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Abstract: 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-1β, 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-1β 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. [Copyright &y& Elsevier]

Neuro-cognitive disabilities are a well-recognized complication of hypothermic circulatory arrest. We and others have reported that prolonged cardiac arrest (CA) produces neuronal death and microglial proliferation and activation that are only partially mitigated by hypothermia. Microglia, and possibly other cells, are suggested to elaborate tumor necrosis factor alpha (TNF-α), which can trigger neuronal death cascades and exacerbate edema after CNS insults. Minocycline is neuroprotective in some brain ischemia models in part by blunting the microglial response. We tested the hypothesis that minocycline would attenuate neuroinflammation as reflected by brain tissue levels of TNF-α after hypothermic CA in rats. Rats were subjected to rapid exsanguination, followed by a 6min normothermic CA. Hypothermia (30°C) was then induced by an aortic saline flush. After a total of 20min CA, resuscitation was achieved via cardiopulmonary bypass (CPB). After 5min reperfusion, minocycline (90mgkg−1; n =6) or vehicle (PBS; n =6) was given. Hypothermia (34°C) was maintained for 6h. Rats were sacrificed at 6 or 24h. TNF-α was quantified (ELISA) in four brain regions (cerebellum, CEREB; cortex, CTX; hippocampus, HIP; striatum, STRI). Naïve rats (n =6) and rats subjected to the same anesthesia and CPB but no CA served as controls (n =6). Immunocytochemistry was used to localize TNF-α. Naïve rats and CPB controls had no detectable TNF-α in any brain region. CA markedly increased brain TNF-α. Regional differences were seen, with the highest TNF-α levels in striatum in CA groups (10-fold higher, P <0.05 vs. all other brain regions). TNF-α was undetectable at 24h. Minocycline attenuated TNF-α levels in CTX, HIP and STRI (P <0.05). TNF-α showed unique co-localization with neurons. In conclusion, we report region-dependent early increases in brain TNF-α levels after prolonged hypothermic CA, with maximal increases in striatum. Surprisingly, TNF-α co-localized in neurons and not microglia. Minocycline attenuated TNF-α by approximately 50% but did not totally ablate its production. That minocycline decreased brain TNF-α levels suggests that it may represent a therapeutic adjunct to hypothermia in CA neuroprotection. University of Pittsburgh IACUC 0809278B-3. [ABSTRACT FROM AUTHOR]

Abstract: Trauma patients who suffer cardiac arrest (CA) from exsanguination rarely survive. Emergency preservation and resuscitation using hypothermia was developed to buy time for resuscitative surgery and delayed resuscitation with cardiopulmonary bypass (CPB), but intact survival is limited by neuronal death associated with microglial proliferation and activation. Pharmacological modulation of microglia may improve outcome following CA. Systemic injection of liposome-encapsulated clodronate (LEC) depletes macrophages. To test the hypothesis that intrahippocampal injection of LEC would attenuate local microglial proliferation after CA in rats, we administered LEC or PBS into the right or left hippocampus, respectively. After rapid exsanguination and 6min no-flow, hypothermia was induced by ice-cold (IC) or room-temperature (RT) flush. Total duration of CA was 20min. Pre-treatment (IC, RTpre) and post-treatment (RTpost) groups were studied, along with shams (cannulation only) and CPB controls. On day 7, shams and CPB groups showed neither neuronal death nor microglial activation. In contrast, the number of microglia in hippocampus in each individual group (IC, RTpre, RTpost) was decreased with LEC vs. PBS by ∼34–46% (P <0.05). Microglial proliferation was attenuated in the IC vs. RT groups (P <0.05). Neuronal death did not differ between hemispheres or IC vs. RT groups. Thus, intrahippocampal injection of LEC attenuated microglial proliferation by ∼40%, but did not alter neuronal death. This suggests that microglia may not play a pivotal role in mediating neuronal death in prolonged hypothermic CA. This novel strategy provides us with a tool to study the specific effects of microglia in hypothermic CA. [Copyright &y& Elsevier]

Abstract: Both ventricular fibrillation cardiac arrest (VFCA) and asphyxial cardiac arrest (ACA) are frequent causes of CA. However, only isolated reports compared cerebral blood flow (CBF) reperfusion patterns after different types of CA, and even fewer reports used methods that allow serial and regional assessment of CBF. We hypothesized that the reperfusion patterns of CBF will differ between individual types of experimental CA. In a prospective block-randomized study, fentanyl-anesthetized adult rats were subjected to 8min VFCA or ACA. Rats were then resuscitated with epinephrine, bicarbonate, manual chest compressions and mechanical ventilation. After the return of spontaneous circulation, CBF was then serially assessed via arterial spin-labeling magnetic resonance imaging (ASL-MRI) in cortex, thalamus, hippocampus and amygdala/piriform complex over 1h resuscitation time (RT). Both ACA and VFCA produced significant temporal and regional differences in CBF. All regions in both models showed significant changes over time (p <0.01), with early hyperperfusion and delayed hypoperfusion. ACA resulted in early hyperperfusion in cortex and thalamus (both p <0.05 vs. amygdala/piriform complex). In contrast, VFCA induced early hyperperfusion only in cortex (p <0.05 vs. other regions). Hyperperfusion was prolonged after ACA, peaking at 7min RT (RT7; 199% vs. BL, Baseline, in cortex and 201% in thalamus, p <0.05), then returning close to BL at ∼RT15. In contrast, VFCA model induced mild hyperemia, peaking at RT7 (141% vs. BL in cortex). Both ACA and VFCA showed delayed hypoperfusion (ACA, ∼30% below BL in hippocampus and amygdala/piriform complex, p <0.05; VFCA, 34–41% below BL in hippocampus and amygdala/piriform complex, p <0.05). In conclusion, both ACA and VFCA in adult rats produced significant regional and temporal differences in CBF. In ACA, hyperperfusion was most pronounced in cortex and thalamus. In VFCA, the changes were more modest, with hyperperfusion seen only in cortex. Both insults resulted in delayed hypoperfusion in all regions. Both early hyperperfusion and delayed hypoperfusion may be important therapeutic targets. This study was approved by the University of Pittsburgh IACUC 1008816-1. [Copyright &y& Elsevier]

Background: Evaluating beneficial effects of potential protective therapies following cardiac arrest in rodent models could be enhanced by exploring behavior and cognitive functions. The Morris Water Maze is a well-known cognitive paradigm to test spatial learning and memory. Results: Behavioral testing with the Morris Water Maze in Sprague-Dawley rats (300 ± 25 g) resuscitated after 8 min of ventricular fibrillation cardiac arrest was carried out 5 and 12 weeks after cardiac arrest (CA) and compared to results of naïve rats (Control). At 5 weeks, within each group latency time to reach the hidden platform (reflecting spatial learning) decreased equally from day 1 to 4 (CA: 105.6 ± 8.2 vs. 8.9 ± 1.2 s, p < 0.001; Control: 75.5 ± 13.2 vs. 17.1 ± 4.5, p < 0.001) with no differences between groups (p = 0.138). In the probe trial 24 h after the last trial, time spent in the target sector (reflecting memory recall) within each group was significantly longer (CA: 25 ± 1.3; Control: 24.7 ± 2.5 s) than in each of the three other sectors (CA: 7.7 ± 0.7, 14.3 ± 2.5, 8.4 ± 0.8 and Control: 7.8 ± 1.2, 11.7 ± 1.5, 10.3 ± 1.6 s) but with no significantly differences between groups. Seven days later (reflecting memory retention), control group animals remained significantly longer in the target sector compared to every other sector, whereas the cardiac arrest group animals did not. Even 12 weeks after cardiac arrest, the single p values showed that the control animals displayed a trend to perform better than the resuscitated animals. Conclusions: Memory recall was impaired early after 8 min of ventricular fibrillation cardiac arrest and might be a more valuable tool for cognitive testing than learning recall after global ischemia due to cardiac arrest. [ABSTRACT FROM AUTHOR]