The authors would like to acknowledge Dr. P. Karagiannis for reading the paper and Professor T. Hoyo for his technical support.

All methods described here have been approved by the local ethical committee (Doshisha University Ethical Committee: 15033), and were in strict accordance with the standards set by the Declaration of Helsinki. Informed consent was obtained from all participants. Participants were male recreational athletes (age, 20.1 &#177; 1.2 years; weight, 69.7 &#177; 6.2 kg; height, 176.1 &#177; 5.8 cm; body fat 13.6 &#177; 1.5% and VO2max, 52.6 &#177; 6.6 mL/min1/kg1).
1. Establishment of the Exercise Protocol
NOTE: The present study consisted of the evaluation of blood hormonal and metabolites dynamics by blood sampling from the antecubital vein during a HIIE protocol.
<ol>
	<li>Ask the subject to perform a warm up with 5 min of cycling in the ergometer at low intensity. After the warm-up, let the subject rest for 5 min.</li>
	<li>Ask the subject to perform the following HIIE in the leg cycle ergometer: three bouts of 30 s high-intensity exercise, separated by 4 min of rest (Figure 1). 
	NOTE: The 30 s bouts should be set at an intensity equal to 90% the power average of a previously performed Wingate test. Refer elsewhere8 for a complete description of the Wingate test.</li>
	<li>To calculate the workload for the 30 s bouts, use the following equations: 
	Work = Force x Distance (equation 1) 
	Force = Mass x Acceleration (equation 2) 
	Work = (Mass x Acceleration) x Distance (equation 3) 
	NOTE: In this model, the mass is the work load (kg), and the acceleration is 9.81 m/s2. The distance is the wheel diameter (1.622 m). Because the wheel rotates 3.7 timed /peal rotation, the distance per pedal rotation is 3.7 x 1.662. Furthermore, because the pedal frequency is 90 rev/min1, we have to multiply one pedal distance by 90. 
	Work = Work load x 9.81 x 3.7 x 1.622 x 90 (equation 4) 
	Work load = Work / (9.81 x 3.7 x 1.622 x 90) (equation 5)</li>
</ol>
2. Catheterization
NOTE: Tubes and vacutainers should be labeled beforehand to avoid losing time.
<ol>
	<li>Wrap a tourniquet to the arm selected for the catheterization. To do this, place an elastic band at the back of the chosen arm. Stretching the band, make a loop with the band around the arm and tie the elastic.</li>
	<li>Select the visible antecubital vein and sterilize the skin with ethanol or alcohol.</li>
	<li>Insert the tip of the sterilized cannula into the vein at an approximate angle of 15&#176; with the skin. 
	NOTE: The cannula should be inserted from the proximal to distal direction. If cannulation is successful, a backflush of blood should be observed.</li>
	<li>Secure the cannula with the hand, and remove the needle softly. 
	NOTE: To maintain the angle of the needle during the exercise, cotton tissues may be used in between the skin and the needle. Fix this structure with a tape.</li>
	<li>Connect a vacutainer with heparin and aspirate for the first blood sample (approximately 2.5 mL). Immediately after, refrigerate the blood sample by putting it in an ice box for no more than 40 minutes. Ask an assistant for help, if blood gases are going to be analyzed.</li>
	<li>Connect a tube to the catheter and inject heparin to the full length of the tube so as to avoid blood coagulation. Close the butterfly.</li>
	<li>Using tape, stabilize the catheter. Ensure that at this point, the arm mobility of the subject does not interfere with the blood collection. To confirm this condition, be sure that the needle position is not affected by any movement.</li>
</ol>
3. Blood Samplings
<ol>
	<li>Remove the heparin by using a vacutainer until there is only blood in the catheter. When blood flashback is observed, collect the sample as described in step 2.5.</li>
	<li>After the sample collection, inject heparin again into the catheter tube to avoid blood coagulation. After this action, step 2.5 may be repeated for several samples collection.</li>
	<li>After the last sample is collected, carefully remove the cannulation tube and clean the skin with alcohol. To avoid possible bleeding, ask the subject to keep sterilized cotton at the place where the catheterization was made.</li>
	<li>After blood is collected in the vacutainer tubes, carefully pass approximately 1.5 mL of blood to a 1.5 mL tube and keep the tubes on ice.</li>
	<li>Measure blood lactate from the remaining blood using a lactate detector. On the same day, obtain the plasma the blood samples (around 1.5 mL of blood).</li>
	<li>For plasma separation, centrifuge the blood samples for 5 min at 5000 x g at 4 oC, and separate the supernatant in a new 1.5 mL tube.</li>
	<li>Repeat the centrifugation one more time to purify the plasma.</li>
	<li>After the plasma separation, store the samples at -80 oC until the hormone analysis.</li>
	<li>Measure the plasma insulin using an ELISA kit as per the manufacturer&#39;s protocol (see Table of Materials).</li>
</ol>

<ol>
	<li>Wahl, P., Mathes, S., K&#246;hler, K., Achtzehn, S., Bloch, W., Mester, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+active+vs.+passive+recovery+during+Wingate-based+training+on+the+acute+hormonal,+metabolic+and+psychological+response.">Effects of active vs. passive recovery during Wingate-based training on the acute hormonal, metabolic and psychological response.</a> Growth Horm IGF Res. 23 (6), 201-208 (2013).</li><li>Wahl, P., Mathes, S., Achtzehn, S., Bloch, W., Mester, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Active+vs.+passive+recovery+during+high-intensity+training+influences+hormonal+response.">Active vs. passive recovery during high-intensity training influences hormonal response.</a> Int J Sports Med. 35 (7), 583-589 (2014).</li><li>Nalbandian, H. M., Radak, Z., Takeda, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+active+recovery+during+interval+training+on+plasma+catecholamines+and+insulin.">Effects of active recovery during interval training on plasma catecholamines and insulin.</a> J Sports Med Phys Fitness. , (2017).</li><li>Stolen, T., Chamari, K., Castagna, C., Wislff, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiology+of+Soccer.">Physiology of Soccer.</a> Sport Med. 35 (6), 501-536 (2005).</li><li>Bangsbo, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+physiology+of+soccer--with+special+reference+to+intense+intermittent+exercise.">The physiology of soccer--with special reference to intense intermittent exercise.</a> Acta Physiol Scand Suppl. 619, 1-155 (1994).</li><li>MacInnis, M. J., Gibala, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+adaptations+to+interval+training+and+the+role+of+exercise+intensity.">Physiological adaptations to interval training and the role of exercise intensity.</a> J Physiol. 595 (9), 2915-2930 (2017).</li><li>Emberts, T., Porcari, J., Dobers-Tein, S., Steffen, J., Foster, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exercise+intensity+and+energy+expenditure+of+a+tabata+workout.">Exercise intensity and energy expenditure of a tabata workout.</a> J Sports Sci Med. 12 (3), 612-613 (2013).</li><li>Smith, J. C., Hill, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+energy+systems+during+a+Wingate+power+test.">Contribution of energy systems during a Wingate power test.</a> Br J Sports Med. 25 (4), 196-199 (1991).</li><li>Dotan, R., Bar-Or, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Load+optimization+for+the+Wingate+Anaerobic+Test.">Load optimization for the Wingate Anaerobic Test.</a> Eur J Appl Physiol Occup Physiol. 51 (3), 409-417 (1983).</li><li>Carrasco-Benso, M. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+adipose+tissue+expresses+intrinsic+circadian+rhythm+in+insulin+sensitivity.">Human adipose tissue expresses intrinsic circadian rhythm in insulin sensitivity.</a> FASEB J. 30 (9), 3117-3123 (2016).</li><li>Hatfield, D. L., Nicoll, J. X., Kraemer, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Circadian+Rhythm+on+Power,+Force,+and+Hormonal+Response+in+Young+Men.">Effects of Circadian Rhythm on Power, Force, and Hormonal Response in Young Men.</a> J Strength Cond Res. 30 (3), 725-732 (2016).</li></ol>

The authors have nothing to disclose.

In this study, the antecubital vein was catheterized, and blood samples were successfully collected during a high interval exercise. As an example of the use of this methodology, blood samples were analyzed for blood lactate and (after plasma separation) for plasma insulin. The main advantage of this method is that it allows the collection of blood samples in short periods of time. At the same time, it does have some limitations. For instance, the limited exercise modalities that allow its use; the number of assistant needed, and specialized trained personnel required; and the risk of low sample volume as a consequence of low blood pressure in the forearms.
For a successful blood sample collection, the subject&#39;s arm should be in static position. Because the forearm mobility is reduced, in its current form the method can be applied only to a few exercises modalities. Here, we described how to apply the method when the exercise is performed in a cycle ergometer, but with modification, it could be used on a treadmill running machine. In both cases, personnel with experience in blood collection is needed.
Regarding the exercise protocol, there are some considerations that should be addressed. If several trials are going to be evaluated (e.g., with and without active recovery between bouts), the moment of decision for the testing is critical. In order to avoid diurnal hormonal variations10,11, it is recommended to perform all trials at the same hour on different days. Furthermore, metabolism is affected by the time of day and meals consumed. Therefore, fasting is recommended before the tests. Additionally, to improve reproducibility, a familiarization period with the exercise protocol by the subjects before testing is recommended. In this period, experimental subjects should perform (progressively) similar exercise to the main test.
In conclusion, we described an effective method to obtain several venous blood samples during exercise. This method can be useful for collecting samples that can be used to study several molecules during exercise (e.g., metabolites, hormones, protein and RNA).

The measurement of metabolic and endocrinal markers during physical activity is of relevance to understanding the physiological implications of different exercise modalities. During some exercise modalities (e.g., high-intensity interval exercise (HIIE)), blood metabolites and hormonal levels fluctuate in relatively short time1,2,3.
Many sports (i.e., soccer, basketball, and rugby) consist of short periods of high-intensity exercise separated by low intense exercise and/or passive periods4,5. HIIE presents similarities with the aforementioned sports and is therefore widely used as a training method for athletes6. Moreover, because of its similarity with sports, HIIE has been used as a model to study exercise physiology7. Blood sampling is critical to measure physiological variables, and it is frequently used for research purposes and the evaluation of the physical condition. However, blood sampling during HIIE can be technically difficult because of the short time between sample collections. As a solution to this problem, here we present a method to obtain blood samples by antecubital venous catheterization in short periods of time while subjects perform HIIE.

<table><tbody><tr><td>Cycle ergometer</td><td>Monark</td><td>MONARK Ergomedic 874 E</td><td></td></tr><tr><td>Catheter</td><td>Terumo</td><td>SR-FF2032</td><td></td></tr><tr><td>Vacutainer</td><td>Terumo</td><td>PZ-D03</td><td></td></tr><tr><td>Tube for catheter</td><td>Terumo</td><td>SP-PTW30L02</td><td></td></tr><tr><td>Heparin</td><td>Terumo</td><td>PF-10HF10UA</td><td></td></tr><tr><td>Insulin ELISA kit</td><td>Abnova</td><td>ELISA Kit KA0921</td><td></td></tr></tbody></table>

evaluation of blood lactate and plasma insulin during high-intensity exercise by antecubital vein catheterization

The measurement of metabolic and endocrinal markers during physical activity is of relevance to understanding the physiological implications of different exercise modalities. During some exercise modalities (e.g., high-intensity interval exercise), blood metabolites and hormonal levels change in short periods of time. In the present study, we describe a method to catheterize the antecubital vein, which allows the collection of several blood samples during exercise. Insulin and venous lactate concentrations were measured during high-intensity exercise by the application of the described method. The exercise consisted of three 30 s bouts of high-intensity exercise separated by 4 min of recovery. After the last recovery period, a Wingate test was performed. Blood samples from the antecubital vein were obtained before and after each 30 s bout and before and after the Wingate test. As a result, it was possible to evaluate the plasma insulin and venous blood lactate variations during the exercise.

As representative results, we used this protocol to measure blood lactate and insulin variation in six subjects during a HIIE in a cycle ergometer. Peak power and Average Power for the Wingate test were 732 &#177; 120 and 549 &#177; 84 Watts, respectively. Average Power was used to calculate the power output (494 &#177; 75 Watts) during the 30 s bouts of the HIIE.
Figure 2 shows plasma insulin and blood lactate variation during the HIIE. Plasma insulin (Figure 2A) decreased during the first and third 30 s bouts but increased during the second bout and the recovery periods. On the other hand, plasma lactate (Figure 2B) decreased during the second and third 30 s bouts, but increased during the first 30 s bout and the recovery periods.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56890/56890fig1.jpg" /> 
Figure 1: Exercise protocol. Red arrows indicate the time of blood samples collection. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56890/56890fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56890/56890fig2.jpg" /> 
Figure 2: Experimental results. (A) Plasma insulin (ulU/mL). (B) Blood lactate (mmol/L). Each color represents a different subject. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56890/56890fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Evaluation of Blood Lactate and Plasma Insulin During High-intensity Exercise by Antecubital Vein Catheterization at JoVE.com

Evaluation of Blood Lactate and Plasma Insulin During High-intensity Exercise by Antecubital Vein Catheterization

Here we present a protocol to obtain several blood samples from the antecubital vein during high-intensity interval training. This protocol may be useful for the measurement of blood metabolites and endocrinal markers during exercise.

The measurement of metabolic and endocrinal markers during physical activity is of relevance to understanding the physiological implications of different ...

evaluation-blood-lactate-plasma-insulin-during-high-intensity

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

6.4K Views.  Kyoto University. Here we present a protocol to obtain several blood samples from the antecubital vein during high-intensity interval training. This protocol may be useful for the measurement of blood metabolites and endocrinal markers during exercise.

Video: Evaluation of Blood Lactate and Plasma Insulin During High-intensity Exercise by Antecubital Vein Catheterization

Hyperinsulinemic-Euglycemic Clamp in the Conscious Rat

Type 2 diabetes (T2D) is rapidly rising in prevalence. Characterized by either inadequate insulin production or the inability to utilize insulin produced, T2D results in elevated blood glucose levels. The "gold-standard" in assessing insulin sensitivity is a hyperinsulinemic-euglycemic clamp or insulin clamp. In this procedure, insulin is infused at a constant rate resulting in a drop in blood glucose. To maintain blood glucose at a constant level, exogenous glucose (D50) is infused into the venous circulation. The amount of glucose infused to maintain homeostasis is indicative of insulin sensitivity. Here, we show the basic clamp procedure in the chronically catheterized, unrestrained, conscious rat. This model allows blood to be collected with minimal stress to the animal. Following the induction of anesthesia, a midline incision is made and the left common carotid artery and right jugular vein are catheterized. Inserted catheters are flushed with heparinized saline, then exteriorized and secured. Animals are allowed to recover for 4-5 days prior to experiments, with weight gain monitored daily. Only those animals who regain weight to pre-surgery levels are used for experiments. On the day of the experiment, rats are fasted and connected to pumps containing insulin and D50. Baseline glucose is assessed from the arterial line and used a benchmark throughout the experiment (euglycemia). Following this, insulin is infused at a constant rate into the venous circulation. To match the drop in blood glucose, D50 is infused. If the rate of D50 infusion is greater than the rate of uptake, a rise in glucose will occur. Similarly, if the rate is insufficient to match whole body glucose uptake, a drop will occur. Titration of glucose continues until stable glucose readings are achieved. Glucose levels and glucose infusion rates during this stable period are recorded and reported. Results provide an index of whole body insulin sensitivity. The technique can be refined to meet specific experimental requirements. It is further enhanced by the use of radioactive tracers that can determine tissue specific insulin-stimulated glucose uptake as well as whole body glucose turnover.

The hyperinsulinemic-euglycemic clamp is the "gold standard" for the assessment of insulin action. Insulin is infused at a constant rate stimulating glucose uptake. The amount of exogenous glucose infused to counter this drop is indicative of insulin sensitivity. Here the procedure is performed on a conscious, unrestrained rat.

Type 2 diabetes (T2D) is rapidly rising in prevalence. Characterized by either inadequate insulin production or the inability to utilize insulin produced, ...

Medicine

Finger-stick Blood Sampling Methodology for the Determination of Exercise-induced Lymphocyte Apoptosis

Exercise is a physiological stimulus capable of inducing apoptosis in immune cells. To date, various limitations have been identified with the measurement of this phenomenon, particularly relating to the amount of time required to isolate and treat a blood sample prior to the assessment of cell death. Because of this, it is difficult to determine whether reported increases in immune cell apoptosis can be contributed to the actual effect of exercise on the system, or are a reflection of the time and processing necessary to eventually obtain this measurement. In this article we demonstrate a rapid and minimally invasive procedure for the analysis of exercise-induced lymphocyte apoptosis. Unlike other techniques, whole blood is added to an antibody panel immediately upon obtaining a sample. Following the incubation period, red blood cells are lysed and samples are ready to be analyzed. The use of a finger-stick sampling procedure reduces the volume of blood required, and minimizes the discomfort to subjects.

Exercise is capable of inducing apoptosis in immune cells. There are various measurement limitations, particularly relating to the amount of time required to isolate and treat a blood sample prior to the assessment. Demonstrated is a rapid and minimally invasive procedure for the analysis of exercise-induced lymphocyte apoptosis.

Exercise is a physiological stimulus capable of inducing apoptosis in immune cells. To date, various limitations have been identified with the measurement ...

Hyperinsulinemic-euglycemic Clamps in Conscious, Unrestrained Mice

Type 2 diabetes is characterized by a defect in insulin action. The hyperinsulinemic-euglycemic clamp, or insulin clamp, is widely considered the "gold standard" method for assessing insulin action in vivo. During an insulin clamp, hyperinsulinemia is achieved by a constant insulin infusion. Euglycemia is maintained via a concomitant glucose infusion at a variable rate. This variable glucose infusion rate (GIR) is determined by measuring blood glucose at brief intervals throughout the experiment and adjusting the GIR accordingly. The GIR is indicative of whole-body insulin action, as mice with enhanced insulin action require a greater GIR. The insulin clamp can incorporate administration of isotopic 2[14C]deoxyglucose to assess tissue-specific glucose uptake and [3-3H]glucose to assess the ability of insulin to suppress the rate of endogenous glucose appearance (endoRa), a marker of hepatic glucose production, and to stimulate the rate of whole-body glucose disappearance (Rd).
The miniaturization of the insulin clamp for use in genetic mouse models of metabolic disease has led to significant advances in diabetes research. Methods for performing insulin clamps vary between laboratories. It is important to note that the manner in which an insulin clamp is performed can significantly affect the results obtained. We have published a comprehensive assessment of different approaches to performing insulin clamps in conscious mice1 as well as an evaluation of the metabolic response of four commonly used inbred mouse strains using various clamp techniques2. Here we present a protocol for performing insulin clamps on conscious, unrestrained mice developed by the Vanderbilt Mouse Metabolic Phenotyping Center (MMPC; URL: www.mc.vanderbilt.edu/mmpc). This includes a description of the method for implanting catheters used during the insulin clamp. The protocol employed by the Vanderbilt MMPC utilizes a unique two-catheter system3. One catheter is inserted into the jugular vein for infusions. A second catheter is inserted into the carotid artery, which allows for blood sampling without the need to restrain or handle the mouse. This technique provides a significant advantage to the most common method for obtaining blood samples during insulin clamps which is to sample from the severed tip of the tail. Unlike this latter method, sampling from an arterial catheter is not stressful to the mouse1. We also describe methods for using isotopic tracer infusions to assess tissue-specific insulin action. We also provide guidelines for the appropriate presentation of results obtained from insulin clamps.

The hyperinsulinemic-euglycemic clamp, or insulin clamp, is the gold standard for assessing insulin action in vivo. A method for performing insulin clamps in mice is described. This includes a method for arterial catheterization that permits experiments to be performed in conscious, unrestrained mice with minimal stress.

Type 2 diabetes is characterized by a defect in insulin action. The hyperinsulinemic-euglycemic clamp, or insulin clamp, is widely considered the "gold ...

Determining the Contribution of the Energy Systems During Exercise

One of the most important aspects of the metabolic demand is the relative contribution of the energy systems to the total energy required for a given physical activity. Although some sports are relatively easy to be reproduced in a laboratory (e.g., running and cycling), a number of sports are much more difficult to be reproduced and studied in controlled situations. This method presents how to assess the differential contribution of the energy systems in sports that are difficult to mimic in controlled laboratory conditions. The concepts shown here can be adapted to virtually any sport.
The following physiologic variables will be needed: rest oxygen consumption, exercise oxygen consumption, post-exercise oxygen consumption, rest plasma lactate concentration and post-exercise plasma peak lactate. To calculate the contribution of the aerobic metabolism, you will need the oxygen consumption at rest and during the exercise. By using the trapezoidal method, calculate the area under the curve of oxygen consumption during exercise, subtracting the area corresponding to the rest oxygen consumption. To calculate the contribution of the alactic anaerobic metabolism, the post-exercise oxygen consumption curve has to be adjusted to a mono or a bi-exponential model (chosen by the one that best fits). Then, use the terms of the fitted equation to calculate anaerobic alactic metabolism, as follows: ATP-CP metabolism = A1 (mL . s-1) x t1 (s). Finally, to calculate the contribution of the lactic anaerobic system, multiply peak plasma lactate by 3 and by the athlete&#x2019;s body mass (the result in mL is then converted to L and into kJ).
The method can be used for both continuous and intermittent exercise. This is a very interesting approach as it can be adapted to exercises and sports that are difficult to be mimicked in controlled environments. Also, this is the only available method capable of distinguishing the contribution of three different energy systems. Thus, the method allows the study of sports with great similarity to real situations, providing desirable ecological validity to the study.

This protocol allows researchers focused on exercise and sports sciences to determine the relative contribution of three different energy systems to the total energy expenditure during a large variety of exercises.

One of the most important aspects of the metabolic demand is the relative contribution of the energy systems to the total energy required for a given ...

Biology

Combined Intravital Microscopy and Contrast-enhanced Ultrasonography of the Mouse Hindlimb to Study Insulin-induced Vasodilation and Muscle Perfusion

It has been demonstrated that insulin's vascular actions contribute to regulation of insulin sensitivity. Insulin's effects on muscle perfusion regulate postprandial delivery of nutrients and hormones to insulin-sensitive tissues. We here describe a technique for combining intravital microscopy (IVM) and contrast-enhanced ultrasonography (CEUS) of the adductor compartment of the mouse hindlimb to simultaneously visualize muscle resistance arteries and perfusion of the microcirculation in vivo. Simultaneously assessing insulin's effect at multiple levels of the vascular tree is important to study relationships between insulin's multiple vasoactive effects and muscle perfusion. Experiments in this study were performed in mice. First, the tail vein cannula is inserted for the infusion of anesthesia, vasoactive compounds and ultrasound contrast agent (lipid-encapsulated microbubbles). Second, a small incision is made in the groin area to expose the arterial tree of the adductor muscle compartment. The ultrasound probe is then positioned at the contralateral upper hindlimb to view the muscles in cross-section. To assess baseline parameters, the arterial diameter is assessed and microbubbles are subsequently infused at a constant rate to estimate muscle blood flow and microvascular blood volume (MBV). When applied before and during a hyperinsulinemic-euglycemic clamp, combined IVM and CEUS allow assessment of insulin-induced changes of arterial diameter, microvascular muscle perfusion and whole-body insulin sensitivity. Moreover, the temporal relationship between responses of the microcirculation and the resistance arteries to insulin can be quantified. It is also possible to follow-up the mice longitudinally in time, making it a valuable tool to study changes in vascular and whole-body insulin sensitivity.

Insulin-induced vasodilation regulates muscle perfusion and increases the microvascular surface area (microvascular recruitment) available for solute exchange between blood and tissue interstitium. Combined intravital microscopy and contrast-enhanced ultrasonography is presented to simultaneously assess insulin&#39;s action at the larger vessels and the microcirculation in vivo.

It has been demonstrated that insulin's vascular actions contribute to regulation of insulin sensitivity. Insulin's effects on muscle perfusion regulate ...

Experimental Protocol of a Three-minute, All-out Arm Crank Exercise Test in Spinal-cord Injured and Able-bodied Individuals

Reliable exercise protocols are required to test changes in exercise performance in elite athletes. Performance improvements in these athletes may be small; therefore, sensitive tools are fundamental to exercise physiology. There are currently many exercise tests that allow for the examination of exercise capacity in able-bodied athletes, with protocols mainly for lower-body or whole-body exercise. There is a trend to test athletes in a sport-specific setting that closely resembles the actions that the participants are used to performing. Only a few protocols test short-term, high-intensity exercise capacity in participants with an impairment of the lower body. Most of these protocols are very sport-specific and are not applicable to a wide range of athletes. One well-known test protocol is the 30 s Wingate test, which is well-established in cycling and in arm crank exercise testing. This test analyzes high-intensity exercise performance over a 30 s time duration. In order to monitor exercise performance over a longer duration, a different method was modified for application to the upper body. The 3 min, all-out arm crank ergometer test allows athletes to be tested in a manner specific to 1,500 m wheelchair racing (in terms of exercise duration), as well as to upper body exercises such as rowing or hand-cycling. In order to increase the reliability with identical test conditions, it is crucial to precisely replicate settings such as the resistance (i.e., torque factor) and the position of the participants (i.e., the height of the crank, the distance between the crank and the participant, and the fixation of the participant). Another important issue concerns the beginning of the exercise test. Fixed revolutions per minute are required to standardize the test conditions for the start of the exercise test. This exercise protocol shows the importance of accurate operations to reproduce identical test conditions and settings.

We present a protocol to test the aerobic and anaerobic power of the upper body muscles over a duration of 3 min in able-bodied as well as in paraplegic and tetraplegic individuals. The protocol presents specific modifications in its application for upper-body exercise in individuals with or without disability.

Reliable exercise protocols are required to test changes in exercise performance in elite athletes. Performance improvements in these athletes may be ...

Postconditioning with Lactate-enriched Blood for Cardioprotection in ST-segment Elevation Myocardial Infarction

The beneficial effects of reperfusion therapy for ST-segment elevation myocardial infarction (STEMI) is attenuated by reperfusion injury. No approach has been proven successful in preventing this injury in the clinical setting to date. Meanwhile, a novel approach for cardioprotection in patients with STEMI, i.e., postconditioning with lactate-enriched blood (PCLeB), has recently been reported. PCLeB is a modification of the original protocol of postconditioning, aimed at increasing the delay in the recovery from tissue acidosis produced during ischemia. This was sought to achieve controlled reperfusion with tissue oxygenation and minimal lactate washout. In this modified postconditioning protocol, the duration of each brief reperfusion is gradually increased in a stepwise manner from 10 to 60 s. Each brief ischemic period lasts for 60 s. At the end of each brief reperfusion, injection of lactated Ringer&#39;s solution (20&#8211;30 mL) is performed directly into the culprit coronary artery immediately before the balloon inflation and the balloon is quickly inflated at the lesion site, so that the lactate is trapped inside the ischemic myocardium during each brief repetitive ischemic period. After seven cycles of balloon inflation and deflation, full reperfusion is performed. Stenting is performed thereafter, and the percutaneous coronary intervention is completed. Excellent in-hospital and 6 month outcomes in a limited number of patients with STEMI treated using PCLeB have already been reported. This method article provides a detailed description of each step of the PCLeB procedures.

Herein, we present a protocol for postconditioning with lactate-enriched blood, which is a novel approach for cardioprotection. This protocol comprises&#160;intermittent reperfusion and timely coronary injections of lactated Ringer&#39;s solution. This can be applied to ST-segment elevation myocardial infarction patients who undergo primary percutaneous coronary intervention.

The beneficial effects of reperfusion therapy for ST-segment elevation myocardial infarction (STEMI) is attenuated by reperfusion injury. No approach has ...

Measurement of Myocardial Lactate Production for Diagnosis of Coronary Microvascular Spasm

In about a quarter of patients with angina and non-obstructive coronary arteries, no epicardial spasm is noted on coronary arteriography during an angina attack. Since the pressure-rate product is almost identical at rest and the onset of attack in those patients, the decrease in coronary blood flow rather than increased myocardial oxygen consumption is likely to explain myocardial ischemia, indicating a substantial involvement with coronary microvascular spasm (MVS). Myocardial lactate production, which could be defined as a negative myocardial lactate extraction ratio (ratio of the coronary arterial-venous difference in lactate concentration to arterial concentration), is considered indicative of objective evidence to support the emerging myocardial ischemia. Thus, monitoring of the myocardial lactate production and the emergence of chest pain and ischemic electrocardiographic changes during acetylcholine (ACh) provocation testing is of significant value for detecting the entity of MVS. Practically, 1 min after incremental doses of ACh (20, 50, and 100 &#956;g) are administered into the left coronary artery (LCA), paired samples of 1 mL of blood are collected from the LCA ostium and coronary sinus for measurement of lactate concentration by a calibrated automatic lactate analyzer. Then, the development of MVS could be confirmed by negative myocardial lactate extraction ratio despite the absence of angiographically demonstrable epicardial coronary spasm or before its occurrence throughout ACh provocation testing. In conclusion, assessment of myocardial lactate production is essential and valuable for the diagnosis of MVS.

Myocardial lactate production (coronary arterial-venous difference in serum lactate level) during coronary spasm provocation testing is considered as a highly sensitive marker that reflects acetylcholine-induced myocardial ischemia due to microvascular spasm. This article presents the procedures to assess myocardial lactate production for the diagnosis of coronary microvascular spasm.

In about a quarter of patients with angina and non-obstructive coronary arteries, no epicardial spasm is noted on coronary arteriography during an angina ...

Using a Combination of Indirect Calorimetry, Infrared Thermography, and Blood Glucose Levels to Measure Brown Adipose Tissue Thermogenesis in Humans

In mammals, brown adipose tissue (BAT) is activated rapidly in response to cold in order to maintain body temperature. Although BAT has been studied greatly in small animals, it is difficult to measure the activity of BAT in humans. Therefore, little is known about the heat-generating capacity and physiological significance of BAT in humans, including the degree to which components of the diet can activate BAT. This is due to the limitations in the currently most used method to assess the activation of BAT-radiolabeled glucose (fluorodeoxyglucose or 18FDG) measured by positron emission tomography-computerized tomography (PET-CT).
This method is usually performed in fasted subjects, as feeding induces glucose uptake by the muscles, which can mask the glucose uptake into the BAT. This paper describes a detailed protocol for quantifying total-body human energy expenditure and substrate utilization from BAT thermogenesis by combining indirect calorimetry, infrared thermography, and blood glucose monitoring in carbohydrate-loaded adult males. To characterize the physiological significance of BAT, measures of the impact of BAT activity on human health are critical. We demonstrate a protocol to achieve this by combining carbohydrate loading and indirect calorimetry with measurements of supraclavicular changes in temperature. This novel approach will help to understand the physiology and pharmacology of BAT thermogenesis in humans.

Here, we present a protocol to quantify the physiological significance of the impact of brown adipose tissue (BAT) activity on human metabolism. This is achieved by combining carbohydrate loading and indirect calorimetry with measurements of supraclavicular changes in temperature. This novel approach can help develop a pharmacological target for BAT thermogenesis in humans.

In mammals, brown adipose tissue (BAT) is activated rapidly in response to cold in order to maintain body temperature. Although BAT has been studied ...

Assessing Strength Exercise Volume and Its Impact on Insulin Sensitivity in Obesity

Randomized Controlled Trial to Study the Acute Effects of Strength Exercise on Insulin Sensitivity in Obese Adults

An acute session of strength exercise (SE) ameliorates insulin sensitivity (IS) for several hours; however, the effects of SE volume (i.e., number of sets) have not been studied thoroughly. Although it is intuitive that some SE is better than none, and more is better than some for the improvement of IS, high-volume sessions might be challenging for diseased populations to complete, especially obese adults, for whom even a brisk walk can be challenging. This protocol details a randomized clinical trial to assess the acute effects of SE on IS in obese adults. The inclusion criteria are body mass index &#62;30 kg/m2, central obesity (waist circumference &#62;88 cm and &#62;102 cm for women and men, respectively), and age &#62;40 years. Participants will be familiarized with the SE (7 exercises targeting major muscle groups) and then will perform three sessions in a randomized order: session 1 - high-volume session (3 sets/exercise); session 2 - low-volume session (1 set/exercise); session 3 - control session (no exercise). Diet will be controlled the day before and on the day of the sessions. Sessions will be completed at night, and an oral glucose tolerance test will be performed the next morning, from which several indexes of IS will be derived, such as the area under the curve (AUC) of glucose and insulin, the Matsuda index, the Cederholm index, the muscle IS index, and the Gutt index. Based on pilot studies, we expect ~15% improvement in IS (insulin AUC, and Matsuda and Cederholm indexes) after the high-volume session, and ~8% improvement after the low-volume session compared to the control session. This study will benefit individuals who find high-volume SE sessions challenging but still aim to improve their IS by investing 1/3 of their time and effort.

Author Spotlight: Exploring the Impact of Reduced Resistance Exercise Volume on Metabolic Health 

This study describes a randomized controlled trial protocol aiming at assessing the acute effects of strength exercise volume on insulin sensitivity in obese individuals.

An acute session of strength exercise (SE) ameliorates insulin sensitivity (IS) for several hours; however, the effects of SE volume (i.e., number of ...