Basic Fibroblast Growth Factor‐Releasing Bioabsorbable Polyglycolic Acid Dura Mater Enhances Neural Progenitor Cell Proliferation and Neuroprotection After Brain Injury

Increased endothelial caveolae leading to transcytosis of plasma proteins is associated with blood-brain barrier (BBB) breakdown and cerebral oedema in brain injury. Increased expression of caveolin-1alpha (Cav-1), an integral caveolar membrane protein, was reported in endothelium of arterioles and veins with BBB breakdown to fibronectin post injury. In this study the phosphorylation state of Cav-1 and its association with BBB breakdown was determined in the rat cortical cold injury model over a period of days 0.5-6 post lesion. Expression of phosphorylated Cav-1 was determined by immunoblotting and dual labelling immunofluorescence for phosphorylated caveolin-1 and fibronectin, a marker of BBB breakdown. A phospho-specific monoclonal antibody that selectively recognizes only tyrosine 14-phosphorylated Cav-1 (PY14Cav-1) was used. Immunoblots showed constitutive expression of PY14Cav-1 in cortex of control rats and a significant increase in PY14Cav-1 expression at the lesion site up to day 4 post lesion. PY14Cav-1 immunostaining was observed in the endothelium of lesion vessels at days 0.5-4 post lesion, in neutrophils at days 0.5 and 2 and in macrophages at day 6 post lesion. Dual labelling showed that 100% of vessels with BBB breakdown to fibronectin showed endothelial PY14Cav-1 on day 0.5, the percentage decreasing to 62% on day 4. On day 6, none of the vessels showed endothelial phosphorylated Cav-1. The presence of phosphorylated Cav-1 in endothelium of vessels showing BBB breakdown suggests that phosphorylated Cav-1 signalling may be one of the factors associated with early BBB breakdown and brain oedema in brain injury.

The present study characterized whether inflammatory leukocytic infiltration is temporally and regionally correlated with neuronal degeneration and/or blood brain barrier (BBB) breakdown resulting from traumatic brain injury. Adult rats were sacrificed at 5 min, 2, 4, 12, 24, and 72 hr after lateral fluid percussion brain injury. BBB breakdown, neuronal degeneration and leukocyte infiltration were assessed using immunocytochemistry, silver impregnation and toluidine blue and eosin staining. BBB breakdown and neuronal degeneration occurred concomitantly in injured cortex, hippocampus, and along the dorsolateral quadrant of the diencephalon. However, neuronal degeneration within deep diencephalic structures transpired in the absence of IgG extravasation. Neutrophils were observed only in regions exhibiting BBB damage and were first apparent in injured cortex and hippocampus between 2-12 hr posttrauma lining the vasculature and filling subarachnoid/subdural spaces. Neutrophils then migrated from damaged vasculature into traumatized cortical and hippocampal parenchyma by 24 hr after lateral fluid percussion injury. Macrophages were also observed within cortical parenchyma at 24 hr and completely filled the cortical lesion site by 72 hr after injury. Macrophages were not as abundant throughout hippocampal parenchyma and were found only in hippocampal regions exhibiting focal hemorrhage at 72 hr. Finally, neutrophils did not migrate to deep diencephalic structures that showed no BBB damage despite extensive neuronal degeneration. Indeed, lateral fluid percussion elicits inflammatory leukocytic recruitment only in regions experiencing concomitant BBB damage and neuronal degeneration. In summary, inflammatory leukocytic recruitment and diffuse neuronal degeneration are separate pathological processes resulting from traumatic brain injury.

Our previous study demonstrated that vascular endothelial growth factor (VEGF), now referred to as VEGF-A, plays a significant role in blood-brain barrier (BBB) breakdown and angiogenesis after brain injury. In this study, VEGF-A expression was compared with that of VEGF-B in the rat cortical cold injury model over a period of 6 hours to 6 days post-injury. VEGF-A and VEGF-B mRNA were detected by in situ hybridization and their protein was detected by immunohistochemistry. The presence of VEGF-A and VEGF-B proteins in endothelium of lesion vessels was related to BBB breakdown by double labeling for either of these growth factors and fibronectin, which was used as a marker of BBB breakdown. Significant induction of both VEGF-A and VEGF-B mRNA occurred at the lesion site during the period of maximal endothelial proliferation. VEGF-A mRNA levels peaked at 3 and 4 days post-injury and returned to basal expression by day 6, while VEGF-B mRNA levels remained elevated up to day 6. VEGF-B protein was constitutively expressed in endothelium of all cerebral vessels. After brain injury, there was increased immunoreactivity for VEGF-B at the lesion site, this protein being present in the endothelium and vascular smooth muscle cells of pial vessels, in inflammatory cells, and later in proliferating endothelial cells, endothelium of neovessels, and astrocytes. Lesion vessels showing BBB breakdown to fibronectin showed endothelial VEGF-A protein but not VEGF-B protein. Constitutive expression of VEGF-B in normal endothelium suggests that it may have a role in maintenance of the BBB in steady states, while its induction at both the gene and protein level post-injury indicates that it has an essential role in angiogenesis and the repair processes after brain injury.

The Protective Mechanisms of Intravenous Immunoglobulin (IVIg) in Ischaemic Stroke

Glial activation is associated with cell proliferation and upregulation of astrocyte marker expression following traumatic injury in the brain. However, the biological significance of these processes remains unclear. In the present study, astrocyte activation was investigated in a murine brain injury model. Brain injury induces blood-brain barrier (BBB) breakdown and immunoglobulin G (IgG) leak into the brain parenchyma. The recovery of BBB breakdown was evaluated by analyzing immunofluorescent staining with mouse IgG antibody. IgG leakage was greatest at 1 day after stab wound injury and decreased thereafter, and almost diminished after 7 days. Bromodeoxy uridine incorporation was used, and astrocyte proliferation rates were examined by coimmunostaining with anti-bromodeoxy uridine and anti-glial fibrillary acid protein antibodies. Consistent with IgG leakage assays, astrocyte activation was the highest at day 3 and decreased after 7 days. Moreover, in reverse transcriptase-quantitative-PCR experiments, genes associated with BBB integrity were downregulated immediately after BBB breakdown and recovered to basal expression levels within 7 days. These data indicated that astrocyte activation correlated with BBB recovery from breakdown following brain injury.

Obesity and insulin resistance elicit blood-brain barrier (BBB) breakdown in humans and animal models, but the relative contributions of the two pathologies remain poorly understood. These studies initially addressed the temporal progression of cerebrovascular dysfunction relative to dietary obesity or diet-induced insulin resistance in male mice. Obesity increased BBB permeability to the low molecular weight fluorophore sodium fluorescein (NaFl), whereas diet-induced insulin resistance increased permeability to both NaFl and Evans blue, which forms a high molecular weight complex with serum albumin. Serial section transmission electron microscopy analysis of hippocampal capillaries revealed that diabetes promotes involution of tight junctions, fenestration of endothelial cells, and pericyte regression. Chronic activation of adenosine receptor 2a (Adora2a) erodes tight junctions between endothelial cells of the cerebral vasculature in other models of chronic neuropathology, and we observed that acute Adora2a antagonism normalized BBB permeability in wild-type mice with diet-induced insulin resistance. Experiments in mice with inducible deletion of Adora2a in endothelial cells revealed protection against BBB breakdown with diet-induced insulin resistance, despite comparable metabolic dysfunction relative to nontransgenic littermates. Protection against BBB breakdown was associated with decreased vascular inflammation, recovery of hippocampal synaptic plasticity, and restoration of hippocampus-dependent memory. These findings indicate that Adora2a-mediated signaling in vascular endothelial cells disrupts the BBB in dietary obesity, and implicate cerebrovascular dysfunction as the underlying mechanism for deficits in synaptic plasticity and cognition with obesity and insulin resistance.SIGNIFICANCE STATEMENT The blood-brain barrier (BBB) restricts the entry of circulating factors into the brain, but obesity promotes BBB breakdown in humans and animal models. We used transgenic mice with resistance to BBB breakdown to investigate the role of neurovascular dysfunction in high-fat diet (HFD)-induced cognitive impairment. Transgenic mice with inducible ablation of Adora2a in endothelial cells were protected against BBB breakdown on HFD, despite comparable metabolic impairments relative to normal mice. Transgenic mice were also resistant to HFD-induced cognitive dysfunction and were protected against deficits in hippocampal synaptic plasticity. These findings indicate that Adora2a-mediated signaling in endothelial cells mediates obesity-induced BBB breakdown, and implicate cerebrovascular dysfunction as the mechanism for deficits in synaptic plasticity and cognition with obesity and diabetes.

The time course of blood-brain barrier (BBB) breakdown after traumatic brain injury (TBI) has important implications for therapy. This study was conducted in order to test post-traumatic BBB dysfunction in a model of fluid-percussion induced TBI in rabbits at 1 and 6 hours after TBI and relate it to white blood cell (WBC) activation. Ten anesthetized rabbits had chronic cranial windows implanted three weeks prior to experimentation. Fluid-percussion injury (3.5 atm.) was induced and animals were followed for 1 or 6 h. Intravital fluorescence videomicroscopy was used to assess BBB permeability and WBC adhesion to pial venules. Na(+)-fluorescein was infused continuously over 30 min at either 30 min (Group I, n = 5) or 5.5 h (Group II, n = 5) after TBI. Microvascular permeability in individual postcapillary venules was assessed qualitatively at 1 and 30 min after start of infusion. TBI led to a transient mean arterial blood pressure (MAP) surge after trauma and a progressive increase in the number of sticking WBCs per mm2 vessel wall. Na(+)-fluorescein extravasation was observed in 4 out of 5 Group I animals and in none of Group II. BBB breakdown was not associated with WBC sticking. We conclude that after fluid-percussion injury the BBB is damaged at 1 h post-trauma and that its function is restored 6 h later. Increased WBC sticking at 6 h is not associated with BBB breakdown. Whether WBCs may cause vascular permeability changes at a later point needs further investigation.

Pericytes, mural cells of brain capillaries, maintain the blood-brain barrier (BBB), regulate cerebral blood flow (CBF), and protect neurons against ischemic damage. To further investigate the role of pericytes in ischemia, we induced stroke by 45-min transient middle cerebral artery occlusion (tMCAo) in 6-month-old pericyte-deficient Pdgfrb + ⁣/− mice and control Pdgfrb+/+ littermates. Compared to controls, Pdgfrb + ⁣/− mice showed a 26% greater loss of CBF during early reperfusion, and 40–50% increase in the infarct and edema volumes and motor neurological score 24 h after tMCAo. These changes were accompanied by 50% increase in both immunoglobulin G and fibrinogen pericapillary deposits in the ischemic cortex 8 h after tMCAo indicating an accelerated BBB breakdown, and 35 and 55% greater losses of pericyte coverage and number of degenerating neurons 24 h after tMCAo, respectively. Treatment of Pdgfrb + ⁣/− mice with 3K3A-activated protein C (APC), a cell-signaling analog of plasma protease APC, administered intravenously 10 min and 4 h after tMCAo normalized CBF during the early reperfusion phase and reduced infarct and edema volume and motor neurological score by 55–60%, with similar reductions in BBB breakdown and number of degenerating neurons. Our data suggest that pericyte deficiency results in greater brain injury, BBB breakdown, and neuronal degeneration in stroked mice and that 3K3A-APC protects the brain from accelerated injury caused by pericyte deficiency. These findings may have implications for treatment of ischemic brain injury in neurological conditions associated with pericyte loss such as those seen during normal aging and in neurodegenerative disorders such as Alzheimer’s disease.

Blood brain barrier (BBB) breakdown and neuroinflammation are key events in ischemic stroke morbidity and mortality. The present study investigated the effects of mast cell deficiency and stabilization on BBB breakdown and neutrophil infiltration in mice after transient middle cerebral artery occlusion (tMCAo). Adult male C57BL6/J wild type (WT) and mast cell-deficient (C57BL6/J Kit(Wsh/Wsh) (Wsh)) mice underwent tMCAo and BBB breakdown, brain edema and neutrophil infiltration were examined after 4 hours of reperfusion. Blood brain barrier breakdown, brain edema, and neutrophil infiltration were significantly reduced in Wsh versus WT mice (P<0.05). These results were reproduced pharmacologically using mast cell stabilizer, cromoglycate. Wild-type mice administered cromoglycate intraventricularly exhibited reduced BBB breakdown, brain edema, and neutrophil infiltration versus vehicle (P<0.05). There was no effect of cromoglycate versus vehicle in Wsh mice, validating specificity of cromoglycate on brain mast cells. Proteomic analysis in Wsh versus WT indicated that effects may be via expression of endoglin, endothelin-1, and matrix metalloproteinase-9. Using an in vivo model of mast cell deficiency, this is the first study showing that mast cells promote BBB breakdown in focal ischemia in mice, and opens up future opportunities for using mice to identify specific mechanisms of mast cell-related BBB injury.

The aim of the present study was to determine the potential therapeutic value of the lazaroid U-83836E on blood brain barrier (BBB) breakdown and edema with respect to the changes in the synaptosomal Na+/K+ and Mg(2+)/Ca(2+)-adenosinetriphosphatase (ATPase) activities, tissue malondialdehyde levels and the neuronal viability in the rat brain subjected to cerebral trauma. Traumatic brain injury (TBI) was introduced by applying a 75 gm. cm force to the right parietal cortex using the weight-drop method. The first set of animals was used for determining time course changes of the synaptosomal Na+/K+ and Mg(2+)/Ca(2+)-ATPase and the malondialdehyde levels and were sacrificed 2, 6 and 24h after lesion production. A group of the animals was treated with U-83836E proir to TBI and sacrificed 24h after cerebral injury. A second set of animals was used for evaluating the alterations in BBB disruption and tissue water content and were sacrificed 2, 6 and 24h after lesion production. Two groups of animals were treated with U-83836E and sacrificed after 2 and 24h following TBI. U-83836E was given intraperitoneally thirty minutes before trauma at a dose of 10 mg/kg. Neuronal necrosis was also evaluated in the groups of U-83836E and physiological saline-treated animals. Extravasation of Evans blue into the traumatized hemisphere was maximum at 2h (p<0.001) and returned close to the control levels at 24h after TBI (p>0.05). Edema had developed progressively over time and reached the maximum degree of 2.1% (p<0.001) at 24h. U-83836E showed no effect on the BBB breakdown and the tissue water content at 2h and still had no effect on the BBB breakdown after 24h following the trauma (p>0.05), although it reduced edema after 24h (p<0.01). The losses of Na+/K+ and Mg(2+)/Ca(2+)-ATPase activities were found as 39.5% (p<0.001) and 29.4% (p<0.01) of the control value, respectively, and remained at the decreased levels throughout the experiment. Malondialdehyde level continued to increase over time reaching up to 209% (p<0.001) of the control value 24h after TBI. Both ATPase activities were improved to near control values (p>.05) by the effect of U-83836E. U-83836E inhibited the increase of lipid peroxidation (p<0.001) and also salvaged neuronal necrosis (p<0.05). U-83836E given prophylactically after cerebral trauma appears to reduce edema, possibly by inhibiting increases in lipid peroxidation and by stabilizing ATPase. Further studies are recommended to verify the similar effects of the brain penetrating lazaroids when they are given after trauma.

Stroke induces blood-brain barrier (BBB) breakdown, which promotes complications like oedema and hemorrhagic transformation. Administration of recombinant tissue plasminogen activator (rtPA) within a therapeutic time window of 4.5h after stroke onset constitutes the only existing treatment. Beyond this time window, rtPA worsens BBB breakdown. Canonical Wnt pathway induces BBB formation and maturation during ontogeny. We hypothesized that the pathway is required to maintain BBB functions after stroke; thus, its activation might improve rtPA therapy. Therefore, we first assessed pathway activity in the brain of mice subjected to transient middle cerebral artery occlusion (MCAo). Next, we evaluated the effect of pathway deactivation early after stroke onset on BBB functions. Finally, we assessed the impact of pathway activation on BBB breakdown associated to delayed administration of rtPA. Our results show that pathway activity is induced predominately in endothelial cells early after ischemic stroke. Early deactivation of the pathway using a potent inhibitor, XAV939, aggravates BBB breakdown and increases hemorrhagic transformation incidence. On the other hand, pathway activation using a potent activator, 6-bromoindirubin-3'-oxime (6-BIO), reduces the incidence of hemorrhagic transformation associated to delayed rtPA administration by attenuating BBB breakdown via promotion of tight junction formation and repressing endothelial basal permeability independently of rtPA proteolytic activity. BBB preservation upon pathway activation limited the deleterious effects of delayed rtPA administration. Our study demonstrates that activation of the canonical Wnt pathway constitutes a clinically relevant strategy to extend the therapeutic time window of rtPA by attenuating BBB breakdown via regulation of BBB-specific mechanisms.

Plasminogen and stroke: more is better

Blood-Brain Barrier Breakdown and Blood-Brain Communication in Neurological and Psychiatric Diseases

The blood brain barrier (BBB), unique to the central nervous system (CNS) vessels, tightly controls the passage of molecules between the blood and the brain. BBB breakdown is documented in many pathological conditions including, stroke, and contributes to the severity of disease and poor outcome. Currently, there is no efficient treatment strategy to prevent BBB breakdown or restore disrupted BBB . Despite BBB’s importance in the CNS function, t he genetic program and mechanism, that regulates BBB formation and maintenance is poorly characterized. To understand the molecular features underlying BBB gene regulation during development we utilize endothelial cell (EC) cultures derived from brain of both embryonic (E-13.5) and adult mice and investigated differences in BBB gene expression and epigenetic status using real time PCR analysis and ChIP-qPCR. We found that a distinct BBB profile exist in the CNS ECs during BBB formation and maintenance . When compared to embryonic ECs, adult ECs showed a significant down regulation in expression of tight junction (TJ) genes such as CLDN-1 , CLDN -11 , ZO-1 and OCLN . Conversely, another important TJ gene CLDN- 5 shows a significant increase in adult ECs. Further, we found that epigenetic-histone modifications are involved in the repression in the TJ gene in adult ECs. To more fully understand the role of Wnt/β-catenin in BBB formation and maintenance we block the downstream of the Wnt/β-catenin signaling pathway using LF3 (block the interaction of β-catenin and transcription factor TCF4) in developing ECs. This results in a adult BBB phenotype in developing ECs indicating, silencing of Wnt/β-catenin pathway is required for BBB maturation . Further, epigenetic component HDAC2 was increased significantly in developing ECs when Wnt/β-catenin signaling is blocked demonstrating this pathway can modulate the epigenetics of CNS ECs . In summary, we describe that BBB genes have distinguished expression pattern during BBB formation and maintenance, guided by the Wnt/β-catenin pathway through epigenetic modifications. Understanding the fundamental epigenetic and regulatory mechanisms of BBB formation and maintenance is critical for developing therapeutic interventions against BBB breakdown.

Historically, the blood-brain barrier (BBB) was considered to be at the level of cerebral endothelium. Currently, the interaction of endothelium with other components of the vessel wall and with neurones and glial cells is considered to constitute a functional unit, termed the neurovascular unit that maintains cerebral homeostasis in steady states and brain injury. The emphasis of this review is on cerebral endothelium, the best-studied component of the neurovascular unit, and its permeability mechanisms in health and acute brain injury. Major advances have been made in unravelling the molecular structure of caveolae and tight junctions, both of which are components of the structural barrier to the entry of plasma proteins into brain. Time course studies suggest that caveolar changes precede junctional changes in acute brain injury. Additional factors modulating BBB permeability in acute brain injury are matrix metalloproteinases-2 and 9 and angiogenic factors, the most notable being vascular endothelial growth factor-A and angiopoietins (Ang) 1 and 2. Vascular endothelial growth factor-A and Ang2 have emerged as potent inducers of BBB breakdown while Ang1 is a potent anti-leakage factor. These factors have the potential to modulate permeability in acute brain injury and this is an area of ongoing research. Overall, a combination of haemodynamic, structural and molecular alterations affecting brain endothelium results in BBB breakdown in acute brain injury.