Experimental Hyperthermia during Cardiac Arrest and CPR Is Associated with Severe Spontaneous Hypothermia in Mice

doi:10.4236/nm.2012.33035

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Neuroscience & Medicine, 2012, 3, 306-313

http://dx.doi.org/10.4236/nm.2012.33035 Published Online September 2012 (http://www.SciRP.org/journal/nm)

Experimental Hyperthermia during Cardiac Arrest and

CPR Is Associated with Severe Spontaneous Hypothermia

in Mice

Ruediger R. Noppens1,2, Julia Kofler2,3, Richard Traystman2,4

1Department of Anesthesiology, Medical Center of the Johannes Guten berg University, Mainz, Germany; 2Department of Anesthe siology

and Peri-Operative Medicine, Ore gon Health & Science Univer sity, Portland, USA; 3Department of Pathology, University of Pittsburgh,

Pittsburgh, USA; 4University of Colorado Denver, Anschutz Medical Center, Colorado, USA.

Email: [email protected]

Received June 1st, 2012; revised July 1st, 2012; accepted July 10th, 2012

ABSTRACT

Background: Since genetically engineered mice are becoming more and more av ailable, these an imals b ecome o f high

interest to study physiologic and pathophysiologic pathways of brain ischemia. The aim of this study was to examine

body temperature (Tb), physical activity variation and neurohistopathology in mice exposed to normothermic and hy-

perthermic cardiac arrest and cardiopulmonary resuscitation (CA/CPR). Methods: Male C57Bl/6 mice weighing 22 -

27 g were implanted intraperitoneally with a radio telemeter and subjected to 10 min cardiac arrest followed by cardio-

pulmonary resuscitation. Normothermia (37.5˚C) or hyperthermia (39.0˚C) was induced by controlling peri-cran ial tem-

perature during the arrest period. Results: Hyperthermia during the arrest resulted in a Tb decrease during early recov-

ery to a nadir of 28˚C ± 0.8˚C (mean ± SE) and partially recovered to 34.4˚C ± 1˚C 36 hrs after CA/CPR. With nor-

mothermia during the arrest, Tb depression was less pronounced (nadir of 32.3˚C ± 0.3˚C) and recovered to physiologic

levels within 24 hrs. Coupling of ph ysical activity and body temperature was absent in all animals after CA/CPR. Neu-

ronal injury in the caudoputamen was greater in the hyperthermia group. Conclusions: This study demonstrates that

CA/CPR eliminates normal connectivity between body temperature and physical activity and induces long-lasting hy-

pothermia, the depth of which is related to severity of brain injury. Long term temperature monitoring is required in

survival murine experiments, if body temperature is a study variable.

Keywords: Cerebral Ischemia; Cardiac Arrest; Cardiopulmonary Resuscitation; Hypothermia; Neuroprotection;

Telemetry; Hyperthermia

1. Introduction

It is now well recognized that body temperature strongly

influences outcome from cerebral ischemia. The benefit

of applied cooling in reducing neuronal damage and

neurological deficits after stroke or cardiac arrest is evi-

dent in many animal injury models and in recent clinical

trials [1,2]. Suppression of hyperthermia, should it de-

velop spontaneously, is thought to improve brain his-

tologic and neuro functional outcome after experimental

and clinical focal cerebral ischemia [3,4].

How body temperature is regulated throughout recov-

ery in animal ischemic injury models is unclear and may

be species, brain region and injury specific. For example,

middle cerebral artery occlusion (MCAO) is associated

with severe hyperthermia in rats (e.g. 39.5˚C) but causes

hypothermia in mice (e.g. 33.1˚C) [5-7]. Global cerebral

ischemia produces mild hyperthermia (38.5˚C) in gerbils

but leads to hypothermia in rats (34˚C) and mice (32˚C)

[8-10]. Although the effect of temperature on neuronal

survival has been widely recognized and temperature is

strictly controlled in animal models of brain injury,

changes of body temperature after cardiac arrest and car-

diopulmonary resuscitation (CA/CPR) in mice are not

well described.

The aims of this study were 1) to characterize physi-

ologic body temperature and physiologic activity in

freely moving mice, 2) examine effects of normothermia

and hyperthermia during global brain ischemia in a well

established model of CA/CPR on body temperature and

activity levels during 3-day recovery and 3) to evaluate

neuronal injury in specific brain regions.

2. Material and Methods

2.1. Animals

Male C57Bl/6 mice (22 - 27 g, Charles River, Hollister,

Experimental Hyperthermia during Cardiac Arrest and CPR Is Associated with Severe Spontaneous

Hypothermia in Mice 307

California) were treated according to NIH guidelines for

the care and use of animals in research, and all experi-

mental protocols were approved by the institutional ani-

mal care and use committee. All animals were housed in

a temperature controlled room (22˚C) under a 12:12 hour

dark:light cycle, room lights were turned on at 7:00 AM

and turned off at 7:00 PM. Mice had free access to food

(RMH 1000, 5P07, Purina Mills, USA) and water

throughout the experiment. Surgical procedures were

performed between 7:30 AM and 1:00 PM in all groups.

2.2. Transient Global Brain Ischemia: Cardiac

Arrest and CPR

Cardiac arrest and CPR were performed as previously

described [11,12]. Briefly, animals were anesthetized

(halothane 1% - 3% with supplemental oxygen), the tra-

chea was intubated with a 20-gauge iv catheter and mice

were mechanically ventilated (Mouse Ventilator Mini-

Vent, Type 845, Hugo Sachs Electronic). Temperature

probes were placed into the left temporal muscle and

rectum, and a PE-10 catheter was inserted into the right

jugular vein for drug ad ministratio n. ECG was monitor ed

using subcutaneous needle electrodes placed on the chest

and a common ECG monitor (Hewlett Packard 78353B).

Animals were allowed to stabilize for 15 minutes before

induction of cardiac arrest. Peri-cranial temperature was

controlled by attaching a circulating warm water coil

around the head five minutes before cardiac arrest [11].

One minute before induction of cardiac arrest, body cool-

ing was begun using an alcohol patch placed on the front

side of the body and a water-filled pad placed underneath

which was chilled by running throu gh an ice-wat er bath.

CA was induced by intravenous injection of cold 0.5

M KCl (50 µl) via the jugular catheter and confirmed by

the presence of isoelectric ECG. Anaesthesia and ventila-

tion were discontinued, and the endotracheal tube was

disconnected from the ventilator. Body temperature was

allowed to fall during the arrest to 27˚C, then the cooling

devices were removed.

CPR was initiated at 10 minutes of CA by i.v.injection

of pre-warmed epinephrine (0.3 µg/kg in 0.5 ml saline),

chest compressions (approximately 300/min) and venti-

lation with 100% oxygen. CPR was concluded once ECG

and cardiac contractions confirmed restoration of spon-

taneous circulation (ROSC). Resuscitation efforts were

ceased, and the animal excluded from the study if ROSC

was not achieved by 2.5 minutes of CPR. The circulating

water coil was removed and the body temperature was

raised to physiologic levels (37˚C) in a controlled man-

ner using an electrical warming pad. A radiotelemeter

(22 × 8 mm) was inserted under aseptic conditions into

the peritoneal cavity for measurement of body tempera-

ture (Tb) and spontaneous physical activity (Activity;

Vital View ER-4000, Mini Mitter, USA) 10 min after

ROSC as previously described and adapted for mice [13].

All probes were cleaned with 70% alcohol and rinsed

with normal saline prior to ethylene oxide gas steriliza-

tion before use in animals. Each mouse was singly

housed on an ER 4000 receiver platform and computer

system for data recording. Tb and Activity were sampled

once a minute (VitalView® software, Mini Mitter, USA).

Activity is measured as a relative count by determining

gross motor activity on the receiver platform. This

method does not allow measurement of e.g. a distance

traveled or the moving speed of the mouse.

After 30 minutes, mice were disconnected from the

ventilator and breathing effort and pattern was evaluated .

When a normal respiratory rate (120/min) with sufficient

strength of the breathing musculature was present, the

endotracheal tube and catheter were removed and skin

wounds closed. The animal was returned to its homecage

35 minutes after CA/CPR for continuous monitoring of

Tb and Activity for the following 3 days. Animals not

surviving the 3-day period were excluded from the study.

2.3. Experimental Groups

2.3.1. Experiment 1: Measurement of Physiological

Body Temperature Variation and Physical

Activity

We first assessed the physiological pattern of body core

temperature and activity variation in healthy mice. Nine

animals were examined in this experiment. A radio-

telemeter was implanted and animals were allowed to

recover 1 week with full access to food and water before

recording of Tb and Activity for 3-days.

2.3.2. Experiment 2: Effect of Normothermia or

Hyperthermia during Brain Ischemia on Body

Temperature Variation, Physi c al Activi ty,

Mortality and Neuronal Survival

To evaluate if Tb and Activity during recovery from

CA/CPR was altered by the level of brain injury, a total

of 26 mice were randomized to receive either nor-

mothermia (peri-cranial temperature during cardiac arrest:

37.5˚C, n = 8), hyperthermia (peri-cranial temperature

39˚C, n = 11) or sham surgery (n = 7, animals followed

the normothermia experimental protocol except receiving

CA/CPR). Radio-telemetric monitoring was performed

for 3-days after CA/CPR.

2.4. Histology

At 3 days post-arrest, mice were deeply anesthetized with

halothane and the brain was perfusion-fixed (10% phos-

phate buffered formalin, as previously described [11].

Tissue analysis was performed by standard hematoxylin

Experimental Hyperthermia during Cardiac Arrest and CPR Is Associated with Severe Spontaneous

Hypothermia in Mice

308

and eosin histology. For analysis, viable and non-viable

neurons were counted by an investigator blinded to

treatment regimen using light microscopy (100×) in the

CA1 region of hippocampus (bregma –1.5 mm), and ros-

tral and caudal caudoputamen (bregma 0.5 and –1 mm,

respectively). The entire length of the CA1 sector was

counted, and six microscopic fields were evaluated fol-

lowing a distinctive pattern in both levels of the cau-

doputamen. Non-viable neurons were identified by pink

hypereosinophilic cytoplasm and a dark pyknotic nucleus,

and the percentage of neurons that were non-viable was

calculated for each region of interest.

2.5. Statistical Analysis

All data are expressed as mean ± SE. Comparison of

treatment groups for physiologic variables and neuro-

histopathology were performed using one-way analysis

of variance (ANOVA) and post-hoc Tukey test or t-Test

where applicable (Sigma Stat 3.0, SPSS Inc.). A one-way

repeated measures analysis of variance and post-hoc test

(Dunnett’s Method) was used for analysis of body tem-

perature in each group. Fisher’s Exact Test was used for

animal survival analysis. Differences were considered

statistically significant with p < 0.05.

3. Results

3.1. Tb and Activity in Healthy Mice

(Experiment 1)

We observed that higher body temperatures and activity

levels occurred during the dark vs light cycle, indicating

strong circadian rhythm variation (Figure 1). Tb and

activity were highest shortly after light changes in the

housing room. Tb and activity appeared coupled in peri-

ods of high spontaneous activity and correlated well with

increased body temperature (r2 = 0.67).

3.2. Physiologic Parameters (Experiment 2)

Body weight at the day of surgery, CPR time and epi-

nephrine dose were not different between groups (Table

1). All mice were successfully resuscitated after CA/CPR.

As intended, peri-cranial temperature during injury was

higher in normothermia vs hyperthermia groups (p <

0.001), however temperature was equivalent in both in-

jury groups by 30 minutes post-arrest (Table 2). Mortal-

ity was not statistically different between normothermia

and hyperthermia groups (p = 0.49, Table 1).

3.3. Body Temperature and Activity after

CA/CPR (Experiment 2)

Tb and Activity decreased spontaneously during recov-

ery from CA/CPR, and this effect was most striking in

(a)

(b)

Figure 1. Physiologic body-temperature (a) and activity (b)

in healthy male C57Bl/6 mice. 12:12 hour light:dark cycle

(7 AM:7 PM) is marked as black bars; n = 9, mean  SE.

Table 1. Body weight, CPR duration, Epinephrine dose and

Animal survival.

Experimen tal Group

ShamNormothermia Hyperthermia

Body Weight [g] 24.8 ± 0.6 24.6 ± 0.5 25.8 ± 0.7

CPR Duration [sec] 59 ± 8 61 ± 8

Epinephrine [µg] 7 ± 0.4 9 ± 0.7

Epinephrine[µg/g/BW] 0.3 ± 0.02 0.3 ± 0.02

Animal Survival 77

(100%)

911

(82%)

Normothermia: 37.5˚C during CA/CPR; Hyperthermia: 39˚C during CA/

CPR. Mean ± SE.

the hyperthermia group (Figure 2(a)). Tb fell during

early recovery in the hyperthermia group and reached a

nadir of 28˚C ± 0.8˚C (p < 0.001 as compared to baseline

values). Tb partially recovered to 34.4˚C ± 1˚C at 34 hrs

post CA (p < 0.001 as compared to baseline valu es), then

Experimental Hyperthermia during Cardiac Arrest and CPR Is Associated with Severe Spontaneous

Hypothermia in Mice 309

again decreased. Phases of high activity levels were not

accompanied by a significant increase of Tb. Although

Tb remained low at all times, spontaneous activity was

evident by 8 hours, increased steadily then again declined

40 hours after CA/CPR (Figure 2(b)). A physiologic

activity variation, as observed in sham operated animals,

was absent during the observation period. In the nor-

mothermia group, Tb was also depressed for hours after

CA/CPR, but the nadir for Tb depression was less

(32.3˚C ± 0.3˚C), and full recovery of Tb did occur. Dur-

ing the first 12 hours activity was higher than in th e other

groups, followed by a decrease in the following observa-

tion period. Nevertheless, the normal coupling of Tb and

spontaneous activity was lost and physiologic variation

was absent in the normothermia group. Despite high

spontaneous activity during the first post-arrest day, Tb

remained low (32.8˚C ± 0.5˚C at 10 hours).

3.4. Neurohistopathological Injury

(Experiment 2)

Neuronal injury was similar in the hippocampal CA1

region in normothermia and hyperthermia groups (Fig-

ure 3). Mice subjected to hyperthermic cardiac arrest had

a higher percentage of injured neurons at the caudal and

rostral level of the caudoputamen than normothermic

cardiac arrest mice (p < 0.001 and p < 0.05, respectively).

Injury was greater in both groups at the caudal caudopu-

tamen than at the more rostral level.

4. Discussion

This study demonstrates that mice undergoing hyper-

Table 2. Body temperature and Peri-Cranial Temperature

at Baseline, end of Cardiac Arrest period, and 10 and 30

min after Restoration of Spontaneous Circulation (ROSC).

Experimental Group

Sham Normothermia Hyperthermia

Rectal Temperature (˚C)

Baseline 36.8 ± 0.1 36.5 ± 0.1 36.6 ± 0.1

10 min Cardiac

Arrest 33.4 ± 0.1 27.3 ± 0 .2 26.8 ± 0.3

10 min ROSC 34.8 ± 0.1 30.6 ± 0. 4 29.5 ± 0.5

30 min ROSC 36.8 ± 0.1 37.2 ± 0.2 36.6 ± 0.3

Peri-Cranial Temperature (˚C)

Baseline 34.7 ± 0.3 34.6 ± 0.2 34.5 ± 0.2

10 min Cardiac

Arrest 37.5 ± 0 37.5 ± 0*** 39 ± 0.1

10 min ROSC 35.6 ± 0.1 31 ± 0.1 31.2 ± 0.4

30 min ROSC 34.6 ± 0.2 34.8 ± 0. 1 34.7 ± 0.1

Figure 2. Body core temperature (a) and Activity (b) in the

housing cage after cardiac arrest and CPR. BI: Brain In-

jury; Mean ± SE.

thermia during CA/CPR have loss of post-ischemic tem-

perature regulation, spontaneous severe hypothermia as

well as uncoupling of spontaneous activity and body

temperature. Normothermia during brain injury resulted

in a milder form of spontaneous hypothermia and a loss

of circadian temperature regulation. Hyperthermia during

CA/CPR resulted in greater neuronal injury in caudopu-

tamen, suggesting that spontaneous marked hypothermia

during recovery is related to the severity of brain injury.

A corollary of the present observations is that long term

temperature monitoring is required in survival murine

experiments, if body temperature is to be controlled as a

study variabl e.

We observed that body temperature and physical ac-

tivity varied in a circadian rhythm under basal conditions,

a phenomenon that has been described in mice and which

differs amongst mouse strains commonly used as ex-

perimental models in neuroscience [14,15]. In mouse,

physiologic temperature ranges from mild hypothermia

during periods of low activity to hyperthermia during

high activity. However, circadian fluctuations are imme

Experimental Hyperthermia during Cardiac Arrest and CPR Is Associated with Severe Spontaneous

Hypothermia in Mice

310

(a)

(b)

(c)

Figure 3. Neuronal injury after cardiac arrest and CPR. A:

CA 1 field of the hippocampus; B: rostral caudoputamen; C:

caudal caudoputamen. *p < 0. 05, **p < 0.01. Mean ± SE.

diately ablated, and not restored, in animals that sustain

injury from global cerebral ischemia. In murine models

of focal ischemia, hyperactivity has been described pre-

viously and its level has been associated with severity of

brain injury [16,17].

The depth of spontaneous hypothermia induced by

CA/CPR, even in the presence of persistent physical ac-

tivity, is surprising. Spontaneous hypothermia after

CA/CPR has been described in a rat model of asphyxial

cardiac arrest, with recovery to control values by 36 hr

ROSC [8]. In another study, rats developed moderate

hypothermia (32˚C) five hours after CA/CPR and recov-

ered within eight hours to normal body temperature [18].

The depth of hypothermia was less in these studies, and

recovery more rapid, than we observed in our mouse

model of CA/CPR. Other rat models of brain ischemia

have also been associated with spontaneous temperature

changes during recovery. The four-vessel occlusion

model in rat causes mild hypothermia in brain [19].

However, results are inconsistent in studies examining

temperature changes in the two-vessel occlusion model

of brain ischemia. One study reports a period of hyper-

thermia lasting up to three days and a loss of circadian

temperature variation after the insult [2 0]. In a later stud y

using the same model of brain ischemia, no post-

ischemic temperature changes could be demonstrated

[21]. In a modified model of two-vessel occlusion, a brief

two hour period of mild hypothermia was followed by

recovery to normothermia within six hours after the in-

sult [22]. In a model of two vessel occlusion in C57Bl/6

mice, spontaneous hypothermia occurred after the in-

sult[10]. In agreement with our results in the normother-

mia during CA/CPR group, the lowest body temperature

in this study was 32˚C and recovered to physiologic lev-

els within 24 hours after brain ischemia [10]. Spontane-

ous hypothermia in patients, especially in pediatric pa-

tients has also been reported anecdotically, however this

finding has not been systematically evaluated [23]. In

part, the lack of attention to endogenous temperature dys-

regulation is due to ongoing interest in therapeutic cool-

ing after CA/CPR or traumatic brain injury. Mild to mo-

derate hypothermia post arrest remains to date the only

successful neuroprotective strategy applied to humans

and is used increasingly in intensiv e care units for select-

ed patients [1,2]. In contrast, post-injury hyperthermia

worsens outcome and increases neuronal injury [24,25].

The hyperthermia during CA/CPR group in our study

exhibited severe hypothermia in the first hours of recov-

ery, with only incomplete recovery during the 3-day ob-

servation period. The mechanism for initiation/mainte-

nance of spontaneous hypothermia is not clear. However,

it is unlikely that low physical activity was responsible

for low temperature in this set of experiments. Mice ex-

hibited high activity levels in the first 12 hours post ar-

rest, yet body temperature failed to rise to basal levels.

Normothermia during CA/CPR mice also showed early

hyperactivity at periods when their lowest temperatures

were observed. The hyperthermia during CA/CPR group

presented body temperatures during early recovery simi-

lar to body temperatures that have been described in tor-

por. Topor is a temporary hibernation-like state that has

been previously shown during times of food restriction

[14,26]. In contrast to topor, which is characterized by a

state of motor inactivity, animals in this study showed a

high level of activity in the early phase of recovery with-

out a corresponding increase of body temperature.

Experimental Hyperthermia during Cardiac Arrest and CPR Is Associated with Severe Spontaneous

Hypothermia in Mice 311

A limitation of our study is that hyperthermia during

CA/CPR was used to induce more severe brain injury.

The temperature manipulations in our model may have

contributed to post-arrest temperature dysregulation.

However, a milder form of spontaneous hypothermia,

albeit in a milder form, has also been described in other

mouse models of global ischemia without intra-ischemic

hyperthermia [10]. Therefore, we believe that the tem-

perature dysregulation in our model is a direct result of

increased neuronal injury and only indirectly related to

the experimental temperature manipulation. We did not

specifically monitor weight changes or food and water

intake in our animals. However, we noted that most mice

in the hyperthermia group did not appear healthy at the

end of the 3-day observation period. Some of these ani-

mals might not have survived much longer than four days

after cardiac arrest and CPR. Several reasons for the poor

health of these animals exist, e.g. multi organ failure,

which might have been responsible for body temperature

and decreased activity two to three days after the insult,

but is unlikely responsible for the Tb and activity altera-

tions during the early recovery period.

The present study does not determine the potentially

complex mechanisms underlying spontaneous post-

ischemic hypothermia. However, the data underscore the

need for long term monitoring of body temperature in

mouse brain injury models and emphasize the extreme

change that occurs in temperature regulation. It is well

recognized that temperature in recovering animals is in-

fluenced by multiple factors, including method of tem-

perature measurement, anesthetic regimen and injury

model [6,8,27,28]. Furthermore, the neuroprotective po-

tential of a number of pharmacologic agents such as

MK-801, NBQX or Magnesium has been linked to tem-

perature [29-31]. Therefore, interactions with brain in-

jury induced spontaneous hypothermia and investiga-

tional neuroprotective drugs must be considered.

No guidelines exist on how body or brain temperature

in small rodents after brain ischemia should be measured

or controlled. Several techniques have been published,

including the use of heating devices such as infrared

lamps or the use of incubators used in human medicine

[8,32]. In a recent review, MacLellan et al recommended

tight temperature measurements of the brain and body

during surgery as well as during recovery after brain

ischemia for at least one day [33]. Telemetry probes are

strongly recommended for measuring temperature in

conscious rodents because this technique allows repeated

measurements without stressing the animal by handling

as is necessary with rectal measurements. It is also im-

portant that the use of a temperature controlled environ-

ment does not obviate the need for frequent temperature

measurements since experimental animals could present

with either hyper- or hypothermia after brain ischemia.

Heating as well as cooling devices must be po sitioned in

such a way that animals are exposed uniformly. The use

of infrared lamps is discouraged as they impose a risk of

non-uniform heating and overheating of an impaired

post-ischemic animal.

In conclusion, the present results demonstrate severe

spontaneous hypothermia after CA/CPR in mice, which

is partially spontaneously reversible. However, future

studies will be necessary to determine which mechanisms

are involved. Our results also underline the necessity for

monitoring body temperature in survival murine studies

since temperature is a major factor influencing outcome.

5. Acknowledgements

This study was supported by National Institutes of Health,

grants NS 46072, NS 20020 and NS 33368. None of the

authors has a financial interest to disclose.

Experiments were conducted at Oregon Health & Sci-

ence University, Department of Anesthesiology and Peri-

Operative Medicine, Portland, Oregon, USA.

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