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

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.