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.remotexs.ntu.edu.sg/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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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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 ...

short-duration-hypothermia-induction-rats-using-models-for-studies

Nanyang Technological University

Research

JoVE Journal

Medicine

2.8K 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

Using Visual and Narrative Methods to Achieve Fair Process in Clinical Care

The Institute of Medicine has targeted patient-centeredness as an important area of quality improvement. A major dimension of patient-centeredness is respect for patient's values, preferences, and expressed needs. Yet specific approaches to gaining this understanding and translating it to quality care in the clinical setting are lacking. From a patient perspective quality is not a simple concept but is best understood in terms of five dimensions: technical outcomes; decision-making efficiency; amenities and convenience; information and emotional support; and overall patient satisfaction. Failure to consider quality from this five-pronged perspective results in a focus on medical outcomes, without considering the processes central to quality from the patient's perspective and vital to achieving good outcomes. In this paper, we argue for applying the concept of fair process in clinical settings. Fair process involves using a collaborative approach to exploring diagnostic issues and treatments with patients, explaining the rationale for decisions, setting expectations about roles and responsibilities, and implementing a core plan and ongoing evaluation. Fair process opens the door to bringing patient expertise into the clinical setting and the work of developing health care goals and strategies. This paper provides a step by step illustration of an innovative visual approach, called photovoice or photo-elicitation, to achieve fair process in clinical work with acquired brain injury survivors and others living with chronic health conditions. Applying this visual tool and methodology in the clinical setting will enhance patient-provider communication; engage patients as partners in identifying challenges, strengths, goals, and strategies; and support evaluation of progress over time. Asking patients to bring visuals of their lives into the clinical interaction can help to illuminate gaps in clinical knowledge, forge better therapeutic relationships with patients living with chronic conditions such as brain injury, and identify patient-centered goals and possibilities for healing. The process illustrated here can be used by clinicians, (primary care physicians, rehabilitation therapists, neurologists, neuropsychologists, psychologists, and others) working with people living with chronic conditions such as acquired brain injury, mental illness, physical disabilities, HIV/AIDS, substance abuse, or post-traumatic stress, and by leaders of support groups for the types of patients described above and their family members or caregivers.

This paper illustrates an innovative visual approach (photovoice or photo-elicitation) to achieve fair process in clinical care for patients living with chronic health conditions, illuminate gaps in clinical knowledge, forge better therapeutic relationships, and identify patient-centered goals and possibilities for healing.

The Institute of Medicine has targeted patient-centeredness as an important area of quality improvement. A major dimension of patient-centeredness is ...

MRI and PET in Mouse Models of Myocardial Infarction

Myocardial infarction is one of the leading causes of death in the Western world. The similarity of the mouse heart to the human heart has made it an ideal model for testing novel therapeutic strategies.
In vivo magnetic resonance imaging (MRI) gives excellent views of the heart noninvasively with clear anatomical detail, which can be used for accurate functional assessment. Contrast agents can provide basic measures of tissue viability but these are nonspecific. Positron emission tomography (PET) is a complementary technique that is highly specific for molecular imaging, but lacks the anatomical detail of MRI. Used together, these techniques offer a sensitive, specific and quantitative tool for the assessment of the heart in disease and recovery following treatment.
In this paper we explain how these methods are carried out in mouse models of acute myocardial infarction. The procedures described here were designed for the assessment of putative protective drug treatments. We used MRI to measure systolic function and infarct size with late gadolinium enhancement, and PET with fluorodeoxyglucose (FDG) to assess metabolic function in the infarcted region. The paper focuses on practical aspects such as slice planning, accurate gating, drug delivery, segmentation of images, and multimodal coregistration. The methods presented here achieve good repeatability and accuracy maintaining a high throughput.

We describe how to perform MRI and PET imaging of the mouse heart. The protocol is tailored to assess treatment efficacy in models of myocardial infarction and heart failure.

Myocardial infarction is one of the leading causes of death in the Western world. The similarity of the mouse heart to the human heart has made it an ...

Gene-environment Interaction Models to Unmask Susceptibility Mechanisms in Parkinson's Disease

Lipoxygenase (LOX) activity has been implicated in neurodegenerative disorders such as Alzheimer&#39;s disease, but its effects in Parkinson&#39;s disease (PD) pathogenesis are less understood. Gene-environment interaction models have utility in unmasking the impact of specific cellular pathways in toxicity that may not be observed using a solely genetic or toxicant disease model alone.&#160;To evaluate if distinct LOX isozymes selectively contribute to PD-related neurodegeneration, transgenic (i.e. 5-LOX and 12/15-LOX deficient) mice can be challenged with a toxin that mimics cell injury and death in the disorder.&#160;Here we describe the use of a neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which produces a nigrostriatal lesion to elucidate the distinct contributions of LOX isozymes to neurodegeneration related to PD. The use of MPTP in mouse, and nonhuman primate, is well-established to recapitulate the nigrostriatal damage in PD.&#160;The extent of MPTP-induced lesioning is measured by HPLC analysis of dopamine and its metabolites and semi-quantitative Western blot analysis of striatum for tyrosine hydroxylase (TH), the rate-limiting enzyme for the synthesis of dopamine. To assess inflammatory markers, which may demonstrate LOX isozyme-selective sensitivity, glial fibrillary acidic protein (GFAP) and Iba-1 immunohistochemistry are performed on brain sections containing substantia nigra, and GFAP Western blot analysis is performed on striatal homogenates.&#160;This experimental approach can provide novel insights into gene-environment interactions underlying nigrostriatal degeneration and PD.

Lipoxygenase (LOX) isozymes can generate products that may increase or decrease neuroinflammation and neurodegeneration.&#160;A gene-environment interaction study could identify LOX isozyme-specific effects. Using the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of nigrostriatal damage in two LOX isozyme-deficient transgenic lines allows for comparison of the contribution of LOX isozymes on dopaminergic integrity and inflammation.

Lipoxygenase (LOX) activity has been implicated in neurodegenerative disorders such as Alzheimer&#39;s disease, but its effects in Parkinson&#39;s disease ...

Handling of the Cotton Rat in Studies for the Pre-clinical Evaluation of Oncolytic Viruses

Oncolytic viruses are a novel anticancer therapy with the ability to target tumor cells, while leaving healthy cells intact. For this strategy to be successful, recent studies have shown that involvement of the host immune system is essential. Therefore, oncolytic virotherapy should be evaluated within the context of an immunocompetent model. Furthermore, the study of antitumor therapies in tolerized animal models may better recapitulate results seen in clinical trials. Cotton rats, commonly used to study respiratory viruses, are an attractive model to study oncolytic virotherapy as syngeneic models of mammary carcinoma and osteosarcoma are well established. However, there is a lack of published information on the proper handling procedure for these highly excitable rodents. The handling and capture approach outlined minimizes animal stress to facilitate experimentation. This technique hinges upon the ability of the researcher to keep calm during handling and perform procedures in a timely fashion. Finally, we describe how to prepare cotton rat mammary tumor cells for consistent subcutaneous tumor formation, and how to perform intratumoral and intraperitoneal injections. These methods can be applied to a wide range of studies furthering the development of the cotton rat as a relevant pre-clinical model to study antitumor therapy.

Cotton rats are extremely excitable and have a strong flight-or-fight response. A handling method optimized to reduce the stress of the animals is described which will make cotton rats more accessible as a preclinical model.

Oncolytic viruses are a novel anticancer therapy with the ability to target tumor cells, while leaving healthy cells intact.  For this strategy to be ...

Ultrasonography in Experimental Reproductive Investigations on Rats

With the development of assisted reproductive technology and the ethical limitations of research on humans, rat animal models have been widely used in reproductive medicine. In the past, the study of reproductive system development in rodents has been based on one-time histological examination of excised tissues. Recently, with the development of high-resolution transabdominal ultrasound, high-quality sonography can now be performed to evaluate the reproductive organs of rats, allowing a new method for studying the reproductive system. Images were obtained using a high-resolution ultrasonographic system. Gynecological ultrasonography was performed on 28 eight-week-old non-pregnant rats and 5 pregnant Sprague-Dawley rats. We describe how to recognize organs of the reproductive system and associated structures in typical views during different phases of the estrus cycle. Color flow Doppler was used to measure uterine artery blood flow and evaluate uterine blood flow pattern changes during different stages of pregnancy. We have demonstrated that ultrasound exploration is a useful method for evaluating changes in internal reproductive organs. Its use raises the possibility of conducting additional experiments, including medical or surgical procedures, and provides the ability to monitor sonographic changes to internal organs without sacrificing animals.

This manuscript describes the utility of ultrasound performed on female rats to design experimental models for reproductive and gynecological investigation. A step-by-step explanation of how to perform ultrasonographic evaluation is shown.

With the development of assisted reproductive technology and the ethical limitations of research on humans, rat animal models have been widely used in ...

Methodology for Sputum Induction and Laboratory Processing

The technique of sputum induction and processing is a recognized non-invasive method allowing the collection and analysis of cells from the airways, which is interesting in various respiratory diseases like asthma, chronic obstructive pulmonary disease (COPD), chronic cough, or idiopathic pulmonary fibrosis. This technique is well tolerated, safe and non-invasive, but is currently limited to research services and specialized centers in clinical practice because it is technically demanding, time-consuming, and requires trained staff. The success rate of sputum induction and analysis is about 80%.
Here, we describe the induction and laboratory processing of sputum samples. Sputum is induced by inhalation of hypertonic or isotonic saline with salbutamol. For the processing, we use the whole sputum technique. Dithiothreitol (DTT) is used to allow mucolysis of sputum samples. The primary aim of sputum processing is to obtain a differential cell count to study the cell types present in the airway lumen. Additional analyses may also be performed on sputum supernatant and sputum cells, which may allow further investigation into inflammatory processes and immune mechanisms. Examples include studying mediators in sputum supernatant and performing a large spectrum of analysis on sputum cells such as flow cytometry, genomics, or proteomics.
Finally, representative results of sputum analysis in healthy controls, asthmatics, and COPD patients are presented.

Here we describe the technique of sputum induction. This protocol also explains the sputum processing to perform a differential cell count and to collect sputum supernatant and cells for further analysis.

The technique of sputum induction and processing is a recognized non-invasive method allowing the collection and analysis of cells from the airways, which ...

Sampling Strategies and Processing of Biobank Tissue Samples from Porcine Biomedical Models

In translational medical research, porcine models have steadily become more popular. Considering the high value of individual animals, particularly of genetically modified pig models, and the often-limited number of available animals of these models, establishment of (biobank) collections of adequately processed tissue samples suited for a broad spectrum of subsequent analyses methods, including analyses not specified at the time point of sampling, represent meaningful approaches to take full advantage of the translational value of the model. With respect to the peculiarities of porcine anatomy, comprehensive guidelines have recently been established for standardized generation of representative, high-quality samples from different porcine organs and tissues. These guidelines are essential prerequisites for the reproducibility of results and their comparability between different studies and investigators. The recording of basic data, such as organ weights and volumes, the determination of the sampling locations and of the numbers of tissue samples to be generated, as well as their orientation, size, processing and trimming directions, are relevant factors determining the generalizability and usability of the specimen for molecular, qualitative, and quantitative morphological analyses. Here, an illustrative, practical, step-by-step demonstration of the most important techniques for generation of representative, multi-purpose biobank specimen from porcine tissues is presented. The methods described here include determination of organ/tissue volumes and densities, the application of a volume-weighted systematic random sampling procedure for parenchymal organs by point-counting, determination of the extent of tissue shrinkage related to histological embedding of samples, and generation of randomly oriented samples for quantitative stereological analyses, such as isotropic uniform random (IUR) sections generated by the &#34;Orientator&#34; and &#34;Isector&#34; methods, and vertical uniform random (VUR) sections.

The practical application and performance of methods for generation of representative tissue samples of porcine animal models for a broad spectrum of downstream analyses in biobank projects are demonstrated, including volumetry, systematic random sampling, and differential processing of tissue samples for qualitative and quantitative morphologic and molecular analyses types.

In translational medical research, porcine models have steadily become more popular. Considering the high value of individual animals, particularly of ...

Assessment of Kidney Function in Mouse Models of Glomerular Disease

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function in diabetic nephropathy and podocyte-specific VEGF-A knock-out mice; therefore, this protocol describes the full kidney work-up used in our lab to assess these mouse models of glomerular disease, enabling a vast amount of information regarding kidney and glomerular function to be obtained from a single mouse. In comparison to alternative methods presented in the literature to assess glomerular function, the use of the method outlined in this paper enables the glomerular phenotype to be fully evaluated from multiple aspects. By using this method, the researcher can determine the kidney phenotype of the model and assess the mechanism as to why the phenotype develops. This vital information on the mechanism of disease is required when examining potential therapeutic avenues in these models. The methods allow for detailed functional assessment of the glomerular filtration barrier through measurement of the urinary albumin creatinine ratio and individual glomerular water permeability, as well as both structural and ultra-structural examination using the Periodic Acid Schiff stain and electron microscopy. Furthermore, analysis of the genes dysregulated at the mRNA and protein level enables mechanistic analysis of glomerular function. This protocol outlines the generic but adaptable methods that can be applied to all mouse models of glomerular disease.

This protocol describes a full kidney work-up that should be carried out in mouse models of glomerular disease. The methods allow for detailed functional, structural, and mechanistic analysis of glomerular function, which can be applied to all mouse models of glomerular disease.

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function ...

Semi-Minimal Invasive Method to Induce Myocardial Infarction in Rats and the Assessment of Cardiac Function by an Isolated Working Heart System

Myocardial infarction (MI) remains the main contributor to morbidity and mortality worldwide. Therefore, research on this topic is mandatory. An easily and highly reproducible MI induction procedure is required to obtain further insight and better understanding of the underlying pathological changes. This procedure can also be used to evaluate the effects or potency of new and promising treatments (as drugs or interventions) in acute MI, subsequent remodeling and heart failure (HF). After intubation and pre-operative preparation of the animal, an anesthetic protocol with isoflurane was performed, and the surgical procedure was conducted&#160;quickly. Using a minimally invasive approach, the left anterior descending artery (LAD) was located and occluded by a ligature. The occlusion can be performed acutely for subsequent reperfusion (ischemia/reperfusion injury). Alternatively, the vessel can be ligated permanently to investigate the development of chronic MI, remodeling or HF. Despite common pitfalls, the drop-out rates are minimal. Various treatments such as remote ischemic conditioning can be examined for their cardioprotective potential pre-, peri- and post-operatively. The post-operative recovery was quick as the anesthesia was precisely controlled and the duration of the operation was short. Post-operative analgesia was administered for three days. The minimally invasive procedure reduces the risk of infection and inflammation. Furthermore, it facilitates rapid recovery. The &#8220;working heart&#8221; measurements were performed ex vivo and enabled precise control of preload, afterload and flow. This procedure requires specific equipment and training for adequate performance. This manuscript provides a detailed step-by-step introduction for conducting these measurements.

This article presents an efficient method to perform myocardial ischemia and subsequent chronic reperfusion in rats using a minimally invasive approach. In addition, left ventricular hemodynamic function of rats is assessed by echocardiography and isolated working heart methods.&#160;

Myocardial infarction (MI) remains the main contributor to morbidity and mortality worldwide. Therefore, research on this topic is mandatory. An easily ...

Ex Vivo and In Vivo Animal Models for Mechanical and Chemical Injuries of Corneal Epithelium

Corneal injury to the ocular surface, including chemical burn and trauma, may cause severe scarring, symblepharon, corneal limbal stem cells deficiency, and result in a large, persistent corneal epithelial defect. Epithelial defect with the following corneal opacity and peripheral neovascularization result in irreversible visual impairment and hinder future management, especially keratoplasty. Since the animal model can be used as an effective drug development platform, models of corneal injury to the mouse and alkali burn to rabbit corneal epithelium are developed here. New Zealand white rabbit is used in the alkali burn model. Different concentrations of sodium hydroxide can be applied onto the central circular area of the cornea for 30 s under intramuscular and topical anesthesia. After copious isotonic normal saline irrigation, residual loose corneal epithelium was removed with corneal burr deep down to the Bowman's layer within this circular area. Wound healing was documented by fluorescein staining under Cobalt blue light. C57BL/6 mice were used in the traumatic model of murine corneal epithelium. The murine central cornea was marked using a skin punch, 2 mm in diameter, and then debrided by a corneal rust ring remover with a 0.5 mm burr under a stereomicroscope. These models can be prospectively used to validate the therapeutic effect of eye drops or mixed agents such as stem cells, which potentially facilitate corneal epithelial regeneration. By observing corneal opacity, peripheral neovascularization, and conjunctival congestion with stereomicroscope and imaging software, therapeutic effects in these animal models can be monitored.

Ex Vivo and In Vivo Animal Models for Mechanical and Chemical Injuries of Corneal Epithelium

Here, animal models based on mouse and rabbit are developed for mechanical and chemical injury of corneal epithelium to screen new therapeutics and the underlying mechanism.

Corneal injury to the ocular surface, including chemical burn and trauma, may cause severe scarring, symblepharon, corneal limbal stem cells deficiency, ...