Mast Cells Dynamically Change And Provide Protective Effects Against The Injury Of Neurons Induced By Cerebral Ischemia

Mast cells are derived from pluripotent progenitor stem cells in the bone marrow. They are present in the peripheral and central nervous system of several species, including human. In the brain, mast cells are predominantly located in the thalamus in the rodents, and in close association to blood vessels and also surrounded by neurons. Mast cells contain a lot of potent preformed substances in their metachromatic granules, including biogenic amines, cytokines, neurotrophins, enzymes, lipid metabolites, nucleotide triphosphates, and reactive oxygen species. These mediators are responsible for the function of mast cells in the brain. Mast cell population and degranulation degree fluctuate under normal physiological conditions, for example, after the courtship and in response to stress. Mast cells are also implicated in the pathogenesis of several neurological diseases, such as multiple sclerosis, Alzheimer's disease and Wernicke's encephalopathy. It seems that the population and the degranulation degree of brain mast cells could change undercertain cerebral pathological conditions.Recent reports suggest that mast cells are involved in the ischemic pathogenesis. In myocardial infarction, they actively participate in postinfarction inflammation, while also regulating fibrous tissue deposition and angiogenesis during the proliferative phase of healing. Mast cells play an important distructive role in ischemia reperfusion injury to skeletal muscle by mast cell-specific protease. And they also have been identified in brain infarcts. There are many mechanisms by which mast cells can influence the cerebral tissue around them. The histamine in the granules contributes to 90% thalamic histamine and 50% whole cerebral histamine. It has known that histamines participate in the impairment of blood-brain barrier and the development of cerebral edema in the early period of reperfusion after ischemia, but protects tissue in late reperfusion. The mast cells, by the mediators like reactive oxygen species, can induce leukocyte adherence/emigration in the inflammatory response, which also occurs following ischemia. Mast cells are also considered to affect the neurons' action, since they are surrounded by elements of the neuropil and the mast cell-derived products can enter neurons by transgranulation. The activated mast cells excite or inhibit the thalamic neurons activity with sex difference. Taken together, mast cells may play a crucial role in the pathological process after cerebral ischemia by their mediators, and may also affect the injury of neurons around them. Therefore, we investigate the change of mast cells after the cerebral ischemia in vivo and in vitro, and the effect of mast cells on the ischemic injury of neurons in vitro.The in vivo transient cerebral ischemia was obtained by four-vessel occlusion, while in vitro ischemia was obtained by oxygen-glucose deprivation (OGD). The mast cells were observed under the light microscopy after toluidine blue staining. The histamine content was measured by the high-performance liquid chromatography, and the LDH activity was determined by an LDH assay kit. 3-[4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide (MTT) assay was performed to determine the survival of neurons and PC12 cells, which are neural cell line used to eliminate the complexity of the heterogeneous mixture of cells in culture.Most of brain mast cells (95-98%) were located in the thalamus in both shamcontrol groups and ischemic groups. Light microscopic observation indicated that metachromatically stained undegranulated mast cells and degranulated mast cells were often adjacent to blood vessels, and also detected in the thalamic neuropil. After cerebral ischemia, the number of mast cells in the thalamus was found to decrease at first. Compared with sham control groups, the number of thalamic mast cell decreased significantly by 56%, 54% and 43% at 1 h, 2 h and 4 h after cerebral ischemia, respectively. Then it gradually increased to the level of sham control groups at 1 d after ischemia. The number of mast cells decreased again by 64% at 7 d after ischemia. On the other hand, the degranulation percentage of thalamic mast cells showed no obvious change until 7 d after ischemia, when the percentage of mast cells degranultion increased significantly by 2.13 times of that of sham control group. In the middle aspect of the thalamus (Bregma - 2.80 to - 3.16 mm), similar change of the number of thalamic mast cells was observed as the changes of whole region (Bregma - 1.80 to - 4.20 mm) in the thalamus after ischemia. At 1 h after ischemia, the number of mast cells decreased throughout the thalamus, but the number in the middle aspect (Bregma - 2.80 to -3.16 mm) did not decrease as markedly as the number in rostral and caudal aspects of the thalamus (including Bregma - 1.80 to - 2.80 mm and - 3.16 to - 4.20mm). However, at 1 d after ischemia, the number of thalamic mast cells in the middle aspect increased markedly and arrived almost twice as the number of mast cells in this region of sham control groups or the number of mast cells per section in both rostral and caudal aspects in the thalamus of ischemic groups. The transient cerebral ischemia also produced a significant increase of histamine content in the thalamus and striatum, but not hypothalamus and cortex at 1 d and 7 d after operation. The ischemia in vitro by OGD increased degranulation percentage of mast cells, becoming significantly different from control after 2 h and increasing further. At 16 h of OGD, it was 71% higher than control. The increase of histamine release began earlier than the degranulation. After 1 h exposure to OGD, histamine release was already significantly elevated, and then gradually increased to 89% above control at 16 h. The LDH release, which indicates the damage of mast cells, accompanied the increase of degranulation and histamine release from 2 h.Rat pheochromocytoma cells (PC12 cells), which are neural cell line, together with neurons, were used in the studies of in vitro ischemic injury. The supernatant collected from mast cells after 1 h OGD protected PC12 cells against OGD-induced injury in an amount-related manner. Complete 100 μl supernatant exposure markedly attenuated the decreased viability about 70%, and 50μl supernatant about 50%. The supernatant also inhibited the injury to PC12 cells following 24 h re-exposure to normoxic glucose-containing DMEM, but the effect was not as strongly as that without re-exposure. Similarly, mast cell-derived protection was demonstrated in neurons. When neurons were exposed to 50 μl supernatant, the decreased viability following 1 h OGD significantly was attenuated by 30%. Pyrilamine, a selective central H₁ receptor antagonist, almost completely reversed the protective effect of supernatant, concentration-dependently. On the other hand, the H₂ receptor antagonist, cimetidine, did not affect the protective action. After the immuno-blocking by anti-histamine serum, the supernatant-mediated protection was significantly reduced. The effective range of histamine content in this protection was from 2 to 20 μM. However, histamine alone, covering the effective range, did not show a clear protective effect on OGD-induced injury in these experiments. NGF, which is also released by mast cells, is known to have neuroprotective effects in the brain. In this study, neither NGF given alone nor together with histamine, significantly increased viability after OGD at doses from 10 to 100 ng/ml.In summary, our studies provide the first evidences that the number and the degranulation percentage of mast cell exhibit dynamic change following transient cerebral ischemia. In the middle aspect of the thalamus, there occurs the congregation of mast cells after 1 d of ischemia. And the histamine content increases in the thalamus and stratium following cerebral ischemia, which may be attribute to the mast cells. Under in vitro ischemic insult, mast cells degranlute to release histamine and LDH, which is supposed to be a cytotoxic response. Mast cell-derived mediators protect against OGD-induced injury in neurons and PC12 cells. This protection is histamine-dependent, and also involves cooperation with other mediators, but notNGF. Therefore, mast cells actively participate in cerebral ischemia-induced injury with the change of the number and degranulation percentage, and may protect the neurons against the injury. The main mediator histamine from mast cells contributes to this protection.