in response to a rise in blood glucose level , the pancreas secretes insulin into the bloodstream where it is quickly distributed to its target tissues such as skeletal muscle , via resistance arteries and capillaries . the delivery of insulin to the skeletal muscle interstitium has been shown to be a rate limiting step for the metabolic actions of insulin that promote glucose disposal234 . within 10 - 15 min , insulin increases the capillary blood volume ( microvascular recruitment ) , an effect that occurs before total blood flow increases56 . microvascular recruitment expands the endothelial surface area available for exchange of nutrients ( and insulin)78 . insulin - mediated microvascular recruitment precedes and is independently associated with changes in skeletal muscle glucose uptake89 . the effect of insulin on the vasculature has been termed ' vascular insulin sensitivity ' . it has been shown that insulin - mediated microvascular recruitment and insulin - induced vasodilation are impaired in obese zucker rats1011 . moreover , lean mice with reduced capillary density display muscle insulin resistance12 . in their influential work , kubota et al . showed that impaired insulin signaling in endothelial cells caused reduction in insulin - induced microvascular  recruitment , which reduced glucose uptake in skeletal muscle by approximately 40%13 . these abnormalities in microvascular function do not only occur in muscle , but also in multiple other tissues and organs such as the heart , retina and kidney141516 . these examples and other studies17181920 suggest that vascular effects of insulin are an important mechanism in the ( patho)physiology of insulin resistance and its complications . while there is substantial evidence that insulin increases microvascular blood volume ( mbv ) in skeletal muscle56 , the mechanisms by which this happens are not completely understood9 . endothelium - dependent vasodilation is essential in many aspects of vascular insulin sensitivity212223 at different levels of the vasculature . vascular insulin sensitivity can manifest itself by insulin - induced relaxation of resistance arteries and by relaxation of pre - capillary arterioles to increase the perfused microvascular exchange surface area72425 . intravital microscopy ( ivm ) has been used in a variety of tissue preparations including skinfold chambers of the mouse dorsum26 , the mesentery of the mouse and rat27 , models of limb ischemia in the mouse28 and the hamster cheek pouch29 . contrast - enhanced ultrasonography ( ceus ) is another imaging technique that allows assessment of the microcirculation in cardiac30 as well as skeletal muscle31 . it utilizes inert gas - filled microbubbles which behave rheologically as red blood cells and remain entirely within the vascular lumen . these microbubbles are infused intravenously at a constant rate to attain a steady state . a high energy ultrasound wave , then the microbubbles ' replenishment speed in the region of interest ( roi ) represents flow velocity ( mfv ) . ceus can be performed repeatedly ( also in humans ) and it has advanced the understanding of vascular dysfunction that occurs in insulin - resistant states ( discussed in barrett et al.32 ) . in the current study we describe a new technique for studying regulation of muscle perfusion , through simultaneous use of both ivm and ceus . here we focus on insulin 's vascular actions in the adductor compartment of the mouse hindlimb . this compartment is one of the largest skeletal muscle groups in the mouse , enabling studies of local glucose uptake in a representative muscle . this compartment is ideal for ivm as the preparation and visualization of the arteries are readily accessible by a standardized surgical procedure28 . moreover , our own group and others have shown that ceus can be used in this compartment3334 . an advantage to the combined ivm and ceus technique is the possibility to assess insulin 's effect at the level of the larger arterioles ( feed or resistance arteries ) and the microcirculation ( capillary beds ) in the same muscle group . in addition , the simultaneous application of the two methods provides insight into the temporal action of insulin at the levels of the resistance arteries and microcirculation . this combined ivm and ceus technique can also be implemented in other vascular biology fields . for example , the role of various proteins and certain pathophysiological conditions affecting the endothelium can be studied using knockout models . moreover , both techniques can be used in one mouse at multiple time points reducing the time and cost of research . all animal experiments have been approved by the local animal care and ethical committee . the entire protocol from induction of anesthesia in the mouse until the end of the hyperinsulinemic - euglycemic clamp takes approximately 2 h. induce anesthesia along with analgesia in  a male mouse weighing 20 - 25 g after a 14 h or overnight fasting with an intraperitoneal injection of a fentanyl ( 0.31 mg / kg ) , midazolam ( 6.25 mg / kg ) and acepromazine ( 6.25 mg / kg ) ( fma anesthesia ) and place it on a rectal - temperature - controlled homeothermic heating pad which maintains body temperature at 37 c . disinfect the operation table and the equipment several times using an alcohol - based solution . attach a 27 g needle to a 10 cm long polyethylene tube ( pe-20 ) and attach the tube to a 4-way connector . this cannula will be used for infusion of microbubbles , insulin , glucose and anesthesia . note : adding heparin ( 5 u / ml ) in sterile saline during the cannulation process to flush the tail vein ( approximately 10 l ) reduces the possibility of clogged cannulas . throughout the surgical procedures and experimental protocols , maintain anesthesia by a continuous intravenous infusion of fma anesthesia  via the tail vein cannula at rate of 33.75 l / kg / min . place the mouse with the ventral side up and fix the feet using a thermostable tape to expose the upper thigh area . use a slight exorotation of the hip joint ( hindlimb paws facing up ) and a 40 - 60 angle at the knee joint to standardize the stretch of the muscle at the adductor compartment of the thigh . remove the hair at the groin and upper thigh areas bilaterally using a depilatory cream . place the mouse under a stereomicroscope and perform the following surgical steps using 10x to 16x magnification . make a 2 cm incision using skin scissors running parallel to the inguinal ligament , just lateral to the abdominal curvature ( figure 1 ) . apply traction on the distal side of the incision distally using a bulldog clamp ( figure 1d ) . this will help to adjust the window as needed and help hold the paraffin oil ( described in 1.12 ) . gently separate the fat pad from the wall instead of dissecting directly through the pad . gentle traction at the fat pad in the distal direction will facilitate the process ( figure 1c ) . identify the femoral artery and follow it down to the first major branches ( the epigastric artery and the gracilis artery ) ( figure 2 ) . the gracilis artery is the first major branch of the femoral artery running on the adductor magnus muscle and then runs deep to the gracilis muscle . the gracilis artery will be used for ivm . identify the transparent deep fascia covering the muscles and the vessels . using sharp forceps , pull the fascia upwards and cut it using a microscissor . cover the exposed muscle with a drop ( 200 l ) of medicinal liquid paraffin ( room temperature , or pre - warmed to 37 c ) to prevent the prepared tissue from drying out . adjust the skin folds of the incision using the bulldog clamp to create a small cavity to hold the paraffin oil that bathes the vessels . place the mouse under a previously calibrated microscope ( 16x optical magnification ) in such a way that the gracilis artery is vertical on the computer screen . attach the microscope to a camera and computer based analysis system that can extract the vessels ' diameter from the image data set . place the light source at a sufficient distance ( minimum 20 cm ) from the hindlimb to reduce head conduction from the light . carefully adjust the angle and direction of the ultrasound probe to get a cross - sectional view of the adductor muscle group . take care to keep the position of the ultrasound probe in relation to the mouse stable to keep the same imaging plane for baseline and hyperinsulinemic measurements . the diameter of the gracilis artery should be stable for 10 min before documenting the baseline diameter . make sure that the baseline gracilis artery diameter is saved by the computer program used for the ivm . prepare the microbubbles in advance as described35 and count with coulter counter to a concentration of 2.5 x 10 bubbles / ml just before the experiment . as the microbubbles can only be viewed in the contrast - mode of the ultrasound machine , control the parameters which affect the image and the contrast data collected ( described in 2.3.1 ) and use consistently during acquisition . use the following settings on the ultrasound machine : contrast gain at 35 decibels ; the time gain compensation to off ; line density to high ; number of focal zones to wide ; transmit power to 4% ; transmit beam width to standard ; sv gate to 4 ; sensitivity to 1 ; persistence to off . level the location of focal zones to the center of the region of interest . use the following settings on the ultrasound machine : contrast gain at 35 decibels ; the time gain compensation to off ; line density to high ; number of focal zones to wide ; transmit power to 4% ; transmit beam width to standard ; sv gate to 4 ; sensitivity to 1 ; persistence to off . level the location of focal zones to the center of the region of interest . start infusing the microbubbles using the tail vein cannula at a 5 l / min rate . place the infusion tube on a vibrating vortex ( 200x per min ) to maintain a uniform suspension of microbubbles . allow 5 min of continuous infusion of microbubbles so that a steady - state level is reached . proceed with obtaining the time - intensity curves of the bubbles using the microbubble destruct function ( mbd ) on the ultrasound machine at 5 min and at 10 min after the start of microbubbles infusion ( figure 3a ) . take the average signal of these two measurements to get the baseline perfusion data . after obtaining the baseline data , start the hyperinsulinemic - euglycemic clamp as described34 . use the tail cannula ( placed in 1.3 ) to administer the insulin and glucose ( and anesthesia ) . 
 in short , induce a hyperinsulinemic state by introducing a 200 mu / kg insulin bolus followed by continuous insulin infusion use a variable infusion of 20% d - glucose to maintain euglycemia.assess blood glucose from the tail vein every 5 min with a glucose monitoring device , and maintain at 5 mm by adjusting the variable glucose infusion rate . determine insulin sensitivity by averaging the mean glucose infusion rate during the last 30 min . 
  in short , induce a hyperinsulinemic state by introducing a 200 mu / kg insulin bolus followed by continuous insulin infusion assess blood glucose from the tail vein every 5 min with a glucose monitoring device , and maintain at 5 mm by adjusting the variable glucose infusion rate . determine insulin sensitivity by averaging the mean glucose infusion rate during the last 30 min . make sure that the diameter of the gracilis artery is documented at the desired time periods ( for example at 10 , 30 and 60 min ) of the start of the hyperinsulinemic - euglycemic clamp with the computer program . after 25 min and/or 55 min of the insulin clamp , start the second ( hyperinsulinemic ) ceus measurement to document the mbv at 30 and/or 60 min , respectively . after completion of ivm and the ceus measurements 60 min after the start of insulin infusion , withdraw blood from the mouse by a heart puncture procedure for later analysis . this will also euthanize the mouse . carefully dissect the gracilis and femoral arteries and store them for further desired experiments ( for e.g. , western blots , immunohistochemistry , ex vivo pressure myography experiments363738 ) . note : the analyses of ivm and ceus measurements should be performed offline by a blinded investigator . ceus offers the possibility to distinguish the microcirculation from larger vessels by temporarily destructing the microbubbles by high intensity ultrasound waves using the mbd function . the signal ( measured in arbitrary units ( a.u ) ) in larger vessels is restored quicker than those in the microcirculation because of the microbubbles speed in the corresponding vessels . use an offline workstation or the software on the ultrasound machine to do the analyses . draw a region of interests ( roi ) to include the microcirculation . draw a separate roi to include the larger femoral vessels ( figure 3a ) . duplicate the microcirculation 's and larger vessels ' rois for the background , baseline and hyperinsulinemic measurements by using the roi copy function built in the software . subtract the intensity signal of the background measurement from the baseline and the hyperinsulinemic measurements . divide the intensity signal of the microcirculation by the intensity signal of the femoral vessels . induce anesthesia along with analgesia in  a male mouse weighing 20 - 25 g after a 14 h or overnight fasting with an intraperitoneal injection of a fentanyl ( 0.31 mg / kg ) , midazolam ( 6.25 mg / kg ) and acepromazine ( 6.25 mg / kg ) ( fma anesthesia ) and place it on a rectal - temperature - controlled homeothermic heating pad which maintains body temperature at 37 c . disinfect the operation table and the equipment several times using an alcohol - based solution . attach a 27 g needle to a 10 cm long polyethylene tube ( pe-20 ) and attach the tube to a 4-way connector . this cannula will be used for infusion of microbubbles , insulin , glucose and anesthesia . note : adding heparin ( 5 u / ml ) in sterile saline during the cannulation process to flush the tail vein ( approximately 10 l ) reduces the possibility of clogged cannulas . throughout the surgical procedures and experimental protocols , maintain anesthesia by a continuous intravenous infusion of fma anesthesia  via the tail vein cannula at rate of 33.75 l / kg / min . place the mouse with the ventral side up and fix the feet using a thermostable tape to expose the upper thigh area . use a slight exorotation of the hip joint ( hindlimb paws facing up ) and a 40 - 60 angle at the knee joint to standardize the stretch of the muscle at the adductor compartment of the thigh . remove the hair at the groin and upper thigh areas bilaterally using a depilatory cream . place the mouse under a stereomicroscope and perform the following surgical steps using 10x to 16x magnification . make a 2 cm incision using skin scissors running parallel to the inguinal ligament , just lateral to the abdominal curvature ( figure 1 ) . apply traction on the distal side of the incision distally using a bulldog clamp ( figure 1d ) . this will help to adjust the window as needed and help hold the paraffin oil ( described in 1.12 ) . dissect the adipose tissue away from the abdominal wall . to avoid bleeding , gently separate the fat pad from the wall instead of dissecting directly through the pad . gentle traction at the fat pad in the distal direction will facilitate the process ( figure 1c ) . identify the femoral artery and follow it down to the first major branches ( the epigastric artery and the gracilis artery ) ( figure 2 ) . the gracilis artery is the first major branch of the femoral artery running on the adductor magnus muscle and then runs deep to the gracilis muscle . the gracilis artery will be used for ivm . identify the transparent deep fascia covering the muscles and the vessels . using sharp forceps , pull the fascia upwards and cut it using a microscissor . cover the exposed muscle with a drop ( 200 l ) of medicinal liquid paraffin ( room temperature , or pre - warmed to 37 c ) to prevent the prepared tissue from drying out . adjust the skin folds of the incision using the bulldog clamp to create a small cavity to hold the paraffin oil that bathes the vessels . place the mouse under a previously calibrated microscope ( 16x optical magnification ) in such a way that the gracilis artery is vertical on the computer screen . attach the microscope to a camera and computer based analysis system that can extract the vessels ' diameter from the image data set . place the light source at a sufficient distance ( minimum 20 cm ) from the hindlimb to reduce head conduction from the light . carefully adjust the angle and direction of the ultrasound probe to get a cross - sectional view of the adductor muscle group . take care to keep the position of the ultrasound probe in relation to the mouse stable to keep the same imaging plane for baseline and hyperinsulinemic measurements . the diameter of the gracilis artery should be stable for 10 min before documenting the baseline diameter . make sure that the baseline gracilis artery diameter is saved by the computer program used for the ivm . prepare the microbubbles in advance as described35 and count with coulter counter to a concentration of 2.5 x 10 bubbles / ml just before the experiment . as the microbubbles can only be viewed in the contrast - mode of the ultrasound machine , control the parameters which affect the image and the contrast data collected ( described in 2.3.1 ) and use consistently during acquisition . use the following settings on the ultrasound machine : contrast gain at 35 decibels ; the time gain compensation to off ; line density to high ; number of focal zones to wide ; transmit power to 4% ; transmit beam width to standard ; sv gate to 4 ; sensitivity to 1 ; persistence to off . level the location of focal zones to the center of the region of interest . 
  use the following settings on the ultrasound machine : contrast gain at 35 decibels ; the time gain compensation to off ; line density to high ; number of focal zones to wide ; transmit power to 4% ; transmit beam width to standard ; sv gate to 4 ; sensitivity to 1 ; persistence to off . start infusing the microbubbles using the tail vein cannula at a 5 l / min rate . place the infusion tube on a vibrating vortex ( 200x per min ) to maintain a uniform suspension of microbubbles . allow 5 min of continuous infusion of microbubbles so that a steady - state level is reached . proceed with obtaining the time - intensity curves of the bubbles using the microbubble destruct function ( mbd ) on the ultrasound machine at 5 min and at 10 min after the start of microbubbles infusion ( figure 3a ) . take the average signal of these two measurements to get the baseline perfusion data . after obtaining the baseline data , start the hyperinsulinemic - euglycemic clamp as described34 . use the tail cannula ( placed in 1.3 ) to administer the insulin and glucose ( and anesthesia ) . 
 in short , induce a hyperinsulinemic state by introducing a 200 mu / kg insulin bolus followed by continuous insulin infusion ( 7.5 mu / kg / min ) for 60 min . use a variable infusion of 20% d - glucose to maintain euglycemia.assess blood glucose from the tail vein every 5 min with a glucose monitoring device , and maintain at 5 mm by adjusting the variable glucose infusion rate . determine insulin sensitivity by averaging the mean glucose infusion rate during the last 30 min . 
  in short , induce a hyperinsulinemic state by introducing a 200 mu / kg insulin bolus followed by continuous insulin infusion assess blood glucose from the tail vein every 5 min with a glucose monitoring device , and maintain at 5 mm by adjusting the variable glucose infusion rate . determine insulin sensitivity by averaging the mean glucose infusion rate during the last 30 min . make sure that the diameter of the gracilis artery is documented at the desired time periods ( for example at 10 , 30 and 60 min ) of the start of the hyperinsulinemic - euglycemic clamp with the computer program . after 25 min and/or 55 min of the insulin clamp , start the second ( hyperinsulinemic ) ceus measurement to document the mbv at 30 and/or 60 min , respectively . after completion of ivm and the ceus measurements 60 min after the start of insulin infusion , withdraw blood from the mouse by a heart puncture procedure for later analysis . this will also euthanize the mouse . carefully dissect the gracilis and femoral arteries and store them for further desired experiments ( for e.g. , western blots , immunohistochemistry , ex vivo pressure myography experiments363738 ) . note : the analyses of ivm and ceus measurements should be performed offline by a blinded investigator . ceus offers the possibility to distinguish the microcirculation from larger vessels by temporarily destructing the microbubbles by high intensity ultrasound waves using the mbd function . the signal ( measured in arbitrary units ( a.u ) ) in larger vessels is restored quicker than those in the microcirculation because of the microbubbles speed in the corresponding vessels . use an offline workstation or the software on the ultrasound machine to do the analyses . draw a region of interests ( roi ) to include the microcirculation . draw a separate roi to include the larger femoral vessels ( figure 3a ) . duplicate the microcirculation 's and larger vessels ' rois for the background , baseline and hyperinsulinemic measurements by using the roi copy function built in the software . subtract the intensity signal of the background measurement from the baseline and the hyperinsulinemic measurements . divide the intensity signal of the microcirculation by the intensity signal of the femoral vessels . glucose infusion rate during the hyperinsulinemic - euglycemic clamp ( insulin sensitivity ) was 180.21  19.81 mol / kg / min . local application of paraffin oil on the adductor muscle compartment to stabilize the vessel did not change the average baseline diameter of the arteries ( 73.6  29.0 m vs. 68.8  17.9 m ; p = 0.58 ) but helped reduce the variation the animals tested ( figure 4a ) . insulin consistently increased the gracilis artery diameter ( by 14.58  6.2% at 60 min ; n = 9 ) which was significantly different ( p < 0.0001 ) from the diameter change caused by saline infusion ( -6.3  4.9% ; n = 6 ) . insulin - induced vasodilation was appreciable after 10 min ( 10.09  5.1% ; p = 0.002 ) and reached approximately 95% of its maximum dilatory capacity after 30 min . using ceus , insulin consistently increased muscle mbv ( figure 5a ) by 33.5% (  31.04% , n = 7 ; p = 0.0009 ) when compared to saline infusion ( -10.63  27.87% , n = 6 ) ( figure 5b ) . the data presented are the signal intensities of the muscle mbv divided by that in the femoral vessels . this reduces experimental variation between different measurements and between different mice ( data not shown ) . the signal intensity in the femoral vessels corresponds linearly with the concentration of microbubbles in the circulation ( figure 3c ) . correcting for the femoral vessels ' signal theoretically corrects for differences in concentrations of microbubbles used ( figure 3d ) . ( a ) an incision is made at the groin , parallel to the direction of the inguinal ligament . ( b ) gentle traction at the fat pad in the distal directions ( black arrows ) will present the connective tissue ( * ) between the fat pad and the abdominal wall . ( c ) the skin folds of the incision can be adjusted using the bulldog clamp to create a small cavity to hold the paraffin oil that bathes the vessels . ( d ) the ultrasound probe is positioned on the contralateral upper hindlimb after the prepared gracilis artery is viewed using a calibrated microscope . the femoral artery ( a ) gives rise to the epigastric artery ( b ) and the gracilis artery ( c ) which runs over the adductor muscle group ( d ) . figure 3:signal intensity of contrast - enhanced ultrasonography in muscle microvascular blood volume and femoral vessels . ( a ) view of the nonlinear contrast imaging mode of the digital imaging platform during microbubbles constant infusion in a male mouse upper hindlimb . right panel : two rois are drawn to represent the muscle mbv and the femoral vessels . only the superficial part of the adductor muscle compartment is included in the roi as the signal intensity decreases with depth . left panel : time - intensity curve from the muscle mbv roi . vertical lines represent the destruction of the microbubbles ( mbd ) with high energy waves . immediately after the mbd , no contrast agent is present in the imaging plane which starts to fill with microbubbles gradually . after 10 - 15 s , the peak of the contrast enhancement has been reached . ( b - d ) after a steady - state signal was reached , the infusion rate of 2.5 x 10 bubbles / ml was doubled ( 5 , 10 , 20 l / min ) . signal intensity from muscle mbv ( b ) and femoral vessels ( c ) paralleled the doubling of the microbubble concentration in the circulation . ( d ) correcting muscle mbv for the femoral vessels signal removes the variability in signal intensity caused by different microbubble concentrations ( n = 9 ; error - bars represent standard deviation ) . ( a ) paraffin oil reduces the variation of the gracilis arteries of different animals ( which is 29.0 m without paraffin vs 17.9 m after applying the oil ) while keeping the average baseline diameter stable ( 73.6 m vs. 68.8 m ; p = 0.58 ) . ( b ) arterial diameters at baseline and after 60 min of insulin or saline infusions . ( c ) insulin - induced vasodilation occurs at 10 min after the start of infusion ( p = 0.002 ) and reaches 95% of the maximum at 30 min . error - bars represent standard deviation ; unpaired student 's t - test is used for statistics . figure 5:microvascular blood volume measurements using contrast - enhanced ultrasonography of the adductor muscle compartment of the mouse hindlimb . ( a ) insulin resulted in a consistent increase in mbv 30 min after the start of insulin infusion . ( b ) the difference between the hyperinsulinemic and the baseline measurements ( mbv change ) is denoted as the insulin - mediated  microvascular recruitment . insulin induced a 33.5% (  31.04% , p = 0.016 ; n = 7 ) microvascular recruitment compared to saline infusion ( -10.63  27.87% , n = 6 ) . error - bars represent standard deviation ; unpaired student 's t - test is used for statistics . we have developed a technique to simultaneously estimate insulin 's vascular actions on the larger arteries ( using ivm ) and the skeletal muscle microcirculation ( using ceus ) . the critical steps for successful and reliable measurements are : 1 ) correctly exposing the gracilis artery without bleeding ; 2 ) preventing leakage of the paraffin oil bathing the artery ; 3 ) having a patent venous access ( tail vein cannula ) for infusion of vasoactive compounds ( insulin ) and contrast agent ( microbubbles ) . the study of microvascular dysfunction in muscle has been gaining attention in the context of obesity and insulin resistance14253940 . the negative impact of obesity and insulin resistance on vascular function is manifested at different levels of the vascular tree . the combined use of the ivm and ceus techniques in the same mouse provides a powerful tool to quantify insulin 's effects at different levels of the vasculature . ivm allows direct visualization and quantitative analysis of the resistance artery and ceus allows for assessment of insulin - induced changes in muscle perfusion . the arteries are readily accessible and the superficial nature of the incision makes it possible to close the skin incision with a 5.0 absorbable sterile suture after the experiment is finished . the animals were injected subcutaneously with buprenorphine after the experiments as an analgesic at a dose of 0.1 mg / kg and allowed to recover in a warm environment . the mice tolerated the procedure very well and we experienced no loss of animals nor infections of the hindlimb in more than 35 animals studied . this makes it possible to follow - up or study the animals in a longitudinal fashion . the animals used in these experiments , however , were anesthetized using 1.8% inhaled isoflurane balanced with oxygen flowing at 0.4 l / min though an anesthesia mask . in contrast to isoflurane anesthesia4142 , fma anesthesia does not disturb peripheral insulin sensitivity . a future plan is to study how well the mice recover from fma anesthesia . the adductor muscle compartment is also useful since various vasoactive compounds mediating local and downstream vascular effects can be evaluated . for example , topical application of these compounds to the target tissue is feasible using superfusion techniques28 or surgical manipulation and implantation of drug - eluting cuffs surrounding the vessels43 . our group and others have gathered substantial experimental evidence using the pressure myograph to document the effects of insulin and other vasoactive compounds on this artery ex vivo363738 . a limitation inherent to the use of the ivm technique is the surgical exposition of the muscle and application of paraffin oil to stabilize the vessels . however , figure 3a shows that the baseline diameter of the gracilis artery bathed in paraffin oil does not change considerably . it has been also shown that mineral oil successfully inhibits the diffusion of oxygen , protecting the tissue from hyperoxic conditions44 . moreover , the oil helps to reduce the variation in the baseline diameter of the arteries . this is why we advocate to use paraffin oil and let the preparation rest for at least 30 min . of note , the use of buffered saline instead of oil  or no oil at all  resulted in highly variable diameters and constriction of the vessel ( data not shown ) . moreover , at the end of the experiments , we isolated the gracilis arteries - bathed in paraffin oil - and tested their reactivity in the pressure myograph ex vivo . the paraffin oil - bathed arteries reacted similarly to control arteries when stimulated with insulin and acetylcholine ( a vasodilator ) ( data not shown ) . the consistent insulin - induced vasodilation clearly shows that the ivm protocol described in this study produces reliable results . the advantage of applying both techniques in the same mouse overcomes some of the intrinsic limitations of one technique by the other : ceus estimates mbv in the undisturbed muscle in vivo , but individual vessels can not be seen ; ivm makes it possible to see individual vessels , albeit it can not estimate mbv . a future plan is to utilize ivm microscopy of the cremaster muscle in combination with ceus of the adductor muscle on the contralateral side . this modification can provide an estimate of the mbv ( using ceus ) and a direct optical access to the capillaries ( using ivm ) . the protocol can be further modified ; the 4-way connector used for the tail cannula can be switched to a 5-way connector . by this , we can avoid detaching the anesthesia tube while performing the second ceus measurement ( described in point 2.9 ) . another modification that can be made to this protocol is the insulin clamp rate used . we used 7.5 mu / kg / min clamp rate which is considered supra - physiological . depending on the study , a lower insulin clamp rate ( for example 3 mu / kg / min ) can be used . while we have found the described protocol reliable , there are specific limitations that need attention . it is crucial that the paraffin oil does not leak from the vessel environment as supplementing it with new oil will disturb the vessel and change the diameter , making it necessary to let the artery rest for another 30 min . in addition , the reflection of the light ( described in step 1.14 of the protocol ) on the surface of the paraffin oil was sometimes too bright , making it difficult to view the artery . this can be counteracted by directing the light source so that the light falls at an angle to the paraffin oil surface and parallel to the artery . in conclusion , the combination of ivm and ceus techniques described in this study makes it possible to quantify different effects of insulin at different levels of the vasculature . ivm of the gracilis artery provides insight into the upstream vascular changes contributing to downstream microvascular perfusion measured using ceus . we advocate the combination of several experimental techniques in the same mouse to better assess the vascular function . visual sonics inc . covered the open access fees , whereas the structure and content of the article remained the full responsibility of the authors .
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 .