Name: supramaximal intensity hypoxic exercise and vascular function assessment in mice
Uploaded: 2019-03-15T12:30:00
Description: Watch this Scientific Journal Video about Supramaximal Intensity Hypoxic Exercise and Vascular Function Assessment in Mice at JoVE.com

The authors would like to thank Danilo Gubian and Stephane Altaus from the Lausanne University Hospital (CHUV) mechanical workshop for helping create the hypoxic setup. The authors would also like to thank Diane Macabrey and Melanie Sipion for their help with training the animals.

The local state&#39;s animal care committee (Service de la Consommation et des Affaires V&#233;t&#233;rinaires [SCAV], Lausanne, Switzerland) approved all experiments (authorization VD3224; 01.06.2017) and all experiments were carried out in accordance with the relevant guidelines and regulations.
1. Animal housing and Preparation
<ol>
	<li>House 6 to 8 week-old C57BL/6J male mice in the animal facility for at least 1 week prior to the beginning of the experiments in order for the mice to ge...

<ol>
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The first objective of this study was to assess the feasibility of hypoxic high-intensity training in mice and to determine the adequate characteristics of the protocol that would be well tolerated by mice. Purposely, since there is no data using supramaximal (i.e., more than Vmax) intensity training in mice, we had to perform trials based on previous protocols developed with athletes, which consisted of four to five sets of five all-out sprints (about 200% of Vmax), interspersed with 20 s active re...

In hypoxia, the decreased inspired fraction of oxygen (O2) leads to hypoxemia (lowered arterial pressure in hypoxia) and an altered O2 transport capacity1. Acute hypoxia induces an increased sympathetic vasoconstrictor activity directed toward skeletal muscle2 and an opposed &#39;compensatory&#39; vasodilatation.
At submaximal intensity in hypoxia, this &#39;compensatory&#39; vasodilatation, relative to the same level of exercise under normoxic conditions, is well established3. This vasodilation is essential to ensure an augmented blood flow and maintenance (or limit the alteration) of oxygen delivery to the active muscles. Adenosine was shown to not have an independent role in this response, while nitric oxide (NO) seems the primary endothelial source since significant blunting of the augmented vasodilatation was reported with nitric oxide synthase (NOS) inhibition during hypoxic exercise4. Several other vasoactive substances are likely playing a role in the compensatory vasodilatation during a hypoxic exercise.
This enhanced hypoxic exercise hyperemia is proportional to the hypoxia-induced fall in arterial O2 content and is larger as the exercise intensity increases, for example during intense incremental exercise in hypoxia.
The NO-mediated component of the compensatory vasodilatation is regulated through different pathways with increasing exercise intensity3: if &#946;-adrenergic receptor-stimulated NO component appears paramount during low-intensity hypoxic exercise, the source of NO contributing to compensatory dilatation seems less dependent on &#946;-adrenergic mechanisms as the exercise intensity increases. There are other candidates for stimulating NO release during higher-intensity hypoxic exercise, such as ATP released from erythrocytes and/or endothelial-derived prostaglandins.
Supramaximal exercise in hypoxia (named repeated sprint training in hypoxia [RSH] in the exercise physiology literature) is a recent training method5 providing performance enhancement in team- or racket-sport players. This method differs from interval training in hypoxia performed at or near maximal speed6 (Vmax) since RSH performed at maximal intensity leads to a greater muscle perfusion and oxygenation7 and specific muscle transcriptional responses8. Several mechanisms have been proposed to explain the effectiveness of RSH: during sprints in hypoxia, the compensatory vasodilation and associated higher blood flow would benefit the fast-twitch fibers more than the slow-twitch fibers. Consequently, RSH efficiency is likely to be fiber-type selective and intensity dependent. We speculate that the improved responsiveness of the vascular system is paramount in RSH.
Exercise training has been extensively studied in mice, both in healthy individuals and in pathological mouse models9,10. The most common way to train mice is using a rodent treadmill, and the traditionally used regimen is low-intensity training, at 40%&#8211;60% of Vmax (determined using an incremental treadmill test11), for 30&#8211;60 min12,13,14,15. Maximal intensity interval training and its impact on pathologies have been widely studied in mice16,17; thus, interval training running protocols for mice have been developed. Those protocols usually consist of about 10 bouts of running at 80%&#8211;100% of Vmax on a rodent motorized treadmill, for 1&#8211;4 min, interspersed with active or passive rest16,18.
The interest in mice exercising at supramaximal intensity (i.e., above the Vmax) in hypoxia comes from previous results that the microvascular vasodilatory compensation and the intermittent exercise performance are both more increased at supramaximal than at maximal or moderate intensities. However, to our knowledge, there is no previous report of a supramaximal training protocol in mice, either in normoxia or in hypoxia.
The first aim of the present study was to test the feasibility of supramaximal intensity training in mice and the determination of a tolerable and adequate protocol (intensity, sprint duration, recovery, etc.). The second aim was to assess the effects of different training regimen in normoxia and hypoxia on the vascular function. Therefore, we test the hypotheses that (1) mice tolerate well supramaximal exercise in hypoxia, and (2) that this protocol induces a larger improvement in vascular function than exercise in normoxia but also than exercise in hypoxia at lower intensities.

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			<td height="21" style="height:21px;width:260px;">Cotton swab</td>
			<td style="width:191px;">Q-tip</td>
			<td style="width:119px;"></td>
			<td style="width:165px;"></td>
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		<tr height="67">
			<td height="67" style="height:67px;width:260px;">Gas mixer Sonimix 7100</td>
			<td style="width:191px;">LSI Swissgas, Geneva, Switzerland</td>
			<td style="width:119px;"></td>
			<td style="width:165px;">Gas-flow: 10 L/min and 1 L/min for O2 and CO2, respectively</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Hypoxic Box&#160;</td>
			<td style="width:191px;">Homemade</td>
			<td style="width:119px;"></td>
			<td>Made in Plexiglas</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Motorized rodents treadmill Panlab LE-8710</td>
			<td style="width:191px;">Bioseb, France</td>
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			<td></td>
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			<td height="63" style="height:63px;width:260px;">Oximeter Greisinger GOX 100</td>
			<td style="width:191px;">GREISINGER electronic Gmbh, Regenstauf, Germany</td>
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			<td></td>
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			<td height="21" style="height:21px;width:260px;">Sedacom software</td>
			<td style="width:191px;">Bioseb, France</td>
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			<td height="42" style="height:42px;width:260px;">Strain gauge</td>
			<td style="width:191px;">PowerLab/8SP; ADInstruments</td>
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supramaximal intensity hypoxic exercise and vascular function assessment in mice

Exercise training is an important strategy for maintaining health and preventing many chronic diseases. It is the first line of treatment recommended by international guidelines for patients suffering from cardiovascular diseases, more specifically, lower extremity artery diseases, where the patients' walking capacity is considerably altered, affecting their quality of life. 
Traditionally, both low continuous exercise and interval training have been used. Recently, supramaximal training has also been shown to improve athletes' performances via vascular adaptations, amongst other mechanisms. The combination of this type of training with hypoxia could bring an additional and/or synergic effect, which could be of interest for certain pathologies. Here, we describe how to perform supramaximal intensity training sessions in hypoxia on healthy mice at 150% of their maximal speed, using a motorized treadmill and a hypoxic box. We also show how to dissect the mouse in order to retrieve organs of interest, particularly the pulmonary artery, the abdominal aorta, and the iliac artery. Finally, we show how to perform ex vivo vascular function assessment on the retrieved vessels, using isometric tension studies.

To our knowledge, the present study is the first to describe a program of supramaximal intensity training in normoxia and in hypoxia for mice. In this protocol, mice ran four sets of five 10 s sprints with a 20 s recovery in between each sprint. The sets were interspersed with 5 min of recovery periods. It was unknown whether the mice would be capable of sustaining such a protocol and complete it properly. However, according to Figure 5, the body weight gain ...

Watch this Scientific Journal Video about Supramaximal Intensity Hypoxic Exercise and Vascular Function Assessment in Mice at JoVE.com

Supramaximal Intensity Hypoxic Exercise and Vascular Function Assessment in Mice

High-intensity training in hypoxia is a protocol that has been proven to induce vascular adaptations potentially beneficial in some patients and to improve athletes&#39; repeated sprint ability. Here, we test the feasibility of training mice using that protocol and identify those vascular adaptations using ex vivo vascular function assessment.

Exercise training is an important strategy for maintaining health and preventing many chronic diseases. It is the first line of treatment recommended by ...

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