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...

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.

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

Ventricular fibrillation in acute experimental myocardial ischemia: protection by magnesium sulfate

The efficacy of magnesium sulfate for prevention of ventricular fibrillation was compared with that of the calcium blocker verapamil and other antiarrhythmic drugs in 54 open-chested anesthetized dogs during a 3-h ligation of the circumflex coronary artery. Latency time to fibrillation, incidence of fibrillation and hemodynamic parameters were assessed. 2. Ventricular fibrillation ocurred in 11 of 14(79%) control dogs, in 2 of 8(25%) dogs treated with magnesium sulfate (100 mg/Kg) and in none of 8 animals treated with verapamil (0.2 mg/Kg) (P = 0.014 and P = 0.0004, respectively, in comparison with controls); lidocaine (60 mg followed by 4 mg/min), amiodarone (5mg/kg) and propafenone (4mg/Kg) had no effect on the incidence of fibrillation. 3. The latency time to fibrillation was 11.6 ñ 9.1 min in controls and it was shortened to 4.0 ñ 3.8 min (P = 0.039) in dogs treated with propafenone, but was unaffected by other drugs. 4. There was no correlation between pre-occlusion heart rate or blood pressure and fibrillation incidence. 5. In this experimental model, magnesium sulfate infusion had a protective effect against ventricular fibrillation that was similar to verapamil, suggesting that magnesium sulfate may be useful as an antifibrillatory agent during acute ischemia

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 < 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 < 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