Cerebral infarct volume is typically smaller in premenopausal females than in age-matched males after ischemic stroke, but the underlying mechanisms are poorly understood. In this study we provide evidence in mice that this gender difference only occurs when the ischemic brain is reperfused. The limited tissue salvage achieved by reperfusion in male mice is associated with increased expression of proinflammatory proteins, including cyclooxygenase-2 (Cox-2), Nox2, and vascular cell adhesion molecule-1 (VCAM-1), and infiltration of Nox2-containing T lymphocytes into the infarcted brain, whereas such changes are minimal in female mice after ischemia-reperfusion (I-R). Infarct volume after I-R was no greater at 72 h than at 24 h in either gender. Infarct development was Nox2 dependent in male but not in female mice, and Nox2 within the infarct was predominantly localized in T lymphocytes. Stroke resulted in an B15-fold increase in Nox2-dependent superoxide production by circulating, but not spleen-derived, T lymphocytes in male mice, and this was Bsevenfold greater than in female mice. These circulating immune cells may thus represent a major and previously unrecognized source of superoxide in the acutely ischemic and reperfused brain of males (and potentially in postmenopausal females). Our findings provide novel insights into mechanisms that could be therapeutically targeted in acute ischemic stroke patients who receive thrombolysis therapy to induce cerebral reperfusion.

Intracellular nucleotide binding and oligomerization domain (NOD) receptors recognize antigens including bacterial peptidoglycans and initiate immune responses by triggering the production of pro-inflammatory cytokines through activating NF-kB and MAP kinases. Receptor interacting protein kinase 2 (RIPK2) is critical for NOD-mediated NF-kB activation and cytokine production. Here we develop and characterize a selective RIPK2 kinase inhibitor, WEHI-345, which delays RIPK2 ubiquitylation and NF-kB activation downstream of NOD engagement. Despite only delaying NF-kB activation on NOD stimulation, WEHI-345 prevents cytokine production in vitro and in vivo and ameliorates experimental autoimmune encephalomyelitis in mice. Our study highlights the importance of the kinase activity of RIPK2 for proper immune responses and demonstrates the therapeutic potential of inhibiting RIPK2 in NOD-driven inflammatory diseases.

Background and Purpose-Toll-like receptors (TLRs) and the scavenger receptor CD36 are key molecular sensors for the innate immune response to invading pathogens. However, these receptors may also recognize endogenous "danger signals" generated during brain injury, such as cerebral ischemia, and trigger a maladaptive inflammatory reaction. Indeed, CD36 and TLR2 and 4 are involved in the inflammation and related tissue damage caused by brain ischemia. Because CD36 may act as a coreceptor for TLR2 heterodimers (TLR2/1 or TLR2/6), we tested whether such interaction plays a role in ischemic brain injury. Methods-The TLR activators FSL-1 (TLR2/6), Pam3 (TLR2/1), or lipopolysaccharide (TLR4) were injected intracerebroventricularly into wild-type or CD36-null mice, and inflammatory gene expression was assessed in the brain. The effect of TLR activators on the infarct produced by transient middle cerebral artery occlusion was also studied. Results-The inflammatory response induced by TLR2/1 activation, but not TLR2/6 or TLR4 activation, was suppressed in CD36-null mice. Similarly, TLR2/1 activation failed to increase infarct volume in CD36-null mice, whereas TLR2/6 or TLR4 activation exacerbated postischemic inflammation and increased infarct volume. In contrast, the systemic inflammatory response evoked by TLR2/6 activation, but not by TLR2/1 activation, was suppressed in CD36-null mice. Conclusions-In the brain, TLR2/1 signaling requires CD36. The cooperative signaling of TLR2/1 and CD36 is a critical factor in the inflammatory response and tissue damage evoked by cerebral ischemia. Thus, suppression of CD36-TLR2/1 signaling could be a valuable approach to minimize postischemic inflammation and the attendant brain injury. (Stroke. 2010;41:898-904.)

1. It is well documented that the incidence and severity of several vascular diseases, such as hypertension, atherosclerosis and stroke, are lower in premenopausal women than men of similar age and post-menopausal women. The mechanisms responsible for gender differences in the incidence and severity of vascular disease are not well understood. However, emerging evidence suggests that sex hormone-dependent differences in vascular oxidative stress may play an important role. The aim of the present brief review is to provide an insight into the effect of gender and sex hormones on vascular oxidative stress. 2. When production of reactive oxygen species (ROS) is enhanced and/or their metabolism by anti-oxidant enzymes is impaired, a condition known as 'oxidative stress' can develop. Oxidative stress is believed to play an important role in both the initiation and progression of a variety of vascular diseases, including hypertension and atherosclerosis. NADPH oxidases are believed to be the major source of vascular ROS. Moreover, excessive production of ROS by NADPH oxidases has been linked to the development of vascular oxidative stress. 3. Increasing evidence suggests that levels of vascular ROS may be lower in women than men during health and disease. Indeed, the activity and expression of vascular NADPH oxidase is lower in female versus male animals under healthy, hypertensive and atherosclerotic conditions. 4. Gonadal sex hormones may play an important role in the regulation of vascular oxidative stress. For example, oestrogens, which are present in highest levels in premenopausal women, have been reported to lower vascular oxidative stress by modulating the expression and function of NADPH oxidases, as well as anti-oxidant enzymes. 5. Further studies are needed to clarify whether lower vascular oxidative stress in women in fact protects against the initiation and development of vascular disease and to further define the roles of gonadal sex hormones in such an effect. Knowledge gained from these studies may potentially lead to advances in the clinical diagnosis and treatment of vascular disease in both genders.

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Significance: The brain has high energetic requirements and is therefore highly dependent on adequate cerebral blood supply. To compensate for dangerous fluctuations in cerebral perfusion, the circulation of the brain has evolved intrinsic safeguarding measures. Recent Advances and Critical Issues: The vascular network of the brain incorporates a high degree of redundancy, allowing the redirection and redistribution of blood flow in the event of vascular occlusion. Furthermore, active responses such as cerebral autoregulation, which acts to maintain constant cerebral blood flow in response to changing blood pressure, and functional hyperemia, which couples blood supply with synaptic activity, allow the brain to maintain adequate cerebral perfusion in the face of varying supply or demand. In the presence of stroke risk factors, such as hypertension and diabetes, these protective processes are impaired and the susceptibility of the brain to ischemic injury is increased. One potential mechanism for the increased injury is that collateral flow arising from the normally perfused brain and supplying blood flow to the ischemic region is suppressed, resulting in more severe ischemia. Future Directions: Approaches to support collateral flow may ameliorate the outcome of focal cerebral ischemia by rescuing cerebral perfusion in potentially viable regions of the ischemic territory.

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Protein modifications cooperatively act to protect the proteome from cellular stress. Focal cerebral ischemia increases protein ubiquitination, resulting in formation of ubiquitin-rich aggregates. A concurrent elevation in small ubiquitin-related modifier (SUMO)-conjugated proteins has also been reported, but a potential connection to ubiquitin remains unexplored. Here we show that SUMO2/3 conjugates are present in postischemic ubiquitin-rich aggregates, physically associated with ubiquitin. The coaggregation of SUMO2/3 and ubiquitin is induced rapidly after ischemia, depends on reperfusion, and is also observed in the absence of ischemic damage. The association between SUMO and ubiquitin suggests overlapping functional roles after ischemia/ reperfusion.