1Department of Anesthesiology and Critical Care, Shriners Hospital for Children, Massachusetts General Hospital, and Harvard Medical School, 2Department of Geriatric Medicine, Graduate School of Medicine, The University of Tokyo
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Asai, A., Sahani, N., Ouchi, Y., Martyn, J., Yasuhara, S. In vivo Micro-circulation Measurement in Skeletal Muscle by Intra-vital Microscopy. J. Vis. Exp. (4), e210, doi:10.3791/210 (2007).
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.
RBC membrane staining
In vivo Microscopic Observation of Skeletal Muscles
We followed Jeff W. Lichtman's method with modification1:
RBC flux analysis
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.
All the procedures related to animal experiments were reviewed and approved by the Subcommittee on Research Animal Care of Massachusetts General Hospital.
We thank JW Lichtman for his advice in in vivo observation of muscles.
|PKH26 dye||Sigma-Aldrich||551/567 nm; Red fluorescent cell linker kit|
|C57BL/10 mice||Animal||Three to six month old males|
|pentobarbital||anesthetic, 50mg/kgBW, i.p.|
1. Lichtman, J. W., Magrassi, L., Purves, D. Visualization of neuromuscular junctions over periods of several months in living mice. J Neurosci 7, 1215-22 (1987).