Platelet activation: ultrastructure and morphometry in platelet-rich plasma of horses

This study was conducted to investigate the activation ability of the platelet-rich plasma (PRP) by pharmacological agents, as well as to verify the need or not of this activation for therapeutic use. The PRP was obtained from four healthy crossbred geldings aged 13 to 16 years (15±1years), and was processed for observation and quantification of the platelet morphology by using the transmission electron microscopy. All PRP samples were activated with 10% calcium chloride (CaCl2) solution, pure bovine thrombin or associated with CaCl2. The control (pure PRP) was not pharmacologically activated. In the pure PRP samples, 49% of the platelets were classified as state of activation uncertain, 41% as resting, 9% as fully activated and 1% as irreversibly damaged. Treatment with 10% CaCl2 provided a distribution of 54% platelets in state of activation uncertain, 24% as fully activated, 20% as resting, and 2% as irreversibly damaged. The platelet morphology of the bovine thrombin treated samples did not fit into classification adopted, as showing irregular shape with emission of large filamentous pseudopods, appearance of ruptured and whole granules in the remaining cytoplasm and extracellular environment. There was effect of the treatment on the platelet morphology (P=0.03). The 10% CaCl2 is an adequate platelet-activating agent. However, in cases the use of PRP under its liquid form is necessary, the use of pure PRP is recommended, since besides presenting an adequate percentage of fully activated platelets it also has significant amount of the resting type, which can be activated by substances found in the injured tissue.

Method to obtain platelet-rich plasma from rabbits (Oryctolagus cuniculus )

Abstract: Platelet-rich plasma (PRP) is a product easy and inxpesnsive, and stands out to for its growth factors in tissue repair. To obtain PRP, centrifugation of whole blood is made with specific time and gravitational forces. Thus, the present work aimed to study a method of double centrifugation to obtain PRP in order to evaluate the effective increase of platelet concentration in the final product, the preparation of PRP gel, and to optimize preparation time of the final sample. Fifteen female White New Zealand rabbits underwent blood sampling for the preparation of PRP. Samples were separated in two sterile tubes containing sodium citrate. Tubes were submitted to the double centrifugation protocol, with lid closed and 1600 revolutions per minute (rpm) for 10 minutes, resulting in the separation of red blood cells, plasma with platelets and leucocytes. After were opened and plasma was pipetted and transferred into another sterile tube. Plasma was centrifuged again at 2000rpm for 10 minutes; as a result it was split into two parts: on the top, consisting of platelet-poor plasma (PPP) and at the bottom of the platelet button. Part of the PPP was discarded so that only 1ml remained in the tube along with the platelet button. This material was gently agitated to promote platelets resuspension and activated when added 0.3ml of calcium gluconate, resulting in PRP gel. Double centrifugation protocol was able to make platelet concentration 3 times higher in relation to the initial blood sample. The volume of calcium gluconate used for platelet activation was 0.3ml, and was sufficient to coagulate the sample. Coagulation time ranged from 8 to 20 minutes, with an average of 17.6 minutes. Therefore, time of blood centrifugation until to obtain PRP gel took only 40 minutes. It was concluded that PRP was successfully obtained by double centrifugation protocol, which is able to increase the platelet concentration in the sample compared with whole blood, allowing its use in surgical procedures. Furthermore, the preparation time is appropriate to obtain PRP in just 40 minutes, and calcium gluconate is able to promote the activation of platelets.

Obtention of injectable platelets rich-fibrin (i-PRF) and its polymerization with bone graft: technical note

The use of autologous platelet concentrates, represent a promising and innovator tools in the medicine and dentistry today. The goal is to accelerate hard and soft tissue healing. Among them, the platelet-rich plasma (PRP) is the main alternative for use in liquid form (injectable). These injectable form ofplatelet concentrates are often used in regenerative procedures and demonstrate good results. The aim of this study is to present an alternative to these platelet concentrates using the platelet-rich fibrin in liquid form (injectable) and its use with particulated bone graft materials in the polymerized form.

Aggregatory behaviour of platelets incubated with subcellular fractions of normal and chagasic human syncytiotrophoblast

The surface of human syncytiotrophoblast does not induce maternal blood platelet aggregation even though it is not an endothelium. It can be surmised that as occurs in endothelial injury the subcellular components of the syncytiotrophoblast may have pro-or antiaggregatory activity. During congenital Chagas' disease which is associated to trophoblast lesions, platelets may play a role in the development of T. cruzi-induced placentitis. In the present work the aggregatory behaviour of normal human blood platelets was recorded after their challenging with subcellular fractions of syncytiotrophoblast isolated from normal and chagasic women. Nuclear, Mitochondrial, Microsomal and Supernatant fractions isolated from normal and chagasic syncytiotrophoblast failed to induce per se any aggregatory reaction on platelets. When samples of platelet-rich plasma (PRP) were preincubated with normal and chagasic nuclear fractions and then stimulated with collagen at threshold level (CT-PRP) an inhibition of the aggregatory response was observed. Treatment of CT-PRP with normal and chagasic mitochondrial fractions induced inhibition of platelet aggregation whereas only chagasic fraction reduced latency time. Microsornal fraction from normal placentas showed no significant effects on platelet aggregation. It is concluded that subcellular fractions of normal human syncytiotrophoblast do not exhibit any effect on platelet aggregation, whereas those subcellular fractions enriched in intracellular membrane components isolated from chagasic placentas inhibit platelet aggregation.

The role of platelet and plasma markers of antioxidant status and oxidative stress in thrombocytopenia among patients with vivax malaria

Malaria remains an important health problem in tropical countries like Brazil. Thrombocytopenia is the most common hematological disturbance seen in malarial infection. Oxidative stress (OS) has been implicated as a possible mediator of thrombocytopenia in patients with malaria. This study aimed to investigate the role of OS in the thrombocytopenia of Plasmodium vivax malaria through the measurement of oxidant and antioxidant biochemical markers in plasma and in isolated platelets. Eighty-six patients with P. vivax malaria were enrolled. Blood samples were analyzed for total antioxidant and oxidant status, albumin, total protein, uric acid, zinc, magnesium, bilirubin, total thiols, glutathione peroxidase (GPx), malondialdehyde (MDA), antibodies against mildly oxidized low-density lipoproteins (LDL-/nLDL ratio) and nitrite/nitrate levels in blood plasma and GPx and MDA in isolated platelets. Plasma MDA levels were higher in thrombocytopenic (TCP) (median 3.47; range 1.55-12.90 µmol/L) compared with the non-thrombocytopenic (NTCP) patients (median 2.57; range 1.95-8.60 µmol/L). Moreover, the LDL-/nLDL autoantibody ratio was lower in TCP (median 3.0; range 1.5-14.8) than in NTCP patients (median 4.0; range 1.9-35.5). Finally, GPx and MDA were higher in the platelets of TPC patients. These results suggest that oxidative damage of platelets might be important in the pathogenesis of thrombocytopenia found in P. vivax malaria as indicated by alterations of GPx and MDA.

Particulate bioglass in the regeneration of alveolar bone in dogs: clinical, surgical and radiographic evaluations

Bone loss, either by trauma or other diseases, generates an increasing need for substitutes of this tissue. This study evaluated Bioglass as a bone substitute in the regeneration of the alveolar bone in mandibles of dogs by clinical, surgical and radiological analysis. Twenty-eight adult dogs were randomly separated into two equal groups. In each animal, a bone defect was created on the vestibular surface of the alveolar bone between the roots of the fourth right premolar tooth. In the treated group, the defect was immediately filled with bioglass, while in the control, it remained unfilled. Clinical evaluations were performed daily for a week, as well as x-rays immediately after surgery and at 8, 14, 21, 42, 60, 90 and 120 days post-operative. Most animals in both groups showed no signs of inflammation and wound healing was similar. Radiographic examination revealed a gradual increase of radiopacity in the region of the defect in the control group. In the treated group, initial radiopacity was higher than that of adjacent bone, decreasing until 21 days after surgery. Then it gradually increased until 120 days after surgery, when the defect became undetectable. The results showed that Bioglass integrates into bone tissue, is biocompatible and reduced the period for complete bone regeneration.

Thrombocytopenia and leptospirosis

The present study has intended to contribute to the elucidation of the pathogenic mechanisms, involved in the thrombocytopenia and in the bleeding diathesis seen in the course of Leptospirosis. The group of cases included in the present prospective study consisted of 30 patients with Leptospirosis, admitted to the Infectious and Parasitic Diseases Ward, Hospital das Clínicas, Faculty of Medicine, University of São Paulo. The following possible mechanisms of thrombocytopenia have been considered and therefore investigated: platelet consumption, due to disseminated intravascular coagulation; immune-mediated platelet destruction, due to platelet-associated antibodies and an inhibited platelet production in the bone marrow. Thrombocytopenia occurred in 86.6% of 30 patients and did not seem to be immune-mediated by platelet-associated antibodies. Furthermore it did not seem to be due to a disseminated intravascular coagulation consumption. Although there was a statistically-significant correlation between bone marrow platelet production and platelet counts we think that the static microscopic examination of a bone marrow aspirate cannot accurately depict the dynamic mechanisms of platelet production when these cells are being consumed in peripheral blood. Vasculitis should be considered as the most important factor for the pathogenesis of the bleeding disturbances in Leptospirosis. However, we believe that thrombocytopenia, uremia and coagulation disorders, individually or as a group, should be included among the contributing factors that lead to and worsen bleeding episodes, which represent the leading cause of death in this disease.

Evaluation of a model for induction of periodontal disease in dogs

There are several methods for inducing periodontal disease in animal models, being the bone defect one of the most reported. This study aimed to evaluate this model, through clinical, radiographic, tomographic and histological analyzes, thus providing standardized data for future regenerative works. Twelve dogs were subjected to the induction protocol. In a first surgical procedure, a mucoperiosteal flap was made on the buccal aspect of the right third and fourth premolars and a defect was produced exposing the furcation and mesial and distal roots, with dimensions: 5mm coronoapical, 5mm mesiodistal, and 3mm buccolingual. Periodontal ligament and cementum were curetted and the defect was filled with molding polyester, which was removed after 21 days on new surgical procedure. Clinical and radiographic examinations were performed after the two surgeries and before the collection of parts for dental tomography and histological analysis. All animals showed grade II furcation exposure in both teeth. Clinical attachment level increased after induction. Defect size did not change for coronoapical and buccolingual measurements, while mesiodistal size was significantly higher than at the time of defect production. Radiographic analysis showed decreased radiopacity and discontinuity of lamina dura in every tooth in the furcation area. The horizontal progression of the disease was evident in micro-computed tomography and defect content in the histological analysis. Therefore, it is concluded that this method promotes the induction of periodontal disease in dogs in a standardized way, thus being a good model for future work.

Lymphocyte subpopulations and neutrophil function in chronic human Chagas' disease

The absolute numbers of total leukocytes, lymphocytes, T cells, helper/inducer, suppressor/cytotoxic and B cells were decreased in the peripheral blood of patients with chronic Chagas' disease. Since antilymphocyte antibodies were present only in a minority of patients they probably cannot account for the abnormalities in lymphocyte subsets. Patient neutrophils stimulated with endotoxin-treated autologous plasma showed depressed chemotactic activity and this seems to be an intrinsic cellular defect rather than plasma inhibition. Random migration of neutrophils was normal. Reduction of nitroblue tetrazolium by endotoxin- stimulated neutrophils was also decreased. These findings further document the presence of immunosuppression in human Chagas' disease. They may be relevant to autoimmunity, defense against microorganisms and against tumor cells at least in a subset of patients with more severe abnormalities.

Susceptibility of Cebus apella monkey (Primates: Cebidae) to experimental Leishmania (L.) infantum chagasi-infection

In Amazonian Brazil, the Cebus apella monkey (Primates: Cebidae) has been associated with the enzootic cycle of Leishmania (V.) shawi, a dermotropic parasite causing American cutaneous leishmaniasis (ACL). It has also been successfully used as animal model for studying cutaneous leishmaniasis. In this work, there has been investigated its susceptibility to experimental Leishmania (L.) infantum chagasi-infection, the etiologic agent of American visceral leishmaniasis (AVL). There were used ten C. apella specimens, eight adult and two young, four males and six females, all born and raised in captivity. Two experimental infection protocols were performed: i) six monkeys were inoculated, intra-dermal via (ID), into the base of the tail with 2 x 10(6) promastigotes forms from the stationary phase culture medium; ii) other four monkeys were inoculated with 3 x 10(7) amastigotes forms from the visceral infection of infected hamsters by two different via: a) two by intravenous via (IV) and, b) other two by intra-peritoneal via (IP). The parameters of infection evaluation included: a) clinical: physical exam of abdomen, weigh and body temperature; b) parasitological: needle aspiration of the bone-marrow for searching of amastigotes (Giemsa-stained smears) and promastigotes forms (culture medium); c) immunological: Indirect fluorescence antibody test (IFAT) and, Delayed-type hypersensitivity (DTH). In the six monkeys ID inoculated (promastigotes forms) all parameters of infection evaluation were negative during the 12 months period of follow-up. Among the four monkeys inoculated with amastigotes forms, two IV inoculated showed the parasite in the bone-marrow from the first toward to the sixth month p.i. and following that they cleared the infection, whereas the other two IP inoculated were totally negative. These four monkeys showed specific IgG-antibody response since the third month p.i. (IP: 1/80 and IV: 1/320 IgG) toward to the 12th month (IP: 1/160 and IV: 1/5120). The DTH-conversion occurred in only one IV inoculated monkey with a strong (30 mm) skin reaction. Considering these results, we do not encourage the use of C. apella monkey as animal model for studying the AVL.

The role of the eosinophil-selective chemokine, eotaxin, in allergic and non-allergic airways inflammation

Blood eosinophilia and tissue infiltration by eosinophils are frequently observed in allergic inflammation and parasitic infections. This selective accumulation of eosinophils suggested the existence of endogenous eosinophil-selective chemoattractants. We have recently discovered a novel eosinophil-selective chemoattractant which we called eotaxin in an animal model of allergic airways disease. Eotaxin is generated in both allergic and non-allergic bronchopulmonary inflammation. The early increase in eotaxin paralled eosinophil infiltration in the lung tissue in both models. An antibody to IL-5 suppressed lung eosinophilia, correlating with an inhibition of eosinophil release from bone marrow, without affecting eotaxin generation. This suggests that endogenous IL-5 is important for eosinophil migration but does not appear to be a stimulus for eotaxin production. Constitutive levels of eotaxin observed in guinea-pig lung may be responsible for the basal lung eosinophilia observed in this species. Allergen-induced eotaxin was present mainly in the epithelium and alveolar macrophages, as detected by immunostaining. In contrast there was no upregulation of eotaxin by the epithelial cells following the injection of Sephadex beads and the alveolar macrophage and mononuclear cells surrounding the granuloma were the predominant positive staining cells. Eotaxin and related chemokines acting through the CCR3 receptor may play a major role in eosinophil recruitment in allergic inflammation and parasitic diseases and thus offer an attractive target for therapeutic intervention.

Atherosclerosis and acute arterial thrombosis in rabbits: a model using balloon desendothelization without dietary intervention

Acute thrombosis can be induced in rabbits by a triggering protocol using Russell's viper venom and histamine given after 8 months of a 1% cholesterol diet and balloon desendothelization. In the present study, we tested the hypothesis that aortic desendothelization performed 4 months before the triggering protocol without a high cholesterol diet is a highly effective and less expensive way of producing arterial atherosclerosis and thrombosis. Nineteen male New Zealand white rabbits on a normal diet were studied. The control group (N = 9) received no intervention during the 4-month observation period, while the other group (N = 10) was submitted to aortic balloon desendothelization using a 4F Fogarty catheter. At the end of this period, all animals were killed 48 h after receiving the first dose of the triggering treatment. Eight of 10 rabbits (80%) in the balloon-trauma group presented platelet-rich arterial thrombosis while none of the animals in the control group had thrombus formation (P<0.01). Thus, this model, using balloon desendothelization without dietary manipulation, induces arterial atherosclerosis and thrombosis and may provide possibilities to test new therapeutic approaches

Impaired regeneration of dystrophin-deficient muscle fibers is caused by exhaustion of myogenic cells

Duchenne muscular dystrophy is one of the most devastating myopathies. Muscle fibers undergo necrosis and lose their ability to regenerate, and this may be related to increased interstitial fibrosis or the exhaustion of satellite cells. In this study, we used mdx mice, an animal model of Duchenne muscular dystrophy, to assess whether muscle fibers lose their ability to regenerate after repeated cycles of degeneration-regeneration and to establish the role of interstitial fibrosis or exhaustion of satellite cells in this process. Repeated degenerative-regenerative cycles were induced by the injection of bupivacaine (33 mg/kg), a myotoxic agent. Bupivacaine was injected weekly into the right tibialis anterior muscle of male, 8-week-old mdx (N = 20) and C57Bl/10 (control, N = 10) mice for 20 and 50 weeks. Three weeks after the last injection, the mice were killed and the proportion of regenerated fibers was counted and reported as a fibrosis index. Twenty weekly bupivacaine injections did not change the ability of mdx muscle to regenerate. However, after 50 weekly bupivacaine injections, there was a significant decrease in the regenerative response. There was no correlation between the inability to regenerate and the increase in interstitial fibrosis. These results show that after prolonged repeated cycles of degeneration-regeneration, mdx muscle loses its ability to regenerate because of the exhaustion of satellite cells, rather than because of an increase in interstitial fibrosis. This finding may be relevant to cell and gene therapy in the treatment of Duchenne muscular dystrophy.

Ultrasound method applied to characterize healthy femoral diaphysis of Wistar rats in vivo

A simple experimental protocol applying a quantitative ultrasound (QUS) pulse-echo technique was used to measure the acoustic parameters of healthy femoral diaphyses of Wistar rats in vivo. Five quantitative parameters [apparent integrated backscatter (AIB), frequency slope of apparent backscatter (FSAB), time slope of apparent backscatter (TSAB), integrated reflection coefficient (IRC), and frequency slope of integrated reflection (FSIR)] were calculated using the echoes from cortical and trabecular bone in the femurs of 14 Wistar rats. Signal acquisition was performed three times in each rat, with the ultrasound signal acquired along the femur's central region from three positions 1 mm apart from each other. The parameters estimated for the three positions were averaged to represent the femur diaphysis. The results showed that AIB, FSAB, TSAB, and IRC values were statistically similar, but the FSIR values from Experiments 1 and 3 were different. Furthermore, Pearson's correlation coefficient showed, in general, strong correlations among the parameters. The proposed protocol and calculated parameters demonstrated the potential to characterize the femur diaphysis of rats in vivo. The results are relevant because rats have a bone structure very similar to humans, and thus are an important step toward preclinical trials and subsequent application of QUS in humans.

Fibrotic sequelae in pulmonary paracoccidioidomycosis: histopathological aspects in BALB/c mice infected with viable and non-viable Paracoccidioides brasiliensis propagules

Patients with paracoccidioidomycosis often present pulmonary fibrosis and exhibit important respiratory limitations. Based on an already established animal model, the contribution of viable and non-viable P. brasiliensis propagules to the development of fibrosis was investigated. BALB/c male mice, 4-6 weeks old were inoculated intranasally either with 4x10(6 )viable conidia (Group I), or 6.5x10(6) fragmented yeast cells (Group II). Control animals received PBS. Six mice per period were sacrificed at 24, 48, 72h (initial) and 1, 2, 4, 8, 12 and 16 weeks post-challenge (late). Paraffin embedded lungs were sectioned and stained with H&E, trichromic (Masson), reticulin and Grocott´s. During the initial period PMNs influx was important in both groups and acute inflammation involving 34% to 45% of the lungs was noticed. Later on, mononuclear cells predominated. In group I, the inflammation progressed and granulomas were formed and by the 12th week they fussed and became loose. Thick collagen I fibers were observed in 66.6% and 83.3% of the animals at 8 and 12 weeks, respectively. Collagen III, thick fibers became apparent in some animals at 4weeks and by 12 weeks, 83% of them exhibited alterations in the organization and thickness of these elements. In group II mice, this pattern was different with stepwise decrease in the number of inflammatory foci and lack of granulomas. Although initially most animals in this group had minor alterations in thin collagen I fibers, they disappeared by the 4th week. Results indicate that tissue response to fragmented yeast cells was transitory while viable conidia evoked a progressive inflammatory reaction leading to granuloma formation and to excess production and/or disarrangement of collagens I and III; the latter led to fibrosis.