Measurement of Strial Blood Flow in Mouse Cochlea Utilizing an Open Vessel-Window and Intravital Fluorescence Microscopy

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 presbycusis^{2,3,4,5,6,7,8,9}. Visualization of intravital CoBF will enable a better understanding of the links between hearing function and cochlear vascular pathology.

Although the complexity and location of the cochlea within the temporal bone precludes direct visualization and measurement of CoBF, various methods have been developed for the assessment of CoBF including laser-doppler flowmetry (LDF)^10,11,12, magnetic resonance imaging (MRI)¹³, fluorescence intravital microscopy (FIVM)¹⁴, fluorescence microendoscopy (FME)¹⁵, endoscopic laser speckle contrast imaging (LSCI)¹⁶, and approaches based on the injection of labeled markers and radioactively tagged microspheres into the bloodstream (optical microangiography, OMAG)^17,18,19,20. However, none of these methods has enabled absolute real-time tracking of changes in CoBF in vivo, with the exception of FIVM. FIVM, in combination with a vessel-window in the lateral cochlear wall, is an approach that has been used and validated in guinea pig under different experimental conditions by various laboratories^14,21,22.

An FIVM method was successfully established for studying the structural and functional changes in the cochlear microcirculation in mouse using fluorescein isothiocyanate (FITC)-dextran as a contrast medium and a fluorescence dye-either DiO (3, 3′-dioctadecyloxacarbocyanine perchlorate, green) or Dil (1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate, red)-for prelabeling blood cells, visualizing vessels, and tracking blood flow velocity. In the present study, the protocol of this method has been described for imaging and quantifying changes in CoBF in mouse under normal and pathological conditions (such as after noise exposure). This technique gives the researcher the tools needed to investigate the underlying mechanisms of CoBF related to hearing dysfunction and pathology in the stria vascularis, especially when applied in conjunction with readily available transgenic mouse models.

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).
   ​NOTE: This anesthesia protocol is very reliable and maintains systemic blood pressure.
2. Lay the mouse on its back, open the skin and expose the chest cavity using tweezers and dissecting scissors. Cut open the diaphragm, grab at the base of the sternum using clamp scissors, cut through the ribcage and lift to expose the heart. Collect 1 mL of blood in heparin (15 IU/mL blood) by cardiac puncture, and centrifuge at 3,000 × g for 3 min at 4 °C. Euthanize the mouse by cervical dislocation after blood collection.
3. Remove the plasma, wash the blood cell pellet with 1 mL of phosphate-buffered saline (PBS), and centrifuge 3x at 3000 × g for 3 min at 4 °C.
4. Label the blood cells with 1 mL of 20 mM DiO or Dil in PBS, and incubate in the dark for 30 min at room temperature^23,24.
5. Centrifuge and wash the labeled blood cells with 1 mL of PBS, centrifuge 3x at 3000 × g for 3 min at 4 °C, and resuspend the cell pellet in 30% hematocrit with ~0.9 mL of PBS (final volume ~1.3 mL) before injection.

2. Surgery to create an open window ²⁵

1. Prepare the sterile surgical instruments and imaging platform, and place a heating pad beneath the drape (Figure 1A). Anesthetize the mice (male C57BL/6J mice aged ~6 weeks) as described in step 1.1, and check the depth of the anesthesia by monitoring the paw reflex and general muscle tone. Place the animal on the warm heating pad, and maintain the rectal temperature at 37 °C.
2. Place the animal tail in the CODATM monitor system for monitoring blood pressure and heartbeat (see the Table of Materials). Record the animal's systolic blood pressure, diastolic blood pressure, and mean blood pressure (MBP) in the anesthetized condition.
   NOTE: No difference is seen in animal MBP in the anesthetized and un-anesthetized state (107 ± 11 mmHg vs. 97 ± 7 mmHg). The animal heart rate is stable under anesthetization, though lower (still within normal range) than in the non-anesthetized condition (357 ± 12 bmp vs. 709 ± 3 bmp). A slightly increased heart beat should be expected in animals placed in restraint²⁶.
3. Open the left tympanic bulla via a lateral and ventral approach under a stereo microscope (see the Table of Materials), leaving the tympanic membrane and ossicles intact²¹.
   1. Make an incision along the midline of the animal's neck with its head immobilized and positioned to minimize movement (Figure 1B). Remove the left submandibular gland and posterior belly of the digastric muscle and cauterize.
      NOTE: Subcutaneously inject buprenorphine at 0.05 mg/kg to reduce the pain during surgery.
   2. Locate and expose the bony bulla by identifying the sternocleidomastoid muscle and facial nerve extending anterior toward the bulla.
   3. Open the bony bulla with a 30 G needle, and carefully remove the surrounding bone with surgical tweezers to provide a clear view of the cochlea and stapedial artery, with its medial margin lying over the edge of the round window niche, and coursing anterior-superior towards the oval window (Figure 1C, D).
      NOTE: Tracheotomy is performed to keep the airway unobstructed and should only be done when the animal has a respiratory issue during surgery. In general, most of the animals under anesthesia have smooth breathing. However, if animals received a tracheotomy during surgery, the recorded blood flow should not be used for any comparison.
4. Use a small knife blade (custom-milled #16 scalpel) to scrape the lateral wall bone at the apex-middle turn of the mouse cochlea, approximately 1.25 mm from the apex until a thin spot is cracked. Remove the bone chips with small wire hooks (Figure 1E).
5. Cover the vessel-window with a cut coverslip to preserve normal physiological conditions and provide an optical view for recording vessel images.
   NOTE: All procedures are to be performed with caution. In addition to monitoring body temperature, the animal's vital signs, including blood pressure and heartbeat, should also be monitored throughout the surgery.

3. Imaging of CoBF under FIVM

1. Make an ~1 cm incision along the right saphenous vein to expose the vessel (Figure 1F).
2. Infuse 100 μL of the FITC-dextran solution (2000 kDa, 40 mg/mL in PBS) and 100 μL of a blood cell suspension (30% hematocrit) successively into the animal through the saphenous vein (Figure 1G) to enable visualization of the blood vessels and tracking of blood flow velocity.
3. Observe the blood flow in real time directly on a video monitor 5 min after the injection. Image the blood vessels using a fluorescence microscope equipped with a long working distance (W.D.) objective (W.D. 30.5 mm, 10x, 0.26 numerical aperture) and a lamp-housing containing a multiple band excitation filter and compatible emission filter (Table of Materials). Record the video using a high-resolution digital black and white charge-coupled device camera (Table of Materials) at 2 frames/s). Acquire more than 350 images per video to ensure successful analysis of the flow velocity.
   NOTE: Blood vessels of both the spiral ligament and stria vascularis can be imaged by adjusting the optical focus (Supplemental Video 1).

4. Video analysis

1. Measure the vessel diameter using appropriate software (Table of Materials), and determine the distance between two fixed points across the vessel in the acquired images.
2. Calculate the blood flow velocity from captured video frames by tracing the movement of labeled blood cellsin the spatial distance between image locations²⁷.
   1. Open the video of blood flow in the software (Fiji [ImageJ] was used in this protocol), and set the scale of the images.
   2. Track the selected DiO-stained blood cells using the tracking function. Use the distance the cells have moved and the interval of time between image frames in the video for auto-calculation of the flow velocity.
3. Calculate the volumetric flow (F) per the following equation: F = V × A. (V: velocity; A: cross-sectional area of the vessel).

5. Noise Exposure

1. Place the animals in wire mesh cages. Expose them to broadband noise at 120 dB sound pressure level in a sound exposure booth for 3 h and for an additional 3 h the next day.
   NOTE: This noise exposure regime, routinely used in this laboratory, produces permanent loss of cochlear sensitivity²⁸.

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 the fluorescence of FITC-dextran mixed with plasma. Individually labeled blood cells distributed in the vascular network are also clearly visible in this image. Two distinct networks-capillaries of the spiral ligament and capillaries of the stria vascularis-are distinguished by location (under an upright microscope, the stria vascularis runs optically beneath the spiral ligament, and it contains more capillary loops and smaller vessels²⁵). Both can be assessed for blood flow with adjustment of the optical focus. As shown in Figure 2B, C, the vessel density of the spiral ligament is sparser than that of the stria vascularis.

Vascular function in the noise-exposed mouse model was compared with vascular function in the control group. CoBF measurement was taken 2 weeks after noise exposure. Figure 3A,B are representative images showing the flow patterns in control and noise-exposed groups. A disturbed pattern of blood flow was seen in the noise-exposed group (Figure 3B). Anomalies included reduced vessel diameter (Figure 3C) and increased variation in vessel diameter (Figure 3D). As illustrated in Figure 4A,B, the blood flow velocities in the control and noise-exposed groups were calculated by tracking the routes of the DiO-labeled blood cells (Supplemental video 2). The results show that blood velocity and volume in the noise-exposed group were significantly lower than in the control group (Figure 4C, D). These data indicate that loud sound notably affects blood circulation and causes reduced and disturbed blood flow.

Figure 1: Preparation of an open vessel-window for IVM imaging in mouse. (A) Preparation of instruments and tools for the surgery. (B) The left bulla was exposed via a lateral and ventral approach. (C) The cochlea was exposed after removing the bulla. (D) Magnified image of the cochlea, from the circle in (C). (E) An open vessel window was created at the apex-middle turn of the cochlear lateral wall (box). (F and G) Intravenous infusion of FITC-dextran and labeled blood cells through the saphenous vein. Abbreviations: OW = oval window; RW = round window; IVM = intravital microscopy; FITC = fluorescein isothiocyanate. Please click here to view a larger version of this figure.

Figure 2: Representative images of cochlear capillaries in the lateral wall. (A) Dil-labeled blood cells (red, arrow) in strial vessels labeled with FITC-dextran (green). (B) DiO-labeled blood cells (green) in spiral ligament vessels labeled with FITC-dextran (arrows, green). Note the vessels are sparse. (C) DiO-labeled blood cells (green) in strial vessels labeled with FITC-dextran (green). Note the vessels are denser. Scale bars: 50 µm and 100 µm. Abbreviations: Dil = 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate; DiO = 3, 3′-dioctadecyloxacarbocyanine perchlorate; FITC = fluorescein isothiocyanate. Please click here to view a larger version of this figure.

Figure 3: Change in vessel diameter in the stria two weeks after noise exposure. (A and B) Representative images of the blood circulation after labeling vessels with FITC-dextran (green) and blood cells with DiO (green) in control and noise-exposed (NE) groups with high-resolution IVM. (C) Meanvessel diameter calculated for control and NE groups. Compared to the control group, the vessel diameter was reduced in the noise-exposed group. (n = 18, t (34) = 2.880, **p = 0.007, Student's t-test, mean ± standard deviation [SD]). (D) Variance of vessel diameter in control and NE groups. The vessel diameter varied much more in the NE group than in the control group. (n = 6, t (10) = 6.630, ****p < 0.0001, Student's t-test, mean ± SD). Scale bars: 100 µm. Abbreviations: DiO = 3, 3′-dioctadecyloxacarbocyanine perchlorate; FITC = fluorescein isothiocyanate; IVM = intravital microscopy. Please click here to view a larger version of this figure.

Figure 4: Blood flow changes in the stria two weeks after noise exposure. (A and B) Representative images show the tracking routes (red, green, and blue lines) of DiO-labeled blood cells (green) for blood flow velocity measurement in control and noise-exposed groups. (C and D) Blood flow velocity (µm/s) and volumetric flow rate (µm³/s) were respectively calculated for control and noise-exposed groups. Blood flow rate and volumetric flow rate in the noise-exposed group were lower than in the control group (n = 54,_tvelocity (106) = 19.705, ****p_velocity < 0.0001; t_volume (106) = 15.342, ****p_volume < 0.0001, Student's t-test, mean ± standard deviation). Scale bars: 100 µm. Abbreviations: DiO = 3, 3′-dioctadecyloxacarbocyanine perchlorate1; FITC = fluorescein isothiocyanate; NE 2W = two-week noise exposure. Please click here to view a larger version of this figure.

Supplemental video 1: Blood vessels of both the spiral ligament and stria vascularis. Please click here to download this video.

Supplemental video 2: Tracking the routes of DiO-labeled blood cellsfor calculation of the blood flow velocities in a control animal. Abbreviations: DiO = 3, 3′-dioctadecyloxacarbocyanine perchlorate. Please click here to download this video.

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 The method provides sufficient resolution for determining the blood flow velocity and volume using fluorescence-labeled blood cells as a tracer (as shown in Figure 3 & Figure 4). This approach can be used for several different applications, such as the assessment of vascular permeability and the examination  of pericyte contractility, and  tracking circulating bone marrow cell migration²⁵. Acute changes in CoBF in response to trauma, infection, noise, foreign bodies, ototoxic drugs, or other agents affecting the lateral wall can also be monitored in real-time^25,29. 
 
In past decades several relatively non-invasive methods were established to assess CoBF without removing the cochlear bony wall, including LDF, MRI, OMAG, endoscopic LSCI, endoscopic FME , and the injection of microspheres into the blood plasma. However, none of these approaches can be used for evaluating  the absolute flow rate in individual vessels. For example, non-invasive endoscopic FME can only be used to image limited regions near the round window but not  blood flow in the stria vascularis.  However, blood circulation in the stria vascularis  has  crucial roles in maintaining the EP, ion transport, and endolymphatic fluid balance, all essential for hearing sensitivity (Zhang et al., 2021). OMAG is also useful for visualization of blood flow in the intact cochlea, however, the resolution is too poor for imaging individual capillaries in the stria vascularis of a mouse model¹⁹.

An open vessel-window, in conjunction with a FIVM, enables real-time visualization of blood flow in the cochlear lateral wall and measurement of vascular diameter and blood flow velocity in the recorded regions. The IVM system has several advantages over other methods. For example, the animal can be conveniently positioned and manipulated as needed. There is flexibility to adjust the contrast of the fluorescence-labeled plasma and blood cells to optimize visualization of vascular architecture and highlight relevant structures. The different molecular weights of FITC-conjugated tracers   can also be selected to optimize the evaluation of vascular permeability. e The choice of whether to use Dil (lex = 550 nm; lem = 564 nm) or Dio (lex = 484 nm; lem = 501 nm) to label the blood cells, in contrast to the FITC-dextran used to label the plasma, is determined by experiment goals. In general, Dil provides better contrast for the visualization of the vessel lumen and individual blood cells, while Dio is preferred when simultaneous imaging is used for visualization of blood cells and lumen in the same acquisition channel. What’s more, tracking is best accomplished with a small number of labeled cells since blood cell fluorescence will mask the vessel lumen fluorescence if all blood cells in the vessel are labeled²¹. In addition, care should be exercised to limit the amount of solution administered to blood,  to minimize dilution and viscosity-related blood flow changes in the cochlea³⁰.

Overall, this method is robust and can be used for investigating structural and functional changes in CoBF under normal and pathological conditions such as inflammation, noise, and aging. Importantly, a constant and normal EP can be maintained during  the surgery, indicating the procedure is harmless to cochlear function when performed carefully enough²⁵. However, the successful establishment of the open vessel-window does require a high degree of surgical skill. For example, care must be exercised in removing the bone of the cochlear lateral wall  to prevent loss of perilymphatic fluid and micro-injury to the outer layer of the cochlear spiral ligament. These could  adversely affect cochlear homeostasis and compromise the imaging.

Materials

Name	Company	Catalog Number	Comments
0.9% Sodium Chloride	Hospira	NDC 0409-1966-02	0.6 mL (for 1 mL)
1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate	Sigma Aldrich	468495	20 µM
3,3′-Dioctadecyloxacarbocyanine perchlorateDio (3,3′-Dioctadecyloxacarbocyanine perchlorate	Sigma Aldrich	D4292	20 µM
CODA Monitor system	Kent scientific		CODA Monitor, for monitoring blood pressure and heartbeat
Coverslip	Fisher Scientific	12-542A
DC Temperature Controller	FHC	40-90-8D
Fiji/ImageJ	NIH		Measurement of vessel diameter
FITC-dextran (2000 kDa)	Sigma Aldrich	FD2000s	40 mg/mL
Heparin Sodium Injection, USP MDV	Mylan	NDC 67457-374-12	5000 USP units/mL
Katathesia (100 mg/mL)	Henry Schein	NDC 11695-0702-1	0.2 mL (for 1 mL)
Microscope Objective	Mitutoyo	378-823-5	Model: M Plan Apo NIR 10x
ORCA-ER Camera	Hamamatsu		Model: C4742-80-12AG
PBS	Gibco	2085387
Xyzaine (100 mg/ml, 5x diluted for use )	Lloyd	LPFL04821	0.2 mL (for 1 mL)
Zoom Stereo Microscope	Olympus		Model: SZ61, fluorescent microscope