Name: protocol for long duration whole body hyperthermia in mice
Uploaded: 2012-08-25T12:00:00.000+00:00
Duration: 7 min 56 s
Description: Watch this Scientific Journal Video about Protocol for Long Duration Whole Body Hyperthermia in Mice at JoVE.com

1. Animal Ethics
Before performing the long duration hyperthermia on mice make sure that the procedure has been approved by Institutional Animal Care and Use Committee. All the procedures discussed in this paper and shown in the accompanying video were approved by the Institutional Animal Ethics Committee of the National Institute of Immunology.
2. Understanding Hyperthermia
Whole Body Hyperthermia (WBH) is delivered by placing the animals in prewarmed cages that are placed in incubator maintained at 39.5&deg;C. During the standardization of protocol, a temperature sensor was surgically implanted in the mice and their body temperature was monitored in different experimental conditions. It was observed that upon placing the animals in heated environment, their temperature gradually increased from 37 &deg;C to up to 42 &deg;C over the duration of 100 min (Figure 1). The resulting "spike" in temperature is intriguing as the internal body temperature shot even higher than the external environmental temperature. 
When the body temperature exceeded 40 &deg;C, the animal would show signs of stress and dehydration such as wetting around the snout. Increase the body temperature beyond 40 &deg;C, will lead to wetting and irreversible stress or even death of animals due to heat stress. So it is important to prevent the temperature spikes during the course of hyperthermia. 
This phenomenon of high temperature spikes is associated with the physical hyperactivity of animal during hyperthermia exposure. Similar temperature spikes were observed by other groups too and were associated with the physical hyperactivity of the animal upon being introduced to environment having higher temperatures 10. 
In our protocol, mice are immobilized during the hyperthermia exposure. As shown in Figure 2, immobilization of animal prevents the temperature spikes.
We have used 8-12 weeks old BALB/Cj mice for demonstrating this protocol. The protocol is not restricted to a particular type/strain of the mice.
3. Preparing for the Experiment
3.1 Incubator
This experiment can be performed in a normal CO2 incubator as they have a good temperature control. Disconnect CO2 gas supply and remove water holding tray of the CO2 incubator. In some models of incubators it is possible to switch off the CO2 controller. Arrange shelves of the incubator so that a mice cage could be placed conveniently inside. Set the temperature of the incubator at 39.5 &deg;C.
3.2 Cages
Typically, the hyperthermia experiment is performed on 4 mice at a time. Two cages are required; one is placed inside the incubator and the other is placed outside at room temperature. Bedding of cages is avoided, as the patches of bedding become wet with time which results in non-uniform temperature inside the cage. The outside cage should have food pellets and water bottle. 
3.3 Mice immobilization tubes
During the course of first 4 cycles of hyperthermia mice are immobilized in perforated tubes. These immobilization tubes can be made from 50 ml 'centrifuge' tubes by piercing 3 mm holes all over its body and four of such tubes will be required. The 50ml tube can conveniently immobilize BALB/Cj mice which are older than 8 weeks. Tubes of appropriate sizes may be required for some unusually different strain of mice or for mice which are very young or older than 50 weeks.
Place immobilization tubes and a cage inside the incubator maintained at 39.5 &deg;C. Wait for at least 2 hour so that the cage and immobilization tubes attain the temperature of the incubator. 
4. First cycle of the Hyperthermia
Mice may experience dehydration during the hyperthermia exposure. Therefore before beginning the experiment, administer 0.5 ml normal saline intraperitoneally to every mouse. Use a 26 G needle with 1 ml syringe.
Quickly take out the cage placed in the incubator, place one animal in each immobilization tubes and place the cage back in the incubator. Record the time. It is essential to keep the cage at the same place in the incubator every time.
After 90 min, take out the cage from the incubator. Release animals from the immobilization tubes and place them in the cage at normal temperature with easy access to food and water. Place empty immobilization tubes back in the incubator. 
After 15 min, the second cycle of hyperthermia is started by delivering 0.5 ml saline intraperitoneally to every mouse. 
They are then immobilized in 50 ml perforated immobilization tubes and placed in the incubator. 
5. Subsequent Hyperthermia Cycles
For first 4 cycles, it is essential to immobilize mice during the hyperthermia exposure and for the first two cycles, saline injection is given before beginning the hyperthermia period. 
After these cycles of hyperthermia followed by rest are completed, mice get acclimatize and do not become hyperactive during the hyperthermia exposure. The intraperitoneal injection of saline may not be necessary if mice readily take food and water during the resting time.
6. After All the Cycles Completed, the Final Step is to Examine the Animals
There should not be any death during the hyperthermia cycles. Every animal has to be watched very carefully in the first few cycles. Rapidly look for any wetting around the snout. This symptom is a signature of dehydration. Mice with such symptoms should immediately be given 0.5 ml normal saline i.p and rest at room temperature. Most of the immobilized mice will not require any additional care and will successfully bear the stress the hyperthermia. 
After completing required cycles, animals may look weak and dehydrated but they will quickly recover within few hours. 
<img src="/files/ftp_upload/3801/3801fig1.jpg" alt="Figure 1" /> 
Figure 1. Variation of the core body temperature of Mice when the cage was placed in an incubator at 39.5 &deg;C. After 90 min the cage was removed from the incubator and placed at 25 &deg;C. Red circles indicate high temperature spikes.
<img src="/files/ftp_upload/3801/3801fig2.jpg" alt="Figure 2" /> 
Figure 2. Variation of the core body temperature of an immobilized Mice during the hyperthermia cycles. After immobilizing the animal, high temperatures spikes were not observe when the cage was placed at 39.5 &deg;C.

<ol>
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We have nothing to disclose.

As far back as 5,000 B.C., Egyptian doctors treated tumors with heat. The Greeks recognized the value of heat in some medical treatments. The most ancient texts of the Law of Moses mention hot springs (Genesis 36:24) to therapeutically elevate body temperature.
The earlier techniques employed either the use of pyrogenic microbial extracts for induction of fever or hot water baths for induction of hyperthermia 1. However none of these approaches could be easily monitored or controlle...

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protocol for long duration whole body hyperthermia in mice

Hyperthermia is a general term used to define the increase in core body temperature above normal. It is often used to describe the increased core body temperature that is observed during fever. The use of hyperthermia as an adjuvant has emerged as a promising procedure for tumor regression in the field of cancer biology. For this purpose, the most important requirement is to have reliable and uniform heating protocols. We have developed a protocol for hyperthermia (whole body) in mice. In this protocol, animals are exposed to cycles of hyperthermia for 90 min followed by a rest period of 15 min. During this period mice have easy access to food and water. High body temperature spikes in the mice during first few hyperthermia exposure cycles are prevented by immobilizing the animal. Additionally, normal saline is administered in first few cycles to minimize the effects of dehydration. This protocol can simulate fever like conditions in mice up to 12-24 hr. We have used 8-12 weeks old BALB/Cj female mice to demonstrate the protocol.

Watch this Scientific Journal Video about Protocol for Long Duration Whole Body Hyperthermia in Mice at JoVE.com

Protocol for Long Duration Whole Body Hyperthermia in Mice

This paper describes a protocol for whole body hyperthermia in mice that can stimulate fever like conditions up to 12-24 hr.

Hyperthermia is a general term used to define the increase in core body temperature above normal. It is often used to describe the increased core body ...

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Research

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Medicine

11.5K Views.  National Institute of Immunology. This paper describes a protocol for whole body hyperthermia in mice that can stimulate fever like conditions up to 12-24 hr.

Video: Protocol for Long Duration Whole Body Hyperthermia in Mice

Magnetic Resonance-Guided High Intensity Focused Ultrasound Generated Hyperthermia: A Feasible Treatment Method in a Murine Rhabdomyosarcoma Model

Magnetic resonance-guided high intensity focused ultrasound (MRgHIFU) is an established method for producing localized hyperthermia. Given the real-time imaging and acoustic energy modulation, this modality enables precise temperature control within a defined area. Many thermal applications are being explored with this noninvasive, nonionizing technology, such as hyperthermia generation, to release drugs from thermosensitive liposomal carriers. These drugs can include chemotherapies such as doxorubicin, for which targeted release is desired due to the dose-limiting systemic side effects, namely cardiotoxicity. Doxorubicin is a mainstay for treating a variety of malignant tumors and is commonly used in relapsed or recurrent rhabdomyosarcoma (RMS). RMS is the most common solid soft tissue extracranial tumor in children and young adults. Despite aggressive, multimodal therapy, RMS survival rates have remained the same for the past 30 years. To explore a solution for addressing this unmet need, an experimental protocol was developed to evaluate the release of thermosensitive liposomal doxorubicin (TLD) in an immunocompetent, syngeneic RMS mouse model using MRgHIFU as the source of hyperthermia for drug release.

Presented here is a protocol to use controlled hyperthermia, generated by magnetic resonance-guided high intensity focused ultrasound, to trigger drug release from temperature-sensitive liposomes in a rhabdomyosarcoma mouse model.

Magnetic resonance-guided high intensity focused ultrasound (MRgHIFU) is an established method for producing localized hyperthermia. Given the real-time ...

Cancer Research

Measuring Skeletal Muscle Thermogenesis in Mice and Rats

Skeletal muscle thermogenesis provides a potential avenue for better understanding metabolic homeostasis and the mechanisms underlying energy expenditure. Surprisingly little evidence is available to link the neural, myocellular, and molecular mechanisms of thermogenesis directly to measurable changes in muscle temperature. This paper describes a method in which temperature transponders are utilized to retrieve direct measurements of mouse and rat skeletal muscle temperature.
Remote transponders are surgically implanted within the muscle of mice and rats, and the animals are given time to recover. Mice and rats must then be repeatedly habituated to the testing environment and procedure. Changes in muscle temperature are measured in response to pharmacological or contextual stimuli in the home cage. Muscle temperature can also be measured during prescribed physical activity (i.e., treadmill walking at a constant speed) to factor out changes in activity as contributors to the changes in muscle temperature induced by these stimuli.
This method has been successfully used to elucidate mechanisms underlying muscle thermogenic control at the level of the brain, sympathetic nervous system, and skeletal muscle. Provided are demonstrations of this success using predator odor (PO; ferret odor) as a contextual stimulus and injections of oxytocin (Oxt) as a pharmacological stimulus, where predator odor induces muscle thermogenesis, and Oxt suppresses muscle temperature. Thus, these datasets display the efficacy of this method in detecting rapid changes in muscle temperature.

Mice and rats are surgically implanted with remote temperature transponders and then habituated to the testing environment and procedure. Changes in muscle temperature are measured in response to pharmacological or contextual stimuli in the home cage or during prescribed physical activity (i.e., treadmill walking at a constant speed).

Skeletal muscle thermogenesis provides a potential avenue for better understanding metabolic homeostasis and the mechanisms underlying energy expenditure. ...

Behavior

Exploring Metabolite Exchange in Brown Adipose Tissue

Arteriovenous Metabolomics to Measure In Vivo Metabolite Exchange in Brown Adipose Tissue

Brown adipose tissue (BAT) plays a crucial role in regulating metabolic homeostasis through a unique energy expenditure process known as non-shivering thermogenesis. To achieve this, BAT utilizes a diverse menu of circulating nutrients to support its high metabolic demand. Additionally, BAT secretes metabolite-derived bioactive factors that can serve as either metabolic fuels or signaling molecules, facilitating BAT-mediated intratissue and/or intertissue communication. This suggests that BAT actively participates in systemic metabolite exchange, an interesting feature that is beginning to be explored. Here, we introduce a protocol for in vivo mouse-level optimized BAT arteriovenous metabolomics. The protocol focuses on relevant methods for thermogenic stimulations and an arteriovenous blood sampling technique using Sulzer's vein, which selectively drains interscapular BAT-derived venous blood and systemic arterial blood. Next, a gas chromatography-based metabolomics protocol using those blood samples is demonstrated. The use of this technique should expand the understanding of BAT-regulated metabolite exchange at the inter-organ level by measuring the net uptake and release of metabolites by BAT.

Author Spotlight: Illuminating New Avenues for Adipose Tissue Metabolism and Disease Prevention 

In this protocol, methods relevant for BAT-optimized arteriovenous metabolomics using GC-MS in a mouse model are outlined. These methods allow for the acquisition of valuable insights into BAT-mediated metabolite exchange at the organismal level.

Brown adipose tissue (BAT) plays a crucial role in regulating metabolic homeostasis through a unique energy expenditure process known as non-shivering ...

Biochemistry

Mouse Body Temperature Measurement Using Infrared Thermometer During Passive Systemic Anaphylaxis and Food Allergy Evaluation

Mouse body temperature measurement is of paramount importance for investigating allergies and anaphylactic symptoms. Rectal probes for temperature readings is common, and they have been proven to be accurate and invaluable in this regard. However, this method of temperature measurement requires the mice to be anesthetized in order to insert the probe without injury to the animal. This limits the ability to observe other phenotypes of the mouse simultaneously. In order to investigate other phenotypes while measuring temperatures, rectal probes are not ideal, and another method is desired. Here, we introduce a noninvasive method of temperature measurement that foregoes the requirement for mouse anesthesia while maintaining equal reliability to rectal probes in measuring body temperature. We use an infrared thermometer that detects body surface temperatures at ranges between 2 and 150 mm. This method of body temperature measurement is successful in reliably replicating temperature change trends during passive system anaphylaxis experiments in mice. We show that body surface temperatures are about 2.0 &#176;C lower than rectal probe measurements, but the degree of temperature drop follows the same trend. Furthermore, we use the same technique to observe mice in a food allergy model to evaluate temperature and activity levels simultaneously.

Here we present a new method to accurately measure body temperature differences in passive systemic anaphylaxis (PSA) and food allergy mouse models using an infrared thermometer. This procedure has been accurately duplicated in previous PSA results.

Mouse body temperature measurement is of paramount importance for investigating allergies and anaphylactic symptoms. Rectal probes for temperature ...

Immunology and Infection

Magnetic-, Acoustic-, and Optical-Triple-Responsive Microbubbles for Magnetic Hyperthermia and Pothotothermal Combination Cancer Therapy

The precision delivery of anti-cancer agents which aim for targeted and deep-penetrated delivery as well as a controlled release at the tumor site has been challenged. Here, we fabricate iron oxide nanoparticle shelled microbubbles (NSMs) through self-assembly, synergizing magnetic, acoustic, and optical responsiveness in one nanotherapeutic platform. Iron oxide nanoparticles serve as both magnetic and photothermal agents. Once intravenously injected, NSMs can be magnetically guided to the tumor site. Ultrasound triggers the release of iron oxide nanoparticles, facilitating the penetration of nanoparticles deep into the tumor due to the cavitation effect of microbubbles. Thereafter, magnetic hyperthermia and photothermal therapy can be performed on the tumor for combinational cancer therapy, a solution for cancer resistance due to the tumor heterogeneity. In this protocol, the synthesis and characterization of NSMs including structural, chemical, magnetic and acoustic properties were performed. In addition, the anti-cancer efficacy by thermal therapy was investigated using in vitro cell cultures. The proposed delivery strategy and combination therapy holds great promise in cancer treatment to improve both delivery and anticancer efficacies.

Presented here is a protocol for the fabrication of iron oxide nanoparticle-shelled microbubbles (NSMs) through self-assembly, synergizing magnetic, acoustic, and optical responsiveness in one nanotherapeutic platform for magnetic hyperthermia and photothermal combination cancer therapy.

The precision delivery of anti-cancer agents which aim for targeted and deep-penetrated delivery as well as a controlled release at the tumor site has ...

Bioengineering

A Behavioral Screen for Heat-Induced Seizures in Mouse Models of Epilepsy

Transgenic mouse models have proved to be powerful tools in studying various aspects of human neurological disorders, including epilepsy. The SCN1A-associated genetic epilepsies comprise a wide spectrum of seizure disorders with incomplete penetrance and clinical variability. SCN1A mutations can result in a large variety of seizure phenotype ranging from simple, self-limited fever-associated febrile seizures (FS), moderate-level genetic epilepsy with febrile seizures plus (GEFS+) to more severe Dravet Syndrome (DS). Although FS are commonly seen in children below 6-7 years of age who do not have genetic epilepsy, FS in GEFS+ patients continue to occur into adulthood. Traditionally, experimental FS have been induced in mice by exposing the animal to a stream of dry air or heating lamps, and the rate of change in body temperature is often not well controlled. Here, we describe a custom-built heating chamber, with a plexiglass front, that is fitted with a digital temperature controller and a heater-equipped electric fan, which can send heated forced air into the test arena in a temperature-controlled manner. The body temperature of a mouse placed in the chamber, monitored through a rectal probe, can be increased to 40-42 &#176;C in a reproducible manner by increasing the temperature inside the chamber. Continual visual monitoring of the animals during the heating period demonstrates induction of heat-induced seizures in mice carrying an FS mutation at a body temperature that does not elicit behavioral seizures in wild-type litter mates. Animals can be easily removed from the chamber and placed on a cooling pad to rapidly return body temperature to normal. This method provides for a simple, rapid, and reproducible screening protocol for the occurrence of heat-induced seizures in epilepsy mouse models.

The goal of the method is to screen for hyperthermia or heat-induced seizures in mouse models. The protocol describes the use of a custom-built chamber with continuous monitoring of the body temperature to determine whether elevated body temperature leads to seizures.

Transgenic mouse models have proved to be powerful tools in studying various aspects of human neurological disorders, including epilepsy. The ...

Neuroscience

Short-Duration Hypothermia Induction in Rats using Models for Studies examining Clinical Relevance and Mechanisms

Therapeutic hypothermia (TH) is a powerful neuroprotective strategy that has provided robust evidence for neuroprotection in pre-clinical studies of neurological disorders. Despite strong pre-clinical evidence, TH has not shown efficacy in clinical trials of most neurological disorders. The only successful trials employing therapeutic hypothermia were related to cardiac arrest in adults and hypoxic ischemic injury in neonates. Further investigations into the parameters of its use, and study design comparisons between pre-clinical and clinical studies, are warranted. This article demonstrates two methods of short-duration hypothermia induction. The first method allows for rapid hypothermia induction in rats using ethanol spray and fans. This method works by cooling the skin, which has been less commonly used in clinical trials and may have different physiological effects. Cooling is much more rapid with this technique than is achievable in human patients due to differences in surface area to volume ratio. Along with this, a second method is also presented, which allows for a clinically achievable cooling rate for short-duration hypothermia. This method is easy to implement, reproducible and does not require active skin cooling.

This article describes two methods of whole-body short-duration hypothermia induction in rats. The first, rapid induction method, employs active cooling using fans and ethanol spray for a rapid decrease in temperature. The second method is a gradual cooling method. This is achieved using the combination of isoflurane anesthesia and the reduction of temperature settings on the homeothermic heat mat. This results in a gradual decrease in core body temperature without the use of any external cooling devices.&#160;

Therapeutic hypothermia (TH) is a powerful neuroprotective strategy that has provided robust evidence for neuroprotection in pre-clinical studies of ...

A Preclinical Model of Exertional Heat Stroke in Mice

Heat stroke is the most severe manifestation of heat-related illnesses. Classic heat stroke (CHS), also known as passive heat stroke, occurs at rest, whereas exertional heat stroke (EHS) occurs during physical activity. EHS differs from CHS in etiology, clinical presentation, and sequelae of multi-organ dysfunction. Until recently, only models of CHS have been well established. This protocol aims to provide guidelines for a refined preclinical mouse model of EHS that is free from major limiting factors such as the use of anesthesia, restraint, rectal probes, or electric shock. Male and female C57Bl/6 mice, instrumented with core temperature (Tc) telemetric probes were utilized in this model. For familiarization with the running mode, mice undergo 3 weeks of training using both voluntary and forced running wheels. Thereafter, mice run on a forced wheel inside a climatic chamber set at 37.5 &#176;C and 40%-50% relative humidity (RH) until displaying symptom limitation (e.g., loss of consciousness) at Tc of 42.1-42.5 &#176;C, although suitable results can be obtained at chamber temperatures between 34.5-39.5 &#176;C and humidity between 30%-90%. Depending on the desired severity, mice are removed from the chamber immediately for recovery in ambient temperature or remain in the heated chamber for a longer duration, inducing a more severe exposure and a higher incidence of mortality. Results are compared with sham-matched exercise controls (EXC) and/or na&#239;ve controls (NC). The model mirrors many of the pathophysiological outcomes observed in human EHS, including loss of consciousness, severe hyperthermia, multi-organ damage as well as inflammatory cytokine release, and acute phase responses of the immune system. This model is ideal for hypothesis-driven research to test preventative and therapeutic strategies that may delay the onset of EHS or reduce the multi-organ damage that characterizes this manifestation.

The protocol describes the development of a standardized, repeatable, preclinical model of exertional heat stroke (EHS) in mice free from adverse external stimuli such as electric shock. The model provides a platform for mechanistic, preventative, and therapeutic studies.

Heat stroke is the most severe manifestation of heat-related illnesses. Classic heat stroke (CHS), also known as passive heat stroke, occurs at rest, ...

Exertional Heat Stroke Mouse Model: A Protocol to Study Mechanisms Underlying Exertional Heat Stroke

Source: King, M. A. et al. <a href="https://www.jove.com/video/62738" target="_blank">A Preclinical Model of Exertional Heat Stroke in Mice</a>. J. Vis. Exp. (2021).
In this video we describe a step-by-step protocol to study exertional heat stroke (EHS) in a mouse model.

Source: King, M. A. et al. A Preclinical Model of Exertional Heat Stroke in Mice. J. Vis. Exp. (2021).
In this video we describe a step-by-step protocol ...

JoVE Encyclopedia of Experiments

Biology

Rodent Models

Adult

Surgical Implantation of Temperature Telemetric Monitoring System in Mouse Model: A Surgical Procedure to Implant Temperature Telemetric Device to Measure Mouse Core Body Temperature

Source: King, M. A. et al.&#160; <a href="https://www.jove.com/video/62738" target="_blank">A Preclinical Model of Exertional Heat Stroke in Mice</a>. J. Vis. Exp. (2021)
In this video, we describe a surgical procedure to implant a temperature telemetric device&#160; in a mouse model to continuously measure its core body temperature.