Name: In vivo Micro-circulation Measurement in Skeletal Muscle by Intra-vital Microscopy (Video) | JoVE Journal
Uploaded: 2007-05-28T00:00:00.000+00:00
Duration: 7 min 31 s
Description: Learn in vivo micro-circulation measurement in skeletal muscle using intra-vital microscopy. This versatile technique enables continuous observation of blood flow parameters including red blood cell flux and velocity.

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5 minutes

We thank JW Lichtman for his advice in in vivo observation of muscles.

RBC membrane staining
<ol>
<li>Mouse red blood cells were corrected from the same mouse strain with heparinization.</li>
<li>Mouse RBC were stained with PKH26 dye (551/567 nm; Red fluorescent cell linker kit, Sigma, U.S.A.).</li>
<li>RBC were washed in PBS and incubated with 2&mu;M solution of PKH26 dye for 5 minutes at room temperature. The reaction was stopped by adding plasma (heat inactivated for 1 hour at 65&deg;C beforehand) and incubated for 1 minute.</li>
<li>The stained RBC were washed 5 times with PBS.</li>
</ol>
Mouse Preparation
<ol>
<li>Three to six month old male C57BL/10 mice were used in this study. We anesthetized mice by intraperitoneal injection of pentobarbital (50mg/kgBW).</li>
<li>Mice were then intubated with a 20-gage polyethylene tube, mechanically ventilated, and warmed at 37&deg;C on a Kapton sheet heater connected to a thermocouple sensor and feedback temperature controller system.</li>
<li>The ventral side of the neck was shaved. The right and left sternomastoid muscles were exposed. The muscles were supported by a stainless steel holder and superfused with sterile Krebs Ringer solution.</li>
</ol>
Injection
<ol>
<li>Stained RBCs were warmed at 37&deg;C, diluted with Krebs Ringer. 50ul of stained RBC (hematocrit 12%) was injected into mouse from the penile vein.</li>
</ol>
In vivo Microscopic Observation of Skeletal Muscles
We followed Jeff W. Lichtman's method with modification1:
<ol>
<li>Animals were placed on a heating pad on top of a hand-made iron stage of a Nikon Eclipse-800 microscope. Water-dipping objective lenses were utilized for live epi-fluorescent observation. We observed PKH26 stained RBC flowing inside of vascular under fluorescent light of a Mercury lamp. We identified primary arterioles, secondary arterioles, capillaries and veins by their RBCs flow directions and architectures.</li>
<li>An intensified SIT Camera (Hamamatsu Photonics, C2400, Japan) and video-frame grabber were installed to capture the low-intensity signal at the real-time video rate (30 frames per second). Images were recorded on DVD as video files.</li>
</ol>
Muscle Stimulation
<ol>
<li>An electrical pulse was generated using a Peripheral Nerve Stimulator (Innervator 252, Fisher &amp; Paykel Healthcare, New Zealand), and delivered to muscle surface via a coated stainless probe. A minus probe end was placed on the proximal part of muscle and a plus end was placed on the distal part of muscle. Tetanus stimulations were given at the rate of 50Hz for 5 seconds. The electrical stimuli did not cause direct myofiber damage on the vicinity of contact area.</li>
</ol>
RBC flux analysis
<ol>
<li>Recorded DVD video images were transferred Video Savant image files through frame grabber software (Video Savant, U.S.A.). Video images were reviewed in adjusted speed in order to visualize individual stained RBC. RBC flux was measured by counting RBC, which flew through a focused primary arteriole per minute.</li>
</ol>

<ol><li>Lichtman, J. W., Magrassi, L., Purves, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Visualization+of+neuromuscular+junctions+over+periods+of+several+months+in+living+mice%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">Visualization of neuromuscular junctions over periods of several months in living mice.</a> J Neurosci. 7, 1215-1222 (1987).</li>
</ol>

All the procedures related to animal experiments were reviewed and approved by the Subcommittee on Research Animal Care of Massachusetts General Hospital.

Important techinical points are as follows: (1) maintenance of physiological status of the animal (ventilation, pH of the perfusative solution, body temperature), (2) injection volume of the stained RBC, and (3) conditions for observation (optimal lens selection, fluorescence intensity). Potential future applications are as follows: (a) combination with pharmacophysiological and/or molecular biological interventions, (b) long-term observation of arterio/arteriolo-sclerosis and neovascularization.

<table><tbody><tr><td>PKH26 dye </td><td></td><td>Sigma-Aldrich</td><td></td><td>551/567 nm; Red fluorescent cell linker kit</td></tr><tr><td>C57BL/10 mice </td><td>Animal</td><td></td><td></td><td>Three to six month old males</td></tr><tr><td>pentobarbital </td><td></td><td></td><td></td><td>anesthetic, 50mg/kgBW, i.p.</td></tr></tbody></table>

in vivo micro-circulation measurement in skeletal muscle by intra-vital microscopy

In vivo Micro-circulation Measurement in Skeletal Muscle by Intra-vital Microscopy

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of imaging had invented. While many macroscopic parameters of blood flow reflect flow velocity, changes in blood flow velocity and red blood cell (RBC) flux does not hold linear relationship in the microscopic observations. There are reports of discrepancy between RBC velocity and RBC flux, RBC flux and plasma flow volume, and of spatial and temporal heterogeneity of flow regulation in the peripheral tissues in microscopic observations, a scientific basis for the requirement of more detailed studies in microcirculatory regulation using intravital microscopy. METHODS: We modified Jeff Lichtman's method of in vivo microscopic observation of mouse sternomastoid muscles. Mice are anesthetized,
ventilated, and injected with PKH26L-fluorescently labeled RBCs for microscopic observation.RESULT &amp; CONCLUSIONS: Fluorescently labeled RBCs are detected and distinguished well by a wide-field microscope. Muscle contraction evoked by electrical stimulation induced increase in RBC flux. Quantification of other parameters including RBC velocity and capillary density were feasible. Mice tolerated well the surgery, injection of stained RBCs, microscopic observation, and electrical stimulation. No muscle or blood vessel damage was observed, suggesting that our method is relatively less invasive and suited for long-term observations.

A new versatile method for observation of microcirculation is presented. It is considered suitable for long-term observation, and for combination with pharmacophysiological or molecular biological interventions.

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of ...

Hi, my name is Aki, A research fellow in Harvard Medical School, Massachusetts General Hospital. In this video journal, I will show a new method of in vivo microscope. With this technique, we can measure quantitative blood flow in micro secretion.The purpose of our experiment is to establish a system for continuative measurement of in vivo micro secretion. We designed this project to visualize blood flow at the level of capillaries and anterior in muscles of live animal under the microscope compared with the conventional method for in vivo ion measurement such as do blood ultrasound or oxygen tension, ours will enable measurement of multiple parameters of blood flow, namely red blood cell flux or obesity velocity and plasma flow. Measuring these parameters will uncover micro circulation changes in more detail.Then our method is applied to detect blood flow changes in response to physiological stimulation in live muscle tissues. Our method is more simple and less invasive than conventional in vivo microscopy so that muscles are directly accessible and we are able to apply electrical physiological stimulation or mouse muscles and to describe the detailed response of blood flow to muscle contraction. General anesthesia was induced by sodium pentobarbital.The hair on the neck was removed by depleting cream. Insert an endotrachial tube. Connect to mechanical ventilator inside the neck.Skin expose sternum mastoid muscle Hold stern mastoid muscle mass was placed on temperature. Controlling pad, Obtain blood From a mouse wash red blood cell with PBS and stay with PKH 26. Incubate five minutes at room temperature, quench the dye with heat inactivated plasma and wash the red blood cells with PBS.The stained red blood cells were injected intravenously in another mouse of The same strain connect probes to electro pulse generator Place probes on muscle place on microscope stage. Observe and record the images under the fluorescent light under the microscope. Observation, normal physiological contraction was induced by pulse generator.The following are several important technical aspect in this experiment to obtain consistent data. First, in order to maintain normal physiological status of the animal, we connected the mouse to a mechanical ventilator tidal volume of one 60 microliter with respiratory rate of one 60 kept the blood gas parameters under consistent physiological status. Body temperature of the animal was controlled with the heating pad and kept at 37 degrees Celsius Second for quantifying blood flow, we counted stain red blood cells, which run through capital E injection.Volume of stain RBC was consistent so that the ratio of stain over non stain RBC should be 0.1 to 0.5%This injection method attained optimal RBC to RBC separation when counting the total number of RBC flow was calculated from the actual count and the ratio of stained to non-ST stained population. Last for in vivo observation under the microscope, an optimal lens selection was critical. An appropriate objective limb was selected based on its ability of water immersion and walking distance.Also, we decrease the fluorescent light intensity with combination of neutral density filters to avoid unexpected light effect on muscle tissue. Further application, we plan to extend this method to study the response of micro circulation to various pharmacological and molecular biological interventions. We will be able to measure how both dilators or vessel constrictors change workflow at the level of capillaries and artery rolls because animals tolerate the procedure and survive well After the experiment, we are able to perform timeless pull up of the same animal in the long term.For example, it is possible to follow subtle changes of arteriosclerosis or angiogenesis at the micro secretory level in the individual vessels of the Same individual mouse.

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Learn in vivo micro-circulation measurement in skeletal muscle using intra-vital microscopy. This versatile technique enables continuous observation of blood flow parameters including red blood cell flux and velocity.

In vivo Micro-circulation Measurement in Skeletal Muscle by Intra-vital Microscopy

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