Visceral adiposity links cerebrovascular dysfunction to cognitive impairment in middle-aged mice.

Midlife cognitive decline is now recognized as a factor of poor prognosis for late-life dementia. Although an epidemiological link has been suggested with high fat diet (HFD)-induced metabolic disorders, the effect of a long period of HFD on midlife cerebrovascular and cognitive functions remains unproven. A cohort of 216 young mice was fed with HFD up to middle age (12 months), and kinetically characterized for metabolic status, including weight, blood lipid profile, hepatic fat accumulation, glucose intolerance, and visceral adiposity. Metabolic disorders were evidenced from 3 months of HFD. Visual recognition memory and flexibility were significantly altered and associated to a visceral adiposity whereas spatial reference memory and working memory did not. Concomitantly, a progressive dysfunction of the vascular endothelium-dependent relaxation was detected in both middle cerebral artery and parenchymal arterioles, with consequences on the regulation of cerebral blood flow, but without any modification of the basal brain tissue MRI perfusion signal. Our data collection empowered us to stratify the mice according to their heterogeneous response to diet, and to propose a statistical prediction model for cognitive impairment, combining visceral adiposity and cerebral vasomotion in a diagnostic perspective of early neurological deficits.

Ferroptosis, a newly characterized form of cell death in Parkinson's disease that is regulated by PKC.

Parkinson's disease (PD) is a complex illness characterized by progressive dopaminergic neuronal loss. Several mechanisms associated with the iron-induced death of dopaminergic cells have been described. Ferroptosis is an iron-dependent, regulated cell death process that was recently described in cancer. Our present work show that ferroptosis is an important cell death pathway for dopaminergic neurons. Ferroptosis was characterized in Lund human mesencephalic cells and then confirmed ex vivo (in organotypic slice cultures) and in vivo (in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model). Some of the observed characteristics of ferroptosis differed from those reported previously. For example, ferroptosis may be initiated by PKCα activation, which then activates MEK in a RAS-independent manner. The present study is the first to emphasize the importance of ferroptosis dysregulation in PD. In neurodegenerative diseases like PD, iron chelators, Fer-1 derivatives and PKC inhibitors may be strong drug candidates to pharmacologically modulate the ferroptotic signaling cascade.

Age and diet modify acute microhemorrhage outcome in the mouse brain.

Cerebral microhemorrhages (CMHs) are considered as asymptomatic lesions, but might impair cognition in non-demented elderly individuals. The aging process includes poor vascular health, enhanced at midlife by metabolic disturbances upon high-fat diet (HFD). The onset of CMHs could thus have more serious consequences in midlife subjects with metabolic disturbances. This hypothesis was tested through the induction of multiple CMHs, using cyclodextrin nanoparticles injection, in mice at midlife (14 month old) or at a younger stage (5 month old) after 12 months or 3 months of normal diet or HFD (40% of animal fat) respectively. When induced at 14 months of age, CMHs were not larger but were more numerous (+25%) in mice on HFD compared with mice on normal diet. They slowed down the locomotor activity significantly but caused neither a change in the working memory nor a difference in the visual recognition memory decline. When induced at 5 months of age, CMHs provoked slighter locomotor and cognitive symptoms, regardless the diet. No spontaneous progression of CMHs toward larger hemorrhages was observed after onset when HFD was prolonged up to midlife. Consistently, no precipitated cognitive decline was observed. Middle-age plus time of metabolic disturbances represent enhanced risk factors for CMH outcome.

Safety of oral anticoagulants on experimental brain microbleeding and cognition.

This study aims at determining the ability of clinical-based doses of four oral anticoagulants to transform the onset of a cerebral microhemorrhages (CMH) burden into a symptomatic intracerebral hemorrhage (ICH) in the healthy brain, and precipitate cognitive impairment. Wild-type mice were anticoagulated for 10 days using apixaban, rivaroxaban or dabigatran as direct oral anticoagulants (DOACs), or warfarin as vitamin K-antagonist. Meanwhile, a burden of ∼20 CMHs was induced in the Sylvian territory by intra-carotid injection of cyclodextrin nanoparticles. At bleeding onset, only warfarin provoked deadly hematoma, and dramatically increased mortality (+45%). All the DOACs enhanced CMH burden through a greater number of intermediate-sized microhemorrhages (+80% to +180%). Although silent at onset, both baseline- and anticoagulant-enhanced CMH burdens increased mortality (+11% to +58%) along the following year without statistical difference among groups, and despite cessation of anticoagulation and absence of CMH progression or transformation into ICH. All survivor mice exhibited reduction in visual recognition memory from 9 months. In the healthy brain, DOACs preserve the onset of microhemorrhages from transformation into ICH, and do not precipitate cognitive impairment despite enhancement of CMH burden. High CMH burdens should however be considered for early detection and preventive memory care apart from anticoagulation decisions.

Neither nefopam nor acetaminophen can be used as postoperative analgesics in a rat model of ischemic stroke.

Analgesics such as opioid agonists are usually not given during the postoperative phase of experimental stroke because they are susceptible to interfere with the evaluation of neuroprotective therapies. Here, we investigate the potential of acetaminophen and nefopam, two nonopioid analgesic drugs, to exert an analgesic effect without inducing neuroprotection in a murine model of ischemic stroke. We demonstrate that acetaminophen (200 mg/kg, PO) induces a significant decrease in the infarct volume, particularly in the cortex (VEHICLE: 200.1 mm3 vs. ACETAMINOPHEN: 140.9 mm3 , P < 0.05), while nefopam (2, 20 or 40 mg/kg, IM), administered at the end of middle cerebral artery occlusion (MCAO), do not influence the infarct size (VEHICLE: 268.6 mm3 vs. NEFOPAM 2: 248.8 mm3 , NEFOPAM 20: 250.6 mm3 and NEFOPAM 40: 215.9 mm3 , P > 0.05). Moreover, we find that nefopam administration (20 mg/kg, IM) in the acute postoperative phase do not change the level of neuroprotection induced by MK801 (3 mg/kg, IV), a well-known neuroprotectant (VEHICLE: 268.6 mm3 vs. MK801: 194.4 mm3 and vs. MK801 + NEFOPAM 20: 195.2 mm3 ). On the other hand, although nefopam induces analgesia in healthy animals, it is not the case when administered during MCAO (behavior scores at 5 min: HEALTHY: 2.1 vs. HEALTHY + NEFOPAM 20: 0.6, P < 0.5; IR: 0.40 vs. IR + NEFOPAM 20: 0.67, P > 0.05). Our data suggest that neither acetaminophen nor nefopam can be used as analgesic agents to meet the needs of limiting rodent pain and distress during experimental stroke surgery.

Pharmacological neutropenia prevents endothelial dysfunction but not smooth muscle functions impairment induced by middle cerebral artery occlusion.

1. The polymorphonuclear neutrophils (PMN) activation and mobilization observed in acute cerebral infarction contribute to the brain tissue damage, but PMN could also be involved in postischemic functional injury of ischemied blood vessel. 2. This study was undertaken to investigate whether pharmacological neutropenia could modify the postischemic endothelial dysfunction in comparison to smooth muscle whose impairment is likely more related to reperfusion and oxidative stress. 3. A cerebral ischemia-reperfusion by endoluminal occlusion of right middle cerebral artery (MCA) was performed 4 days after intravenous administration of vinblastine or 12 h after RP-3 anti-rat neutrophils monoclonal antibody (mAb RP-3) injection into the peritoneal cavity, on male Wistar rats with 1-h ischemia then followed by 24-h reperfusion period. Brain infarct volume was measured by histomorphometric analysis and vascular endothelial and smooth muscle reactivity of MCA was analysed using Halpern myograph. 4. Neutropenia induced a neuroprotective effect as demonstrated by a significant decrease of brain infarct size. In parallel to neuroprotection, neutropenia prevented postischemic impairment of endothelium-dependent relaxing response to acetylcholine. In contrast, smooth muscle functional alterations were not prevented by neutropenia. Ischemia-reperfusion-induced myogenic tone impairment remained unchanged in vinblastine and mAb RP-3-treated rats. Postischemic Kir2.x-dependent relaxation impairment was not prevented in neutropenic conditions. The fully relaxation of smooth muscle response to sodium nitroprusside was similar in all groups. 5. Our results evidenced the dissociate prevention of pharmacologically induced neutropenia on postischemic vascular endothelial and smooth muscle impairment. The selective endothelial protection by neutropenia is parallel to a neuroprotective effect suggesting a possible relationship between the two phenomena.

Repolarization abnormalities and their arrhythmogenic consequences in porcine tachycardia-induced cardiomyopathy.

OBJECTIVES: Action potential prolongation related to the alteration of several membrane currents is constantly reported in heart failure (HF) but reports about its role in arrhythmogenesis are sparse. Our aim was to determine, by analogy with long QT syndromes, whether prolonged repolarization is associated with increased dispersion or linked to bradycardia-dependent ventricular arrhythmias in pacing-induced cardiomyopathy. METHODS: QT intervals, action potentials and transmural activation-to-recovery intervals (ARIs) along with whole-cell delayed rectifier (I(K)) and transient outward (I(to1)) K+ currents were recorded in left ventricle from pigs with HF and controls. HF was obtained after 14 days of rapid pacing at 250 ms. RESULTS: Repolarization was delayed as indexed by corrected QT intervals (13.7% increase, P<0.01) or ARIs (252+/-4 to 340+/-7 ms, P<0.01). ARIs were uniformly prolonged with disappearance of the transmural gradient, spatial dispersion of repolarization decreased by 50% (P<0.05). I(to1) density was reduced in HF from 1.35+/-0.1 to 0.57+/-0.04 pA/pF subepicardially, from 1.05+/-0.19 to 0.55+/-0.08 pA/pF midmyocardially and from 1.04+/-0.1 to 0.48+/-0.04 pA/pF subendocardially. I(K) density was significantly decreased in HF pigs vs. controls: subepicardially from 0.46+/-0.04 to 0.22+/-0.02 pA/pF; midmyocardially from 0.46+/-0.05 to 0.25+/-0.03 pA/pF; and subendocardially from 0.49+/-0.04 to 0.20+/-0.04 pA/pF following depolarization at +50 mV. Electrocardiogram (ECG) monitoring at the time of death did not disclose any polymorphic ventricular tachyarrhythmia. CONCLUSION: Despite a profound alteration in K+ currents, repolarization is uniformly prolonged in this model with no proclivity to develop bradycardia-dependent arrhythmias.

Delayed cerebrovascular protective effect of lipopolysaccharide in parallel to brain ischemic tolerance.

Cerebrovascular abnormalities, in endothelium and smooth muscle compartments, occur in the course of cerebral ischemia-reperfusion as evidenced by the impairment of endothelium-dependent relaxation and decrease in potassium inward rectifier density in occluded middle cerebral arteries (MCAs). The authors investigated whether a delayed vascular protection occurred in a model of brain ischemic tolerance. A low dose of lipopolysaccharide (0.3 mg/kg) administered 72 h before MCA occlusion induced a significant decrease in infarct volume. In parallel to this delayed neuroprotective effect, lipopolysaccharide prevented the ischemia-reperfusion-induced impairment of endothelium relaxation. In addition, lipopolysaccharide prevented the postischemic alteration of potassium inward rectifier-dependent smooth muscle relaxation as well as the decrease in potassium inward rectifier density measured by patch-clamp in dissociated vascular smooth muscle cells originated from the occluded MCA. These results suggest that during brain ischemic tolerance, lipopolysaccharide is able to induce both a delayed neuroprotective and vasculoprotective effect.

Risperidone prolongs cardiac action potential through reduction of K+ currents in rabbit myocytes.

Prolongation of QT interval by antipsychotic drugs is an unwanted side effect that may lead to ventricular arrhythmias. The antipsychotic agent risperidone has been shown to cause QT prolongation, especially in case of overdosage. We investigated risperidone effects on action potentials recorded from rabbit Purkinje fibers and ventricular myocardium and on potassium currents recorded from atrial and ventricular rabbit isolated myocytes. The results showed that (1) risperidone (0.1-3 microM) exerted potent lengthening effects on action potential duration in both tissues with higher potency in Purkinje fibers and caused the development of early afterdepolarizations at low stimulation rate; (2) risperidone (0.03-0.3 microM) reduced significantly the current density of the delayed rectifier current and at 30 microM decreased the transient outward and the inward rectifier currents. This study might explain QT prolongation observed in some patients treated with risperidone and gives enlightenment on the risk of cardiac adverse events.

Risperidone reduces K+ currents in human atrial myocytes and prolongs repolarization in human myocardium.

The antipsychotic agent risperidone has been shown to cause QT prolongation. In rabbit heart preparations, we have demonstrated that risperidone markedly lengthened action potential duration and blocked the delayed rectifier current, I(Kr.) The current study was designed to investigate the risperidone effects: (i) on the main K(+) repolarizing currents on human atrial myocytes, using whole-cell patch clamp recordings; (ii) on action potentials recorded from human atrial and ventricular myocardium using conventional microelectrodes. We found that: (1) risperidone (3-30 microM) reduced significantly the sustained current, I(sus), and 30 microM decreased significantly the transient outward current I(to) but was without effect on the inward rectifier current I(K1); (2) risperidone (0.3-10 microM) lengthened significantly the final repolarization of the atrial action potential and risperidone (10 microM) markedly lengthened the final repolarization in ventricular myocardium. This study showed that risperidone exerts direct electrophysiological effects on human preparations but only at relatively high concentration.

The neuroprotective effect of the antioxidant flavonoid derivate di-tert-butylhydroxyphenyl is parallel to the preventive effect on post-ischemic Kir2.x impairment but not to post-ischemic endothelial dysfunction.

In the rat model of transient cerebral ischemia induced by intraluminal occlusion of the middle cerebral artery, we investigated the respective roles of ischemia and reperfusion in endothelium-dependent relaxation and smooth muscle relaxation related to the inward rectifier potassium current (Kir2.x), using the Halpern arteriography technique and/or patch-clamp technique. We first demonstrated that reperfusion is necessary to induce a significant impairment of smooth muscle Kir2.x, since ischemia alone has no effect on Kir2.x current density and function. In addition, we demonstrated that both ischemia and reperfusion are necessary for the occurrence of maximal post-ischemic endothelial dysfunction. The crucial role of reperfusion in post-ischemic vascular impairment prompted us to characterize the effect of a new antioxidant synthetic flavonoid derivate, 3'5'di- tert-butylhydroxyphenyl (dt-BC), on both neuronal and vascular injuries. Dt-BC (10 mg/kg) induced a neuroprotective effect as demonstrated by a significant decrease in infarct size, while there was no protective effect with the doses of 3 mg/kg and 30 mg/kg. Parallel to neuroprotection, dt-BC at a dose of 10 mg/kg, but not with doses of 3 mg/kg and 30 mg/kg, prevented post-ischemic impairment of smooth muscle Kir2.x current density and function, while dt-BC had no effect on the post-ischemic alteration of endothelial function whatever doses are used. These data demonstrate the potential of a new synthetic flavonoid derivate to induce neurovascular protection and support a possible relationship between vascular and neuronal protection via pharmacological modulation of oxidative stress.

Clarithromycin reduces Isus and Ito currents in human atrial myocytes with minor repercussions on action potential duration.

The macrolide antibacterial agent clarithromycin has been shown to cause QT interval prolongation on the electrocardiogram. In rabbit heart preparations clarithromycin (concentration dependently) lengthened the action potential duration and blocked the delayed rectifier current. The aim of the present study was to investigate the clarithromycin effects: (i) on the Ca2+ L-type and the main K+ repolarizing currents on human atrial myocytes, using whole-cell patch clamp recordings and (ii) on action potentials recorded from human atrial and ventricular myocardium using conventional microelectrodes. It has been found that (i) 10-30 microM clarithromycin reduced the sustained current Isus significantly and that a 100 microM concentration was needed to cause a significant reduction in the transient outward current Ito, whereas clarithomycin did not affect the calcium current and (ii) clarithromycin (10-100 microM) prolonged the action potential duration in atrial preparations but did not alter the different parameters of the ventricular action potential. It is concluded that clarithromycin exerts direct cardiac electrophysiological effects that may contribute to pro-arrythmic potential.

Effects of the PPAR-α agonist fenofibrate on acute and short-term consequences of brain ischemia.

In stroke, there is an imperative need to develop disease-modifying drugs able to (1) induce neuroprotection and vasculoprotection, (2) modulate recovery and brain plasticity, and (3) limit the short-term motor and cognitive consequences. We hypothesized that fenofibrate, a peroxisome proliferator-activated receptor-α (PPAR-α) agonist, could exert a beneficial effect on immediate and short-term poststroke consequences related to its pleiotropic mechanisms. Rats or mice were subjected to focal ischemia to determine the effects of acute treatment by fenofibrate on (i) motor and memory impairment, (2) both cerebral and vascular compartments, (3) inflammation, (4) neurogenesis, and (5) amyloid cascade. We show that fenofibrate administration results in both neuronal and vascular protection and prevents the short-term motor and cognitive poststroke consequences by interaction with several mechanisms. Modulation of PPAR-α generates beneficial effects in the immediate poststroke consequences by mechanisms involving the interactions between polynuclear neutrophils and the vessel wall, and microglial activation. Fenofibrate modulates mechanisms involved in neurorepair and amyloid cascade. Our results suggest that PPAR-α agonists could check the key points of a potential disease-modifying effect in stroke.

Time-induced progressive alteration of kir current in cerebral smooth muscle cells of stroke-prone spontaneously hypertensive rats.

We investigated the involvement of potassium inward rectifier current (Kir) impairment in smooth muscle cells of cerebral arteries under the condition of increased susceptibility of stroke, in spontaneously hypertensive stroke-prone (SHRsp) rats compared to spontaneously hypertensive (SHR) ones as well as to controls (WKY). Kir current was studied with whole-cell patch-clamp techniques on freshly isolated single smooth muscle cells (SMC) of middle cerebral artery (MCA) from SHRsp, SHR, and WKY male rats (are range 12-32 weeks). A significant and progressive Kir current density reduction was observed on SMC of SHRsp rats from the 22nd week of age on, as opposed to the Kir current density stability observed over the same time in the SMC of WKY and SHR rats. The Kir density alteration was correlated to the age of the SHRsp animals. These results suggest that in the cerebral vascular smooth muscle cells of SHRsp rats, there is a progressive Kir channel impairment, leading to a reduction of Kir current density. This impairment may underpin a lack of vasodilation of the MCA and be implicated in the stroke-proneness observed on SHRsp animals.

PPARs: a potential target for a disease-modifying strategy in stroke.

Stroke is one of the major causes of mortality and disability in adults in industrialized countries. Despite numerous preclinical studies and clinical trials in the field of cerebral ischemia, no pharmacological agent has been validated in the treatment of acute ischemic, except thrombolysis. Cerebral ischemia is not only a neuronal disease but it affects the entire neurovascular unit. The therapeutic strategy in stroke should be more global and combine preventive approaches, acute phase treatment and long-term care to improve recovery and prevent or treat affective and cognitive post-stroke consequences. There is an imperative need to develop disease-modifying drugs, which should be able to induce neuroprotection, to serve as adjuvants for thrombolysis by decreasing the hemorrhagic risk and to limit the long-term post-stroke consequences. This review presents the potential effects of Peroxisome Proliferator-Activated Receptors (PPARs) and of their agonists in stroke. We focus on each PPAR receptor and detail their implication in stroke. PPARs are nuclear receptors, acting as ligand-dependent transcription factors. They are expressed in the neurovascular unit that suggests that PPARs could play a role in stroke. Indeed, it has been shown that they are able to interfere with pathways implicated in the pathophysiology of stroke. They could be an answer to this disease-modifying drug concept, being able to act on the different phases of ischemia.

Withdrawal of fenofibrate treatment partially abrogates preventive neuroprotection in stroke via loss of vascular protection.

To explore the mechanisms of action of preventive neuroprotection induced by PPAR-alpha activation, we have evaluated the neuronal, vascular effects of preventive treatment with fenofibrate up until the induction of experimental brain ischaemia and fenofibrate treatment withdrawn 3days before ischaemia induction. Fenofibrate (200mg/kg/day) was administered in rats for 14days or withdrawn 3days before induction of cerebral ischaemia. Animals underwent a 1-hour middle cerebral artery occlusion (MCAo), followed by reperfusion for 24h. The MCA's vasoreactivity was analyzed and brain sections were used to assess infarct size, inflammatory and oxidative stress markers. Fenofibrate administration significantly decreases the cerebral infarct volume. This effect was associated with partial prevention of post-ischaemic endothelial dysfunction. However, withdrawal of the fenofibrate treatment 3days before the induction of ischaemia reduced the neuroprotection and was less beneficial in preventing endothelial dysfunction as well as superoxide anion production. In contrast, fenofibrate significantly reduced microglial activation and neutrophil infiltration into the ischaemic zone to a similar extent in both treatment modes. Our results show that the fenofibrate-induced cerebral protective effect may be related to both an acute effect and a preconditioning-like mechanism. The vascular protective effect appears rather to translate the acute effects of fenofibrate administration.