Comment on "Inflammasome inhibition prevents α-synuclein pathology and dopaminergic neurodegeneration in mice".

We highlight potential issues with a Western blot assay using mouse anti-caspase-1 antibody to detect cleaved caspase-1 p20 subunit in mouse brain tissue lysates.

An In Vitro Bone Model to Investigate the Role of Triggering Receptor Expressed on Myeloid Cells-2 in Bone Homeostasis.

Triggering receptor expressed on myeloid cells-2 (TREM-2), a transmembrane receptor expressed by macrophages, microglia, and osteoclasts (OCs), plays a protective role in late-onset Alzheimer Disease (AD). To validate TREM-2 as a therapeutic target in AD, its potential secondary parallel effect on bone homeostasis should be clarified. However, animal models and monolayer cultures of human cells were shown poorly predictive of TREM-2 function in human. Therefore, this study aimed to engineer a tridimensional in vitro model using human progenitors differentiated into osteoblasts and OCs, recapitulating physiological bone homeostasis. Human bone marrow-derived mesenchymal cells were seeded and cultured under perfusion inside a collagen type I scaffold for 3 weeks, generating osteoblasts and mineralized matrix. Human peripheral blood-derived CD14+ monocytes were subsequently seeded through the generated tissue, thanks to perfusion flow, and further cultured for up to 3 weeks with an inductive medium, generating mature OCs. This culture system supported collagenous matrix deposition and resorption, allowing for the investigation of kinetic of soluble TREM-2 over the coculture time. Agonistic activation of TREM-2 in this model had no effect on OC activity or on mineralized matrix turnover. In conclusion, the engineered culture system represents a tridimensional, in vitro human bone model for drug testing and suggested no effect of TREM-2 agonist on bone resorption.

The FTD-like syndrome causing TREM2 T66M mutation impairs microglia function, brain perfusion, and glucose metabolism.

Genetic variants in the triggering receptor expressed on myeloid cells 2 (TREM2) increase the risk for several neurodegenerative diseases including Alzheimer's disease and frontotemporal dementia (FTD). Homozygous TREM2 missense mutations, such as p.T66M, lead to the FTD-like syndrome, but how they cause pathology is unknown. Using CRISPR/Cas9 genome editing, we generated a knock-in mouse model for the disease-associated Trem2 p.T66M mutation. Consistent with a loss-of-function mutation, we observe an intracellular accumulation of immature mutant Trem2 and reduced generation of soluble Trem2 similar to patients with the homozygous p.T66M mutation. Trem2 p.T66M knock-in mice show delayed resolution of inflammation upon in vivo lipopolysaccharide stimulation and cultured macrophages display significantly reduced phagocytic activity. Immunohistochemistry together with in vivo TSPO small animal positron emission tomography (µPET) demonstrates an age-dependent reduction in microglial activity. Surprisingly, perfusion magnetic resonance imaging and FDG-µPET imaging reveal a significant reduction in cerebral blood flow and brain glucose metabolism. Thus, we demonstrate that a TREM2 loss-of-function mutation causes brain-wide metabolic alterations pointing toward a possible function of microglia in regulating brain glucose metabolism.

Genome-Wide Analysis of the Circulating miRNome After Cerebral Ischemia Reveals a Reperfusion-Induced MicroRNA Cluster.

BACKGROUND AND PURPOSE: Circulating microRNAs (miRNAs) are emerging biomarkers for stroke because of their high stability in the bloodstream and association with pathophysiologic conditions. However, the circulating whole-genome miRNAs (miRNome) has not been characterized comprehensively in the acute phase of stroke. METHODS: We profiled the circulating miRNome in mouse models of acute ischemic and hemorrhagic stroke by next-generation sequencing. Stroke models were compared with sham-operated and naive mice to identify deregulated circulating miRNAs. Top-ranked miRNAs were validated and further characterized by quantitative reverse transcription polymerase chain reaction. RESULTS: We discovered 24 circulating miRNAs with an altered abundance in the circulation 3 hours after ischemia, whereas the circulating miRNome was not altered after intracerebral hemorrhage compared with sham-operated mice. Among the upregulated miRNAs in ischemia, the top-listed miR-1264/1298/448 cluster was strongly dependent on reperfusion in different ischemia models. A time course experiment revealed that the miR-1264/1298/448 cluster peaked in the circulation around 3 hours after reperfusion and gradually decreased thereafter. CONCLUSIONS: Alteration of the miRNome in the circulation is associated with cerebral ischemia/reperfusion, but not hemorrhage, suggesting a potential to serve as biomarkers for reperfusion in the acute phase. The pathophysiological role of reperfusion-inducible miR-1264/1298/448 cluster, which is located on chromosome X within the introns of the serotonin receptor HTR2C, requires further investigation.

A Mouse Model of Post-Stroke Pneumonia Induced by Intra-Tracheal Inoculation with Streptococcus pneumoniae.

BACKGROUND: Stroke-induced immunodeficiency increases the risk of infectious complications, which adversely affects neurological outcome. Among those, pneumonia affects as many as one third of stroke patients and is the main contributor to mortality in the post-acute phase of stroke. Experimental findings on post-stroke susceptibility to spontaneous pneumonia in mice are contradictory. Here, we established a mouse model inducing standardized bacterial pneumonia and characterized the impaired pulmonary cellular and humoral immune responses after experimental stroke. METHODS: Bacterial pneumonia was induced by intra-tracheal inoculation with Streptococcus pneumoniae at different time points after transient middle cerebral artery occlusion (MCAO). Bacterial counts in lungs and blood, histological changes, and cytokine production in the lungs were assessed. Furthermore, we investigated the effect of pneumonia on stroke outcome. RESULTS: Intra-tracheal inoculation resulted in reproducible pneumonia and bacteraemia, and demonstrated post-stroke susceptibility to streptococcal pneumonia developing with a delay of at least 24 h after MCAO. Higher bacterial counts in mice infected 3 days after stroke induction correlated with reduced neutrophil and macrophage infiltration in the lungs and lower levels of pro-inflammatory cytokines in the broncho-alveolar lavage compared to sham-operated animals. Pneumonia increased mortality without affecting brain-infiltrating leukocytes. CONCLUSIONS: In this standardized mouse model of post-stroke pneumonia, we describe attenuated leukocyte infiltration and cytokine production in response to bacterial infection in the lungs that has a profound effect on outcome.

Idarucizumab Improves Outcome in Murine Brain Hemorrhage Related to Dabigatran.

Lack of specific antidotes is a major concern in intracerebral hemorrhage (ICH) related to direct anticoagulants including dabigatran (OAC-ICH). We examined the efficacy of idarucizumab, an antibody fragment binding to dabigatran, in a mouse model of OAC-ICH. Dabigatran etexilate (DE) dose-dependently prolonged diluted thrombin time and tail-vein bleeding time, which were reversed by idarucizumab. Pretreatment with DE increased intracerebral hematoma volume and cerebral hemoglobin content. Idarucizumab in equimolar dose prevented excess hematoma expansion for both DE doses. In more extensive ICH, idarucizumab significantly reduced mortality. Thus, idarucizumab prevents excess intracerebral hematoma formation in mice anticoagulated with dabigatran and reduces mortality.

DAMP signaling is a key pathway inducing immune modulation after brain injury.

Acute brain lesions induce profound alterations of the peripheral immune response comprising the opposing phenomena of early immune activation and subsequent immunosuppression. The mechanisms underlying this brain-immune signaling are largely unknown. We used animal models for experimental brain ischemia as a paradigm of acute brain lesions and additionally investigated a large cohort of stroke patients. We analyzed release of HMGB1 isoforms by mass spectrometry and investigated its inflammatory potency and signaling pathways by immunological in vivo and in vitro techniques. Features of the complex behavioral sickness behavior syndrome were characterized by homecage behavior analysis. HMGB1 downstream signaling, particularly with RAGE, was studied in various transgenic animal models and by pharmacological blockade. Our results indicate that the cytokine-inducing, fully reduced isoform of HMGB1 was released from the ischemic brain in the hyperacute phase of stroke in mice and patients. Cytokines secreted in the periphery in response to brain injury induced sickness behavior, which could be abrogated by inhibition of the HMGB1-RAGE pathway or direct cytokine neutralization. Subsequently, HMGB1-release induced bone marrow egress and splenic proliferation of bone marrow-derived suppressor cells, inhibiting the adaptive immune responses in vivo and vitro. Furthermore, HMGB1-RAGE signaling resulted in functional exhaustion of mature monocytes and lymphopenia, the hallmarks of immune suppression after extensive ischemia. This study introduces the HMGB1-RAGE-mediated pathway as a key mechanism explaining the complex postischemic brain-immune interactions.

Amplification of regulatory T cells using a CD28 superagonist reduces brain damage after ischemic stroke in mice.

BACKGROUND AND PURPOSE: Neuroinflammation plays an important role in ischemic brain injury. Regulatory T cells (Treg) are important endogenous immune modulators. We tested the hypothesis that Treg amplification with a CD28 superagonistic monoclonal antibody (CD28SA) reduces brain damage in murine cerebral ischemia. METHODS: Cerebral ischemia was induced by coagulation of the distal middle cerebral artery or by 60 minutes filament occlusion of the proximal middle cerebral artery in C57BL6 mice. 150 µg CD28SA was injected intraperitoneally 3 or 6 hours after ischemia onset. Outcome was determined by infarct volumetry and behavioral testing. Brain-infiltrating leukocyte subpopulations were analyzed by flow cytometry and immunohistochemistry 3 and 7 days after middle cerebral artery occlusion. RESULTS: CD28SA reduced infarct size in both models and attenuated functional deficit 7 days after stroke induction. Mice treated with CD28SA increased numbers of Treg in spleen and brain. Tregs were functionally active and migrated into the brain where they accumulated and proliferated in the peri-infarct area. More than 60% of brain infiltrating Treg produced interleukin-10 in CD28SA compared with 30% in control. CONCLUSIONS: In vivo expansion and amplification of Treg by CD28SA attenuates the inflammatory response and improves outcome after experimental stroke.

Neuroinflammation after intracerebral hemorrhage.

Spontaneous intracerebral hemorrhage (ICH) is a particularly severe type of stroke for which no specific treatment has been established yet. Although preclinical models of ICH have substantial methodological limitations, important insight into the pathophysiology has been gained. Mounting evidence suggests an important contribution of inflammatory mechanisms to brain damage and potential repair. Neuroinflammation evoked by intracerebral blood involves the activation of resident microglia, the infiltration of systemic immune cells and the production of cytokines, chemokines, extracellular proteases and reactive oxygen species (ROS). Previous studies focused on innate immunity including microglia, monocytes and granulocytes. More recently, the role of adaptive immune cells has received increasing attention. Little is currently known about the interactions among different immune cell populations in the setting of ICH. Nevertheless, immunomodulatory strategies are already being explored in ICH. To improve the chances of translation from preclinical models to patients, a better characterization of the neuroinflammation in patients is desirable.

Antigen dependently activated cluster of differentiation 8-positive T cells cause perforin-mediated neurotoxicity in experimental stroke.

Neuroinflammation plays a key role in secondary brain damage after stroke. Although deleterious effects of proinflammatory cytokines are well characterized, direct cytotoxic effects of invading immune cells on the ischemic brain and the importance of their antigen-dependent activation are essentially unknown. Here we examined the effects of adaptive and innate immune cells-cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells-that share the direct perforin-mediated cytotoxic pathway on outcome after cerebral ischemia in mice. Although CTLs and NK cells both invaded the ischemic brain, only brain-infiltrating CTLs but not NK cells were more activated than their splenic counterparts. Depletion of CTLs decreased infarct volumes and behavioral deficit in two ischemia models, whereas NK cell depletion had no effect. Correspondingly, adoptive CTL transfer from wild-type into Rag1 knock-out mice increased infarct size. Adoptive CTL transfer from perforin knock-out or interferon-γ knock-out mice into Rag1 knock-out mice revealed that CTL neurotoxicity was mediated by perforin. Accordingly, CTLs isolated from wild-type or interferon-γ knock-out but not from perforin knock-out mice induced neuronal cell death in vitro. CTLs derived from ovalbumin-specific T-cell receptor transgenic mice were not activated and infiltrated less into the ischemic brain compared with wild-type CTLs. Their transfer did not increase the infarct size of Rag1 knock-out mice, indicating antigen-dependent activation as an essential component of CTL neurotoxicity. Our findings underscore the importance of antigen-dependent, direct cytotoxic immune responses in stroke and suggest modulation of CTLs and their effector pathways as a potential new strategy for stroke therapy.

Differential effects of sympathetic nervous system and hypothalamic-pituitary-adrenal axis on systemic immune cells after severe experimental stroke.

Infectious complications are the leading cause of death in the post-acute phase of stroke. Post-stroke immunodeficiency is believed to result from neurohormonal dysregulation of the sympathetic nervous system (SNS) and hypothalamic-pituitary-adrenal (HPA) axis. However, the differential effects of these neuroendocrine systems on the peripheral immune cells are only partially understood. Here, we determined the impact of the hormones of the SNS and HPA on distinct immune cell populations and characterized their interactions after stroke. At various time points after cortical or extensive hemispheric cerebral ischemia, plasma cortisone, corticosterone, metanephrine and adrenocorticotropic hormone (ACTH) levels were measured in mice. Leukocyte subpopulations were flow cytometrically analyzed in spleen and blood. To investigate their differential sensitivity to stress hormones, splenocytes were incubated in vitro with prednisolone, epinephrine and their respective receptor blockers. Glucocorticoid receptor (GCR) and beta2-adrenergic receptor (ß2-AR) on leukocyte subpopulations were quantified by flow cytometry. In vivo effects of GCR and selective ß2-AR blockade, respectively, were defined on serum hormone concentrations, lymphopenia and interferon-γ production after severe ischemia. We found elevated cortisone, corticosterone and metanephrine levels and associated lymphocytopenia only after extensive brain infarction. Prednisolone resulted in a 5 times higher cell death rate of splenocytes than epinephrine in vitro. Prednisolone and epinephrine-induced leukocyte cell death was prevented by GCR and ß2-AR blockade, respectively. In vivo, only GCR blockade prevented post ischemic lymphopenia whereas ß2-AR preserved interferon-γ secretion by lymphocytes. GCR blockade increased metanephrine levels in vivo and prednisolone, in turn, decreased ß2-AR expression on lymphocytes. In conclusion, mediators of the SNS and the HPA axis differentially affect the systemic immune system after stroke. Moreover, our findings suggest a negative-feedback of corticosteroids on the sympathetic axis which may control the post-stroke stress-reaction. This complex interplay between the HPA and the SNS after stroke has to be considered when targeting the neurohormonal systems in the post acute phase of severe stroke.

Leukocyte invasion of the brain after experimental intracerebral hemorrhage in mice.

BACKGROUND AND PURPOSE: Neuroinflammatory processes contribute to secondary neuronal damage after intracerebral hemorrhage. We aimed to characterize the time course of brain immigration of different leukocyte subsets after striatal injection of either autologous blood or collagenase in mice. METHODS: Intracerebral hemorrhage was induced by injection of either autologous blood (20 µL) or collagenase (0.03 U) in C57Bl/6J mice. Hematoma volumetry was performed on cryosections. Blood volume was measured by hemoglobin spectrophotometry. Leukocytes were isolated from hemorrhagic hemisphere 1, 3, 5, and 14 days after intracerebral hemorrhage, stained for leukocyte markers, and measured by flow cytometry. Heterologous blood injection from CD45.1 mice was used to investigate the origin of brain-invading leukocytes. RESULTS: Collagenase injection induced a larger hematoma volume but a similar blood content compared with blood injection. Cerebral leukocyte infiltration in the hemorrhagic hemisphere was similar in both models. The majority of leukocytes isolated from the brain originated from the circulation. CD4+ T lymphocytes were the predominant brain leukocyte population in both models. However, cerebral granulocyte counts were higher after collagenase compared with blood injection. CONCLUSIONS: Brain infiltration of systemic immune cells is similar in both murine intracerebral hemorrhage models. The pathophysiological impact of invading leukocytes and, in particular, of T cells requires further investigation.

Boosting regulatory T cells limits neuroinflammation in permanent cortical stroke.

Inflammatory mechanisms contribute substantially to secondary tissue injury after brain ischemia. Regulatory T cells (Tregs) are key endogenous modulators of postischemic neuroinflammation. We investigated the potential of histone deacetylase inhibition (HDACi) to enhance Treg potency for experimental stroke in mice. HDACi using trichostatin A increased the number of Tregs and boosted their immunosuppressive capacity and interleukin (IL)-10 expression. In vivo treatment reduced infarct volumes and behavioral deficits after cortical brain ischemia, attenuated cerebral proinflammatory cytokine expression, and increased numbers of brain-invading Tregs. A similar effect was obtained using tubastatin, a specific inhibitor of HDAC6 and a key HDAC in Foxp3 regulation. The neuroprotective effect of HDACi depended on the presence of Foxp3(+) Tregs, and in vivo and in vitro studies showed that the anti-inflammatory cytokine IL-10 was their main mediator. In summary, modulation of Treg function by HDACi is a novel and potent target to intervene at the center of neuroinflammation. Furthermore, this novel concept of modulating endogenous immune mechanisms might be translated to a broad spectrum of diseases, including primary neuroinflammatory and neurodegenerative disorders.

Hydrogen supplemented air inhalation reduces changes of prooxidant enzyme and gap junction protein levels after transient global cerebral ischemia in the rat hippocampus.

Transient global cerebral ischemia (TGCI) occurs during acute severe hypotension depriving the brain of oxygen and glucose for a short period of time. During reperfusion, several mechanisms can induce secondary neuronal damage, including the increased production of reactive oxygen species (ROS). Hydrogen gas-enriched air inhalation is a neuroprotective approach with proven antioxidant potential, which has not yet been examined in TGCI. Accordingly, we set out to describe the effect of inhalation of 2.1% hydrogen supplemented room air (H(2)-RA) in comparison with a well studied neuroprotective agent, rosiglitazone (RSG) in a TGCI rat model. Male Wistar rats were exposed to TGCI (n=26) or sham operation (n=26), while a third group served as intact control (naive, n=5). The operated groups were further divided into non-treated, H(2)-RA, RSG (6 mg/kg i.v.) and vehicle treated animals. Tissue samples from the hippocampus and frontal cortex were taken 3 days following surgery. Western blot analysis was applied to determine the expressions of cyclooxygenase-2 (COX-2), neuronal and endothelial nitric oxide synthase (nNOS and eNOS, respectively), manganese superoxide dismutase (MnSOD) and glial connexin proteins: connexin 30 and connexin 43. The expressions of COX-2, and connexin proteins were upregulated, while nNOS was downregulated 3 days after TGCI. Both RSG and H(2)-RA prevented the changes of enzyme and connexin levels. Considering the lack of harmful side effects, inhalation of H(2)-RA can be a promising approach to reduce neuronal damage after TGCI.

FTY720 reduces post-ischemic brain lymphocyte influx but does not improve outcome in permanent murine cerebral ischemia.

BACKGROUND: The contribution of neuroinflammation and specifically brain lymphocyte invasion is increasingly recognised as a substantial pathophysiological mechanism after stroke. FTY720 is a potent treatment for primary neuroinflammatory diseases by inhibiting lymphocyte circulation and brain immigration. Previous studies using transient focal ischemia models showed a protective effect of FTY720 but did only partially characterize the involved pathways. We tested the neuroprotective properties of FTY720 in permanent and transient cortical ischemia and analyzed the underlying neuroimmunological mechanisms. METHODOLOGY/PRINCIPAL FINDINGS: FTY720 treatment resulted in substantial reduction of circulating lymphocytes while blood monocyte counts were significantly increased. The number of histologically and flow cytometrically analyzed brain invading T- and B lymphocytes was significantly reduced in FTY720 treated mice. However, despite testing a variety of treatment protocols, infarct volume and behavioural dysfunction were not reduced 7d after permanent occlusion of the distal middle cerebral artery (MCAO). Additionally, we did not measure a significant reduction in infarct volume at 24 h after 60 min filament-induced MCAO, and did not see differences in brain edema between PBS and FTY720 treatment. Analysis of brain cytokine expression revealed complex effects of FTY720 on postischemic neuroinflammation comprising a substantial reduction of delayed proinflammatory cytokine expression at 3d but an early increase of IL-1ß and IFN-γ at 24 h after MCAO. Also, serum cytokine levels of IL-6 and TNF-α were increased in FTY720 treated animals compared to controls. CONCLUSIONS/SIGNIFICANCE: In the present study we were able to detect a reduction of lymphocyte brain invasion by FTY720 but could not achieve a significant reduction of infarct volumes and behavioural dysfunction. This lack of neuroprotection despite effective lymphopenia might be attributed to a divergent impact of FTY720 on cytokine expression and possible activation of innate immune cells after brain ischemia.

Inhibition of lymphocyte trafficking shields the brain against deleterious neuroinflammation after stroke.

T lymphocytes are increasingly recognized as key modulators of detrimental inflammatory cascades in acute ischaemic stroke, but the potential of T cell-targeted therapy in brain ischaemia is largely unexplored. Here, we characterize the effect of inhibiting leukocyte very late antigen-4 and endothelial vascular cell adhesion molecule-1-mediated brain invasion-currently the most effective strategy in primary neuroinflammatory brain disease in murine ischaemic stroke models. Very late antigen-4 blockade by monoclonal antibodies improved outcome in models of moderate stroke lesions by inhibiting cerebral leukocyte invasion and neurotoxic cytokine production without increasing the susceptibility to bacterial infections. Gene silencing of the endothelial very late antigen-4 counterpart vascular cell adhesion molecule-1 by in vivo small interfering RNA injection resulted in an equally potent reduction of infarct volume and post-ischaemic neuroinflammation. Furthermore, very late antigen-4-inhibition effectively reduced the post-ischaemic vascular cell adhesion molecule-1 upregulation, suggesting an additional cross-signalling between invading leukocytes and the cerebral endothelium. Dissecting the specific impact of leukocyte subpopulations showed that invading T cells, via their humoral secretion (interferon-γ) and immediate cytotoxic mechanisms (perforin), were the principal pathways for delayed post-ischaemic tissue injury. Thus, targeting T lymphocyte-migration represents a promising therapeutic approach for ischaemic stroke.

Changes in pro-oxidant and antioxidant enzyme levels during cerebral hypoperfusion in rats.

Chronic cerebral hypoperfusion is a mild ischemic condition associated with a cognitive decline which is prevalent during senescence or Alzheimer's disease. Its experimental animal model compromises permanent occlusion of the common carotid arteries (2VO) in rats, which results in neuronal damage and microglia activation. Various mechanisms, including oxidative stress, have been proposed to be involved in this process. Accordingly, we set out to characterize the changes induced in the expressions of several pro-oxidant and antioxidant enzymes in cerebral hypoperfusion. Male Wistar rats were exposed to 2VO (n=30) or sham operation (n=33), while a third group served as absolute control (naive, n=16). Tissue samples from the hippocampus and frontal cortex were taken 1 and 3 days, 1 and 2 weeks and 3, 6 and 12 months following surgery. Western blot analysis was applied to determine the expressions of cyclooxygenase-2 (COX-2), endothelial, neuronal and inducible nitric oxide synthase (eNOS, nNOS and iNOS, respectively) and manganese superoxide dismutase (MnSOD). During the early phase of hypoperfusion, the COX-2 and eNOS enzyme levels increased in both the hippocampus and the frontal cortex, indicating the presence of excitotoxicity and vascular reactions caused by ischemia, while the expressions of nNOS, iNOS and MnSOD were less affected. There were significant reductions in most of the investigated enzyme levels 2 weeks and 3 months after 2VO induction, which may be a sign of neuronal loss. One year following 2VO onset, the eNOS expression was upregulated, which may strengthen the adaptation of the brain to cerebral ischemia.

Capillary injury in the ischemic brain of hyperlipidemic, apolipoprotein B-100 transgenic mice.

AIMS: Apolipoprotein B-100 (apoB-100) has been implicated in hyperlipidemia, which contributes to the pathogenesis of vascular disorders. Our aim was to investigate whether the expression of human apoB-100 in transgenic mice and/or a high-cholesterol diet cause cerebral microvascular lesions, and whether these conditions augment ischemia-related capillary damage. MAIN METHODS: Human apoB-100 overexpressing transgenic (Tg(apoB-100), n=23) and wild-type mice (C5/B6, Wt, n=26) were supplied with standard or 2% cholesterol-enriched diet for 17-19 weeks. Cerebral ischemia was induced by unilateral common carotid artery occlusion. Cortical samples were embedded for electron microscopy. Microvascular density (number of microvascular profiles/examined area), lumen diameter, the swelling of astrocytic endfeet, the occurrence of endothelial microvilli (affected capillaries expressed as ratio of all capillaries encountered), and the ratio of intact capillaries (devoid of all the above pathology) were calculated. KEY FINDINGS: The expression of apoB-100 coincided with decreased cortical microvascular density (195+/-7 vs. 223+/-8 vessels/mm(2), vs. Wt; P<0.008) and increased capillary lumen diameter (3.16+/-0.5 vs. 2.88+/-0.6 microm, vs. Wt; P<0.001). Cerebral ischemia promoted the swelling of perivascular astrocytes (62.1+/-4.2 vs. 36.5+/-4.0%, vs. contralateral, Wt; P<0.001), and reduced the ratio of intact capillaries (32.1+/-5.6 vs. 65.2+/-3.7%, vs. contralateral, Wt; P<0.001). Hyperlipidemia did not exacerbate the injury. SIGNIFICANCE: The overexpression of human apoB-100 alters the density of the microvascular network and the diameter of capillaries, which may compromise cerebrovascular reactivity during ischemia.

Pre-treatment and post-treatment with alpha-tocopherol attenuates hippocampal neuronal damage in experimental cerebral hypoperfusion.

Alpha-tocopherol, a potent antioxidant, has been widely investigated as a dietary supplement with which to reduce the risk of atherosclerosis, and has recently been considered as a potential supplement to moderate oxidative neuronal damage in Alzheimer's disease patients. Since alpha-tocopherol appears beneficial in vascular and neurodegenerative disorders, we set out to identify its neuroprotective action in a rat model of chronic cerebral hypoperfusion-induced brain injury. The bilateral common carotid arteries of male Wistar rats were permanently occluded (2VO). Sham-operated animals served as controls. Half of the animals were pre- or post-treated repeatedly with alpha-tocopherol (5x100 mg/kg daily, i.p.), the other half receiving only soybean oil, the alpha-tocopherol vehicle. One week after the onset of 2VO, the spatial learning capacity of the animals was assessed in the Morris water maze. After testing, hippocampal slices were stained with cresyl violet in order to examine the pyramidal cell layer integrity. The density of microtubule-associated protein-2 (MAP-2)-positive dendrites and the OX-42-labeled microglial activation level were determined immunocytochemically. Finally, alpha-tocopherol was determined in the peripheral tissues, blood and brain. Alpha-tocopherol moderated the 2VO-induced learning impairment. The various forms of alpha-tocopherol treatment, and particularly the post-treatment, prevented the 2VO-induced pyramidal cell death and the activation of microglia in the hippocampus CA1 region, and the degeneration of MAP-2-positive dendrites in the CA3 region. The alpha-tocopherol concentration was elevated in the peripheral tissues and the blood, but not in the brain. The data indicate that alpha-tocopherol, particularly when administered as post-treatment, is neuroprotective in chronic cerebral hypoperfusion.