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

Summary

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.

Abstract

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 & 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.

Protocol

RBC membrane staining Mouse red blood cells were corrected from the same mouse strain with heparinization. Mouse RBC were stained with PKH26 dye (551/567 nm; Red fluorescent cell linker kit, Sigma, U.S.A.). RBC were washed in PBS and incubated with 2μM solution of PKH26 dye for 5 minutes at room temperature. The reaction was stopped by adding plasma (heat inactivated for 1 hour at 65°C beforehand) and incubated for 1 minute. The stained RBC were washed 5 times with PBS. </…

Discussion

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.

Materials

Material Name	Type	Company	Catalogue Number	Comment
PKH26 dye		Sigma		551/567 nm; Red fluorescent cell linker kit
C57BL/10 mice	Animal			Three to six month old males
pentobarbital				anesthetic, 50mg/kgBW, i.p.