Shedding light on human cerebral lipofuscin: An explorative study on identification and quantification.

Increased oxidative stress has been associated with several neurodegenerative diseases such as Alzheimer's disease, but also with neurological diseases sharing pathophysiological pathways like epilepsy. Lipofuscin is a nondegradable end-product of oxidative stress; its cerebral presence reflects the cumulative amount of oxidative stress the brain has endured. In this study, we have observed prominent autofluorescent particles in the pial arterial wall and in neocortical parenchyma of young, drug-resistant epilepsy patients (18-28 years old) who underwent resective brain surgery (n = 6), as well as in older control patients (n = 3). With fluorescence spectroscopic imaging, brightfield microscopy, histochemistry and fluorescence lifetime imaging, these autofluorescent particles were identified as the age pigment lipofuscin. An evaluation of these lipofuscin particles using Imaris© software allowed robust quantification, while the 3D properties allowed visualization of the complex configuration. We elaborate on the usefulness of lipofuscin as a marker of cumulative oxidative stress in the brain. Furthermore, we speculate on the observed differences in particle size and density that we found between young patients and older controls, which could imply a role for lipofuscin in the pathophysiology of epilepsy and possibly other neurological diseases.

Cerebral amyloid-ß-related angiitis without cerebral microbleeds in a patient with subarachnoid hemorrhage.

Amyloid-ß-related angiitis (ABRA), a subtype of cerebral amyloid angiopathy (CAA), is vasculitis occurring in relation to amyloid-ß (Aß) deposition in the walls of intracranial blood vessels. ABRA is presumed to be caused by some immune response to the deposited Aß. An 81-year-old man on oral anticoagulant therapy complained of headache, nausea, and difficulty with standing after a head injury. Head computed tomography revealed subcortical bleeding in the right temporoparietal lobe, and 3 days after admission, magnetic resonance imaging (MRI) showed subarachnoid hemorrhage (SAH) around the hematoma. Cerebral microbleeds, a characteristic of CAA, were not detected on MRI. On worsening of his symptoms, intracranial brain biopsy and hematoma removal were performed. Intraoperative rapid diagnosis with a frozen section suspected vasculitis, which enabled the prompt initiation of steroid therapy. He was pathologically diagnosed with ABRA (granulomatous angiitis) using a formalin-fixed paraffin-embedded section. Vasculitis was prominent around blood vessels in the pia matter covering the cerebrum. In this case, the inflammatory cells seemed to appear via the subarachnoid space following cerebral hemorrhage and SAH. ABRA seemed to be developed by intracranial hemorrhage in this case.

CSD-Induced Arterial Dilatation and Plasma Protein Extravasation Are Unaffected by Fremanezumab: Implications for CGRP's Role in Migraine with Aura.

Cortical spreading depression (CSD) is a wave of neuronal depolarization thought to underlie migraine aura. Calcitonin gene-related peptide (CGRP) is a potent vasodilator involved in migraine pathophysiology. Evidence for functional connectivity between CSD and CGRP has triggered scientific interest in the possibility that CGRP antagonism may disrupt vascular responses to CSD and the ensuing plasma protein extravasation (PPE). Using imaging tools that allow us to generate continuous, live, high-resolution views of spatial and temporal changes that affect arteries and veins in the dura and pia, we determined the extent to which CGRP contributes to the induction of arterial dilatation or PPE by CSD in female rats, and how these events are affected by the anti-CGRP monoclonal antibody (anti-CGRP-mAb) fremanezumab. We found that the CSD-induced brief dilatation and prolonged constriction of pial arteries, prolonged dilatation of dural arteries and PPE are all unaffected by fremanezumab, whereas the brief constriction and prolonged dilatation of pial veins are affected. In comparison, although CGRP infusion gave rise to the expected dilatation of dural arteries, which was effectively blocked by fremanezumab, it did not induce dilatation in pial arteries, pial veins, or dural veins. It also failed to induce PPE. Regardless of whether the nociceptors become active before or after the induction of arterial dilatation or PPE by CSD, the inability of fremanezumab to prevent them suggests that these events are not mediated by CGRP, a conclusion with important implications for our understanding of the mechanism of action of anti-CGRP-mAbs in migraine prevention.SIGNIFICANCE STATEMENT The current study identifies fundamental differences between two commonly used models of migraine, CSD induction and systemic CGRP infusion. It raises the possibility that conclusions drawn from one model may not be true or relevant to the other. It sharpens the need to accept the view that there is more than one truth to migraine pathophysiology and that it is unlikely that one theory will explain all types of migraine headache or the mechanisms of action of drugs that prevent it. Regarding the latter, it is concluded that not all vascular responses in the meninges are born alike and, consequently, that drugs that prevent vascular dilatation through different molecular pathways may have different therapeutic outcomes in different types of migraine.

Expression of Connexin43 in Cerebral Arteries of Patients with Moyamoya Disease.

BACKGROUND AND PURPOSE: Moyamoya disease has a high incidence of cerebral vascular accident in children and adolescents, which can endanger the physical and mental health of children and adults seriously. However, the etiology and the pathogenesis of moyamoya disease remain unclear. Connexin43 (Cx43) is a predominant intercellular gap junction protein that plays an important role in the normal function of arteries and the development of several cardiovascular diseases. We aimed to preliminarily investigate pathological changes and the expression of Cx43 in cerebral arteries of patients with moyamoya disease. MATERIALS AND METHODS: This study collected 10 experimental cerebral artery specimens from patients with moyamoya disease and 10 control cerebral artery specimens from patients without moyamoya disease during surgery, then pathological changes and change in Cx43 expression of cerebral artery specimens were investigated in the 2 groups by hematoxylin and eosin staining and immunofluorescence. RESULTS: The intima of cerebral arteries was thin with monolayer endothelial cells in the control group but had asymmetrical thickening for the cerebral arteries in the experimental group. The mean ± standard deviation of the mean optical density of Cx43 in the experimental group was .065 ± .011 (range, .045-.081), whereas that in the control group was .035 ± .005 (range, .028-.042). The expression of Cx43 in the experimental group was statistically significantly higher in comparison with the control group (P < .01). CONCLUSION: The abnormal expression of Cx43 in the cerebral arteries may play an important role in the formation of vascular intima thickening in patients with moyamoya disease.

Ischemic Stroke: Histological Thrombus Composition and Pre-Interventional CT Attenuation Are Associated with Intervention Time and Rate of Secondary Embolism.

BACKGROUND AND PURPOSE: The introduction of stent retrievers has made the complete extraction and histological analysis of human thrombi possible. A number of large randomized trials have proven the efficacy of thrombectomy for ischemic stroke; however, thrombus composition could have an impact on the efficacy and risk of the intervention. We therefore investigated the impact of histologic thrombus features on interventional outcome and procedure-related embolisms. For a pre-interventional estimation of histologic features and outcome parameters, we assessed the pre-interventional CT attenuation of the thrombi. METHODS: We prospectively included all consecutive patients with occlusion of the middle cerebral artery who underwent thrombectomy between December 2013 and February 2016 at our university medical center. Samples were histologically analyzed (H&E, Elastica van Gieson, Prussian blue); additionally, immunohistochemistry for CD3, CD20, and CD68/KiM1P was performed. Main thrombus components (fibrin, erythrocytes, and white blood cells) were determined and compared to intervention time, frequency of secondary embolisms, as well as additional clinical and interventional parameters. Additionally, we assessed the pre-interventional CT attenuation of the thrombi in relation to the unaffected side (rHU) and their association with histologic features. RESULTS: One hundred eighty patients were included; of these, in 168 patients (93.4%), complete recanalization was achieved and 27 patients (15%) showed secondary embolism in the control angiogram. We observed a significant association of high amounts of fibrin (p < 0.001), low percentage of red blood cells (p < 0.001), and lower rHU (p < 0.001) with secondary embolism. Higher rHU values were significantly associated with higher amounts of fibrin (p ≤ 0.001) and low percentage of red blood cells (p ≤ 0.001). Additionally, high amounts of fibrin were associated with longer intervention times (p ≤ 0.001), whereas thrombi with high amounts of erythrocytes correlated with shorter intervention times (p ≤ 0.001). ROC analysis revealed reliable prediction of secondary embolisms for low rHU (AUC = 0.746; p ≤ 0.0001), low amounts of RBC (AUC = 0.764; p ≤ 0.0001), and high amounts of fibrin (AUC = 0.773; p ≤ 0.0001). CONCLUSIONS: Fibrin-rich thrombi with low erythrocyte percentage are significantly associated with longer intervention times. Embolisms in the thrombectomy process occur more often in thrombi with a small fraction of red blood cells and a low CT-density, suggesting a higher fragility of these thrombi.

In vivo Imaging of the Cerebral Endothelial Glycocalyx in Mice.

BACKGROUND/AIMS: Endothelial glycocalyx refers to the proteoglycan or glycoprotein layer of vessel walls and has critical physiological functions. Cerebral glycocalyx may have additional functions considering the blood-brain barrier and other features. However, the assessment of it has only been performed ex vivo, which includes processes presumably damaging the glycocalyx layer. Here we visualize and characterize the cerebral endothelial glycocalyx in vivo. METHODS: We visualized and quantified the cerebral endothelial glycocalyx in vivo under a 2-photon microscope by tagging glycocalyx and vessel lumen with wheat germ agglutinin lectin and dextran, respectively. The radial intensity was analyzed to measure the thickness of the cerebral endothelial glycocalyx in each vessel type. RESULTS: Cerebral arteries and capillaries have an intact endothelial glycocalyx, but veins and venules do not. The thickness of the glycocalyx layer in pial arteries, penetrating arteries, and capillaries was different; however, it was not correlated with the vessel diameter within each vessel type. CONCLUSION: We characterized the distribution of the cerebral endothelial glycocalyx in vivo. Compared to the results from ex vivo studies, the layer is thicker, indicating that the layer may be damaged in ex vivo systems. We also observed an inhomogeneous cerebral endothelial glycocalyx distribution that might reflect the functional heterogeneity of the vessel type.

Proteomic differences in brain vessels of Alzheimer's disease mice: Normalization by PPARγ agonist pioglitazone.

Cerebrovascular insufficiency appears years prior to clinical symptoms in Alzheimer's disease. The soluble, highly toxic amyloid-ß species, generated from the amyloidogenic processing of amyloid precursor protein, are known instigators of the chronic cerebrovascular insufficiency observed in both Alzheimer's disease patients and transgenic mouse models. We have previously demonstrated that pioglitazone potently reverses cerebrovascular impairments in a mouse model of Alzheimer's disease overexpressing amyloid-ß. In this study, we sought to characterize the effects of amyloid-ß overproduction on the cerebrovascular proteome; determine how pioglitazone treatment affected the altered proteome; and analyze the relationship between normalized protein levels and recovery of cerebrovascular function. Three-month-old wildtype and amyloid precursor protein mice were treated with pioglitazone- (20 mg/kg/day, 14 weeks) or control-diet. Cerebral arteries were surgically isolated, and extracted proteins analyzed by gel-free and gel-based mass spectrometry. 193 cerebrovascular proteins were abnormally expressed in amyloid precursor protein mice. Pioglitazone treatment rescued a third of these proteins, mainly those associated with oxidative stress, promotion of cerebrovascular vasocontractile tone, and vascular compliance. Our results demonstrate that amyloid-ß overproduction perturbs the cerebrovascular proteome. Recovery of cerebrovascular function with pioglitazone is associated with normalized levels of key proteins in brain vessel function, suggesting that pioglitazone-responsive cerebrovascular proteins could be early biomarkers of Alzheimer's disease.

Clinical Significance of the Cerebral Perforating Arteries.

There is scarce data in the contemporary literature regarding the correlation of the microanatomy of the perforating arteries, their atherosclerosis, and the ischemia in their territory. In order to examine, at least partially, those parameters, the perforating arteries of 12 brains were microdissected or their vascular casts were obtained. In addition, 30 specimens of the perforators were used for a histological and immunohistochemical study. Finally, radiological images of 14 patients with deep cerebral infarcts were examined following a selection among 62 subjects. It was found out that certain groups of the perforators ranged in number between 0 to 11 (1.1-8.4 on average). In addition to the origin from the parent vessels, some of the perforators also arose from the leptomeningeal branches. Occlusion of such a branch may result in both a superficial and a deep ischemic lesion. Besides, the common stems of certain perforators supplied both right and left portions of the corresponding brain regions. Occlusion of such a common trunk leads to bilateral infarction. The atherosclerosis of the perforating vessels, which was found in one third of the specimens, is the basis for the ischemic lesions development on their territory. Among the 62 patients with ischemic lesions, 14 had a deep cerebral infarcts, most often within the thalamus, as well as on the territory of the middle cerebral and the anterior choroidal artery perforators of the hemispheres. Our study showed that a strong correlation exists between certain microanatomical features, atherosclerosis, and region of supply of the perforating arteries, on the one hand, and location of the ischemic lesions on the other hand.

Difference in the intratumoral distributions of extracellular-fluid and intravascular MR contrast agents in glioblastoma growth.

Contrast enhancement by an extracellular-fluid contrast agent (CA) (Gd-DOTA) depends primarily on the blood-brain-barrier permeability (bp), and transverse-relaxation change caused by intravascular T2 CA (superparamagnetic iron oxide nanoparticles, SPIONs) is closely associated with the blood volume (BV). Pharmacokinetic (PK) vascular characterization based on single-CA-using dynamic contrast-enhanced MRI (DCE-MRI) has shown significant measurement variation according to the molecular size of the CA. Based on this recognition, this study used a dual injection of Gd-DOTA and SPIONs for tracing the changes of bp and BV in C6 glioma growth (Days 1 and 7 after the tumor volume reached 2 mL). bp was quantified according to the non-PK parameters of Gd-DOTA-using DCE-MRI (wash-in rate, maximum enhancement ratio and initial area under the enhancement curve (IAUC)). BV was estimated by SPION-induced ΔR2 * and ΔR2 . With validated measurement reliability of all the parameters (coefficients of variation ≤10%), dual-contrast MRI demonstrated a different region-oriented distribution between Gd-DOTA and SPIONs within a tumor as follows: (a) the BV increased stepwise from the tumor center to the periphery; (b) the tumor periphery maintained the augmented BV to support continuous tumor expansion from Day 1 to Day 7; (c) the internal tumor area underwent significant vascular shrinkage (i.e. decreased ΔR2 and ΔR2 ) as the tumor increased in size; (d) the tumor center showed greater bp-indicating parameters, i.e. wash-in rate, maximum enhancement ratio and IAUC, than the periphery on both Days 1 and 7 and (e) the tumor center showed a greater increase of bp than the tumor periphery in tumor growth, as suggested to support tumor viability when there is insufficient blood supply. In the MRI-histologic correlation, a prominent BV increase in the tumor periphery seen in MRI was verified with increased fluorescein isothiocyanate-dextran signals and up-regulated immunoreactivity of CD31-VEGFR. In conclusion, the spatiotemporal alterations of BV and bp in glioblastoma growth, i.e. augmented BV in the tumor periphery and increased bp in the center, can be sufficiently evaluated by MRI with dual injection of extracellular-fluid Gd chelates and intravascular SPION.

Clinico-Radiological Characteristics and Pathological Diagnosis of Cerebral Amyloid Angiopathy-Related Intracerebral Hemorrhage.

OBJECTIVE: We aim to clarify the clinico-radiological characteristics of cerebral amyloid angiopathy-related intracerebral hemorrhage and to investigate the efficacy of pathological diagnosis using biopsy specimens. METHOD: We retrospectively reviewed 253 consecutive patients with cortico-subcortical hemorrhage who had been admitted to Aizawa Hospital between January 2006 and July 2013. We had performed craniotomy and hematoma evacuation in 48 patients, as well as biopsy of the evacuated hematoma, cerebral parenchyma adjacent to the hematoma, or both, and they were classified according to the histological results (positive or negative for vascular amyloid deposition) and to the Boston criteria. We compared the clinico-radiological characteristics of cerebral amyloid angiopathy-related intracerebral hemorrhage. We also investigated the detection rate of cerebral amyloid angiopathy with respect to the origins of the specimens. RESULTS: Pathological examination revealed that 22 subjects were positive for vascular amyloid. The number of the cerebral microbleeds located in the deep or infratentorial region was significantly larger in the negative group than in the positive group (P <.05). There was no significant difference in the distribution of lobar cerebral microbleeds and in the prevalence of hypertension. In the probable cerebral amyloid angiopathy-related intracerebral hemorrhage patients, the probability of having vascular amyloid detected by biopsy of both hematoma and parenchyma was 100%. Rebleeding in the postoperative periods was observed in 2 cases (9.1%) of the positive group. CONCLUSIONS: Our results demonstrate the importance and safety of biopsy simultaneously performed with hematoma evacuation. Deep or infratentorial microbleeds are less correlated with cerebral amyloid angiopathy-related intracerebral hemorrhage than with noncerebral amyloid angiopathy-related intracerebral hemorrhage.

Deficiency of endothelium-specific transcription factor Sox17 induces intracranial aneurysm.

BACKGROUND: Intracranial aneurysm (IA) is a common vascular disorder that frequently leads to fatal vascular rupture. Although various acquired risk factors associated with IA have been identified, the hereditary basis of IA remains poorly understood. As a result, genetically modified animals accurately modeling IA and related pathogenesis have been lacking, and subsequent drug development has been delayed. METHODS AND RESULTS: The transcription factor Sox17 is robustly expressed in endothelial cells of normal intracerebral arteries. The combination of Sox17 deficiency and angiotensin II infusion in mice induces vascular abnormalities closely resembling the cardinal features of IA such as luminal dilation, wall thinning, tortuosity, and subarachnoid hemorrhages. This combination impairs junctional assembly, cell-matrix adhesion, regeneration capacity, and paracrine secretion in endothelial cells of intracerebral arteries, highlighting key endothelial dysfunctions that lead to IA pathogenesis. Moreover, human IA samples showed reduced Sox17 expression and impaired endothelial integrity, further strengthening the applicability of this animal model to clinical settings. CONCLUSIONS: Our findings demonstrate that Sox17 deficiency in mouse can induce IA under hypertensive conditions, suggesting Sox17 deficiency as a potential genetic factor for IA formation. The Sox17-deficient mouse model provides a novel platform to develop therapeutics for incurable IA.

Proteomic identification of differentially expressed proteins in vascular wall of patients with ruptured intracranial aneurysms.

OBJECTIVE: Intracranial aneurysms (IA) are serious cerebral vascular abnormalities, however, little is known about the mechanisms underlying IA formation, progression and rupture. Therefore, this study aimed to assess protein expression specific to the vascular tissues of IA patients. METHODS: IA samples were intraoperatively collected from 14 patients after microneurosurgical clipping and pooled. Matched superficial temporal artery (STA) tissues collected from the same patients were used as controls. Differentially expressed proteins were identified using isobaric tags for relative and absolute quantification (iTRAQ) and two-dimensional liquid chromatography-tandem mass spectrometry (2D LC-MS/MS), validated by immunoblot. RESULTS: 816 proteins were found to be differently expressed in IA and healthy tissues. The expression level of 162 proteins differed by at least two fold. Expression of 80 proteins was up-regulated and expression of 82 was down-regulated. According to PANTHER, these proteins were involved in immune responses, cell adhesion, cellular component organization and developmental processes. Azurocidin-1 (AZU1, a known antimicrobial) and Transmembrane 9 superfamily member 1 (TM9SF1, a novel autophagy-related protein) were 8.0 and 8.6 fold up-regulated, respectively, in IA, while Sorbin and SH3 domain-containing protein 2 (SORBS2), involved in signaling complex assembly, was 12.1 fold down-regulated. CONCLUSION: These findings suggest that IA formation and rupture might be related to autophagy and immune responses, which possibly accounts for proteolytic degradation of vessel wall connective tissues and cytoskeleton components.

Cerebral arterial pulsation drives paravascular CSF-interstitial fluid exchange in the murine brain.

CSF from the subarachnoid space moves rapidly into the brain along paravascular routes surrounding penetrating cerebral arteries, exchanging with brain interstitial fluid (ISF) and facilitating the clearance of interstitial solutes, such as amyloid ß, in a pathway that we have termed the "glymphatic" system. Prior reports have suggested that paravascular bulk flow of CSF or ISF may be driven by arterial pulsation. However, cerebral arterial pulsation could not be directly assessed. In the present study, we use in vivo two-photon microscopy in mice to visualize vascular wall pulsatility in penetrating intracortical arteries. We observed that unilateral ligation of the internal carotid artery significantly reduced arterial pulsatility by ~50%, while systemic administration of the adrenergic agonist dobutamine increased pulsatility of penetrating arteries by ~60%. When paravascular CSF-ISF exchange was evaluated in real time using in vivo two-photon and ex vivo fluorescence imaging, we observed that internal carotid artery ligation slowed the rate of paravascular CSF-ISF exchange, while dobutamine increased the rate of paravascular CSF-ISF exchange. These findings demonstrate that cerebral arterial pulsatility is a key driver of paravascular CSF influx into and through the brain parenchyma, and suggest that changes in arterial pulsatility may contribute to accumulation and deposition of toxic solutes, including amyloid ß, in the aging brain.

Giant intracranial aneurysm embolization with a yield stress fluid material: insights from CFD analysis.

The endovascular treatment of intracranial aneurysms remains a challenge, especially when the aneurysm is large in size and has irregular, non-spherical geometry. In this paper, we use computational fluid dynamics to simulate blood flow in a vertebro-basilar junction giant aneurysm for the following three cases: (1) an empty aneurysm, (2) an aneurysm filled with platinum coils, and (3) an aneurysm filled with a yield stress fluid material. In the computational model, blood and the coil-filled region are treated as a non-Newtonian fluid and an isotropic porous medium, respectively. The results show that yield stress fluids can be used for aneurysm embolization provided the yield stress value is 20 Pa or higher. Specifically, flow recirculation in the aneurysm and the size of the inflow jet impingement zone on the aneurysm wall are substantially reduced by yield stress fluid treatment. Overall, this study opens up the possibility of using yield stress fluids for effective embolization of large-volume intracranial aneurysms.

Search for varicella zoster virus and herpes simplex virus-1 in normal human cerebral arteries.

Virological confirmation of varicella zoster virus (VZV) vasculopathy is provided by presence of virus in the cerebral arteries, frequently associated with inflammation. Yet, cerebral arteries from normal subjects have never been studied for VZV DNA or antigen. We analyzed 63 human cerebral arteries from 45 subjects for VZV DNA and antigen, control herpes simplex virus (HSV)-1 DNA and antigen, and leukocyte-specific CD45 antigen. No cerebral arteries contained VZV or HSV-1 DNA or antigen; eight arteries from seven subjects contained leukocytes expressing CD45. Thus, the presence of VZV antigen in cerebral arteries of patients with stroke is likely to be clinically significant.

Effects of selective head cooling on cerebral blood flow and metabolism in newborn piglets after hypoxia-ischemia.

AIM: the effect of selective head cooling on cerebral blood flow (CBF) and cerebral metabolism rate (CMR) was investigated in newborn piglets. METHODS: seven days old newborn piglets were randomly assigned to one of the following three groups: Selective head cooling in normal piglets (n=4), selective head cooling after HI (n=6) and normal temperature after HI (n=6). CBF was measured with color microspheres. Cerebral oxygenation metabolism rate (CMRO(2)), Cerebral glucose consumption (CMR(Glu)) and Cerebral lactate production (CMR(lac)) were calculated. RESULT: in normal piglets, CBF, CMRO(2) and CMR(glu) were significantly decreased at both 35°C (P<0.05) and 32°C (P<0.01), while CMR(lac) did not change. Compared to baseline, CBF and CMRO(2) were significantly reduced (P<0.05), while CMR(glu) and CMR(lac) were significantly increased (P<0.01), AVDO(2) was decreased (P<0.05), while AVD(glu) and AVD(lac) were significantly increased (P<0.01 respectively) in HI piglets with normal temperature respectively. Compared to normal temperature after HI, selective head cooling after HI significantly reduced CMR(glu) and CMR(lac), and AVDO(2), AVD(glu), AVD(lac) were improved at 35°C. CONCLUSION: selective head cooling not only reduced energy consumption, but also improve brain oxygen metabolism in newborn after HI.

Subtype identification and functional characterization of ryanodine receptors in rat cerebral artery myocytes.

Ryanodine receptors (RyRs) regulate contractility in resistance-size cerebral artery smooth muscle, yet their molecular identity, subcellular location, and phenotype in this tissue remain unknown. Following rat resistance-size cerebral artery myocyte sarcoplasmic reticulum (SR) purification and incorporation into POPE-POPS-POPC (5:3:2; wt/wt) bilayers, unitary conductances of 110 +/- 8, 334 +/- 15, and 441 +/- 27 pS in symmetric 300 mM Cs(+) were usually detected. The most frequent (34/40 bilayers) conductance (334 pS) decreased to

Tumor necrosis factor-alpha does not mediate diabetes-induced vascular inflammation in mice.

OBJECTIVE: Vascular inflammation is a key feature of both micro- and macrovascular complications in diabetes. Several lines of evidence have implicated the cytokine tumor necrosis factor (TNF) alpha as an important mediator of inflammation in diabetes. In the present study we evaluated the role of TNF alpha in streptozotocin (STZ)-induced diabetes on vascular inflammation in C57BL/6 wild-type and apoE-/- mice. METHODS AND RESULTS: Diabetes increased the expression of vascular cell adhesion molecule (VCAM)-1 in cerebral arteries 150 m in diameter as well as the macrophage accumulation in aortic root atherosclerotic plaques in apoE-/- mice. A more pronounced vascular inflammatory response was observed in diabetic TNF alpha-deficient apoE-/- mice. These mice were also characterized by increased accumulation of IgG and IgM autoantibodies in atherosclerotic lesions. Diabetes also increased VCAM-1 expression and plaque formation in apoE-competent TNF alpha -/- mice, whereas no such effects were observed in C57BL/6 wild-type mice. CONCLUSIONS: The present findings suggest that TNF alpha does not mediate diabetic-induced vascular inflammation in mice and reveal an unexpected protective role for TNF alpha. These effects are partly attributable to a direct antiinflammatory role of TNF alpha, but may also reflect a defective development of the immune system in these mice.

Occludin oligomeric assemblies at tight junctions of the blood-brain barrier are altered by hypoxia and reoxygenation stress.

Hypoxic (low oxygen) and reperfusion (post-hypoxic reoxygenation) phases of stroke promote an increase in microvascular permeability at tight junctions (TJs) of the blood-brain barrier (BBB) that may lead to cerebral edema. To investigate the effect of hypoxia (Hx) and reoxygenation on oligomeric assemblies of the transmembrane TJ protein occludin, rats were subjected to either normoxia (Nx, 21% O(2), 60 min), Hx (6% O(2), 60 min), or hypoxia/reoxygenation (H/R, 6% O(2), 60 min followed by 21% O(2), 10 min). After treatment, cerebral microvessels were isolated, fractionated by detergent-free density gradient centrifugation, and occludin oligomeric assemblies associated with plasma membrane lipid rafts were solubilized by perfluoro-octanoic acid (PFO) exclusively as high molecular weight protein complexes. Analysis by non-reducing and reducing sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis/western blot of PFO-solubilized occludin revealed that occludin oligomeric assemblies co-localizing with 'TJ-associated' raft domains contained a high molecular weight 'structural core' that was resistant to disassembly by either SDS or a hydrophilic reducing agent ex vivo, and by Hx and H/R conditions in vivo. However, exposure of PFO-solubilized occludin oligomeric assemblies to SDS ex vivo revealed the non-covalent association of a significant amount of dimeric and monomeric occludin isoforms to the disulfide-bonded inner core, and dispersal of these non-covalently attached occludin subunits to lipid rafts of higher density in vivo was differentially promoted by Hx and H/R. Our data suggest a model of isoform interaction within occludin oligomeric assemblies at the BBB that enables occludin to simultaneously perform a structural role in inhibiting paracellular diffusion, and a signaling role involving interactions of dimeric and monomeric occludin isoforms with a variety of regulatory molecules within different plasma membrane lipid raft domains.

Altered structure of small cerebral arteries in patients with essential hypertension.

OBJECTIVE: Structural alterations in the microcirculation may be considered an important mechanism of organ damage. An increased media-to-lumen ratio of subcutaneous small resistance arteries has been demonstrated to predict the development of cardiocerebrovascular events in hypertensive patients. Alterations in the structure of small cerebral arteries have been demonstrated in animal models of experimental or genetic hypertension. However, no evaluation with reliable techniques has ever been performed in humans. DESIGN AND METHODS: Twenty-eight participants were included in the present study: they were 13 hypertensive patients and 15 normotensive individuals. All participants underwent a neurosurgical intervention for benign or malign tumors. A small portion of morphologically normal cerebral tissue was excised from surgical samples and examined. Cerebral small resistance arteries (relaxed diameter around 200 mum) were dissected and mounted on an isometric and isobaric myograph, and the tunica media to internal lumen ratio was measured. In addition, cerebral cortical microvessel density (MVD) was also evaluated. The tissue was sectioned and stained for CD31, and MVD was measured with an automated image analyzer (percentage of area stained). Blood pressure values were evaluated, before surgical intervention, by standard sphygmomanometry. RESULTS: M/L was significantly greater and MVD significantly lower in hypertensive patients than that in normotensive individuals. No difference between groups in collagen content or mechanical properties of cerebral small arteries was observed. CONCLUSION: Our results indicate that structural alterations of small cerebral vessels are present in hypertensive patients compared with normotensive individuals, similar to those previously observed in subcutaneous small arteries.