a b s t r a c t

Background: The purpose of this study was to investigate the effect of prolonged cooling on cardiac and Cerebral injury in animals under cardiac arrest.

Methods: Adult male Wistar rats were equally randomized to normothermia, 5H1, 5H2, 7H1, 7H2, and 7H4 groups. The first number in the group name indicated ventricular fibrillation duration (minutes), the middle H indicated hypothermia, and the last number signified hypothermia duration (hours). Ventricular fibrillation was induced and untreated for 5 minutes (normothermia, 5H1, and 5H2) or 7 minutes (7H1, 7H2, and 7H4) followed by 1 minute of cardiopulmonary resuscitation followed by Electric shocks. Hypothermia was initiated simultaneously with cardiopulmonary resuscitation initiation and maintained for 1 hour (5H1 and 7H1), 2 hours (5H2 and 7H2) or 4 hours (7H4).

Results: There were 12 rats in each group. Compared with the 7H1 group, the 7H4 group had significantly better systolic function (dp/dt₄₀) and cardiac output within the early postcardiac arrest period. histologic examination disclosed less myocardial and hippocampal damage in the 7H4 group than the 7H1 group and in the 5H2 group than the 5H1 group. Plasma troponin I, fatty acid-binding protein, and S-100? concentrations were significantly lower in the 7H4 and 5H2 groups. The 7H4 and 5H2 groups survived statistically longer than the groups with shorter cooling duration.

Conclusion: Slightly prolonging hypothermia may mitigate myocardial and cerebral damage and improve survival and neurologic outcomes in a rat model of ventricular fibrillation cardiac arrest.

(C) 2015

1. Introduction

About postcardiac care, therapeutic hypothermia is the only therapy known to improve myocardial function, neurologic outcomes, and sur- vival. Based on 2 prospective randomized clinical trials demonstrated by Bernard et al [1] and the Hypothermia after Cardiac Arrest Study Group [2], the American Heart Association recommends cooling at 32?C to 34?C for 12 to 24 hours in unconscious ventricular fibrillation (VF) cardiac arrest survivors [3]. Recently, a randomized trial disclosed

? Disclosure of funding: The study was supported by a research funding from the Na- tional Taiwan University Hospital (Taipei, Taiwan) NTUH.102-S2132.

?? Conflict of interest: none.

? The study was performed at Department of Emergency Medicine, National Taiwan

University Medical College and Hospital, Taipei, Taiwan.

* Corresponding author at: Department of Emergency Medicine, National Taiwan Uni- versity Hospital, No. 7, Chung-Shan S. Road, Taipei, Taiwan, 100. Tel.: +886 2 23562831;

fax: +886 2 23223150.

E-mail address: [email protected] (W.-J. Chen).

no significant difference in survival and neurologic outcomes between patients with target temperature management at 33?C and 36?C [4] and raised several questions about the benefit of hypothermia. Un- doubtedly, target temperature management remains an important component in postcardiac arrest care [5].

Several animal studies have suggested that cooling should be initiated as early as possible, even during cardiopulmonary resuscitation (CPR) [6]. The beneficial effect of hypothermia disappears if cooling is initiated after 20 minutes of delay after return of spontaneous circulation (ROSC) and is maintained for less than 1 hour [7,8]. Prolonging hypothermia duration to 48 hours reportedly resulted in greater hippocampal neuron survival on the seventh day after ROSC compared with 24 hours [9]. However, several adverse effects may limit the Therapeutic effect of hypothermia including increased coagulation time, immunosuppression, increased infection, and altered drug pharmacokinetics and pharmacodynamics [10,11]. Ye et al

[12] demonstrated that postcardiac arrest cooling for 2 hours had equiv- alent or better tissue microcirculation, myocardial, and cerebral function than cooling for 5 or 8 hours.

http://dx.doi.org/10.1016/j.ajem.2015.07.030

0735-6757/(C) 2015

Biochemical parameters“>Whether slightly prolonging hypothermia duration could benefit cardiac and neurologic outcomes has not been clarified. In addition, whether hypothermia duration should be adjusted according to the du- ration of cardiac arrest to mitigate myocardial and cerebral damage also remains unclear. In the current study, we hypothesized that slight pro- longation of therapeutic hypothermia could improve VF Cardiac arrest outcomes, and we investigated the effect of prolonged cooling on cardi- ac and cerebral injury in animals receiving different cardiac arrest dura- tions to clarify the potential beneficial effect of prolonged cooling in prolonged cardiac arrest.

1. Materials and methods

The study was approved by the Animal Care and Use Committee of National Taiwan University. Male Wistar rats (14 weeks old) were anesthetized with an intraperitoneal sodium pentobarbital injection (50 mg/kg body weight). The animals were prepared as described pre- viously [13]. Before the experiment, body temperature was maintained at 37?C +- 0.5?C using an incandescent heating lamp.

• 1. Current-induced cardiac arrest animal model and experimental design

Ventricular fibrillation cardiac arrest was induced as described in de- tail previously [13,14]. After VF induction, the animals were equally ran- domized to normothermia (NormoT), 5H1, 5H2, 7H1, 7H2, and 7H4 groups (Fig. 1). The first number in the group name indicated VF dura- tion (minutes), the middle H indicated hypothermia, and, finally, the last number indicated hypothermia duration (hours). Ventricular fibril- lation was left untreated for 5 minutes in the NormoT, 5H1, and 5H2 groups or 7 minutes in the 7H1, 7H2, and 7H4 groups. After VF, chest compression (300 beats per minutes) and mechanical ventilation were delivered with synchronized CPR (compression/ventilation ratio, 3:1). After 1 minute of CPR, one 3-J monophasic electric shock was given followed by a sequence of 30 seconds of CPR and one 5-J electric shock. A maximum of 4 shocks were given in each resuscitation to achieve ROSC; otherwise, failure would be declared. Hypothermia was initiated with intravenous administration of 1-mL 4?C saline and main- tained as described in detail previously with a target temperature of 32?C. The target temperature was usually achieved within 30 minutes [14]. After ROSC, hypothermia was maintained for 1 hour in the 5H1 and 7H1 groups, 2 hours in the 5H2 and 7H2 groups, and 4 hours in

the 7H4 group. During the rewarming process, the temperature was kept at the rate of 0.5?C/h when intubated and rewarmed naturally after extubation with temperature monitored hourly. Body temperature of the NormoT group was kept at 37?C. There was a Sham group receiv- ing preparation but without inducing cardiac arrest and hypothermia. The successfully Resuscitated animals were closely monitored for 6 hours after ROSC and subsequently extubated. Survival was monitored hourly until 72 hours after ROSC, and mortality was verified by loss of heart beat and spontaneous respiratory movement for 2 minutes. Ani- mals that survived 72 hours were humanely sacrificed with an intraper- itoneal pentobarbital injection of 250 mg/kg for further histologic examinations.

• 1. Evaluation of myocardial functions

Left ventricle -positive dp/dt₄₀ and maximal LV-negative dp/dt (-dp/dt_max), blood pressure, central venous pressure, body temperature, and needle-probe electrocardiogram monitoring data were recorded with a personal computer-based data-acquisition system (ADInstruments, Sydney, Australia). The dp/dt₄₀ and -dp/dt_max reflected systolic and Diastolic function, respectively. Cardiac output (CO) was measured with 0.2-mL isotonic saline indicator that had been injected intravenously at Room temperature into the right atrium, and the temperature change was recorded by the thermodilution-tipped catheter (ADInstruments) in the abdominal aorta. Cardiac output was calculated using a Cardio- Max II computer (Columbus Instruments, Columbus, OH).

• 1. Biochemical parameters

Blood was sampled 2 hours after ROSC. Plasma cardiac troponin I, fatty acid-binding protein (FABP), and S-100? levels were measured using enzyme-linked immunosorbent assay kits (Life Diagnostics Inc, West Chester, PA; Hycult Biotechnology, Uden, Netherlands; Kamiya Biomedical Company, Seattle, WA), respectively, and the absorbance was measured at 450 nm.

• 1. Histologic examinations

To investigate the myocardial and cerebral damage, morphologic and histologic results of the hearts were examined at the end of the fourth and 72nd hours, respectively, and the brains were examined at

Fig. 1. Study design.

the end of the 72nd hour. Histologic examinations were performed by 2 independent pathologists who had been blinded to the groups. An inde- pendent assessment was undertaken by a third investigator, and the majority view was chosen if there were any discrepancies.

• • 1. Hematoxylin and eosin staining of the heart

The apex, septum, and lateral wall of the LV were selected. Myocytolysis was counted in 5 independent, randomly selected micro- scopic fields at x200 magnification in each specimen. Fibrosis was eval- uated as percent area in 5 independent, randomly selected microscopic fields at x40 magnification in each specimen. In total, 6 specimens were counted per animal.

• • 1. Histologic examination of the brain

To evaluate neuronal death, methyl green pyronine Y (MGPY) stain- ing, cresyl violet staining, and Fluoro-Jade C staining were used [15-17]. The brains were removed, embedded in paraffin, and cut into coronal sections (5 um for MGPY and cresyl violet staining and 10 um for Fluoro-Jade C staining) on a rotary microtome. The hippocampus was selected. In methyl green pyronine Y-stained sections, viable cells exhibited a light blue nucleus with intact nuclear integrity and a pink cytoplasm. In cresyl violet-stained sections, healthy cells had oval nuclei with prominent nucleoli lacking eosinophilic cytoplasm. In Fluoro-Jade C-stained sections, degenerating neurons and their den- drites exhibit green fluorescence, whereas healthy neurons are un- stained. Positive Fluoro-Jade C-stained neurons were counted in 3 independent, randomly selected microscopic fields at x200 magnification in the Cornu amonis (CA1, CA2, CA3), and dentate gyrus of each brain specimen. In total, 3 hippocampal specimens were counted per animal.

• • 1. Terminal deoxynucleotidyl transferase staining of heart and brain

DNA fragmentation was visualized with a DNA fragmentation detec- tion kit (colorimetric-terminal deoxynucleotidyl transferase enzyme) by fluorescence (Calbiochem, San Diego, CA) using 4?,6-diamidino-2- phenylindole as a nuclei counterstain [18]. Positive terminal deoxynucleotidyl transferase (TUNEL) staining cells were counted in 5 independent, randomly selected microscopic fields at x400 magnifica- tion in each heart specimen and 3 independent, randomly selected mi- croscopic fields at x 200 magnification in the CA1, CA2, CA3, and dendate gyrus of each brain specimen. In total, 6 myocardial specimens and 3 hippocampal specimens were counted per animal.

• 1. Evaluation of neurologic outcomes

Neurologic outcomes were evaluated at the end of the sixth, 24th, 48th, and 72nd hours after ROSC. As described in detail previously [14], neurologic function scoring included consciousness level, corneal reflex, respiration, righting reflex, coordination, and movement/activity. Assessments were performed independently by 2 investigators. Any discrepancies were resolved by an independent assessment by a third investigator, and the majority score was accepted. The neurologic out- come of the animals that died was scored 0. Neurologic function scoring greater than or equal to 10 was classified as a Good neurologic outcome.

• 1. Statistical analysis

Hypothesizing that prolonging hypothermia might increase the 72- hour survival rate from 10% in the 7H1 group to 60% in the 7H4 group, the required sample size to achieve 80% power at ? = .05 for correctly detecting such a difference was 12. Therefore, we used 12 animals for each group with block randomization. Binomial variables were analyzed with the Fisher exact test. Continuous values were presented as the mean +- SD. The difference in hemodynamics was analyzed by the gen- eral linear model. The significance of the differences in histologic analy- sis, biomarkers, and neurologic function scoring was evaluated with the Mann-Whitney U test. survival curves were determined by the Kaplan-

Meier method and compared using the log-rank test. P values b .05 were considered to be statistically significant. All of the statistical analyses were performed with SPSS 19.0 software (SPSS Inc, Chicago, IL).

1. Results

There were 72 rats for the survival study and an extra 28 rats for his- tologic examination. Of these, 10 of 12 (83%) animals in both the 5H1 and 5H2 groups got ROSC, and the ROSC rates in the 7H1, 7H2, 7H4, and NormoT groups were 58% (7/12), 67% (8/12), 67% (8/12), and 33%(4/12), respectively. The numbers of electric shocks and CPR dura- tion did not differ between 5H1 and 5H2 groups or among the 7H1, 7H2, and 7H4 groups (Table). The body temperatures at ROSC were

32.6?C +- 0.7?C in animals that received 5 minutes of VF and 32.8?C +- 0.9?C in animals that received 7 minutes of VF.

• 1. Prolonged hypothermia improved hemodynamics

At ROSC, the animals receiving 5 minutes of VF had better dp/dt₄₀ (4718.5 +- 1079.5 vs 3159.6 +- 1425.3 mm Hg/s, P b .01) and -dp/dt_max than those with 7 minutes of VF (-4300.7 +- 1333.0 vs -3282.8 +- 1712.0 mm Hg/s, P b .05). Within 6 hours, there was no significant dif- ference in dp/dt₄₀, -dp/dt_max, and CO between the 5H1 and 5H2 groups. When compared with the 7H1 group, the dp/dt₄₀ and CO in the 7H4 group improved significantly, as the cooling duration increased. The difference between the 7H4 and 5H2 groups in cardiac systolic function and CO reduced, as cooling duration increased (dp/dt₄₀, P =

.01 at ROSC; P = not significant at sixth hour; CO, P = .069 at ROSC; P = not significant at sixth hour) (Fig. 2).

• 1. Prolonged hypothermia decreased myocardial damage, apoptosis, and fibrosis

Compared with the sham group, the hematoxylin and eosin-stained LV sections at the fourth hour exhibited myocytolysis, waving and transverse contraction bands in all of the groups (Fig. 3A). The 5H2 group had less myocytolysis than the 5H1 group (P b .01). The 7H4 group also had reduced myocytolysis compared with the 7H1 and 7H2 groups (P b .01) (Fig. 3A and B). At the 72nd hour, there was significant- ly less myocardial fibrosis in the 5H2 group than the 5H1 group (P =

.001) and in the 7H4 group than the 7H2 group (P b .001) (Fig. 3A and

C). Using TUNEL staining as apoptosis markers, the 5H2 group demon- strated less myocardial apoptosis at the fourth hour than the 5H1 group (P = .027). There was also less myocardial apoptosis in the 7H4 group than the 7H2 group (P = .005) (Fig. 3A and D). There were no sig- nificant differences in myocytolysis, fibrosis, and apoptosis between the 5H1 and 7H2 groups or between the 5H2 and 7H4 groups.

• 1. Prolonged hypothermia reduced troponin I and FABP release

At the second hour, plasma troponin I and FABP concentrations were significantly lower in the 5H2 group than in the 5H1 group (troponin I, 4.18 +- 1.24 vs 7.32 +- 1.86 ng/mL; P = .001) (FABP, 4230 +- 1377 vs

5968 +- 1318 pg/mL; P = .003). Both the 7H2 and 7H4 groups had lower troponin I and FABP concentrations than the 7H1 group (Fig. 4A and B). There were no differences in troponin I and FABP levels between the 7H2 and 7H4 groups. Plasma troponin I and FABP concentrations did not differ between the 5H1 and 7H2 groups or between the 5H2 and 7H4 groups.

• 1. Prolonged hypothermia decreased hippocampus damage and apoptosis as well as S-100? release

At the 72nd hour, the 5H2 group demonstrated less neuronal death (P = .002) and apoptosis (P = .027) in the hippocampus compared with the 5H1 group. The 7H4 group had significantly less neuronal

Table

Baseline characteristics and Resuscitation events of survival study

5H1

5H2

7H1

7H2

7H4

NormoT

n = 12

n = 12

n = 12

n = 12

n = 12

n = 12

Baseline characteristics

Weight (g)

417.8 +- 62.4

421.7 +- 47.2

412.1 +- 48.3

434.4 +- 57.5

413.1 +- 53.8

417.8 +- 62.4

Heart rate (/min)

423.7 +- 30.3

416.5 +- 22.2

403.5 +- 43.7

415.0 +- 32.0

403.3 +- 36.5

409.1 +- 28.1

dp/dt 40 (x1000 mm Hg/s)

9.16 +- 0.93

9.46 +- 0.91

9.45 +- 1.17

9.19 +- 0.99

9.50 +-1.16

9.17 +- 1.17

-dp/dt max (x1000 mm Hg/s) Resuscitation events

-10.70 +- 2.37

-11.17 +- 1.46

-11.44 +- 1.59

-10.89 +- 1.26

-10.91 +- 2.06

-10.29 +- 2.15

ROSC (%)

10 (83.3%)a

10 (83.3%)a

7 (58.3%)

8 (66.7%)

8 (66.7%)

4 (33.3%)

CPR duration (s) in successfully resuscitated animals

85. 0 +- 15.0a

88.2 +- 18.0a

97.3 +- 18.0

105.8 +- 14.4

103.1 +- 23.3

115.0 +- 12.5

CPP (mm Hg) after 1 min of CPR

27.2 +- 6.9

26.5 +- 7.1

23.4 +- 7.2

23.4 +- 7.0

22.5 +- 8.3

21.6 +- 6.8

ETCO2 after 1 min of CPR

14.83 +- 4.39

15.47 +- 3.12a

13.59 +- 1.71

13.82 +- 2.20

12.94 +- 2.12

11.03 +- 1.35

Electric shock no. in successfully resuscitated animals

1.4 +- 0.5a

1.5 +- 0.7a

2.0 +- 0.8

2.1 +- 0.6

2.1 +- 0.8

2.5 +- 0.6

pH at ROSC

7.10 +- 0.14a

7.11 +- 0.10a

6.99 +- 0.13

7.01 +- 0.08

7.06 +- 0.10

6.98 +- 0.11

HCO3 (meq/L) at ROSC

15.00 +- 6.02a

15.22 +- 5.26a

9.84 +- 3.76

9.63 +- 3.89

9.73 +- 4.71

8.75 +- 6.40

pH at 2 h

7.26 +- 0.05a

7.26 +- 0.07a

7.14 +- 0.09

7.23 +- 0.04a

7.23 +- 0.05a

7.14 +- 0.08

HCO3 (meq/L) at 2 h

20.38 +- 0.51a

23.70 +- 3.95a

17.36 +- 5.44

20.13 +- 2.92a

20.25 +- 5.10a

14.35 +- 3.25

Abbreviations: CPP, coronary perfusion pressure; ETCO₂ = end-tidal CO₂; HCO₃, bicarbonate.

^a P b .05 vs the NormoT group.

death (P b .001) and apoptosis (P = .003) than the 7H2 groups (Fig. 5A- C). There were no significant differences in apoptosis between the 5H1 and 7H2 groups or between the 5H2 and 7H4 groups. The S-100? con- centration at the second hour was also significantly lower in the 5H2 group than in the 5H1 groups (1.68 +- 0.52 vs 2.31 +- 0.51 ng/mL, P =

.029). The 7H2 and 7H4 groups also had lower S-100? concentrations than the 7H1 group (Fig. 5D). No significant difference in S-100? was noted between the 7H2 and 7H4 groups. The S-100? concentration did not differ between the 5H1 and 7H2 groups or between the 5H2 and 7H4 groups.

• 1. Prolonged hypothermia improved survival and neurologic outcomes

Prolonged hypothermia groups such as 5H2 and 7H4 had statistical- ly longer survival durations than the groups with shorter cooling dura- tion. Of the animals, 6 of 12 in both the 5H2 and 7H4 groups survived 72 hours, whereas 3 of 12 animals in the 5H1 and 7H2 groups and only 1 of 12 animals in the 7H1 and NormoT groups survived (Fig. 6A). In total, 6 of 12 animals in the 5H2 and 7H4 groups had good neurologic outcomes at the 72nd hour, whereas only 3 of 12 animals in the 5H1 and 7H2 groups and only 1 of 12 animals in the 7H1 and NormoT groups had a

Fig. 2. Hemodynamics in the early postcardiac arrest period. There was no significant difference of dp/dt₄₀, -dp/dt_max, and CO between the 5H1 and 5H2 groups. When compared with the 7H1 group, the 7H4 group had significant better dp/dt₄₀ and CO but not -dp/dt_max. The dp/dt₄₀ and CO were better in the 5H2 group than in the 7H4 group, but the difference decreased along with the cooling duration increase.

Fig. 3. Histologic examinations of heart. A, Representative pictures of hematoxylin and eosin staining at fourth and 72nd hour and TUNEL staining at 72nd hour. A and B, The 5H2 group had less myocytolysis than the 5H1 group. The 7H4 group also had reduced myocytolysis when compared with the 7H1 and 7H2 groups. A, C, and D, At the 72nd hour, there was significant less myocardial fibrosis and apoptosis in the 5H2 group (compared with the 5H1 group) and in the 7H4 group (compared with the 7H2 group). There was no significant difference in myocytolysis, fibrosis, and apoptosis between 5H1 and 7H2 groups and between 5H2 and 7H4 groups. HE stain, hematoxylin and eosin stain. ?P b .05.

favorable outcome. The 7H4 groups had significantly better neurologic outcomes than the 7H1 groups at the sixth and 72nd hour after cardiac arrest (Fig. 6B).

1. Discussion

This study systematically evaluated whether mild prolongation of cooling duration mitigated postcardiac arrest damage and demonstrat- ed that slightly prolonging hypothermia could decrease myocardial and cerebral damage and improve myocardial function as well as neurologic outcomes and survival.

The suggested hypothermia duration for cardiac arrest was based on 2 major clinical trials in which cooling durations were 12 and 24 hours

[1,2]. However, the difference in hypothermia durations raised the question of what the optimal hypothermia duration might be. Several studies demonstrated superior protection when therapeutic hypother- mia was prolonged [1,2,19,20]. In local ischemic or hemorrhagic brain lesions, longer cooling may be required to maximally reduce the dam- Age SIze [21,22]. Better pyramidal neuronal survival in the hippocampus and greater cerebellar Purkinje cell density were reported in rats that were cooled for 48 hours than those cooled for 24 hours [9,23]. Howev- er, in an animal study in which cooling duration was prolonged from 2 to 8 hours, tissue microcirculation as well as myocardial and cerebral functions did not expand accordingly [12]. In our study, prolonging cooling duration slightly in animals with the same cardiac arrest dura- tion resulted in improved myocardial function and less myocardial

Fig. 4. Plasma troponin I and FABP concentrations. The plasma troponin I and FABP concentrations were lower in the 5H2 group than in the 5H1 group and lower in the 7H4 and 7H2 groups than the 7H1 group. The plasma troponin I and FABP concentrations did not differ between 5H1 and 7H2 groups and between 5H2 and 7H4 groups. ?P b .05.

Fig. 5. Histologic examinations of brain and plasma S-100? concentration. A, Representative pictures of MGPY, cresyl violet, Fluro-Jade C, and TUNEL stainings of hippocampus at 72nd hour. A and B, The 5H2 group showed less neuron death and apoptosis compared with the 5H1 group. The 7H4 group had significantly less neuron apoptosis than the 7H2 group. There was no significant difference in apoptosis between 5H1 and 7H2 groups and between 5H2 and 7H4 groups. C, The S-100? concentration in the 5H2 group was lower than in the

5H1 group. The 7H2 and 7H4 groups displayed lower S-100? concentration than the 7H1 group. The S-100? concentration did not differ between 5H1 and 7H2 groups and between 5H2 and 7H4 groups. ?P b .05.

Fig. 6. survival analysis and neurologic scaling scores. Compared with the 7H1 group, the 7H4 group had better survival rate at 72nd hour and neurologic outcomes at the sixth and 72nd hour. ?P b .05.

and cerebral damage as well as better neurologic outcomes and survival. Those findings were consistent in animals with both 5- and 7-minute VF durations.

In the current study, longer cardiac arrest duration resulted in more myocardial and cerebral damage. When cooling duration was prolonged to 4 hours in the animals receiving 7-minute VF, myocardial and cerebral damage was significantly reduced, and the difference in cardiac systolic function and CO between the 7H4 and 5H2 groups was also reduced. The biomarkers, myocardial and cerebral damage, neurologic outcomes, and survival also did not differ between these 2 groups. These findings raised speculation that cooling duration

may need to be optimally prolonged in animals with longer cardiac arrest duration.

A number of adverse effects have been reported in hypothermia. In trying to prolong the cooling duration to maximize beneficial effects, it would be a major concern as to whether prolonging hypothermia would increase the risks of adverse effects or cause detrimental effects other than those we already know. Common reported adverse effects included pneumonia, sepsis, arrhythmia, electrolyte imbalance, bleed- ing, and coagulopathy [11,24]. However, those adverse effects could be detected and managed by Close monitoring and appropriate treat- ments. Hypothermia also reportedly affects the pharmacokinetics and

pharmacodynamics of several drugs including clopidogrel, dopamine, and phenytoin [10,25-28]. Continuous observation and careful adjust- ment of drug dosage may be necessary during therapeutic hypothermia. Silasi et al [28] demonstrated no compromised neuroplasticity after 7 days of cooling. However, microcirculation was reduced during prolonged cooling [12]. Extensive and systematic evaluation should be conducted to clarify the potential influence of prolonged cooling.

1. Limitations

There were certain limitations in the current study. First, based on our previous study, we chose 1 to 4 hours as the cooling duration for small animals, but the shorter (compared with humans) cooling dura- tion may limit the interpretation of the cooling effect and fail to evaluate potential harmful influences. Second, only 1 animal surviving 72 hours in the 7H1 group limited the analysis of myocardial and cerebral dam- age. Third, the temperature probe placed in the abdominal aorta may not reflect temperature changes in the brain. Fourth, the assumption of neurologic scores for dead animals might not reflect true neurologic outcomes. However, removal of these dead animals from the neurologic outcome analysis would significantly disguise the results. Another lim- itation is that we used healthy small male animals without comorbidi- ties for the cardiac arrest model. However, in clinical practice, VF cardiac arrest usually accompanies acute coronary syndromes and acute myocardial infarction and occurs in both sexes. Besides, the cur- rent study used a laboratory animal model, and the results did not re- flect a translational effect in human cardiac arrest. Extrapolation should not be made, until prolonging cooling is studied in humans. The electric shock doses chosen in the present study were also based on rat resuscitation models in other laboratories and our previous pilot studies. The strength of these shocks is larger than that used on humans in clinical practice on a per weight basis. Therefore, the electric shock-induced injury may be accentuated in the present study, which could potentially affect the injury mechanisms.

1. Conclusion

We concluded that slightly prolonging hypothermia may improve myocardial function in the early postcardiac arrest period, mitigate myocardial and cerebral damage, and result in better survival and neu- rologic outcomes in a rat model of VF cardiac arrest and intra-arrest cooling. In prolonged cardiac arrest, the cooling duration may be prolonged in accordance to reduce excess cardiac and cerebral damage.

References

1. Bernard SA, Gray TW, Buist MD, Jones BM, Silvester W, Gutteridge G, et al. Treatment of comatose survivors of out-of-hospital cardiac arrest with induced hypothermia. N Engl J Med 2002;346:557-63.
2. Hypothermia after Cardiac Arrest Study Group. Mild therapeutic hypothermia to im- prove the neurologic outcome after cardiac arrest. N Engl J Med 2002;346:549-56.
3. Peberdy MA, Callaway CW, Neumar RW, Geocadin RG, Zimmerman J. Part 9: post- cardiac arrest care: 2010 American Heart Association Guidelines for Cardiopulmo- nary Resuscitation and Emergency Cardiovascular Care. Circulation 2010;122: S768-86.
4. Nielsen N, Wetterslev J, Cronberg T, Erlinge D, Gasche Y, Hassager C, et al. Targeted temperature management at 33?C versus 36?C after cardiac arrest. N Engl J Med 2013;369:2197-206.
5. International Liaison Committee–ILCOR. ILCOR update: targeted temperature man- agement. Targeted temperature management following cardiac arrest: an update. ILCOR; 2013[Available from: http://www.ilcor.org/data/TTM-ILCOR-update-Dec- 2013.pdf].
6. Abella BS, Zhao D, Alvarado J, Hamann K, Vanden Hoek TL. Intra-arrest cooling im- proves outcomes in a murine cardiac arrest model. Circulation 2004;109:2786-91.
7. Takata K, Takeda Y, Sato T, Nakatsuka H, Yokoyama M, Morita K. Effects of hypother- mia for a short period on histologic outcome and extracellular glutamate concentra- tion during and after cardiac arrest in rats. Crit Care Med 2005;33:1340-5.
8. Kuboyama K, Safar P, Radovsky A, Tisherman SA, Stezoski SW, Alexander H. Delay in cooling negates the beneficial effect of mild resuscitative cerebral hypothermia after cardiac arrest in dogs: a prospective, randomized study. Crit Care Med 1993;21: 1348-58.
9. Che D, Li L, Kopil CM, Liu Z, Guo W, Neumar RW. Impact of therapeutic hypothermia onset and duration on survival, neurologic function, and neurodegeneration after cardiac arrest. Crit Care Med 2011;39:1423-30.
10. van den Broek MP, Groenendaal F, Egberts AC, Rademaker CM. Effects of hypother- mia on pharmacokinetics and pharmacodynamics: a systematic review of preclinical and clinical studies. Clin Pharmacokinet 2010;49:277-94.
11. Lampe JW, Becker LB. State of the art in therapeutic hypothermia. Annu Rev Med 2011;62:79-93.
12. Ye S, Weng Y, Sun S, Chen W, Wu X. Comparison of the durations of mild therapeutic hypothermia on outcome after cardiopulmonary resuscitation in the rat. Circulation 2012;125:123-9.
13. Tsai MS, Huang CH, Tsai CY, Chen HW, Lee HC, Cheng HJ, et al. Ascorbic acid miti- gates the myocardial injury after cardiac arrest and Electrical shock. Intensive Care Med 2011;37:2033-40.
14. Tsai MS, Huang CH, Tsai CY, Chen HW, Cheng HJ, Hsu CY, et al. Combination of intra- venous ascorbic acid administration and hypothermia after resuscitation improves myocardial function and survival in a ventricular fibrillation cardiac arrest model in the rat. Acad Emerg Med 2014;21:257-65.
15. Moffitt P. A methyl green-pyronin technique for demonstrating cell death in the mu- rine tumour S180. Cell Biol Int 1994;18:677-9.
16. Bennett SA, Stevenson B, Staines WA, Roberts DC. Periodic acid-Schiff (PAS)-positive deposits in brain following kainic acid induced seizures: relationships to fos induc- tion, neuronal necrosis, reactive gliosis, and blood-brain barrier breakdown. Acta Neuropathol 1995;89:126-38.
17. Schmued LC, Albertson C, Slikker Jr W. Fluoro-Jade. A novel fluorochrome for the sensitive and reliable histochemical localization of neuronal degeneration. Brain Res 1997;751:37-46.
18. Huang CH, Chen HW, Tsai MS, Hsu CY, Peng RH, et al. Antiapoptotic cardioprotective effect of hypothermia treatment against oxidative stress injuries. Acad Emerg Med 2009;16:872-80.
19. Shankaran S, Laptook AR, Ehrenkranz RA, Tyson JE, McDonald SA. Whole-body hy- pothermia for neonates with Hypoxic-ischemic encephalopathy. N Engl J Med 2005;353:1574-84.
20. Clark DL, Penner M, Orellana-Jordan IM, Colbourne F. Comparison of 12, 24 and 48 hours of systemic hypothermia on outcome after permanent focal ischemia in rat. Exp Neurol 2008;212:386-92.
21. Florian B, Vintilescu R, Balseanu AT, Buga AM, Grisk O, Walker LC, et al. Long-term hypothermia reduces infarct volume in aged rats after focal ischemia. Neurosci Lett 2008;438:180-5.
22. Gasser S, Khan N, Yonekawa Y, Imhof HG, Keller E. Long-term hypothermia in pa- tients with severe Brain edema after poor-grade subarachnoid hemorrhage: feasibil- ity and intensive care complications. J Neurosurg Anesthesiol 2003;15:240-8.
23. Paine MG, Che D, Li L, Neumar RW. Cerebellar Purkinje cell neurodegeneration after cardiac arrest: effect of therapeutic hypothermia. Resuscitation 2012;83:1511-6.
24. Bjelland TW, Hjertner O, Klepstad P, Kaisen K, Dale O, Haugen BO. Antiplatelet effect of clopidogrel is reduced in patients treated with therapeutic hypothermia after car- diac arrest. Resuscitation 2010;81:1627-31.
25. Hogberg C, Erlinge D, Braun OO. Mild hypothermia does not attenuate platelet ag- gregation and may even increase ADP-stimulated platelet aggregation after clopidogrel treatment. Thromb J 2009;7:2.
26. Filseth OM, How OJ, Kondratiev T, Gamst TM, Sager G, et al. Changes in cardiovascu- lar effects of dopamine in response to graded hypothermia in vivo. Crit Care Med 2012;40:178-86.
27. Empey PE, de Mendizabal NV, Bell MJ, Bies RR, Anderson KB, Kochanek PM, et al. Therapeutic hypothermia decreases phenytoin elimination in children with trau- matic brain injury. Crit Care Med 2013;41:2379-87.
28. Silasi G, Klahr AC, Hackett MJ, Auriat AM, Nichol H, Colbourne F. Prolonged thera- peutic hypothermia does not adversely impact neuroplasticity after global ischemia in rats. J Cereb Blood Flow Metab 2012;32:1525-34.