Ultrastructural studies on blood-brain barrier dysfunction after cerebral air embolism in the rat

Summary Male albino rats were anaesthetized with diazepam, injected with horseradish peroxidase and Evans blue-labeled albumin and given an embolus of 0.01 ml air in the right common carotid artery after ligation of the external carotid branch.The pial arteries of the right cerebral hemisphere were stained blue, particularly the middle cerebral artery and its main arterial branchlets. Ultrastructurally, some endothelial cells in the right middle cerebral artery, small arteries and arterioles showed a diffuse distribution of horseradish peroxidase in their cytoplasm, although these vessels only occasionally showed peroxidase in their basement membranes. Other endothelial cells in these arterial branchlets showed few if any signs of a diffuse distribution of peroxidase but displayed several pinocytotic vesicles and occasionally trans-endothelial channels filled with peroxidase, sometimes with a slight leakage of peroxidase into adjacent basement membranes and neuropil. Scattered pinocytotic vesicles were observed in capillaries and venules, but there was usually no extravasation of peroxidase around these vessels.     

Reactivity of the cerebrovascular bed to carbon dioxide in patients with primary high haematocrit before and after venesection

Paired cerebral blood flow measurements at 2 levels of arterial carbon dioxide tension (PaCO2) were made in 10 subjects with an elevated haematocrit and blood viscosity. A value of 32 was obtained for the percentage change in flow per kPa change in PaCO2 (CO2 reactivity). The haematocrit was then lowered by venesection from 0.536 to 0.458 and the measurements were repeated. The post-venesection CO2 reactivity was 29, which was not significantly different from the pre-venesection value. Thus CO2 reactivity is not comprised in patients with low cerebral blood flow secondary to an elevated haematocrit.     

The blood-brain barrier and epilepsy

During the past several years, there has been increasing interest in the role of the blood-brain barrier (BBB) in epilepsy. Advances in neuroradiology have enhanced our ability to image and study the human cerebrovasculature, and further developments in the research of metabolic deficiencies linked to seizure disorders (e.g., GLUT1 deficiency), neuroinflammation, and multiple drug resistance to antiepileptic drugs (AEDs) have amplified the significance of the BBB's relationship to epilepsy. Prior to 1986, BBB research in epilepsy focused on three main areas: ultrastructural studies, brain glucose availability and transport, and clinical uses of AEDs. However, contrast-based imaging techniques and medical procedures such as BBB disruption provided a framework that demonstrated that the BBB could be reversibly disrupted by pathologic or iatrogenic manipulations, with important implications in terms of CNS drug delivery to "multiple drug resistant" brain. This concept of BBB breakdown for therapeutic purposes has also unveiled a previously unrecognized role for BBB failure as a possible etiologic mechanism in epileptogenesis. Finally, a growing body of evidence has shown that inflammatory mechanisms may participate in the pathological changes observed in epileptic brain, with increasing awareness that blood-borne cells or signals may participate in epileptogenesis by virtue of a leaky BBB. In this article we will review the relationships between BBB function and epilepsy. In particular, we will illustrate consensus and divergence between clinical reality and animal studies.     

Enhanced blood-brain barrier leakage to evans blue-labelled albumin after air embolism in ethanol-intoxicated rats

Summary Control and ethanol-intoxicated rats were given a small air embolus in the right common carotid artery after ligation of the external carotid branch. The extravasation of Evans blue-labelled albumin (EBA) was studied. Control rats displayed a slight extravasation of EBA through arteries and arterioles at the surface of the right hemisphere with a slight spread into adjacent brain parenchyma. Additionally, ethanol-intoxicated rats showed a great leakage through small blood vessels even deep in the right hemisphere and a blue-staining of most of the embolized hemisphere.Thus it was shown that during embolization, ethanol increases the vascular permeability to macromolecules. It is proposed that this phenomenon mainly is due to injury to the endothelial cells.     

The biphasic opening of the blood-brain barrier to proteins following temporary middle cerebral artery occlusion

Summary The behavior of the blood-brain barrier (BBB) was studied in cats following release after 1-h middle cerebral artery (MCA) occlusion. The regional cerebral blood flow (rCBF) was determined by hydrogen clearance method in the caudate nucleus and the cerebral cortex. The BBB was assayed with Evans blue (EB) tracer and by immunohistochemical peroxidaseantiperoxidase (PAP) method. Following release of MCA occlusion, there were two openings of the BBB, separated by a refractory period. The first opening, occurred shortly after recirculation; this was associated with rCBF below 15 ml/100 g/min during the ischemic period and a pronounced reactive hyperemia promptly following release of MCA occlusion. A refractory period of the BBB was indicated by the absence of EB leakage in cats injected with the tracer 30 min before killing at 3 h after recirculation, although the rCBF values in these animals were even lower (6±1 ml/100 g/min) during occlusion, and all of them showed a pronounced hyperemia after recirculation. The occurrence of the previous BBB opening in these animals was confirmed by the PAP staining. The second opening of the BBB was observed at 5 and 72 h after recirculation in cats which were injected with EB 30 min before killing, and which showed rCBF below 15 ml/100 g/min during occlusion, followed by a pronounced reactive hyperemia. No EB extravasations were observed at any time in cats in which the rCBF during occlusion was above 15 ml/100 g/min and which failed to show a marked reactive hyperemia.     

Cerebral microembolization in the rat: Changes in blood-brain barrier permeability and cerebral blood flow as related to the degree of ischemia

Unilateral cerebral microembolism was performed in the rat by injecting calibrated, 50 micrometers in diameter, carbonized microspheres into the internal carotid artery. The events that follow brain ischemia due to cerebral embolization were studied by the analysis of the blood-brain barrier (BBB) function, the degree of regional cerebral blood flow (CBF) and the development of brain edema. Two hours after embolization there was no change in the brain water content. The local CBF (14C-ethanol technique) was only reduced in the ipsilateral hemisphere. Twenty-four hours after embolization the brain water content was increased significantly in the ipsilateral, but not in the contralateral hemisphere. Local CBF further decreased in the ipsilateral hemisphere and a reduction in flow was also observed in the contralateral hemisphere. Embolization led to an increase in the BBB permeability, analysed as regional penetrability of 3H-dextran and of Evans blue-albumin complexes, which was restricted to the side of the injection of the microspheres.     

大鼠蛛网膜下腔出血后脑局部血流量与血脑屏障动态变化

目的：探讨大鼠蛛网膜下腔出血（SAH）后局部脑血流（rCBF）与血脑屏障（BBB）超微结构的动态变化规律和尼莫地平（Nim）的作用特点。方法：通过大鼠枕大池自体血注入法制备SAH后脑血管痉挛（CVS）模型，在SAH后1h、4h、12、24h、3d、7d、14d、及21d等不同时相点，利用激光普勒技术和电镜对各组大鼠局部脑血流与血脑屏障超微结构变化进行观察。结果：SAH后大鼠的rCBF明显下降且具有“两期”反应，在SAH后1h后rCBF有所恢复，4h后再度降低，并可持续至21d。BBB损害改变在SAH后早期即可观察到。Nim对rCBF降低和BBB损害均有改善作用。结论：SAH后rCBF与BBB超微结构的动态变化符合CVS的发展规律，可以反映该时期CVS的状态。早期应用Nim可以改善SAH后CVS，并对BBB的损害有一定保护作用。     

Cerebral blood flow, haematocrit and viscosity in subjects with a high oxygen affinity haemoglobin variant

The cerebral blood flow is low in primary polycythaemia, and it has been suggested that this is due to the increase in viscosity which accompanies the elevated haematocrit. In the present study cerebral blood flow has been measured by a non-invasive ¹³³Xenon technique in six subjects with an elevated haematocrit secondary to a haemoglobin variant with increased oxygen affinity. Flow was significantly higher than normal and 81 % higher than in 11 subjects of comparable age, matched for haematocrit and viscosity, but without the haemoglobin variant. In patients with this unusual type of polycythaemia, cerebral blood flow is high despite the elevated blood viscosity and the implications of these results are discussed.     

The effects of sodium bicarbonate on brain blood flow,brain water content,and blood-brain barrier in the neonatal dog

Summary To explore the relationship between cerebral hemorrhage in the newborn and administration of sodium bicarbonate, we gave a standard dose of sodium bicarbonate (5 mEq/kg) to neonatal dogs and then assessed changes in cerebral blood flow, brain water content, and the blood-brain barrier. This dose of sodium bicarbonate produced no increase in blood pressure or cerebral blood flow and no alteration in blood-brain barrier. However, infusion of sodium bicarbonate did cause hyperosmolality and hypernatremia and a significant decrease in brain water content. Cerebral hemorrhage in the neonate associated with infusions of sodium bicarbonate may be related to shifts in brain water rather than to changes in blood pressure or cerebral blood flow.Supported in part by NIH grant NS 18039 (RSKY)     

Cerebral blood flow changes during pilocarpine-induced status epilepticus activity in the rat hippocampus

Introduction

There is a known relationship between convulsive status epilepticus (SE) and hippocampal injury. Although the precise causes of this hippocampal vulnerability remains uncertain, potential mechanisms include excitotoxicity and ischaemia. It has been hypothesised that during the early phase of seizures, cerebral blood flow (CBF) increases in the cortex to meet energy demand, but it is unclear whether these compensatory mechanisms occur in the hippocampus. In this study we investigated CBF changes using perfusion MRI during SE in the pilocarpine rat.

Methods

First, we determined whether SE could be induced under anaesthesia. Two anaesthetic protocols were investigated: isoflurane (n = 6) and fentanyl/medetomidine (n = 7). Intrahippocampal EEG electrodes were used to determine seizure activity and reflex behaviours were used to assess anaesthesia. Pilocarpine was administered to induce status epilepticus. For CBF measurements, MRI arterial spin labelling was performed continuously for up to 3 h. Either pilocarpine (375 mg/kg) (n = 7) for induction of SE or saline (n = 6) was administered. Diazepam (10 mg/kg) was administered i.p. 90 min after the onset of SE.

Results and discussion

We demonstrated time-dependent significant (p < 0.05) differences between the CBF responses in the parietal cortex and the hippocampus during SE. This regional response indicates a preferential distribution of flow to certain regions of the brain and may contribute to the selective vulnerability observed in the hippocampus in humans.     

Cerebral blood flow during chronic hypoglycemia in the rat

Regional cerebral blood flow (rCBF) was measured in normoglycemic and chronically hypoglycemic rats. Chronic hypoglycemia was produced by continuously infusing insulin for 6-7 days. During chronic hypoglycemia (plasma glucose = 1.97 mumol/ml), rCBF increased in all regions except the cerebellum and hypothalamus. Blood flow increases present during chronic hypoglycemia were not as great as those previously measured during acute hypoglycemia. Therefore, adjustments in the regulation of rCBF occurred during chronic hypoglycemia compared to acute hypoglycemia.     

Cerebral hemodynamic response induced by the Tower of Hanoi puzzle and the Wisconsin Card Sorting test

Transcranial Doppler sonography (TCD) was applied in normal subjects to investigate the effect of prefrontal functions like the Tower of Hanoi (TOH) task and the Wisconsin Card Sorting test (WCST) on cerebral hemodynamics. In 20 healthy volunteers, left and right middle cerebral artery (MCA) and anterior cerebral artery (ACA) were insonated. The TOH task and the WCST were administered while cerebral blood flow velocity (CBFV) was registered. Each test was repeated once per artery pair. There was a visuomotor test to control the motor and visual stimulations. Three phases of CBFV time course were detected: an initial peak within 5 s, a following decrease within 25 s and a steady state beginning at 40 s. The TOH task, WCST and visuomotor tests had different mean CBFV during the initial peak (MCA: P<0.05; ACA: P<0.05) as well as for the decrease (ACA: P<0.01) and the steady state (MCA: P<0.01; ACA: P<0.01). The TOH showed an increased mean CBFV as compared with the WCST during the steady state (MCA: P<0.01; ACA: P<0.05). However, temporal modulation of mean CBFV during category shift of the WCST resulted in significantly increased values after category shift (MCA: P<0.001; ACA: P<0.01) as compared with CBFV before the category shift. These findings showed a different CBFV pattern during the TOH task and WCST than during the visuomotor test. In conclusion, TCD was able to assess CBFV in prefrontal functions, using a high resolution in time.     

A new dynamic in vitro model for the multidimensional study of astrocyte-endothelial cell interactions at the blood-brain barrier

Blood-brain barrier endothelial cells are characterized by the presence of tight intercellular junctions, the absence of fenestrations, and a paucity of pinocytotic vesicles. The in vitro study of the BBB has progressed rapidly over the past several years as new cell culture techniques and improved technologies to monitor BBB function became available. Studies carried out on viable in vitro models are set to accelerate the design of drugs that selectively and aggressively can target the CNS. Several systems in vitro attempt to reproduce the physical and biochemical behavior of intact BBB, but most fail to reproduce the three-dimensional nature of the in vivo barrier and do not allow concomitant exposure of endothelial cells to abluminal (glia) and lumenal (flow) influences. For this purpose, we have developed a new dynamic in vitro BBB model (NDIV-BBB) designed to allow for extensive pharmacological, morphological and physiological studies. Bovine aortic endothelial cells (BAEC) developed robust growth and differentiation when co-cultured alone. In the presence of glial cells, BAEC developed elevated Trans-Endothelial Electrical Resistance (TEER). Excision of individual capillaries proportionally decreased TEER; the remaining bundles were populated with healthy cells. Flow played an essential role in EC differentiation by decreasing cell division. In conclusion, this new dynamic model of the BBB allows for longitudinal studies of the effects of flow and co-culture in a controlled and fully recyclable environment that also permits visual inspection of the abluminal compartment and manipulation of individual capillaries.     

Transfer of bromocriptine across the blood-brain barrier in man

The transfer of ¹⁴C-bromocriptine across the blood-brain barrier was studied in 10 patients using the double indicator single injection method. The extraction (E) of bromocriptine was 8 % (quartiles: 6 and 14 %). Based on the results the cerebral uptake velocity of bromocriptine was calculated assuming constant arterial drug concentration. Its T_1/2 was found to be 12–24 minutes, depending on the tissue-blood partition coefficient.     

梗塞性脑血管病认知电位P300与局部脑血流量的相关研究

本研究应用P300和SPECT观察了30例梗塞性脑血管病人,分别进行P300潜伏期,脑血流显像,rCBF比值测定。研究表明,在梗塞性脑血管病ERP测试的内源成分P300 PL即显示出有意义的变化,与对照组比较PL明显延长(P<0.001)。SPECT脑显像则显示出梗塞区及其周围放射性稀疏区,病变范围大于XCT,rCBF比值小于0.9。经统计处理显示脑血流量与P300 PL延长呈负相关。反映了脑梗塞患者认知功能障碍与CBF的关系。     

Effect of histamine and antagonists on electrical resistance across the blood-brain barrier in rat brain-surface microvessels

The effect of histamine on blood-brain barrier permeability was investigated using in situ measurement of transendothelial electrical resistance in brain-surface microvessels of anaesthetized rats. Mean resistance of vessels superfused with artificial cerebrospinal fluid was 1500 omega.cm2, indicating a tight barrier with low ion permeability. The addition of 10(-4) M histamine resulted in a 75% decrease in resistance, in both arterial and venous vessels, indicating a marked increase in barrier permeability. To determine the nature of the response to histamine, rats were given presurgical intraperitoneal injections of promethazine (H1 receptor antagonist), cimetidine (H2 receptor antagonist) or indomethacin (cyclo-oxygenase inhibitor), singularly and in combinations. Cimetidine completely blocked the histamine-mediated increase in barrier permeability whereas promethazine only had a small effect and indomethacin was ineffective. In addition, cimetidine treatment resulted in a 100% increase in basal resistance in both arterial and venous vessels, suggesting endogenous histamine was acting to increase blood-brain barrier permeability. It is concluded that histamine causes an increase in blood-brain barrier permeability which is mediated via endothelial H2 receptors, and that the electrical resistance in cimetidine-treated rats most closely represents the true permeability of the blood-brain barrier.     

脑梗死急性期使用丁苯酞注射液对患者脑血流灌注和认知功能的影响

目的 研究伴有认知障碍的脑梗死急性期患者使用丁苯酞注射液对脑血流灌注和蒙特利尔认知 评估量表（Montreal cognitive assessment scale,MoCA）评分的影响。      

Cerebral circulation and oxygen metabolism in Moyamoya disease of ischemic type in children

Cerebral rCBF, rOEF, rCMRO₂, and rCBV in moyamoya disease were studied by means of positron emmission tomography (PET), using ¹⁵O as a tracer. Steady-state methods with C¹⁵O₂ and ¹⁵O₂ were used to obtain the functional images of rCBF, rCMRO₂, and rOEF. The ¹⁵O single-inhalation method was used to obtain the rCBV image. Five children (two boys and three girls) with mean age of 11 years and eight normal volunteers with mean age of 31 years were included in the study. The symptoms of moyamoya disease were due to cerebral ischemia, such as transient ischemic attack (TIA), reversible ischemic neurological deficit (RIND), and minor stroke. The interval between the latest ictus and PET scan ranged from 3 days to 3 years 6 months. Physiological parameters (rCBF, rCMRO₂ etc.) in cerebral gray matter, cerebral white matter and basal ganglia were calculated from the single functional images. Any, low density areas appearing in X-ray-CT performed just prior to the PET study were carefully excluded from the analysis. The parameters of moyamoya disease were statistically compared with normal control parameters. Though the value of rCBF was slightly higher in moyamoya disease, this difference was not statistically significant. On the other hand, in moyamoya disease rCBV increased significantly in gray matter, white matter, and basal ganglia. The ratio of CBF to CBV is considered to be the index of perfusion pressure and reciprocal of cerebral mean transit time under the normal autoregulation of CBF. This ratio was calculated and compared with the normal value for each tissue. The ratio was significantly decreased in each tissue in moyamoya disease, indicating the presence of a low perfusion pressure in the moyamoya brain. In general, when the reduction of perfusion pressure becomes profound the decrease in the CBF-to-CBV ratio is followed by an increase in rOEF. In spite of the reduction in the CBF-to-CBV ratio there was no significant increase in rOEF in moyamoya disease. Thus, the cerebral circulation in childhood moyamoya disease of ischemic type was characterized by a mild decrease in perfusion pressure and a prolonged circulation time.     

Effects of transient loss of shear stress on blood-brain barrier endothelium: role of nitric oxide and IL-6

Loss of blood-brain barrier (BBB) function may contribute to post-ischemic cerebral injury by yet unknown mechanisms. Ischemia is associated with anoxia, aglycemia and loss of flow (i.e. shearing forces). We tested the hypothesis that loss of shear stress alone does not acutely affect BBB function due to a protective cascade of mechanisms involving cytokines and nitric oxide (NO). To determine the relative contribution of shear stress on BBB integrity we used a dynamic in vitro BBB model based on co-culture of rat brain microvascular endothelial cells (RBMEC) and astrocytes. Trans-endothelial electrical resistance (TEER), IL-6 release and NO levels were measured from the lumenal and ablumenal compartments throughout the experiment. Flow-exposed RBMEC were challenged with 1 h of normoxic-normoglycemic flow cessation (NNFC) followed by reperfusion for 2 to 24 h. NNFC caused a progressive drop in nitric oxide production during flow cessation followed by a time-dependent increase in ablumenal IL-6 associated with a prolonged NO increase during reperfusion. The nitric oxide synthetase (NOS) inhibitor L-NAME (10 microM) abrogated all effects of NNFC, including changes in NO and cytokine production. BBB permeability did not increase during or after NNFC/reperfusion, but was increased by treatment with L-NAME or when the effects of IL-6 were blocked. Flow adapted RBMEC and astrocytes respond to NNFC/reperfusion by overproduction of IL-6, possibly secondary to increased production of NO during the reperfusion. Maintenance of BBB function during and following NNFC appears to depend on intact NO signaling and IL-6 release.