Ischemic Cell Death in the CNS - applications of a new in vitro model

Ischemic brain damage is a common cause of death and disability. A global ischemic insult is usually the result of a transient cardiac arrest while occlusion of cerebral blood vessels leads to focal ischemic lesions, commonly termed stroke. During the last decades our knowledge about the metabolic and cellular events leading to cell death following ischemia has expanded mainly due to experimental studies in vivo and in vitro. Important findings concerning the relevance of body temperature and blood glucose levels have been confirmed in humans. However, a vast number of pharmacological agents with protective effects in animal models of ischemia have failed in subsequent clinical trials. This illustrates that our knowledge of the mechanisms of ischemic cell death is still incomplete and that we need to question the models we use to mimic the human disorders. We have used the organotypic tissue culture from mouse hippocampus to establish a new model of in vitro ischemia (IVI). Similar to previous models we combine anoxia and aglycemia but in addition we apply a combination of ions similar to what is found in the brain extracellular fluid during ischemia. We found that the combination of a high potassium level (70mM), a low calcium level (0.3mM) and acidosis (pH 6.8) during IVI made the pattern of cell death more similar to what is found following global ischemia in vivo in that became more delayed and selective. A high level of glucose was found to increase cell death in contrast to what had previously been found in other cell culture models of ischemia but in similarity to what is found in vivo. While cell death following IVI could be completely prevented by the withdrawal of extracellular calcium during the insult or antagonists of glutamatergic NMDA-receptors, no effect of either was found in the hyperglycaemic IVI paradigm. On the other hand, intracellular calcium chelation prevented against cell death following hyperglycaemic IVI but not IVI. Inhibition of free radicals was ineffective in both paradigms. These findings illustrates that IVI and hyperglycaemic IVI induces two different patterns of cell death both of which may be important during ischemia in vivo. Intraischemic acidosis protected neurons in the CA3-subregion of hippocampus more than in CA1. NMDA-receptors in both regions were inhibited by acidosis but they recovered significantly slower in the CA3-region. This prolonged inhibition could explain the sparing of the CA3-neurons following IVI and global ischemia. The neuromodulator adenosine inhibits glutamate release through presynaptic A1-receptors. We used transgenic A1-receptor knock-out mice to study the importance of this receptor for the development of cell death following IVI and global ischemia in vivo. No effect of the knock-out was found in any of the two paradigms. The A1-receptor antagonist, 8-CPT increased damage in vivo but had no effect in vitro. This discrepancy between the models could be explained by a less importance of vesicular glutamate release in vitro or an undiscovered systemic side-effect of 8-CPT in vivo. The described models of IVI and hyperglycaemic IVI are well suited for further studies on the pathophysiology of cerebral ischemia using transgenic, pharmacological, electrophysiological and imaging techniques

β-Adrenoceptor activation depresses brain inflammation and is neuroprotective in lipopolysaccharide-induced sensitization to oxygen-glucose deprivation in organotypic hippocampal slices

<p>Abstract</p> <p>Background</p> <p>Inflammation acting in synergy with brain ischemia aggravates perinatal ischemic brain damage. The sensitizing effect of pro-inflammatory exposure prior to hypoxia is dependent on signaling by TNF-α through TNF receptor (TNFR) 1. Adrenoceptor (AR) activation is known to modulate the immune response and synaptic transmission. The possible protective effect of <inline-formula><m:math xmlns:m="http://www.w3.org/1998/Math/MathML" name="1742-2094-7-94-i1"><m:mover accent="true"><m:mi>α</m:mi><m:mo>˜</m:mo></m:mover></m:math></inline-formula> and <inline-formula><m:math xmlns:m="http://www.w3.org/1998/Math/MathML" name="1742-2094-7-94-i2"><m:mover accent="true"><m:mi>β</m:mi><m:mo>˜</m:mo></m:mover></m:math></inline-formula>AR activation against neuronal damage caused by tissue ischemia and inflammation, acting in concert, was evaluated in murine hippocampal organotypic slices treated with lipopolysaccharide (LPS) and subsequently subjected to oxygen-glucose deprivation (OGD).</p> <p>Method</p> <p>Hippocampal slices from mice were obtained at P6, and were grown <it>in vitro </it>for 9 days on nitrocellulose membranes. Slices were treated with β1(dobutamine)-, β2(terbutaline)-, α1(phenylephrine)- and α2(clonidine)-AR agonists (5 and 50 μM, respectively) during LPS (1 μg/mL, 24 h) -exposure followed by exposure to OGD (15 min) in a hypoxic chamber. Cell death in the slice CA1 region was assessed by propidium iodide staining of dead cells.</p> <p>Results</p> <p>Exposure to LPS + OGD caused extensive cell death from 4 up to 48 h after reoxygenation. Co-incubation with β1-agonist (50 μM) during LPS exposure before OGD conferred complete protection from cell death (P < 0.001) whereas the β2-agonist (50 μM) was partially protective (p < 0.01). Phenylephrine was weakly protective while no protection was attained by clonidine. Exposure to both β1- and β2-agonist during LPS exposure decreased the levels of secreted TNF-α, IL-6 and monocyte chemoattractant protein-1 and prevented microglia activation in the slices. Dobutamine remained neuroprotective in slices exposed to pure OGD as well as in TNFR1^-/-and TNFR2^-/-slices exposed to LPS followed by OGD.</p> <p>Conclusions</p> <p>Our data demonstrate that activation of both β1- and β2-receptors is neuroprotective and may offer mechanistic insights valuable for development of neuro-protective strategies in neonates.</p

The brain-enriched microRNA miR-124 in plasma predicts neurological outcome after cardiac arrest

Introduction: Early prognostication after successful cardiopulmonary resuscitation is difficult, and there is a need for novel methods to estimate the extent of brain injury and predict outcome. In this study, we evaluated the impact of the cardiac arrest syndrome on the plasma levels of selected tissue-specific microRNAs (miRNAs) and assessed their ability to prognosticate death and neurological disability. Methods: We included 65 patients treated with hypothermia after cardiac arrest in the study. Blood samples were obtained at 24 hours and at 48 hours. For miRNA-screening purposes, custom quantitative polymerase chain reaction (qPCR) panels were first used. Thereafter individual miRNAs were assessed at 48 hours with qPCR. miRNAs that successfully predicted prognosis at 48 hours were further analysed at 24 hours. Outcomes were measured according to the Cerebral Performance Category (CPC) score at 6 months after cardiac arrest and stratified into good (CPC score 1 or 2) or poor (CPC scores 3 to 5). Results: At 48 hours, miR-146a, miR-122, miR-208b, miR-21, miR-9 and miR-128 did not differ between the good and poor neurological outcome groups. In contrast, miR-124 was significantly elevated in patients with poor outcomes compared with those with favourable outcomes (P < 0.0001) at 24 hours and 48 hours after cardiac arrest. Analysis of receiver operating characteristic curves at 24 and 48 hours after cardiac arrest showed areas under the curve of 0.87 (95% confidence interval (CI) = 0.79 to 0.96) and 0.89 (95% CI = 0.80 to 0.97), respectively. Conclusions: The brain-enriched miRNA miR-124 is a promising novel biomarker for prediction of neurological prognosis following cardiac arrest

Cognitive function after cardiac arrest and temperature management; rationale and description of a sub-study in the Target Temperature Management trial

Background: Mild to moderate cognitive impairment is common amongst long-term survivors of cardiac arrest. In the Target Temperature Management trial (TTM-trial) comatose survivors were randomized to 33 degrees C or 36 degrees C temperature control for 24 hours after cardiac arrest and the effects on survival and neurological outcome assessed. This protocol describes a sub-study of the TTM-trial investigating cognitive dysfunction and its consequences for patients' and relatives' daily life. Methods/Design: Sub-study sites in five European countries included surviving TTM patients 180 days after cardiac arrest. In addition to the instruments for neurological function used in the main trial, sub-study patients were specifically tested for difficulties with memory (Rivermead Behavioural Memory Test), attention (Symbol Digit Modalities Test) and executive function (Frontal Assessment Battery). Cognitive impairments will be related to the patients' degree of participation in society (Mayo-Portland Adaptability Inventory-4), health related quality of life (Short Form Questionnaire-36v2 (c)), and the caregivers' situation (Zarit Burden Interview (c)). The two intervention groups (33 degrees C and 36 degrees C) will be compared with a group of myocardial infarction controls. Discussion: This large international sub-study of a randomized controlled trial will focus on mild to moderate cognitive impairment and its consequences for cardiac arrest survivors and their caregivers. By using an additional battery of tests we may be able to detect more subtle differences in cognitive function between the two intervention groups than identified in the main study. The results of the study could be used to develop a relevant screening model for cognitive dysfunction after cardiac arrest

Serum neurofilament light levels are correlated to long-term neurocognitive outcome measures after cardiac arrest

OBJECTIVE: To explore associations between four methods assessing long-term neurocognitive outcome after out-of-hospital cardiac arrest and early hypoxic-ischemic neuronal brain injury assessed by the biomarker serum neurofilament light (NFL), and to compare the agreement for the outcome methods. METHODS: An explorative post-hoc study was conducted on survivor data from the international Target Temperature Management after Out-of-hospital Cardiac Arrest trial, investigating serum NFL sampled 48/72-hours post-arrest and neurocognitive outcome 6 months post-arrest. RESULTS: Among the long-term surviving participants (N = 457), serum NFL (n = 384) was associated to all outcome instruments, also when controlling for demographic and cardiovascular risk factors. Associations between NFL and the patient-reported Two Simple Questions (TSQ) were however attenuated when adjusting for vitality and mental health. NFL predicted results on the outcome instruments to varying degrees, with an excellent area under the curve for the clinician-report Cerebral Performance Category (CPC 1-2: 0.90). Most participants were classified as CPC 1 (79%). Outcome instrument correlations ranged from small (Mini-Mental State Examination [MMSE]-TSQ) to strong (CPC-MMSE). CONCLUSIONS: The clinician-reported CPC was mostly related to hypoxic-ischemic brain injury, but with a ceiling effect. These results may be useful when selecting methods and instruments for clinical follow-up models

Neurofilament light as an outcome predictor after cardiac arrest : a post hoc analysis of the COMACARE trial

Purpose Neurofilament light (NfL) is a biomarker reflecting neurodegeneration and acute neuronal injury, and an increase is found following hypoxic brain damage. We assessed the ability of plasma NfL to predict outcome in comatose patients after out-of-hospital cardiac arrest (OHCA). We also compared plasma NfL concentrations between patients treated with two different targets of arterial carbon dioxide tension (PaCO2), arterial oxygen tension (PaO2), and mean arterial pressure (MAP). Methods We measured NfL concentrations in plasma obtained at intensive care unit admission and at 24, 48, and 72 h after OHCA. We assessed neurological outcome at 6 months and defined a good outcome as Cerebral Performance Category (CPC) 1-2 and poor outcome as CPC 3-5. Results Six-month outcome was good in 73/112 (65%) patients. Forty-eight hours after OHCA, the median NfL concentration was 19 (interquartile range [IQR] 11-31) pg/ml in patients with good outcome and 2343 (587-5829) pg/ml in those with poor outcome,p <0.001. NfL predicted poor outcome with an area under the receiver operating characteristic curve (AUROC) of 0.98 (95% confidence interval [CI] 0.97-1.00) at 24 h, 0.98 (0.97-1.00) at 48 h, and 0.98 (0.95-1.00) at 72 h. NfL concentrations were lower in the higher MAP (80-100 mmHg) group than in the lower MAP (65-75 mmHg) group at 48 h (median, 23 vs. 43 pg/ml,p = 0.04). PaCO(2)and PaO(2)targets did not associate with NfL levels. Conclusions NfL demonstrated excellent prognostic accuracy after OHCA. Higher MAP was associated with lower NfL concentrations.Peer reviewe

GFAp and tau protein as predictors of neurological outcome after out-of-hospital cardiac arrest: A post hoc analysis of the COMACARE trial

Aim: To determine the ability of serum glial fibrillary acidic protein (GFAp) and tau protein to predict neurological outcome after out-of-hospital cardiac arrest (OHCA). Methods: We measured plasma concentrations of GFAp and tau of patients included in the previously published COMACARE trial (NCT02698917) on intensive care unit admission and at 24, 48, and 72 h after OHCA, and compared them to neuron specific enolase (NSE). NSE concentrations were determined already during the original trial. We defined unfavourable outcome as a cerebral performance category (CPC) score of 3-5 six months after OHCA. We determined the prognostic accuracy of GFAp and tau using the receiver operating characteristic curve and area under the curve (AUROC). Results: Overall, 39/112 (35%) patients had unfavourable outcomes. Over time, both markers were evidently higher in the unfavourable outcome group (p < 0.001). At 48 h, the median (interquartile range) GFAp concentration was 1514 (886-4995) in the unfavourable versus 238 (135-463) pg/ ml in the favourable outcome group (p < 0.001). The corresponding tau concentrations were 99.6 (14.5-352) and 3.0 (2.2-4.8) pg/ml (p < 0.001). AUROCs at 48 and 72 h were 0.91 (95% confidence interval 0.85-0.97) and 0.91 (0.85-0.96) for GFAp and 0.93 (0.86-0.99) and 0.95 (0.89-1.00) for tau. Corresponding AUROCs for NSE were 0.86 (0.79-0.94) and 0.90 (0.82-0.97). The difference between the prognostic accuracies of GFAp or tau and NSE were not statistically significant. Conclusions: At 48 and 72 h, serum both GFAp and tau demonstrated excellent accuracy in predicting outcomes after OHCA but were not superior to NSE. Clinical trial registration: NCT02698917 (https://www.clinicaltrials.gov/ct2/show/NCT02698917).Peer reviewe

The association of partial pressures of oxygen and carbon dioxide with neurological outcome after out-of-hospital cardiac arrest: an explorative International Cardiac Arrest Registry 2.0 study

Background Exposure to extreme arterial partial pressures of oxygen (PaO2) and carbon dioxide (PaCO2) following the return of spontaneous circulation (ROSC) after out-of-hospital cardiac arrest (OHCA) is common and may affect neurological outcome but results of previous studies are conflicting. Methods Exploratory study of the International Cardiac Arrest Registry (INTCAR) 2.0 database, including 2162 OHCA patients with ROSC in 22 intensive care units in North America and Europe. We tested the hypothesis that exposure to extreme PaO2 or PaCO2 values within 24 h after OHCA is associated with poor neurological outcome at discharge. Our primary analyses investigated the association between extreme PaO2 and PaCO2 values, defined as hyperoxemia (PaO2 > 40 kPa), hypoxemia (PaO2  6.7 kPa) and hypocapnemia (PaCO2  40 kPa with PaCO2  6.7 kPa and neurological outcome. To define a cut point for the onset of poor neurological outcome, we tested a model with increasing and decreasing PaO2 levels and decreasing PaCO2 levels. Cerebral Performance Category (CPC), dichotomized to good (CPC 1–2) and poor (CPC 3–5) was used as outcome measure. Results Of 2135 patients eligible for analysis, 700 were exposed to hyperoxemia or hypoxemia and 1128 to hypercapnemia or hypocapnemia. Our primary analyses did not reveal significant associations between exposure to extreme PaO2 or PaCO2 values and neurological outcome (P = 0.13–0.49). Our secondary analyses showed no significant associations between combinations of PaO2 and PaCO2 and neurological outcome (P = 0.11–0.86). There was no PaO2 or PaCO2 level significantly associated with poor neurological outcome. All analyses were adjusted for relevant co-variates. Conclusions Exposure to extreme PaO2 or PaCO2 values in the first 24 h after OHCA was common, but not independently associated with neurological outcome at discharge.publishedVersio

ERC-ESICM guidelines on temperature control after cardiac arrest in adults

The aim of these guidelines is to provide evidence based guidance for temperature control in adults who are comatose after resuscitation from either in-hospital or out-of-hospital cardiac arrest, regardless of the underlying cardiac rhythm. These guidelines replace the recommendations on temperature management after cardiac arrest included in the 2021 post-resuscitation care guidelines co-issued by the European Resuscitation Council (ERC) and the European Society of Intensive Care Medicine (ESICM). The guideline panel included thirteen international clinical experts who authored the 2021 ERC-ESICM guidelines and two methodologists who participated in the evidence review completed on behalf of the International Liaison Committee on Resuscitation (ILCOR) of whom ERC is a member society. We followed the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) approach to assess the certainty of evidence and grade recommendations. The panel provided suggestions on guideline implementation and identified priorities for future research. The certainty of evidence ranged from moderate to low. In patients who remain comatose after cardiac arrest, we recommend continuous monitoring of core temperature and actively preventing fever (defined as a temperature > 37.7 degrees C) for at least 72 hours. There was insufficient evidence to recommend for or against temperature control at 32-36 degrees C or early cooling after cardiac arrest. We recommend not actively rewarming comatose patients with mild hypothermia after return of spontaneous circulation (ROSC) to achieve normothermia. We recommend not using prehospital cooling with rapid infusion of large volumes of cold intravenous fluids immediately after ROSC.Peer reviewe