Published: March 3rd, 2021
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.
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.
TH is the practice of cooling body or brain temperature in order to preserve the viability and function of the organ/system1,2. The role of hypothermia in neuroprotection has been investigated and has shown benefits in a range of pre-clinical models of neurological diseases such as stroke3, subarachnoid hemorrhage4, and traumatic brain injury5. In terms of clinical applications, TH has shown efficacy in patients post-cardiac arrest and in neonatal hypoxic-ischemic injury6.
TH induction is achieved using either surface or endovascular cooling methods. The majority of pre-clinical hypothermia studies perform surface cooling by applying water or ethanol to the animal's fur, or by using a cooling blanket to achieve target temperature1. In humans, systemic surface cooling is achieved by using ice packs and cooling blankets7,8. More rapid cooling has been shown in patients using endovascular methods, which couple an induction infusion of cold saline through an intravenous or intra-arterial catheter, with the placement of an endovascular cooling device within the inferior vena cava9,10. For example, a moderate target temperature of 33 °C can be reached in 1.5 h with endovascular cooling compared to 3-4 h with surface cooling in patients11. The endovascular approach has also become more popular in recent years because it has been reported to reduce some of the side effects seen in systemic surface cooling, such as shivering12,13. The European multicenter, randomized phase III clinical trial of hypothermia for ischemic stroke (EUROHYP-1) used mostly surface cooling14. Results recently published from this trial showed that shivering was a major complication and might have limited the ability to achieve the target temperature10. The shivering response is known to be primarily driven by skin temperature. Some efforts have been made to develop a rodent endovascular cooling method15, but the highly invasive nature of the technique compared to that used in humans, risks confounding any results obtained from that model.
Temperature is the key modulator of biological processes in the body and is tightly regulated by homeostasis. Therefore, any manipulation of the body temperature can have associated risks. Cooling duration is a factor that may have limited the success of hypothermia clinical trials. These trials use a long-duration cooling method, with many maintaining hypothermia from 24-72 h11. This extended duration poses a risk for infection during the cooling protocol. Pneumonia is the most common complication from hypothermia, affecting between 40-50% of patients who undergo the procedure13. This is in contrast to what is normally seen in animal studies of hypothermia where a short-duration paradigm is used (1-6 h)3. The success of these pre-clinical animal studies will likely result in the adaptation of short-duration hypothermia for the use in clinical trials. As a result, it is necessary to have an animal model of short-duration hypothermia that resembles the cooling rates of future clinical trials. Further details pertaining to other temperature parameters and the validity of short-duration hypothermia have been discussed in several review articles1,16,17,18.
Demonstrated here is a gradual model of cooling that is more clinically achievable than current experimental hypothermia models. This novel method has a much slower rate of cooling and therefore, the time to target temperature is closer to the range of those seen in clinical trials of hypothermia11. It also avoids direct surface cooling, which has specific physiological effects, and may, therefore, be more comparable to endovascular cooling, which has been the most commonly used cooling method in clinical trials9,12. This model allows animals to be cooled gradually over 2 h followed by a short period of maintenance at target temperature. Additionally, the rapid cooling short-duration hypothermia method19 is also demonstrated. The fast-cooling method allows target temperature to be achieved rapidly after hypothermia onset. While this approach is not as clinically relevant as the gradual cooling method, it is useful for studies that aim to explore the mechanisms of hypothermia neuroprotection to potentially mimic its powerful neuroprotective effects pharmacologically. This method also has potential applications outside of neuroscience and could be adapted to any number of pre-clinical studies. Another advantage of both methods compared to other approaches is that they are inexpensive and do not require specialist equipment. Finally, this protocol also demonstrates implantation of temperature dataloggers, since post-operative warming and monitoring thereof are important to prevent inadvertent post-operative hypothermia, with its potential to confound study results20.
All experimental procedures were in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes and were approved by the Animal Care and Ethics Committee of the University of Newcastle (A-2013-343 and A-2020-003). In addition to the hypothermia induction methods described below, the following protocols are routinely done in conjunction with hypothermia: femoral line cannulation to monitor blood pressure and heart rate21, and experimental stroke22.
1. Datalogger implantation
NOTE: The datalogger device used in this protocol was not capable of providing real time read-out of body temperature. Read out is possible once the datalogger has been removed from the animal and connected back to the computer. As a result, the rectal temperature probe is used to provide real time information during the cooling and rewarming process. Additionally, the rectal probe is also vital to this method because the surgical heat mat on which the animal is placed during the procedure is regulated by the rectal probe system. The datalogger also serves a valuable purpose of providing temperature data in freely moving, awake rats and is important for ensuring that normal body temperature is maintained after rewarming. Therefore, both temperature-monitoring devices are important for this protocol.
Figure 1: Implantation of datalogger device. (A) Panels left to right show an incision of approximately 2 cm being made on the right side of the lower abdomen of the rat. (B) Temperature monitoring datalogger was inserted subcutaneously into the pocket incision. (C) The incision was closed with nylon sutures. Please click here to view a larger version of this figure.
2. Induction of active (fast) hypothermia for mechanistic studies
3. Induction of clinically achievable gradual onset hypothermia without active skin cooling
Figure 3A is a representation of how a Wistar rat responds to hypothermia using the rapid cooling approach. Hypothermia induction is achieved by the use of fans and 70% ethanol spray. Hypothermia to a target of 32.5 °C is achieved in 15 min. Care must be taken to ensure a delicate interplay between the use of the fans/ heat lamp and ethanol spray to maintain target temperature. As can be seen from Figure 3A, a slight temperature overshoot is observed, which may occur if cooling is not ceased from about 0.5 °C above target temperature. Target is maintained and stabilized at the 30 min mark and rewarming is initiated at 1.5 h.
Figure 3B shows the gradual protocol in which target temperature to 33 °C is reached at 2 h and maintained for 30 min before rewarming at 2.5 h. Here, the temperature is adjusted in increments which prolongs the duration necessary to reach target temperature. Vertical dotted lines in both graphs represent the duration of cooling.
Figure 3A and Figure 3B are obtained from the datalogger device. At the start of the experiment, the datalogger is programmed to initiate recording prior to implantation. At the end of the experiment, the datalogger is removed from the animal and connected to the provided temperature reader via USB port. The software (e.g., eTemperature) reads and generates the data, which can then be exported to a spreadsheet software.
Figure 2: Set up of rapid cooling protocol. (A) Two fans (black arrow) were situated over the lower back region of the rat. At hypothermia initiation, both fans were turned on and ethanol spray was applied to the lower back. The combination of ethanol and the fan facilitates and accelerates hypothermia to rapidly achieve target temperature. (B) A heat lamp (white arrow) was used to prevent hypothermia overshoot. Once target temperature was reached, the heat lamp was used to prevent the rat core temperature from dropping any lower. Once target had stabilized, the heat lamp and/or remaining fan was turned off. Please click here to view a larger version of this figure.
Figure 3: Hypothermia induction using active (A) and gradual (B) methods. (A) Target temperature was reached in 15 min using the active cooling process and was maintained for 60 min in the above example before the animal was rewarmed. (B) Target temperature was reached in 2 h using the gradual cooling method and was maintained for 30 min before the animal was rewarmed. Shaded regions in both graphs represent time points in which target temperature was maintained. Dotted perpendicular lines in both graphs refer to the overall cooling duration. Please click here to view a larger version of this figure.
The procedures described here are easily implemented, non-invasive, and provide reliable and reproducible decreases in core body temperature to a desired target temperature.
There are several critical steps in the rapid cooling method which include the following. Do not oversaturate ethanol spray - care must be taken not to soak the animal in ethanol, as this will interfere with results. Monitor the animal during hypothermia induction- care must be taken to closely monitor animal responses to rapid hypothermia induction. A close watch of rectal temperature is important to ensure that temperature does not go below the desired target- if this happens, turn off the fans and allow the heat lamp to gently rewarm the animal back up to the required target.
In both methods, physiological monitoring is important to ensure appropriate adjustment of anesthetic dose. For prolonged cooling, inadequate anesthetic dose may prolong the duration of cooling. In this case, isoflurane concentration can be increased until an adequate cooling rate is achieved. Another critical step is the cross-calibration of temperature devices. When using a temperature probe regulated heat mat and a datalogger in the same experiment, it is best practice to cross-calibrate the datalogger with the rectal probe, in vivo, since there may be minor variations in the recorded temperature of the two devices.
These methods are suitable for studies that wish to explore the use of hypothermia as a potential treatment for neurological disorders. The specific aim of the study should dictate which method is used. Both methods can be classed as systemic surface cooling, however the second method does not require any active cooling. The gradual cooling model described above has important potential applications for the use of hypothermia in ischemic stroke treatment. Long-duration hypothermia and their resulting complications pose a challenge to elderly stroke patients. Moreover, the shivering response makes it difficult to achieve target temperature in some patients10. While anti-shivering medication can help to reduce the shivering response, short-duration gradual cooling could more effectively ameliorate the issue. Having a shorter cooling period is also likely to reduce the incidence of pneumonia often reported in trials. Another potential benefit of this short-duration method is that the speed of rewarming might not be as important when compared to long-duration cooling. Very early clinical studies of long-duration cooling in stroke patients with large infarcts found that fast rewarming led to large elevations in intracranial pressure (ICP), which worsened outcome and was often fatal. This led to the development of gradual rewarming paradigms, which further extended the overall duration of cooling. Short duration cooling only maintains target temperature for a short period and may less likely result in rebound ICP. Previous work which has investigated hypothermia treatment for ICP elevation, using a similar rapid cooling and rewarming protocol as the ones described here, have shown no rebound ICP elevation after rewarming23,24.
Clinical trials of hypothermia for ischemic stroke treatment have not been able to translate the benefits of hypothermia that are reported in experimental studies. The mismatch in cooling rates and duration between experimental models and patients, are important variables that may account for this discrepancy. Having an experimental model of hypothermia that better resembles the clinical rate of cooling will allow for a more informed investigation into the benefits of hypothermia as a treatment measure for stroke patients.
The authors have nothing to disclose.
This project was funded by the University of Newcastle, Hunter Medical Research Institute (HMRI) Dalara Early Research Career Researcher Fellowship, NSW Health Early-Mid Career Research Fellowship, and National Health and Medical Research Council (NHMRC) Australia.
|ThermoFisher Scientific/ Ajax Finechem
|Diluted with deionized water to give 70 % ethanol
|Antiseptic solution (Chlorhexidine)
|Brushless fan motor
|2 x 12 V/130 mA
|240 V 50/60 Hz
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