Kiyoshi Niwa

SOD1 rescues cerebral endothelial dysfunction in mice overexpressing amyloid precursor protein.

Peptides derived from proteolytic processing of the β–amyloid precursor protein (APP), including the amyloid–β peptide, are important for the pathogenesis of Alzheimers dementia. We found that transgenic mice overexpressing APP have a profound and selective impairment in endothelium–dependent regulation of the neocortical microcirculation. Such endothelial dysfunction was not found in transgenic mice expressing both APP and superoxide dismutase–1 (SOD1) or in APP transgenics in which SOD was topically applied to the cerebral cortex. These cerebrovascular effects of peptides derived from APP processing may contribute to the alterations in cerebral blood flow and to neuronal dysfunction in Alzheimers dementia.

Cyclooxygenase-1 Participates in Selected Vasodilator Responses of the Cerebral Circulation

Abstract— Cyclooxygenase (COX) is a prostanoid-synthesizing enzyme present in 2 isoforms: COX-1 and COX-2. Although it has long been hypothesized that prostanoids participate in cerebrovascular regulation, the lack of adequate pharmacological tools has led to conflicting results and has not permitted investigators to define the relative contribution of COX-1 and COX-2. We used the COX-1 inhibitor SC-560 and COX-1-null (COX-1−/−) mice to investigate whether COX-1 plays a role in cerebrovascular regulation. Mice were anesthetized (urethane and chloralose) and equipped with a cranial window. Cerebral blood flow (CBF) was measured by laser Doppler flowmetry or by the 14C-iodoantipyrine technique with quantitative autoradiography. In wild-type mice, SC-560 (25 &mgr;mol/L) reduced resting CBF by 21±4% and attenuated the CBF increase produced by topical application of bradykinin (−59%) or calcium ionophore A23187 (−49%) and by systemic hypercapnia (−58%) (P <0.05 to 0.01). However, SC-560 did not reduce responses to acetylcholine or the increase in somatosensory cortex blood flow produced by vibrissal stimulation. In COX-1−/− mice, resting CBF assessed by 14C-iodoantipyrine was reduced (−13% to −20%) in cerebral cortex and other telencephalic regions (P <0.05). The CBF increase produced by bradykinin, A23187, and hypercapnia, but not acetylcholine or vibrissal stimulation, were attenuated (P <0.05 to 0.01). The free radical scavenger superoxide dismutase attenuated responses to bradykinin and A23187 in wild-type mice but not in COX-1−/− mice, suggesting that COX-1 is the source of the reactive oxygen species known to mediate these responses. The data provide evidence for a critical role of COX-1 in maintaining resting vascular tone and in selected vasodilator responses of the cerebral microcirculation.

Exogenous Aβ1–40 Reproduces Cerebrovascular Alterations Resulting from Amyloid Precursor Protein Overexpression in Mice

Transgenic mice overexpressing the amyloid precursor protein (APP) have a profound impairment in endothelium-dependent cerebrovascular responses that is counteracted by the superoxide scavenger superoxide dismutase (SOD). The authors investigated whether the amyloid-β peptide (Aβ) is responsible for the cerebrovascular effects of APP overexpression. Cerebral blood flow (CBF) was monitored by a laser—Doppler flowmeter in anesthetized-ventilated mice equipped with a cranial window. Superfusion of Aβ1–40 on the neocortex reduced resting CBF in a dose-dependent fashion (−29% ± 7% at 5 μmol/L) and attenuated the increase in CBF produced by the endothelium-dependent vasodilators acetylcholine (−41% ± 8%), bradykinin (−39% ± 9%), and the calcium ionophore A23187 (−37% ± 5%). Aβ1–40 did not influence the CBF increases produced by the endothelium-independent vasodilators S-nitroso-N-acetylpenicillamine and hypercapnia. In contrast, Aβ1–42 did not attenuate resting CBF or the CBF increases produced by endothelium-dependent vasodilators. Cerebrovascular effects of Aβ1–40 were reversed by the superoxide scavengers SOD or MnTBAP. Furthermore, substitution of methionine 35 with norleucine, a mutation that blocks the ability of Aβ to generate reactive oxygen species, abolished Aβ1–40 vasoactivity. The authors conclude that Aβ1–40, but not Aβ1–42, reproduces the cerebrovascular alterations observed in APP transgenics, Thus, Aβ1–40 could play a role in the cerebrovascular alterations observed in Alzheimers dementia.

Alterations in cerebral blood flow and glucose utilization in mice overexpressing the amyloid precursor protein.

We have used quantitative autoradiographic techniques to study the relationship between cerebral blood flow (CBF) and glucose utilization (CGU) in two lines of transgenic mice overexpressing Swedish mutant amyloid precursor protein (APP) and APP-derived Abeta peptides. Mice were studied at an age when there are no amyloid plaques. In the 2123 line, CBF was reduced only in telencephalic regions with no corresponding decrease in CGU. In 2576 transgenics, a line with higher levels of Abeta peptide, both CBF and CGU were reduced throughout the brain. The data indicate that Abeta induces alterations in resting CBF that are either associated with or independent of alterations in CGU and that occur in the absence of amyloid deposition in neuropil of blood vessels. These observations support the hypothesis that cerebrovascular and metabolic abnormalities are early events in the pathogenesis of Alzheimers disease.

The Cyclooxygenase-2 Inhibitor NS-398 Ameliorates Ischemic Brain Injury in Wild-Type Mice but not in Mice with Deletion of the Inducible Nitric Oxide Synthase Gene

The authors investigated the role of the prostaglandin-synthesizing enzyme cyclooxygenase-2 (COX-2) in the mechanisms of focal cerebral ischemia and its interaction with inducible nitric oxide synthase (iNOS). Focal cerebral ischemia was produced by permanent occlusion of the middle cerebral artery (MCA) in mice. Infarct volume was measured 96 hours later by computer-assisted planimetry in thionin-stained brain sections. The highly selective COX-2 inhibitor NS398 (20 mg/kg; intraperitoneally), administered twice a day starting 6 hours after MCA occlusion, reduced total infarct volume in C57BL/6 (–23%) and 129/SVeV mice (–21%), and ameliorated the motor deficits produced by MCA occlusion (P < .05). However, NS398 did not influence infarct volume in mice with deletion of the iNOS gene (P > .05). In contrast, the neuronal NOS inhibitor 7-NI (50 mg/kg; intraperitoneally), administered once 5 minutes after MCA occlusion, reduced neocortical infarct volume by 20% in iNOS −/− mice (P < .05). NS398 did not affect arterial pressure, resting CBF or the CBF reactivity to hypercapnia in anesthetized iNOS null mice (P > .05). The data suggest that COX-2 reaction products, in mouse as in rat, contribute to ischemic brain injury. However, the failure of NS398 to reduce infarct volume in iNOS null mice suggests that iNOS-derived NO is required for the deleterious effects of COX-2 to occur. Thus, COX-2 reaction products may be another mechanism by which iNOS-derived NO contributes to ischemic brain injury.

Increased susceptibility to ischemic brain injury in cyclooxygenase-1-deficient mice.

Cyclooxygenase-1 (COX-1), a rate-limiting enzyme in the synthesis of prostanoids, is involved in selected vasodilatatory responses of the cerebral circulation. Cyclooxygenase-1–null mice were used to determine whether COX-1 influences cerebral ischemic damage. The middle cerebral artery was occluded in COX-1 −/− and +/+ mice (n = 9/group), and lesion volume was determined in thionin-stained sections 24 or 96 hours later. Middle cerebral artery occlusion produced larger infarcts in COX-1 −/− mice, both at 24 (35 ± 17%;P < 0.05) and 96 hours (41 ± 16%;P < 0.05) after ischemia. The enlargement was not due to increased susceptibility to glutamate excitotoxicity, because microinjection of N-methl- d -asparatate or kainate in the parietal cortex produced comparable lesions in COX-1 +/+ and −/− mice (P > 0.05; n = 8/group). To examine the contribution of hemodynamic factors to the enlargement of the infarct, cerebral blood flow was monitored by laser-Doppler flowmetry in the ischemic territory (n = 6/group). Although the reduction in cerebral blood flow was comparable in the ischemic core (P > 0.05), at the periphery of the ischemic territory the reduction was greater in COX-1 −/− mice (−58 ± 4%) than in COX-1 +/+ mice (−34 ± 5%;P < 0.05). It is concluded that mice lacking COX-1 are more susceptible to focal cerebral ischemia, an effect that can be attributed to a more severe cerebral blood flow reduction in vulnerable regions at the periphery of the ischemic territory. Thus, the vascular effects of COX-1 may contribute to maintain cerebral blood flow in the postischemic brain and, as such, play a protective role in ischemic brain injury.

Neural Regulation of the Cerebral Circulation

It was once believed that cerebral blood vessels could not dilate and constrict independently and that the cerebral circulation was entirely under the control of the systemic circulation [see ref 1 for a historical review]. However, evidence accumulated over the past 100 years indicates that cerebral blood vessels are in a dynamic state. Thus, the cerebral vasculature is endowed with complex regulatory systems that allow the brain to finely regulate its own blood supply. One of the major factors that regulates cerebral blood flow (CBF) is neuronal activity. In this chapter, we will focus on the mechanisms governing the relationship between neural activity and blood flow with emphasis on the role of nitric oxide (NO) and cyclooxygenase-2 (COX-2).

Neurovascular coupling in health and disease: lessons from transgenic mice

Abstract Neuronal activity is one of the major factors regulating the cerebral circulation. Synaptic activity produces increases in blood flow that are spatially restricted to the activated region and temporally related to the period of activation, a phenomenon termed functional hyperemia. The cellular and molecular factors linking synaptic activity to cerebral blood flow (CBF) and the mechanisms of their alteration in disease states are not well understood. The introduction of genetically engineered mice with overexpression or deletion of selected gene products provides a powerful tool to investigate the mechanisms of functional hyperemia. In this work, we will review the contributions to this field provided by transgenic mouse models, focusing on the role of nitric oxide (NO) and cyclooxygenase (COX) in cerebral cortex and/or cerebellum. Furthermore, the alterations in functional hyperemia produced by β-amyloid, a peptide that accumulates in the brain of patients with Alzheimers disease, will also be examined.

Role of Inducible Nitric Oxide Synthase and Cyclooxygenase-2 in the Mechanisms of Ischemic Brain Injury

There is a growing body of evidence indicating that cerebral ischemic damage is associated with a local inflammatory reaction, which contributes to the development of ischemic brain injury. We review evidence that inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) are involved in the mechanisms by which ischemia-induced inflammation contributes to tissue damage. In rodents, as in humans, iNOS is expressed in inflammatory cells infiltrating the ischemic brain and in cerebral blood vessels. Administration of iNOS inhibitors 24h after ischemia reduces the size of the infarct produced by occlusion of the rat middle cerebral artery (MCA). Furthermore, “knockout” mice lacking the iNOS gene have smaller infarcts than wild-type mice. COX-2 is expressed in the postischemic brain with a time course similar to that of iNOS; and it is present in ischemic neurons, inflammatory cells, and blood vessels. Administration of the selective COX-2 inhibitor NS-398 reduces infarct size. Furthermore, NO produced by iNOS activates COX-2, thereby increasing the production of toxic prostanoids and free radicals. The evidence suggests that NO synthesized by iNOS, in part through reaction products of COX-2, contributes to the expansion of the infarct that occurs during the late postischemic period. Thus, the interaction between iNOS and COX-2 plays an important role in the late stages of cerebral ischemic damage. Delayed administration of iNOS and COX-2 inhibitors may be a useful therapeutic strategy to target selectively the progression of ischemic brain injury.