João José Lachat

Evaluation of the biocompatibility of a new biomembrane

Biocompatibility has been considered one of the most important items to validate a biomaterial for its application in human organisms. The present work evaluates the biocompatibility of a new biomembrane using in vivo assay in different animal species. The experiments to evaluate the cellular reaction were carried out through the implantation of the material into the subcutaneous tissue of animals and the results showed a good reaction of the host tissue without any signal of fibrosis or rejection. The cell adhesion experiments were done by means of the measure of the DNA content on the material surface after its implantation into the subcutaneous tissue of animals and the results showed a growing number of DNA that was proportional to the time of implantation. The healing process was evaluated using a dermal ulcer model and the results showed a good tissue repair resembling a physiologic process. The overall results presented here lead to the conclusion that this new biomembrane is a biocompatible material but more research must be done, as it is a new material desired for medical use.

Increased Vascular Permeability, Angiogenesis and Wound Healing Induced by the Serum of Natural Latex of the Rubber Tree Hevea brasiliensis

Increases in vascular permeability and angiogenesis are crucial events to wound repair, tumoral growth and revascularization of tissues submitted to ischemia. An increased vascular permeability allows a variety of cytokines and growth factors to reach the damaged tissue. Nevertheless, the angiogenesis supply tissues with a wide variety of nutrients and is also important to metabolites clearance. It has been suggested that the natural latex from Hevea brasiliensis showed wound healing properties and angiogenic activity. Thus, the purpose of this work was to characterize its angiogenic activity and its effects on vascular permeability and wound healing. The serum fraction of the latex was separated from the rubber with reduction of the pH. The activity of the dialyzed serum fraction on the vascular permeability injected in subcutaneous tissue was assayed according Miles method. The angiogenic activity was determined using a chick embryo chorioallantoic membrane assay and its effects on the wound‐healing process was determined by the rabbit ear dermal ulcer model. The serum fraction showed evident angiogenic effect and it was effective in enhancing vascular permeability. In dermal ulcers, this material significantly accelerated wound healing. Moreover, the serum fraction boiled and treated with proteases lost these activities. These results are in accordance with the enhancement of wound healing observed in clinical trials carried out with a biomembrane prepared with the same natural latex. Copyright

Prótese vascular derivada do látex

BACKGROUND: The development of vascular grafts has been crucial for advances and achievements in reconstructive vascular surgery over the past 5 decades. OBJECTIVES: To develop a new model of microperforated vascular graft using fabric covered with a natural latex-derived polymer taken from Hevea brasiliensis and assess its patency, thrombogenicity, biocompatibility and healing process, besides some mechanical properties (adaptability, elasticity, impermeability and possibility of suture), using expanded polytetrafluoroethylene graft as control. METHODS: Fifteen dogs were divided into three groups of five animals. The microperforated latex graft was implanted in all dogs and the expanded polytetrafluoroethylene graft was implanted in the contralateral pelvic limb. Postoperative follow-up was 4, 8 and 12 weeks. Analysis of results was performed according to clinical evaluation of pulses, complications (fluid collection, dehiscence, granuloma and infection), arteriography, macroscopic analysis and scanning electron micrography. RESULTS: Statistical tests revealed no significant differences (p > 0.05) concerning post-operative complications and graft patency. Both grafts were properly integrated to surrounding tissues, with connective tissue formed by collagen fibers. A neointimal layer covering all extension of the luminal surface was observed in the microperforated latex graft. Conversely, the endothelial development over the neointimal surface was limited to regions adjacent to the anastomoses in the expanded polytetrafluoroethylene graft. CONCLUSIONS: The microperforated latex graft showed satisfactory structural qualities (adaptability, elasticity, impermeability and possibility of suture) as a vascular substitute. It stimulated endothelial growth beyond contact regions with the artery in anastomoses and was biocompatible in the dogs arterial system, presenting adequate tissue integration.

Avaliação da isquemia cerebral focal induzida pela oclusão da artéria cerebral média e a ação neuroprotetora do cetoprofeno em ratos

Cerebral ischaemia is eventualy observed during neurosurgical procedures and in several clinical entities that may cause severe neurological deficits and even death. Because it is a severe and complex problem, several studies have been done aiming to elucidate the mechanisms of the ischemic phenomenon and aiming to abolish or to diminish its effects, using drugs that protect the neurons from ischaemia-induced damage. Several neurotransmitters play a role in cerebral ischaemia with emphasis to glutamate by its high concentration in the central nervous system. The purpose of this study was to evaluate the effect of focal cerebral ischaemia in the rat through the dosage of the glutamate and morphological findings, and to evaluate a possible protective effect of the ketoprofen to ischemic neurons. Thirty-six rats Wistar were divided into four groups. The first was a control group, the second a sham group and the animals of the third and fourth groups were submitted to induced cerebral ischaemia through selective obstruction of the midlle cerebral artery during 15, 30 and 45 minutes. Animals of the fourth group were previously treated with ketoprofen 15 minutes before the ischaemia. The ischaemia was evaluated through the histopathological examination and through dosage of the extracellular glutamate in vitro. The histopathological examination showed that there was no difference between the animals of the control and of the sham groups. In the animals submitted to ischemia histopathological alterations appeared at 30 minutes and become more intense at 45 minutes of ischaemia. The main findings were interstitial edema, chromatinic disorganization, vacuolization and nuclear desintegration. The animals treated with ketoprofen showed similar alterations, but they were less intense. Decrease in the dosage of glutamate in the parietal cortex of the animals submitted to ischaemia started at 30 minutes and became more intense at 45 minutes of ischaemia and was similar for animals previously treated or not with ketoprofen, indicating that this drug seems not to interfere with the metabolism of the glutamate at the synapses. The morphological findings in the parietal cortex of the animals submitted to ischaemia, previously treated or not with ketoprofen, suggest that this drug has a neuroprotective effect.

Changes caused by hidrocephalus, induced by kaolin, in the corpus callosum of adult dogs

PURPOSE: To analyze the ventricular enlargement and myelination of the corpus callosum in adult dogs after four and eight weeks of kaolin-induction of hydrocephalus. METHODS: 36 dogs were randomly divided into 3 groups: 1 - without hydrocephalus, 2 - kaolin-induction of hydrocephalus until the fourth week, and 3 - kaolin-induction of hydrocephalus until the eighth week. Ventricular ratios and volumes were calculated using magnetic resonance images, and myelination of the corpus callosum were histologically evaluated using solocromo-cianin stain. RESULTS: Radiological hydrocephalus was observed in 93.75% and overall mortality was 38.4%. Ventricular volumes and ratios were higher in groups 2 and 3 compared to group 1 and similar when measures in the fourth and eighth weeks were compared in the group 3. Indices of luminescence in the knee and in the splenium of the corpus callosum were higher in group 2 than in group 1 indicating that there was loss of myelin in group 2, and similar in groups 1 and 3, showing a tendency to remyelination after 8 weeks. CONCLUSION: The corpus callosum of dogs with kaolin-induced hydrocephalus responds with demyelination of the knee and splenium by the fourth week with a tendency to remyelination by the eighth week.

Ultrastructural study of the lateral ventricle choroid plexus in experimental hydrocephalus in Wistar rats

Hydrocephalus is one of the most frequent and complex neurological diseases characterized by the abnormal buildup of cerebrospinal fluid (CSF) in the ventricles of the brain, due to an altered CSF dynamics. To detect possible ultrastructural alterations of the lateral ventricles choroid plexus (responsible for the CSF production), rats seven days after birth were submitted to an intracisternal injection of 20% kaolim (hydrated aluminum silicate) for the hydrocephalus induction. Twenty-eight or 35 days after injection, injected animals and respective controls were processed for observation under a transmission electron microscopy. Alterations found: presence of concentric cell membrane fragments, larger number of primary and secondary lysossomes, vacuoles, and cytoplasmic vesicles, and an enlargement of the intercellular space and between the basolateral interdigitation of the choroid epithelium. The alterations observed are probably associated to an increase of the ventricular pressure, inducing morpho-functional effects on the choroid plexus integrity.

Oral treatment with Hev b 13 ameliorates experimental colitis in mice.

Hev b 13 is an allergenic esterase obtained from the rubber tree Hevea brasiliensis, which has been shown recently to induce human mononuclear cells to release interleukin (IL)‐10 in vitro. This immunoregulatory cytokine appears to play an important role in preventing inflammation and mucosal damage in animal models of colitis and in Crohns disease patients. The aim of this study was to evaluate the therapeutic effect of Hev b 13 in mice with 2,4,6‐trinitrobenzene sulphonic acid (TNBS)‐induced colitis. Two hours following colonic instillation of the haptenizing agent, and daily thereafter for 5 days, Hev b 13 was administered by oral gavage. In mice treated with daily doses of either 0·5u2003mg/kg or 5·0u2003mg/kg of Hev b 13, the clinical signs of diarrhoea, rectal prolapse and body weight loss and also histological damage of the distal colon, were reduced significantly, in comparison with water‐treated diseased mice. These findings suggest a potent anti‐inflammatory activity of Hev b 13; this activity is speculated to be related to its interaction with cells from the immune system.

Oral treatment with Hev b 13 prevents experimental arthritis in mice

Hev b 13 is an allergenic esterase obtained from the rubber tree Hevea brasiliensis, which has been shown recently to induce human monocytes to release interleukin (IL)‐10 in vitro, and to exert a potent anti‐inflammatory effect in vivo. Moreover, Hev b 13 has been shown to reduce clinical signs of inflammation and also histological damage to the distal colon of mice with 2,4,6‐trinitrobenze sulphonic acid (TNBS)‐induced colitis after its oral administration. The aim of this study was to investigate the effect of Hev b 13 on human mononuclear cells, as well as its therapeutic use in the methylated bovine serum albumin (mBSA) model of antigen‐induced arthritis. Five days before the intra‐articular challenge, and daily thereafter for 8 days, Hev b 13 was administered by oral gavage. In mice treated with a dose of 0·5u2003mg/kg of Hev b 13, the severity of oedema, leucocyte infiltration, pannus formation and cartilage erosion were reduced significantly. These findings underscore the anti‐inflammatory activity suggested previously for Hev b 13, an activity speculated to be related to its interaction with monocytes/macrophages and the consequent stimulation of IL‐10 release and reduction of tumour necrosis factor (TNF) release. The study also opens a wide range of possible applications in the field of immune‐mediated inflammatory diseases.

Proteção farmacológica da medula espinal isquémica

Ketamine (30 mg/kg, IV) and chlorpromazine (2mg/kg, IV) were evaluated as pharmacological agents, for protection of the ischemic spinal cord in a rat model. A thirty-minute period of ischemia obtained by occlusion of the proximal descending thoracic aorta was followed by reperfusion. In 70 animals (medium weght-380g) distributed in 7 equal groups, the results regarding the complete motor and sensitivity function recovery were as follows: 1) Sham-operation: 100%; 2) Ischemia-reperfusion: 0%; 3) Ketamine, 1 minute before ischemia: 30%; 4) Ketamine, 10 minutes before ischemia: 50%; 5) Chlorpromazine, 1 minute before isquemia: 50%; 6) Chlorpromazine, 1 minute before reperfusion: 10%; 7) Ketamine+chlorpromazine, 1 minute before ischemia: 60%. Both pharmacological agents were effective in the protection of the ischemic spinal cord, as confirmed by the microscopic study. However, comparison of several groups showed statistical significant difference for groups 6 and 7, only. Perfusion of the subarachnoid space revealed excessive amounts of neuro-excitatories amino-acids, L-aspartate and L-glutamate.

Chronic effect of vagotomy in the morphometry of the myenteric plexus of rats' duodenum

BACKGROUND: The gastrointestinal disorders have been associated with morphological alterations in the myenteric nervous plexus. AIM: To evaluate, through morphometric studies, the chronic effects of the subdiaphragmatic trunk vagotomy on the nervous plexus. METHODS: Fifteen male exemplars of Wistar Rattus novergicus weighing about 150g, distributed into three groups, have been used: control (n=5), Sham (n=5) and vagotomized (n=5). The animals were sacrificed after 30 and 90 days post surgery. Fragments of duodenum were fixed in Bouin solution, embedded into paraffin and stained with HE and PAS. Morphometric analysis was performed by a Carl Zeiss KM 450 image system. The following aspects were observed: the density of nervous cells per linear micrometer (µm) (ND); the area of perikarya (µm2) (NA); the number of satellite cells per µm (SCD); and the number of satellite cells per neuron (SC/N). The averages were compared with the help of software program Sigma Plus through two way - ANOVA and Tuckey post-test. RESULTS: Denervation increased SC/N (p<0,05) and NA (p<0,05), in a time-dependent denervation way (p<0,05). However ND and SCD, decreased, which significantly with the animals age (p<0,001). CONCLUSION: Vagotomy altered the myenteric plexus morphology in a time-dependent way.