Name: Video: Short-Duration Hypothermia Induction in Rats using Models for Studies examining Clinical Relevance and Mechanisms
Uploaded: 2021-03-03T15:00:00.000+00:00
Duration: 5 min

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,&#160;and National Health and Medical Research Council (NHMRC) Australia.

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.
<ol>
	<li>Anesthetize 10-12-week-old male outbred Wistar rat with isoflurane (5% for induction and 2-2.5% for maintenance) in a 50% N2 and 50% O2 mixture.</li>
	<li>Following induction, place the rat in the prone position on a surgical heat mat.</li>
	<li>Position the rat so that the nose is sitting in the nose cone. Secure the nose with surgical tape to ensure no gases are escaping.</li>
	<li>Shave the fur from the lower right abdomen and inject the site subcutaneously with a local anesthetic, Bupivacaine 0.2 mL, 0.05%.</li>
	<li>Apply antiseptic solution to the freshly shaved region.</li>
	<li>Using sterilized surgical tools, make a 2 cm longitudinal incision along the right abdominal region, proximal to the right thigh. Make the incision deep enough to expose the space at the ventral thigh crease.</li>
	<li>Use hemostats and forceps to create a &#39;pocket&#39; under the skin that is large enough to hold the device.</li>
	<li>Insert the temperature monitoring datalogger device into the pocket and close the muscle and skin using 5-0 silk sutures. The subcutaneous method described here is preferred to the intra-peritoneal method as it is less invasive and allows for better recovery after the procedure.</li>
	<li>Ensure datalogger and rectal probe are cross calibrated for temperature monitoring (see Discussion).</li>
	<li>Ensure the datalogger is not resting against the animal&#39;s heat mat after insertion, as this will influence temperature readings.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62325/62325fig01.jpg" /> 
Figure 1:&#160;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. <a href="https://www.jove.com/files/ftp_upload/62325/62325fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. Induction of active (fast) hypothermia for mechanistic studies
<ol>
	<li>Set up for hypothermia (see Figure 2). Place two retort stands with clamps at either side of the rat&#39;s body.</li>
	<li>Attach a 60 mm 12 v/130 mA fan to each retort stand ensuring that the fans are aimed towards the lower back of the rat. The distance between the clamp and the rat is approximately 20 cm. The fan used must have a speed of 4,000 rpm.</li>
	<li>Have an animal heat lamp ready either at the side or&#160;on a third retort stand.</li>
	<li>Commence hypothermia by adjusting the animal heat mat to the desired target temperature. In this example, 32.5 &#176;C is the target temperature (3.75 on the temperature control unit).</li>
	<li>Turn both fans on and apply three to four sprays of 70% ethanol (standard plastic spray bottle) on the lower back of the rat. Ruffle animal fur when spraying for faster cooling induction. 
	NOTE: Ethanol is used as a preferred solution over water because it has a faster rate of evaporation and therefore results in more rapid hypothermia induction.</li>
	<li>Take care not to oversaturate the fur, since this can contribute to overshooting the target temperature. 
	NOTE: The fans will accelerate ethanol evaporation and the cooling process.</li>
	<li>Allow short intervals between ethanol applications while keeping a close eye on the rectal temperature of the rat.</li>
	<li>Cease any further application of ethanol once rectal temperature reaches within 1 &#176;C of target temperature.</li>
	<li>Turn off both fans once the temperature has reached within 0.5 &#176;C of target temperature (33 &#176;C in this case). 
	&#8203;NOTE: Turning off fans before the target temperature is reached helps prevent the rat from overcooling beyond required temperature.</li>
	<li>Allow the temperature to drop to 32.5 &#176;C.
	<ol>
		<li>If overcooling does occur, use animal heat lamp to mildly warm the animal back up to target. The assistance of one fan may be used to prevent a rewarming overshoot.</li>
	</ol>
	</li>
	<li>Once target temperature is reached and has stabilized, continue to monitor temperature. Temperature will usually remain very stable for the remainder of the hypothermia period without the need for spraying, use of fans or use of heat lamp.</li>
	<li>To rewarm the animal at the end of hypothermia, adjust the heat mat temperature back to 37 &#176;C (6 on the temperature control unit used in this example) and allow the animal to thermoregulate over a 30 min period. 
	&#8203;NOTE: Temperature settings on temperature control units may vary and it may, therefore, be necessary to determine settings for target hypothermia and normothermia on individual devices.</li>
</ol>
3. Induction of clinically achievable gradual onset hypothermia without active skin cooling
<ol>
	<li>Achieve hypothermia by reducing the temperature of the core temperature regulated homeothermic heat mat in small increments to the required target temperature. In the illustrated example (Figure 3B), a 1 &#176;C increment every 30 min was used.</li>
	<li>Cool the animal to target temperature over the desired period (2 h in the example described). Once cooled, maintain at target for the desired interval. Usually, no further intervention is required if they are maintained on the homeothermic heat mat set for the desired target temperature.</li>
	<li>No external cooling is necessary with this protocol as anesthesia prevents the normal regulation of core body temperature. 
	&#8203;NOTE: Isoflurane requirements decline with hypothermia. In most animals, a starting isoflurane concentration of 2% can be reduced in 0.1% increments every 20-30 min to 1.5% to maintain stable respiratory rate (&#62;50 breaths/min), heart rate and blood pressure, and preserve suppression of reflex responses.</li>
	<li>To rewarm the animal after hypothermia, adjust the heat mat to allow the animal to rewarm over the desired interval. In the example, rewarming with a single adjustment to 37 &#176;C (6 on the FHC temperature control unit used in the example) was achieved over a 30 min period.
	<ol>
		<li>For longer-term studies that require animal recovery, keep animals in a cage that is placed half over a heat mat to allow the animal to thermoregulate and to avoid inadvertent post-operative hypothermia.</li>
		<li>Rectal paracetamol (250 mg/kg) can be administered for recovery and ovrenight pain relief. Subcutaneous saline injections (2 x 1.5 mL) can also be given to prevent dehydration from anesthesia and surgical procedures.</li>
	</ol>
	</li>
</ol>

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<li>Lyden, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Results+of+the+ICTuS+2+Trial+%28Intravascular+cooling+in+the+treatment+of+stroke+2%29%5BTitle%5D)+AND+%22Stroke%22%5BJournal%5D)">Results of the ICTuS 2 Trial (Intravascular cooling in the treatment of stroke 2).</a> Stroke. 47 (12), 2888-2895 (2016).</li>
<li>vander Worp, H. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Therapeutic+hypothermia+for+acute+ischaemic+stroke.+Results+of+a+European+multicentre%2C+randomised%2C+phase+III+clinical+trial%5BTitle%5D)+AND+%22European+Stroke+Journal%22%5BJournal%5D)">Therapeutic hypothermia for acute ischaemic stroke. Results of a European multicentre, randomised, phase III clinical trial.</a> European Stroke Journal. 4 (3), 254-262 (2019).</li>
<li>Wu, T. C., Grotta, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hypothermia+for+acute+ischaemic+stroke%5BTitle%5D)+AND+%22Lancet+Neurology%22%5BJournal%5D)">Hypothermia for acute ischaemic stroke.</a> Lancet Neurology. 12 (3), 275-284 (2013).</li>
<li>Hemmen, T. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intravenous+thrombolysis+plus+hypothermia+for+acute+treatment+of+ischemic+stroke+%28ICTuS-L%29%3A+final+results%5BTitle%5D)+AND+%22Stroke%22%5BJournal%5D)">Intravenous thrombolysis plus hypothermia for acute treatment of ischemic stroke (ICTuS-L): final results.</a> Stroke. 41 (10), 2265-2270 (2010).</li>
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<li>Lamb, J. A., Rajput, P. S., Lyden, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Novel+method+for+inducing+rapid%2C+controllable+therapeutic+hypothermia+in+rats+using+a+perivascular+implanted+closed-loop+cooling+circuit%5BTitle%5D)+AND+%22Journal+of+Neuroscience+Methods%22%5BJournal%5D)">Novel method for inducing rapid, controllable therapeutic hypothermia in rats using a perivascular implanted closed-loop cooling circuit.</a> Journal of Neuroscience Methods. 267, 55-61 (2016).</li>
<li>Dumitrascu, O. M., Lamb, J., Lyden, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Still+cooling+after+all+these+years%3A+Meta-analysis+od+pre-clinical+trials+of+therapeutic+hypothermia+for+acute+ischemic+stroke%5BTitle%5D)+AND+%22Journal+of+Cerebral+Blood+Flow+and+Metabolism%22%5BJournal%5D)">Still cooling after all these years: Meta-analysis od pre-clinical trials of therapeutic hypothermia for acute ischemic stroke.</a> Journal of Cerebral Blood Flow and Metabolism. 36 (7), 1157-1164 (2016).</li>
<li>Wu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hypothermia+neuroprotection+against+ischemic+stroke%3A+The+2019+update%5BTitle%5D)+AND+%22Journal+of+Cerebral+Blood+Flow+and+Metabolism%22%5BJournal%5D)">Hypothermia neuroprotection against ischemic stroke: The 2019 update.</a> Journal of Cerebral Blood Flow and Metabolism. 40 (3), 461-481 (2020).</li>
<li>Hemmen, T. M., Lyden, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hypothermia+after+acute+ischemic+stroke%5BTitle%5D)+AND+%22Journal+of+Neurotrauma%22%5BJournal%5D)">Hypothermia after acute ischemic stroke.</a> Journal of Neurotrauma. 26 (3), 387-391 (2009).</li>
<li>Colbourne, F., Sutherland, G. R., Auer, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+automated+system+for+regulating+brain+temperature+in+awake+and+freely+moving+rodents%5BTitle%5D)+AND+%22Journal+of+Neuroscience+Methods%22%5BJournal%5D)">An automated system for regulating brain temperature in awake and freely moving rodents.</a> Journal of Neuroscience Methods. 67 (2), 185-190 (1996).</li>
<li>Campbell, K., Meloni, B. P., Zhu, H., Knuckey, N. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Magnesium+treatment+and+spontaneous+mild+hypothermia+after+transient+focal+cerebral+ischemia+in+the+rat%5BTitle%5D)+AND+%22Brain+Research+Bulletin%22%5BJournal%5D)">Magnesium treatment and spontaneous mild hypothermia after transient focal cerebral ischemia in the rat.</a> Brain Research Bulletin. 77 (5), 320-322 (2008).</li>
<li>Jespersen, B., Knupp, L., Northcott, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Femoral+arterial+and+venous+catheterization+for+blood+sampling%2C+drug+administration+and+conscious+blood+pressure+and+heart+rate+measurements%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Femoral arterial and venous catheterization for blood sampling, drug administration and conscious blood pressure and heart rate measurements.</a> Journal of Visualized Experiments. (59), e3496(2012).</li>
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</ol>

The authors have nothing to disclose.

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.

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&#39;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 &#176;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.

<table><tbody><tr><td>Absolute ethanol</td><td>ThermoFisher Scientific/ Ajax Finechem</td><td>AJA214-20LPL</td><td>Diluted with deionized water to give 70 % ethanol</td></tr><tr><td>Antiseptic solution (Chlorhexidine)</td><td>David Craig</td><td>A2957</td><td></td></tr><tr><td>Anaesthetic (Marcain)</td><td>Aspen</td><td>PS13977</td><td></td></tr><tr><td>Brushless fan motor</td><td>Sirocco</td><td>YX2505</td><td>2 x 12 V/130 mA</td></tr><tr><td>Heat lamp</td><td>Reptile One</td><td>AC220</td><td>240 V 50/60 Hz</td></tr><tr><td>Heat pad</td><td>FHC, Inc</td><td>40-90-2</td><td></td></tr><tr><td>Rectal probe</td><td>FHC, Inc</td><td>40-90-5D-02</td><td></td></tr><tr><td>Temperature controller</td><td>FHC, Inc</td><td>40-90-8D</td><td></td></tr><tr><td>Temperature Datalogger</td><td>Maxim</td><td>DS1922L-F5</td><td></td></tr></tbody></table>

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.

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 &#176;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 &#176;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 &#176;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.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62325/62325fig02.jpg" /> 
Figure 2:&#160;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. <a href="https://www.jove.com/files/ftp_upload/62325/62325fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62325/62325fig03.jpg" /> 
Figure 3:&#160;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. <a href="https://www.jove.com/files/ftp_upload/62325/62325fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

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 ...

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Research

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Medicine

2.9K Views.  University of Newcastle. 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 us...

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

Strategies for Study of Neuroprotection from Cold-preconditioning

Neurological injury is a frequent cause of morbidity and mortality from general anesthesia and related surgical procedures that could be alleviated by development of effective, easy to administer and safe preconditioning treatments. We seek to define the neural immune signaling responsible for cold-preconditioning as means to identify novel targets for therapeutics development to protect brain before injury onset. Low-level pro-inflammatory mediator signaling changes over time are essential for cold-preconditioning neuroprotection. This signaling is consistent with the basic tenets of physiological conditioning hormesis, which require that irritative stimuli reach a threshold magnitude with sufficient time for adaptation to the stimuli for protection to become evident.
Accordingly, delineation of the immune signaling involved in cold-preconditioning neuroprotection requires that biological systems and experimental manipulations plus technical capacities are highly reproducible and sensitive. Our approach is to use hippocampal slice cultures as an in vitro model that closely reflects their in vivo counterparts with multi-synaptic neural networks influenced by mature and quiescent macroglia / microglia. This glial state is particularly important for microglia since they are the principal source of cytokines, which are operative in the femtomolar range. Also, slice cultures can be maintained in vitro for several weeks, which is sufficient time to evoke activating stimuli and assess adaptive responses. Finally, environmental conditions can be accurately controlled using slice cultures so that cytokine signaling of cold-preconditioning can be measured, mimicked, and modulated to dissect the critical node aspects. Cytokine signaling system analyses require the use of sensitive and reproducible multiplexed techniques. We use quantitative PCR for TNF-&alpha; to screen for microglial activation followed by quantitative real-time qPCR array screening to assess tissue-wide cytokine changes. The latter is a most sensitive and reproducible means to measure multiple cytokine system signaling changes simultaneously. Significant changes are confirmed with targeted qPCR and then protein detection. We probe for tissue-based cytokine protein changes using multiplexed microsphere flow cytometric assays using Luminex technology. Cell-specific cytokine production is determined with double-label immunohistochemistry. Taken together, this brain tissue preparation and style of use, coupled to the suggested investigative strategies, may be an optimal approach for identifying potential targets for the development of novel therapeutics that could mimic the advantages of cold-preconditioning.

We seek to define the neural immune signaling responsible for cold-preconditioning as means to identify novel targets for therapeutics development to protect brain before injury onset. We present strategies for such work that require biological systems, experimental manipulations plus technical capacities that are highly reproducible and sensitive.

Neurological injury is a frequent cause of morbidity and mortality from general anesthesia and related surgical procedures that could be alleviated by ...

Neuroscience

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.

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 ...

The Hypoxic Ischemic Encephalopathy Model of Perinatal Ischemia

Hypoxic-Ischemic Encephalopathy (HIE) is the consequence of systemic asphyxia occurring at birth. Twenty five percent of neonates with HIE develop severe and permanent neuropsychological sequelae, including mental retardation, cerebral palsy, and epilepsy. The outcomes of HIE are devastating and permanent, making it critical to identify and develop therapeutic strategies to reduce brain injury in newborns with HIE. To that end, the neonatal rat model for hypoxic-ischemic brain injury has been developed to model this human condition. The HIE model was first validated by Vannucci et al 1 and has since been extensively used to identify mechanisms of brain injury resulting from perinatal hypoxia-ischemia 2 and to test potential therapeutic interventions 3,4. The HIE model is a two step process and involves the ligation of the left common carotid artery followed by exposure to a hypoxic environment. Cerebral blood flow (CBF) in the hemisphere ipsilateral to the ligated carotid artery does not decrease because of the collateral blood flow via the circle of Willis; however with lower oxygen tension, the CBF in the ipsilateral hemisphere decreases significantly and results in unilateral ischemic injury. The use of 2,3,5-triphenyltetrazolium chloride (TTC) to stain and identify ischemic brain tissue was originally developed for adult models of rodent cerebral ischemia 5, and is used to evaluate the extent of cerebral infarctin at early time points up to 72 hours after the ischemic event 6. In this video, we demonstrate the hypoxic-ischemic injury model in postnatal rat brain and the evaluation of the infarct size using TTC staining.

The postnatal rat model for hypoxic-ischemic brain injury is a well-established model of human neonatal hypoxic ischemic encephalopathy (HIE). In this article, we describe the model of HIE in post-natal rat pups.

Hypoxic-Ischemic Encephalopathy (HIE) is the consequence of systemic asphyxia occurring at birth. Twenty five percent of neonates with HIE develop severe ...

Biology

2-Vessel Occlusion/Hypotension: A Rat Model of Global Brain Ischemia

Cardiac arrest followed by resuscitation often results in dramatic brain damage caused by ischemia and subsequent reperfusion of the brain. Global brain ischemia produces damage to specific brain regions shown to be highly sensitive to ischemia 1. Hippocampal neurons have higher sensitivity to ischemic insults compared to other cell populations, and specifically, the CA1 region of the hippocampus is particularly vulnerable to ischemia/reperfusion 2.
The design of therapeutic interventions, or study of mechanisms involved in cerebral damage, requires a model that produces damage similar to the clinical condition and in a reproducible manner. Bilateral carotid vessel occlusion with hypotension (2VOH) is a model that produces reversible forebrain ischemia, emulating the cerebral events that can occur during cardiac arrest and resuscitation. We describe a model modified from Smith et al. (1984) 2, as first presented in its current form in Sanderson, et al. (2008) 3, which produces reproducible injury to selectively vulnerable brain regions 3-6. The reliability of this model is dictated by precise control of systemic blood pressure during applied hypotension, the duration of ischemia, close temperature control, a specific anesthesia regimen, and diligent post-operative care. An 8-minute ischemic insult produces cell death of CA1 hippocampal neurons that progresses over the course of 6 to 24 hr of reperfusion, while less vulnerable brain regions are spared. This progressive cell death is easily quantified after 7-14 days of reperfusion, as a near complete loss of CA1 neurons is evident at this time.
In addition to this brain injury model, we present a method for CA1 damage quantification using a simple, yet thorough, methodology. Importantly, quantification can be accomplished using a simple camera-mounted microscope, and a free ImageJ (NIH) software plugin, obviating the need for cost-prohibitive stereology software programs and a motorized microscopic stage for damage assessment.

Bilateral carotid occlusion coupled with systemic hypotension produces global brain ischemia in the rat, resulting in damage to the hippocampus with reproducible severity. Animal subjects are impaired with predictable patterns of brain damage, they recover expediently, and mortality rates are comparatively low.

Cardiac arrest followed by resuscitation often results in dramatic brain damage caused by ischemia and subsequent reperfusion of the brain. Global brain ...

Remote Limb Ischemic Preconditioning:  A Neuroprotective Technique in Rodents

Sublethal ischemia protects tissues against subsequent, more severe ischemia through the upregulation of endogenous mechanisms in the affected tissue. Sublethal ischemia has also been shown to upregulate protective mechanisms in remote tissues. A brief period of ischemia (5-10 min) in the hind limb of mammals induces self-protective responses in the brain, lung, heart and retina. The effect is known as remote ischemic preconditioning (RIP). It is a therapeutically promising way of protecting vital organs, and is already under clinical trials for heart and brain injuries. This publication demonstrates a controlled, minimally invasive method of making a limb &#8211; specifically the hind limb of a rat &#8211; ischemic. A blood pressure cuff developed for use in human neonates is connected to a manual sphygmomanometer and used to apply 160 mmHg pressure around the upper part of the hind limb. A probe designed to detect skin temperature is used to verify the ischemia, by recording the drop in skin temperature caused by pressure-induced occlusion of the leg arteries, and the rise in temperature which follows release of the cuff. This method of RIP affords protection to the rat retina against bright light-induced damage and degeneration.

Remote ischemic preconditioning (RIP) is a method of conditioning tissues against damaging stress. We have established a method of remote ischemia at the hind limb, by inflating a sphygmomanometer cuff for 5-10 min. The neuroprotective capabilities of RIP have been demonstrated in a model of retinal degeneration in rodents.

Sublethal ischemia protects tissues against subsequent, more severe ischemia through the upregulation of endogenous mechanisms in the affected tissue. ...

A Recovery Cardiopulmonary Bypass Model Without Transfusion or Inotropic Agents in Rats

Cardiopulmonary bypass (CPB) is indispensable in cardiovascular surgery. Despite the dramatic refinement of CPB technique and devices, multi-organ complications related to prolonged CPB still compromise the outcome of cardiovascular surgeries, and may worsen postoperative morbidity and mortality. Animal models recapitulating the clinical usage of CPB enable the clarification of the pathophysiological processes that occur during CPB, and facilitate pre-clinical studies to develop strategies protecting against these complications. Rat CPB models are advantageous because of their greater cost-effectiveness, convenient experimental processes, abundant testing methods at the genetic or protein levels, and genetic consistency. They can be used for investigating the immune system activation and synthesis of proinflammatory cytokines, compliment activation, and production of oxygen free radicals. The rat models have been refined and have gradually taken the place of large-animal models. Here, we describe a simple CPB model without transfusion and/or inotropic agents in a rat. This recovery model allows the study of the long-term multiple organ sequelae of CPB.

Here, we present a protocol to describe a simple recovery cardiopulmonary bypass model without transfusion or inotropic agents in a rat. This model allows the study of the long-term multiple organ sequelae of cardiopulmonary bypass.

Cardiopulmonary bypass (CPB) is indispensable in cardiovascular surgery. Despite the dramatic refinement of CPB technique and devices, multi-organ ...

Immunology and Infection

Esophageal Heat Transfer for Patient Temperature Control and Targeted Temperature Management

Controlling patient temperature is important for a wide variety of clinical conditions. Cooling to normal or below normal body temperature is often performed for neuroprotection after ischemic insult (e.g. hemorrhagic stroke, subarachnoid hemorrhage, cardiac arrest, or other hypoxic injury). Cooling from febrile states treats fever and reduces the negative effects of hyperthermia on injured neurons. Patients are warmed in the operating room to prevent inadvertent perioperative hypothermia, which is known to cause increased blood loss, wound infections, and myocardial injury, while also prolonging recovery time. There are many reported approaches for temperature management, including improvised methods that repurpose standard supplies (e.g., ice, chilled saline, fans, blankets) but more sophisticated technologies designed for temperature management are typically more successful in delivering an optimized protocol. Over the last decade, advanced technologies have developed around two heat transfer methods: surface devices (water blankets, forced-air warmers) or intravascular devices (sterile catheters requiring vascular placement). Recently, a novel device became available that is placed in the esophagus, analogous to a standard orogastric tube, that provides efficient heat transfer through the patient&#39;s core. The device connects to existing heat exchange units to allow automatic patient temperature management via a servo mechanism, using patient temperature from standard temperature sensors (rectal, Foley, or other core temperature sensors) as the input variable. This approach eliminates vascular placement complications (deep venous thrombosis, central line associated bloodstream infection), reduces obstruction to patient access, and causes less shivering when compared to surface approaches. Published data have also shown a high degree of accuracy and maintenance of target temperature using the esophageal approach to temperature management. Therefore, the purpose of this method is to provide a low-risk alternative method for controlling patient temperature in critical care settings.

This study presents a novel method to provide efficient patient temperature control for cooling or warming patients. A single use, triple lumen device is placed into the esophagus, analogous to a standard orogastric tube, and connects to existing heat exchange units to perform automatic patient temperature management.

Controlling patient temperature is important for a wide variety of clinical conditions. Cooling to normal or below normal body temperature is often ...

Establishment of Deep Hypothermic Circulatory Arrest in Rats

Deep hypothermic circulatory arrest (DHCA) is routinely applied during surgeries for complex congenital heart disease and aortic arch disease. The present study aims to provide a method for establishing DHCA in rats. To evaluate the impact of the DHCA process on vital signs, a normal temperature cardiopulmonary bypass (CPB) rat model without circulatory arrest was used as a control. As expected, DHCA led to a significant decrease in body temperature and mean arterial blood pressure. The blood gas analysis indicated that DHCA increased lactic acid levels but did not influence the blood pH and the concentrations of hemoglobin, hematocrit, Na+, Cl&#8722;, K+, and glucose. Furthermore, compared with the normal temperature CPB rats, the results of the transmission electron microscopy showed a mild increase in hippocampal autophagosomes in the DHCA rats.

This protocol presents the establishment of deep hypothermic circulatory arrest in rats, which can be applied to investigate systemic inflammatory response syndrome, ischemia/reperfusion injury, oxidative stress, neuroinflammation, etc.

Deep hypothermic circulatory arrest (DHCA) is routinely applied during surgeries for complex congenital heart disease and aortic arch disease. The present ...

In vitro Assessment of Myocardial Protection following Hypothermia-Preconditioning in a Human Cardiac Myocytes Model

Ischemia/reperfusion-derived myocardial dysfunction is a common clinical scenario in patients after cardiac surgery. In particular, the sensitivity of cardiomyocytes to ischemic injury is higher than that of other cell populations. At present, hypothermia affords considerable protection against an expected ischemic insult. However, investigations into complex hypothermia-induced molecular changes remain limited. Therefore, it is essential to identify a culture condition similar to in vivo conditions that can induce damage similar to that observed in the clinical condition in a reproducible manner. To mimic ischemia-like conditions in vitro, the cells in these models were treated by oxygen/glucose deprivation (OGD). In addition, we applied a standard time-temperature protocol used during cardiac surgery. Furthermore, we propose an approach to use a simple but comprehensive method for the quantitative analysis of myocardial injury. Apoptosis and expression levels of apoptosis-associated proteins were assessed by flow cytometry and using an ELISA kit. In this model, we tested a hypothesis regarding the effects of different temperature conditions on cardiomyocyte apoptosis in vitro. The reliability of this model depends on strict temperature control, controllable experimental procedures, and stable experimental results. Additionally, this model can be used to study the molecular mechanism of hypothermic cardioprotection, which may have important implications for the development of complementary therapies for use with hypothermia.

The distinct effects of different degrees of hypothermia on myocardial protection have not been thoroughly evaluated. The goal of the present study was to quantify the levels of cell death following different hypothermia treatments in a human cardiomyocyte-based model, laying the foundation for future in-depth molecular research.

Ischemia/reperfusion-derived myocardial dysfunction is a common clinical scenario in patients after cardiac surgery. In particular, the sensitivity of ...

Early Pathological and Magnetic Resonance Detection of Cerebral Injury Using a Rat Model of Neonatal Hypoxic Ischemic Encephalopathy

Perinatal hypoxic-ischemic encephalopathy (HIE) is an acute disease that may afflict newborns, resulting in variable long- and short-term neurodevelopmental outcomes. Early diagnosis is critical to identifying infants who may benefit from intervention; however, early diagnosis relies heavily on clinical criteria. No molecular or radiological tests have shown promise in detecting early cerebral injury. Studies have shown that magnetic resonance imaging (MRI) can show changes in both blood flow/ischemia and metabolic disruption. However, they have all been used to evaluate the secondary phase of the disease (&gt;12 h) after the onset of the injury. Early diagnosis is critical to rapidly starting therapeutic hypothermia in eligible infants, which is currently recommended to be initiated within 6 h of birth. The rat model of hypoxic-ischemic injury was developed in 1981 and has been validated and used extensively to study changes in brain perfusion, cerebral injury markers, and morphology. However, it has primarily been used as a "late model", evaluating injury several days after the initial ischemic insult. The model has been known to have poor sensitivity in evaluating reliable and reproducible early cerebral changes. The objective of this study was to develop a reliable model to study early gross morphological and radiological markers of HIE using pathological staining and cerebral magnetic resonance imaging/magnetic resonance spectroscopy.

The present protocol describes a rodent model of newborn hypoxic-ischemic injury for identifying early changes in cerebral tissue by gross morphology and magnetic resonance imaging. This has benefits over existing models, which can be used to study late injury but do not allow the evaluation of reproducible early changes.