Research reportAcute stress increases permeability of the blood–brain-barrier through activation of brain mast cells
Introduction
The BBB is formed by a complex system of endothelial cells [32], astroglia, pericytes, perivascular macrophages and basal lamina [73]. Under normal conditions, the BBB tightly regulates the entry of any compound into the brain [12]. The protective function of the BBB can be altered during various diseases of the CNS, specifically during cerebral inflammation [12] such as that present in multiple sclerosis (MS) [35]. In this case, leukocyte infiltration in the brain parenchyma [65] follows a decrease in the integrity of the BBB [35]. This increase in BBB permeability may possibly be mediated through the action of vasoactive mediators, such as histamine [58], released from perivascular brain mast cells [35], [69].
Stress activates the hypothalamic–pituitary–adrenal (HPA) axis through the release of CRH leading to secretion of catecholamines and glucocorticoids [7]. Stress, however, seems to precipitate or worsen a number of neuroinflammatory disorders [55], such as relapsing–remitting multiple sclerosis [24], [45], [74]. This is a paradoxical finding given the fact that glucocorticoids released during HPA activation are the primary mode of therapy for such conditions [44]. A possible explanation could be that CRH released from the hypothalamus or elsewhere in the brain either affects the BBB directly, or through mast cell activation. Support for this premise comes from the recent evidence that CRH also has proinflammatory effects [33], apparently mediated through mast cell activation [71]. Acute stress by immobilization was shown to induce intracranial rat mast cell degranulation and elevate in the cerebrospinal fluid (CSF) the connective type mast cell marker rat mast cell protease (RMCP); both actions were CRH-dependent [72]. Moreover, CRH [71] and its structurally related urocortin [62] induced mast cell degranulation and Evans blue extravasation in rodent skin, a phenomenon duplicated by acute immobilization stress [63].
Mast cells are ubiquitous in the body and are critical for the development of allergic reactions [21]. In the brain, they are predominantly located perivascularly, especially in the thalamus and hypothalamus [16], [17], [23], [30], [51], [52] where they have been definitively characterized [50]. Increasing evidence indicates that mast cells may also be involved in neuroimmune interactions [8], [20], [22], [67], especially in the dura [14], [56], including the development of neuroinflammatory processes [70]. However, the function of brain mast cells remains unclear [37]. As many mast cell mediators are vasoactive, it has been suggested that mast cells may regulate the permeability of the blood–brain-barrier (BBB) [69]. This proposal was recently supported using a chemical trigger of mast cells, compound 48/80, in the habenula of pigeons which is rich in mast cells [75].
This study investigated whether acute non-traumatic stress in rats can alter the permeability of the BBB and whether such an effect involves brain mast cell activation. Extravasation of intravenous 99Tc was used to evaluate BBB permeability in different brain regions and mast cell degranulation was determined histochemically after staining with toluidine blue.
Section snippets
Mast cell histochemistry
After 99-Technetium gluceptate (99Tc) radioactivity was measured, tissue collected from both control and stressed animals which had been washed intracardially with formalin, was placed in formalin for 2 days. Brain tissue was then immersed in tissue freezing medium and 10 μ sections were cut using a cryostat (Jung CM3000, Leica, Deerfield, IL). Mast cell degranulation was evaluated using a light microscope (Nikon, Don Santo, MA), after toluidine blue (0.5%, pH 2.5) staining of brain sections at
BBB permeability documented by Evans blue extravasation
BBB permeability was first evaluated by extravasation of Evans Blue in the diencephalon (Table 1), the brain region which contains the highest number of mast cells. Immediately following the 30-min acute immobilization stress, dye extravasation measured in arbitrary fluorescence units increased (P=0.0004) from 0.019±0.048 (control n=11 rats) to 0.187±0.052 (stress n=12 rats).
Effect of immobilization stress on serum corticosterone levels
Serum corticosterone levels were increased due to stress, in all experiments. Representative control rats had serum
Discussion
Acute stress by immobilization induced a significant increase in BBB permeability as evidenced by Evans blue and 99Tc extravasation in the diencephalon, cerebellum and brainstem. The cerebral cortex showed an increase that was not statistically significant due to the large standard deviation. This may require further investigation using a different study design (and power) and ultimately a larger sample size (to avoid a possible type 2 error). 99Technetium-gluceptate was used because the
Acknowledgements
This work was supported in part by NIH grant NS38326 to TCT. Thanks are due to Mr. Dominic Siewko for his help with making 99Tc timely available and Jerry Harmatz for his assistance with the statistical analysis. We also appreciate Ms. Sharon Titus’ word processing skills.
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