Carmen Capone

Nox2-derived radicals contribute to neurovascular and behavioral dysfunction in mice overexpressing the amyloid precursor protein

Alterations in cerebrovascular regulation related to vascular oxidative stress have been implicated in the mechanisms of Alzheimers disease (AD), but their role in the amyloid deposition and cognitive impairment associated with AD remains unclear. We used mice overexpressing the Swedish mutation of the amyloid precursor protein (Tg2576) as a model of AD to examine the role of reactive oxygen species produced by NADPH oxidase in the cerebrovascular alterations, amyloid deposition, and behavioral deficits observed in these mice. We found that 12- to 15-month-old Tg2576 mice lacking the catalytic subunit Nox2 of NADPH oxidase do not develop oxidative stress, cerebrovascular dysfunction, or behavioral deficits. These improvements occurred without reductions in brain amyloid-β peptide (Aβ) levels or amyloid plaques. The findings unveil a previously unrecognized role of Nox2-derived radicals in the behavioral deficits of Tg2576 mice and provide a link between the neurovascular dysfunction and cognitive decline associated with amyloid pathology.

Threats to the Mind Aging, Amyloid, and Hypertension

Aging, Alzheimer disease, and hypertension, major determinants of cognitive dysfunction, are associated with profound alterations in the structure and function of cerebral blood vessels. These vascular alterations may impair the delivery of energy substrates and nutrients to the active brain, and impede the clearance of potentially toxic metabolic byproducts. Reactive oxygen species derived form the enzyme NADPH oxidase are key pathogenic effectors of the cerebrovascular dysregulation. The resulting alterations in the homeostasis of the cerebral microenvironment may lead to cellular dysfunction and death and to cognitive impairment. The prominent role that cerebrovascular oxidative stress plays in conditions associated with cognitive impairment suggests new therapeutic opportunities to counteract and, possibly, reverse the devastating effects of cerebrovascular dysfunction on the brain.

Neurosphere-Derived Cells Exert a Neuroprotective Action by Changing the Ischemic Microenvironment

Background Neurosphere-derived cells (NC), containing neural stem cells, various progenitors and more differentiated cells, were obtained from newborn C57/BL6 mice and infused in a murine model of focal ischemia with reperfusion to investigate if: 1) they decreased ischemic injury and restored brain function; 2) they induced changes in the environment in which they are infused; 3) changes in brain environment consequent to transient ischemia were relevant for NC action. Methodology/Principal Findings NC were infused intracerebroventricularly 4 h or 7 d after 30 min middle cerebral artery occlusion. In ischemic mice receiving cells at 4 h, impairment of open field performance was significantly improved and neuronal loss significantly reduced 7–14 d after ischemia compared to controls and to ischemic mice receiving cells at 7 d. Infusion of murine foetal fibroblast in the same experimental conditions was not effective. Assessment of infused cell distribution revealed that they migrated from the ventricle to the parenchyma, progressively decreased in number but they were observable up to 14 d. In mice receiving NC at 7 d and in sham-operated mice, few cells could be observed only at 24 h, indicating that the survival of these cells in brain tissue relates to the ischemic environment. The mRNA expression of trophic factors such as Insulin Growth Factor-1, Vascular Endothelial Growth Factor-A, Transforming Growth Factor-β1, Brain Derived Neurotrophic Factor and Stromal Derived Factor−1α, as well as microglia/macrophage activation, increased 24 h after NC infusion in ischemic mice treated at 4 h compared to sham-operated and to mice receiving cells at 7 d. Conclusions/Significance NC reduce functional impairment and neuronal damage after ischemia/reperfusion injury. Several lines of evidence indicate that the reciprocal interaction between NC and the ischemic environment is crucial for NC protective actions. Based on these results we propose that a bystander control of the ischemic environment may be the mechanism used by NC to rapidly restore acutely injured brain function.

Central Cardiovascular Circuits Contribute to the Neurovascular Dysfunction in Angiotensin II Hypertension

Hypertension, a powerful risk factor for stroke and dementia, has damaging effects on the brain and its vessels. In particular, hypertension alters vital cerebrovascular control mechanisms linking neural activity to cerebral perfusion. In experimental models of slow-developing hypertension, free radical signaling in the subfornical organ (SFO), one of the forebrain circumventricular organs, is critical for the hormonal release and sympathetic activation driving the elevation in arterial pressure. However, the contribution of this central mechanism to the cerebrovascular alterations induced by hypertension remains uncertain. We tested the hypothesis that free radical production in the SFO is involved in the alterations in cerebrovascular regulation produced by hypertension. In a mouse model of gradual hypertension induced by chronic administration of subpressor doses of angiotensin II (AngII), suppression of free radicals in the SFO by overexpression of CuZn-superoxide dismutase (CuZnSOD) prevented the alteration in neurovascular coupling and endothelium-dependent responses in somatosensory cortex induced by hypertension. The SFO mediates the dysfunction via two signaling pathways. One involves SFO-dependent activation of the paraventricular hypothalamic nucleus, elevations in plasma vasopressin, upregulation of endothelin-1 in cerebral resistance arterioles and activation of endothelin type A receptors. The other pathway depends on activation of cerebrovascular AngII type 1 (AT1) receptors by AngII. Both pathways mediate vasomotor dysfunction by inducing vascular oxidative stress. The findings implicate for the first time the SFO and its efferent hypothalamic pathways in the cerebrovascular alterations induced by AngII, and identify vasopressin and endothelin-1 as potential therapeutic targets to counteract the devastating effects of hypertension on the brain.

Cyclooxygenase 1–Derived Prostaglandin E2 and EP1 Receptors Are Required for the Cerebrovascular Dysfunction Induced by Angiotensin II

Prostaglandin E2 (PGE2) EP1 receptors (EP1Rs) may contribute to hypertension and related end-organ damage. Because of the key role of angiotensin II (Ang II) in hypertension, we investigated the role of EP1R in the cerebrovascular alterations induced by Ang II. Mice were equipped with a cranial window, and cerebral blood flow was monitored by laser-Doppler flowmetry. The attenuation in cerebral blood flow responses to whisker stimulation (−46±4%) and the endothelium-dependent vasodilator acetylcholine (−40±4%) induced by acute administration of Ang II (250 ng/kg per minute; IV for 30 to 40 minutes) were not observed after cyclooxygenase 1 or EP1R inhibition or in cyclooxygenase 1 or EP1-null mice. In contrast, cyclooxygenase 2 inhibition or genetic inactivation did not prevent the attenuation. Ang II–induced oxidative stress was not observed after cyclooxygenase 1 or EP1R inhibition or in EP1R-null mice. Prostaglandin E2 reinstated the Ang II–induced cerebrovascular dysfunction and oxidative stress after cyclooxygenase 1 inhibition. Brain prostaglandin E2 levels were not increased by Ang II but were attenuated by cyclooxygenase 1 and not cyclooxygenase 2 inhibition. The cerebrovascular dysfunction induced by 14-day administration of “slow-pressor” doses of Ang II (600 ng/kg per minute) was attenuated by neocortical application of SC51089. Cyclooxygenase 1 immunoreactivity was observed in microglia and EP1R in endothelial cells. We conclude that the cerebrovascular dysfunction induced by Ang II requires activation of EP1R by constitutive production of prostaglandin E2 derived from cyclooxygenase 1. The findings provide the first evidence that EP1Rs are involved in the deleterious cerebrovascular effects of Ang II and suggest new therapeutic approaches to counteract them.

Progranulin deficiency promotes post-ischemic blood-brain barrier disruption.

Loss-of-function mutations of progranulin (PGRN) have been linked to frontotemporal dementia, but little is known about the effects of PGRN deficiency on the brain in health and disease. PGRN has been implicated in neurovascular development, inflammation, and Wnt signaling, a pathway involved in the formation of the blood–brain barrier (BBB). Because BBB alterations and inflammation contribute to ischemic brain injury, we examined the role of PGRN in the brain damage produced by ischemia-reperfusion. PGRN+/− and PGRN−/− mice underwent middle cerebral artery occlusion (MCAO) with monitoring of cerebral blood flow. Infarct volume and motor deficits were assessed 72 h later. Post-ischemic inflammation was examined by expression of inflammatory genes and flow cytometry. BBB structure and permeability were examined by electron microscopy (EM) and Evans blue (EB) extravasation, respectively. MCAO resulted in ∼60% larger infarcts in PGRN+/− and PGRN−/− mice, an effect independent of hemodynamic factors or post-ischemic inflammation. Rather, massive hemorrhages and post-ischemic BBB disruption were observed, unrelated to degradation of tight junction (TJ) proteins or matrix metalloproteinases (MMPs). By EM, TJ were 30–52% shorter, fewer, and less interlocking, suggesting a weaker seal between endothelial cells. Intracerebral injection of platelet-derived growth factor-CC (PDGF-CC), which increases BBB permeability, resulted in a more severe BBB breakdown in PGRN+/− and PGRN−/− than wild-type mice. We describe a previously unrecognized involvement of PGRN in the expression of key ultrastructural features of the BBB. Such a novel vasoprotective role of PGRN may contribute to brain dysfunction and damage in conditions associated with reduced PGRN function.

Endothelin 1–Dependent Neurovascular Dysfunction in Chronic Intermittent Hypoxia

Obstructive sleep apnea, a condition resulting in chronic intermittent hypoxia (CIH), is an independent risk factor for stroke and dementia, but the mechanisms of the effect are unknown. We tested the hypothesis that CIH increases cerebrovascular risk by altering critical mechanisms regulating cerebral blood flow thereby lowering cerebrovascular reserves. Male C57Bl6/J mice were subjected to CIH (10% O2 for 90 seconds/room air for 90 seconds; during sleep hours) or sham treatment for 35 days. Somatosensory cortex blood flow was assessed by laser Doppler flowmetry in anesthetized mice equipped with a cranial window. CIH increased mean arterial pressure (from 74±2 to 83±3 mm Hg; P<0.05) and attenuated the blood flow increase produced by neural activity (whisker stimulation; −39±2%; P<0.05) or neocortical application of endothelium-dependent vasodilators (acetylcholine response: −41±3%; P<0.05). The cerebrovascular dysfunction was associated with oxidative stress in cerebral resistance arterioles and was abrogated by free radical scavenging or NADPH oxidase inhibition. Furthermore, cerebrovascular dysfunction and free radical increase were not observed in mice lacking the NOX2 subunit of NADPH oxidase. CIH markedly increased endothelin 1 in cerebral blood vessels, whereas cerebrovascular dysfunction and oxidative stress were abrogated by neocortical application of the endothelin type A receptor antagonist BQ123. These data demonstrate for the first time that CIH alters key regulatory mechanisms of the cerebral circulation through endothelin 1 and NADPH oxidase–derived radicals. The ensuing cerebrovascular dysfunction may increase stroke risk in patients with sleep apnea by reducing cerebrovascular reserves and increasing the brains susceptibility to cerebral ischemia.

Estrous cycle-dependent neurovascular dysfunction induced by angiotensin II in the mouse neocortex.

Female mice are protected from the cerebrovascular dysfunction induced by angiotensin II (Ang II), an effect attributed to estrogen. We examined whether such cerebrovascular protection from Ang II is related to the estrous cycle. Cerebral blood flow was monitored by laser-Doppler flowmetry in anesthetized (urethane-chloralose) C57BL/6 female mice equipped with a cranial window. The phase of the estrous cycle was determined by vaginal smear cytology and plasma estrogen measurement. Ang II (0.25 &mgr;g/kg per minute, IV, 30 to 45 minutes) elevated arterial pressure (15 to 20 mm Hg) equally across the estrous cycle. However, in proestrus and estrus, phases in which estrogen is relatively high, Ang II did not impair the increase in the cerebral blood flow induced by neural activity or by endothelium-dependent vasodilators (P>0.05 from vehicle). In contrast, in diestrus (lower estrogen), Ang II induced a marked cerebrovascular dysfunction comparable to that of male mice. For example, the cerebral blood flow responses to whisker stimulation and to the endothelium-dependent vasodilator acetylcholine were attenuated by 41±12% and 49±12%, respectively (P<0.05; n=6 per group). The protection from the cerebrovascular effects of Ang II in proestrus was abolished by the estrogen receptor inhibitor ICI182,780. Ang II also increased production of free radicals in cerebral blood vessels in diestrus (+116±13%; P<0.05) but not in proestrus and estrus (P>0.05 from control). Topical treatment with ICI182,780 reestablished Ang II–induced oxidative stress in proestrus (P>0.05 from diestrus). We conclude that the protection from the neurovascular dysfunction induced by acute administration of Ang II in females depends on the estrous cycle and may underlie the increased propensity to cerebrovascular damage associated with low estrogen states.

Prostaglandin E2 type 1 receptors contribute to neuronal apoptosis after transient forebrain ischemia.

Cyclooxygenase-2-derived prostaglandin E2 (PGE2) contributes to excitotoxic and ischemic neuronal cell death by engaging neuronal PGE2 type 1 receptors (EP1R). Our previous studies have shown that EP1R signaling resulted in disturbances of intracellular Ca2 + homeostasis and suppression of the pro-survival protein kinase AKT. The aim of this study was to investigate whether these pathophysiological mechanism have a role in the neuronal cell death after transient forebrain ischemia. Mice were subjected to ischemia/reperfusion by bilateral common carotid artery occlusion. Hippocampal cornu ammonis area 1 (CA1) neuronal cell death was determined 5 days after reperfusion. Animals treated with the EP1R antagonist SC51089 or EP1R-deficient mice (EP1 –/–) showed significantly less neuronal injury as compared to vehicle-treated wild-type controls. Benefits of EP1R blockage were still evident 14 days after injury. Better neuronal survival was correlated with reduced neuronal caspase-3 activity and decreased nuclear translocation of the apoptosis-inducing factor. Neuroprotection could be reverted by intracerebroventricular administration of the phosphoinositide 3-kinase inhibitor LY294002 and was not further increased by the calcineurin inhibitor FK506. These data implicate EP1R in postischemic neuronal apoptosis possibly by facilitating AKT inhibition.

2-Aminotetraline Derivative Protects from Ischemia/Reperfusion Brain Injury with a Broad Therapeutic Window

The effect of ST1942, a 2-aminotetraline derivative with anti-inflammatory properties, was evaluated in ischemia/reperfusion injury in CD1 and C57BL/6 mice. ST1942 or saline were injected intraperitoneally 30 min and 6, 24, 36 h after ischemia. Forty-eight hours after ischemia, ST1942 (25 mg/kg) reduced the infarct volume by 50% in CD1 and 61% in C57BL/6 mice. All subsequent data were obtained from the latter strain. The ischemic lesion was significantly reduced by 30% when the first injection was administered 6 h after ischemia, revealing a broad effective window. Degenerating neurons in striatum, cortex and hippocampus of ischemic mice were markedly decreased by ST1942. Also examined was the effect of ST1942 on general and focal neurological deficits for 4 days after ischemia. Mice receiving the drug twice daily showed constantly reduced deficits. We then investigated the cortical mRNA expression of some inflammatory and apoptotic genes by real-time PCR. Forty-eight hours after ischemia ST1942 treatment significantly counteracted ischemia-induced activation of IL-1β, TNFα, and Bax, and enhanced the expression of the antiapoptotic gene, Bcl-2, showing in vivo anti-inflammatory and antiapoptotic actions. The microglial activation/macrophage recruitment in the ischemic lesion was strongly prevented in mice receiving ST1942. In neuron-microglia cocultures, ST1942 significantly counteracted LPS-induced cytotoxicity. Binding data and experiments on microglial cell cultures indicate that the anti-inflammatory effect of ST1942 may be due to its action on 5-HT2B receptors, thus highlighting the possibility that this 5-HT receptor subtype may represent a novel target for neuroprotective drugs in ischemic injury.