Mechanism of action of collagenase on the blood-brain barrier permeability. Increase of endothelial cell pinocytotic activity as shown with horse-radish peroxidase as a tracer.

The ultrastructural mechanism of the protease induced blood-brain barrier permeability-increase was studied with horse-radish peroxidase as a tracer. After intravenous injection of collagenase or pronase, a significantly increased number of pinocytotic vesicles was found in brain capillary endothelial cells. alpha-Chymotrypsine did not exert such an action.     

Studies on blood-brain barrier permeability after microwave-radiation

Summary Since the reported alterations of permeability of the blood-brain barrier by microwave radiation have implications for safety considerations in man, studies were conducted to replicate some of the initial investigations. No transfer of parenterally-administered fluorescein across the blood-brain barrier of rats after 30 min of 1.2-GHz radiation at power densities from 2–75 mW/cm² was noted. Increased fluorescein uptake was seen only when the rats were made hyperthermic in a warm-air environment. Similarly, no increase of brain uptake of¹⁴C-mannitol using the Oldendorf dual isotope technique was seen as a result of exposure to pulsed 1.3-GHz radiation at peak power densities up to 20 mW/cm², or in the continuous wave mode from 0.1–50 mW/cm². An attempt to alter the permeability of the blood-brain barrier for serotonin with microwave radiation was unsuccessful. From these studies it would appear that the brain must be made hyperthermic for changes in permeability of the barrier induced by microwave radiation to occur.The research reported in this paper was conducted by personnel of the Radiation Sciences Division, USAF School of Aerospace Medicine, Brooks AFB, Tex. 78235. The animals involved in the study were procured, maintained, and used in accordance with the Animal Welfare Act of 1970 and the Guide for the Care und Use of Laboratory Animals prepared by the Institute of Laboratory Animal Resources, National Research Council     

Effect of atrial natriuretic factor on blood-brain barrier permeability

Recent studies have demonstrated receptors for atrial natriuretic factor on endothelium of intracerebral vessels. The physiological role of these receptors is not known. The present study was undertaken to determine whether atrial natriuretic factor has an effect on blood-brain barrier permeability to protein and ions using horseradish peroxidase and lanthanum as markers of permeability alterations. This study does not demonstrate a significant effect of atrial natriuretic factor on blood-brain barrier permeability mechanisms in steady states.     

Effect of chromocarb diethylamine on the permeability of the blood-brain barrier

Intravenously injected collagenase, detectable in brain microvessels by immunological methods, partially degrades the constituents of the vascular wall and so increases the permeability of the blood-brain barrier (BBB). Intravenous administration of collagenase is a model for diseases in which the concentration of endogenous proteases is increased. Peroral treatment of rats with chromocarb diethylamine (CD) significantly reduced the degradation of the vascular wall by intravenous collagenase, as demonstrated by a lesser permeability increase of the BBB, a shorter recovery time, lower hydroxyproline levels in the cerebrospinal fluid and a lesser decrease of the collagen content of the brain capillary basal lamina.     

Reproductive hormones regulate the selective permeability of the blood-brain barrier

Reproductive hormones have been demonstrated to modulate both gap and tight junction protein expression in the ovary and other reproductive tissues, however the effects of changes in reproductive hormones on the selective permeability of the blood-brain barrier (BBB) remain unclear. Age-related declines in BBB integrity correlate with the loss of serum sex steroids and increase in gonadotropins with menopause/andropause. To examine the effect of reproductive senescence on BBB permeability and gap and tight junction protein expression/localization, female mice at 3 months of age were either sham operated (normal serum E2 and gonadotropins), ovariectomized (low serum E2 and high serum gonadotropins) or ovariectomized and treated with the GnRH agonist leuprolide acetate (low serum E2 and gonadotropins). Ovariectomy induced a 2.2-fold increase in Evan's blue dye extravasation into the brain. The expression and localization of the cytoplasmic membrane-associated tight junction protein zona occludens 1 (ZO-1) in microvessels was not altered among groups indicating that the increased paracellular permeability was not due to changes in this tight junction protein. However, ovariectomy induced a redistribution of the gap junction protein connexin-43 (Cx43) such that immunoreactivity relocalized from along the extracellular microvascular endothelium to become associated with endothelial cells. An increase in Cx43 expression in the mouse brain following ovariectomy was suppressed in ovariectomized animals treated with leuprolide acetate, indicating that serum gonadotropins rather than sex steroids were modulating Cx43 expression. These results suggest that elevated serum gonadotropins following reproductive senescence may be one possible cause of the loss of selective permeability of the BBB at this time. Furthermore, these findings implicate Cx43 in mediating changes in BBB permeability, and serum gonadotropins in the cerebropathophysiology of age-related neurodegenerative diseases such as stroke and Alzheimer's disease.     

Effect of losartan on the blood-brain barrier permeability in diabetic hypertensive rats

Our previous publication has stressed the benefits of losartan, an angiotensin II receptor blocker, on the permeability of blood-brain barrier (BBB) and blood pressure during L-NAME-induced hypertension. This study reports the impacts of anti-hypertensive treatment by losartan on the brain endothelial barrier function and the arterial blood pressure, during acute hypertension episode, in experimentally diabetic hypertensive rats. Systolic blood pressure measurements were taken with tail cuff method before and during administration of L-NAME (0.5 mg/ml). We induced diabetes by using alloxan (50 mg/kg, i.p). Losartan (3 mg/kg, i.v) was given to rats following the L-NAME treatment. Acute hypertensive vascular injury was induced by epinephrine (40 microg/kg). The BBB disruption was quantified according to the extravasation of the Evans blue (EB) dye. L-NAME induced a significant increase in arterial blood pressure on day 14 in normoglycemic and hyperglycemic rats (p < 0.05). Losartan significantly reduced the increased blood pressure in hypertensive and diabetic hypertensive rats (p < 0.01). Epinephrine-induced acute hypertension in diabetic hypertensive rats increased the content of EB dye dramatically in cerebellum and diencephalon (p < 0.01) and slightly in both cerebral cortex (p < 0.05). Losartan treatment reduced the increased BBB permeability to EB dye in the brain regions of diabetic hypertensive rats treated with epinephrine (p < 0.05). This study indicates that, in diabetic hypertensive rats, epinephrine administration leads to an increase in microvascular-EB-albumin efflux to brain, however losartan treatment significantly attenuates this protein's transport to brain tissue.     

Influence of surgical pain stress on the blood-brain barrier permeability in rats

Effect of surgical pain stress on the blood-brain barrier permeability was investigated in rats. The animals were divided into four groups: Group 1: control, Group 2: immobilization stress, Group 3: acute hypertension, Group 4: immobilization stress + surgical pain stress.Bilateral hid paw surgical wounds for cannulations were applied in animals' inguinal regions under diethyl-ether anesthesia, then the animals were awaken from anesthesia to produce surgical pain stress. Evans-blue was used as a blood-brain barrier tracer. There is no significantly blood-brain barrier breakdown after short-time immobilization stress, but after adrenalin hypertension blood-brain barrier permeability was increased especially on frontal and occipital cortices in 50% of the animals. Surgical pain stress increased blood-brain barrier permeabiliy in comparison to acute adrenalin-induced hypertension (p < 0.01). In surgical pain stress-induced animals distinct Evans-blue leakage was observed in the occipital, frontal and parieto-temporal cortices.     

Flavonoid permeability across an in situ model of the blood-brain barrier

Understanding mechanisms associated with flavonoid neuroprotection is complicated by the lack of information on their ability to enter the CNS. This study examined naringenin and quercetin permeability across the blood-brain barrier (BBB), using in vitro (ECV304/C6 coculture) and in situ (rat) models. We report measurable permeabilities (P(app)) for both flavonoids across the in vitro BBB model, consistent with their lipophilicity. Both flavonoids showed measurable in situ BBB permeability. The rates of uptake (K(in)) into the right cerebral hemisphere were 0.145 and 0.019 ml min(-1) g(-1) for naringenin and quercetin, respectively. Quercetin K(in) was comparable to that of colchicine (0.006 ml min(-1) g(-1)), a substrate for P-glycoprotein (P-gp). Preadministration of the P-gp inhibitor PSC833 or GF120918 (10 mg/kg body wt) significantly increased colchicine K(in), but only GF120918 (able to inhibit breast cancer resistance protein, BCRP) affected K(in) for quercetin. Naringenin K(in) was not affected. The influence of efflux transporters on flavonoid permeability at the BBB was further studied using MDCK-MDR1 and immortalized rat brain endothelial cells (RBE4). Colchicine, quercetin, and naringenin all showed measurable accumulation (distribution volume, V(d) (microl/mg protein)) in both cell types. The V(d) for colchicine increased significantly in both cell lines following coincubation with either PSC833 (25 microM) or GF120918 (25 microM). Both inhibitors also caused an increase in naringenin V(d); by contrast only GF120918 coincubation significantly increased quercetin V(d). In conclusion, the results demonstrate that flavonoids are able to traverse the BBB in vivo. However, the permeability of certain flavonoids in vivo is influenced by their lipophilicity and interactions with efflux transporters.     

Ineffectiveness of dimethyl sulfoxide in altering the permeability of the blood-brain barrier

The failure of DMSO to alter the permeability of the blood-brain barrier has been studied using several polar, nonpolar, hydrophilic, and hydrophobic compounds labeled with selected radioactive isotopes. The metabolites were Na¹³¹I, ¹³¹I-iodinated human serum albumin, l-[³⁵S]methionine, dl-[ring-2-¹⁴C]tryptophan, [U-¹⁴C]sucrose, d-[6-¹⁴C]glucose, and [4-¹⁴C]cholesterol. DMSO was injected intraperitoneally at a dose of 1 g/kg followed after 1 hr by the intracarotid injection of the labeled metabolite. An appropriate volume of saline was substituted for the DMSO in control animals. The brain and one gastrocnemius muscle were removed at selected intervals up to 30 min and the uptake into these tissues was measured.It was found that the permeability of neither the blood-brain barrier nor skeletal muscle was altered by this concentration of DMSO. This dose of DMSO, administered intravenously, frequently caused death and, intraperitoneally, caused muscular twitching, lethargy, and hematuria.     

Separation methods that are capable of revealing blood-brain barrier permeability

The objective of this review is to emphasize the application of separation science in evaluating the blood-brain barrier (BBB) permeability to drugs and bioactive agents. Several techniques have been utilized to quantitate the BBB permeability. These methods can be classified into two major categories: in vitro or in vivo. The in vivo methods used include brain homogenization, cerebrospinal fluid (CSF) sampling, voltametry, autoradiography, nuclear magnetic resonance (NMR) spectroscopy, positron emission tomography (PET), intracerebral microdialysis, and brain uptake index (BUI) determination. The in vitro methods include tissue culture and immobilized artificial membrane (IAM) technology. Separation methods have always played an important role as adjunct methods to the methods outlined above for the quantitation of BBB permeability and have been utilized the most with brain homogenization, in situ brain perfusion, CSF sampling, intracerebral microdialysis, in vitro tissue culture and IAM chromatography. However, the literature published to date indicates that the separation method has been used the most in conjunction with intracerebral microdialysis and CSF sampling methods. The major advantages of microdialysis sampling in BBB permeability studies is the possibility of online separation and quantitation as well as the need for only a small sample volume for such an analysis. Separation methods are preferred over non-separation methods in BBB permeability evaluation for two main reasons. First, when the selectivity of a determination method is insufficient, interfering substances must be separated from the analyte of interest prior to determination. Secondly, when large number of analytes is to be detected and quantitated by a single analytical procedure, the mixture must be separated to each individual component prior to determination. Chiral separation in particular can be essential to evaluate the stereo-selective permeation and distribution of agents into the brain. In conclusion, the usefulness of separation methods during BBB permeability evaluation is immense and more application of these methods is foreseen in the future.     

Increased blood-brain barrier permeability of amino acids in chronic hypertension

A previous communication from this laboratory reported that brain uptake of libenzapril, a small polar molecule, was enhanced in chronic hypertension (1). The objective of this investigation was to determine if this was a more generalized phenomenon. Therefore, experiments were undertaken to examine the effect of chronic hypertension on the brain uptake of tryptophan (an amino acid with high brain permeability) and glutamic acid (one with low permeability). Brain concentrations of these two amino acids were 5- to 12-fold greater in chronic hypertensive rats, as compared to normotensive rats; the corresponding brain uptake index (BUI) values were 2- to 5-fold higher in the former group. Since blood-brain barrier transport of amino acids involve both saturable (carrier) and non-saturable (most likely, diffusion via pores) mechanisms, data from this study show that hypertension can enhance BBB transport of amino acids by affecting one or both of these pathways.     

An electrodiffusion model for the blood-brain barrier permeability to charged molecules

The endothelial surface glycocalyx layer (SGL) and the basement membrane (BM) are two important components of the blood-brain barrier (BBB). They provide large resistance to solute transport across the BBB in addition to the tight junctions in the cleft between adjacent endothelial cells. Due to their glycosaminoglycan compositions, they carry negative charge under physiological conditions. To investigate the charge effect of the SGL and BM on the BBB permeability to charged solutes, we developed an electrodiffusion model for the transport of charged molecules across the BBB. In this model, constant charge densities were assumed in the SGL and in the BM. Both electrostatic and steric interaction and exclusion to charged molecules were considered within the SGL and the BM and at their interfaces with noncharged regions of the BBB. On the basis of permeability data for the positively charged ribonuclease (+4,radius=2.01?nm) and negatively charged α-lactalbumin (-10,radius=2.08?nm) measured in intact rat mesenteric and pial microvessels, our model predicted that the charge density in both SGL and BM would be ～30?mEq/L, which is comparable to that in the SGL of mesenteric microvessels. Interestingly, our model also revealed that due to the largest concentration drop in the BM, there is a region with a higher concentration of negatively charged α-lactalbumin in the uncharged inter-endothelial cleft, although the concentration of α-lactalbumin is always lower than that of positively charged ribonuclease and that of a neutral solute in the charged SGL and BM.     

Signalling pathways regulating the tight junction permeability in the blood-brain barrier.

Tight junctions (TJs) of the cerebral endothelial cells play a crucial role in the regulation of BBB permeability under physiological, as well as pathological conditions. The regulation of the junctional proteins is under a complex control. In these regulatory processes signalling molecules, some of them localized to the TJ, play an important role. Among the best characterized second messengers which regulate TJ function are the cyclic nucleotides, which, as shown in our experiments, as well, decrease paracellular permeability. Another important signalling molecule involved in TJ regulation is protein kinase C, which may affect differently the formation of TJ and the function of mature TJ. Further signalling molecules known to regulate paracellular permeability are G-proteins, both conventional and small G-proteins, MAP kinases and other protein kinases. Much of our knowledge concerning second messenger regulation of TJ arises fon the study of epithelial cells of different origin, mostly from kidney, therefore the specific regulation of the junctional complex of the BBB still remains to be elucidated.