Paul A. Fraser

Distribution of extracellular tracers in perivascular spaces of the rat brain

Large molecular weight tracers (india ink or albumin labeled with colloidal gold, Evans blue or rhodamine) were micro-injected into the perivascular space of an artery or vein on the brain surface, or within the cerebral cortex or the subarachnoid space of anesthetized rats. The subsequent distribution was followed both under intravital microscopy, in order to outline the pathways and direction of tracer movement, and in histological section, in order to describe the pathways of flow at the light and electron microscopic level. The tracers remained largely in the perivascular spaces and in the interconnecting network of extracellular channels, including the subpial space and the core of subarachnoid trabeculae. Tracer also leaked across the pia into subarachnoid CSF. Bulk flow of fluid within the perivascular space, around both arteries and veins, was suggested from video-densitometric measurements of fluorescently labeled albumin. However, this flow was slow, and its direction varied in an unpredictable way. These results confirm that perivascular spaces may serve as channels for fluid exchange between brain and CSF, but do not support the idea that CSF circulates rapidly through brain tissue via perivascular spaces.

The role of free radical generation in increasing cerebrovascular permeability

The brain endothelium constitutes a barrier to the passive movement of substances from the blood into the cerebral microenvironment, and disruption of this barrier after a stroke or trauma has potentially fatal consequences. Reactive oxygen species (ROS), which are formed during these cerebrovascular accidents, have a key role in this disruption. ROS are formed constitutively by mitochondria and also by the activation of cell receptors that transduce signals from inflammatory mediators, e.g., activated phospholipase A₂ forms arachidonic acid that interacts with cyclooxygenase and lipoxygenase to generate ROS. Endothelial NADPH oxidase, activated by cytokines, also contributes to ROS. There is a surge in ROS following reperfusion after cerebral ischemia and the interaction of the signaling pathways plays a role in this. This review critically evaluates the literature and concludes that the ischemic penumbra is a consequence of the initial edema resulting from the ROS surge after reperfusion.

Sulforaphane preconditioning of the Nrf2/HO-1 defense pathway protects the cerebral vasculature against blood-brain barrier disruption and neurological deficits in stroke

Disruption of the blood-brain barrier (BBB) and cerebral edema are the major pathogenic mechanisms leading to neurological dysfunction and death after ischemic stroke. The brain protects itself against infarction via activation of endogenous antioxidant defense mechanisms, and we here report the first evidence that sulforaphane-mediated preactivation of nuclear factor erythroid 2-related factor 2 (Nrf2) and its downstream target heme oxygenase-1 (HO-1) in the cerebral vasculature protects the brain against stroke. To induce ischemic stroke, Sprague-Dawley rats were subjected to 70 min middle cerebral artery occlusion (MCAo) followed by 4, 24, or 72 h reperfusion. Nrf2 and HO-1 protein expression was upregulated in cerebral microvessels of peri-infarct regions after 4-72 h, with HO-1 preferentially associated with perivascular astrocytes rather than the cerebrovascular endothelium. In naïve rats, treatment with sulforaphane increased Nrf2 expression in cerebral microvessels after 24h. Upregulation of Nrf2 by sulforaphane treatment prior to transient MCAo (1h) was associated with increased HO-1 expression in perivascular astrocytes in peri-infarct regions and cerebral endothelium in the infarct core. BBB disruption, lesion progression, as analyzed by MRI, and neurological deficits were reduced by sulforaphane pretreatment. As sulforaphane pretreatment led to a moderate increase in peroxynitrite generation, we suggest that hormetic preconditioning underlies sulforaphane-mediated protection against stroke. In conclusion, we propose that pharmacological or dietary interventions aimed to precondition the brain via activation of the Nrf2 defense pathway in the cerebral microvasculature provide a novel therapeutic approach for preventing BBB breakdown and neurological dysfunction in stroke.

Two components of blood‐brain barrier disruption in the rat

1 Permeability of pial venular capillaries to Lucifer Yellow (Ply) was measured using the single microvessel occlusion technique. 2 P ly was extremely low, when measured shortly after the removal of the meninges, consistent with an intact blood‐brain barrier, but rose spontaneously to (1.65 ± 060)× 10−6cms−1 (mean ±s.d.) within 20–‐60 min. This first phase of spontaneous disruption lasted 44–164 min. A second phase started when Ply rose sharply, and was characterized by rapid permeability fluctuations with a mean of (12.31 ± 15.14) × 1CT−6 cm s−1. 3 The first phase could be mimicked by applying the divalent cation ionophore A23187 in the presence of Ca2+, when Ply rose by (1.47±0–25) × 10−6 cm s−1 (mean±s.e.m.). Application of histamine (10 μm) to tight vessels increased Ply by (2.41 ± 0.22) × 10−6 cm s−1. 4 Substances that raised intraendothelial cAMP of vessels during the first phase of disruption reduced Ply to the initial blood‐brain barrier level. 5 The second phase could be prevented by applying catalase. Similar high and fluctuating Ply values could be produced reversibly by applying arachidonic acid or NH4C1. 6 This is the first report of two distinct types of permeability increase in the cerebral microvasculature, and reasons for this are discussed.

Acute effects of bradykinin on cerebral microvascular permeability in the anaesthetized rat

1 The permeability response to acutely applied bradykinin and [des‐Arg9]‐bradykinin on single cerebral venular capillaries has been investigated using the low molecular mass fluorescent dyes Lucifer Yellow and Sulforhodamine B with the single vessel occlusion technique. 2 When bradykinin was applied repeatedly for up to 2 h, the permeability increase was small and reversible for concentrations that ranged from 5 nm to 50 μm. 3 The logEC50 of the permeability response to bradykinin was −5.3 ± 0.15 (logm; mean ±s.e.m.). This was reduced to −6.37 ± 0.24 with the angiotensin‐converting enzyme inhibitor captopril, to −6.33 ± 0.19 with the neutral endopeptidase inhibitor phosphoramidon and to −7.3 ± 0.20 with captopril and phosphoramidon combined. 4 The permeability response to bradykinin was blocked by the bradykinin B2 receptor antagonist HOE 140, by inhibition of the Ca2+‐independent phospholipase A2, by the scavenging of free radicals, or by inhibition of both cyclo‐oxygenase and lipoxygenase in combination. Block of Ca2+ entry channels with SKF 96365 had no effect on the response. 5 Application of [des‐Arg9]‐bradykinin also increased permeability over the concentration range 5 nm to 50 μm, with a logEC50 of −5.6 ± 0.37. This response was not affected by free radical scavenging, but was completely blocked by the histamine H2 receptor blocker cimetidine. 6 These results imply that the acute permeability response to bradykinin is mediated via the release of arachidonic acid, which is acted on by cyclo‐oxygenase and lipoxygenase resulting in the formation of free radicals, and that the response to [des‐Arg9]‐bradykinin is mediated via histamine.

Regulation of cerebral microvascular permeability by histamine in the anaesthetized rat

1 The permeability response of slightly leaky pial venular capillaries to histamine was investigated using the single microvessel occlusion technique. 2 Histamine dose‐response curves showed that concentrations between 5 nm and 5 μM increased permeability, while concentrations from 50 μM to 5 mM reduced it. 3 The H2 receptor antagonist cimetidine (2 μM) blocked the effects of lower concentrations of histamine, while the H1 receptor antagonist mepyramine (3 nM) blocked those of higher concentrations of histamine. 4 The effects of lower doses of histamine were mimicked by the H2 receptor agonist dimaprit, and the effects of higher doses of histamine were mimicked by the H1 receptor agonist α‐2‐(2‐aminoethyl)pyridine (AEP). 5 Low concentrations of histamine, which normally increase the permeability of Lucifer Yellow (PLY), reduced it when co‐applied with the phosphodiesterase 4 (PDE4) inhibitor rolipram. Rolipram also potentiated the response to AEP, but had no effect on that to dimaprit. 6 The effects of dimaprit were blocked by reducing extracellular Ca2+ from 2.5 mM to nominally Ca2+ free, or by applying the calcium entry blocker SKF 96365.

Measurement of filtration coefficient in single cerebral microvessels of the frog.

1. This study reports the first results of measurements of filtration coefficient (Lp) and osmotic reflection coefficient to sucrose (sigma suc) in single brain microvessels. 2. Microvessels on the surface of frog brain were cannulated with a micropipette and perfused with an artificial cerebrospinal fluid (CSF) containing the low molecular weight impermeant dye carboxyfluorescein (MW 376). The superfusing solution was a similar CSF which could be made hypertonic by the addition of 40‐125 mmol l‐1 sucrose. 3. Vessels were assessed for dye retention using video‐intensified microscopy after occlusion with a glass microneedle. Only six vessels out of a total of ninety‐five were tight under the experimental conditions used. Those vessels which were tight were occluded while an osmotic load was applied across them. When this load was 50 mosmol l‐1 and less, the steady‐state dye concentration within the vessel lumen was similar to that predicted assuming the endothelium behaves as a perfect semipermeable membrane, with concentration polarization of solute. 4. The product Lp sigma was estimated in two ways: (i) from the fitted monoexponential function that described the rising dye concentration within the occluded segment, and (ii) from the initial rate of increase in dye concentration. The two values obtained were similar and it was concluded that sigma NaCl = sigma suc = 1, and the best estimate for filtration coefficient Lp = 2.0 x 10(‐9) cm (cmH2O s)‐1. 5. At the osmotic loads of 100 mosmol l‐1 and more, the initial rate of increase estimate of Lp sigma was less than half of the whole curve estimate, the axial dye distributions were dissimilar from those predicted by a mathematical model based on the perfect semipermeable membrane, and the steady‐state concentration was less than 70% of that expected. These findings are consistent with a diffusive pathway having opened. The model was modified to include patches of vessel wall which had developed leaks and a good fit to the data was obtained with a sucrose permeability and an Lp similar to skeletal muscle endothelium. 6. The possibility that water passes through a paracellular pathway across the intact blood‐brain barrier is discussed. It is concluded that this pathway could not be detected by the methods used and can carry no more than 50% of the water driven by a hydrostatic pressure gradient.

Modulation of blood-brain barrier permeability by neutrophils: in vitro and in vivo studies

The blood-brain barrier (BBB) restricts solute permeability across healthy cerebral endothelial cells. However, during inflammation, permeability is increased and can lead to deleterious cerebral edema. Neutrophils are early cellular participants in acute inflammation, but their effect on BBB permeability is unclear. To study this, neutrophils were applied in a resting and activated state to in vitro and in vivo models of the BBB. In vitro, human neutrophils (5 x 10(6)/ml) were activated with tumor necrosis factor (100 U/ml) and leukotriene B(4) (10(-7) mol/l). Untreated neutrophils reduced permeability across the human brain endothelial cell line hCMEC/D3. Activated neutrophils returned permeability to baseline, an effect blocked by the reactive oxygen scavengers superoxide dismutase (10 U/ml) and catalase (1000 U/ml). In vivo, human neutrophils (2.5 x1 0(5) in 4 microl) were injected into the striatum of anesthetized juvenile Wistar rats, and BBB permeability measured 30 min later. This was compared to control injections (4 microl) of vehicle (0.9% saline) and arachidonic acid (10(-3) mol/l). The injection generated a small hematoma around the injection tract (<3 microl). Untreated neutrophils induced significantly lower permeability in their vicinity than activated neutrophils, with a trend to lowered permeability compared to the vehicle control. Neither untreated nor activated neutrophils induced permeability increases, while arachidonic acid increased permeability as a positive control. This study further delineates the effect of neutrophils on the BBB, and demonstrates that resting neutrophils induce acute reductions in permeability while activated neutrophils have a neutral effect. The in vivo model reiterates some aspects of acute intracerebral hemorrhage.

MRI measurement of blood-brain barrier permeability following spontaneous reperfusion in the starch microsphere model of ischemia

Quantification of the acute increases in blood-brain barrier (BBB) permeability that occur subsequent to experimental ischemic injury has been limited to single time-point, invasive methodologies. Although permeability can be qualitatively assessed to visualise regional changes during sequential studies on the same animal using contrast-enhanced magnetic resonance imaging (MRI), quantitative information on the magnitude of change is required to compare barrier function during sequential studies on the same animal or between different animals. Recently, improvements in MRI tracer kinetic models and in MR hardware design mean that an estimate of permeability in vivo can now be obtained with acceptable accuracy and precision. We report here the use of such methods to study acute changes following spontaneous reperfusion in an animal model of ischemia. We have obtained estimates of BBB permeability following spontaneous reperfusion, subsequent to forebrain ischemia by unilateral carotid injection of starch microspheres in the rat. T2*-weighted and diffusion-trace imaging were used to monitor the initial reduction in CBF and the time-course of ischemia, respectively. Following reperfusion, an intraveneous bolus of dimeglumine gadopentetate (Gd-DTPA) and horseradish peroxidase (HRP) was given during a continuous acquisition of T1 maps with a 48 s temporal resolution. Permeability maps were constructed using a 4-compartment model; K(trans), the permeability-surface area product of the capillary walls was estimated to be 9.2 +/- 0.6 x 10(-4) min(-1) in the cortex. Visualisation of the regional extent of HRP extravasation on histological sections following termination of the experiment demonstrated very little correspondence to the region of Gd-DTPA leakage. Quantitative MRI assessment of BBB permeability following ischemia-reperfusion is consistent with published values obtained by invasive methods. Differences between Gd-DTPA-enhancement and HRP may reflect differences in the molecular size of the tracers.

Role of NADPH Oxidase in Retinal Microvascular Permeability Increase by RAGE Activation

PURPOSE The accumulation of advanced glycation end products (AGEs) within the retina in diabetes is associated with a chronic increase in retinal microvascular permeability. Isolated perfused retinas were used to examine the acute effects of AGEs on retinal microvascular permeability. METHODS Retinas were dissected from eyes obtained from male Wistar rats, pinned out flat, and perfused with the low-molecular-weight fluorescent dye sulforhodamine B. Microvascular permeability was determined from the rate of decrease in fluorescence gradient across a vessel under conditions of zero flow. The production of reactive oxygen species (ROS) in JG2.1 retinal endothelial cells was also assessed with a fluorescent probe working solution. RESULTS A 30-second application of AGE-modified bovine serum albumin (AGE-BSA) to the abluminal surface of the retinal vasculature produced a rapid dose-dependent increase in retinal capillary permeability that was inhibited by pretreatment with anti-RAGE IgG. The permeability response also required ROS generated by NADPH oxidase because pretreatment with apocynin and the free radical scavengers superoxide dismutase and catalase significantly reduced the response. Pretreatment with calphostin C, SKF-96365, and U-73122 also significantly reduced the permeability response. In addition, the permeability response to bradykinin increased permeability through ROS and was potentiated after pretreatment with AGE-BSA. This potentiation was blocked by apocynin. CONCLUSIONS Acute activation of NADPH oxidase by phospholipase C-mediated activation of Ca(2+)-dependent PKC occurs downstream of RAGE activation to acutely increase retinal capillary permeability in the isolated perfused rat retina.