Early appearance of activated matrix metalloproteinase-9 and blood–brain barrier disruption in mice after focal cerebral ischemia and reperfusion

Blood–brain barrier (BBB) disruption is thought to play a critical role in the pathophysiology of ischemia/reperfusion. Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes that can degrade all the components of the extracellular matrix when they are activated. Gelatinase A (MMP-2) and gelatinase B (MMP-9) are able to digest the endothelial basal lamina, which plays a major role in maintaining BBB impermeability. The present study examined the expression and activation of gelatinases before and after transient focal cerebral ischemia (FCI) in mice. Adult male CD1 mice were subjected to 60 min FCI and reperfusion. Zymography was performed from 1 to 23 h after reperfusion using the protein extraction method with detergent extraction and affinity-support purification. MMP-9 expression was also examined by both immunohistochemistry and Western blot analysis, and tissue inhibitors to metalloproteinase-1 was measured by reverse zymography. The BBB opening was evaluated by the Evans blue extravasation method. The 88-kDa activated MMP-9 was absent from the control specimens, while it appeared 3 h after transient ischemia by zymography. At this time point, the BBB permeability alteration was detected in the ischemic brain. Both pro-MMP-9 (96 kDa) and pro-MMP-2 (72 kDa) were seen in the control specimens, and were markedly increased after FCI. A significant induction of MMP-9 was confirmed by both immunohistochemistry and Western blot analysis. The early appearance of activated MMP-9, associated with evidence of BBB permeability alteration, suggests that activation of MMP-9 contributes to the early formation of vasogenic edema after transient FCI.     

Activated Macrophages and the Blood–Brain Barrier: Inflammation after CNS Injury Leads to Increases in Putative Inhibitory Molecules

The cellular responses to spinal cord or brain injury include the production of molecules that modulate wound healing. This study examined the upregulation of chondroitin sulfate proteoglycans, a family of molecules present in the wound healing matrix that may inhibit axon regeneration in the central nervous system (CNS) after trauma. We have demonstrated increases in these putative inhibitory molecules in brain and spinal cord injury models, and we observed a close correlation between the tissue distribution of their upregulation and the presence of inflammation and a compromised blood–brain barrier. We determined that the presence of degenerating and dying axons injured by direct trauma does not provide a sufficient signal to induce the increases in proteoglycans observed after injury. Activated macrophages, their products, or other serum components that cross a compromised blood–brain barrier may provide a stimulus for changes in extracellular matrix molecules after CNS injury. While gliosis is associated with increased levels of proteoglycans, not all reactive astrocytes are associated with augmented amounts of these extracellular matrix molecules, which suggests a heterogeneity among glial cells that exhibit a reactive phenotype. Chondroitin sulfate also demarcates developing cavities of secondary necrosis, implicating these types of boundary molecules in the protective response of the CNS to trauma.     

Kinetics and distribution of [Fe–I]transferrin injected into the ventricular system of the rat

We examined the kinetics and distribution of [⁵⁹Fe–¹²⁵I] rat Tf and unlabelled human Tf injected into a lateral cerebral ventricle (i.c.v. injection) in the rat. [⁵⁶Fe–¹³¹I]Tf injected intravenously served as a control of blood–brain barrier (BBB) integrity. In CSF of adult rats, ⁵⁹Fe and [¹²⁵I]Tf decreased to only 2.5% of the dose injected after 4 h. In brain parenchyma, [¹²⁵I]Tf had disappeared after 24 h, whereas approximately 18% of i.c.v.-injected ⁵⁹Fe was retained even after 72 h. The elimination pattern of [¹²⁵I]Tf from the CSF corresponded to that of [¹³¹I]albumin injected i.c.v., suggesting a nonselective washout of CSF proteins. [¹³¹I]Tf was hardly detectable in the brain, reflecting an unimpaired BBB during the experiments. Morphologically, ⁵⁹Fe and i.c.v. injected human Tf were confined to the ventricular surface and meningeal areas, whereas grey matter regions at distances more than 2–3 mm from the ventricles and the subarachnoid space were unlabelled. However, accumulation of ⁵⁹Fe was observed in the anterior thalamic and the medial habenular nuclei, and in brain regions with synaptic communications to these areas. In the newborn rats aged 7 days (P7) injected i.c.v. with [⁵⁹Fe–¹²⁵I]Tf and examined after 24 h, the amounts of [¹²⁵I]Tf in CSF were approximately 3.5 times higher than in adult rats collected after the same time interval, whereas the amounts of ⁵⁹Fe in CSF were at the same level in P7 and adult rats. In the brain tissue of the i.c.v. injected P7 rats, both [¹²⁵I]Tf and ⁵⁹Fe were retained to a significantly higher degree compared to that seen in adult brains. The rapid washout and lack of capability for i.c.v. injected [¹²⁵I]Tf to penetrate deeply into the brain parenchyma of the adult brain question the importance of Tf of the CSF, and choroid plexus-derived Tf, for Fe neutralization and delivery of Fe–Tf to TfR-containing neurons and other cells in the CNS. However, it may serve these functions in young animals due to a lower rate of turnover of CSF.     

Effects of hypothermia on thrombin-induced brain edema formation

Recent studies have shown that thrombin plays an important role in brain edema formation after intracerebral hemorrhage (ICH). The possible mechanisms of thrombin-induced brain edema formation include blood-brain barrier (BBB) disruption and inflammatory response involving polymorphonuclear (PMN) leukocyte. Animal experiments have revealed that moderate therapeutic hypothermia improves pathological and functional outcome in various models of brain injury. In this study, we examined the effect of hypothermia on thrombin-induced brain edema formation. Effects of hypothermia on BBB permeability and the accumulation of PMN leukocytes were also determined to clarify the protective mechanism of hypothermia in this model. Anesthetized adult rats received an injection of 10 Units of thrombin into the basal ganglia. Animals were separated into the normothermic and hypothermic groups, which were housed in a room maintained at 25 degrees C and in a cold room maintained at 5 degrees C, respectively, for 24 h after the thrombin injection. The brain temperature in rats housed in a cold room reduced temporarily to approximately 30 degrees C and then gradually recovered to 35 degrees C by the end of the observation. Brain water content in the basal ganglia was significantly reduced in rats treated with hypothermia compared to the normothermic rats (84.3+/-0.2 vs. 82.4+/-0.1%; P<0.01). The decrease of brain water content was accompanied with a significant reduction in BBB permeability to Evan's blue dye and in accumulation of PMN leukocytes. This study indicates that hypothermic treatment significantly reduces thrombin-induced brain edema formation in the rat. Inhibition of thrombin-induced BBB breakdown and inflammatory response by hypothermia appear to contribute to brain protection in this model. Hypothermic treatment may provide an approach to potentially reduce ongoing edema after ICH.     

Carrier-mediated processes in blood–brain barrier penetration and neural uptake of paraquat

Due to the structural similarity to N-methyl-4-phenyl pyridinium (MPP⁺), paraquat might induce dopaminergic toxicity in the brain. However, its blood–brain barrier (BBB) penetration has not been well documented. We studied the manner of BBB penetration and neural cell uptake of paraquat using a brain microdialysis technique with HPLC/UV detection in rats. After subcutaneous administration, paraquat appeared dose-dependently in the dialysate. In contrast, MPP⁺ could not penetrate the BBB in either control or paraquat pre-treated rats. These data indicated that the penetration of paraquat into the brain would be mediated by a specific carrier process, not resulting from the destruction of BBB function by paraquat itself or a paraquat radical. To examine whether paraquat was carried across the BBB by a certain amino acid transporter, -valine or -lysine was pre-administered as a co-substrate. The pre-treatment of -valine, which is a high affinity substrate for the neutral amino acid transporter, markedly reduced the BBB penetration of paraquat. When paraquat was administered to the striatum through a microdialysis probe, a significant amount of paraquat was detected in the striatal cells after a sequential 180-min washout with Ringer’s solution. This uptake was significantly inhibited by a low Na⁺ condition, but not by treatment with putrescine, a potent uptake inhibitor of paraquat into lung tissue. These findings indicated that paraquat is possibly taken up into the brain by the neutral amino acid transport system, then transported into striatal, possibly neuronal, cells in a Na⁺-dependent manner.     

Complete inhibition of ABCB1 and ABCG2 at the blood–brain barrier by co-infusion of erlotinib and tariquidar to improve brain delivery of the model ABCB1/ABCG2 substrate [11C]erlotinib

P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) restrict at the blood–brain barrier (BBB) the brain distribution of the majority of currently known molecularly targeted anticancer drugs. To improve brain delivery of dual ABCB1/ABCG2 substrates, both ABCB1 and ABCG2 need to be inhibited simultaneously at the BBB. We examined the feasibility of simultaneous ABCB1/ABCG2 inhibition with i.v. co-infusion of erlotinib and tariquidar by studying brain distribution of the model ABCB1/ABCG2 substrate [¹¹C]erlotinib in mice and rhesus macaques with PET. Tolerability of the erlotinib/tariquidar combination was assessed in human embryonic stem cell-derived cerebral organoids. In mice and macaques, baseline brain distribution of [¹¹C]erlotinib was low (brain distribution volume, V_T,brain < 0.3 mL/cm³). Co-infusion of erlotinib and tariquidar increased V_T,brain in mice by 3.0-fold and in macaques by 3.4- to 5.0-fold, while infusion of erlotinib alone or tariquidar alone led to less pronounced V_T,brain increases in both species. Treatment of cerebral organoids with erlotinib/tariquidar led to an induction of Caspase-3-dependent apoptosis. Co-infusion of erlotinib/tariquidar may potentially allow for complete ABCB1/ABCG2 inhibition at the BBB, while simultaneously achieving brain-targeted EGFR inhibition. Our protocol may be applicable to enhance brain delivery of molecularly targeted anticancer drugs for a more effective treatment of brain tumors.     

Regulation of Multidrug Resistance P‐Glycoprotein in the Developing Blood–Brain Barrier: Interplay between Glucocorticoids and Cytokines

P‐glycoprotein (P‐gp) encoded by Abcb1 provides protection to the developing brain from xenobiotics. P‐gp in brain endothelial cells (BECs) derived from the developing brain microvasculature is up‐regulated by glucocorticoids and inhibited by pro‐inflammatory cytokines in vitro. However, little is known about how prenatal maternal glucocorticoid treatment can affect Abcb1/P‐gp function and subsequent cytokine regulation in foetal BECs. We hypothesised that glucocorticoid exposure increases Abcb1/P‐gp in the foetal brain microvasculature and enhances the sensitivity of Abcb1/P‐gp in BECs to the inhibitory effects of cytokines. BECs isolated from dexamethasone‐ or vehicle‐exposed foetal guinea pigs were cultured and treated with interleukin‐1β, interleukin‐6 or tumour necrosis factor‐α, and Abcb1/P‐gp expression and function were assessed. Prenatal dexamethasone exposure significantly increased Abcb1/P‐gp expression/activity and cytokine receptor levels in BECs of the foetal brain microvasculature. Foetal dexamethasone exposure in vivo also increased the subsequent responsiveness of BECs to pro‐inflammatory cytokines in vitro. In conclusion, maternal treatment with synthetic glucocorticoids appears to prematurely mature P‐gp mediated drug resistance at the foetal BBB in vivo and profoundly impact the subsequent responsiveness of P‐gp to pro‐inflammatory cytokines in the foetal BEC. The significance of these findings to foetal brain protection against xenobiotics and other P‐gp substrates in vivo requires further elaboration. However, the results of the present study may have implications for human pregnancy and foetal brain protection, particularly in cases of preterm birth combined with infection.     

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.     

An improved low-permeability in vitro-model of the blood–brain barrier: transport studies on retinoids, sucrose, haloperidol, caffeine and mannitol

Primary cultures of porcine brain capillary endothelial cells grown on collagen coated polycarbonate membranes were used to build up an in vitro-model for the blood–brain barrier. Improved cultivation techniques allowed cell-storage and experiments under serum-free conditions. We employed this model to perform permeability studies in vitro with the radioactively labelled marker substances sucrose, retinoic acid, retinol, haloperidol, caffeine, and mannitol. Permeability values obtained with this blood–brain barrier model (1.0×10⁻⁶ cm/s for sucrose, 6.2×10⁻⁶ cm/s for retinoic acid, 4.8×10⁻⁶ cm/s for retinol, 49.5×10⁻⁶ cm/s for haloperidol, 62.4×10⁻⁶ cm/s for caffeine, and 1.8×10⁻⁶ cm/s for mannitol) show a good correlation to data which are already known from in vivo-experiments. As judged by the sucrose permeability our blood–brain barrier model is less permeable than numerous other models published so far. Therefore it represents a powerful tool for in vitro-prediction of blood–brain barrier permeability of drugs and offers the possibility to scan a large quantity of drugs for their potential to enter the brain.     

去骨瓣减压术对大鼠局灶性脑缺血后脑水肿、血脑屏障、脑血流变化的影响

目的:观察去骨瓣减压术对局灶性脑缺血大鼠脑水肿、血脑屏障(BBB)及脑血流(CBF)的影响,探讨该手术对缺血性脑损害的保护机制。方法:改良Koizumi′s法制作脑缺血大鼠动物模型,脑缺血后6h行去骨瓣减压术,分别在术后3和7d观察脑水肿和血脑屏障的变化,另持续观察缺血1h 去骨瓣减压2h的CBF变化。结果:去骨瓣减压术后半暗区CBF增加更加持久和有效,在术后第3天时BBB破坏范围明显缩小,术后第7天时脑水含量明显降低。结论:去骨瓣减压术对局灶性脑缺血的保护作用可能与增加半暗区CBF、改善BBB破坏和减轻脑水肿有关。