and that it can be accomplished during at least six weeks.