Microglial vesicles improve post-stroke recovery by preventing immune cell senescence and favoring oligodendrogenesis.

Contrasting myelin damage through the generation of new myelinating oligodendrocytes represents a promising approach to promote functional recovery after stroke. Here, we asked whether activation of microglia and monocyte-derived macrophages affects the regenerative process sustained by G protein-coupled receptor 17 (GPR17)-expressing oligodendrocyte precursor cells (OPCs), a subpopulation of OPCs specifically reacting to ischemic injury. GPR17-iCreERT2:CAG-eGFP reporter mice were employed to trace the fate of GPR17-expressing OPCs, labeled by the green fluorescent protein (GFP), after permanent middle cerebral artery occlusion. By microglia/macrophages pharmacological depletion studies, we show that innate immune cells favor GFP+ OPC reaction and limit myelin damage early after injury, whereas they lose their pro-resolving capacity and acquire a dystrophic "senescent-like" phenotype at later stages. Intracerebral infusion of regenerative microglia-derived extracellular vesicles (EVs) restores protective microglia/macrophages functions, limiting their senescence during the post-stroke phase, and enhances the maturation of GFP+ OPCs at lesion borders, resulting in ameliorated neurological functionality. In vitro experiments show that EV-carried transmembrane tumor necrosis factor (tmTNF) mediates the pro-differentiating effects on OPCs, with future implications for regenerative therapies.

Improvement of fiber connectivity and functional recovery after stroke by montelukast, an available and safe anti-asthmatic drug.

Stroke is one of the main causes of death, neurological dysfunctions or disability in elderly. Neuroprotective drugs have been proposed to improve long-term recovery after stroke, but failed to reach clinical effectiveness. Hence, recent studies suggested that restorative therapies should combine neuroprotection and remyelination. Montelukast, an anti-asthmatic drug, was shown to exert neuroprotection in animal models of CNS injuries, but its ability to affect oligodendrocytes, restoring fiber connectivity, remains to be determined. In this study, we evaluated whether montelukast induces long-term repair by promoting fiber connectivity up to 8 weeks after middle cerebral artery occlusion (MCAo), using different experimental approaches such as in vivo diffusion magnetic resonance imaging (MRI), electrophysiological techniques, ex vivo diffusion tensor imaging (DTI)-based fiber tracking and immunohistochemistry. We found that, in parallel with a reduced evolution of ischemic lesion and atrophy, montelukast increased the DTI-derived axial diffusivity and number of myelin fibers, the density of myelin binding protein (MBP) and the number of GSTpi+ mature oligodendrocytes. Together with the rescue of MCAo-induced impairments of local field potentials in ischemic cortex, the data suggest that montelukast may improve fibers reorganization. Thus, to ascertain whether this effect involved changes of oligodendrocyte precursor cells (OPCs) activation and maturation, we used the reporter GPR17iCreERT2:CAG-eGreen florescent protein (GFP) mice that allowed us to trace the fate of OPCs throughout animal's life. Our results showed that montelukast enhanced the OPC recruitment and proliferation at acute phase, and increased their differentiation to mature oligodendrocytes at chronic phase after MCAo. Considering the crosstalk between OPCs and microglia has been widely reported in the context of demyelinating insults, we also assessed microglia activation. We observed that montelukast influenced the phenotype of microglial cells, increasing the number of M2 polarized microglia/macrophages, over the M1 phenotype, at acute phase after MCAo. In conclusion, we demonstrated that montelukast improves fiber re-organization and long-term functional recovery after brain ischemia, enhancing recruitment and maturation of OPCs. The present data suggest that montelukast, an already approved drug, could be "repositioned "as a protective drug in stroke acting also on fiber re-organization.

GPR17 expressing NG2-Glia: Oligodendrocyte progenitors serving as a reserve pool after injury.

In the adult brain NG2-glia continuously generate mature, myelinating oligodendrocytes. To which extent the differentiation process is common to all NG2-glia and whether distinct pools are recruited for repair under physiological and pathological conditions still needs clarification. Here, we aimed at investigating the differentiation potential of adult NG2-glia that specifically express the G-protein coupled receptor 17 (GPR17), a membrane receptor that regulates the differentiation of these cells at postnatal stages. To this aim, we generated the first BAC transgenic GPR17-iCreER(T2) mouse line for fate mapping studies. In these mice, under physiological conditions, GPR17(+) cells--in contrast to GPR17(-) NG2-glia--did not differentiate within 3 months, a peculiarity that was overcome after cerebral damage induced by acute injury or ischemia. After these insults, GPR17(+) NG2-glia rapidly reacted to the damage and underwent maturation, suggesting that they represent a 'reserve pool' of adult progenitors maintained for repair purposes.

Peroxisome proliferator-activated receptor {alpha} agonism prevents renal damage and the oxidative stress and inflammatory processes affecting the brains of stroke-prone rats.

A growing body of evidence suggests that chronic kidney disease is a significant risk for cardiovascular events and stroke regardless of traditional risk factors. The aim of this study was to examine the effects of peroxisome proliferator-activated receptor (PPAR) agonists on the tissue damage affecting salt-loaded spontaneously hypertensive stroke-prone rats ( SHRSPs), an animal model that develops a complex pathology characterized by systemic inflammation, hypertension, and proteinuria and leads to end-organ injury (initially renal and subsequently cerebral). Compared with the PPARγ agonist rosiglitazone, the PPARα ligands fenofibrate and clofibrate significantly increased survival (p < 0.001) by delaying the occurrence of brain lesions monitored by magnetic resonance imaging (p < 0.001) and delaying increased proteinuria (p < 0.001). Fenofibrate completely prevented the renal disorder characterized by severe vascular lesions, tubular damage, and glomerular sclerosis, reduced the number of ED-1-positive cells and collagen accumulation, and decreased the renal expression of interleukin-1ß, transforming growth factor ß, and monocyte chemoattractant protein 1. It also prevented the plasma and urine accumulation of acute-phase and oxidized proteins, suggesting that the protection induced by PPARα agonists was at least partially caused by their anti-inflammatory and antioxidative properties. The results of this study demonstrate that PPAR agonism has beneficial effects on spontaneous brain and renal damage in SHRSPs by inhibiting systemic inflammation and oxidative stress, and they support carrying out future studies aimed at evaluating the effect of PPARα agonists on proteinuria and clinical outcomes in hypertensive patients with renal disease at increased risk of stroke.

Treatment with LXR agonists after focal cerebral ischemia prevents brain damage.

Stroke is characterized by massive inflammation in areas surrounding the injury that magnifies damage to the brain. The liver X receptors (LXRs) are nuclear receptors that regulate cholesterol, lipid, and glucose metabolism. Synthetic LXR agonists have potent anti-inflammatory properties in a variety of settings, including neuroinflammation. However, the ability of LXR agonists to suppress stroke-associated inflammation has not been evaluated. Here, we have used time-lapse magnetic resonance imaging (MRI) to show that a single dose of an LXR ligand administered post-injury dramatically reduces brain damage in a model of acute brain ischemia. Neuroprotection was associated with suppression of neuroinflammation.

Sex-Specific Features of Microglia from Adult Mice.

Sex has a role in the incidence and outcome of neurological illnesses, also influencing the response to treatments. Neuroinflammation is involved in the onset and progression of several neurological diseases, and the fact that estrogens have anti-inflammatory activity suggests that these hormones may be a determinant in the sex-dependent manifestation of brain pathologies. We describe significant differences in the transcriptome of adult male and female microglia, possibly originating from perinatal exposure to sex steroids. Microglia isolated from adult brains maintain the sex-specific features when put in culture or transplanted in the brain of the opposite sex. Female microglia are neuroprotective because they restrict the damage caused by acute focal cerebral ischemia. This study therefore provides insight into a distinct perspective on the mechanisms underscoring a sexual bias in the susceptibility to brain diseases.

Statins: multiple mechanisms of action in the ischemic brain.

Although substantial epidemiological studies have failed to find a correlation between cholesterol levels and stroke, clinical trials have shown that HMG-CoA reductase inhibitors (or statins, the most potent hypocholesterolemic drugs available) greatly reduce the incidence of stroke. These clinical observations have opened the way to a number of studies of the non-cholesterol-dependent (or pleiotropic) effects in animal models of stroke, indicating that the neuroprotection is attributable to multiple activities. One of the main protective mechanisms elicited by statin administration is the increase in nitric oxide bioavailability that regulates cerebral perfusion and improves endothelial function, but others include antioxidant properties, the inhibition of inflammatory responses, immunomodulatory actions, the regulation of progenitor cells, and the stabilization of atherosclerotic plaques. Many of these effects are due to the inhibited synthesis of isoprenoid intermediates, which serve as lipid attachments for a variety of intracellular signaling molecules. This article describes the mechanisms involved in the neuroprotective effects of statins.

Novel 3-O-glycosyl-3-demethylthiocolchicines as ligands for glycine and gamma-aminobutyric acid receptors.

New 3-O-glycosyl-3-demethylthiocolchicines containing natural and unnatural sugar moieties were prepared and tested on gamma-aminobutyric acid (GABA) and strychnine-sensitive glycine receptors present in rat brain and spinal cord. Two different synthetic approaches were used with the readily available 3-O-demethylthiocolchicine (1b) and thiocolchicoside (2a). Glycosyl compounds 2a-g were obtained from 1b and 1-fluorosugars 4. 6'-Heterosubstituted glycosyl compounds 6-12 and the 6'-desoxy derivative 2h were prepared from 2a.

The role of HMG-CoA reductase inhibition in endothelial dysfunction and inflammation.

Statin-induced inhibition ofHMG-CoA reductase reduces cholesterol production and prevents the formation of many non-steroidal isoprenoid compounds, such as farnesylpyrophosphate and geranylgeranylpyrophosphate, that act as lipid attachments for the post-translational modification of various proteins, including the G-proteins and transcription factors involved in a number of cell processes. However, the blockade of isoprenylation elicited by statin treatment also has biological effects on cell function that go beyond the decrease in cholesterol synthesis: these are the so-called "pleiotropic" effects that mainly relate to vascular function. Endothelial dysfunction is an independent predictor of cardiovascular events that correlates with inflammation markers/mediators and robust predictors of cardiovascular diseases such as increased high-sensitivity C-reactive protein levels. The results of in vivo and in vitro studies indicate that the statins have beneficial effects unrelated to cholesterol lowering, such as improving endothelial function, increasing myocardial perfusion, and enhancing the availability of nitric oxide. This review describes the pleiotropic effects of statins that may be involved in modulating/preventing endothelial dysfunction and inflammatory processes, as well as the cellular and molecular mechanisms through which they improve endothelial function.

The role of oligodendrocyte precursor cells expressing the GPR17 receptor in brain remodeling after stroke.

Following stroke-induced neuronal damage, quiescent oligodendrocyte precursors (OPCs) are activated to proliferate and later to differentiate to myelin-producing cells. GPR17, a receptor transiently expressed on early OPCs, has emerged as a target to implement stroke repair through stimulation of OPC maturation. However, being GPR17 completely downregulated in myelin-producing oligodendrocytes, its actual role in determining the final fate of OPCs after cerebral ischemia is still uncertain. Here, to univocally define the spatiotemporal changes and final fate of GPR17-expressing OPCs, we induced ischemia by middle cerebral artery occlusion (MCAo) in reporter GPR17iCreERT2:CAG-eGreen florescent protein (GFP) mice, in which, upon tamoxifen treatment, cells expressing GPR17 become green and traceable for their entire life. Starting from 3 days and up to 2 weeks after MCAo, GFP+ cells markedly accumulated in regions surrounding the ischemic lesion; several of them proliferated, as shown by co-labeling of the DNA synthesis marker 5-Bromo-2'-deoxyuridine (BrdU). Almost all GFP+/BrdU+ cells expressed the OPC early marker neural/glial antigen 2 (NG2), indicating that they were still precursors. Accumulation of GFP+ cells was also because of OPC recruitment from surrounding areas, as suggested in vivo by acquisition of typical features of migrating OPCs, shown in vitro in presence of the chemoattractant PDGF-AA and confirmed by transplantation of GFP+-OPCs in wild-type MCAo mice. Eight weeks after MCAo, only some of these precociously recruited cells had undergone maturation as shown by NG2 loss and acquisition of mature myelinating markers like GSTpi. A pool of recruited GFP+-OPCs was kept at a precursor stage to likely make it available for further insults. Thus, very early after ischemia, GFP+-OPCs proliferate and migrate toward the lesion; however, most of these cells remain undifferentiated, suggesting functional roles other than myelination.

3-demethoxy-3-glycosylaminothiocolchicines: Synthesis of a new class of putative muscle relaxant compounds.

A novel class of 3-demethoxy-3-glycosylaminothiocolchicines (7) was prepared and tested for muscle relaxant activity. The syntheses were performed starting from the new 3-amino-3-demethoxythiocolchicine (5) prepared in good yield from 3-O-demethylthiocolchicine (1c) using the Buchwald-Hartwig reaction. The condensation of 5 with a series of pentose and hexose sugars (6) gave a series of 3-demethoxy-3-glycosylaminothiocolchicines (7). Their preparation was accomplished by adapting and improving a previous procedure for the preparation of N-arylglycosylamines. In particular, replacing traditional heating with microwave irradiation represents the key improvement of the process. The biological activity of the 3-demethoxy-3-glycosylaminothiocolchicines (7) was evaluated on GABA and strychnine-sensitive glycine receptors present in rat brain and spinal cord.

Treatment with statins after induction of focal ischemia in rats reduces the extent of brain damage.

OBJECTIVE: In the present study, MRI has been used to investigate therapeutic intervention with statins in a model of permanent focal cerebral ischemia in rat. METHODS AND RESULTS: Brain ischemia was induced in rats by the permanent occlusion of middle cerebral artery (MCAO) and the brain infarct size followed up in alive animals 2, 24, and 48 hours after MCAO, using the trace of apparent diffusion coefficient [Tr(D)] maps and T2-weighted images. In vehicle-treated rats, the infarct volumes increased by 38.5% and 89% after 24 and 48 hours, respectively, compared with the damage detected at 2 hours after MCAO. Treatment with simvastatin (20 mg/kg) after MCAO prevented the increase in brain infarct volume occurring at 24 hours and induced a 46.6% reduction after 48 hours. This effect was similar to that observed when simvastatin was administered before the induction of focal ischemia. T2W-MRI images confirmed these findings. The neuroprotective effects of simvastatin were paralleled by an increase in endothelial NO synthase immunoreactivity, detectable in the brain of simvastatin-treated rats. CONCLUSIONS: Statins, in addition to their preventive effect on cerebral ischemia, exert a neuroprotective role in the attenuation of brain damage after acute stroke.

Prophylactic but not delayed administration of simvastatin protects against long-lasting cognitive and morphological consequences of neonatal hypoxic-ischemic brain injury, reduces interleukin-1beta and tumor necrosis factor-alpha mRNA induction, and does not affect endothelial nitric oxide synthase expression.

BACKGROUND AND PURPOSE: Prophylactic administration of simvastatin has been shown to protect against brain damage and its long-lasting behavioral consequences in neonatal rats. To establish the drug treatment window, we evaluated the effectiveness of simvastatin administered at different intervals before and after stroke. Furthermore, we determined whether simvastatin affected endothelial nitric oxide synthase (eNOS) or inflammatory cytokines in brain tissue or cholesterol levels in serum. METHODS: On postnatal day 7, male rats were subjected to hypoxia-ischemia (HI). The experiment included sham-operated controls and HI animals receiving daily saline or activated simvastatin (20 mg/kg) injections from postnatal day 1 to day 7 (HI-simvastatin 1-7 group), from postnatal day 4 to day 11 (HI-simvastatin 4-11 group), or from postnatal day 7 to day 14 (HI-simvastatin 7-14 group). The neuroprotective effect of simvastatin was evaluated at adulthood by means of behavioral and histological analyses. Cytokines and eNOS expression were assessed by reverse transcriptase-polymerase chain reaction and Western blotting. RESULTS: Animals in both the HI-simvastatin 1-7 and HI-simvastatin 4-11 groups performed better than HI rats in either the T-maze or the circular water maze and showed significantly attenuated brain damage. Expression of interleukin-1beta and tumor necrosis factor-alpha mRNA in cortex was significantly increased in HI but not in HI-simvastatin 1-7 animals. In the same brain area, simvastatin treatment did not affect the increase of eNOS expression observed after HI. CONCLUSIONS: These findings indicate that prophylactic but not delayed administration of simvastatin improves functional outcome in neonatal rat stroke. The reduced induction of cytokines suggests that the neuroprotective effect of simvastatin may be related to a dampening of the inflammatory response.

New insights into brain damage in stroke-prone rats: a nuclear magnetic imaging study.

BACKGROUND AND PURPOSE: The spontaneously hypertensive stroke-prone rat (SHRSP) is an animal model for a complex form of cerebrovascular pathology. MRI provides an efficient and noninvasive tool for studying the time course of brain damage. The aim of this study was to gain new insights into the pathological phenomena responsible for the occurrence of brain injury in SHRSP with the use of the apparent diffusion coefficient of water (ADC), one of the most efficient MRI parameters for detecting brain abnormalities. To this end, the pattern of ADC variation observed in SHRSP was compared with that of focal ischemia induced in both SHRSP and Sprague-Dawley rats. METHODS: Four groups of animals were studied: SHRSP developing spontaneous brain lesions fed with a salt-loaded (n=15, group 1) or standard diet (n=3, group 2) and Sprague-Dawley rats (n=8, group 3) and SHRSP (n=8, group 4) with permanent middle cerebral artery occlusion. ADC maps and T2-weighted images of brains were performed by MRI. After the rats were killed, the brains were removed and histologically processed. RESULTS: There was no decrease in ADC during spontaneous stroke in the SHRSP fed with a normal or salt-enriched diet, while both the SHRSP and Sprague-Dawley rats with middle cerebral artery occlusion showed a marked decrease that lasted for 24 to 48 hours. CONCLUSIONS: Cerebral ischemia cannot be considered a major factor in the onset of spontaneous brain lesions in SHRSP, which show only vasogenic edema after the beginning of the damage with no evidence of metabolic impairment.

Ventral tegmental area/substantia nigra and prefrontal cortex rodent organotypic brain slices as an integrated model to study the cellular changes induced by oxygen/glucose deprivation and reperfusion: effect of neuroprotective agents.

Unveiling the roles of distinct cell types in brain response to insults is a partially unsolved challenge and a key issue for new neuroreparative approaches. In vivo models are not able to dissect the contribution of residential microglia and infiltrating blood-borne monocytes/macrophages, which are fundamentally undistinguishable; conversely, cultured cells lack original tissue anatomical and functional complexity, which profoundly alters reactivity. Here, we tested whether rodent organotypic co-cultures from mesencephalic ventral tegmental area/substantia nigra and prefrontal cortex (VTA/SN-PFC) represent a suitable model to study changes induced by oxygen/glucose deprivation and reperfusion (OGD/R). OGD/R induced cytotoxicity to both VTA/SN and PFC slices, with higher VTA/SN susceptibility. Neurons were highly affected, with astrocytes and oligodendrocytes undergoing very mild damage. Marked reactive astrogliosis was also evident. Notably, OGD/R triggered the activation of CD68-expressing microglia and increased expression of Ym1 and Arg1, two markers of "alternatively" activated beneficial microglia. Treatment with two well-known neuroprotective drugs, the anticonvulsant agent valproic acid and the purinergic P2-antagonist PPADS, prevented neuronal damage. Thus, VTA/SN-PFC cultures are an integrated model to investigate OGD/R-induced effects on distinct cells and easily screen neuroprotective agents. The model is particularly adequate to dissect the microglia phenotypic shift in the lack of a functional vascular compartment.

Microglia is a key player in the reduction of stroke damage promoted by the new antithrombotic agent ticagrelor.

The ADP-responsive P2Y12 receptor is expressed on both platelets and microglia. Clinical data show that ticagrelor, a direct-acting, reversibly binding P2Y12-receptor antagonist, reduces total cardiovascular events, including stroke. In our present study, we investigated the expression of P2Y12 receptors and the effects of ticagrelor on brain injury in Sprague-Dawley rats subjected to a permanent middle cerebral artery occlusion (MCAo). Rats were treated per os with ticagrelor 3 mg/kg or vehicle at 10 minutes, 22, and 36 hours after MCAo and killed after 48 hours. Immunofluorescence analysis showed an ischemia-related modulation of the P2Y12 receptor, which is constitutively expressed in Iba1(+) resting microglia. After MCAo, activated microglia was mainly concentrated around the lesion, with fewer cells present inside the ischemic core. Ticagrelor significantly attenuated the evolution of ischemic damage-evaluated by magnetic resonance imaging (MRI) at 2, 24, and 48 hours after MCAo-, the number of infiltrating cells expressing the microglia/monocyte marker ED-1, the cerebral expression of proinflammatory mediators (interleukin 1 (IL-1), monocyte chemoattractant protein 1 (MCP-1), nitric oxide synthase (iNOS)) and the associated neurologic impairment. In transgenic fluorescent reporter CX3CR1-green fluorescent protein (GFP) mice, 72 hours after MCAo, ticagrelor markedly reduced GFP(+) microglia and both early and late infiltrating blood-borne cells. Finally, in primary cultured microglia, ticagrelor fully inhibited ADP-induced chemotaxis (P<0.01). Our results show that ticagrelor is protective against ischemia-induced cerebral injury and this effect is mediated, at least partly, by inhibition of P2Y12-mediated microglia activation and chemotaxis.

Triflusal reduces cerebral ischemia induced inflammation in a combined mouse model of Alzheimer's disease and stroke.

Clinical data has shown that stroke exacerbates dementia in Alzheimer's disease (AD) patients. Previous work, combining rat models of AD and stroke have shown that neuroinflammation may be the common mediator between AD and stroke toxicity. This study examined the effects of triflusal (2-acetoxy-4-trifluoromethylbenzoic acid) in APP(23) transgenic mice receiving strokes. Six month-old APP(23) mice over-expressing mutant human amyloid precursor protein (APP) were used to model AD in this study. Unilateral injections of a potent vasoconstrictor, endothelin-1, into the striatum were used to mimic small lacunar infarcts. Immunohistochemical analysis was performed to examine AD-like pathology and inflammatory correlates of stroke and AD. APP(23) mice showed increases in AD-like pathology and inflammatory markers of AD in the cortex and hippocampus. Endothelin-induced ischemia triggered an inflammatory response along with increases in AD pathological markers in the region of the infarct. Triflusal reduced inflammation surrounding the endothelin-induced infarct only. At the dose used, anti-inflammatory treatment may be beneficial in reducing the AD and inflammatory correlates of stroke in a combined AD-stroke mouse model.

Prevention of ischemic brain injury by treatment with the membrane penetrating apoptosis inhibitor, TAT-BH4.

In acute thromboembolic stroke, neurological damage is due to ischemia-induced apoptotic death of neuronal cells and the surrounding vascular network. Here, we demonstrate that the BH4 domain of the anti-apoptotic protein, Bcl-x(L), attached to the membrane transport peptide, TAT, reduces stroke injury after intracerebroventricular infusion into immature rats subjected to carotid artery ligation and additional exposure to hypoxia. The injected TAT-BH4 entered neuron bodies, maintained brain architecture, protected neuronal and endothelial cells from apoptosis and promoted neuronal stem cell recruitment. In vitro, TAT-BH4 enhanced the survival of endothelial cells exposed to H(2)O(2), increased neuronal differentiation, and induced axonal remodelling of adult neuronal stem cells. These findings indicate that TAT-BH4 administration protects against acute hypoxia/ischemia injury in the brain by preventing endothelial and neuron cell apoptosis and by inducing neuronal plasticity.