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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

An open vessel-window approach using fluorescent tracers provides sufficient resolution for cochlear blood flow (CoBF) measurement. The method facilitates the study of structural and functional changes in CoBF in mouse under normal and pathological conditions.

Abstract

Transduction of sound is metabolically demanding, and the normal function of the microvasculature in the lateral wall is critical for maintaining endocochlear potential, ion transport, and fluid balance. Different forms of hearing disorders are reported to involve abnormal microcirculation in the cochlea. Investigation of how cochlear blood flow (CoBF) pathology affects hearing function is challenging due to the lack of feasible interrogation methods and the difficulty in accessing the inner ear. An open vessel-window in the lateral cochlear wall, combined with fluorescence intravital microscopy, has been used for studying CoBF changes in vivo, but mostly in guinea pig and only recently in the mouse. This paper and the associated video describe the open vessel-window method for visualizing blood flow in the mouse cochlea. Details include 1) preparation of the fluorescent-labeled blood cell suspension from mice; 2) construction of an open vessel-window for intravital microscopy in an anesthetized mouse, and 3) measurement of blood flow velocity and volume using an offline recording of the imaging. The method is presented in video format to show how to use the open window approach in mouse to investigate structural and functional changes in the cochlear microcirculation under normal and pathological conditions.

Introduction

Normal function of the microcirculation in the lateral cochlear wall (comprising the majority of the capillaries in the spiral ligament and stria vascularis) is critically important for maintaining hearing function1. Abnormal CoBF is implicated in the pathophysiology of many inner ear disorders including noise-induced hearing loss, ear hydrops, and presbycusis2,3,4,5,6,7,8,9. ....

Protocol

NOTE: This is a non-survival surgery. All procedures involving the use of animals were reviewed and approved by the Institutional Animal Care and Use Committee at Oregon Health & Science University (IACUC approval number: TR01_IP00000968).

1. Preparation of the fluorescent-labeled blood cells

  1. Anesthetize the donor mice (male C57BL/6J mice aged ~6 weeks) with an intraperitoneal (i.p.) injection of ketamine/xylazine anesthetic solution (5 mL/kg, see the Table of Materials.......

Representative Results

After surgical exposure of the cochlear capillaries in the lateral wall (Figure 1), intravital high-resolution fluorescence microscopic observation of Dil-labeled blood cells in FITC-dextran-labeled vessels was feasible through an open vessel-window. Figure 2A is a representative image taken under FIVM that shows the capillaries of the mouse cochlear apex-middle turn lateral wall. The lumina of these vessels is made visible by th.......

Discussion

This paper demonstrates how capillaries in the cochlear lateral wall (and in the stria vascularis) of a mouse model can be visualized with fluorophore labeling in an open vessel-window preparation under a FIVM system. Mouse model is widely used and preferred as a mammalian model for investigating  human health and disease. The protocol described here is a feasible approach for imaging and investigating CoBF in the mouse lateral wall (particularly in the stria vascularis) using an open vessel-window under FIVM system.......

Acknowledgements

This research was supported by NIH/NIDCD R21 DC016157 (X.Shi), NIH/NIDCD R01 DC015781 (X.Shi), NIH/NIDCD R01-DC010844 (X.Shi), and Medical Research Foundation from Oregon Health and Science University (OHSU) (X.Shi).

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Materials

NameCompanyCatalog NumberComments
0.9% Sodium ChlorideHospiraNDC 0409-1966-020.6 mL (for 1 mL)
1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorateSigma Aldrich46849520 µM
3,3′-Dioctadecyloxacarbocyanine perchlorateDio (3,3′-Dioctadecyloxacarbocyanine perchlorateSigma AldrichD429220 µM
CODA Monitor systemKent scientificCODA Monitor, for monitoring blood pressure and heartbeat
CoverslipFisher Scientific12-542A
DC Temperature ControllerFHC40-90-8D
Fiji/ImageJNIHMeasurement of vessel diameter
FITC-dextran (2000 kDa)Sigma AldrichFD2000s40 mg/mL
Heparin Sodium Injection, USP MDVMylanNDC 67457-374-125000 USP units/mL
Katathesia (100 mg/mL)Henry ScheinNDC 11695-0702-10.2 mL (for 1 mL)
Microscope ObjectiveMitutoyo378-823-5Model: M Plan Apo NIR 10x
ORCA-ER CameraHamamatsuModel: C4742-80-12AG
PBSGibco2085387
Xyzaine (100 mg/ml, 5x diluted for use )LloydLPFL048210.2 mL (for 1 mL)
Zoom Stereo MicroscopeOlympusModel: SZ61, fluorescent microscope

References

  1. Shi, X. Physiopathology of the cochlear microcirculation. Hearing Research. 282 (1), 10-24 (2011).
  2. Brown, J. N., Miller, J. M., Nuttall, A. L. Age-related changes in cochlear vascular conductance in mice. Hea....

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