Journal of Cerebral Blood Flow & Metabolism

T₂^*-weighted MRI of transient oxygen challenge (OC) showed exaggerated OC percent changes in the ischemic tissue at risk compared to normal tissue. One ambiguity is that regions with high vascular density also showed exaggerated OC percent changes. This study explored time-to-peak (TTP) of the OC percent changes to improve the utility of T₂^*-weighted OC MRI. Experiments were performed longitudinally at 30 min, 150 min and 24 h after transient (60-min) stroke in rats. Ischemic core, normal, and mismatch tissue were classified pixel-by-pixel based on apparent diffusion coefficient and cerebral blood flow. Major findings were: (i) Delayed OC TTP was localized to and corresponded well with the perfusion-diffusion mismatch. (ii) By contrast, the exaggerated OC percent changes were less localized, with changes not only in the at-risk tissue but also in some areas of the contralesional hemisphere with venous vessel origins. (iii) The OC time-course of the mismatch tissue was biphasic, with a faster initial increase followed by a slower increase. (iv) At-risk tissue with delayed TTP and exaggerated OC was normal after reperfusion and the at-risk tissue was mostly (83 ± 18%) rescued by reperfusion as indicated by normal 24-h T₂. OC TTP offers unique information toward better characterization of at-risk tissue in ischemic stroke.

Glutamate excitotoxicity is a primary contributor of ischemic neuronal death and other cellular components of the neurovascular unit. Several strategies have been developed against glutamate excitotoxicity, however none of them have not shown positive results in the clinical practice so far. Nowadays, the concept of blood/brain glutamate grabbing or scavenging is well recognized as a novel and attractive protective strategy to reduce the excitotoxic effect of excess extracellular glutamate that accumulates in the brain following an ischemic stroke. The main advantage of this novel therapeutic strategy is that it occurs in the blood circulation and therefore does not affect the normal brain neurophysiology, as it has been described for other drug treatments used against glutamate excitotoxicity. In this work we report all experimental data from the beginning of our studies, focused on stroke pathology, and we describe new findings about the potential application of this therapy. Future clinical trials will allow to know the real efficacy of this novel therapeutic strategy in stroke patients.

Cerebral small vessel disease (SVD) gives rise to one in five strokes worldwide and constitutes a major source of cognitive decline in the elderly. SVD is known to occur in relation to hypertension, diabetes, smoking, radiation therapy and in a range of inherited and genetic disorders, autoimmune disorders, connective tissue disorders, and infections. Until recently, changes in capillary patency and blood viscosity have received little attention in the aetiopathogenesis of SVD and the high risk of subsequent stroke and cognitive decline. Capillary flow patterns were, however, recently shown to limit the extraction efficacy of oxygen in tissue and capillary dysfunction therefore proposed as a source of stroke-like symptoms and neurodegeneration, even in the absence of physical flow-limiting vascular pathology. In this review, we examine whether capillary flow disturbances may be a shared feature of conditions that represent risk factors for SVD. We then discuss aspects of capillary dysfunction that could be prevented or alleviated and therefore might be of general benefit to patients at risk of SVD, stroke or cognitive decline.

Full kinetic modeling of dynamic PET images requires the measurement of radioligand concentrations in the arterial plasma. The unchanged parent radioligand must, however, be separated from its radiometabolites by chromatographic methods. Thus, only few samples can usually be analyzed and the resulting measurements are often noisy. Therefore, the measurements must be fitted with a mathematical model. This work presents a comprehensive analysis of the different models proposed in the literature to describe the plasma parent fraction (PPf) and of the alternative approaches for radiometabolite correction. Finally, we used a dataset of [¹¹C]PBR28 brain PET data as a case study to guide the reader through the PPf model selection process.

Normal brain homeostasis depends on the integrity of the blood–brain barrier that controls the access of nutrients, humoral factors, and immune cells to the CNS. The blood–brain barrier is composed mainly of brain endothelial cells. Forming the interface between two compartments, they are highly polarized. Apical/luminal and basolateral/abluminal membranes differ in their lipid and (glyco-)protein composition, allowing brain endothelial cells to secrete or transport soluble factors in a polarized manner and to maintain blood flow. Here, we summarize the basic concepts of apicobasal cell polarity in brain endothelial cells. To address potential molecular mechanisms underlying apicobasal polarity in brain endothelial cells, we draw on investigations in epithelial cells and discuss how polarity may go awry in neurological diseases.

The clinical relevance of the transient intraluminal filament model of middle cerebral artery occlusion (tMCAO) has been questioned due to distinct cerebral blood flow profiles upon reperfusion between tMCAO (abrupt reperfusion) and alteplase treatment (gradual reperfusion), resulting in differing pathophysiologies. Positive results from recent endovascular thrombectomy trials, where the occluding clot is mechanically removed, could revolutionize stroke treatment. The rapid cerebral blood flow restoration in both tMCAO and endovascular thrombectomy provides clinical relevance for this pre-clinical model. Any future clinical trials of neuroprotective agents as adjuncts to endovascular thrombectomy should consider tMCAO as the model of choice to determine pre-clinical efficacy.

Many MRI techniques require prior knowledge of the T1-relaxation time of blood (T_1bl). An assumed/fixed value is often used; however, T_1bl is sensitive to magnetic field (B₀), haematocrit (Hct), and oxygen saturation (Y). We aimed to combine data from previous in vitro measurements into a mathematical model, to estimate T_1bl as a function of B₀, Hct, and Y. The model was shown to predict T_1bl from in vivo studies with a good accuracy (±87 ms). This model allows for improved estimation of T_1bl between 1.5–7.0 T while accounting for variations in Hct and Y, leading to improved accuracy of MRI-derived perfusion measurements.

This Brief Communication reports early homogenization of capillary network flow during somatosensory activation in the rat cerebral cortex. We used optical coherence tomography and statistical intensity variation analysis for tracing changes in the red blood cell flux over hundreds of capillaries nearly at the same time with 1-s resolution. We observed that while the mean capillary flux exhibited a typical increase during activation, the standard deviation of the capillary flux exhibited an early decrease that happened before the mean flux increase. This network-level data is consistent with the theoretical hypothesis that capillary flow homogenizes during activation to improve oxygen delivery.

This is the first study to assess the influence of sex on the evolution of ischaemic injury and penumbra. Permanent middle cerebral artery occlusion was induced in male (n = 9) and female (n = 10) Sprague-Dawley rats. Diffusion-weighted imaging was acquired over 4 h and infarct determined from T₂ images at 24 h post-permanent middle cerebral artery occlusion. Penumbra was determined retrospectively from serial apparent diffusion coefficient lesions and T₂-defined infarct. Apparent diffusion coefficient lesion volume was significantly smaller in females from 0.5 to 4 h post permanent middle cerebral artery occlusion as was infarct volume. Penumbral volume, and its loss over time, was not significantly different despite the sex difference in acute and final lesion volumes.

The blood–brain barrier is composed of cerebrovascular endothelial cells and tight junctions, and maintaining its integrity is crucial for the homeostasis of the neuronal environment. Recently, we discovered that mitochondria play a critical role in maintaining blood–brain barrier integrity. We report for the first time a novel mechanism underlying blood–brain barrier integrity: miR-34a mediated regulation of blood–brain barrier through a mitochondrial mechanism. Bioinformatics analysis suggests miR-34a targets several mitochondria-associated gene candidates. We demonstrated that miR-34a triggers the breakdown of blood–brain barrier in cerebrovascular endothelial cell monolayer in vitro, paralleled by reduction of mitochondrial oxidative phosphorylation and adenosine triphosphate production, and decreased cytochrome c levels.

To evaluate the effect of age on the response of brains to an ischemic challenge, we subjected young and aged mice to transient forebrain ischemia, and analyzed the heat shock response and unfolded protein response, ubiquitin conjugation and SUMO conjugation, and O-linked β-N-acetylglucosamine modification of proteins (O-GlcNAcylation). The most prominent age-related difference was an inability of aged mice to activate O-GlcNAcylation. Considering many reports on the protective role of O-GlcNAcylation in various stress conditions including myocardial ischemia, this pathway could be a promising target for therapeutic intervention to improve functional recovery of aged patients following brain ischemia.

Hypertension and cerebral amyloid angiopathy (CAA) are major risk factors for intracerebral hemorrhage (ICH); however the mechanisms of interplay between the two are not fully understood. We investigated the effect of hypertension in a transgenic mouse model with Alzheimer’s-like pathology (Tg2576) treating them with angiontensin II and L-N^G-nitroarginine methyl ester. A similar increase in systolic blood pressure was observed in both Tg2576 and control mice; however Tg2576 mice developed signs of stroke with a markedly shorter latency. Cerebral deposition of amyloid beta promotes the hypertension-induced ICH, thus supporting the notion that hypertension is a risk factor for ICH among patients with CAA.

We tested whether nanoliposomes containing phosphatidylcholine, cholesterol and phosphatidic acid (NLPA) prevent β-amyloid 1-42 (Aβ42) fibrillation and Aβ42-induced human arteriole endothelial dysfunction. NLPA abolished Aβ42 fibril formation (thioflavin-T fluorescence/electron microscopy). In ex-vivo human adipose and leptomeningeal arterioles, Aβ42 impaired dilator response to acetylcholine that was reversed by NLPA; this protection was abolished by L-NG-nitroarginine methyl ester. Aβ42 reduced human umbilical vein endothelial cell NO production that was restored by NLPA. Nanoliposomes prevented Aβ42 amyloid formation, reversed Aβ42-induced human microvascular endothelial dysfunction and may be useful in Alzheimer’s disease.

Following ischemia, the blood–brain barrier is compromised in the peri-infarct zone leading to secondary injury and dysfunction that can limit recovery. Currently, it is uncertain what structural changes could account for blood–brain barrier permeability, particularly with aging. Here we examined the ultrastructure of early and delayed changes (3 versus 72 h) to the blood–brain barrier in young adult and aged mice (3–4 versus 18 months) subjected to photothrombotic stroke. At both time points and ages, permeability was associated with a striking increase in endothelial caveolae and vacuoles. Tight junctions were generally intact although small spaces were detected in a few cases. In young mice, ischemia led to a significant increase in pericyte process area and vessel coverage whereas these changes were attenuated with aging. Stroke led to an expansion of the basement membrane region that peaked at 3 h and partially recovered by 72 h in both age groups. Astrocyte endfeet and their mitochondria were severely swollen at both times points and ages. Our results suggest that blood–brain barrier permeability in young and aged animals is mediated by transcellular pathways (caveolae/vacuoles), rather than tight junction loss. Further, our data indicate that the effects of ischemia on pericytes and basement membrane are affected by aging.

The conjugation/de-conjugation of Small Ubiquitin-like Modifier (SUMO) has been shown to be associated with a diverse set of physiologic/pathologic conditions. The clinical significance and ostensible therapeutic utility offered via the selective control of the global SUMOylation process has become readily apparent in ischemic pathophysiology. Herein, we describe the development of a novel quantitative high-throughput screening (qHTS) system designed to identify small molecules capable of increasing SUMOylation via the regulation/inhibition of members of the microRNA (miRNA)-182 family. This assay employs a SHSY5Y human neuroblastoma cell line stably transfected with a dual firefly-Renilla luciferase reporter system for identification of specific inhibitors of either miR-182 or miR-183. In this study, we have identified small molecules capable of inducing increased global conjugation of SUMO in both SHSY5Y cells and rat E18-derived primary cortical neurons. The protective effects of a number of the identified compounds were confirmed via an in vitro ischemic model (oxygen/glucose deprivation). Of note, this assay can be easily repurposed to allow high-throughput analyses of the potential drugability of other relevant miRNA(s) in ischemic pathobiology.

Brain uptake of [¹⁴C]acetate has been reported to be a useful marker of astrocytic energy metabolism. In addition to uptake values, the rate of radiolabeled acetate washout from the brain appears to reflect CO₂ exhaustion and oxygen consumption in astrocytes. We measured the time–radioactivity curves of benzyl [1-¹⁴C]acetate ([1-¹⁴C]BA), a lipophilic probe of [1-¹⁴C]acetate, and compared it with that of benzyl [2-¹⁴C]acetate ([2-¹⁴C]BA) in rat brains. The highest brain uptake was observed immediately after injecting either [1-¹⁴C]BA or [2-¹⁴C]BA, and both subsequently disappeared from the brain in a single-exponential manner. Estimated [1-¹⁴C]BA washout rates in the cerebral cortex and cerebellum were higher than those of [2-¹⁴C]BA. These results suggested that [1-¹⁴C]BA could be a useful probe for estimating the astrocytic oxidative metabolism. The [1-¹⁴C]BA washout rate in the cerebral cortex of immature rats was lower than that of mature rats. An autoradiographic study showed that the washout rates of [1-¹⁴C]BA from the rat brains of a lithium–pilocarpine-induced status epilepticus model were not significantly different from the values in control rat brains except for the medial septal nucleus. These results implied that the enhancement of amino acid turnover rate rather than astrocytic oxidative metabolism was increased in status epilepticus.

Pericytes, spatially isolated contractile cells on capillaries, have been reported to control cerebral blood flow physiologically, and to limit blood flow after ischaemia by constricting capillaries and then dying. Paradoxically, a recent paper dismisses the idea of pericytes controlling cerebral blood flow, despite confirming earlier data showing a role for pericytes. We show that these discrepancies are apparent rather than real, and depend on the new paper defining pericytes differently from previous reports. An objective definition of different sub-classes of pericyte along the capillary bed is needed to develop novel therapeutic approaches for stroke and disorders caused by pericyte malfunction.

Pyroptotic neuronal cell death mediated by the AIM2 inflammasome

Adamczak SE, de Rivero Vaccari JP, Dale G, Brand FJ 3rd, Nonner D, Bullock MR, Dahl GP, Dietrich WD, Keane RW

Journal of Cerebral Blood Flow & Metabolism 2014; 34: 621–629.

The TNF-α/NF-κB signaling pathway has a key role in methamphetamine–induced blood–brain barrier dysfunction

Vanessa Coelho-Santos, Ricardo A Leitão, Filipa L Cardoso, Inês Palmela, Manuel Rito, Marcos Barbosa, Maria A Brito, Carlos A Fontes-Ribeiro and Ana P Silva

Journal of Cerebral Blood Flow & Metabolism 2015; 35: 1260–1271.