After ischemic stroke, in the lesion core as well as in the ischemic penumbra, evolution of tissue damage and repair is strongly affected by neuroinflammatory events that involve activation of local ...specialized glial cells, release of inflammatory mediators, recruiting of systemic cells and vascular remodelling. To take advantage of this intricate response in the quest to devise new protective therapeutic strategies we need a better understanding of the territorial and temporal interplay between stroke-triggered inflammatory and cell death-inducing processes in both parenchymal and vascular brain cells. Our goal is to describe structural rearrangements and functional modifications occurring in glial and vascular cells early after an acute ischemic stroke. Low and high scale mapping of the glial activation on brain sections of mice subjected to 30 minutes middle cerebral artery occlusion (MCAO) was correlated with that of the neuronal cell death, with markers for microvascular changes and with markers for pro-inflammatory (IL-1β) and reparative (TGFβ1) cytokines. Our results illustrate a time-course of the neuroinflammatory response starting at early time-points (1 h) and up to one week after MCAO injury in mice, with an accurate spatial distribution of the observed phenomena.
A hallmark of stroke is water accumulation (edema) resulting from dysregulation of osmotic homeostasis. Brain edema contributes to tissue demise and may lead to increased intracranial pressure and ...lethal herniation. Currently, there are only limited treatments to prevent edema formation following stroke. Aquaporin 4 (AQP4), a brain water channel, has become a focus of interest for therapeutic approaches targeting edema. At present, there are no pharmacological tools to block AQP4. The role of AQP4 in edema after brain injury remains unclear with conflicting results from studies using AQP4−/− mice and of AQP4 expression following stroke. Here, we studied AQP4 and its role in edema formation by testing AQP4−/− mice in a model of middle cerebral artery occlusion using novel quantitative MRI water content measurements, histology and behavioral changes as outcome measures. Absence of AQP4 was associated with decreased mortality and increased motor recovery 3 to 14 days after stroke. Behavioral improvement was associated with decreased lesion volume, neuronal cell death and neuroinflammation in AQP4−/− compared to wild type mice. Our data suggest that the lack of AQP4 confers an overall beneficial role at long term with improved neuronal survival and reduced neuroinflammation, but without a direct effect on edema formation.
Lactate has been shown to offer neuroprotection in several pathologic conditions. This beneficial effect has been attributed to its use as an alternative energy substrate. However, recent description ...of the expression of the HCA1 receptor for lactate in the central nervous system calls for reassessment of the mechanism by which lactate exerts its neuroprotective effects. Here, we show that HCA1 receptor expression is enhanced 24 hours after reperfusion in an middle cerebral artery occlusion stroke model, in the ischemic cortex. Interestingly, intravenous injection of L-lactate at reperfusion led to further enhancement of HCA1 receptor expression in the cortex and striatum. Using an in vitro oxygen-glucose deprivation model, we show that the HCA1 receptor agonist 3,5-dihydroxybenzoic acid reduces cell death. We also observed that D-lactate, a reputedly non-metabolizable substrate but partial HCA1 receptor agonist, also provided neuroprotection in both in vitro and in vivo ischemia models. Quite unexpectedly, we show D-lactate to be partly extracted and oxidized by the rodent brain. Finally, pyruvate offered neuroprotection in vitro whereas acetate was ineffective. Our data suggest that L- and D-lactate offer neuroprotection in ischemia most likely by acting as both an HCA1 receptor agonist for non-astrocytic (most likely neuronal) cells as well as an energy substrate.
It is well established that lactate can be used as an energy substrate by the brain by conversion to pyruvate and a subsequent oxidation in the mitochondria. Knowing the need for readily ...metabolizable substrates directly after ischemia and the protective effect of lactate after excitotoxicity, the aim of this study was to investigate whether lactate administration directly after ischemia could be neuroprotective. In vitro, the addition of 4 mmol/L l-lactate to the medium of rat organotypic hippocampal slices, directly after oxygen and glucose deprivation (OGD), protected against neuronal death, whereas a higher dose of 20 mmol/L was toxic. In vivo, after middle cerebral artery occlusion in the mouse, an intracerebroventricular injection of 2 μL of 100 mmol/L l-lactate, immediately after reperfusion, led to a significant decrease in lesion size, which was more pronounced in the striatum, and an improvement in neurologic outcome. A later injection 1 h after reperfusion did not reduce lesion size, but significantly improved neurologic outcome, which is an important point in the context of a potential clinical application. Therefore, a moderate increase in lactate after ischemia may be a therapeutic tool.
Cerebral metabolism, which can be monitored by magnetic resonance spectroscopy (MRS), changes rapidly after brain ischaemic injury. Hyperpolarisation techniques boost
C MRS sensitivity by several ...orders of magnitude, thereby enabling in vivo monitoring of biochemical transformations of hyperpolarised (HP)
C-labelled precursors with a time resolution of seconds. The exogenous administration of the metabolite L-lactate was shown to decrease lesion size and ameliorate neurological outcome in preclinical studies in rodent stroke models, as well as influencing brain metabolism in clinical pilot studies of acute brain injury patients. The aim of this study was to demonstrate the feasibility of measuring HP 1-
C L-lactate metabolism in real-time in the mouse brain after ischaemic stroke when administered after reperfusion at a therapeutic dose. We showed a rapid, time-after-reperfusion-dependent conversion of 1-
C L-lactate to 1-
C pyruvate and
C bicarbonate that brings new insights into the neuroprotection mechanism of L-lactate. Moreover, this study paves the way for the use of HP 1-
C L-lactate as a sensitive molecular-imaging biosensor in ischaemic stroke patients after endovascular clot removal.
Evolution of the neurochemical profile consisting of 19 metabolites after 30 mins of middle cerebral artery occlusion was longitudinally assessed at 3, 8 and 24 h in 6 to 8 µL volumes in the striatum ...using localized 1H-magnetic resonance spectroscopy at 14.1 T. Profound changes were detected as early as 3 h after ischemia, which include elevated lactate levels in the presence of significant glucose concentrations, decreases in glutamate and a transient twofold glutamine increase, likely to be linked to the excitotoxic release of glutamate and conversion into glial glutamine. Interestingly, decreases in N-acetyl-aspartate (NAA), as well as in taurine, exceeded those in neuronal glutamate, suggesting that the putative neuronal marker NAA is rather a sensitive marker of neuronal viability. With further ischemia evolution, additional, more profound concentration decreases were detected, reflecting a disruption of cellular functions. We conclude that early changes in markers of energy metabolism, glutamate excitotoxicity and neuronal viability can be detected with high precision noninvasively in mice after stroke. Such investigations should lead to a better understanding and insight into the sequential early changes in the brain parenchyma after ischemia, which could be used for identifying new targets for neuroprotection.
Lactate protects mice against the ischaemic damage resulting from transient middle cerebral artery occlusion (MCAO) when administered intracerebroventricularly at reperfusion, yielding smaller lesion ...sizes and a better neurological outcome 48 h after ischaemia. We have now tested whether the beneficial effect of lactate is long-lasting and if lactate can be administered intravenously.
Male ICR-CD1 mice were subjected to 15-min suture MCAO under xylazine + ketamine anaesthesia. Na L-lactate (2 µl of 100 mmol/l) or vehicle was administered intracerebroventricularly at reperfusion. The neurological deficit was evaluated using a composite deficit score based on the neurological score, the rotarod test and the beam walking test. Mice were sacrificed at 14 days. In a second set of experiments, Na L-lactate (1 µmol/g body weight) was administered intravenously into the tail vein at reperfusion. The neurological deficit and the lesion volume were measured at 48 h.
Intracerebroventricularly injected lactate induced sustained neuroprotection shown by smaller neurological deficits at 7 days (median = 0, min = 0, max = 3, n = 7 vs. median = 2, min = 1, max = 4.5, n = 5, p < 0.05) and 14 days after ischaemia (median = 0, min = 0, max = 3, n = 7 vs. median = 3, min = 0.5, max = 3, n = 7, p = 0.05). Reduced tissue damage was demonstrated by attenuated hemispheric atrophy at 14 days (1.3 ± 4.0 mm(3), n = 7 vs. 12.1 ± 3.8 mm(3), n = 5, p < 0.05) in lactate-treated animals. Systemic intravenous lactate administration was also neuroprotective and attenuated the deficit (median = 1, min = 0, max = 2.5, n = 12) compared to vehicle treatment (median = 1.5, min = 1, max = 8, n = 12, p < 0.05) as well as the lesion volume at 48 h (13.7 ± 12.2 mm(3), n = 12 vs. 29.6 ± 25.4 mm(3), n = 12, p < 0.05).
The beneficial effect of lactate is long-lasting: lactate protects the mouse brain against ischaemic damage when supplied intracerebroventricularly during reperfusion with behavioural and histological benefits persisting 2 weeks after ischaemia. Importantly, lactate also protects after systemic intravenous administration, a more suitable route of administration in a clinical emergency setting. These findings provide further steps to bring this physiological, commonly available and inexpensive neuroprotectant closer to clinical translation for stroke.
Lactate is an intriguing molecule with emerging physiological roles in the brain. It has beneficial effects in animal models of acute brain injuries and traumatic brain injury or subarachnoid ...hemorrhage patients. However, the mechanism by which lactate provides protection is unclear. While there is evidence of a metabolic effect of lactate providing energy to deprived neurons, it can also activate the hydroxycarboxylic acid receptor 1 (HCAR1), a Gi-coupled protein receptor that modulates neuronal firing rates. After cerebral hypoxia-ischemia, endogenously produced brain lactate is largely increased, and the exogenous administration of more lactate can decrease lesion size and ameliorate the neurological outcome. To test whether HCAR1 plays a role in lactate-induced neuroprotection, we injected the agonists 3-chloro-5-hydroxybenzoic acid and 3,5-dihydroxybenzoic acid into mice subjected to 30-min middle cerebral artery occlusion. The
in vivo
administration of HCAR1 agonists at reperfusion did not appear to exert any relevant protective effect as seen with lactate administration. Our results suggest that the protective effects of lactate after hypoxia-ischemia come rather from the metabolic effects of lactate than its signaling through HCAR1.
Abstract The c-Jun-N-terminal kinase signaling pathway (JNK) is highly activated during ischemia and plays an important role in apoptosis and inflammation. We have previously demonstrated that ...D-JNKI1, a specific JNK inhibitor, is strongly neuroprotective in animal models of stroke. We presently evaluated if D-JNKI1 modulates post-ischemic inflammation such as the activation and accumulation of microglial cells. Outbred CD1 mice were subjected to 45 min middle cerebral artery occlusion (MCAo). D-JNKI1 (0.1 mg/kg) or vehicle (saline) was administered intravenously 3 h after MCAo onset. Lesion size at 48 h was significantly reduced, from 28.2 ± 8.5 mm3 ( n = 7) to 13.9 ± 6.2 mm3 in the treated group ( n = 6). Activation of the JNK pathway (phosphorylation of c-Jun) was observed in neurons as well as in Isolectin B4 positive microglia. We quantified activated microglia (CD11b) by measuring the average intensity of CD11b labelling (infra-red emission) within the ischemic tissue. No significant difference was found between groups. Cerebral ischemia was modelled in vitro by subjecting rat organotypic hippocampal slice cultures to oxygen (5%) and glucose deprivation for 30 min. In vitro , D-JNKI1 was found predominantly in NeuN positive neurons of the CA1 region and in few Isolectin B4 positive microglia. Furthermore, 48 h after OGD, microglia were activated whereas resting microglia were found in controls and in D-JNKI1-treated slices. Our study shows that D-JNKI1 reduces the infarct volume 48 h after transient MCAo and does not act on the activation and accumulation of microglia at this time point. In contrast, in vitro data show an indirect effect of D-JNKI1 on the modulation of microglial activation.
Neuronal death in cerebral ischemia is largely due to excitotoxic mechanisms, which are known to activate the c-Jun N-terminal kinase (JNK) pathway. We have evaluated the neuroprotective power of a ...cell-penetrating, protease-resistant peptide that blocks the access of JNK to many of its targets. We obtained strong protection in two models of middle cerebral artery occlusion (MCAO): transient occlusion in adult mice and permanent occlusion in 14-d-old rat pups. In the first model, intraventricular administration as late as 6 h after occlusion reduced the lesion volume by more than 90% for at least 14 d and prevented behavioral consequences. In the second model, systemic delivery reduced the lesion by 78% and 49% at 6 and 12 h after ischemia, respectively. Protection correlated with prevention of an increase in c-Jun activation and c-Fos transcription. In view of its potency and long therapeutic window, this protease-resistant peptide is a promising neuroprotective agent for stroke.
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