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Significance One of the hallmarks of Alzheimer’s disease (AD) is cerebral amyloid angiopathy (CAA), which is a strong and independent risk factor for cerebral hemorrhage, ischemic stroke, and dementia. However, the mechanisms by which CAA contributes to these conditions are poorly understood. Results from the present study provide strong evidence that vascular oxidative stress plays a causal role in CAA-induced cerebrovascular dysfunction, CAA-induced cerebral hemorrhage, and CAA formation, itself. They also suggest that NADPH oxidase is the source of this oxidative stress and that strategies to inhibit NADPH oxidase may have therapeutic potential in patients with AD and CAA. Cerebral amyloid angiopathy (CAA) is characterized by deposition of amyloid β peptide (Aβ) within walls of cerebral arteries and is an important cause of intracerebral hemorrhage, ischemic stroke, and cognitive dysfunction in elderly patients with and without Alzheimer’s Disease (AD). NADPH oxidase-derived oxidative stress plays a key role in soluble Aβ-induced vessel dysfunction, but the mechanisms by which insoluble Aβ in the form of CAA causes cerebrovascular (CV) dysfunction are not clear. Here, we demonstrate evidence that reactive oxygen species (ROS) and, in particular, NADPH oxidase-derived ROS are a key mediator of CAA-induced CV deficits. First, the NADPH oxidase inhibitor, apocynin, and the nonspecific ROS scavenger, tempol, are shown to reduce oxidative stress and improve CV reactivity in aged Tg2576 mice. Second, the observed improvement in CV function is attributed both to a reduction in CAA formation and a decrease in CAA-induced vasomotor impairment. Third, anti-ROS therapy attenuates CAA-related microhemorrhage. A potential mechanism by which ROS contribute to CAA pathogenesis is also identified because apocynin substantially reduces expression levels of ApoE—a factor known to promote CAA formation. In total, these data indicate that ROS are a key contributor to CAA formation, CAA-induced vessel dysfunction, and CAA-related microhemorrhage. Thus, ROS and, in particular, NADPH oxidase-derived ROS are a promising therapeutic target for patients with CAA and AD.

B cells play a variety of immunoregulatory roles through their antigen-presentation ability and through cytokine and chemokine production. Innate immune activation of B cells may play a beneficial role through the generation of natural cross-reactive antibodies, by maintaining B cell memory and by exercising immunomodulatory functions that may provide protection against autoimmunity. In this article, we review human B cell populations and their functional properties, with a particular focus on a population of inherently autoreactive B cells, which seem to play an important physiological role in innate immunity, but which, if selected into adaptive immune responses, appear to become pathogenic agents in systemic lupus erythematosus.

Cognitive dysfunction is the primary driver of poor long-term outcome in aneurysmal subarachnoid hemorrhage (SAH) survivors; modeling such deficits preclinically is thus key for mechanistic and translational investigation. Although rat SAH causes long-term deficits in learning and memory, it remains unknown whether similar deficits are seen in the mouse, a species particularly amenable to powerful, targeted genetic manipulation. We thus subjected mice to endovascular perforation SAH and assessed long-term cognitive outcome via the Morris water maze (MWM), the most commonly used metric for rodent neurocognition. No significant differences in MWM performance (by either of two protocols) were seen in SAH versus sham mice. Moreover, SAH caused negligible hippocampal CA1 injury. These results undercut the potential of commonly used methods (of SAH induction and assessment of long-term neurocognitive outcome) for use in targeted molecular studies of SAH-induced cognitive deficits in the mouse.

Various techniques have been developed to study changes in the cerebral vasculature in numerous neuropathological processes including subarachnoid hemorrhage (SAH). One of the most widely employed techniques uses India ink-gelatin casting, which presents numerous challenges due to its high viscosity, rapid solidification, and its impact on immunohistochemical analysis. To overcome these limitations, we developed a novel technique for assessing cerebral vasospasm using cerebrovascular perfusion with ROX, SE (5-Carboxy-X-Rhodamine, Succinimidyl Ester), a fluorescent labeling dye. We found that ROX SE perfusion achieves excellent delineation of the cerebral vasculature, was qualitatively and quantitatively superior to India ink-gelatin casting for the assessment of cerebral vasospasm, permits outstanding immunohistochemical examination of non-vasospasm components of secondary brain injury, and is a more efficient and cost-effective experimental technique. ROX SE perfusion is therefore a novel and highly useful technique for studying cerebrovascular pathology following experimental SAH.

Abstract BACKGROUND Cerebral vasospasm is an independent predictor of poor outcome after subarachnoid hemorrhage (SAH). The nitric oxide–cyclic guanosine monophosphate (NO-cGMP) vasodilatory pathway is strongly implicated in its pathophysiology. Preliminary studies suggest that phosphodiesterase 5 (PDE5), an enzyme that degrades cGMP, may play a role because the PDE5 inhibitor sildenafil was found to reduce vasospasm after SAH. However, several questions that are critical when considering translational studies remain unanswered. OBJECTIVE To elucidate the mechanism of action of sildenafil against vasospasm and to assess whether sildenafil attenuates SAH-induced neuronal cell death, improves functional outcome after SAH, or causes significant physiological side effects when administered at therapeutically relevant doses. METHODS SAH was induced via endovascular perforation in male C57BL6 mice. Beginning 2 hours later, mice received sildenafil citrate (0.7, 2 or 5 mg/kg orally twice daily) or vehicle. Neurological outcome was assessed daily. Vasospasm was determined on post-SAH day 3. Brain PDE5 expression and activity, cGMP content, neuronal cell death, arterial blood pressure, and intracranial pressure were examined. RESULTS We found that PDE5 activity (but not expression) is increased after SAH, leading to decreased cGMP levels. Sildenafil attenuates this increase in PDE5 activity and restores cGMP levels after SAH. Post-SAH initiation of sildenafil was found to decrease vasospasm and neuronal cell death and markedly improve neurological outcome without causing significant physiological side effects. CONCLUSION Sildenafil, a US Food and Drug Administration–approved drug with a proven track record of safety in humans, is a promising new therapy for vasospasm and neurological deficits after SAH.

Background Cerebral amyloid angiopathy (CAA) is characterized by deposition of fibrillar amyloid β (Aβ) within cerebral vessels. It is commonly seen in the elderly and almost universally present in patients with Alzheimer's Disease (AD). In both patient populations, CAA is an independent risk factor for lobar hemorrhage, ischemic stroke, and dementia. To date, definitive diagnosis of CAA requires obtaining pathological tissues via brain biopsy (which is rarely clinically indicated) or at autopsy. Though amyloid tracers labeled with positron-emitting radioligands such as [ 11 C]PIB have shown promise for non-invasive amyloid imaging in AD patients, to date they have been unable to clarify whether the observed amyloid load represents neuritic plaques versus CAA due in large part to the low resolution of PET imaging and the almost equal affinity of these tracers for both vascular and parenchymal amyloid. Therefore, the development of a precise and specific non-invasive technique for diagnosing CAA in live patients is desired. Results We found that the phenoxazine derivative resorufin preferentially bound cerebrovascular amyloid deposits over neuritic plaques in the aged Tg2576 transgenic mouse model of AD/CAA, whereas the congophilic amyloid dye methoxy-X34 bound both cerebrovascular amyloid deposits and neuritic plaques. Similarly, resorufin-positive staining was predominantly noted in fibrillar Aβ-laden vessels in postmortem AD brain tissues. Fluorescent labeling and multi-photon microscopy further revealed that both resorufin- and methoxy-X34-positive staining is colocalized to the vascular smooth muscle (VSMC) layer of vessel segments that have severe disruption of VSMC arrangement, a characteristic feature of CAA. Resorufin also selectively visualized vascular amyloid deposits in live Tg2576 mice when administered topically, though not systemically. Resorufin derivatives with chemical modification at the 7-OH position of resorufin also displayed a marked preferential binding affinity for CAA, but with enhanced lipid solubility that indicates their use as a non-invasive imaging tracer for CAA is feasible. Conclusions To our knowledge, resorufin analogs are the fist class of amyloid dye that can discriminate between cerebrovascular and neuritic forms of amyloid. This unique binding selectivity suggests that this class of dye has great potential as a CAA-specific amyloid tracer that will permit non-invasive detection and quantification of CAA in live patients.

Exposure to ethanol in neonatal rats results in reduced neuronal numbers in the cerebellar cortex and deep nuclei of juvenile and adult animals. This reduction in cell numbers is correlated with impaired delay eyeblink conditioning (EBC), a simple motor learning task in which a neutral conditioned stimulus (CS; tone) is repeatedly paired with a co-terminating unconditioned stimulus (US; periorbital shock). Across training, cell populations in the interpositus (IP) nucleus model the temporal form of the eyeblink-conditioned response (CR). The hippocampus, though not required for delay EBC, also shows learning-dependent increases in CA1 and CA3 unit activity. In the present study, rat pups were exposed to 0, 3, 4, or 5 mg/kg/day of ethanol during postnatal days (PD) 4–9. As adults, CR acquisition and timing were assessed during 6 training sessions of delay EBC with a short (280 ms) interstimulus interval (ISI; time from CS onset to US onset) followed by another 6 sessions with a long (880 ms) ISI. Neuronal activity was recorded in the IP and area CA1 during all 12 sessions. The high-dose rats learned the most slowly and, with the moderate-dose rats, produced the longest CR peak latencies over training to the short ISI. The low dose of alcohol impaired CR performance to the long ISI only. The 3E (3 mg/kg/day of ethanol) and 5E (5 mg/kg/day of ethanol) rats also showed slower-than-normal increases in learning-dependent excitatory unit activity in the IP and CA1. The 4E (4 mg/kg/day of ethanol) rats showed a higher rate of CR production to the long ISI and enhanced IP and CA1 activation when compared to the 3E and 5E rats. The results indicate that binge-like ethanol exposure in neonatal rats induces long-lasting, dose-dependent deficits in CR acquisition and timing and diminishes conditioning-related neuronal excitation in both the cerebellum and hippocampus.

The adult repertoire of antibody specificities is acquired in a developmentally programed fashion that, in mouse and man, parallels the ordered rearrangement of a limited number of germ-line heavy chain variable region (VH) gene segments during development. It has been hypothesized that this developmental bias is a consequence of gene organization. In the mouse, rearrangement of VHgene segments proximal to the heavy chain joining region (JH) locus precedes rearrangement of genes located more distal to the JHlocus. Similarly, in man, two VHelements located proximal to JHare expressed during fetal development. To test further this hypothesis in man, we have determined in a single individual the positions of an additional eight distinct VHelements known to comprise a significant fraction of the human developmental repertoire. These developmentally expressed VHelements were found to be dispersed over a region of 890 kilobases of the VHlocus and were interspersed with other VHelements that are not known to be developmentally expressed. Thus, the ordered developmental expression of VHgene segments in man must involve mechanisms beyond physical proximity to the JHlocus. Further, these results support the notion that fetal expression of VHgene segments is a regulated process and suggest that this regulation is important in the acquisition of immunocompetence.

Vasospasm-induced delayed cerebral ischemia (DCI) remains a major source of morbidity in patients with aneurysmal subarachnoid hemorrhage (SAH). Moreover, cognitive dysfunction is the primary driver of poor long-term outcome in SAH survivors; modeling such deficits preclinically is thus key for mechanistic and translational investigation. We hypothesized that activating innate neurovascular protective mechanisms by conditioning may represent a novel therapeutic approach against SAH-induced DCI, short-term, neurological deficits, and long-term neurocognitive deficits; and, secondarily, that the neurovascular protection it provides is mediated by endothelial nitric oxide synthase (eNOS) and hypoxia-inducible factor 1α (HIF-1α). In Experiment 1, wild-type C57BL/6 mice were subjected to hypoxic preconditioning (PC) or normoxia followed 24 hours later by endovascular-perforation SAH. Neurological function was analyzed daily via sensorimotor scoring; vasospasm was assessed on post-surgery day 2. Nitric oxide availability, eNOS expression, and eNOS activity were also assessed. In a separate experiment, wild-type and eNOS-null mice were subjected to hypoxic PC or normoxia followed by SAH and assessed for vasospasm and neurological deficits. All experiments were performed in a randomized and blinded fashion. PC nearly completely prevented SAH-induced vasospasm and neurological deficits. It also prevented SAH-induced reduction in nitric oxide availability and increased eNOS activity in mice with and without SAH. PC-induced protection against vasospasm and neurological deficits was lost in wild-type mice treated with the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester and in eNOS-null mice. From these results, we conclude that endogenous protective mechanisms against vasospasm exist, are powerful, and can be induced by PC. eNOS-derived nitric oxide is a critical mediator of this neurovascular protection. These “proof-of-principle” results suggest that conditioning represents a promising new strategy to mitigate SAH-induced neurovascular dysfunction. In Experiment 2, we sought to determine whether these innate protective mechanisms are induced by a more clinically relevant conditioning paradigm, and whether the cerebrovascular protection extends to non-vasospasm contributors to DCI, microthrombosis and microvessel dysfunction. Adult male mice were subjected to sham surgery, SAH surgery, or SAH and subsequently postconditioned with isoflurane (2% for 1h, starting 1h after surgery). Contributors to DCI – vasospasm of the ipsilateral middle cerebral artery, cortical microthrombosis as assessed by fibrinogen immunohistochemistry, and cerebrovascular vasodilatory function was assessed in pial vessels through a closed cranial window – were measured 3 days post-SAH. Neurological outcome was assessed daily. Moreover, isoflurane-induced changes in HIF-1α-–dependent genes (glucose transporter-1, GLUT1; BNIP3) and HIF-2α-driven erythropoietin (EPO) were assessed via quantitative-PCR. HIF-1α was inhibited either pharmacologically (2-methoxyestradiol, 2ME2) or genetically in endothelial cells (EC-HIF-1-null). All experiments were performed in a randomized and blinded fashion. We found the following: first, isoflurane postconditioning markedly reduced SAH-induced DCI in wild-type mice: vasospasm was attenuated, microthrombosis was significantly reduced, and microvessel function was restored. Neurological deficits were also significantly attenuated. Second, isoflurane modulated HIF-1α- and HIF-2α-dependent genes; these changes were abolished in 2ME2-treated wild-type mice and in EC-HIF-1-null mice (HIF-1α-dependent genes only in the latter). Third, postconditioning-induced protection against vasospasm and neurological deficits was attenuated in 2ME2-treated wild-type mice and in EC-HIF-1-null mice. In Experiment 3, we sought to assess whether the protection afforded by conditioning extended to long-term neurocognitive outcomes. Whereas rat SAH causes long-term deficits in learning and memory, it remains unknown whether similar deficits are seen in the mouse, a species particularly amenable to powerful, targeted genetic manipulation. We thus subjected mice to SAH and assessed long-term cognitive outcome via the Morris water maze (MWM), the most commonly used metric for rodent neurocognition. No significant differences in MWM performance (by either of two protocols) were seen in SAH versus sham mice. Moreover, SAH caused negligible hippocampal CA1 injury. These results undercut the potential of commonly used methods (of SAH induction and assessment of long-term neurocognitive outcome) for use in targeted molecular studies of SAH-induced cognitive deficits in the mouse. From these results, we concluded that endogenous protective mechanisms against DCI exist, are powerful, and can be induced by hypoxic PC or isoflurane postconditioning. This protection depends critically on eNOS-derived nitric oxide and endothelial cell–derived HIF-1α. These studies provide strong evidence that conditioning – especially isoflurane postconditioning – represents a promising new strategy to reduce DCI after SAH. Future studies examining long-term neurocognitive deficits should utilize rat models of SAH.

Objective Outcome after aneurysmal subarachnoid hemorrhage (SAH) depends critically on delayed cerebral ischemia (DCI) – a process driven primarily by vascular events including cerebral vasospasm, microvessel thrombosis, and microvascular dysfunction. This study sought to determine the impact of postconditioning – the phenomenon whereby endogenous protection against severe injury is enhanced by subsequent exposure to a mild stressor – on SAH‐induced DCI. Methods Adult male C57BL/6 mice were subjected to sham, SAH, or SAH plus isoflurane postconditioning. Neurological outcome was assessed daily via sensorimotor scoring. Contributors to DCI including cerebral vasospasm, microvessel thrombosis, and microvascular dysfunction were measured 3 days later. Isoflurane‐induced changes in hypoxia‐inducible factor 1alpha (HIF‐1α)‐dependent genes were assessed via quantitative polymerase chain reaction. HIF‐1α was inhibited pharmacologically via 2‐methoxyestradiol (2ME2) or genetically via endothelial cell HIF‐1α‐null mice (EC‐HIF‐1α‐null). All experiments were performed in a randomized and blinded fashion. Results Isoflurane postconditioning initiated at clinically relevant time points after SAH significantly reduced cerebral vasospasm, microvessel thrombosis, microvascular dysfunction, and neurological deficits in wild‐type (WT) mice. Isoflurane modulated HIF‐1α‐dependent genes – changes that were abolished in 2ME2‐treated WT mice and EC‐HIF‐1α‐null mice. Isoflurane‐induced DCI protection was attenuated in 2ME2‐treated WT mice and EC‐HIF‐1α‐null mice. Interpretation Isoflurane postconditioning provides strong HIF‐1α‐mediated macro‐ and microvascular protection in SAH, leading to improved neurological outcome. These results implicate cerebral vessels as a key target for the brain protection afforded by isoflurane postconditioning, and HIF‐1α as a critical mediator of this vascular protection. They also identify isoflurane postconditioning as a promising novel therapeutic for SAH.