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Resuscitation 83 (2012) 1145– 1151

Contents lists available at SciVerse ScienceDirect

Resuscitation

jo u rn al hom epage : www.elsev ier .com/ locate / resusc i ta t ion

xperimental paper

annabinoid 1 (CB1) receptor mediates WIN55, 212-2 induced hypothermia andmproved survival in a rat post-cardiac arrest model�

inlun Wenga,c, Shijie Suna,b, Jeonghyun Parka, Sen Yea, Max Harry Weila,b, Wanchun Tanga,b,c,∗

Weil Institute of Critical Care Medicine, Rancho Mirage, CA, United StatesKeck School of Medicine of the University of Southern California, Los Angeles, CA, United StatesThe Sun Yat-Sen Memorial Hospital of Sun Yat-Sen University, Guangzhou, China

r t i c l e i n f o

rticle history:eceived 9 September 2011eceived in revised form3 December 2011ccepted 16 January 2012

eywords:annabinoid receptorardiopulmonary resuscitationypothermiayocardial–neurological function

a b s t r a c t

Aim: The nonselective Cannabinoid (CB) receptor agonist, WIN55, 212-2, was demonstrated to inducehypothermia and improve post-resuscitation outcomes in a rat post-cardiac arrest model. The presentstudy was to explore the potential mechanisms of WIN55, 212-2 on thermoregulation following resusci-tation and to investigate which class of CB receptors was involved in WIN55, 212-2-induced hypothermia.Methods: Ventricular fibrillation (VF) was induced and untreated for 6 min in 20 male Sprague-Dawley rats. Defibrillation was attempted after 8 min of Cardiopulmonary resuscitation (CPR). Five minpost-resuscitation, resuscitated animals were randomized to receive an intramuscular injection of selec-tive CB1 receptors antagonist, SR141716A (5 mg kg−1); selective CB2 receptors antagonist SR144528(5 mg kg−1); or placebo. Thirty min after injection, animals received continuous intravenous infusion ofWIN55, 212-2 (1.0 mg kg−1 h−1) for 4 h while control animals received placebo. The identical temperatureenvironment was maintained in all animals.Results: In animals treated with WIN55, 212-2, blood temperatures decreased progressively from 37 ◦Cto 34 ◦C within 4 h. This hypothermic effect was completely blocked by CB1 but not CB2 antagonist.Accordingly, significantly better cardiac output, ejection fraction and myocardial performance index,reduced neurological deficit scores, improved microcirculation and longer duration of survival were

observed in WIN55, 212-2-treated animals, which were also completely abolished by pretreatment withCB1 antagonist.Conclusions: Pharmacologically induced hypothermia with WIN55, 212-2 improved post-resuscitationmyocardial and cerebral function, associated with a significantly increased duration of survival in arat post-cardiac arrest model. The hypothermic and resulted beneficial effects of WIN55, 212-2 were

cepto

mediated through CB1 re

. Introduction

Though the initial success of cardiopulmonary resuscitationCPR) is as much as 40%, the majority of these initially resus-itated patients die in the hospital due to post-resuscitationyocardial and cerebral dysfunction, yielding a functional survival

ate of less than 10%.1,2 Both experimental and clinical stud-

es have demonstrated that therapeutic hypothermia is one ofhe post-resuscitation therapies to reduce neurological disabil-ty and improve survival after cardiac arrest.3–6 Current available

� A Spanish translated version of the summary of this article appears as Appendixn the final online version at doi:10.1016/j.resuscitation.2012.01.022.∗ Corresponding author at: Weil Institute of Critical Care Medicine, 35100 Bobope Drive, Rancho Mirage, CA 92270, United States. Tel.: +1 760 778 4911;

ax: +1 760 778 3468.E-mail address: drsheart@aol.com (W. Tang).

300-9572/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.resuscitation.2012.01.022

rs.© 2012 Elsevier Ireland Ltd. All rights reserved.

techniques for inducing hypothermia all use physical methods.These techniques are either less effective or limited to in-hospitaluse, such as, the physical dimensions of ice pads or blankets, orcomplexity of intravenous devices. Even the widely used infu-sion of chilled fluid requires additional methods to maintain mildhypothermia.7 These are key limitations that prevent the wide-spread adoption of early application of this life-saving intervention.

A constant “core” temperature is maintained through the tem-perature feedback regulating center located in the preoptic anteriorhypothalamus (POAH). Accordingly, pharmacological blockade ofthe temperature feedback regulating center may reduce the “core”temperature. The nonselective Cannabinoid (CB) receptors ago-nist, WIN55, 212-2, has been shown to induce dose-dependenthypothermia through the major CB1 receptors in POAH in nor-

mal rats. It has also been demonstrated to have a short meandistribution half-life (0.12 h) and long mean terminal elimina-tion half-life (4.93 h), supporting its rapid onset and long lastingeffect.9 Our preliminary study in a rat post-cardiac arrest model also

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146 Y. Weng et al. / Resusci

emonstrated that WIN55, 212-2 significantly induced mildypothermia, therefore improving outcomes of CPR.10

The present study extended our previous observations andested the hypothesis that CB1 receptors were involved primarilyn WIN55, 212-2-induced hypothermia and improved outcomes ofPR in a rat post-cardiac arrest model.

. Methods

All animals received humane care in compliance with the Guideor the Care and Use of Laboratory Animals.11 The protocol waspproved by the Institutional Animal Care and Use Committee ofhe Weil Institute of Critical Care Medicine.

.1. Animal preparation

Male Sprague-Dawley rats, weighing between 450 and 550 g,ere fasted overnight except for free access to water. The animalsere anesthetized by intraperitoneal injection of pentobarbital

45 mg kg−1) and additional doses (10 mg kg−1) were administeredt hourly intervals to maintain anesthesia. The trachea was orallyntubated with a 14-gauge cannula mounted on a blunt-tipped nee-le (Abbocath-T, Abbott Hospital Products Division, North Chicago,

L) with a 145◦ angled tip.12 End-tidal CO2 (ETCO2) was contin-ously measured with a side-stream infrared CO2 analyzer (200,

nstrumentation Laboratories, Lexington, MA) interposed betweenhe tracheal cannula and the ventilator. Three 23-gauge PE-50atheters (Becton-Dickinson, Franklin Lakes, NJ) were advancedhrough the left external jugular vein into the right atrium, throughhe left femoral artery into the descending aorta and through theeft femoral vein into the inferior vena cava for measurement ofight atrial pressures, aortic pressure, and the infusion of WIN55,12-2 or placebo. A thermocouple microprobe 0.5 mm in diam-ter (9030-12-D-34, Columbus Instruments, Columbus, OH) wasdvanced through right femoral artery into the descending aortaor measurement of blood temperature. A 3-Fr PE catheter (C-PMS-01J, Cook Critical Care, Bloomington, IN) was advanced throughhe right external jugular vein into the right atrium. A pre-curveduide wire supplied with the catheter was then advanced throughhe catheter into the right ventricle until endocardial electrocar-iogram was confirmed. All catheters were flushed intermittentlyith saline containing 2.5 IU mL−1 of crystalline bovine heparin. A

onventional electrocardiogram lead II was continuously recorded.

.2. Experimental procedures

Ventricular fibrillation (VF) was electrically induced with arogressive increase in 60-Hz current to a maximum of 3.5 mAelivered to the right ventricular endocardium. The current flowas continued for 3 min to prevent spontaneous defibrillation.echanical ventilation was discontinued after onset of VF. Pre-

ordial compression was begun after 6 min of untreated VF with pneumatically driven mechanical chest compressor as previouslyescribed.13 Coincident with the start of precordial compres-ion, mechanical ventilation was resumed at a tide volume of.65 mL/100 g of body weight and a frequency of 100/min with

FiO2 of 1.0. Precordial compression at a rate of 200/min wasynchronized to provide a compression-ventilation ratio of 2:1ith equal compression–relaxation duration. Depth of compres-

ion was adjusted to maintain a coronary perfusion pressure (CPP)f 22 ± 2 mm Hg. Resuscitation was attempted with up to three 2-J

ountershocks after 8 min of CPR. Resuscitation was defined as theeturn of supraventricular rhythm with a mean aortic pressure of0 mm Hg for a minimum of 5 min. No pharmacology agent wassed during CPR.

83 (2012) 1145– 1151

Resuscitated animals were randomized into 4 groups of 5: (1)control; (2) WIN55, 212-2 alone; (3) selective CB1 receptors antag-onist plus WIN55, 212-2; (4) selective CB2 receptors antagonistplus WIN55, 212-2. The principal investigator was blinded to therandomization.

The CB1 or 2 receptors antagonists, SR141716A or SR144528(Cayman Chemicals, Ann Arbor, MI), (5 mg kg−1, dissolved in0.1 mL dimethylsulfoxide), or vehicle placebo (dimethylsulfoxide),were injected intramuscularly at 5 min post-resuscitation. Thirtymin after injection, either 1.0 mg kg−1 h−1 of WIN55, 212-2 (Cay-man Chemicals, Ann Arbor, MI) or vehicle placebo (2% Tween-80in 0.9% NaCl solution) was intravenously infused at a rate of1.4 mL kg−1 h−1 for 4 h.

As demonstrated in our previous studies, the temperature in thecontrol group was titrated to maintain at 37.0 ± 0.2 ◦C with the aidof a heating lamp to prevent spontaneous hypothermia.10,14 Thisexternal warming was maintained consistent in all groups.

Following resuscitation, mechanical ventilation was continuedwith 100% oxygen for 1 h and 21% for the following 3 h. All catheterswere then removed after 4 h of infusion. The animals were thenreturned to their cages where the temperature was maintained at26 ◦C with a heated pet mat (Allied Precision Industries, Inc., Elburn,IL) and closely observed for 72 h, and then were euthanized withsodium pentobarbital (150 mg kg−1, IP).

An autopsy was performed to inspect for gross abnormalities,including evidence of traumatic injuries consequent to cannulation,airway management or precordial compression.

2.3. Measurements

Aortic and right atrial pressures, electrocardiogram, and ETCO2were continuously recorded for 4 h on a personal computer-based data-acquisition system supported by WINDAQ software(DATAQ, Akron, OH). CPP was calculated as the differencebetween decompression diastolic aortic and time-coincident rightatrial pressure measured at the end of each min of precordialcompression.

Myocardial function, including cardiac output (CO), ejectionfraction (EF) and myocardial performance index (MPI), wasassessed at baseline and hourly during infusion using echocardio-graphy (HD 11 XE, Philips Healthcare, Endover, MA). MPI, whichcombines time intervals related to systolic and diastolic func-tion and reflects the global cardiac function, was also calculatedusing the formula (a − b)/b, where a = mitral closure-to-openinginterval (time interval from cessation to onset of mitral inflow)and b = ET (aortic flow ejection time, obtained at the left ven-tricle outflow tract).15 EF served as an indicator of myocardialcontractility.

Systemic vascular resistance (SVR) was calculated using the for-mula: (mean artery pressure − right atrial pressure)/CO.

Sublingual microcirculations were measured at baseline andhourly during infusion using a side stream dark field imaging device(MicroScan, MicroVision Medical, Amsterdam, Netherlands).Microcirculation flow index (MFI) was utilized to level the micro-circulation from 0 (no flow), 1 (markedly reduced flow), 2 (reducedflow), to 3 (normal flow).16 Density of the small vessel (VDs) wasmeasured as proportional to the number of vessels crossing arbi-trary lines. The proportion of perfused small vessels (PPVs) wasdefined as the ratio of the number of perfused vessels to the totalvessels. Perfused vessel density (PVDs), an estimate of functional

Neurological deficit scores (NDS) ranging from 0 (normal) to500 (coma/death) was measured to assess levels of consciousness,brain stem function and overall performance at 24, 48 and 72 hpost-resuscitation.18

Y. Weng et al. / Resuscitation 83 (2012) 1145– 1151 1147

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Fig. 2. Cardiac output (CO), mL/min and Ejection fraction (EF), %. BL = baseline.PR = post-resuscitation. WIN = WIN55, 212-2. **p < 0.01 vs. control and

or SR144528-pretreated animals when compared to the other twogroups whether at 24, 48 or 72 h of post-resuscitation (p < 0.05)(Table 1).

ig. 1. Blood temperature, C. BL = baseline. PR = post-resuscitation. WIN = WIN55,12-2. **p < 0.01 vs. control and SR141716A + WIN55, 212-2. Based on one wayNOVA test and Bonferroni test.

.4. Statistical analysis

One way analysis of variance (ANOVA) was the primaryethod for quantitative data. Dependent variables were assumed

o be fixed to normal distribution and equal variances by theolmogorov–Smirno test and homogeneity of variance test. If theyere not fixed, the Kruskal–Wallis test was applied. If there was

ignificant difference in overall comparison of groups, compar-sons between any other two groups were made by the Bonferroniest. Comparisons between time-based measurements within eachroup were performed with repeated-measurement analysis ofariance. Survival rate was compared by Fisher’s exact probabil-ties test. All data were presented as mean ± SD. All the statisticalnalyses were performed by SPSS version 15.0 (SPSS, Chicago, IL).

value of p < 0.05 was considered significant.

. Results

Twenty-five rats were used for this study, five of which werexcluded because of instrumentation or technical failure. No dif-erences in baseline status were observed among the 4 groupsFigs. 1–3, Tables 1 and 2).

Blood temperature decreased progressively from 37.1 ± 0.1 ◦C to5.8 ± 0.3 ◦C during the first h of infusion and reached 33.9 ± 0.1 ◦Ct 3.5 h of infusion in animals treated with WIN55, 212-2 alone.emperature was maintained constant after 3.5 h of infusion, and ateast 5 h were needed to return to 37 ◦C after stopping infusion. Sim-lar reduction in temperature was observed in animals pretreated

ith SR144528. There was no significant reduction in blood tem-erature in animals pretreated with SR141716A, which was similaro the control animals (Fig. 1).

Mean aortic pressure (MAP) decreased by approximately2 mm Hg in WIN55, 212-2 group. Pretreatment with SR141716Aartially inhibited the hypotensive effect, thus decreasing the meanortic pressure approximately 15 mm Hg, while SR144528 did notlock the hypotension (Table 1).

Myocardial function was significantly impaired 20 min post-esuscitation in all animals. In animals treated with WIN55, 212-2lone or pretreated with SR144528, myocardial function was sig-ificantly improved with a recovery to within 80% of baseline in CO,F and MPI at 4 h of infusion, while no improvement was observedhen pretreated with SR141716A, resulting in similar myocar-ial function as the control group (WIN55, 212-2 vs control: CO,

2.8 ± 9.1 vs 68.0 ± 4.5, p < 0.01; EF, 62.6 ± 4.6 vs 44.8 ± 5.8, p < 0.01;PI, 0.78 ± 0.06 vs 1.28 ± 0.12, p < 0.01) (Figs. 2 and 3).Mild reduction in SVR was observed post-resuscitation in

IN55, 212-2 alone and SR144528 plus WIN55, 212-2 group, while

SR141716A + WIN55, 212-2. Based on one way ANOVA test and Bonferronitest.

a significant increase in SVR was observed in the control group(Fig. 3).

Significantly lower NDS was observed in WIN55, 212-2 alone

Fig. 3. Myocardial performance index (MPI) and systemic vascular resistance (SVR),mm Hg/L/min. BL = baseline. PR = post-resuscitation. WIN = WIN55, 212-2. **p < 0.01vs. control and SR141716A + WIN55, 212-2. Based on one way ANOVA test andBonferroni test.

1148 Y. Weng et al. / Resuscitation 83 (2012) 1145– 1151

Table 1Characteristics in body weight, PETCO2, uretic output, survival and NDS.

Control WIN55, 212-2 SR141716A + WIN55, 212-2 SR144528 + WIN55, 212-2

Body weight, ga 524 ± 11 525 ± 10 528 ± 5 527 ± 13ETCO2, mm Hga

Prior to VF 39.4 ± 1.8 40.2 ± 0.4 40.8 ± 1.8 40.0 ± 1.9Prior to infusion 42.4 ± 3.5 43.4 ± 3.0 43.4 ± 1.1 42.8 ± 4.6PR 4 h 35.2 ± 1.1 40.6 ± 4.3* 42.0 ± 3.5* 39.0 ± 4.3

Uretic outputb

PR 4 h 1.1 ± 1.0 2.4 ± 0.9*,# 1.5 ± 1.2 2.9 ± 1.6*,#

Survival durationb 43 ± 27 72 ± 0.0*,# 45 ± 27 63 ± 19.7*,#

Survival ratec

24 h 3/5 5/5 4/5 5/548 h 2/5 5/5 3/5 4/572 h 2/5 5/5 2/5 4/5

NDSb

24 h 314 ± 191 64 ± 28*,# 256 ± 134 105 ± 101*,#

48 h 338 ± 223 40 ± 31*,# 334 ± 228 134 ± 205*,#

72 h 324 ± 241 20 ± 28*,# 321 ± 245 120 ± 213*,#

MAPPrior to VF 138 ± 3 142 ± 4 141 ± 4 142 ± 4Prior to infusion 106 ± 6 107 ± 6 101 ± 7 105 ± 2PR 4 h 111 ± 7 89 ± 5**,# 98 ± 7 91 ± 5**

PR, post-resuscitation; NDS, neurological deficit score.a Based on ANOVA test and Bonferroni test.b Based on Kruskal–Wallis test and Bonferroni test.c Based on Fisher’s exact probabilities test and Bonferroni test.* p < 0.05.

** p < 0.01 vs. control group.

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# p < 0.05.#p < 0.01 vs. SR141716A + WIN55, 212-2.

All the animals treated with WIN55, 212-2 alone survived over2 h, and 4 of 5 animals pretreated with SR144528 survived over2 h with an average duration of survival of 63 ± 20 h. This con-rasted with the other two groups, in which 2 of 5 animals survivedor more than 72 h with an average duration of survival of 43 ± 27 hn the control group and 45 ± 27 h in the SR141716A plus WIN55,12-2 group (p < 0.05) (Table 1).

In control animals, microcirculation blood flow presented at aow-density and inadequate perfused state following resuscitation,

hich was also observed in SR141716A plus WIN55, 212-2 group.

owever, in the WIN55, 212-2 alone or pretreated with SR144528roups, there were significant improvements in MFI, VDs, PPVs, andVDs (WIN55, 212-2 vs. control: MFI, 2.28 ± 0.37 vs. 1.59 ± 0.32,

< 0.05; VDs, 8.58 ± 2.76 vs. 4.57 ± 2.14, p < 0.05; PPVs, 0.78 ± 0.18

able 2ublingual microcirculation variables.

Control WIN55, 212-2

MFIa

Prior to VF 3.0 ± 0.0 3.0 ± 0.0

PR 1 h 1.3 ± 0.3 1.2 ± 0.1#

PR 4 h 1.6 ± 0.3 2.3 ± 0.4*,##

VDs (mm−1)a

Prior to VF 8.2 ± 2.0 7.4 ± 0.9

PR 1 h 6.3 ± 3.0 7.0 ± 3.1

PR 4 h 4.6 ± 2.1 8.6 ± 2.8*,##

PPVs (%)a

Prior to VF 1.0 ± 0.0 0.9 ± 0.2

PR 1 h 0.3 ± 0.3 0.4 ± 0.2

PR 4 h 0.2 ± 0.3 0.8 ± 0.2**,##

PVDs (mm−1)a

Prior to VF 8.2 ± 2.0 6.2 ± 1.3

PR 1 h 1.3 ± 0.6 3.0 ± 1.4#

PR 4 h 1.1 ± 1.5 6.8 ± 3.3*,#

R, post-resuscitation; MFI, microcirculation flow index; VDs, small vessels density; PPVsa Based on ANOVA test and Bonferroni test.* p < 0.05.

** p < 0.01 vs. control group.# p < 0.05.

## p < 0.01 vs. SR141716A + WIN55, 212-2.

vs. 0.20 ± 0.28, p < 0.01; PVDs, 6.82 ± 3.33 vs. 1.06 ± 1.46, p < 0.05)(Fig. 4, Table 2).

No gross abnormalities were observed at necropsy in all animals.

4. Discussion

The present study demonstrated that the non-selectiveCB receptors agonist, WIN55, 212-2, induced mild hypother-mia. Therefore improving the post-resuscitation microcirculation,myocardial and neurological function together with prolonged

duration of survival in a rat model. These beneficial effects werecompletely blocked by CB1 receptors antagonist.

Pharmacological agents, such as 3-iodothyronamine, neu-rotensin, and hydrogen sulfide could induce hypothermia in rats,

SR141716A + WIN55, 212-2 SR144528 + WIN55, 212-2

3.0 ± 0.0 3.0 ± 0.01.1 ± 0.1 1.3 ± 0.4**,##

1.6 ± 0.3 2.0 ± 0.3*,#

9.2 ± 5.0 9.6 ± 3.46.2 ± 2.4 7.5 ± 2.44.7 ± 2.0 7.1 ± 0.6*,#

1.0 ± 0.0 1.0 ± 0.10.2 ± 0.2 0.4 ± 0.10.3 ± 0.1 0.8 ± 0.2*,##

9.0 ± 5.0 9.3 ± 2.80.9 ± 0.6 3.2 ± 1.4*,#

1.6 ± 1.1 5.4 ± 1.6**,##

, proportion of perfusion small vessels; PVDs, perfusion small vessels density.

Y. Weng et al. / Resuscitation 83 (2012) 1145– 1151 1149

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ig. 4. Sublingual microcirculation. BL = baseline. PR = post-resuscitation. WIN = Wesuscitation. (C) Control group at baseline. (D) Control group at 4 h post resuscitati

ut so far there were still lack of data scaling up the hypother-ic effect into large animals, therefore raising needs to explore

ther potential hypothermia-inducing agents.19–21 We previ-usly demonstrated that WIN55, 212-2 induced hypothermia andmproved outcomes of CPR in a rat model.10 This finding providedlternative means to facilitate or supplement already establishedethods of therapeutic hypothermia. However, the mechanisms byhich WIN55, 212-2-induced-hypothermia improved outcomes

f CPR remain to be investigated. Rawls et al. demonstrated thatn normal anesthetized rats, WIN55, 212-2-induced hypothermia

as associated with activation of CB1 receptors located mainlyn POAH, serving as the central locus for thermoregulation, orhose partially located in extra-hypothalamus sites by interact-ng with other hypothermia-inducing neurotransmitter systems.,22

SR141716A and SR144528 have high affinity to CB1 and 2eceptors with a Ki of 1.8 nM and 0.6 nM, respectively.23,24 Periph-ral administration of these two antagonists could take effect inentral nerve system because of their lipid solubility and penetra-ion across the blood–brain barrier.8,25 Based on previous studies,

mg kg−1 of SR141716A or SR144528 completely blocked the cen-ral effects of CB1 or 2 receptors with a long lasting (>12 h) effectn normal rats, respectively, supporting their effectiveness fornteraction with WIN55, 212-2 within duration of action.8,10 More-ver, neither SR141716A nor SR144528 altered body temperature,uggesting that the endocannabinoid system does not tonicallyodulate body temperature without cannabimimetic activity in

hermoregulation.8,26 This provides evidence that SR141716A andR144528 were valuable tools to investigate the role of CB1 and 2eceptors in the treatment of cardiac arrest with WIN55, 212-2.

Considering the potential compromising hemodynamics byingle high-dose injection, we adopted continuous intravenousnfusion of WIN55, 212-2 (1.0 mg kg−1 h−1), which differed fromhe Rawls’s study (5 mg kg−1). Based on the current method ofdministration, WIN55, 212-2 produced a rapid, progressive androlonged hypothermia, which correlated well with our previ-

us study.8 Similarly, SR141716A, but not SR144528 completelybolished the WIN55, 212-2-induced hypothermia, indicating thatB1 receptors mediated WIN55, 212-2-induced hypothermia post-esuscitation.

212-2. (A) WIN55, 212-2 group at baseline. (B) WIN55, 212-2 group at 4 h post

Under the circumstance of external warming, WIN55, 212-2 decreased blood temperature to 34 ◦C within 4 h and tookat least 5 h more to return to 37 ◦C after discontinuing infu-sion. Although there is overwhelming evidence indicating thatprompt initiation and prolonged hypothermia would augment theeffectiveness, Dietrich et al.27 demonstrated in rats that 3 h ofhypothermia (30 ◦C) after global brain ischemia still produced shortterm protection. However, long lasting protection was present inextended hypothermia of 32 ◦C from 12 to 24 h in gerbils and 32to 34 ◦C for 48 h in rats.28,29 Even spontaneous hypothermia, whichwas initiated after several hours and recovered to normothermiawithin 24–36 h, was found neuroprotective.30 Besides, the onsetof hypothermia was expected to accelerate if external warmingsetting was removed.

Consistently, when the hypothermic effect of WIN55, 212-2 was blocked by SR141716A, the other beneficial effects inmyocardial and neurological function no longer existed. However,SR144528 did not alter those effects of WIN55, 212-2. We there-fore hypothesized that the beneficial effects of WIN55, 212-2 onoutcomes of CPR was predominantly associated with therapeu-tic hypothermia. Though WIN55, 212-2 has been demonstrated tohave anti-inflammatory and anti-oxidative effects in inflammatoryor ischemia diseases independent of its hypothermic effects,31–33

we could not exclude other effects based on the current studydesign.

Our results indicated that WIN55, 212-2 produced a slightlydecreased MAP, which was partially blocked by SR141716A ratherthan SR144528. Niederhoffer et al.34 demonstrated four more car-diovascular regulatory mechanisms, namely: sympatho-inhibition,prejunctional inhibition of noradrenaline release from postgan-glionic sympathetic neurones, vagal cardiac efferent activation,or even central sympatho-excitation. The complex cardiovascu-lar effects of WIN55, 212-2 were influenced by the nature of theexperiment, the state of the experimental animals and the routeof administration. Our experimental protocol did not permit us toascertain which of the vasodilation mechanisms was responsible.

Physical hypothermia might cause reduction in cardiac output,increase in peripheral vasoconstriction and then systemic vascularresistance.35 However, the opposite phenomenon was observed inour study in which systemic vascular resistance was decreased with

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n increasing cardiac output following administration of WIN55,12-2. This is consistent with the finding that WIN55, 212-2 pro-uced peripheral vasodilation by activating CB1 receptors.36 In ourtudy, the slight reduction in blood pressure, heart rate and sys-emic vascular resistance may have positive effects since thosehanges decrease myocardial oxygen consumption, reduce after-oad, and improve tissue perfusion.

Fries et al. revealed that in a swine model of CPR, macrocircula-ion was highly correlated with microcirculation.37 WIN55, 212-2as found to mildly reduce blood pressure, which raised the need

o explore the effect of WIN55, 212-2 in microcirculation. In ourtudy, the mild reduction in blood pressure did not affect sublin-ual microcirculation, which reflected blood flow supplied by thearotid artery with the implication of being partially the source oferebral blood flow. Instead, microcirculation after administrationf WIN55, 212-2 was significantly increased when compared toontrol animals. Since hypothermia itself decreases microcircula-ion, the increased microcirculation following infusion of WIN55,12-2 may be the result of direct peripheral vasodilatation afterctivation of CB1 receptors.36

.1. Study limitations

This study has several limitations: (1) Our study was performedn rats free of heart disease, while cardiac arrest often occursn patients with heart disease; (2) Based on the current exper-ment protocol, it is unable to exclude the inherent protectiveffect of WIN55, 212-2 independent of hypothermia; (3) Becausef a smaller surface area to volume ratio in rats, it needs morexploration in larger mammals to scale up these findings frommall animals to larger mammals; (4) Toxicity analysis needs toe investigated before it is proposed for future human clinicalse.

. Conclusion

WIN55, 212-2 significantly induced hypothermia, and resultedn improved myocardial and neurological function, and survivaln a rat post-cardiac arrest model. The hypothermia and resultedeneficial effects of WIN55, 212-2 were mediated by the CB1 recep-ors. Our findings may provide an alternative option for earlynd effective induction of hypothermia after cardiac arrest byharmacological means alone or in combination with physicalooling.

onflict of interest statement

The authors have no conflicts of interest to declare.

cknowledgement

This study was supported in part by American Heart Associationrant 11IRG 4870001.

eferences

1. Oddo M, Ribordy V, Feihl F, et al. Early predictors of outcome in comatosesurvivors of ventricular fibrillation and non-ventricular fibrillation car-diac arrest treated with hypothermia: a prospective study. Crit Care Med2008;36:2296–301.

2. Aufderheide TP, Nichol G, Rea TD, et al. A trial of an impedance threshold devicein out-of-hospital cardiac arrest. N Engl J Med 2011;365:798–806.

3. Mild therapeutic hypothermia to improve the neurologic outcome after cardiac

arrest. N Engl J Med 2002;346:549–56.

4. Tsai MS, Barbut D, Tang W, et al. Rapid head cooling initiated coincident withcardiopulmonary resuscitation improves success of defibrillation and post-resuscitation myocardial function in a porcine model of prolonged cardiac arrest.J Am Coll Cardiol 2008;51:1988–90.

3

83 (2012) 1145– 1151

5. Bernard SA, Gray TW, Buist MD, et al. Treatment of comatose survivorsof out-of-hospital cardiac arrest with induced hypothermia. N Engl J Med2002;346:557–63.

6. Boddicker KA, Zhang Y, Zimmerman MB, Davies LR, Kerber RE. Hypothermiaimproves defibrillation success and resuscitation outcomes from ventricularfibrillation. Circulation 2005;111:3195–201.

7. Lampe JW, Becker LB. Rapid cooling for saving lives: a bioengineering opportu-nity. Expert Rev Med Devices 2007;4:441–6.

8. Rawls SM, Cabassa J, Geller EB, Adler MW. Cb1 receptors inthe preoptic anterior hypothalamus regulate win 55, 212-2[(4,5-dihydro-2-methyl-4(4-morpholinylmethyl)-1-(1-naphthalenyl-carbonyl)-6h-pyrrolo[3,2,1ij]quinolin-6-one]-induced hypothermia. JPharmacol Exp Ther 2002;301:963–8.

9. Valiveti S, Hammell DC, Earles DC, Stinchcomb AL. Transdermal delivery of thesynthetic cannabinoid win 55, 212-2: in vitro/in vivo correlation. Pharm Res2004;21:1137–45.

0. Sun S, Tang W, Song F, et al. Pharmacologically induced hypothermia withcannabinoid receptor agonist win55, 212-2 after cardiopulmonary resuscitation.Crit Care Med 2010;38:2282–6.

1. Institute of Laboratory Animal Resources (U.S.), ebrary Inc. Guide for the careand use of laboratory animals; 1996.

2. Stark RA, Nahrwold ML, Cohen PJ. Blind oral tracheal intubation of rats. J ApplPhysiol 1981;51:1355–6.

3. Sun S, Weil MH, Tang W, Kamohara T, Klouche K. Delta-opioid receptor agonistreduces severity of postresuscitation myocardial dysfunction. Am J Physiol HeartCirc Physiol 2004;287:H969–74.

4. Weng Y, Sun S, Song F, et al. Cholecystokinin octapeptide induces hypothermiaand improves outcomes in a rat model of cardiopulmonary resuscitation. CritCare Med 2011;39:2407–12.

5. Tei C. New non-invasive index for combined systolic and diastolic ventricularfunction. J Cardiol 1995;26:135–6.

6. Spronk PE, Ince C, Gardien MJ, et al. Nitroglycerin in septic shock after intravas-cular volume resuscitation. Lancet 2002;360:1395–6.

7. Rosenbluth MJ, Lam WA, Fletcher DA. Analyzing cell mechanics in hema-tologic diseases with microfluidic biophysical flow cytometry. Lab Chip2008;8:1062–70.

8. Hendrickx HH, Rao GR, Safar P, Gisvold SE. Asphyxia, cardiac arrestand resuscitation in rats. I: short term recovery. Resuscitation 1984;12:97–116.

9. Doyle KP, Suchland KL, Ciesielski TM, et al. Novel thyroxine deriva-tives, thyronamine and 3-iodothyronamine, induce transient hypother-mia and marked neuroprotection against stroke injury. Stroke 2007;38:2569–76.

0. Katz LM, Young A, Frank JE, Wang Y, Park K. Neurotensin-induced hypother-mia improves neurologic outcome after hypoxic-ischemia. Crit Care Med2004;32:806–10.

1. Knapp J, Heinzmann A, Schneider A, et al. Hypothermia and neuroprotectionby sulfide after cardiac arrest and cardiopulmonary resuscitation. Resuscitation2011;82:1076–80.

2. Nava F, Carta G, Gessa GL. Permissive role of dopamine d(2) receptors inthe hypothermia induced by delta(9)-tetrahydrocannabinol in rats. PharmacolBiochem Behav 2000;66:183–7.

3. Rinaldi-Carmona M, Barth F, Millan J, et al. Sr 144528, the first potent andselective antagonist of the cb2 cannabinoid receptor. J Pharmacol Exp Ther1998;284:644–50.

4. Rinaldi-Carmona M, Barth F, Heaulme M, et al. Sr 141716a, a potent andselective antagonist of the brain cannabinoid receptor. FEBS Lett 1994;350:240–4.

5. Rinaldi-Carmona M, Barth F, Heaulme M, et al. Biochemical and pharmacologicalcharacterisation of sr 141716a, the first potent and selective brain cannabinoidreceptor antagonist. Life Sci 1995;56:1941–7.

6. Compton DR, Aceto MD, Lowe J, Martin BR. In vivo characterization of aspecific cannabinoid receptor antagonist (sr 141716a): inhibition of delta9-tetrahydrocannabinol-induced responses and apparent agonist activity. JPharmacol Exp Ther 1996;277:586–94.

7. Dietrich WD, Busto R, Alonso O, Globus MY, Ginsberg MD. Intraischemic but notpostischemic brain hypothermia protects chronically following global forebrainischemia in rats. J Cereb Blood Flow Metab 1993;13:541–9.

8. Colbourne F, Corbett D. Delayed postischemic hypothermia: a six month sur-vival study using behavioral and histological assessments of neuroprotection. JNeurosci 1995;15:7250–60.

9. Colbourne F, Corbett D. Delayed and prolonged post-ischemic hypothermia isneuroprotective in the gerbil. Brain Res 1994;654:265–72.

0. Hickey RW, Ferimer H, Alexander HL, et al. Delayed, spontaneous hypothermiareduces neuronal damage after asphyxial cardiac arrest in rats. Crit Care Med2000;28:3511–6.

1. Pacher P, Hasko G. Endocannabinoids and cannabinoid receptors inischaemia-reperfusion injury and preconditioning. Br J Pharmacol 2008;153:252–62.

2. Chen Y, Chen SY, Wang Q, et al. Neuroprotective effect of preconditioning withcannabinoid receptor agonist win 55, 212-2 on focal cerebral ischemia: experi-

ment with rats. Zhonghua Yi Xue Za Zhi 2008;88:2219–22.

3. Fernandez-Lopez D, Martinez-Orgado J, Nunez E, et al. Characterization ofthe neuroprotective effect of the cannabinoid agonist win-55212 in anin vitro model of hypoxic-ischemic brain damage in newborn rats. Pediatr Res2006;60:169–73.

tation

3

3

36. Gardiner SM, March JE, Kemp PA, Bennett T. Regional haemodynamic responses

Y. Weng et al. / Resusci

4. Niederhoffer N, Hansen HH, Fernandez-Ruiz JJ, Szabo B. Effects of cannabi-

noids on adrenaline release from adrenal medullary cells. Br J Pharmacol2001;134:1319–27.

5. Nishimura Y, Naito Y, Nishioka T, Okamura Y. The effects of cardiac coolingunder surface-induced hypothermia on the cardiac function in the in situ heart.Interact Cardiovasc Thorac Surg 2005;4:101–5.

3

83 (2012) 1145– 1151 1151

to the cannabinoid agonist, win 55, 212-2, in conscious, normotensive rats, andin hypertensive, transgenic rats. Br J Pharmacol 2001;133:445–53.

7. Fries M, Weil MH, Chang YT, Castillo C, Tang W. Microcirculation during cardiacarrest and resuscitation. Crit Care Med 2006;34:S454–7.