Preliminary evaluation of mast cells in rats with an experimental fibrosarcoma induced by 3-methylcholanthrene

The aim of the study was the evaluation of mast cells in experimental fibrosarcoma induced in the rats' skin. Experiments were carried out on 50 male Wistar rats divided into three groups: 1. The cancer was induced in 34 rats' by onefold subcutaneous injection of 0.2 mg 3-methylcholanthrene in 0. 25 ml of olive oil; 2.--8 rats received subcutaneous injection of 0.25 ml olive oil; and 3.--8 rats, no treatment. The tumors developed after 14-35 weeks. The examined tumors had the mass of 1 g to 176 g--mean 15 g. Tissue material was fixed in Carnoy's or Bouin's fluid. Paraffin sections were stained with hematoxylin and eosin and using Azan methods. Mast cells were stained with alcjan blue+saphranin and with toluidine blue in pH 1.5. Immunohistochemical reactions detecting tryptase in mast cells and von Willebrand Factor in endothelial cells were also performed--using specific antibodies (DAKO) and ABC complex. We have found a very significant growth of the quantity of mast cells in the connective tissue of tumours. The distinct increase in immunostaining was found for tryptase in mast cells of the tumor periphery zone, where most areas of strong neoangiogenesis were found.     

Morphometric evaluation of murine pulmonary mast cells in experimental hemorrhagic shock

Respiratory failure resulting frequently in death is one of the complications in the course of post-hemorrhagic changes. A systemic inflammatory reaction plays a significant role in the pathogenesis of this syndrome. Mast cells also contribute to this effect. To broaden our knowledge of the pathogenesis of respiratory insufficiency, we evaluated morphometrically lung mast cells in hemorrhagically shocked rats. Lung sections were stained with alcian blue and safranin, and four separate locations were distinguished: under the lung pleura, around the bronchi and the large vessels, and in the interalveolar septa. A decrease in the area and volume of mast cells and an increase in their circularity index in interalveolar septa and around the bronchi was observed. An enlargement of mast cells around lung vessels was also found. There were no changes in the morphometric parameters of mast cells under pleura. The results suggest an activation and degranulation of mast cells and a role in the inflammatory process causing acute lung injury in hemorrhagic shock.     

The evaluation of quantity and distribution of murine pulmonary mast cells in experimental hemorrhagic shock

Respiratory failure is one of the most serious clinical complications in the course of post-hemorrhagic changes. Cascade-like, systemic inflammatory reaction including the axis: intestines-liver-lung-immune system has a special significance in the pathogenesis of this syndrome. In order to broaden the knowledge of the respiratory insufficiency pathogenesis in hemorrhagic shock, the attempt was made to evaluate quantitatively rat mast cells localized under the pulmonary pleura, around the bronchi, around large vessels and placed in the interalveolar septa. The examinations were conducted on 24 young female Wistar rats, divided into two groups (n=12): (I) sham-operated and (II) shocked. The hemorrhagic shock was evoked by the withdrawal of 25% of the circulating blood. The shock duration was 75 min. The obtained lung sections were stained with toluidine blue and examined in a light microscope. After hemorrhagic shock, sections of lung samples revealed about two-fold increase in mast cell number/mm2 compared to controls. Mast cells were concentrated mostly around the bronchi and blood vessels. Hyperplasia and migration of mast cells may suggest their role in the modulation of inflammatory process causing acute lung injury in the hemorrhagic shock.     

Preliminary evaluation of gastric endocrine cells in rats with experimental uremia

Chronic renal failure can be the cause of various disturbances in hormonal and electrolyte metabolism, including calcium and phosphate metabolism. The aim of this study was the evaluation of pyloric endocrine cells in Wistar rats with experimental uremia. Fragments of gastric pylorus were collected 30 days after nephrectomy. Paraffin embedded sections were stained with H+E and by silver impregnation. We also performed immunohistochemical reactions with the use of specific antibodies against calcitonin gene-related peptide (CGRP), synaptophysin (SY), somatostatin (ST), and neuronal specific enolase (NSE). The rats with experimental uremia showed an increase in the number of endocrine cells and in intensity of all the examined reactions.     

Intestinal mast cells during experimental ancylostomiasis.

The alterations of mast cell counts induced in the gut of swiss albino mice by infection with Ancylostoma caninum larvae have been studied, both in single and multiple infections. It was found that mast cell rise was greater in females than males. In singly infected animals, mast cell increase was influenced by the infective dose of inoculated larvae. In case of repeatedly infected mice, mast cell counts were decreased because of their earlier degranulation.     

Preliminary evaluation of neuroendocrine cells in the respiratory tract in rats with experimental uremia

The goal of this study was to investigate the influence of experimetally induced chronic renal failure on endocrine cells in the respiratory tract in rats. After 30 days of uremia, the fragments of rat lungs were collected. Paraffin sections were stained using H+E, silver impregnation and immunohistochemistry with specific antibodies against calcitonin (CT), synaptophysin (SY), somatostatin (ST), and neuron-specific enolase (NSE). A large number of endocrine cells with a strong calcitonin immunoreactivity were observed in the respiratory tract of rats with experimental uremia, as compared with the control group. Other immunoreactions were weakened.     

Isolation of cardiac mast cells in experimental Trypanosoma cruzi infection

Mast cells (MC) secrete diverse pre-stored chemical mediators that are pivotal in inflammatory and fibrotic etiologies, such as Trypanosoma cruzi-induced myocardiopathy. However, due to reduced number of cardiac MC, in situ and in vitro identification, and difficult tissue isolation, these cells are rarely addressed. In this work we optimized the identification of cardiac and peritoneal MC and developed an enzymatic method for MC isolation using control and T. cruzi-infected mice. MC were identified by: toluidine blue (TB); alcian blue (AB)/safranin (S); AB or a mixed solution composed by AB/S/TB. Previous evaluations of cardiac MC in T. cruzi infection were based on TB staining and our results using AB/S/TB solution showed an increase in, at least, five times the detection of MC. This mixed solution may improve the identification of MC populations also from skin, mucosa and tissues that are infected by other pathogens or under the influence of chronic inflammation, leading to more precise results. Furthermore, the appropriate combination of samples (frozen/unfixed/thick slices) and staining protocols can assure the best evaluation of MC. We have also isolated cardiac MC using collagenase and developed a highly efficient 60%/70% Percoll-graded protocol that enriched in, at least, 95% the population of cardiac MC.     

Heparin and mast cells in the dynamics of experimental extrahepatic cholestasis]

The content of heparin in the blood and mast cell count in rat tissues were studied during different periods of experimental extrahepatic cholestasis induced in rats by ligation of the common bile duct. During cholestasis the blood heparin level proved to increase and the mast cell count fell on the 3rd day, increased on the 7th day and fell again on the 14th day. Correlation between the degranulated and nondegranulated forms of mast cells altered in favour of the latter. The fluctuation of mast cell counts and increase of degranulated forms is considered to be the best result of mast cell irritation produced by bile acids and pigments which accumulated in the organism.     

Visualization of exocytotic secretory processes of mast cells by fluorescence techniques

Secretory processes via exocytosis in rat peritoneal mast cells were visualized by two complementary fluorescence techniques; one staining pre-exocytotic granules with a basic probe and the other staining post-exocytotic granules with acidic probes. Granules within mast cells were selectively stained with acridine orange and emitted orange yellow fluorescence. Upon stimulation with compound 48/80, release of acridine orange from granules was observed both in population and single cell measurements. This release was seen in some localized area of mast cells. Opening of pores between plasma membranes and granule membranes was monitored using acidic fluorescence probes such as 6-carboxyfluorescein or lucifer yellow CH. Not only granules located at peripheral region, but also granules near the core region participated in exocytosis. The existence of junctions between these granules was suggested. TMA-DPH, a lipophilic membrane probe, which was localized at plasma membrane before stimulation, diffused into granule membranes after stimulation. This shows that after stimulation, some constituents of plasma and granule membranes were mixed. Even after extensive degranulation, mast cells extruded acidic probes, indicating the plasma membranes still play a role of barrier. Activation of lateral motion of granules preceding to exocytosis was not observed. It was concluded that the visualization of secretory processes by fluorescence and image processing techniques will be useful for the study of molecular mechanisms underlying exocytosis.     

The role of mast cells in the elicitation of experimental allergic encephalomyelitis

Experimental allergic encephalomyelitis (EAE), a T cell-mediated autoimmune disease can be transferred with lymphoid cells from actively immunized rats into naive recipients. In the mouse, previous studies have suggested a role for histamine/serotonin in the development of active EAE. We have found that myelin basic protein-reactive cells transfer a biphasic skin test response to naive rats analogous to what has been described in the mouse contact dermatitis system, where mast cell sensitization by Ag-specific T cell factors is required for the induction of skin test responses. Treatment of cell recipients with the serotonin receptor antagonists, cyproheptadine or methysergide, blocked or significantly reduced the development of EAE. Furthermore, it was found that treatment with cyproheptadine was effective in blocking clinical disease when administered day 3 to day 6 after cell transfer. In contrast, cyproheptadine treatments before induction of paralysis day 0 to 3, failed to alter the course of clinical disease. The inhibitor of mast cell degranulation, proxicromil, was also found to effectively block the elicitation of adoptively transferred EAE and was also found to be effective when administered just before the onset of clinical disease. Reserpine, a compound known to deplete mast cells of vasoactive amines by forcing granule contents into the cytoplasm where they are degraded by cell enzymes, was also effective in blocking both active and adoptively transferred EAE. Disease inhibition was found to be partially reversed with pargyline, an inhibitor of monoamine oxidase. In addition lymphocytes from treated animals were capable of transferring disease to naive recipients and appeared to have normal activity as assessed by Ag-or mitogen-driven proliferation in addition to IL-2 production.     

Activation of mast cells after experimental myocardial infarction in 3 week-old rats

It is known that mast cells (MC) take an active part in regeneration processes in postinfarction heart in adult rats and humans. Behaviour of population of cardial MCs has been studied 20, 60, 75 and 90 days after experimental myocardial infraction induced in 3 week-old and adult rats by ligation of left coronary artery. The density of MC of different degrees of maturity was estimated in atrium and ventricle on paraffin sections stained with Alcian blue - Safranin. Findings were compared with MC density obtained in hearts of intact rats. The MC density in intact 1.5-2.5 month-old rats in atrium and ventricle was about 0.6 cells/mm2, in intact 3.5-4.0 month-old rats in atrium--1.2 cells/mm2, in ventricle--0.6 cells/mm2. The MC density in 3 week-old rats with infarction was significantly higher than in intact rats: 5-fold increase in 20 and 60 days in atrium, and 2-fold increase in 60 and 75 days in ventricle. In 60 days after infarction the MC density in adult rats was 3 times lower in atrium and 2 times lower in ventricle than in the same heart compartments of 3 week-old rats with infarction. After infarction in 3 week-old rats, a relative share of young cells with alcian-positive granules sharply increased in 20 days and then decreased by 60-75 days. This indicates a migration of immature MCs to infracted myocardium and their subsequent differentiation. The MC activation after infraction in young rats may result from a more active immune reaction in younger rats and/or functional peculiarities of their MC.     

Changing processes from bone marrow-derived cultured mast cells to connective tissue-type mast cells in the peritoneal cavity of mast cell-deficient w/wv mice: association of proliferation arrest and differentiation

Connective tissue-type mast cells (CTMC) and mast cells grown in vitro exhibit many differences in morphology, biochemistry, and function. When cultured mast cells of WBB6F1-+/+ mouse origin were injected into the peritoneal cavity of genetically mast cell-deficient WBB6F1-W/Wv mice, however, the cultured mast cells acquired characteristics similar to CTMC. In this study, we analyzed the changing process. When the density of the cultured mast cells was measured by Percoll density gradient centrifugation, the proportion of dense mast cells increased after injection into the peritoneal cavity. Because the increase in proportion of dense mast cells paralleled the increase in proportion of heparin-containing mast cells, both parameters may be used as an index for differentiation activity of cultured mast cells into CTMC. When proliferation activity of mast cells was estimated by the incorporation of bromodeoxyuridine, the proliferation activity decreased after the i.p. transfer. Moreover, when cultured mast cells were recovered 10 wk after the i.p. transfer, the mast cells almost lost proliferation activity in the same culture condition that had been used for establishment of cultured mast cells from the bone marrow of WBB6F1-+/+ mice. These results demonstrate that the proliferation arrest and the acquisition of CTMC-like characters are associated after i.p. transfer of cultured mast cells.     

Interleukin-1beta-mediated inhibition of the processes of angiogenesis in cardiac microvascular endothelial cells

Angiogenesis, the formation of new capillaries from preexisting vessels, plays an essential role in revascularization of the myocardium following myocardial infarction (MI). Interleukin-1beta (IL-1beta), a proinflammatory cytokine increased in the heart following MI, is shown to be essential for angiogenesis in the invasiveness of tumor cells, the progression of arthritic conditions and endometriosis, and the promotion of wound healing. Here we studied the steps of angiogenesis in response to IL-1beta in cardiac microvascular endothelial cells (CMECs) and aortic tissue. Cell cycle progression analysis using flow cytometry indicated a G0/G1 phase cell cycle arrest in IL-1beta-stimulated cells. IL-1beta significantly reduced levels of fibrillar actin in the cytoskeleton, a pre-requisite for tube formation, as indicated by phalloidin-FITC staining. Wound healing assays demonstrated IL-1beta prevents cell-to-cell contact formation. On the other hand, vascular endothelial growth factor-D (VEGF-D) initiated restoration of the cell monolayer. IL-1beta significantly inhibited in vitro tube formation as analyzed by three-dimensional collagen matrix assay. Aortic ring assay demonstrated that IL-1beta inhibits basal and VEGF-D-stimulated microvessel sprouting from aortic rings. The data presented here are novel and of significant interest, providing evidence that IL-1beta impedes the process of angiogenesis in myocardial endothelial cells.     

The evaluation of murine pleural lavage fluid cellular composition in experimental hemorrhagic shock with special regard to mast cells morphometry.

In the course of a hemorrhagic shock, pathological changes occur, which result in intensifying the insufficiency of various vital organs. It can also lead to the development of the multiorgan dysfunction syndrome (MODS) that is the cause of high posthemorrhagic mortality. As a result of the ischemia in the lung there appear proinflammatory factors that mobilize and activate mast cells, inducing degranulation in them. The aim of the study was the analysis of cellular composition and cytomorphometric evaluation of mast cells present in the lavage fluid from the pleural cavity of rats in a sham operated group and in the group presenting hemorrhagic shock. The results revealed an increase of the total cell count in the lavage fluid from the pleural cavity. In the cytological smears a statistically significant accumulation of inflammatory cells was present, especially neutrophils. The increase in mast cells and eosinophils number was not statistically significant. There was not a change in the morphometric parameters of mast cells except the circularity index. A decline of the circularity index indirectly may suggest the degranulation of mast cells, which reflects an inflammatory process in the lungs.