Differential Effects of Targeted Temperature Management on Sex-Dependent Outcomes After Experimental Asphyxial Cardiac Arrest.

Asphyxial cardiac arrest (ACA) survivors face lasting neurological disability from hypoxic ischemic brain injury. Sex differences in long-term outcomes after cardiac arrest (CA) are grossly understudied and underreported. We used rigorous targeted temperature management (TTM) to understand its influence on survival and lasting sex-specific neurological and neuropathological outcomes in a rodent ACA model. Adult male and female rats underwent either sham or 5-minute no-flow ACA with 18 hours TTM at either ∼37°C (normothermia) or ∼36°C (mild hypothermia). Survival, temperature, and body weight (BW) were recorded over the 14-day study duration. All rats underwent neurological deficit score (NDS) assessment on days 1-3 and day 14. Hippocampal pathology was assessed for cell death, degenerating neurons, and microglia on day 14. Although ACA females were less likely to achieve return of spontaneous circulation (ROSC), post-ROSC physiology and biochemical profiles were similar between sexes. ACA females had significantly greater 14-day survival, NDS, and BW recovery than ACA males at normothermia (56% vs. 29%). TTM at 36°C versus 37°C improved 14-day survival in males, producing similar survival in male (63%) versus female (50%). There were no sex or temperature effects on CA1 histopathology. We conclude that at normothermic conditions, sex differences favoring females were observed after ACA in survival, NDS, and BW recovery. We achieved a clinically relevant ACA model using TTM at 36°C to improve long-term survival. This model can be used to more fully characterize sex differences in long-term outcomes and test novel acute and chronic therapies.

FGF21 modulates hippocampal cold-shock proteins and CA2-subregion proteins in neonatal mice with hypoxia-ischemia.

BACKGROUND: Fibroblast growth factor 21 (FGF21) is a neuroprotectant with cognitive enhancing effects but with poorly characterized mechanism(s) of action, particularly in females. Prior studies suggest that FGF21 may regulate cold-shock proteins (CSPs) and CA2-marker proteins in the hippocampus but empirical evidence is lacking. METHODS: We assessed in normothermic postnatal day (PND) 10 female mice, if hypoxic-ischemic (HI) brain injury (25 min 8% O2/92% N2) altered endogenous levels of FGF21 in serum or in the hippocampus, or its receptor ß-klotho. We also tested if systemic administration of FGF21 (1.5 mg/kg) modulated hippocampal CSPs or CA2 proteins. Finally, we measured if FGF21 therapy altered markers of acute hippocampal injury. RESULTS: HI increased endogenous serum FGF21 (24 h), hippocampal tissue FGF21 (4d), and decreased hippocampal ß-klotho levels (4d). Exogenous FGF21 therapy modulated hippocampal CSP levels, and dynamically altered hippocampal CA2 marker expression (24 h and 4d). Finally, FGF21 ameliorated neuronal damage markers at 24 h but did not affect GFAP (astrogliosis) or Iba1 (microgliosis) levels at 4d. CONCLUSIONS: FGF21 therapy modulates CSP and CA2 protein levels in the injured hippocampus. These proteins serve different biological functions, but our findings suggest that FGF21 administration modulates them in a homeostatic manner after HI. IMPACT: Hypoxic-ischemic (HI) injury in female post-natal day (PND) 10 mice decreases hippocampal RNA binding motif 3 (RBM3) levels in the normothermic newborn brain. HI injury in normothermic newborn female mice alters serum and hippocampal fibroblast growth factor 21 (FGF21) levels 24 h post-injury. HI injury in normothermic newborn female mice alters hippocampal levels of N-terminal EF-hand calcium binding protein 2 (NECAB2) in a time-dependent manner. Exogenous FGF21 therapy ameliorates the HI-mediated loss of hippocampal cold-induced RNA-binding protein (CIRBP). Exogenous FGF21 therapy modulates hippocampal levels of CA2-marker proteins after HI.

PHLPP Inhibitor NSC74429 Is Neuroprotective in Rodent Models of Cardiac Arrest and Traumatic Brain Injury.

Pleckstrin homology domain and leucine rich repeat protein phosphatase (PHLPP) knockout mice have improved outcomes after a stroke, traumatic brain injury (TBI), and decreased maladaptive vascular remodeling following vascular injury. Thus, small-molecule PHLPP inhibitors have the potential to improve neurological outcomes in a variety of conditions. There is a paucity of data on the efficacy of the known experimental PHLPP inhibitors, and not all may be suited for targeting acute brain injury. Here, we assessed several PHLPP inhibitors not previously explored for neuroprotection (NSC13378, NSC25247, and NSC74429) that had favorable predicted chemistries for targeting the central nervous system (CNS). Neuronal culture studies in staurosporine (apoptosis), glutamate (excitotoxicity), and hydrogen peroxide (necrosis/oxidative stress) revealed that NSC74429 at micromolar concentrations was the most neuroprotective. Subsequent testing in a rat model of asphyxial cardiac arrest, and in a mouse model of severe TBI, showed that serial dosing of 1 mg/kg of NSC74429 over 3 days improved hippocampal survival in both models. Taken together, NSC74429 is neuroprotective across multiple insult mechanisms. Future pharmacokinetic and pharmacodynamic (PK/PD) studies are warranted to optimize dosing, and mechanistic studies are needed to determine the percentage of neuroprotection mediated by PHLPP1/2 inhibition, or potentially from the modulation of PHLPP-independent targets.

Targeting TNFα-mediated cytotoxicity using thalidomide after experimental cardiac arrest in rats: An exploratory study.

Cardiac arrest (CA) results in a central and systemic cytokine and inflammatory response. Thalidomide has been reported to be neuroprotective by selectively decreasing TNFα synthesis. We hypothesized that thalidomide would decrease the systemic and organ-specific TNFα/cytokine response and biomarkers of injury in rats subjected to 10 min CA. Naïves, CA treated with vehicle (CA) and CA treated with thalidomide (50 mg/kg; CA+T) were studied (n=6 per group). TNFα and key cytokines were assessed at 3 h after resuscitation in the cortex, hippocampus, striatum, cerebellum, plasma, heart and lung. Neuron specific enolase (NSE), S100b, cardiac troponin T (cTnT) and intestinal fatty acid binding protein (IFABP) were used to assess neuronal, glial, cardiac and intestinal damage, respectively. CA increased TNFα and multiple pro-inflammatory cytokines in plasma and selected tissues with no differences between the CA and CA+T groups in any region. NSE, S100b, cTnT and IFABP were increased after CA or CA+T vs. in the naïve group (all P<0.05) without significant differences between the CA and CA+T groups. In conclusion, CA resulted in a TNFα and cytokine response, with increased biomarkers of organ injury. Notably, thalidomide at a dose reported to improve the outcome in in vivo models of brain ischemia did not decrease TNFα or cytokine levels in plasma, brain or extracerebral organs, or biomarkers of injury. Although CA at 3 h post resuscitation produces a robust TNFα response, it cannot be ruled out that an alternative dosing regimen or assessment at other time-points might yield different results. The marked systemic and regional cytokine response to CA remains a potential therapeutic target.

Hypoxia-ischemia-mediated effects on neurodevelopmentally regulated cold-shock proteins in neonatal mice under strict temperature control.

BACKGROUND: Neonates have high levels of cold-shock proteins (CSPs) in the normothermic brain for a limited period following birth. Hypoxic-ischemic (HI) insults in term infants produce neonatal encephalopathy (NE), and it remains unclear whether HI-induced pathology alters baseline CSP expression in the normothermic brain. METHODS: Here we established a version of the Rice-Vannucci model in PND 10 mice that incorporates rigorous temperature control. RESULTS: Common carotid artery (CCA)-ligation plus 25 min hypoxia (8% O2) in pups with targeted normothermia resulted in classic histopathological changes including increased hippocampal degeneration, astrogliosis, microgliosis, white matter changes, and cell signaling perturbations. Serial assessment of cortical, thalamic, and hippocampal RNA-binding motif 3 (RBM3), cold-inducible RNA binding protein (CIRBP), and reticulon-3 (RTN3) revealed a rapid age-dependent decrease in levels in sham and injured pups. CSPs were minimally affected by HI and the age point of lowest expression (PND 18) coincided with the timing at which heat-generating mechanisms mature in mice. CONCLUSIONS: The findings suggest the need to determine whether optimized therapeutic hypothermia (depth and duration) can prevent the age-related decline in neuroprotective CSPs like RBM3 in the brain, and improve outcomes during critical phases of secondary injury and recovery after NE. IMPACT: The rapid decrease in endogenous neuroprotective cold-shock proteins (CSPs) in the normothermic cortex, thalamus, and hippocampus from postnatal day (PND) 11-18, coincides with the timing of thermogenesis maturation in neonatal mice. Hypoxia-ischemia (HI) has a minor impact on the normal age-dependent decline in brain CSP levels in neonates maintained normothermic post-injury. HI robustly disrupts the expected correlation in RNA-binding motif 3 (RBM3) and reticulon-3 (RTN3). The potent neuroprotectant RBM3 is not increased 1-4 days after HI in a mouse model of neonatal encephalopathy (NE) in the term newborn and in which rigorous temperature control prevents the manifestation of endogenous post-insult hypothermia.

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.

Minocycline fails to improve neurologic and histologic outcome after ventricular fibrillation cardiac arrest in rats.

BACKGROUND: Prolonged cardiac arrest (CA) produces extensive neuronal death and microglial proliferation and activation resulting in neuro-cognitive disabilities. Among other potential mechanisms, microglia have been implicated as triggers of neuronal death after hypoxic-ischemic insults. Minocycline is neuroprotective in some brain ischemia models, either by blunting the microglial response or by a direct effect on neurons. AIM: To improve survival, attenuate neurologic deficits, neuroinflammation, and histological damage after ventricular fibrillation (VF) CA in rats. METHODS: Adult male isoflurane-anesthetized rats were subjected to 6 min VF CA followed by 2 min resuscitation including chest compression, epinephrine, bicarbonate, and defibrillation. After return of spontaneous circulation (ROSC), rats were randomized to two groups: (1) Minocycline 90 mg/kg intraperitoneally (i.p.) at 15 min ROSC, followed by 22.5 mg/kg i.p. every 12 h for 72 h; and (2) Controls, receiving the same volume of vehicle (phosphate-buffered saline). The rats were kept normothermic during the postoperative course. Neurologic injury was assessed daily using Overall Performance Category (OPC; 1 = normal, 5 = dead) and Neurologic Deficit Score (NDS; 0% = normal, 100% = dead). Rats were sacrificed at 72 h. Neuronal degeneration (Fluoro-Jade C staining) and microglia proliferation (anti-Iba-1 staining) were quantified in four selectively vulnerable brain regions (hippocampus, striatum, cerebellum, cortex) by three independent reviewers masked to the group assignment. RESULTS: In the minocycline group, 8 out of 14 rats survived to 72 h compared to 8 out of 19 rats in the control group (P = 0.46). The degree of neurologic deficit at 72 h [median, (interquartile range)] was not different between survivors in minocycline vs controls: OPC 1.5 (1-2.75) vs 2 (1.25-3), P = 0.442; NDS 12 (2-20) vs 17 (7-51), P = 0.328) or between all studied rats. The number of degenerating neurons (minocycline vs controls, mean ± SEM: Hippocampus 58 ± 8 vs 76 ± 8; striatum 121 ± 43 vs 153 ± 32; cerebellum 20 ± 7 vs 22 ± 8; cortex 0 ± 0 vs 0 ± 0) or proliferating microglia (hippocampus 157 ± 15 vs 193 cortex 0 ± 0 vs 0 ± 0; 16; striatum 150 ± 22 vs 161 ± 23; cerebellum 20 ± 7 vs 22 ± 8; cortex 26 ± 6 vs 31 ± 7) was not different between groups in any region (all P > 0.05). Numerically, there were approximately 20% less degenerating neurons and proliferating microglia in the hippocampus and striatum in the minocycline group, with a consistent pattern of histological damage across the individual regions of interest. CONCLUSION: Minocycline did not improve survival and failed to confer substantial benefits on neurologic function, neuronal loss or microglial proliferation across multiple brain regions in our model of rat VF CA.

Nitrite pharmacokinetics, safety and efficacy after experimental ventricular fibrillation cardiac arrest.

INTRODUCTION: Besides therapeutic hypothermia or targeted temperature management no novel therapies have been developed to improve outcomes of patients after cardiac arrest (CA). Recent studies suggest that nitrite reduces neurological damage after asphyxial CA. Nitrite is also implicated as a new mediator of remote post conditioning produced by tourniquet inflation-deflation, which is under active investigation in CA. However, little is known about brain penetration or pharmacokinetics (PK). Therefore, to define the optimal use of this agent, studies on the PK of nitrite in experimental ventricular fibrillation (VF) are needed. We tested the hypothesis that nitrite administered after resuscitation from VF is detectable in cerebrospinal fluid (CSF), brain and other organ tissues, produces no adverse hemodynamic effects, and improves neurologic outcome in rats. METHODS: After return of spontaneous circulation (ROSC) of 5 min untreated VF, adult male Sprague-Dawley rats were given intravenous nitrite (8 µM, 0.13 mg/kg) or placebo as a 5 min infusion beginning at 5 min after CA. Additionally, sham groups with and without nitrite treatment were also studied. Whole blood nitrite levels were serially measured. After 15 min, CSF, brain, heart and liver tissue were collected. In a second series, using a randomized and blinded treatment protocol, rats were treated with nitrite or placebo after arrest. Neurological deficit scoring (NDS) was performed daily and eight days after resuscitation, fear conditioning testing (FCT) and brain histology were assessed. RESULTS: In an initial series of experiments, rats (n = 21) were randomized to 4 groups: VF-CPR and nitrite therapy (n = 6), VF-CPR and placebo therapy (n = 5), sham (n = 5), or sham plus nitrite therapy (n = 5). Whole blood nitrite levels increased during drug infusion to 57.14 ±â€¯10.82 µM at 11 min post-resuscitation time (1 min after dose completion) in the VF nitrite group vs. 0.94 ±â€¯0.58 µM in the VF placebo group (p < 0.001). There was a significant difference between the treatment and placebo groups in nitrite levels in blood between 7.5 and 15 min after CPR start and between groups with respect to nitrite levels in CSF, brain, heart and liver. In a second series (n = 25 including 5 shams), 19 out of 20 animals survived until day 8. However, NDS, FCT and brain histology did not show any statistically significant difference between groups. CONCLUSIONS: Nitrite, administered early after ROSC from VF, was shown to cross the blood brain barrier after a 5 min VF cardiac arrest. We characterized the PK of intravenous nitrite administration after VF and were able to demonstrate nitrite safety in this feasibility study.

Phenotyping Cardiac Arrest: Bench and Bedside Characterization of Brain and Heart Injury Based on Etiology.

OBJECTIVES: Cardiac arrest etiology may be an important source of between-patient heterogeneity, but the impact of etiology on organ injury is unknown. We tested the hypothesis that asphyxial cardiac arrest results in greater neurologic injury than cardiac etiology cardiac arrest (ventricular fibrillation cardiac arrest), whereas ventricular fibrillation cardiac arrest results in greater cardiovascular dysfunction after return of spontaneous circulation. DESIGN: Prospective observational human and randomized animal study. SETTING: University laboratory and ICUs. PATIENTS: Five-hundred forty-three cardiac arrest patients admitted to ICU. SUBJECTS: Seventy-five male Sprague-Dawley rats. INTERVENTIONS: We examined neurologic and cardiovascular injury in Isoflurane-anesthetized rat cardiac arrest models matched by ischemic time. Hemodynamic and neurologic outcomes were assessed after 5 minutes no flow asphyxial cardiac arrest or ventricular fibrillation cardiac arrest. Comparison was made to injury patterns observed after human asphyxial cardiac arrest or ventricular fibrillation cardiac arrest. MEASUREMENTS AND MAIN RESULTS: In rats, cardiac output (20 ± 10 vs 45 ± 9 mL/min) and pH were lower and lactate higher (9.5 ± 1.0 vs 6.4 ± 1.3 mmol/L) after return of spontaneous circulation from ventricular fibrillation cardiac arrest versus asphyxial cardiac arrest (all p < 0.01). Asphyxial cardiac arrest resulted in greater early neurologic deficits, 7-day neuronal loss, and reduced freezing time (memory) after conditioned fear (all p < 0.05). Brain antioxidant reserves were more depleted following asphyxial cardiac arrest. In adjusted analyses, human ventricular fibrillation cardiac arrest was associated with greater cardiovascular injury based on peak troponin (7.8 ng/mL [0.8-57 ng/mL] vs 0.3 ng/mL [0.0-1.5 ng/mL]) and ejection fraction by echocardiography (20% vs 55%; all p < 0.0001), whereas asphyxial cardiac arrest was associated with worse early neurologic injury and poor functional outcome at hospital discharge (n = 46 [18%] vs 102 [44%]; p < 0.0001). Most ventricular fibrillation cardiac arrest deaths (54%) were the result of cardiovascular instability, whereas most asphyxial cardiac arrest deaths (75%) resulted from neurologic injury (p < 0.0001). CONCLUSIONS: In transcending rat and human studies, we find a consistent phenotype of heart and brain injury after cardiac arrest based on etiology: ventricular fibrillation cardiac arrest produces worse cardiovascular dysfunction, whereas asphyxial cardiac arrest produces worsened neurologic injury associated with greater oxidative stress.

Ventricular fibrillation cardiac arrest produces a chronic striatal hyperdopaminergic state that is worsened by methylphenidate treatment.

Cardiac arrest survival rates have improved with modern resuscitation techniques, but many survivors experience impairments associated with hypoxic-ischemic brain injury (HIBI). Currently, little is understood about chronic changes in striatal dopamine (DA) systems after HIBI. Given the common empiric clinical use of DA enhancing agents in neurorehabilitation, investigation evaluating dopaminergic alterations after cardiac arrest (CA) is necessary to optimize rehabilitation approaches. We hypothesized that striatal DA neurotransmission would be altered chronically after ventricular fibrillation cardiac arrest (VF-CA). Fast-scan cyclic voltammetry was used with median forebrain bundle (MFB) maximal electrical stimulations (60Hz, 10s) in rats to characterize presynaptic components of DA neurotransmission in the dorsal striatum (D-Str) and nucleus accumbens 14 days after a 5-min VF-CA when compared to Sham or Naïve. VF-CA increased D-Str-evoked overflow [DA], total [DA] released, and initial DA release rate versus controls, despite also increasing maximal velocity of DA reuptake (Vmax ). Methylphenidate (10 mg/kg), a DA transporter inhibitor, was administered to VF-CA and Shams after establishing a baseline, pre-drug 60 Hz, 5 s stimulation response. Methylphenidate increased initial evoked overflow [DA] more-so in VF-CA versus Sham and reduced D-Str Vmax in VF-CA but not Shams; these findings are consistent with upregulated striatal DA transporter in VF-CA versus Sham. Our work demonstrates that 5-min VF-CA increases electrically stimulated DA release with concomitant upregulation of DA reuptake 2 weeks after brief VF-CA insult. Future work should elucidate how CA insult duration, time after insult, and insult type influence striatal DA neurotransmission and related cognitive and motor functions.

Unique brain region-dependent cytokine signatures after prolonged hypothermic cardiac arrest in rats.

We previously showed that prolonged cardiac arrest (CA) produces neuronal death with microglial proliferation. Microglial proliferation, but not neuronal death, was attenuated by deeper hypothermia. Microglia are reportedly a major source of cytokines. In this study, we tested the hypotheses that (1) CA will result in highly specific regional and temporal increases in brain cytokines; and (2) these increases will be attenuated by deep hypothermia. Adult male Sprague-Dawley rats were subjected to rapid exsanguination. After 6 minutes of normothermic no-flow, different levels of hypothermia were induced by either ice-cold (IC) or room-temperature (RT) aortic flush. After 20 minutes CA, rats were resuscitated with cardiopulmonary bypass (CPB), and sacrificed at 6 or 24 hours. Rats subjected to CPB only (without CA) and shams (no CPB or CA) served as controls (n=6 per group). Cytokines were analyzed in cerebellum, cortex, hippocampus, and striatum. Immunofluorescence was used to identify cell types associated with individual cytokines. Intra-CA temperature was lower after IC versus RT flush (21°C vs. 28°C, p<0.05). At 6 hours, striatum showed a massive increase in interleukin (IL)-1α and tumor necrosis factor-alpha (TNF-α) (>100-fold higher than in hippocampus), which was attenuated by deeper hypothermia in the IC versus RT group. In contrast, IL-12 was 50-fold higher in hippocampus versus striatum. At 24 hours, cytokines decreased. In striatum, IL-1α colocalized with astrocytes while TNF-α colocalized with neurons. In hippocampus, IL-12 colocalized with hippocampal hilar neurons, the only region where neuronal degeneration was observed at 24 hours at both IC and RT groups. We report important temporo-spatial differences in the brain cytokine response to hypothermic CA, with a novel role of striatum. Astrocytes and neurons, but not microglia colocalized with individual cytokines. Hypothermia showed protective effects. These neuroinflammatory reactions precede neuronal death. New therapeutic strategies may need to target early regional neuroinflammation.

Global and regional differences in cerebral blood flow after asphyxial versus ventricular fibrillation cardiac arrest in rats using ASL-MRI.

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.

Minocycline attenuates brain tissue levels of TNF-α produced by neurons after prolonged hypothermic cardiac arrest in rats.

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 6 min normothermic CA. Hypothermia (30 °C) was then induced by an aortic saline flush. After a total of 20 min CA, resuscitation was achieved via cardiopulmonary bypass (CPB). After 5 min reperfusion, minocycline (90 mg kg−1; n = 6) or vehicle (PBS; n = 6) was given. Hypothermia (34 °C) was maintained for 6 h. Rats were sacrificed at 6 or 24 h. 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 24 h. 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.

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.

Extracorporeal versus conventional cardiopulmonary resuscitation after ventricular fibrillation cardiac arrest in rats: a feasibility trial.

OBJECTIVES: Extracorporeal cardiopulmonary resuscitation with cardiopulmonary bypass potentially provides cerebral reperfusion, cardiovascular support, and temperature control for resuscitation from cardiac arrest. We hypothesized that extracorporeal cardiopulmonary resuscitation is feasible after ventricular fibrillation cardiac arrest in rats and improves outcome versus conventional cardiopulmonary resuscitation. DESIGN: Prospective randomized study. SETTING: University laboratory. SUBJECTS: Adult male Sprague-Dawley rats. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Rats (intubated, instrumented with arterial and venous catheters and cardiopulmonary bypass cannulae) were randomized to conventional cardiopulmonary resuscitation, extracorporeal cardiopulmonary resuscitation with/without therapeutic hypothermia, or sham groups. After 6 minutes of ventricular fibrillation cardiac arrest, resuscitation was performed with drugs (epinephrine, sodium bicarbonate, and heparin), ventilation, either cardiopulmonary resuscitation or extracorporeal cardiopulmonary resuscitation, and defibrillation. Temperature was maintained at 37.0°C or 33.0°C for 12 hours after restoration of spontaneous circulation. Neurologic deficit scores, overall performance category, histological damage scores (viable neuron counts in CA1 hippocampus at 14 days; % of sham), and microglia proliferation and activation (Iba-1 immunohistochemistry) were assessed. RESULTS: Extracorporeal cardiopulmonary resuscitation induced hypothermia more rapidly than surface cooling (p<0.05), although heart rate was lowest in the extracorporeal cardiopulmonary resuscitation hypothermia group (p<0.05). Survival, neurologic deficit scores, overall performance category, and surviving neurons in CA1 did not differ between groups. Hypothermia significantly reduced neuronal damage in subiculum and thalamus and increased the microglial response in CA1 at 14 days (all p<0.05). There was no benefit from extracorporeal cardiopulmonary resuscitation versus cardiopulmonary resuscitation on damage in any brain region and no synergistic benefit from extracorporeal cardiopulmonary resuscitation with hypothermia. CONCLUSIONS: In a rat model of 6-minute ventricular fibrillation cardiac arrest, cardiopulmonary resuscitation or extracorporeal cardiopulmonary resuscitation leads to survival with intact neurologic outcomes. Twelve hours of mild hypothermia attenuated neuronal death in subiculum and thalamus but not CA1 and, surprisingly, increased the microglial response. Resuscitation from ventricular fibrillation cardiac arrest and rigorous temperature control with extracorporeal cardiopulmonary resuscitation in a rat model is feasible, regionally neuroprotective, and alters neuroinflammation versus standard resuscitation. The use of experimental extracorporeal cardiopulmonary resuscitation should be explored using longer insult durations.

Microglial depletion using intrahippocampal injection of liposome-encapsulated clodronate in prolonged hypothermic cardiac arrest in rats.

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.

Intravenous hydrogen sulfide does not induce hypothermia or improve survival from hemorrhagic shock in pigs.

Several laboratory studies suggested that induced hypothermia during hemorrhagic shock improves survival. Inhaled hydrogen sulfide (H2S) induced hypothermia and decreased metabolism in mice and rats but not in piglets. We tested the hypothesis that i.v. H2S will induce hypothermia, reduce oxygen consumption (VO2), and improve outcome in prolonged hemorrhagic shock in pigs. We also assessed markers of organ injury (alanine aminotransferase, aspartate aminotransferase, creatine phosphokinase, creatinine, and troponin) and level of protein thiols to monitor H2S metabolism. In a prospective randomized study, pigs were subjected to volume-controlled hemorrhagic shock with limited fluid resuscitation to maintain MAP 30 mmHg or greater. The study group received infusion of H2S at 5 mg·kg·h; the control group received vehicle (n = 8 per group). Dose was based on the highest tolerated dose in pilot studies. Full resuscitation was initiated after 3 h. There were no differences in survival at 24 h between groups (2/8 in H2S vs. 3/8 in control group). Heart rate increased similarly during hemorrhagic shock in both groups. Cardiac output was better preserved in the delayed phase of hemorrhagic shock in the control group. Temperature and VO2 were similar in both groups during hemorrhagic shock and resuscitation. Markers of organ injury and protein thiols markedly increased in both groups with no differences between groups. In conclusion, we were not able to demonstrate the hypothermia-inducing effect or a reduction in VO2 from H2S infusion in our model of hemorrhagic shock in pigs. Our data mirror those seen in piglets and provide additional evidence of difficulty in translating the hypothermia effect of H2S to large animals in a clinically relevant postinsult paradigm.

Deep hypothermia attenuates microglial proliferation independent of neuronal death after prolonged cardiac arrest in rats.

INTRODUCTION: Conventional resuscitation of exsanguination cardiac arrest (CA) victims is generally unsuccessful. Emergency preservation and resuscitation is a novel approach that uses an aortic flush to induce deep hypothermia during CA, followed by delayed resuscitation with cardiopulmonary bypass. Minocycline has been shown to be neuroprotective across a number of brain injury models via attenuating microglial activation. We hypothesized that deep hypothermia and minocycline would attenuate neuronal death and microglial activation and improve outcome after exsanguination CA in rats. METHODS: Using isoflurane anesthesia, rats were subjected to a lethal hemorrhagic shock. After 5 min of no flow, hypothermia was induced with an aortic flush. Three groups were studied: ice-cold (IC) flush, room-temperature (RT) flush, and RT flush followed by minocycline treatment (RT-M). After 20 min of CA, resuscitation was achieved via cardiopulmonary bypass. Survival, Overall Performance Category (1 = normal, 5 = death), Neurologic Deficit Score (0%-10% = normal, 100% = max deficit), neuronal death (Fluoro-Jade C), and microglial proliferation (Iba1 immunostaining) in hippocampus were assessed at 72 h. RESULTS: Rats in the IC group had lower tympanic temperature during CA versus other groups (IC, 20.9 degrees C +/- 1.3 degrees C; RT, 28.4 degrees C +/- 0.6 degrees C; RT-M, 28.3 degrees C +/- 0.7 degrees C; P < 0.001). Although survival was similar in all groups (RT, 6/9; IC, 6/7; RT-M, 6/11), neurological outcome was better in the IC group versus other groups (Overall Performance Category: IC, 1 +/- 1; RT, 3 +/- 1; RT-M, 2 +/- 1; P < 0.05; Neurologic Deficit Score: IC, 8% +/- 9%; RT, 55% +/- 19%; RT-M, 27% +/- 16%; P < 0.05). Histological damage assessed in survivors showed selective neuronal death in CA1 and dentate gyrus, similar in all groups (P = 0.15). In contrast, microglial proliferation was attenuated in the IC group versus all other groups (P < 0.01). CONCLUSIONS: Deeper levels of hypothermia induced by the IC versus RT flush resulted in better neurological outcome in survivors. Surprisingly, deep hypothermia attenuated microglial activation but not hippocampal neuronal death. Minocycline had modest benefit on neurologic outcome in survivors but did not attenuate microglial activation in brain. Our findings suggest a novel effect of deep hypothermia on microglial proliferation during exsanguination CA.

Blood-brain barrier integrity in a rat model of emergency preservation and resuscitation.

UNLABELLED: Emergency Preservation and Resuscitation (EPR) represents a novel approach to treat exsanguination cardiac arrest (CA) victims, using an aortic flush to induce hypothermia during circulatory arrest, followed by delayed resuscitation with cardiopulmonary bypass (CPB). The status of the blood-brain barrier (BBB) integrity after prolonged hypothermic CA is unclear. The objective of this study was to assess BBB permeability in two EPR models in rats, associated with poor outcome. Rats subjected to traumatic brain injury (TBI) and naïve rats served as positive and negative controls, respectively. HYPOTHESIS: The BBB will be disrupted after TBI, but intact after prolonged hypothermic CA. METHODS: Four groups were studied: (1) EPR-IC (ice cold)-75 min CA at 15 degrees C; (2) EPR-RT (room temperature)-20 min CA at 28 degrees C; (3) TBI; (4) sham. Rats in EPR groups were subjected to rapid hemorrhage, followed by CA. Rats in the TBI group had a controlled cortical impact to the left hemisphere. Naïves were subjected to the same anesthesia and surgery. 1h after insult, rats were injected with Evans Blue (EB), a marker of BBB permeability for albumin. Rats were sacrificed after 5h and EB absorbance was quantified in brain samples. RESULTS: TBI produced an approximately 10-fold increase in EB absorbance in the left (injured) hemisphere vs. left hemisphere for all other groups (p=0.001). In contrast, EB absorbance in either EPR group did not differ from sham. CONCLUSION: BBB integrity to albumin is not disrupted early after resuscitation from prolonged CA treated with EPR. Neuroprotective adjuncts to hypothermia in this setting should focus on agents that penetrate the BBB. These findings also have implications for deep hypothermic circulatory arrest.

Protein nitration and poly-ADP-ribosylation in brain after rapid exsanguination cardiac arrest in a rat model of emergency preservation and resuscitation.

BACKGROUND: Emergency preservation and resuscitation (EPR) of 60 min in rats is achievable with favorable outcome, while 75 min is associated with substantial mortality and impaired neurological outcome in survivors. We hypothesized that 75 min but not 60 min of EPR would be associated with activation of two potential secondary injury cascades in brain as reflected by protein nitration and poly (ADP-ribose) polymerase (PARP) activation. METHODS: Rats were rapidly exsanguinated over 5 min. After 1 min of cardiac arrest (CA), rats were cooled to a target tympanic temperature of 15 degrees C. After either 60 min or 75 min of CA, resuscitation was achieved via cardiopulmonary bypass (CPB). Rats subjected to CPB only served as controls. Overall performance category (OPC) and neurologic deficit score (NDS) were assessed at 24 h. Protein nitration and poly-ADP-ribosylation were assessed by Western blotting and immunohistochemistry for 3-nitrotyrosine and poly-ADP ribose polymers, respectively, in multiple brain regions. RESULTS: Neurologic outcome was better in the 60 min vs. the 75 min EPR group (OPC, P<0.001; NDS, P=0.001). Densitometric analysis of the major 64 kD band showed that nitration and PARP activation were significantly increased in hippocampus, cortex and striatum in the 75 min EPR group vs. other groups. However, there were no differences in cerebellum. Analysis of the full protein spectrum showed significantly increased PARP activation only in hippocampus in the 75 min EPR group vs. other groups. CONCLUSIONS: Extending the duration of EPR beyond the limit that can yield favorable recovery in rats was associated with increased nitration and ribosylation of selected proteins in selectively vulnerable brain regions. The impact of these mechanisms on the outcome remains to be determined.