Cardiac Arrest Induced by Asphyxia Versus Ventricular Fibrillation Elicits Comparable Early Changes in Cytokine Levels in the Rat Brain, Heart, and Serum.

Background Current postresuscitative care after cardiac arrest (CA) does not address the cause of CA. We previously reported that asphyxial CA (ACA) and ventricular fibrillation CA (VFCA) elicit unique injury signatures. We hypothesized that the early cytokine profiles of the serum, heart, and brain differ in response to ACA versus VFCA. Methods and Results Adult male rats were subjected to 10 minutes of either ACA or VFCA. Naives and shams (anesthesia and surgery without CA) served as controls (n=12/group). Asphyxiation produced an ≈4-minute period of progressive hypoxemia followed by a no-flow duration of ≈6±1 minute. Ventricular fibrillation immediately induced no flow. Return of spontaneous circulation was achieved earlier after ACA compared with VFCA (42±18 versus 105±22 seconds; P<0.001). Brain cytokines in naives were, in general, low or undetectable. Shams exhibited a modest effect on select cytokines. Both ACA and VFCA resulted in robust cytokine responses in serum, heart, and brain at 3 hours. Significant regional differences pinpointed the striatum as a key location of neuroinflammation. No significant differences in cytokines, neuron-specific enolase, S100b, and troponin T were observed across CA models. Conclusions Both models of CA resulted in marked systemic, heart, and brain cytokine responses, with similar degrees of change across the 2 CA insults. Changes in cytokine levels after CA were most pronounced in the striatum compared with other brain regions. These collective observations suggest that the amplitude of the changes in cytokine levels after ACA versus VFCA may not mediate the differences in secondary injuries between these 2 CA phenotypes.

Regional TNFα mapping in the brain reveals the striatum as a neuroinflammatory target after ventricular fibrillation cardiac arrest in rats.

Cardiac arrest (CA) triggers neuroinflammation that could play a role in a delayed neuronal death. In our previously established rat model of ventricular fibrillation (VF) CA characterized by extensive neuronal death, we tested the hypothesis that individual brain regions have specific neuroinflammatory responses, as reflected by regional brain tissue levels of tumor necrosis factor (TNF)α and other cytokines. In a prospective study, rats were randomized to 6min (CA6), 8min (CA8) or 10min (CA10) of VF CA, or sham group. Cortex, striatum, hippocampus and cerebellum were evaluated for TNFα and interleukin (IL)-1α, IL-1ß, IL-2, IL-4, IL-6, IL-10, IL-12 and interferon gamma at 3h, 6h or 14 d after CA by ELISA and Luminex. Immunohistochemistry was used to determine the cell source of TNFα. CA resulted in a selective TNFα response with significant regional and temporal differences. At 3h after CA, TNFα-levels increased in all regions depending on the duration of the insult. The most pronounced increase was observed in striatum that showed 20-fold increase in CA10 vs. sham, and 3-fold increase vs. CA6 or CA8 group, respectively (p<0.01). TNFα levels in striatum decreased between 3h and 6h, but increased in other regions between 3h and 14 d. TNFα levels remained twofold higher in CA6 vs. shams across brain regions at 14 d (p<0.01). In contrast to pronounced TNFα response, other cytokines showed only a minimal increase in CA6 and CA8 groups vs. sham in all brain regions with the exception that IL-1ß increased twofold in cerebellum and striatum (p<0.01). TNFα colocalized with neurons. In conclusion, CA produced a duration-dependent acute TNFα response, with dramatic increase in the striatum where TNFα colocalized with neurons. Increased TNFα levels persist for at least two weeks. This TNFα surge contrasts the lack of an acute increase in other cytokines in brain after CA. Given that striatum is a selectively vulnerable brain region, our data suggest possible role of neuronal TNFα in striatum after CA and identify therapeutic targets for future experiments. This study was approved by the University of Pittsburgh IACUC 1002340A-3.

Pharmacological inhibition of pleckstrin homology domain leucine-rich repeat protein phosphatase is neuroprotective: differential effects on astrocytes.

Pleckstrin homology domain and leucine-rich repeat protein phosphatase 1 (PHLPP1) inhibits protein kinase B (AKT) survival signaling in neurons. Small molecule pan-PHLPP inhibitors (selective for PHLPP1 and PHLPP2) may offer a translatable method to induce AKT neuroprotection. We tested several recently discovered PHLPP inhibitors (NSC117079 and NSC45586; benzoic acid, 5-[2-[4-[2-(2,4-diamino-5-methylphenyl)diazenyl]phenyl]diazenyl]-2-hydroxy-,sodium salt.) in rat cortical neurons and astrocytes and compared the biochemical response of these agents with short hairpin RNA (shRNA)-mediated PHLPP1 knockdown (KD). In neurons, both PHLPP1 KD and experimental PHLPP inhibitors activated AKT and ameliorated staurosporine (STS)-induced cell death. Unexpectedly, in astrocytes, both inhibitors blocked AKT activation, and NSC117079 reduced viability. Only PHLPP2 KD mimicked PHLPP inhibitors on astrocyte biochemistry. This suggests that these inhibitors could have possible detrimental effects on astrocytes by blocking novel PHLPP2-mediated prosurvival signaling mechanisms. Finally, because PHLPP1 levels are reportedly high in the hippocampus (a region prone to ischemic death), we characterized hippocampal changes in PHLPP and several AKT targeting prodeath phosphatases after cardiac arrest (CA)-induced brain injury. PHLPP1 levels increased in rat brains subjected to CA. None of the other AKT inhibitory phosphatases increased after global ischemia (i.e., PHLPP2, PTEN, PP2A, and PP1). Selective PHLPP1 inhibition (such as by shRNA KD) activates AKT survival signaling in neurons and astrocytes. Nonspecific PHLPP inhibition (by NSC117079 and NSC45586) only activates AKT in neurons. Taken together, these results suggest that selective PHLPP1 inhibitors should be developed and may yield optimal strategies to protect injured hippocampal neurons and astrocytes-namely from global brain ischemia.

Hemostasis in cardiac arrest patients treated with mild hypothermia initiated by cold fluids.

AIM OF THE STUDY: Application of mild hypothermia (32-33 degrees C) has been shown to improve neurological outcome in patients with cardiac arrest. However, hypothermia affects hemostasis, and even mild hypothermia is associated with bleeding and increased transfusion requirements in surgery patients. On the other hand, crystalloid hemodilution has been shown to induce a hypercoagulable state. The study aim was to elucidate in which way the induction of mild therapeutic hypothermia by a bolus infusion of cold crystalloids affects the coagulation system of patients with cardiac arrest. METHODS: This was a prospective pilot study in 18 patients with cardiac arrest and return of spontaneous circulation (ROSC). Mild hypothermia was initiated by a bolus infusion of cold 0.9% saline fluid (4 degrees C; 30ml/kg/30min) and maintained for 24h. At 0h (before hypothermia), 1, 6 and 24h we assessed coagulation parameters (PT, APPT), platelet count and performed thrombelastography (ROTEM) after in vitro addition of heparinase. RESULTS: A total amount of 2528 (+/-528)ml of 0.9% saline fluid was given. Hematocrit (p<0.01) and platelet count (-27%; p<0.05) declined, whereas APTT increased (2.7-fold; p<0.01) during the observation period. All ROTEM parameters besides clotting time (CT) after 1h (-20%; p<0.05) did not significantly change. CONCLUSION: Mild hypothermia only slightly prolonged clotting time as measured by rotation thrombelastography. Therefore, therapeutic hypothermia initiated by cold crystalloid fluids has only minor overall effects on coagulation in patients with cardiac arrest.

Pronounced platelet hyperfunction in patients with cardiac arrest achieving restoration of spontaneous circulation.

OBJECTIVE: Markers of platelet activation are increased in patients undergoing cardiopulmonary resuscitation. Hyperfunctional platelets may contribute to impairment of microcirculatory function and overall poor outcome despite restoration of spontaneous circulation (ROSC). Patients with myocardial infarction have hyperfunctional platelets, which predict the degree of myocardial necrosis. Thus, we hypothesized that platelets may be even more activated in patients whose myocardial infarction leads to cardiac arrest and compared them with patients whose cardiac arrest was due to a noncardiac origin. DESIGN: Prospective observational study. SETTING: Emergency department of a tertiary care hospital. PATIENTS: One hundred four patients with witnessed cardiac arrest who achieved ROSC. INTERVENTIONS: Blood sampling. MEASUREMENTS AND MAIN RESULTS: We assessed collagen adenosine diphosphate closure time with the platelet function analyzer-100, and measured plasma levels of von Willebrand factor: ristocetin cofactor activity levels by turbidometry. Independent physicians diagnosed the origin of cardiac arrest. The majority of cardiac arrests were caused by myocardial ischemia. Invariably, collagen adenosine diphosphate closure time values (55 seconds; 95% confidence interval: 52-58 seconds) were much shorter in these patients compared with patients with other causes of cardiac arrest (110 seconds; 95% confidence interval: 84-135 seconds, p < 0.001). von Willebrand factor: ristocetin cofactor activity plasma levels were more than three-fold above normal values in both groups. CONCLUSIONS: Patients with myocardial ischemia-triggered cardiac arrest had the highest degree of platelet hyperfunction under high shear rates, which was not solely due to increased von Willebrand factor. Future trials are necessary to clarify whether rapid, more aggressive antiplatelet therapy improves outcome after cardiac arrest.

Automated external defibrillators do not recommend false positive shocks under the influence of electromagnetic fields present at public locations.

Electromagnetic fields (EMF) reduce the signal quality of electrocardiograms and may lead to the misinterpretation by automated external defibrillators (AED). We designed this investigation as a prospective study, with a randomized sequence of AED applications on healthy volunteers. We chose busy public places where public access defibrillation was possible as test locations. Strong EMF were sought and found at train stations next to accelerating and decelerating trains. The primary outcome variable was the absolute number of shocks advised in the presence of sinus rhythm by five commonly used AED in Austria. For data analysis, the statistician was blinded in regard to the AED models tested. Data analysis was based on a per protocol evaluation. Of 390 tests run, 0 cases of false positive results occurred (95% CI: 0-0.77). AED can be regarded as safe, even with the interference of EMF present at train stations.