Multi-molecular hyperspectral PRM-SRS microscopy.

Lipids play crucial roles in many biological processes. Mapping spatial distributions and examining the metabolic dynamics of different lipid subtypes in cells and tissues are critical to better understanding their roles in aging and diseases. Commonly used imaging methods (such as mass spectrometry-based, fluorescence labeling, conventional optical imaging) can disrupt the native environment of cells/tissues, have limited spatial or spectral resolution, or cannot distinguish different lipid subtypes. Here we present a hyperspectral imaging platform that integrates a Penalized Reference Matching algorithm with Stimulated Raman Scattering (PRM-SRS) microscopy. Using this platform, we visualize and identify high density lipoprotein particles in human kidney, a high cholesterol to phosphatidylethanolamine ratio inside granule cells of mouse hippocampus, and subcellular distributions of sphingosine and cardiolipin in human brain. Our PRM-SRS displays unique advantages of enhanced chemical specificity, subcellular resolution, and fast data processing in distinguishing lipid subtypes in different organs and species.

Intracarotid Infusion of Redox-Active Manganese Porphyrin, MnTnBuOE-2-PyP5+, following Reperfusion Improves Long-Term, 28-Day Post-Stroke Outcomes in Rats.

Endovascular mechanical thrombectomy, combined with a tissue plasminogen activator (t-PA), is efficacious as a standard care for qualifying ischemic stroke patients. However, > 50% of thrombectomy patients still have poor outcomes. Manganese porphyrins, commonly known as mimics of superoxide dismutases, are potent redox-active catalytic compounds that decrease oxidative/nitrosative stress and in turn decrease inflammatory responses, mitigating therefore the secondary injury of the ischemic brain. This study investigates the effect of intracarotid MnTnBuOE-2-PyP5+ (BMX-001) administration on long-term, 28-day post-stroke recovery in a clinically relevant setting. The 90 min of transient middle cerebral artery occlusion was performed in young, aged, male, female, and spontaneous hypertension rats. All physiological parameters, including blood pressure, blood gas, glucose, and temperature, were well controlled during ischemia. Either BMX-001 or a vehicle solution was infused through the carotid artery immediately after the removal of filament, mimicking endovascular thrombectomy, and was followed by 7 days of subcutaneous injection. Neurologic deficits and infarct volume were assessed at 28 days in a blinded manner. The effects of BMX-001 on the carotid arterial wall and blood-brain barrier permeability and its interaction with t-PA were assessed in normal rats. There were no intra-group differences in physiological variables. BMX-001-treated stroke rats regained body weight earlier, performed better in behavioral tests, and had smaller brain infarct size compared to the vehicle-treated group. No vascular wall damage and blood-brain barrier permeability changes were detected after the BMX-001 infusion. There was no drug interaction between BMX-001 and t-PA. Intracarotid BMX-001 infusion was safe, and it significantly improved stroke outcomes in rats. These findings indicate that BMX-001 is a candidate drug as an adjunct treatment for thrombectomy procedure to further improve the neurologic outcomes of thrombectomy patients. This study warrants further clinical investigation of BMX-001 as a new stroke therapy.

Mouse Cardiac Arrest Model for Brain Imaging and Brain Physiology Monitoring During Ischemia and Resuscitation.

Most cardiac arrest (CA) survivors experience varying degrees of neurologic deficits. To understand the mechanisms that underpin CA-induced brain injury and, subsequently, develop effective treatments, experimental CA research is essential. To this end, a few mouse CA models have been established. In most of these models, the mice are placed in the supine position in order to perform chest compression for cardiopulmonary resuscitation (CPR). However, this resuscitation procedure makes the real-time imaging/monitoring of brain physiology during CA and resuscitation challenging. To obtain such critical knowledge, the present protocol presents a mouse asphyxia CA model that does not require the chest compression CPR step. This model allows for the study of dynamic changes in blood flow, vascular structure, electrical potentials, and brain tissue oxygen from the pre-CA baseline to early post-CA reperfusion. Importantly, this model applies to aged mice. Thus, this mouse CA model is expected to be a critical tool for deciphering the impact of CA on brain physiology.

A Modified Transcranial Middle Cerebral Artery Occlusion Model to Study Stroke Outcomes in Aged Mice.

In experimental stroke research, middle cerebral artery occlusion (MCAO) with an intraluminal filament is widely used to model ischemic stroke in mice. The filament MCAO model typically exhibits a massive cerebral infarction in C57Bl/6 mice that sometimes includes brain tissue in the territory supplied by the posterior cerebral artery, which is largely due to a high incidence of posterior communicating artery atresia. This phenomenon is considered a major contributor to the high mortality rate observed in C57Bl/6 mice during long-term stroke recovery after filament MCAO. Thus, many chronic stroke studies exploit distal MCAO models. However, these models usually produce infarction only in the cortex area, and consequently, the assessment of post-stroke neurologic deficits could be a challenge. This study has established a modified transcranial MCAO model in which the MCA at the trunk is partially occluded either permanently or transiently via a small cranial window. Since the occlusion location is relatively proximal to the origin of the MCA, this model generates brain damage in both the cortex and striatum. Extensive characterization of this model has demonstrated an excellent long-term survival rate, even in aged mice, as well as readily detectable neurologic deficits. Therefore, the MCAO mouse model described here represents a valuable tool for experimental stroke research.

Epinephrine-induced Effects on Cerebral Microcirculation and Oxygenation Dynamics Using Multimodal Monitoring and Functional Photoacoustic Microscopy.

BACKGROUND: The administration of epinephrine after severe refractory hypotension, shock, or cardiac arrest restores systemic blood flow and major vessel perfusion but may worsen cerebral microvascular perfusion and oxygen delivery through vasoconstriction. The authors hypothesized that epinephrine induces significant microvascular constriction in the brain, with increased severity after repetitive dosing and in the aged brain, eventually leading to tissue hypoxia. METHODS: The authors investigated the effects of intravenous epinephrine administration in healthy young and aged C57Bl/6 mice on cerebral microvascular blood flow and oxygen delivery using multimodal in vivo imaging, including functional photoacoustic microscopy, brain tissue oxygen sensing, and follow-up histologic assessment. RESULTS: The authors report three main findings. First, after epinephrine administration, microvessels exhibited severe immediate vasoconstriction (57 ± 6% of baseline at 6 min, P < 0.0001, n = 6) that outlasted the concurrent increase in arterial blood pressure, while larger vessels demonstrated an initial increase in flow (108 ± 6% of baseline at 6 min, P = 0.02, n = 6). Second, oxyhemoglobin decreased significantly within cerebral vessels with a more pronounced effect in smaller vessels (microvessels to 69 ± 8% of baseline at 6 min, P < 0.0001, n = 6). Third, oxyhemoglobin desaturation did not indicate brain hypoxia; on the contrary, brain tissue oxygen increased after epinephrine application (from 31 ± 11 mmHg at baseline to 56 ± 12 mmHg, 80% increase, P = 0.01, n = 12). In the aged brains, microvascular constriction was less prominent yet slower to recover compared to young brains, but tissue oxygenation was increased, confirming relative hyperoxia. CONCLUSIONS: Intravenous application of epinephrine induced marked cerebral microvascular constriction, intravascular hemoglobin desaturation, and paradoxically, an increase in brain tissue oxygen levels, likely due to reduced transit time heterogeneity.

A mast cell-thermoregulatory neuron circuit axis regulates hypothermia in anaphylaxis.

IgE-mediated anaphylaxis is an acute life-threatening systemic reaction to allergens, including certain foods and venoms. Anaphylaxis is triggered when blood-borne allergens activate IgE-bound perivascular mast cells (MCs) throughout the body, causing an extensive systemic release of MC mediators. Through precipitating vasodilatation and vascular leakage, these mediators are believed to trigger a sharp drop in blood pressure in humans and in core body temperature in animals. We report that the IgE/MC-mediated drop in body temperature in mice associated with anaphylaxis also requires the body's thermoregulatory neural circuit. This circuit is activated when granule-borne chymase from MCs is deposited on proximal TRPV1+ sensory neurons and stimulates them via protease-activated receptor-1. This triggers the activation of the body's thermoregulatory neural network, which rapidly attenuates brown adipose tissue thermogenesis to cause hypothermia. Mice deficient in either chymase or TRPV1 exhibited limited IgE-mediated anaphylaxis, and, in wild-type mice, anaphylaxis could be recapitulated simply by systemically activating TRPV1+ sensory neurons. Thus, in addition to their well-known effects on the vasculature, MC products, especially chymase, promote IgE-mediated anaphylaxis by activating the thermoregulatory neural circuit.

SIRT1 Activation Promotes Long-Term Functional Recovery After Subarachnoid Hemorrhage in Rats.

BACKGROUND: An increase in sirtuin 1 (SIRT1) reportedly attenuates early brain injury, delayed cerebral ischemia, and short-term neurologic deficits in rodent models of subarachnoid hemorrhage (SAH). This study investigates the effect of resveratrol, a SIRT1 activator, on long-term functional recovery in a clinically relevant rat model of SAH. METHODS: Thirty male Wistar rats were subjected to fresh arterial blood injection into the prechiasmatic space and randomized to receive 7 days of intraperitoneal resveratrol (20 mg/kg) or vehicle injections. Body weight and rotarod performance were measured on days 0, 3, 7, and 34 post SAH. The neurologic score was assessed 7 and 34 days post SAH. Morris water maze performance was evaluated 29-33 days post SAH. Brain SIRT1 activity and CA1 neuronal survival were also assessed. RESULTS: Blood pressure rapidly increased in all SAH rats, and no between-group differences in blood pressure, blood gases, or glucose were detected. SAH induced weight loss during the first 7 days, which gradually recovered in both groups. Neurologic score and rotarod performance were significantly improved after resveratrol treatment at 34 days post SAH (p = 0.01 and 0.04, respectively). Latency to find the Morris water maze hidden platform was shortened (p = 0.02). In the resveratrol group, more CA1 neurons survived following SAH (p = 0.1). An increase in brain SIRT1 activity was confirmed in the resveratrol group (p < 0.05). CONCLUSIONS: Treatment with resveratrol for 1 week significantly improved the neurologic score, rotarod performance, and latency to find the Morris water maze hidden platform 34 days post SAH. These findings indicate that SIRT1 activation warrants further investigation as a mechanistic target for SAH therapy.

Deep-tissue SWIR imaging using rationally designed small red-shifted near-infrared fluorescent protein.

Applying rational design, we developed 17 kDa cyanobacteriochrome-based near-infrared (NIR-I) fluorescent protein, miRFP718nano. miRFP718nano efficiently binds endogenous biliverdin chromophore and brightly fluoresces in mammalian cells and tissues. miRFP718nano has maximal emission at 718 nm and an emission tail in the short-wave infrared (SWIR) region, allowing deep-penetrating off-peak fluorescence imaging in vivo. The miRFP718nano structure reveals the molecular basis of its red shift. We demonstrate superiority of miRFP718nano-enabled SWIR imaging over NIR-I imaging of microbes in the mouse digestive tract, mammalian cells injected into the mouse mammary gland and NF-kB activity in a mouse model of liver inflammation.

Beneficial effects of neuronal ATF6 activation in permanent ischemic stroke.

Objective: Brain ischemia leads to the accumulation of unfolded/misfolded proteins in the endoplasmic reticulum (ER) lumen and consequently, ER stress. To help cells restore ER function, a series of adaptive stress response pathways, collectively termed the unfolded protein response (UPR), are activated. We have previously demonstrated that the UPR pathway initiated by ATF6 is pro-survival in transient ischemic stroke. However, the effect of ATF6 activation on the outcome after permanent ischemic stroke remains unknown. Here, we addressed this knowledge gap. Method: sATF6-KI mice with functional short-form ATF6 (sATF6) predominantly expressed in forebrain neurons were subjected to two ischemic stroke models: photothrombotic stroke and permanent middle cerebral artery occlusion (pMCAO). Both short-term and long-term functional outcomes were evaluated. Changes in neuroinflammation and cerebrovascular density after pMCAO were also assessed. Results: Compared to littermate controls, sATF6-KI mice performed significantly better in open field, cylinder, and foot fault tests on day 1 or 3 after photothrombotic stroke. However, on days 7 and 14 after stroke, the performance of these functional tests was not significantly different between groups, which is likely related to mild brain damage associated with this stroke model. Thus, to evaluate the long-term effects of ATF6 activation in permanent stroke, we turned to our pMCAO model. We first found that on day 4 after pMCAO, functional outcome was better, and infarct volumes were smaller in sATF6-KI mice vs controls. Next, the 15-day stroke outcome study indicated that compared to control mice, sATF6-KI mice consistently exhibited improved performance in neurologic scoring, tight rope test, and tape removal test, after pMCAO. Moreover, sATF6-KI mice showed higher vascular density and lower activation of both astrocytes and microglia around stroke regions on day 16 after pMCAO. Conclusions: Here, we presented the first evidence that activation of the ATF6 UPR branch is protective in permanent ischemic stroke, which further supports the therapeutic potential of targeting the ATF6 pathway in stroke.

Manganese Porphyrin Promotes Post Cardiac Arrest Recovery in Mice and Rats.

Introduction Cardiac arrest (CA) and resuscitation induces global cerebral ischemia and reperfusion, causing neurologic deficits or death. Manganese porphyrins, superoxide dismutase mimics, are reportedly able to effectively reduce ischemic injury in brain, kidney, and other tissues. This study evaluates the efficacy of a third generation lipophilic Mn porphyrin, MnTnBuOE-2-PyP5+, Mn(III) ortho meso-tetrakis (N-n-butoxyethylpyridinium-2-yl)porphyrin (MnBuOE, BMX-001), in both mouse and rat models of CA. Methods Forty-eight animals were subjected to 8 min of CA and resuscitated subsequently by chest compression and epinephrine infusion. Vehicle or MnBuOE was given immediately after resuscitation followed by daily subcutaneous injections. Body weight, spontaneous activity, neurologic deficits, rotarod performance, and neuronal death were assessed. Kidney tubular injury was assessed in CA mice. Data were collected by the investigators who were blinded to the treatment groups. Results Vehicle mice had a mortality of 20%, which was reduced by 50% by MnBuOE. All CA mice had body weight loss, spontaneous activity decline, neurologic deficits, and decreased rotarod performance that were significantly improved at three days post MnBuOE daily treatment. MnBuOE treatment reduced cortical neuronal death and kidney tubular injury in mice (p < 0.05) but not hippocampus neuronal death (23% MnBuOE vs. 34% vehicle group, p = 0.49). In rats, they had a better body-weight recovery and increased rotarod latency after MnBuOE treatment when compared to vehicle group (p < 0.01 vs. vehicle). MnBuOE-treated rats had a low percentage of hippocampus neuronal death (39% MnBuOE vs. 49% vehicle group, p = 0.21) and less tubular injury (p < 0.05) relative to vehicle group. Conclusions We demonstrated the ability of MnBuOE to improve post-CA survival, as well as functional outcomes in both mice and rats, which jointly account for the improvement not only of brain function but also of the overall wellbeing of the animals. While MnBuOE bears therapeutic potential for treating CA patients, the females and the animals with comorbidities must be further evaluated before advancing toward clinical trials.

Myocardial Injury Caused by Chronic Alcohol Exposure-A Pilot Study Based on Proteomics.

Chronic alcohol exposure can cause myocardial degenerative diseases, manifested as cardiac insufficiency, arrhythmia, etc. These are defined as alcoholic cardiomyopathy (ACM). Alcohol-mediated myocardial injury has previously been studied through metabolomics, and it has been proved to be involved in the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway concerning unsaturated fatty acids biosynthesis and oxidative phosphorylation, which tentatively explored the mechanism of ACM induced by chronic drinking. To further study alcohol-induced myocardial injury, myocardial specimens from a previously successfully established mouse model of ACM were subjected to histological, echocardiographic, and proteomic analyses, and validated by real-time quantitative polymerase chain reaction (qPCR). Results of histopathology and echocardiography showed the hypertrophy of cardiomyocytes, the dilation of ventricles, and decreased cardiac function. Proteomic results, available via ProteomeXchange with identifier PXD032949, revealed 56 differentially expressed proteins (DEPs) were identified, which have the potential to be involved in the KEGG pathway related to fatty acid biosynthesis disorders, lipid metabolism disorders, oxidative stress, and, ultimately, in the development of dilated cardiomyopathy (DCM). The present study further elucidates the underlying effects of myocardial injury due to chronic alcohol intake, laying a foundation for further studies to clarify the potential mechanisms of ACM.

Real-time whole-brain imaging of hemodynamics and oxygenation at micro-vessel resolution with ultrafast wide-field photoacoustic microscopy.

High-speed high-resolution imaging of the whole-brain hemodynamics is critically important to facilitating neurovascular research. High imaging speed and image quality are crucial to visualizing real-time hemodynamics in complex brain vascular networks, and tracking fast pathophysiological activities at the microvessel level, which will enable advances in current queries in neurovascular and brain metabolism research, including stroke, dementia, and acute brain injury. Further, real-time imaging of oxygen saturation of hemoglobin (sO2) can capture fast-paced oxygen delivery dynamics, which is needed to solve pertinent questions in these fields and beyond. Here, we present a novel ultrafast functional photoacoustic microscopy (UFF-PAM) to image the whole-brain hemodynamics and oxygenation. UFF-PAM takes advantage of several key engineering innovations, including stimulated Raman scattering (SRS) based dual-wavelength laser excitation, water-immersible 12-facet-polygon scanner, high-sensitivity ultrasound transducer, and deep-learning-based image upsampling. A volumetric imaging rate of 2 Hz has been achieved over a field of view (FOV) of 11 × 7.5 × 1.5 mm3 with a high spatial resolution of ~10 µm. Using the UFF-PAM system, we have demonstrated proof-of-concept studies on the mouse brains in response to systemic hypoxia, sodium nitroprusside, and stroke. We observed the mouse brain's fast morphological and functional changes over the entire cortex, including vasoconstriction, vasodilation, and deoxygenation. More interestingly, for the first time, with the whole-brain FOV and micro-vessel resolution, we captured the vasoconstriction and hypoxia simultaneously in the spreading depolarization (SD) wave. We expect the new imaging technology will provide a great potential for fundamental brain research under various pathological and physiological conditions.

Single-cell transcriptomic analysis of the immune cell landscape in the aged mouse brain after ischemic stroke.

BACKGROUND: Ischemic stroke is a medical emergency that primarily affects the elderly. A complex immune response in the post-stroke brain constitutes a key component of stroke pathophysiology. This study aimed to determine how stroke affects immune cell populations in the aged brain based on molecular profiles of individual cells. METHODS: Single-cell RNA sequencing and a new transient ischemic stroke mouse model with late reperfusion were used. RESULTS: We generated, for the first time, a composite picture of immune cell populations in the stroke aged brain at single-cell resolution. We discovered at least 6 microglial subsets in the stroke aged brain, including a potentially stroke-specific subtype. Moreover, we identified major cell subpopulations formed by infiltrated myeloid cells after stroke, and revealed their unique molecular profiles. CONCLUSIONS: This study provided the first scRNA-seq data set for immune cells in the stroke aged brain, and offered novel insights into post-stroke immune cell heterogeneity.

Effects of Long-Term Vagus Nerve Electrical Stimulation Therapy on Acute Cerebral Infarction and Neurological Function Recovery in Post MCAO Mice.

Background: Vagus nerve stimulation therapy is proven to produce neuroprotective effects against central nervous system diseases and reduce neurological injury, having a positive effect on the recovery of neurological functions in mouse model of stroke. Objective: This study was aimed at exploring a wider time window for VNS treatment, investigating the long-term behavioral improvement of long-term VNS in mice after pMCAO, and exploring the antiapoptotic properties of VNS and its role in autophagy, all of which may be a permanent deficiency potential mechanism of neuroprotection in hemorrhagic stroke. Methods: Permanent focal cerebral ischemia and implantation of vagus nerve stimulator were performed through intracavitary occlusion of the right middle cerebral artery (MCA). The vagus nerve stimulation group received five times vagus nerve stimulation from 6 h after surgery for 5 days. Adhesive removal test and NSS neurological score were used to evaluate the neurological deficit of mice. TTC staining of mouse brain tissue was performed one week after surgery in order to assess the area of cerebral infarction. Additionally, frozen sections were stained with Fluoro-Jade B to observe the apoptotic cells in the ischemic penumbra of brain tissue. Finally, Western blot was used to detect the changes in the levels of apoptosis-related proteins such as cleaved-caspase3 and Bcl-2 and autophagy-related proteins such as mTOR, Beclin-1, and LC3-II in brain tissue. Results: VNS can effectively reduce the behavioral score of pMCAO mice; TTC results showed that VNS could effectively reduce the infarct area after pMCAO (P < 0.05). After VNS intervention of the pMCAO group compared with the pMCAO+VNI group, the FJB-positive cells in the VNS group were significantly decreased (P < 0.05); Western Blot analysis showed that the expression of cleaved-caspase3 in the brain tissue of mice increased after pMCAO (P < 0.05), and the expression of Bcl-2 decreased (P < 0.05). This change could be effectively reversed after VNS intervention (P < 0.05). Conclusion: VNS could effectively improve the behavioral performance of mice after permanent stroke in addition to significantly reducing the infarct size of the brain tissue. The mechanism may be related to the effective reduction of cell apoptosis and excessive autophagy after pMCAO by VNS.

Post-ischemia common carotid artery occlusion worsens memory loss, but not sensorimotor deficits, in long-term survived stroke mice.

Ischemic stroke in rodents is usually induced by intraluminal occlusion of the middle cerebral artery (MCA) via the external carotid artery (ECA) or the common carotid artery (CCA). The latter route requires permanent CCA occlusion after ischemia, and here, we assess its effects on long-term outcomes. Transient occlusion of MCA and CCA was performed at normal body temperature. After 90 min of ischemia, mice were randomized to permanent CCA occlusion or no occlusion (control group). Body weight, and motor and sensory functions, ie, pole test, adhesive tape removal, and elevated plus maze, were evaluated at 24 h, and at 7 and 28 days after stroke. Infarct volume, apoptosis, and activation of astrocytes and microglia were assessed at 4 weeks by an investigator blinded to groups. The Morris water maze test was performed at 3 weeks in the second experiment. One mouse died at 4 days, and the other mice survived with persistent neurologic deficits. CCA-occluded mice exhibited delayed turn on the pole at 24 h and decreased responses to the von Frey filament, and spent more time on the pole at 7 and 28 days than the control group. Infarction, hemispheric atrophy, glial activation, and apoptotic neuronal death were present in all mice, and no intra-group difference was found. However, CCA-occluded mice had a significantly poorer performance in the Morris water maze compared to the control group, which showed an adverse effect of post-ischemia CCA occlusion on cognition. Thus, the model selection should be well considered in preclinical efficacy studies on stroke-induced vascular dementia and stroke with Alzheimer's disease.

Cervical Vagus Nerve Stimulation Improves Neurologic Outcome After Cardiac Arrest in Mice by Attenuating Oxidative Stress and Excessive Autophagy.

BACKGROUND: Cerebral ischemia and reperfusion (I/R) induces oxidative stress and activates autophagy, leading to brain injury and neurologic deficits. Cervical vagus nerve stimulation (VNS) increases cerebral blood flow (CBF). In this study, we investigate the effect of VNS-induced CBF increase on neurologic outcomes after cardiac arrest (CA). MATERIALS AND METHODS: A total of 40 male C57Bl/6 mice were subjected to ten minutes of asphyxia CA and randomized to vagus nerve isolation (VNI) or VNS treatment group. Eight mice received sham surgery and VNI. Immediately after resuscitation, 20 minutes of electrical stimulation (1 mA, 1 ms, and 10 Hz) was started in the VNS group. Electrocardiogram, blood pressure, and CBF were monitored. Neurologic and histologic outcomes were evaluated at 72 hours. Oxidative stress and autophagy were assessed at 3 hours and 24 hours after CA. RESULTS: Baseline characteristics were not different among groups. VNS mice had better behavioral performance (ie, open field, rotarod, and neurologic score) and less neuronal death (p < 0.05, vs VNI) in the hippocampus. CBF was significantly increased in VNS-treated mice at 20 minutes after return of spontaneous circulation (ROSC) (p < 0.05). Furthermore, levels of 8-hydroxy-2'-deoxyguanosine in the blood and autophagy-related proteins (ie, LC-3Ⅱ/Ⅰ, Beclin-1, and p62) in the brain were significantly decreased in VNS mice. Aconitase activity was also reduced, and the p-mTOR/mTOR ratio was increased in VNS mice. CONCLUSIONS: Oxidative stress induced by global brain I/R following CA/ROSC leads to early excessive autophagy and impaired autophagic flux. VNS promoted CBF recovery, ameliorating these changes. Neurologic and histologic outcomes were also improved.

Activation of the XBP1s/O-GlcNAcylation Pathway Improves Functional Outcome After Cardiac Arrest and Resuscitation in Young and Aged Mice.

ABSTRACT: After cardiac arrest (CA) and resuscitation, the unfolded protein response (UPR) is activated in various organs including the brain. However, the role of the UPR in CA outcome remains largely unknown. One UPR branch involves spliced X-box-binding protein-1 (XBP1s). Notably, XBP1s, a transcriptional factor, can upregulate expression of specific enzymes related to glucose metabolism, and subsequently boost O-linked ß-N-acetylglucosamine modification (O-GlcNAcylation). The current study is focused on effects of the XBP1 UPR branch and its downstream O-GlcNAcylation on CA outcome. Using both loss-of-function and gain-of-function mouse genetic tools, we provide the first evidence that activation of the XBP1 UPR branch in the post-CA brain is neuroprotective. Specifically, neuron-specific Xbp1 knockout mice had worse CA outcome, while mice with neuron-specific expression of Xbp1s in the brain had better CA outcome. Since it has been shown that the protective role of the XBP1s signaling pathway under ischemic conditions is mediated by increasing O-GlcNAcylation, we then treated young mice with glucosamine, and found that functional deficits were mitigated on day 3 post CA. Finally, after confirming that glucosamine can boost O-GlcNAcylation in the aged brain, we subjected aged mice to 8 min CA, and then treated them with glucosamine. We found that glucosamine-treated aged mice performed significantly better in behavioral tests. Together, our data indicate that the XBP1s/O-GlcNAc pathway is a promising target for CA therapy.

Activation of the ATF6 (Activating Transcription Factor 6) Signaling Pathway in Neurons Improves Outcome After Cardiac Arrest in Mice.

Background Ischemia/reperfusion injury impairs proteostasis, and triggers adaptive cellular responses, such as the unfolded protein response (UPR), which functions to restore endoplasmic reticulum homeostasis. After cardiac arrest (CA) and resuscitation, the UPR is activated in various organs including the brain. However, the role of the UPR in CA has remained largely unknown. Here we aimed to investigate effects of activation of the ATF6 (activating transcription factor 6) UPR branch in CA. Methods and Results Conditional and inducible sATF6-KI (short-form ATF6 knock-in) mice and a selective ATF6 pathway activator 147 were used. CA was induced in mice by KCl injection, followed by cardiopulmonary resuscitation. We first found that neurologic function was significantly improved, and neuronal damage was mitigated after the ATF6 pathway was activated in neurons of sATF6-KI mice subjected to CA/cardiopulmonary resuscitation. Further RNA sequencing analysis indicated that such beneficial effects were likely attributable to increased expression of pro-proteostatic genes regulated by ATF6. Especially, key components of the endoplasmic reticulum-associated degradation process, which clears potentially toxic unfolded/misfolded proteins in the endoplasmic reticulum, were upregulated in the sATF6-KI brain. Accordingly, the CA-induced increase in K48-linked polyubiquitin in the brain was higher in sATF6-KI mice relative to control mice. Finally, CA outcome, including the survival rate, was significantly improved in mice treated with compound 147. Conclusions This is the first experimental study to determine the role of the ATF6 UPR branch in CA outcome. Our data indicate that the ATF6 UPR branch is a prosurvival pathway and may be considered as a therapeutic target for CA.

Development and Evaluation of a Novel Mouse Model of Asphyxial Cardiac Arrest Revealed Severely Impaired Lymphopoiesis After Resuscitation.

Background Animal disease models represent the cornerstone in basic cardiac arrest (CA) research. However, current experimental models of CA and resuscitation in mice are limited. In this study, we aimed to develop a mouse model of asphyxial CA followed by cardiopulmonary resuscitation (CPR), and to characterize the immune response after asphyxial CA/CPR. Methods and Results CA was induced in mice by switching from an O2/N2 mixture to 100% N2 gas for mechanical ventilation under anesthesia. Real-time measurements of blood pressure, brain tissue oxygen, cerebral blood flow, and ECG confirmed asphyxia and ensuing CA. After a defined CA period, mice were resuscitated with intravenous epinephrine administration and chest compression. We subjected young adult and aged mice to this model, and found that after CA/CPR, mice from both groups exhibited significant neurologic deficits compared with sham mice. Analysis of post-CA brain confirmed neuroinflammation. Detailed characterization of the post-CA immune response in the peripheral organs of both young adult and aged mice revealed that at the subacute phase following asphyxial CA/CPR, the immune system was markedly suppressed as manifested by drastic atrophy of the spleen and thymus, and profound lymphopenia. Finally, our data showed that post-CA systemic lymphopenia was accompanied with impaired T and B lymphopoiesis in the thymus and bone marrow, respectively. Conclusions In this study, we established a novel validated asphyxial CA model in mice. Using this new model, we further demonstrated that asphyxial CA/CPR markedly affects both the nervous and immune systems, and notably impairs lymphopoiesis of T and B cells.