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Representative brain histology 3 days post-cardiac arrest. (A) hippocampal CA1, (B) cortex, (C) dentate gyrus, (D) thalamus. Arrows point to dead neurons.
Source publication
Experimental cardiac arrest (CA) in aging research is infrequently studied in part due to the limitation of animal models. We aimed to develop an easily performed mouse CA model to meet this need. A standard mouse KCl-induced CA model using chest compressions and intravenous epinephrine for resuscitation was modified by blood withdrawal prior to CA...
Context in source publication
Context 1
... locomotor activity gradually declined over the 3-day recovery interval and was worse in the aged versus young adult mice (Fig. 4D, p= 0.04 in activity time). Travel distance declined as well (p< 0.01). No difference was present in velocity (p= 0.18). Neuronal necrosis was found in multiple areas including cortex, CA1, dentate gyrus, and thalamus (Fig. 5). There was no intra-group histologic difference in either cortex (aged 8 ± 4 dead neurons/field, young adult 6 ± 3 dead neurons/field, p= 0.42) and CA1 (aged 6 ± 3 %, young adult 6 ± 4%, p= 0.93). Amongst the 5 aged mice for which rectal temperature control was extended to 30 min post-CA, one died at 48 h. The remaining 4 mice survived ...
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ELife digest
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Citations
... Furthermore, neurologic outcome and locomotor activity appear to be similar between male and female animals without cardiac arrest. A previous report 29 showed a significant decline in locomotor function after 8 min of cardiac arrest in mice. After cardiac arrest, indoleamine 2,3-dioxygenase-deleted mice showed a higher spontaneous locomotor activity and a significant increase in the total distance traveled compared to wild-type animals. ...
Background:
The catabolism of the essential amino acid tryptophan to kynurenine is emerging as a potential key pathway involved in post-cardiac arrest brain injury. The aim of this study was to evaluate the effects of the modulation of kynurenine pathway on cardiac arrest outcome, through genetic deletion of the rate-limiting enzyme of the pathway, indoleamine-2,3-dyoxygenase(IDO).
Methods:
Wild-type (WT) and IDO-deleted (IDO -/-) mice were subjected to 8 min cardiac arrest. Survival, neurological outcome, and locomotor activity were evaluated following resuscitation. Brain magnetic resonance imaging with diffusion tensor and diffusion-weighted imaging sequences was performed, together with microglia/macrophage activation and neurofilament light chain measurements.
Results:
IDO-/- mice showed higher survival compared to WT mice (IDO-/- 11/16, WT 6/16, log-rank p=0.036). Neurological function was higher in IDO-/- mice than in WT mice following cardiac arrest (IDO-/- 9±1, WT 7±1, p=0.012, n=16). IDO-deletion preserved locomotor function while maintaining physiologic circadian rhythm after cardiac arrest. Brain magnetic resonance imaging with diffusion tensor imaging showed an increase in mean fractional anisotropy in the corpus callosum (IDO-/- 0.68±0.01, WT 0.65±0.01, p=0.010, n=5-4) and in the external capsule (IDO -/- 0.47±0.01, WT 0.45±0.01, p=0.006, n=5-4) in IDO-/- mice compared to WT ones. Increased release of neurofilament light chain was observed in WT mice compared to IDO-/- (median concentrations (IQR), pg/mL: WT 1138 (678-1384), IDO-/- 267 (157-550), p<0.001, n=3-4).Brain magnetic resonance imaging with diffusion-weighted imaging revealed restriction of water diffusivity 24 hours after cardiac arrest in WT mice, IDO-deletion prevented water diffusion abnormalities, which was reverted in IDO-/- mice receiving L-kynurenine (Apparent Diffusion Coefficient, μm2/ms: WT 0.48±0.07, IDO-/- 0.59±0.02, IDO-/- +L-Kynurenine 0.47±0.08, p=0.007, n=6).
Conclusions:
Kynurenine pathway represents a novel target to prevent post-cardiac arrest brain injury. The neuroprotective effects of IDO-deletion were associated with preservation of brain white matter microintegrity and with reduction of cerebral cytotoxic edema.
... Chest compressions were performed with a single finger at 300 stroke/min until sinus rhythm of electrocardiograph (ECG) appeared. [17] If the rescue time exceeded 10 min, the mice could not return to spontaneous circulation (ROSC), and the subsequent resuscitation was terminated. When spontaneous ventilation was sufficient, the ventilator was disconnected and the trachea was extubated. ...
... Cardiac arrest and cardiopulmonary resuscitation animal model CA/CPR operation was performed as previously described (Liu et al., 2018). Briefly, tracheal intubation was given after anesthesia induction (4%-5% isoflurane), and anesthesia was maintained in mice by mechanical ventilation with 1.0%-1.5% isoflurane. ...
Ischemia/reperfusion caused by cardiac arrest (CA) disturbs endoplasmic reticulum (ER) homeostasis and redox balance in neurons. AA147, originally developed as a pharmacologic activator of the activating transcription factor 6 (ATF6), can protect multiple tissues from ischemia/reperfusion injury (IRI) by decreasing reactive oxygen species (ROS) and restoring ER function. However, it is unclear whether pharmacologic treatment of AA147 could ameliorate post-CA cerebral IRI and whether it is associated with proteostasis regulation and anti-oxidative stress mechanism. In the present study, mice were subjected to 9 min-CA surgery followed by cardiopulmonary resuscitation (CPR). AA147 or vehicle was administered 1 day before the operation and 15 min after the return of spontaneous circulation. We found that AA147 restored neurological function and reduced dead neurons in mice suffering from CA. Moreover, AA147 inhibited CA/CPR-caused neuronal apoptosis and ER stress, indicated by reduced TUNEL-positive neurons, surged expression of Bcl-2/Bax, and down expression of cleaved caspase-3, caspase-12, C/EBP homologous protein (CHOP). The expression of ATF6 and its regulated gene glucose-regulated protein 78 (GRP78) increased significantly after the administration of AA147, suggesting the activation of the ATF6 pathway. In addition, AA147 also alleviated the upsurge of the ROS generation and MDA levels as well as increased SOD activity, accompanied by enhancement of the nuclear factor E2-related factor 2 (Nrf2) and its modulated heme-oxygenase-1 (HO-1) expressions. Cotreatment of AA147 with inhibitors of the ATF6 or Nrf2 significantly suppressed AA147-dependent reductions in ROS scavenging and neuronal death after CA/CPR. The results suggested that AA147 could confer neuroprotection against post-CA cerebral IRI through inhibition of oxidative stress along with ER stress-associated apoptosis, which is attributed to the coregulation of both ATF6 and Nrf2 signaling pathways activity. Our findings support the potential for AA147 as a therapeutic approach to improve post-CA brain injury.
... Then, a PE-10 tube for mice or silicone tube for rats was placed. After 15 min of physiological stabilization, cardiac arrest (CA) was induced by solution of potassium chloride (KCl) as described previously [39,40]. In brief, 300 µL or 5 mL venous blood was withdrawn from the jugular vein in mice or rats into the heparin syringe. ...
... Following resuscitation, animals were randomly assigned to vehicle or MnBuOE treatment using the online software GraphPad QuickCalcs (n = 10 in the CA vehicle group and n = 10 in the CA MnBuOE treatment group for each species, 20 mice and 20 rats total). The sample size was calculated using a power analysis based on the neuroscore data from a previous study [39]. In mice, an additional 4 mice received sham surgery and vehicle treatment. ...
... Neuroscore Neurologic deficits were examined in mice 3 days post CA and in rats 1 and 7 days after CA using the scoring system as previously reported [39]. The scoring system includes the ability to climb on a vertical screen, walk on a horizontal wooden rod, and hang on horizontal rope. ...
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.
... Due to systemic reactions or perfusion timing errors in the systemic damage model, obtaining constant and uniform damage within the brain is challenging (Brücken et al., 2018;Jia et al., 2021;Vognsen et al., 2017). In addition, intensive systemic management that mimics clinical situations is a prerequisite, which is complicated to apply in small animals (Dave et al., 2013;Ettl et al., 2018;Liu et al., 2018). In the brain damage-type models, traditional 4VO models tend to have insufficient brain damage because cell death exclusively occurs in the hippocampal CA1 area, which does not reflect the widespread damage observed in the brain of cardiac arrest patients (Motolese et al., 2015). ...
Background
Post-cardiac arrest brain injury (PCABI) is a major cause of disability and death in patients with post-cardiac arrest syndrome (PCAS). However, there have been no suitable animal models with characteristic behaviors and cerebral damage adequately mimicking clinical PCABI.
New Method
We established a chimeric model by increasing transient middle cerebral artery occlusion-mediated ipsilateral hemisphere damage in the 4-vessel occlusion (4VO) model, thereby inducing global forebrain asymmetric hemisphere ischemia. Severity of brain damage was then evaluated by behavioral and histological approaches. Neuroprotection was assessed by performing targeted temperature management (TTM) for two hours.
Results
Comatose behaviors were observed in both groups. Compared to the 4VO group, the chimeric group exhibited a higher neurological deficit score (NDS) (70.5±17.6 vs. 139.5±16.8, p=0.0002), decreased brain cell viability (88.6±18.0% vs. 5.7±2.7%, p<0.0001), and increased inflammation in the cortex (10.3±1.6% vs. 16.9±1.1%, p=0.0061). After TTM neuroprotection, the chimeric-TTM group showed improvement in NDS (139.5±16.8 vs. 0.0±0.0, p<0.0001), cortex and hippocampus cell viability (5.7±2.7% vs. 72.8±10.0%, p<0.0001; and 2.5±1.5% vs. 75.5±10.3%, p<0.0001, respectively) and inflammation (16.9±1.1% vs. 11.0±2.3%, p=0.190; and 30.9±1.7% vs. 16.6±1.2%, p<0.0001, respectively) compared to the chimeric group.
Comparison with Existing Method
Unlike the extensive brain damage found in clinical PCAS settings, the existing 4VO models showed only global forebrain damage involving CA1 lesions on both hippocampi. Our model induced global forebrain and additional asymmetric hemisphere ischemic damages, which resulted in simulating PCABI-specific clinical manifestations than conventional models.
Conclusions
Our model adequately simulates clinical PCABI and reflects appropriate neuroprotective effects of TTM.
... Neurologic scoring. A 9-point scoring system, a wellestablished neurologic testing system designed to detect motor deficits in the rat, was modified and used to assess overall neurologic deficits in mice (32). The final score ranked from zero to nine, with normal mice being awarded 9 points and the severely injured ones 0 point. ...
Background
Cardiac arrest/cardiopulmonary resuscitation (CA/CPR) represents one of the devastating medical emergencies and is associated with high mortality and neuro-disability. Post-cardiac arrest syndrome (PCAS) is mechanistically ascribed to acute systemic ischemia/reperfusion(I/R) injury. The lncRNA/microRNA/mRNA networks have been found to play crucial roles in the pathogenesis of the hypoxia-responsive diseases. Nonetheless, the precise molecular mechanisms by which lncRNA/miRNA/mRNA axes are involved in the astrocyte–microglia crosstalk in CA/CPR have not been fully elucidated.
Methods
We collected and purified the exosomes from the blood of CA/CPR patients and supernatant of OGD/R-stimulated astrocytes. On the basis of microarray analysis, bioinformatic study, and luciferase activity determination, we speculated that lncRNA GAS5/miR-137 is implicated in the astrocyte–microglia crosstalk under the insult of systemic I/R injury. The regulation of lncRNA GAS5/miR-137 on INPP4B was examined by cellular transfection in OGD/R cell culture and by lateral ventricle injection with miR-137 agomir in CA/CPR mice model. Flow cytometry and immunofluorescence staining were performed to detect the microglial apoptosis, M1/M2 phenotype transformation, and neuroinflammation. Neurological scoring and behavior tests were conducted in CA/CPR group, with miR-137 agomir lateral-ventricle infusion and in their controls.
Results
In all the micRNAs, miR-137 was among the top 10 micRNAs that experienced greatest changes, in both the blood of CA/CPR patients and supernatant of OGD/R-stimulated astrocytes. Bioinformatic analysis revealed that miR-137 was sponged by lncRNA GAS5, targeting INPP4B, and the result was confirmed by Luciferase activity assay. qRT-PCR and Western blotting showed that lncRNA GAS5 and INPP4B were over-expressed whereas miR-137 was downregulated in the blood of CA/CPR patients, OGD/R-stimulated astrocytes, and brain tissue of CA/CPR mice. Silencing lncRNA GAS5 suppressed INPP4B expression, but over-expression of miR-137 negatively modulated its expression. Western blotting exhibited that PI3K and Akt phosphorylation was increased when lncRNA GAS5 was silenced or miR-137 was over-expressed. However, PI3K and Akt phosphorylation was notably suppressed in the absence of miR-137, almost reversing their phosphorylation in the silencing lncRNA GAS5 group. Then we found that GAS5 siRNA or miR-137 mimic significantly increased cell viability and alleviated apoptosis after OGD/R injury. Furthermore, over-expression of miR-137 attenuated microglial apoptosis and neuroinflammation in CA/CPR mice model, exhibiting significantly better behavioral tests after CA/CPR.
Conclusion
LncRNA GAS5/miR-137 may be involved in the astrocyte–microglia communication that inhibits PI3K/Akt signaling activation via regulation of INPP4B during CA/CPR.
... All tests were conducted in the light phase, and the procedures were essentially the same as described preivouly. 13,[19][20][21] Briefly, a 9-point scoring system was used to evaluate general functional deficits (9 points=normal, and 0 points=severe injury). 20 The duration that each mouse was able to remain on the vertical screen, horizontal rod, and horizontal rope was recorded and then a score was assigned (0-3 points for each test). ...
... 13,[19][20][21] Briefly, a 9-point scoring system was used to evaluate general functional deficits (9 points=normal, and 0 points=severe injury). 20 The duration that each mouse was able to remain on the vertical screen, horizontal rod, and horizontal rope was recorded and then a score was assigned (0-3 points for each test). The rotarod test evaluated balance, grip strength, and motor coordination using a device with an accelerating rotating rod (4-40 rpm; Med Associates). ...
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.
... Our group previously developed a modified KCl-induced CA model by reducing the KCl dosage, which shortens the duration of CPR necessary to achieve ROSC, and substantially improves survival rates while still producing detectable neurologic deficits. 24 In addition to KCl models, few other murine CA/ CPR models have been reported, but their utility in experimental CA research has not been well established. 25,26 In one such model, CA was induced by bolus injections of vecuronium (neuromuscular blockade) and esmolol (a β-adrenergic receptor blocker) followed by cessation of mechanical ventilation. ...
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 O 2 /N 2 mixture to 100% N 2 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.
... Young male mice (3-4 months) and aged male mice (20-22 months) were used. CA/CPR surgery was performed as previously described (6,16), with minor modifications. Briefly, following anesthesia induction and endotracheal intubation, mice were maintained on 1.5% −1.7% isoflurane with a body temperature of 37.0℃ ± 0.2℃ (rectal probe) before CA induction. ...
... All tests were conducted by experimenters who were blinded to genotypes and treatments, using our standard protocols (6). Sham groups have been evaluated in our previous studies (16,19), and thus were not included in these tests. ...
... To address this critical question, we first used neuron-specific Xbp1 knockout mice, ie, Xbp1-cKO (8). Our mouse CA/CPR model has been well-characterized in previous studies (6,16,19,20). Using laser speckle contrast imaging (LSCI), we further showed here that in our model, the cortical CBF decreased dramatically, and reached a zero level during CA (Fig. 1A). ...
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 β-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.
... While data is limited concerning procedure times in the traditional model, Abella et al. describe 50 minutes of anesthesia induction and up to 40 min of venous instrumentation prior to induction of asystole, followed by 2 hours of invasive monitoring [14]. More recently, Liu et al. report venous instrumentation followed by 15 min of stabilization, followed by asystole and CPR and then 30 min of invasive monitoring [15]. ...
... Following extubation, body temperature was maintained with active heating in a recovery cage for 2 hours. However, body temperatures fell significantly after active heating was stopped, which is consistent with post-arrest changes in humans [28] as well as previous mouse models of SCA [15,29]. The SCA mice continued to show significant temperature dysregulation and depressed HR at one day (Fig 1, Table 1). ...
... While some groups are able to demonstrate neurologic injury with as little as 6-minutes of cardiac arrest [31], others have required extended arrest time of 12-14 minutes to detect neurologic changes [22,29]. The 8-min time point was chosen as it is a well-established time-point in this field [9,15,32], however the procedure could be modified to allow for prolonged arrest time to study neurologic insult. ...
Aim
Mouse models of sudden cardiac arrest are limited by challenges with surgical technique and obtaining reliable venous access. To overcome this limitation, we sought to develop a simplified method in the mouse that uses ultrasound-guided injection of potassium chloride directly into the heart.
Methods
Potassium chloride was delivered directly into the left ventricular cavity under ultrasound guidance in intubated mice, resulting in immediate asystole. Mice were resuscitated with injection of epinephrine and manual chest compressions and evaluated for survival, body temperature, cardiac function, kidney damage, and diffuse tissue injury.
Results
The direct injection sudden cardiac arrest model causes rapid asystole with high surgical survival rates and short surgical duration. Sudden cardiac arrest mice with 8-min of asystole have significant cardiac dysfunction at 24 hours and high lethality within the first seven days, where after cardiac function begins to improve. Sudden cardiac arrest mice have secondary organ damage, including significant kidney injury but no significant change to neurologic function.
Conclusions
Ultrasound-guided direct injection of potassium chloride allows for rapid and reliable cardiac arrest in the mouse that mirrors human pathology without the need for intravenous access. This technique will improve investigators’ ability to study the mechanisms underlying post-arrest changes in a mouse model.
