Neural Vasculature Research Articles

Single-cell analysis of mesenchymal cells in permeable neural vasculature reveals novel diverse subpopulations of fibroblasts

BackgroundIn the choroid plexus and pituitary gland, vasculature is known to have a permeable, fenestrated phenotype which allows for the free passage of molecules in contrast to the blood brain barrier observed in the rest of the CNS. The endothelium of these compartments, along with secretory, neural-lineage cells (choroid epithelium and pituitary endocrine cells) have been studied in detail, but less attention has been given to the perivascular mesenchymal cells of these compartments.MethodsThe Hic1CreERT2 Rosa26LSL−TdTomato mouse model was used in conjunction with a PdgfraH2B−EGFP mouse model to examine mesenchymal cells, which can be subdivided into Pdgfra+ fibroblasts and Pdgfra− pericytes within the choroid plexus (CP) and pituitary gland (PG), by histological, immunofluorescence staining and single-cell RNA-sequencing analyses.ResultsWe found that both CP and PG possess substantial populations of distinct Hic1+ mesenchymal cells, including an abundance of Pdgfra+ fibroblasts. Within the pituitary, we identified distinct subpopulations of Hic1+ fibroblasts in the glandular anterior pituitary and the neurosecretory posterior pituitary. We also identified multiple distinct markers of CP, PG, and the meningeal mesenchymal compartment, including alkaline phosphatase, indole-n-methyltransferase and CD34.ConclusionsNovel, distinct subpopulations of mesenchymal cells can be found in permeable vascular interfaces, including the CP, PG, and meninges, and make distinct contributions to both organs through the production of structural proteins, enzymes, transporters, and trophic molecules.

The alleviative effect of Calendula officinalis L. extract against Parkinson's disease-like pathology in zebrafish via the involvement of autophagy activation.

Parkinson's disease (PD) is the second most prevalent neurodegenerative disorder. However, effective preventative or therapeutic agents for PD remain largely limited. Marigold Calendula officinalis L. (CoL) has been reported to possess a wide range of biological activities, but its neuroprotective activity including anti-neurodegenerative diseases is unclear. Here, we aim to investigate whether the extract of CoL (ECoL) has therapeutic activity on PD. We identified the chemical composition of flavonoid, an important active ingredient in ECoL, by a targeted HPLC-Q-TOF-MS analysis. Subsequently, we evaluated the anti-PD effect of ECoL by using zebrafish PD model induced by 1-methyl-4-phenyl-1-1,2,3,6-tetrahydropyridine (MPTP). After ECoL+MPTP co-treatments, the changes of dopaminergic neurons, neural vasculature, nervous system, and locomotor activity were examined, respectively. The expressions of genes related to neurodevelopment and autophagy were detected by RT-qPCR. Further, the interaction between autophagy regulators and ECoL flavonoids was predicted using molecular docking method. As a result, 5 kinds of flavonoid were identified in ECoL, consisting of 121 flavones and flavonols, 32 flavanones, 22 isoflavonoids, 11 chalcones and dihydrochalcones, and 17 anthocyanins. ECoL significantly ameliorated the loss of dopaminergic neurons and neural vasculature, restored the injury of nervous system, and remarkably reversed the abnormal expressions of neurodevelopment-related genes. Besides, ECoL notably inhibited the locomotor impairment in MPTP-induced PD-like zebrafish. The underlying anti-PD effect of ECoL may be implicated in activating autophagy, as ECoL significantly upregulated the expressions of genes related to autophagy, which contributes to the degradation of α-synuclein aggregation and dysfunctional mitochondria. Molecular docking simulation showed the stable interaction between autophagy regulators (Pink, Ulk2, Atg7, and Lc3b) and 10 main compounds of flavonoid in ECoL, further affirming the involvement of autophagy activation by ECoL in anti-PD action. Our results suggested that ECoL has the anti-PD effect, and ECoL might be a promising therapeutic candidate for PD treatment.

Maternal immune activation leads to defective brain-blood vessels and intracerebral hemorrhages in male offspring.

Intracerebral hemorrhages are recognized risk factors for neurodevelopmental disorders and represent early biomarkers for cognitive dysfunction and mental disability, but the pathways leading to their occurrence are not well defined. We report that a single intrauterine exposure of the immunostimulant Poly I:C to pregnant mice at gestational day 9, which models a prenatal viral infection and the consequent maternal immune activation, induces the defective formation of brain vessels and causes intracerebral hemorrhagic events, specifically in male offspring. We demonstrate that maternal immune activation promotes the production of the TGF-β1 active form and the consequent enhancement of pSMAD1-5 in males' brain endothelial cells. TGF-β1, in combination with IL-1β, reduces the endothelial expression of CD146 and claudin-5, alters the endothelium-pericyte interplay resulting in low pericyte coverage, and increases hemorrhagic events in the adult offspring. By showing that exposure to Poly I:C at the beginning of fetal cerebral angiogenesis results in sex-specific alterations of brain vessels, we provide a mechanistic framework for the association between intragravidic infections and anomalies of the neural vasculature, which may contribute to neuropsychiatric disorders.

Injectable Prevascularized Mature Adipose Tissues (iPAT) to Achieve Long-Term Survival in Soft Tissue Regeneration.

Soft tissue regeneration remains a challenge in reconstructive surgery. So far, both autologous fat implantations and artificial implants methods used in clinical applications lead to various disadvantages and limited lifespan. To overcome these limitations and improve the graft volume maintenance, reproducing a mature adipose tissue already including vasculature structure before implantation can be the solution. Therefore, injectable prevascularized adipose tissues (iPAT) are made from physiological collagen microfibers mixed with human mature adipocytes, adipose-derived stem cells, and human umbilical vein endothelial cells, embedded in fibrin gel. Following murine subcutaneous implantation, the iPAT show a higher cell survival (84% ± 6% viability) and volume maintenance after 3 months (up to twice heavier) when compared to non-prevascularized balls and liposuctioned fat implanted controls. This higher survival can be explained by the greater amount of blood vessels found (up to 1.6-fold increase), with balanced host anastomosis (51% ± 1% of human/mouse lumens), also involving infiltration by the lymphatic and neural vasculature networks. Furthermore, with the cryopreservation possibility enabling their later reinjection, the iPAT technology has the merit to allow noninvasive soft tissue regeneration for long-term outcomes.

Intravenous injection of cyclophilin A realizes the transient and reversible opening of barrier of neural vasculature through basigin in endothelial cells

Neural vasculature forms the blood–brain barrier against the delivery of systemically administered therapeutic drugs into parenchyma of neural tissues. Therefore, procedures to open the blood–brain barrier with minimal damage to tissues would lead to the great progress in therapeutic strategy for intractable neural diseases. In this study, through analyses with mouse in vitro brain microvascular endothelial cells and in vivo neural vasculature, we demonstrate that the administration of cyclophilin A (CypA), a ligand of basigin which is expressed in barrier-forming endothelial cells, realizes the artificial opening of blood–brain barrier. Monolayers of endothelial cells lost their barrier properties through the disappearance of claudin-5, an integral tight junction molecule, from cell membranes in a transient and reversible manner. Furthermore, the intravenous injection of a single dose of CypA into mice resulted in the opening of blood–brain barrier for a certain period which enabled the enhanced delivery of systemically administered doxorubicin into the parenchyma of neural tissues. These findings that the pre-injection of a single dose of CypA realizes an artificial, transient as well as reversible opening of blood–brain barrier are considered to be a great step toward the establishment of therapeutic protocols to overcome the intractability of neural diseases.

284-OR: Characterization of Cognitive Changes in Type 1 Diabetes of Extreme Duration by Retinal Neural Structure and Vasculature

The risk of cognitive dysfunction in aging populations with type 1 diabetes (T1D) may be similar to that of people with type 2 diabetes, but it has not been well-characterized clinically. In the Joslin Medalist Study, a cohort of people with ≥ 50 years of T1D, cognitive dysfunction was previously shown to correlate with cardiovascular disease (CVD) and proliferative diabetic retinopathy (PDR). In a Medalist subset (n=129), we extended these findings by assessing the relationships of cognitive changes and other complications with vascular abnormalities in the retina, a developmental extension of the central nervous system. Medalists were evaluated with validated cognitive tests to assess psychomotor speed (PS), immediate memory (IM), delayed memory (DM), working memory (WM), and executive function (EF). Retinal optical coherence tomography (OCT) and OCT angiography were performed to obtain neural retinal layer thickness and vascular density for the superficial (SCP) and deep (DCP) retinal capillary plexuses. CVD and PDR were associated with worse IM (p=0.03) and decline in PS (p=0.01), respectively, adjusting for age, A1c, BMI, triglycerides and hypertension. Thinning of the retinal outer nuclear layer (ONL) was associated with PDR (p=0.04), CVD (p=0.02), worse IM (p=0.05) and PS (p=0.01), adjusted for sex, diabetes duration, PDR and visual acuity. CVD, PDR and DM severity were associated with decreased DCP density (p=0.04, p=0.0004, and p=0.002, respectively). Decreased density of the SCP was associated with worse dominant hand PS (p=0.01), adjusted for PDR, T1D duration, and visual acuity. In summary, cognitive decline in people with long duration of T1D is associated with PDR and CVD, which is supported by shared specific retinal vascular imaging abnormalities, suggesting a potential common pathogenesis underlying these complications in T1D. Disclosure E. Wolfson: None. W. Fickweiler: None. K. Sampani: None. H. Shah: None. L.P. Aiello: Advisory Panel; Self; Novo Nordisk Inc. Consultant; Self; KalVista Pharmaceuticals, Inc., Novo Nordisk Inc. Stock/Shareholder; Self; KalVista Pharmaceuticals, Inc. Other Relationship; Self; Optos. J. Sun: Research Support; Self; Adaptive Sensory Technology, Boehringer Ingelheim Pharmaceuticals, Inc., KalVista Pharmaceuticals, Inc., Novo Nordisk Inc., Optovue, Inc., Roche Pharma. Other Relationship; Self; Merck Foundation, Novartis Pharmaceuticals Corporation, Novo Nordisk Inc., Roche Pharma. G.L. King: Research Support; Self; Janssen Pharmaceuticals, Inc. Funding National Institute of Diabetes and Digestive and Kidney Diseases (P30DK036836, UL1RR025758-03, R24283, DK083957-01DP3, DK094333-01, T32DK007260); JDRF (17-2013-310)

The intracranial vasculature of canines represents a model for neurovascular ischemia and training residents and fellows in endovascular neurosurgery.

We describe use of a canine model to evaluate physiological effects and neuroprotective strategies in the setting of cerebral ischemia and endovascular neurosurgery training. We performed transfemoral digital subtraction cerebral and cervical angiography on eight anesthetized dogs. Angiographic images of cerebral arteries were obtained following cannulation of the femoral artery. Cerebral ischemia models were made after angiography. The canine cerebral vasculature exhibited extensive tortuosity of the carotid and vertebral arteries. Conversely, the bilateral anterior spinal arteries were easily catheterized using microcatheters and microguidewires. The basilar artery and its branches were facilely cannulable. Circle of Willis continuity sans hypoplasia or aplasia of its constitutive segments was appreciated in all animals. The middle cerebral arteries could be easily accessed via the posterior communicating arteries. We generated an empirically evaluable therapeutically interventional experimental animal model of cerebral ischemia by occluding the middle cerebral artery using small coils for a duration between 15 and 60 min. Unique amenability of the canine intracranial vasculature to selective and microcatheter cannulation renders experimentally induced cerebral, cerebellar, and brainstem via occlusion of the supratentorial and infratentorial arteries a simple matter. The neural vasculature irrigating the canine cerebrum, brainstem, and cerebellum may consequently prove useful in helping young and nascent endovascular neurosurgeons in developing and refining their skills of microcatheter navigation and manipulation and deployment of therapeutic devices to achieve effective occlusion of aneurysms, arteriovenous malformations, arteriovenous fistulas, and neoplasms of the intracranial cavity.

Anti-Parkinson's disease activity of phenolic acids from Eucommia ulmoides Oliver leaf extracts and their autophagy activation mechanism.

Although Parkinson's disease (PD) is the second most common neurodegenerative disorder, the preventative or therapeutic agents for the treatment of PD are limited. Eucommia ulmoides Oliver (EuO) is widely used as a traditional herb to treat various diseases. EuO bark extracts have been reported to possess anti-PD activity. Here, we investigated whether extracts of EuO leaves (EEuOL) also have therapeutic effects on PD since similar components and clinical applications have been found between barks and leaves of this tree. We identified the chemical composition of EEuOL by HPLC-Q-TOF-MS and tested the anti-PD effect of EEuOL using the zebrafish PD model. As a result, 28 compounds including 3 phenolic acids, 7 flavonoids, and 9 iridoids were identified. EEuOL significantly reversed the loss of dopaminergic neurons and neural vasculature and reduced the number of apoptotic cells in zebrafish brain in a concentration-dependent manner. Moreover, EEuOL relieved locomotor impairments in MPTP-modeled PD zebrafish. We also investigated the underlying mechanism and found that EEuOL may activate autophagy, contributing to α-synuclein degradation, therefore alleviating PD-like symptoms. Molecular docking simulation implied the interaction between autophagy regulators (Pink1, Beclin1, Ulk2, and Atg5) and phenolic acids of EEuOL, affirming the involvement of autophagy in EEuOL-exerted anti-PD action. The overall results indicated the anti-PD effect of EEuOL, opening the possibility to use the extract in PD treatment.

86-OR: Association of Cognitive Function with Retinal Neural Structure and Vasculature in Type 1 Diabetes

Early cognitive decline, a dreaded complication of diabetes, is difficult and time consuming to diagnose. The Joslin Medalist Study, comprising individuals with ≥50 years of type 1 diabetes (T1D), reported that cognitive dysfunction was greater in T1D patients as compared to age-matched nondiabetic controls and was associated with proliferative diabetic retinopathy (PDR). We examined a larger Medalist sample to determine whether cognitive dysfunction is correlated with specific structural and vascular abnormalities in the retina, which embryonically is an extension of the central nervous system. Retinal imaging techniques such as optical coherence tomography (OCT) and OCT angiography (OCTA) can quantitate structural changes in individual retinal layers and the superficial and deep capillary plexus of the retinal microvasculature. We assessed Medalists (n=110) who underwent cognitive testing consisting of 5 validated measures in the following domains: executive functioning (EF), motor skills (MS), and immediate (IM), delayed (DM) and working memory (WM). Thinning of the neuroretinal ganglion cell layer (β: 0.69, P: 0.02) and inner plexiform layer (β: 0.56, P: 0.01) was associated with worse EF. PDR was associated with worse cognitive function (MS, dominant hand: OR: 1.02, P: 0.01; MS, non-dominant hand: OR: 1.01, P: 0.02; EF: OR: 0.83, P: 0.03) and decreased superficial retinal capillary plexus density (p=0.001). Decreased vessel density of the deep capillary plexus was associated with worse cognitive function including DM (β: 0.10, P: 0.01), IM (β: 0.10, P: 0.02), MS (dominant β: -0.07, P: 0.01; non-dominant β: -0.04, P: 0.03), even after adjusting for DR severity, T1D duration and visual acuity. These findings suggest that noninvasive ocular imaging techniques have potential to identify T1D individuals with early cognitive impairment, and provide insights into pathophysiological connections between cognitive function, diabetes and microvascular complications. Disclosure S.M. Paniagua: None. W. Fickweiler: None. V. Bahnam: None. K. Sampani: None. I. Wu: None. H. Shah: None. G. Musen: None. L.P. Aiello: Advisory Panel; Self; Novo Nordisk A/S. Consultant; Self; Decision Resources Group, KalVista Pharmaceuticals, Inc., Mingsight, Retinal Solutions. Stock/Shareholder; Self; KalVista Pharmaceuticals, Inc. Other Relationship; Self; Optos. J.K. Sun: Research Support; Self; Adaptive Sensory Technology, Boston Micromachines Corporation, Genentech, Inc., KalVista Pharmaceuticals, Inc., Optovue, Incorporated. Other Relationship; Self; Genentech, Inc., Novartis Pharmaceuticals Corporation, Novo Nordisk Inc. G.L. King: Research Support; Self; Sanofi. Funding National Institute of Diabetes and Digestive and Kidney Diseases (P30DK036836, UL1RR025758-03, R24283DK083957-01, DP3DK094333-01, T32DK007260); JDRF (17-2013-310); Thomas J. Beatson, Jr. Foundation Inc.

Cross-talk between blood vessels and neural progenitors in the developing brain.

The formation of the central nervous system (CNS) involves multiple cellular and molecular interactions between neural progenitor cells (NPCs) and blood vessels to establish extensive and complex neural networks and attract a vascular supply that support their function. In this review, we discuss studies that have performed genetic manipulations of chick, fish and mouse embryos to define the spatiotemporal roles of molecules that mediate the reciprocal regulation of NPCs and blood vessels. These experiments have highlighted core functions of NPC-expressed ligands in initiating vascular growth into and within the neural tube as well as establishing the blood–brain barrier. More recent findings have also revealed indispensable roles of blood vessels in regulating NPC expansion and eventual differentiation, and specific regional differences in the effect of angiocrine signals. Accordingly, NPCs initially stimulate blood vessel growth and maturation to nourish the brain, but blood vessels subsequently also regulate NPC behaviour to promote the formation of a sufficient number and diversity of neural cells. A greater understanding of the molecular cross-talk between NPCs and blood vessels will improve our knowledge of how the vertebrate nervous system forms and likely help in the design of novel therapies aimed at regenerating neurons and neural vasculature following CNS disease or injury.

ADAM12 and ADAM17 are essential molecules for hypoxia-induced impairment of neural vascular barrier function.

Neural vascular barrier is essential for the life of multicellular organisms, and its impairment by tissue hypoxia is known to be a central of pathophysiology accelerating the progression of various intractable neural diseases. Therefore, the molecules involved in hypoxia-induced impairment of vascular barrier can be the targets to establish new therapies for intractable diseases. Here, we demonstrate that a disintegrin and metalloproteinases (ADAMs) 12 and 17 expressed in endothelial cells are the molecules responsible for the impairment of neural vascular barrier by hypoxia. Brain microvascular endothelial cells in vitro lost their barrier properties immediately after hypoxic stimulation through diminished localization of claudin-5, a tight junction molecule, on cell membranes. Hypoxic disappearance of claudin-5 from cell membranes and the consequent loss of barrier properties were completely suppressed by inhibition of the metalloproteinase activity which was found to be attributed to ADAM12 and ADAM17. Inhibition of either ADAM12 or ADAM17 was sufficient to rescue the in vivo neural vasculature under hypoxia from the loss of barrier function. This is the first report to specify the molecules which are responsible for hypoxia-induced impairment of neural vascular barrier and furthermore can be the targets of new therapeutic strategies for intractable neural diseases.

Resistance of LDL against Oxidation is Related to Macular Pigment Density and Cognition in Healthy Older Adults

Lutein, a dietary carotenoid, is selectively taken up and accumulated in neural tissues. Macular pigment (MP) is considered a surrogate biomarker of lutein levels in the macula and brain. MP density and lutein brain concentrations have been related to better cognitive function, and lutein has also been associated with the reduction of atherosclerotic lesions. Lutein's antioxidant properties may be one of the mechanisms by which it is protective of neural tissue, vasculature, and cognition. Our objective was to evaluate the relationships among MP density, oxidative stress, and cognition in healthy older adults (50 - 84 y, n = 44). MP density was measured using heterochromatic flicker photometry. The resistance of LDL against oxidation (rLDLox), a biomarker of oxidative stress and vascular health, was assessed using lag time of Cu2+induced oxidation. A computerized assessment battery (CANTAB) was used for measures of cognition. Cognitive data were normalized to z-scores; subjects were designated as higher o...

Ultrasound assessment of the median nerve: a biomarker that can help in setting a treat to target approach tailored for carpal tunnel syndrome patients.

Ultrasonography (US) is a valuable tool for confirming the diagnosis of carpal tunnel syndrome (CTS) as it enables the detection of changes in the median nerve shape and rule out anatomic variants as well as space-occupying lesions such as ganglion cysts or tenosynovitis. This work was carried out aiming at: 1. Ultrasonography assessment of the median nerve and its neurovascular blood-flow in CTS patients before and after management. 2. Verify the possibility of using baseline US parameters as a biomarker to predict likely outcomes and frame a treatment plan for CTS patients.233 CTS subjects diagnosed based on clinical and electrophysiological (NCS) testing were included in this work. US measures at the tunnel inlet included: cross sectional area, flattening ratio and neural Power Doppler (PD) signals. Patients who had severe NCS outcomes or neurological deficit were referred for open surgical decompression; the remaining patients were given the choice of either conservative or surgical management. The main outcome variable was improvement >70% in CTS symptoms. Assessments were carried out at baseline, 1-week, 1-month and 6-months post treatment. Results revealed an inverse relation between the neural vasculature and CTS severity defined by NCS (r = − 0.648). In CTS cases treated conservatively, the US measures started to improve within 1-week, whereas in the surgically treated cohort there was an initial phase of post-operative nerve measures increase, before settling at 1-month time of follow-up. The risk of poor outcomes was significantly higher (RR 3.3) in patients with high median nerve flattening ratio. This risk was most marked in the cohort with nerve flattening associated with longer duration of illness (RR 4.3) and low PD signal (RR 4.1). The results revealed that in addition to the diagnostic value of US in CTS, the detection of increased median nerve neuro-vasculature has a good prognostic value as an indicator of early median nerve affection.

Neural alterations from lead exposure in zebrafish

Lead was used extensively as a gas additive and pesticide, in paints, batteries, lead shot, pipes, canning and toy manufacturing. Although uses of lead have been restricted, lead persists in our environment especially in older homes, and generally in soil and water. Although extensive studies have determined that fetal and childhood exposures to lead have been associated with childhood and adolescent memory impairments and learning disabilities, there are limited studies investigating early neural and morphological effects that may lead to these behavioral and learning abnormalities. Here we utilize the zebrafish vertebrate model system to study early effects of lead exposure on the brain. We treat embryos with 0.2mM lead for 24, 48 and 72h and analyze neural structures through live imagery and transgenic approaches. We find structural abnormalities in the hindbrain region as well as changes in branchiomotor neuron development and altered neural vasculature. Additionally, we find areas of increased apoptosis. We conclude that lead is developmentally neurotoxic to a specific region of the brain, the hindbrain and is toxic to branchiomotor neurons residing in rhombomeres 2 through 7 of the hindbrain and hindbrain central artery vasculature.

Sciatic Nerve of Diabetic Rat Treated With Epoetin Delta: Effects on C-Fibers and Blood Vessels Including Pericytes

In diabetes mellitus (DM) reduced motor and sensory properties of peripheral nerves are linked with the dysfunction of neural vasculature. We investigated C-fibers and microvessels of sciatic nerve of normal, DM, and DM + epoetin delta-treated rats. C-fibers immunoreactive for calcitonin gene-related peptide (CGRP), tyrosine hydroxylase (TH), epoetin receptor (EpoR), and common beta receptor subunit of the interleukin 3 receptor (IL-3Rbeta) were present in all rats, whereas in DM and epoetin-treated rats C-fibers also showed neuronal (nNOS) and inducible (iNOS) nitric oxide synthases. The cross-sectional area of CGRP-positive C-fibers was decreased in DM, but it recovered after epoetin treatment. In all conditions, vascular endothelium showed scarce immunolabeling for endothelial nitric oxide synthase (eNOS); the profound immunoreactivity for eNOS, EpoR, and IL-3Rbeta was in pericytes. Some perivascular autonomic nerves were damaged and IL-3Rbeta positive. Findings are discussed in terms of declined sensory conduction velocity in DM, its improvement after epoetin treatment, and the possible vascular contribution to these phenomena.

Sustained hypercapnia induces cerebral microvascular degeneration in the immature brain through induction of nitrative stress

Hypercapnia is regularly observed in chronic lung disease, such as bronchopulmonary dysplasia in preterm infants. Hypercapnia results in increased nitric oxide synthase activity and in vitro formation of nitrates. Neural vasculature of the immature subject is particularly sensitive to nitrative stress. We investigated whether exposure to clinically relevant sustained high CO(2) causes microvascular degeneration in the newborn brain by inducing nitrative stress, and whether this microvascular degeneration has an impact on brain growth. Newborn rat pups were exposed to 10% CO(2) as inspired gas (Pa(CO(2)) = 60-70 mmHg) starting within 24 h of birth until postnatal day 7 (P7). Brains were notably collected at different time points to measure vascular density, determine brain cortical nitrite/nitrate, and trans-arachidonic acids (TAAs; products of nitration) levels as effectors of vessel damage. Chronic exposure of rat pups to high CO(2) (Pa(CO(2)) approximately 65 mmHg) induced a 20% loss in cerebrovascular density at P3 and a 15% decrease in brain mass at P7; at P30, brain mass remained lower in CO(2)-exposed animals. Within 24 h of exposure to CO(2), brain eNOS expression and production of nitrite/nitrate doubled, lipid nitration products (TAAs) increased, and protein nitration (3-nitrotyrosine immunoreactivity) was also coincidently augmented on brain microvessels (lectin positive). Intracerebroventricular injection of TAAs (10 microM) replicated cerebrovascular degeneration. Treatment of rat pups with NOS inhibitor (L-N(omega)-nitroarginine methyl ester) or a peroxynitrite decomposition catalyst (FeTPPS) prevented hypercapnia-induced microvascular degeneration and preserved brain mass. Cytotoxic effects of high CO(2) were reproduced in vitro/ex vivo on cultured endothelial cells and sprouting microvessels. In summary, hypercapnia at values frequently observed in preterm infants with chronic lung disease results in increased nitrative stress, which leads to cerebral cortical microvascular degeneration and curtails brain growth.

Glucose Transporter Distribution in the Vessels of the Central Nervous System of the Axolotl Ambystoma mexicanum (Urodela: Ambystomatidae)

The GLUT-1 isoform of the glucose transporter is commonly considered a reliable molecular marker of blood-brain barrier endothelia in the neural vasculature organized in a three-dimensional network of single vessels. The central nervous system of the axolotl Ambystoma mexicanum is characterized by a vascular architecture that contains both single and paired vessels. The presence and distribution of the GLUT-1 transporter are studied in this urodele using both immunoperoxidase histochemistry and immunogold technique. Light microscopy reveals immunopositivity in both parenchymal and meningeal vessels. The transverse-sectioned pairs of vessels do not show the same size. Furthermore, in the same pair, the two elements often differ in diameter. The main regions of the central nervous system show a different percentage of the paired structures. Only immunogold cytochemistry reveals different staining intensity in the two adjoined elements of a vascular pair. Colloidal gold particles show an asymmetric distribution in the endothelia of both single and paired vessels. These particles are more numerous on the abluminal surface than on the luminal one. The particle density is calculated in both vascular types. The different values could indicate functional differences between single and paired vessels and between the two adjoined elements of a pair, regarding glucose transport.

Hypoxia Disrupts the Barrier Function of Neural Blood Vessels through Changes in the Expression of Claudin-5 in Endothelial Cells

The mechanisms underlying the hypoxia-induced disruption of the barrier function of neural vasculature were analyzed with reference to the expression of claudin-5, a component of tight junctions between neural endothelial cells. The movement of claudin-5 from the cytoplasm to the plasma membrane of cultured confluent brain-derived endothelial (bEND.3) cells was closely correlated with the increase in the transendothelial electrical resistance. Inhibition of the expression of claudin-5 by RNAi resulted in a reduction of transendothelial electrical resistance, indicating a critical role of claudin-5 in the barrier property. Hypoxia (1% O(2)) altered the location of claudin-5 in the plasma membrane and the level of claudin-5 protein in bEND.3 cells, and these changes were accompanied by a decrease in the transendothelial electrical resistance. In vivo the claudin-5 molecules were expressed under normoxia in the plasma membrane of retinal microvascular endothelial cells but were significantly reduced under hypoxic conditions. Tracer experiments revealed that the barrier function of hypoxic retinal vasculature with depressed claudin-5 expression was selectively disrupted against small molecules, which is very similar to the phenotype of claudin-5-deficient mice. These in vitro and in vivo data indicate that claudin-5 is a target molecule of hypoxia leading to the disruption of the barrier function of neural vasculature.

Glucose transporter (GLUT-1) distribution in the brain vessels of the adult Italian wall lizard, Podarcis sicula

The GLUT-1 isoform of the glucose transporter is commonly accepted as a reliable molecular marker of blood-brain barrier endothelia in neural vasculature organized in a three-dimensional network of single vessels. The brain of the lizard Podarcis sicula is characterized by a vascular architecture based on a pattern of paired vessels. The presence and distribution of GLUT-1 were studied in adult lizards using both light and transmission electron microscopic techniques. Immunoperoxidase histochemistry was applied to sections from paraffin-embedded brain using gold-conjugated secondary antibodies to localize this antigen on ultrathin sections. The transverse sectioned pairs of vessels did not show the same size and, in particular, the two elements of the same pair often differed in their diameters. Light microscopy revealed immunopositivity in both parenchymal and meningeal vessels. In each transverse-sectioned vascular pair, one element was intensely labelled, and the adjacent one showed only slight or negligible reaction. Colloidal gold particles were restricted to endothelial cells, showing an asymmetric labelling pattern, which was always characterized by markedly higher density of immunolabelling of the abluminal rather than the luminal plasmalemma. Moreover, in every vascular pair, one profile had lower amounts of scantier labelling by gold particles than the adjacent element. This pattern indicates functional differences between the adjacent vascular limbs regarding glucose transport.

Spatiotemporal Pattern of Macrophage Recruitment after Chronic Nerve Compression Injury

The contribution of macrophages to the pathogenesis of chronic nerve compression (CNC) injuries is presently unclear. We examined the time course and spatial localization of macrophage invasion from 24 hours to 28 days post-CNC injury with immunohistochemistry (IHC) and electron microscopy (EM). To clarify the differences in macrophage activity between different peripheral nerve injuries, we compared CNC injury to a nerve crush (CR) injury at similar time points. Entire counts of macrophages with ED1-immunoreactivity (IR) showed a slow, gradual increase in macrophage number from 24 hours to 28 days post-operatively in compressed sections. ED1-IR was greatest at the site of compression and in distal nerve segments with minimal immunostaining in proximal and normal sections. Quantitative analysis of ED1-IR after crush injury demonstrated a rapid time course of macrophage recruitment with ED1-IR peaking at 48 hours and declining to normal values as early as 21 days post-CR injury. Ultrastructural analysis with EM 14 days post-CNC injury revealed greater macrophage localization in the inner one-third region of normal nerves relative to the outer region. Differences in macrophage localization within inner and outer regions of compressed sections were negligible, as macrophages were found diffusely throughout the endoneurium by day 14. Our findings suggest that macrophage recruitment is dependent upon proximity to neural vasculature with relative macrophage density highest specifically around endoneurial blood vessels in both normal and compressed sections. Taken together, our results detail the unique spatiotemporal dynamics of macrophage recruitment early after CNC injury as distinct from a crush injury.