scholarly journals Nanobody-Mediated Inhibition of P2X7 on Microglia Improves Stroke Outcome

2021 ◽  
Author(s):  
Maximilian Wilmes ◽  
Carolina Pinto Espinoza ◽  
Peter Ludewig ◽  
Arthur Liesz ◽  
Annette Nicke ◽  
...  
Keyword(s):  
Ischemic Stroke ◽  
Transgenic Mice ◽  
Tissue Damage ◽  
Calcium Influx ◽  
Atp Release ◽  
Artery Occlusion ◽  
Membrane Pores ◽  
Caspase 1 ◽  
Ischemic Tissue

Abstract BackgroundPrevious studies have demonstrated that purinergic receptors could be therapeutic targets to modulate the inflammatory response in multiple brain disease models. However, tools for the selective and efficient targeting of these receptors are scarce. The new development of P2X7-specific nanobodies (nbs) enables us to effectively block the P2X7-channel.MethodsTemporary middle cerebral artery occlusion (tMCAO) in wildtype and P2X7-transgenic mice was used as a model for ischemic stroke. ATP release was assessed in transgenic ATP sensor mice. Stroke size was measured without treatment and after injection of P2X7-specific nbs i.v. and i.c.v. directly before tMCAO-surgery. P2X7-GFP expressing transgenic mice were used to show immunhistochemically P2X7 distribution in the brain. In vitro cultured microglia were used to investigate calcium-influx, pore-formation via DAPI uptake, caspase 1 activation and IL-1b release after incubation with P2X7-specific nbs. ResultsATP sensor mice showed an increase of ATP-release in the ischemic hemisphere compared to the contralateral hemisphere or sham mice up to 24 h after stroke. We could further verify the role of the ATP-P2X7 axis in P2X7-overexpressing mice, which showed significantly greater stroke volumes after 24 h. In vitro experiments with primary microglia cells showed that P2X7-specific nanobodies were capable of dampening the ATP-trigged calcium-influx and formation of membrane pores measured by Fluo4 fluorescence or DAPI uptake. We found a lower caspase 1 activity and a subsequently lower IL-1b release. However, the intravenous (i.v.) injection of P2X7-specific nanobodies compared to isotype controls before the tMCAO-surgery did not result in smaller stroke size compared to isotype controls. As demonstrated by FACS, nbs had only reached brain infiltrating macrophages but not microglia. To reach microglia, we injected the P2X7-spezific nbs or the isotype directly intraventricularly (icv). 30 mg of P2X7-specific nbs proved efficient for microglial targeting, reducing post-stroke microglia activation and stroke size significantly.ConclusionHere, we demonstrate the importance of locally produced ATP for the tissue damage observed in ischemic stroke and we show the potential of icv injected P2X7-specific nbs to reduce ischemic tissue damage.

scholarly journals Supplemental N-3 Polyunsaturated Fatty Acids Limit A1-Specific Astrocyte Polarization via Attenuating Mitochondrial Dysfunction in Ischemic Stroke in Mice

2021 ◽  
Vol 2021 ◽  
pp. 1-13
Author(s):  
Jun Cao ◽  
Lijun Dong ◽  
Jialiang Luo ◽  
Fanning Zeng ◽  
Zexuan Hong ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Ischemic Stroke ◽  
Polyunsaturated Fatty Acids ◽  
Mitochondrial Dysfunction ◽  
Artery Occlusion ◽  
Mice Model ◽  
Neuron Death ◽  
The Impact

Ischemic stroke is one of the leading causes of death and disability for adults, which lacks effective treatments. Dietary intake of n-3 polyunsaturated fatty acids (n-3 PUFAs) exerts beneficial effects on ischemic stroke by attenuating neuron death and inflammation induced by microglial activation. However, the impact and mechanism of n-3 PUFAs on astrocyte function during stroke have not yet been well investigated. Our current study found that dietary n-3 PUFAs decreased the infarction volume and improved the neurofunction in the mice model of transient middle cerebral artery occlusion (tMCAO). Notably, n-3 PUFAs reduced the stroke-induced A1 astrocyte polarization both in vivo and in vitro. We have demonstrated that exogenous n-3 PUFAs attenuated mitochondrial oxidative stress and increased the mitophagy of astrocytes in the condition of hypoxia. Furthermore, we provided evidence that treatment with the mitochondrial-derived antioxidant, mito-TEMPO, abrogated the n-3 PUFA-mediated regulation of A1 astrocyte polarization upon hypoxia treatment. Together, this study highlighted that n-3 PUFAs prevent mitochondrial dysfunction, thereby limiting A1-specific astrocyte polarization and subsequently improving the neurological outcomes of mice with ischemic stroke.

scholarly journals AB276. SPR-03 In vitro evaluation of hydrostatic pressure on ATP release and Caspase-1 activation in rat urothelial cells

2016 ◽  
Vol 5 (S2) ◽  
pp. AB276-AB276
Author(s):  
Cody L. Dunton ◽  
F. Monty Hughes ◽  
J. Todd Purves ◽  
Jiro Nagatomi
Keyword(s):  
Hydrostatic Pressure ◽  
Atp Release ◽  
Urothelial Cells ◽  
In Vitro Evaluation ◽  
Caspase 1

scholarly journals Neutrophil extracellular traps promote tPA-induced brain hemorrhage via cGAS in mice with stroke

Blood ◽  
2021 ◽  
Author(s):  
Ranran Wang ◽  
Yuanbo Zhu ◽  
Zhongwang Liu ◽  
Luping Chang ◽  
Xiaofei Bai ◽  
...  
Keyword(s):  
Ischemic Stroke ◽  
Thrombolytic Therapy ◽  
Clinical Problem ◽  
Neutrophil Extracellular Traps ◽  
Artery Occlusion ◽  
Type I ◽  
Brain Hemorrhage ◽  
Extracellular Traps ◽  
Dnase 1

Intracerebral hemorrhage associated with thrombolytic therapy with tissue plasminogen activator (tPA) in acute ischemic stroke continues to present a major clinical problem. Here, we report that infusion of tPA resulted in a significant increase in markers of neutrophil extracellular traps (NETs) in the ischemic cortex and plasma of mice subjected to photothrombotic middle cerebral artery occlusion. Peptidylarginine deiminase 4 (PAD4), a critical enzyme for NET formation, is also significantly upregulated in the ischemic brains in tPA-treated mice. Blood-brain barrier (BBB) disruption following ischemic challenge in an in vitro model of BBB was exacerbated after exposure to NETs. Importantly, disruption of NETs by DNase 1 or inhibition of NET production by PAD4 deficiency restored tPA-induced loss of BBB integrity and consequently decreased tPA-associated brain hemorrhage after ischemic stroke. Furthermore, either DNase 1 or PAD4 deficiency reversed tPA-mediated upregulation of the DNA sensor cyclic GMP-AMP (cGAMP) synthase (cGAS). Administration of cGAMP after stroke abolished DNase 1-mediated downregulation of the STING pathway and type I interferon (IFN) production, and blocked the antihemorrhagic effect of DNase 1 in tPA-treated mice. We also show that tPA-associated brain hemorrhage after ischemic stroke was significantly reduced in cGas-/- mice. Collectively, these findings demonstrate that NETs significantly contribute to tPA-induced BBB breakdown in ischemic brain, and suggest that targeting NETs or cGAS may ameliorate thrombolytic therapy for ischemic stroke by reducing tPA-associated hemorrhage.

scholarly journals Ras-like Gem GTPase induced by Npas4 promotes activity-dependent neuronal tolerance for ischemic stroke

2021 ◽  
Vol 118 (32) ◽  
pp. e2018850118
Author(s):  
Hiroo Takahashi ◽  
Ryo Asahina ◽  
Masayuki Fujioka ◽  
Takeshi K. Matsui ◽  
Shigeki Kato ◽  
...  
Keyword(s):  
Ischemic Stroke ◽  
Cortical Neurons ◽  
Glucose Deprivation ◽  
Artery Occlusion ◽  
Small Gtpase ◽  
Neuroprotective Effects ◽  
Necessary And Sufficient ◽  
Activity Dependent

Ischemic stroke, which results in loss of neurological function, initiates a complex cascade of pathological events in the brain, largely driven by excitotoxic Ca2+ influx in neurons. This leads to cortical spreading depolarization, which induces expression of genes involved in both neuronal death and survival; yet, the functions of these genes remain poorly understood. Here, we profiled gene expression changes that are common to ischemia (modeled by middle cerebral artery occlusion [MCAO]) and to experience-dependent activation (modeled by exposure to an enriched environment [EE]), which also induces Ca2+ transients that trigger transcriptional programs. We found that the activity-dependent transcription factor Npas4 was up-regulated under MCAO and EE conditions and that transient activation of cortical neurons in the healthy brain by the EE decreased cell death after stroke. Furthermore, both MCAO in vivo and oxygen-glucose deprivation in vitro revealed that Npas4 is necessary and sufficient for neuroprotection. We also found that this protection involves the inhibition of L-type voltage-gated Ca2+ channels (VGCCs). Next, our systematic search for Npas4-downstream genes identified Gem, which encodes a Ras-related small GTPase that mediates neuroprotective effects of Npas4. Gem suppresses the membrane localization of L-type VGCCs to inhibit excess Ca2+ influx, thereby protecting neurons from excitotoxic death after in vitro and in vivo ischemia. Collectively, our findings indicate that Gem expression via Npas4 is necessary and sufficient to promote neuroprotection in the injured brain. Importantly, Gem is also induced in human cerebral organoids cultured under an ischemic condition, revealing Gem as a new target for drug discovery.

scholarly journals Dual Role of Caspase-11 in Mediating Activation of Caspase-1 and Caspase-3 under Pathological Conditions

2000 ◽  
Vol 149 (3) ◽  
pp. 613-622 ◽  
Author(s):  
Shin-Jung Kang ◽  
Suyue Wang ◽  
Hideaki Hara ◽  
Erin P. Peterson ◽  
Shobu Namura ◽  
...  
Keyword(s):  
Caspase 3 ◽  
Artery Occlusion ◽  
Caspase 1 ◽  
Pathological Conditions ◽  
Positional Scanning ◽  
Caspase Family ◽  
Cerebral Artery Occlusion ◽  
Deficient Mice

Caspase-11, a member of the murine caspase family, has been shown to be an upstream activator of caspase-1 in regulating cytokine maturation. We demonstrate here that in addition to its defect in cytokine maturation, caspase-11–deficient mice have a reduced number of apoptotic cells and a defect in caspase-3 activation after middle cerebral artery occlusion (MCAO), a mouse model of stroke. Recombinant procaspase-11 can autoprocess itself in vitro. Purified active recombinant caspase-11 cleaves and activates procaspase-3 very efficiently. Using a positional scanning combinatorial library method, we found that the optimal cleavage site of caspase-11 was (I/L/V/P)EHD, similar to that of upstream caspases such as caspase-8 and -9. Our results suggest that caspase-11 is a critical initiator caspase responsible for the activation of caspase-3, as well as caspase-1 under certain pathological conditions.

scholarly journals Optogenetic Inhibition of Striatal Neuronal Activity Improves the Survival of Transplanted Neural Stem Cells and Neurological Outcomes after Ischemic Stroke in Mice

2017 ◽  
Vol 2017 ◽  
pp. 1-11 ◽  
Author(s):  
Yifan Lu ◽  
Lu Jiang ◽  
Wanlu Li ◽  
Meijie Qu ◽  
Yaying Song ◽  
...  
Keyword(s):  
Ischemic Stroke ◽  
Neuronal Activity ◽  
Brain Ischemia ◽  
Fluorescent Protein ◽  
Infarct Volume ◽  
Artery Occlusion ◽  
Control Group ◽  
Striatal Neurons ◽  
And Migration

Neural stem cell (NSC) transplantation is a promising treatment to improve the recovery after brain ischemia. However, how the survival, proliferation, migration, and differentiation of implanted NSC are influenced by endogenous neuronal activity remains unclear. In this work, we used optogenetic techniques to control the activity of striatal neurons and investigated how their activity affected the survival and migration of transplanted NSCs and overall neurological outcome after ischemic stroke. NSCs cultured from transgenic mice expressing fluorescent protein were transplanted into the peri-infarct region of the striatum after transient middle cerebral artery occlusion (tMCAO) surgery. The striatal neurons were excited or inhibited for 15 minutes daily via implanted optical fiber after tMCAO. The results revealed that mice which received NSC transplantation and optogenetic inhibition had smaller brain infarct volume and increased NSC migration compared to the NSC alone or PBS group (p<0.05). In contrast, mice which received NSC transplantation and optogenetic excitation showed no difference in infarct volume and neurological behavior improvement compared to the PBS control group. In vitro experiments further revealed that the conditioned media from excited GABAergic neurons reduced NSC viability through paracrine mechanisms.Conclusion. Optogenetic inhibition of striatal neuronal activity further improved neurological recovery after NSC transplantation at the subacute phase after brain ischemia.

scholarly journals Role for nuclear interleukin-37 in the suppression of innate immunity

2019 ◽  
Vol 116 (10) ◽  
pp. 4456-4461 ◽  
Author(s):  
Suzhao Li ◽  
Jesus Amo-Aparicio ◽  
Charles P. Neff ◽  
Isak W. Tengesdal ◽  
Tania Azam ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Nuclear Translocation ◽  
Surface Membrane ◽  
Peritoneal Macrophages ◽  
Membrane Receptors ◽  
Dual Function ◽  
Nuclear Function ◽  
Caspase 1

The IL-1 family member IL-37 broadly suppresses innate inflammation and acquired immunity. Similar to IL-1α and IL-33, IL-37 is a dual-function cytokine in that IL-37 translocates to the nucleus but also transmits a signal via surface membrane receptors. The role of nuclear IL-37 remains unknown on the ability of this cytokine to inhibit innate inflammation. Here, we compared suppression of innate inflammation in transgenic mice expressing native human IL-37 (IL-37Tg) with those of transgenic mice carrying the mutation of aspartic acid (D) to alanine (A) at amino acid 20 (IL-37D20ATg). The mutation D20A prevents cleavage of caspase-1, a step required for IL-37 nuclear translocation. In vitro, peritoneal macrophages from IL-37Tg mice reduced LPS-induced IL-1β, IL-6, TNFα and IFNγ by 40–50% whereas in macrophages from IL-37D20ATg mice this suppression was not observed, consistent with loss of nuclear function. Compared with macrophages from IL-37Tg mice, significantly less or no suppression of LPS-induced MAP kinase and NFκB activation was also observed in macrophages from IL-37D20ATg mice. In vivo, levels of IL-1β, IL-6, and TNFα in the lungs and liver were markedly reduced during endotoxemia in IL-37Tg mice but not observed in IL-37D20ATg mice. However, suppression of innate inflammation remains intact in the IL-37D20A mice once the cytokine is released from the cell and binds to its receptor. These studies reveal a nuclear function for suppression of innate inflammation and are consistent with the dual function of IL-37 and a role for caspase-1 in limiting inflammation.

scholarly journals Restoration of Polyamine Metabolic Patterns inIn VivoandIn VitroModel of Ischemic Stroke following Human Mesenchymal Stem Cell Treatment

2016 ◽  
Vol 2016 ◽  
pp. 1-11 ◽  
Author(s):  
Tae Hwan Shin ◽  
Geetika Phukan ◽  
Jeom Soon Shim ◽  
Duc-Toan Nguyen ◽  
Yongman Kim ◽  
...  
Keyword(s):  
Ischemic Stroke ◽  
Cell Model ◽  
Glucose Deprivation ◽  
Human Mesenchymal Stem Cell ◽  
Artery Occlusion ◽  
Control Group ◽  
Stem Cell Treatment ◽  
Cerebral Artery Occlusion

We investigated changes in PA levels by the treatment of human bone-marrow-derived mesenchymal stem cells (hBM-MSCs) in ischemic stroke in rat brain model and in cultured neuronal SH-SY5Y cells exposed to oxygen-glucose deprivation (OGD). In ischemic rat model, transient middle cerebral artery occlusion (MCAo) was performed for 2 h, followed by intravenous transplantation of hBM-MSCs or phosphate-buffered saline (PBS) the day following MCAo. Metabolic profiling analysis of PAs was examined in brains from three groups: control rats, PBS-treated MCAo rats (MCAo), and hBM-MSCs-treated MCAo rats (MCAo + hBM-MSCs). In ischemic cell model, SH-SY5Y cells were exposed to OGD for 24 h, treated with hBM-MSCs (OGD + hBM-MSCs) prior to continued aerobic incubation, and then samples were collected after coculture for 72 h. In thein vivoMCAo ischemic model, levels of some PAs in brain samples of the MCAo and MCAo + hBM-MSCs groups were significantly different from those of the control group. In particular, putrescine, cadaverine, and spermidine in brain tissues of the MCAo + hBM-MSCs group were significantly reduced in comparison to those in the MCAo group. In thein vitroOGD system,N1-acetylspermidine, spermidine,N1-acetylspermine, and spermine in cells of the OGD + hBM-MSCs group were significantly reduced compared to those of OGD group.

scholarly journals A review of experimental models of focal cerebral ischemia focusing on the middle cerebral artery occlusion model

F1000Research ◽  
2021 ◽  
Vol 10 ◽  
pp. 242
Author(s):  
Melissa Trotman-Lucas ◽  
Claire L. Gibson
Keyword(s):  
Ischemic Stroke ◽  
Middle Cerebral Artery ◽  
Middle Cerebral Artery Occlusion ◽  
Cerebral Artery ◽  
Artery Occlusion ◽  
Lesion Volume ◽  
Mechanistic Investigation ◽  
Cerebral Artery Occlusion

Cerebral ischemic stroke is a leading cause of death and disability, but current pharmacological therapies are limited in their utility and effectiveness. In vitro and in vivo models of ischemic stroke have been developed which allow us to further elucidate the pathophysiological mechanisms of injury and investigate potential drug targets. In vitro models permit mechanistic investigation of the biochemical and molecular mechanisms of injury but are reductionist and do not mimic the complexity of clinical stroke. In vivo models of ischemic stroke directly replicate the reduction in blood flow and the resulting impact on nervous tissue. The most frequently used in vivo model of ischemic stroke is the intraluminal suture middle cerebral artery occlusion (iMCAO) model, which has been fundamental in revealing various aspects of stroke pathology. However, the iMCAO model produces lesion volumes with large standard deviations even though rigid surgical and data collection protocols are followed. There is a need to refine the MCAO model to reduce variability in the standard outcome measure of lesion volume. The typical approach to produce vessel occlusion is to induce an obstruction at the origin of the middle cerebral artery and reperfusion is reliant on the Circle of Willis (CoW). However, in rodents the CoW is anatomically highly variable which could account for variations in lesion volume. Thus, we developed a refined approach whereby reliance on the CoW for reperfusion was removed. This approach improved reperfusion to the ischemic hemisphere, reduced variability in lesion volume by 30%, and reduced group sizes required to determine an effective treatment response by almost 40%. This refinement involves a methodological adaptation of the original surgical approach which we have shared with the scientific community via publication of a visualised methods article and providing hands-on training to other experimental stroke researchers.

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