The effect of cardiac arrest on the permeability of the mouse blood-brain and blood-spinal cord barrier to pituitary adenylate cyclase activating polypeptide (PACAP)

Time-dependent changes in peptide transport system (PTS-6), which transports the 38 amino acid pituitary adenylate cyclase activating polypeptide (PACAP) across the blood-brain barrier (BBB) and blood-spinal cord barrier (BSCB), were studied in mice in a cardiac arrest model. The permeability of the BSCB to radioactivity labeled I131 showed a reversible increase on Day 2-(24 h) after cardiac arrest. The BBB showed no such increase. The increase in BSCB permeability was primarily located within the thoracic region of the spinal cord. We conclude that the ischemia occurring with cardiac arrest results in a transient increase in PTS-6 activity located primarily in the thoracic region of the spinal cord.     

Evidence of compromised blood-spinal cord barrier in early and late symptomatic SOD1 mice modeling ALS

Background

The blood-brain barrier (BBB), blood-spinal cord barrier (BSCB), and blood-cerebrospinal fluid barrier (BCSFB) control cerebral/spinal cord homeostasis by selective transport of molecules and cells from the systemic compartment. In the spinal cord and brain of both ALS patients and animal models, infiltration of T-cell lymphocytes, monocyte-derived macrophages and dendritic cells, and IgG deposits have been observed that may have a critical role in motor neuron damage. Additionally, increased levels of albumin and IgG have been found in the cerebrospinal fluid in ALS patients. These findings suggest altered barrier permeability in ALS. Recently, we showed disruption of the BBB and BSCB in areas of motor neuron degeneration in the brain and spinal cord in G93A SOD1 mice modeling ALS at both early and late stages of disease using electron microscopy. Examination of capillary ultrastructure revealed endothelial cell degeneration, which, along with astrocyte alteration, compromised the BBB and BSCB. However, the effect of these alterations upon barrier function in ALS is still unclear. The aim of this study was to determine the functional competence of the BSCB in G93A mice at different stages of disease.

Methodology/Principal Findings

Evans Blue (EB) dye was intravenously injected into ALS mice at early or late stage disease. Vascular leakage and the condition of basement membranes, endothelial cells, and astrocytes were investigated in cervical and lumbar spinal cords using immunohistochemistry. Results showed EB leakage in spinal cord microvessels from all G93A mice, indicating dysfunction in endothelia and basement membranes and confirming our previous ultrastructural findings on BSCB disruption. Additionally, downregulation of Glut-1 and CD146 expressions in the endothelial cells of the BSCB were found which may relate to vascular leakage.

Conclusions/Significance

Results suggest that the BSCB is compromised in areas of motor neuron degeneration in ALS mice at both early and late stages of the disease.     

Retinoic Acid Prevents Disruption of Blood-Spinal Cord Barrier by Inducing Autophagic Flux After Spinal Cord Injury

Spinal cord injury (SCI) induces the disruption of the blood-spinal cord barrier (BSCB), which leads to infiltration of blood cells, inflammatory responses and neuronal cell death, with subsequent development of spinal cord secondary damage. Recent reports pointed to an important role of retinoic acid (RA), the active metabolite of the vitamin A, in the induction of the blood–brain barrier (BBB) during human and mouse development, however, it is unknown whether RA plays a role in maintaining BSCB integrity under the pathological conditions such as SCI. In this study, we investigated the BSCB protective role of RA both in vivo and in vitro and demonstrated that autophagy are involved in the BSCB protective effect of RA. Our data show that RA attenuated BSCB permeability and also attenuated the loss of tight junction molecules such as P120, β-catenin, Occludin and Claudin5 after injury in vivo as well as in brain microvascular endothelial cells. In addition, RA administration improved functional recovery of the rat model of trauma. We also found that RA could significantly increase the expression of LC3-II and decrease the expression of p62 both in vivo and in vitro. Furthermore, combining RA with the autophagy inhibitor chloroquine (CQ) partially abolished its protective effect on the BSCB and exacerbated the loss of tight junctions. Together, our studies indicate that RA improved functional recovery in part by the prevention of BSCB disruption via the activation of autophagic flux after SCI.     

The angiopoietin1–Akt pathway regulates barrier function of the cultured spinal cord microvascular endothelial cells through Eps8

In mammalian central nervous system (CNS), the integrity of the blood–spinal cord barrier (BSCB), formed by tight junctions (TJs) between adjacent microvascular endothelial cells near the basement membrane of capillaries and the accessory structures, is important for relatively independent activities of the cellular constituents inside the spinal cord. The barrier function of the BSCB are tightly regulated and coordinated by a variety of physiological or pathological factors, similar with but not quite the same as its counterpart, the blood–brain barrier (BBB). Herein, angiopoietin 1 (Ang1), an identified ligand of the endothelium-specific tyrosine kinase receptor Tie-2, was verified to regulate barrier functions, including permeability, junction protein interactions and F-actin organization, in cultured spinal cord microvascular endothelial cells (SCMEC) of rat through the activity of Akt. Besides, these roles of Ang1 in the BSCB in vitro were found to be accompanied with an increasing expression of epidermal growth factor receptor pathway substrate 8 (Eps8), an F-actin bundling protein. Furthermore, the silencing of Eps8 by lentiviral shRNA resulted in an antagonistic effect vs. Ang1 on the endothelial barrier function of SCMEC. In summary, the Ang1–Akt pathway serves as a regulator in the barrier function modulation of SCMEC via the actin-binding protein Eps8.     

Permeation of blood-borne IL15 across the blood-brain barrier and the effect of LPS

Interleukin15 (IL 15) is a proinflammatory cytokine with elevated concentrations in autoimmune diseases involving the periphery (e.g. rheumatoid arthritis) and CNS (e.g. multiple sclerosis). Its interactions with the blood-brain barrier (BBB) were studied in normal and lipopolysaccharide (LPS)-treated mice. ¹²⁵I-IL15 remained intact for at least 10 min after i.v. injection and reached CNS parenchyma with regional differences between brain and spinal cord. Both in vivo and in situ brain perfusion of ¹²⁵I-IL15 showed that its permeation of the BBB was non-saturable. LPS induced a significant increase of IL15 uptake by the brain and spinal cord, partly related to a higher general permeability of the BBB. The results suggest that the BBB is an interface for blood-borne IL15 to interact with the CNS in the basal state and during inflammation.     

Upregulation of the transport system for TNFalpha at the blood-brain barrier

The transport system for the cytokine tumor necrosis factor-alpha (TNFalpha) at the blood-brain barrier (BBB) enables an enhanced yet saturable entry of TNFalpha from blood to the CNS. This review focuses on the selective upregulation of the transport system for TNFalpha at the BBB that is specific for type of pathology, region, and time. The upregulation is reflected by increased CNS tissue uptake of radiolabeled TNFalpha after iv injection in mice and by inhibition of this increase with excess non-radiolabeled TNFalpha. (1) Spinal cord injury (SCI): upregulation of TNFalpha uptake after thoracic transection is seen in the delayed phase of BBB disruption at the lumbar spinal cord. Thoracic SCI by compression, however, has a longer lasting impact on TNFalpha transport that involves thoracic and lumbar spinal cord, in contrast to the upregulation confined to the lumbar region in lumbar SCI by compression. Regardless, the uptake of TNFalpha by spinal cord does not parallel BBB disruption as measured by the leakage of radiolabeled albumin. (2) Experimental autoimmune encephalomyelitis (EAE): the increase in the differential permeability to TNFalpha is seen in all CNS regions (brain and cervical, thoracic, and lumbar spinal cord) and has a distinct time course and reversibility. Exogenous TNFalpha has biphasic effects in modulating functional scores. The BBB, a dynamically regulated barrier, is actively involved in disease processes.     

Effects of acute hyperosmolality on blood-brain barrier function in ovine fetuses and lambs

We examined the effects of hyperosmolality on blood-brain barrier (BBB) permeability during development to test the vulnerability of the immature barrier to stress. The BBB response to hyperosmolality was quantified using the blood-to-brain transfer constant (Ki) with alpha-aminoisobutyric acid in fetuses at 60% and 90% gestation, premature, newborn, and older lambs. Ki plotted against osmolality increased as a function of increases in osmolality in all groups and brain regions. The relationship was described (P < 0.05) by a segmented regression model. At lower osmolalities, changes in Ki were minimal, but after a break point (threshold) was reached, the increase (P < 0.05) was linear. We examined the responses of Ki to hyperosmolality within each brain region by comparing the thresholds and slopes of the second regression segment. Lower thresholds and higher slopes imply greater vulnerability to hyperosmolality in the younger groups. Thresholds increased (P < 0.05) with development in the thalamus, superior colliculus, pons, and spinal cord, and slopes of the second regression segment decreased (P < 0.05) in the cerebellum, hippocampus, inferior colliculus, medulla, and spinal cord. BBB resistance to hyperosmolality increased (P < 0.05) with development in most brain regions. The pattern of the Ki plotted against osmolality was (P < 0.05) heterogenous among brain regions in fetuses and premature and newborn lambs, but not in older lambs. We conclude that 1) BBB permeability increased as a function of changes in osmolality, 2) the barrier becomes more resistant to hyperosmolality during development, and 3) the permeability response to hyperosmolality is heterogenous among brain regions in fetuses and premature and newborn lambs.     

Retinoic Acid Induced-Autophagic Flux Inhibits ER-Stress Dependent Apoptosis and Prevents Disruption of Blood-Spinal Cord Barrier after Spinal Cord Injury

Spinal cord injury (SCI) induces the disruption of the blood-spinal cord barrier (BSCB) which leads to infiltration of blood cells, an inflammatory response, and neuronal cell death, resulting spinal cord secondary damage. Retinoic acid (RA) has a neuroprotective effect in both ischemic brain injury and SCI, however the relationship between BSCB disruption and RA in SCI is still unclear. In this study, we demonstrated that autophagy and ER stress are involved in the protective effect of RA on the BSCB. RA attenuated BSCB permeability and decreased the loss of tight junction (TJ) molecules such as P120, β-catenin, Occludin and Claudin5 after injury in vivo as well as in Brain Microvascular Endothelial Cells (BMECs). Moreover, RA administration improved functional recovery in the rat model of SCI. RA inhibited the expression of CHOP and caspase-12 by induction of autophagic flux. However, RA had no significant effect on protein expression of GRP78 and PDI. Furthermore, combining RA with the autophagy inhibitor chloroquine (CQ) partially abolished its protective effect on the BSCB via exacerbated ER stress and subsequent loss of tight junctions. Taken together, the neuroprotective role of RA in recovery from SCI is related to prevention of of BSCB disruption via the activation of autophagic flux and the inhibition of ER stress-induced cell apoptosis. These findings lay the groundwork for future translational studies of RA for CNS diseases, especially those related to BSCB disruption.     

NK1 Receptor Blockade Is Ineffective in Improving Outcome following a Balloon Compression Model of Spinal Cord Injury

The neuropeptide substance P (SP) is a well-known mediator of neurogenic inflammation following a variety of CNS disorders. Indeed, inhibition of SP through antagonism of its receptor, the tachykinin NK1 receptor, has been shown to be beneficial following both traumatic brain injury and stroke. Such studies demonstrated that administration of an NK1 receptor antagonist reduced blood-brain-barrier permeability, edema development and improved functional outcome. Furthermore, our recent studies have demonstrated a potential role for SP in mediating neurogenic inflammation following traumatic spinal cord injury (SCI). Accordingly, the present study investigates whether inhibition of SP may similarly play a neuroprotective role following traumatic SCI. A closed balloon compression injury was induced at T10 in New Zealand White rabbits. At 30 minutes post-injury an NK1 receptor antagonist was administered intravenously. Animals were thereafter assessed for blood spinal cord barrier (BSCB) permeability, spinal water content (edema), intrathecal pressure (ITP), and histological and functional outcome from 5 hours to 2 weeks post-SCI. Administration of an NK1 receptor antagonist was not effective in reducing BSCB permeability, edema, ITP, or functional deficits following SCI. We conclude that SP mediated neurogenic inflammation does not seem to play a major role in BSCB disruption, edema development and consequential tissue damage seen in acute traumatic SCI. Rather it is likely that the severe primary insult and subsequent hemorrhage may be the key contributing factors to ongoing SCI injury.     

Epidermal growth factor attenuates blood‐spinal cord barrier disruption via PI3K/Akt/Rac1 pathway after acute spinal cord injury

After spinal cord injury (SCI), disruption of blood–spinal cord barrier (BSCB) elicits blood cell infiltration such as neutrophils and macrophages, contributing to permanent neurological disability. Previous studies show that epidermal growth factor (EGF) produces potent neuroprotective effects in SCI models. However, little is known that whether EGF contributes to the integrity of BSCB. The present study is performed to explore the mechanism of BSCB permeability changes which are induced by EGF treatment after SCI in rats. In this study, we demonstrate that EGF administration inhibits the disruption of BSCB permeability and improves the locomotor activity in SCI model rats. Inhibition of the PI3K/Akt pathways by a specific inhibitor, LY294002, suppresses EGF‐induced Rac1 activation as well as tight junction (TJ) and adherens junction (AJ) expression. Furthermore, the protective effect of EGF on BSCB is related to the activation of Rac1 both in vivo and in vitro. Blockade of Rac1 activation with Rac1 siRNA downregulates EGF‐induced TJ and AJ proteins expression in endothelial cells. Taken together, our results indicate that EGF treatment preserves BSCB integrity and improves functional recovery after SCI via PI3K‐Akt‐Rac1 signalling pathway.     

Lithium chloride contributes to blood–spinal cord barrier integrity and functional recovery from spinal cord injury by stimulating autophagic flux

Blood–spinal cord barrier (BSCB) disruption following spinal cord injury (SCI) significantly compromises functional neuronal recovery. Autophagy is a potential therapeutic target when seeking to protect the BSCB. We explored the effects of lithium chloride (LiCl) on BSCB permeability and autophagy-induced SCI both in a rat model of SCI and in endothelial cells subjected to oxygen–glucose deprivation. We evaluated BSCB status using the Evans Blue dye extravasation test and measurement of tight junction (TJ) protein levels; we also assessed functional locomotor recovery. We detected autophagy-associated proteins in vivo and in vitro using both Western blotting and immunofluorescence staining. We found that, in a rat model of SCI, LiCl attenuated the elevation in BSCB permeability, improved locomotor recovery, and inhibited the degradation of TJ proteins including occludin and claudin-5. LiCl significantly induced the extent of autophagic flux after SCI by increasing LC3-II and ATG-5 levels, and abolishing p62 accumulation. In addition, a combination of LiCl and the autophagy inhibitor chloroquine not only partially eliminated the BSCB-protective effect of LiCl, but also exacerbated TJ protein degradation both in vivo and in vitro. Together, these findings suggest that LiCl treatment alleviates BSCB disruption and promotes locomotor recovery after SCI, partly by stimulating autophagic flux.     

Neuregulin-1-beta1 enters brain and spinal cord by receptor-mediated transport

Proteins of the neuregulin (NRG) family play important regulatory roles in neuronal survival and synaptic activity. NRG-1-beta1 has particular potential as a therapeutic agent because it enhances myelination of neurites in spinal cord explants. In this study, we determined the permeation of NRG-1-beta1 across the blood-brain and blood-spinal cord barriers (BBB and BSCB respectively). Intact radioactively labeled NRG-1-beta1 had a saturable and relatively rapid influx rate from blood to the CNS in mice. Capillary depletion studies showed that NRG-1-beta1 entered the parenchyma of the brain and spinal cord rather than being trapped in the capillaries that compose the BBB. The possible mechanism of receptor-mediated transport was shown by the ability of antibodies to erbB3 and erbB4 receptors to inhibit the influx. Lipophilicity, less important for such saturable transport mechanisms, was measured by the octanol : buffer partition coefficient and found to be low. The results indicate that NRG-1-beta1 enters spinal cord and brain by a saturable receptor-mediated mechanism, which provides the opportunity for possible therapeutic manipulation at the BBB level.     

Diabetes-Related Alteration of Occludin Expression in Rat Blood–Spinal Cord Barrier

Occludin is an essential component of tight junctions, which are involved in controlling the integrity of the blood–brain barrier and blood–spinal cord barrier (BSCB). Diabetes-induced alteration of occludin in rat BSCB and the relationship between occludin level and disease course was examined. Diabetes was induced using streptozotocin. Occludin rat spinal cord mRNA levels were assessed by real-time quantitative RT-PCR. Protein levels were examined by western blot. Occludin expression in 1-month diabetic rats was significantly reduced compared to the controls (0.20 ± 0.01 vs 1.00 ± 0.01, respectively; P < 0.05). Expression was also significantly lower in the 3-month diabetic group (0.06 ± 0.02; P < 0.01). Occludin protein levels of 1-month (0.53 ± 0.01) and 3-month (0.31 ± 0.01) diabetic rats were also significantly reduced compared to controls (0.91 ± 0.06; P < 0.01 for both). Diabetes decreased BSCB occludin expression at the mRNA and protein level. This down-regulation appears to correlate with the course of the disease, and may be a causal factor of diabetes-induced increase of BSCB permeability.     

A new antioxidant compound H-290/51 modulates glutamate and GABA immunoreactivity in the rat spinal cord following trauma

Summary.  The involvement of the excitatory amino acid glutamate and the inhibitory amino acid gamma-amino butyric acid (GABA) in the pathophysiology of spinal cord injury is not known in details. This investigation is focused on the role of glutamate and GABA in a rat model of spinal cord trauma using immunohistochemistry. Spinal cord injury produced by a longitudinal incision of the right dorsal horn of the T10–11 segments resulted in profound edema and cell damage in the adjacent T9 segment at 5 h. Pretreatment with H-290/51 (50 mg/kg, p.o.), a potent antioxidant compound, effectively reduced the blood-spinal cord barrier (BSCB) permeability, edema formation and cell injury following trauma. At this time, untreated traumatised rats exhibited a marked increase in glutamate immunoreactivity along with a distinct decrease in GABA immunostaining in the T9 segment. These changes in glutamate and GABA immunoreactivity in traumatised rats were considerably attenuated by pretreatment with H-290/51. These results suggest that (i) oxidative stress contributes to alterations in glutamate and GABA in spinal cord injury, (ii) glutamate and GABA are important factors in the breakdown of the BSCB, edema formation and cell changes, and (iii) the antioxidant compound H-290/51 has a potential therapeutic value in the treatment of spinal cord injuries. Received July 3, 2001 Accepted August 6, 2001 Published online July 31, 2002     

Metformin ameliorates BSCB disruption by inhibiting neutrophil infiltration and MMP‐9 expression but not direct TJ proteins expression regulation

Blood‐spinal cord barrier (BSCB) disruption is a major process for the secondary injury of spinal cord injury (SCI) and is considered to be a therapeutic target for SCI. Previously, we demonstrated that metformin could improve functional recovery after SCI; however, the effect of metformin on BSCB is still unknown. In this study, we found that metformin could prevent the loss of tight junction (TJ) proteins at day 3 after SCI in vivo, but in vitro there was no significant difference of these proteins between control and metformin treatment in endothelial cells. This indicated that metformin‐induced BSCB protection might not be mediated by up‐regulating TJ proteins directly, but by inhibiting TJ proteins degradation. Thus, we investigated the role of metformin on MMP‐9 and neutrophils infiltration. Neutrophils infiltration is the major source of the enhanced MMP‐9 in SCI. Our results showed that metformin decreased MMP‐9 production and blocked neutrophils infiltration at day 1 after injury, which might be related to ICAM‐1 down‐regulation. Also, our in vitro study showed that metformin inhibited TNF‐α‐induced MMP‐9 up‐regulation in neutrophils, which might be mediated via an AMPK‐dependent pathway. Together, it illustrated that metformin prevented the breakdown of BSCB by inhibiting neutrophils infiltration and MMP‐9 production, but not by up‐regulating TJ proteins expression. Our study may help to better understand the working mechanism of metformin on SCI.     

Pericytes from Brain Microvessels Strengthen the Barrier Integrity in Primary Cultures of Rat Brain Endothelial Cells

(1) The blood–brain barrier (BBB) characteristics of cerebral endothelial cells are induced by organ-specific local signals. Brain endothelial cells lose their phenotype in cultures without cross-talk with neighboring cells. (2) In contrast to astrocytes, pericytes, another neighboring cell of endothelial cells in brain capillaries, are rarely used in BBB co-culture systems. (3) Seven different types of BBB models, mono-culture, double and triple co-cultures, were constructed from primary rat brain endothelial cells, astrocytes and pericytes on culture inserts. The barrier integrity of the models were compared by measurement of transendothelial electrical resistance and permeability for the small molecular weight marker fluorescein. (4) We could confirm that brain endothelial monolayers in mono-culture do not form tight barrier. Pericytes induced higher electrical resistance and lower permeability for fluorescein than type I astrocytes in co-culture conditions. In triple co-culture models the tightest barrier was observed when endothelial cells and pericytes were positioned on the two sides of the porous filter membrane of the inserts and astrocytes at the bottom of the culture dish. (5) For the first time a rat primary culture based syngeneic triple co-culture BBB model has been constructed using brain pericytes beside brain endothelial cells and astrocytes. This model, mimicking closely the anatomical position of the cells at the BBB in vivo, was superior to the other BBB models tested. (6) The influence of pericytes on the BBB properties of brain endothelial cells may be as important as that of astrocytes and could be exploited in the construction of better BBB models.     

TNF-alpha induced NFκB signaling and p65 (RelA) overexpression repress Cldn5 promoter in mouse brain endothelial cells

Inflammatory cytokine TNFα enhances permeability of brain capillaries constituting blood brain barrier (BBB). In the monoculture endothelial models of BBB TNFα alters tight junction (TJ) structure and protein content. Claudin-5 (Cldn5) is a key TJ protein whose expression in the brain endothelial cells is critical to the function of BBB. TNFα reduces Cldn5 promoter activity and mRNA expression in mouse brain derived endothelial cells but the regulatory elements and signaling mechanism involved are not defined. Here we report that TNFα acts through NFκB signaling and requires a conserved promoter region for the down-regulation of Cldn5 expression. Overexpression of the NFκB subunit p65 (RelA) alone repressed Cldn5 promoter activity in mouse brain endothelial cells. We observed partial loss of Cldn5 protein expression after prolonged TNFα treatment in primary endothelial culture isolated from C56BL/6 mice brain. Taken together, our results confirm and extend previous observations of TNFα induced down-regulation of Cldn5 expression in mouse brain endothelial cells.     

Biologic TNFα-inhibitors that cross the human blood-brain barrier

Tumor necrosis factor (TNF)α inhibitors (TNFI) are a major class of biologic therapeutics, and include decoy receptor and monoclonal antibody (MAb) therapeutics that block TNFα action. TNFα is a pro-inflammatory cytokine in brain disease, such as stroke, brain or spinal cord injury, or Alzheimer disease. However, the biologic TNFIs cannot be developed for the brain, because these large molecules do not cross the blood-brain barrier (BBB). Brain penetrating forms of TNFα decoy receptors or anti-TNFα antibody therapeutics can be re-engineered as IgG fusion proteins with a BBB molecular Trojan horse, such as the mAb against the human insulin receptor (HIR). The HIRMAb undergoes receptor-mediated transport across the BBB via the endogenous insulin receptor, and carries into brain the fused biologic TNFI. A fusion protein of the HIRMAb and the type II TNF receptor (TNFR) extracellular domain, designated the HIRMAb-TNFR fusion protein, has been engineered and expressed in stably transfected Chinese hamster ovary (CHO) cells. The HIRMAb-TNFR fusion protein binds both the HIR and TNFα with low nM affinity. The HIRMAb cross reacts with the Rhesus monkey insulin receptor, and the HIRMAb-TNFR is rapidly, and selectively, taken up by primate brain at concentrations that inhibit TNFα. In addition, a fusion protein of the HIRMAb and a therapeutic single chain Fv (ScFv) antibody has been engineered and also expressed in stably transfected CHO cells. The BBB molecular Trojan horse platform technology allows for the engineering of brain-penetrating recombinant proteins as new biologic therapeutics for the human brain.     

One year follow up in ischemic brain injury and the role of Alzheimer factors

Ongoing interest in brain ischemia research has provided data showing that ischemia may be involved in the pathogenesis of Alzheimer disease. Brain ischemia in the rat produces a stereotyped pattern of selective neuronal degeneration, which mimics early Alzheimer disease pathology. The objective of this study was to further develop and characterize cardiac arrest model in rats, which provides practical way to analyze Alzheimer-type neurodegeneration. Rats were made ischemic by cardiac arrest. Blood-brain barrier (BBB) insufficiency, accumulation of different parts of amyloid precursor protein (APP) and platelets inside and outside BBB vessels were investigated in ischemic brain up to 1-year survival. Ischemic brain tissue demonstrated haphazard BBB changes. Toxic fragments of APP deposits were associated with the BBB vessels. Moreover our study revealed platelet aggregates in- and outside BBB vessels. Toxic parts of APP and platelet aggregates correlated very well with BBB permeability. Progressive injury of the ischemic brain parenchyma may be caused not only by a degeneration of neurons destroyed during ischemia but also by chronic damage in BBB. Chronic ischemic BBB insufficiency with accumulation of toxic components of APP in the brain tissue perivascular space, may gradually over a lifetime, progress to brain atrophy and to full blown Alzheimer-type pathology.     

Early radiation-induced endothelial cell loss and blood-spinal cord barrier breakdown in the rat spinal cord

Using a rat spinal cord model, this study was designed to characterize radiation-induced vascular endothelial cell loss and its relationship to early blood-brain barrier disruption in the central nervous system. Adult rats were given a single dose of 0, 2, 8, 19.5, 22, 30 or 50 Gy to the cervical spinal cord. At various times up to 2 weeks after irradiation, the spinal cord was processed for histological and immunohistochemical analysis. Radiation-induced apoptosis was assessed by morphology and TdT-mediated dUTP nick end labeling combined with immunohistochemical markers for endothelial and glial cells. Image analysis was performed to determine endothelial cell and microvessel density using immunohistochemistry with endothelial markers, namely endothelial barrier antigen, glucose transporter isoform 1, laminin and zonula occludens 1. Blood-spinal cord barrier permeability was assessed using immunohistochemistry for albumin and (99m)Tc-diethylenetriamine pentaacetic acid as a vascular tracer. Endothelial cell proliferation was assessed using in vivo BrdU labeling. During the first 24 h after irradiation, apoptotic endothelial cells were observed in the rat spinal cord. The decrease in endothelial cell density at 24 h after irradiation was associated with an increase in albumin immunostaining around microvessels. The decrease in the number of endothelial cells persisted for 7 days and recovery of endothelial density was apparent by day 14. A similar pattern of blood-spinal cord barrier disruption and recovery of permeability was observed over the 2 weeks, and an increase in BrdU-labeled endothelial cells was seen at day 3. These results are consistent with an association between endothelial cell death and acute blood-spinal cord barrier disruption in the rat spinal cord after irradiation.