Normal blood supply to cochlea is critically important for cochlea Meniere's Disease especially for maintaining indoor cochlear potential which is the essential driving force for hair cells transaction. This function of blood circulation has been closely associated with different forms of hearing loss. Better understanding of cochlear blood flow will enable more effective management of hearing disorders resulting from blood flow dysfunction.
However, direct blood flow measurement is very very challenging due to difficulty to assess the inner ear. Techniques for assessing cochlear blood flow is currently still under development. Here we demonstrate one of the recent methods established in our lab open muscle window in accommodation with high resolution fluorescence microscope to narrow cochlear blood flow in small animal species such as mouse.
Application of this technique with transgenetic mass models has the potential for advancing research to unravel the links between hearing function and the pathology related to cochlear blood flow in intravascular risk. Our procedures should be conducted with caution. In addition to momentary body hemorrhage, animal's vital signs, including blood pressure and heartbeat, must be monitored during your surgery.
Begin by collecting approximately one millimeter of blood in heparin by cardiac puncture. Centrifuge the blood at 3000 times G for three minutes at four degrees Celsius. Remove the plasma, then wash the blood cell pellet with one milliliter of PBS three times by centrifugation at 3000 times G for three minutes at four degrees Celsius.
Label the blood cells with one milliliter of 20 micromolar DiO or Dil in PBS, and incubate in the dark for 30 minutes at room temperature. Wash the labeled blood cells with one milliliter of PBS and centrifuge three times at 3000 times G for three minutes. Resuspend the cell pellet in 30%hematocrit with approximately 0.9 milliliters of PBS before injection.
Prepare sterile surgical instruments and imaging platform and place a heating pad beneath the drape. Monitor the poor reflex and general muscle tone of the anesthetized mice to check the depth of the anesthesia. Place the animal on the warm heating pad and its tail in the monitor system for monitoring blood pressure and heartbeat.
Maintain the rectal temperature at 37 degrees Celsius and record the animal's systolic blood pressure, diastolic blood pressure, and mean blood pressure in the anesthetized condition. Open the left tympanic bulla via a lateral and vental approach under a stereo microscope leaving the tympanic membrane and ossicles intact. Make an incision along the midline of the animal's neck with its head immobilized to minimize movement.
Remove the left sub mandibular gland and posterior belly of the digastric muscle and cauterize. Locate and expose the bony bulla by identifying the sternocleidomastoid muscle and facial nerve extending anterior toward the bulla. Open the bony bulla with a 30 gauge needle and carefully remove the surrounding bone with surgical tweezers to provide a clear view of the cochlear and stapedial artery with its medial margin lying over the edge of the round window niche and causing anterior superior towards the oval window.
Use a small knife blade to scrape the lateral wall bone at the apex middle turn of the mouse cochlea approximately 1.25 millimeters from the apex until a thin spot is cracked. Remove the bone chips with small wire hooks. Cover the vessel window with a cut cover slip to preserve normal physiological conditions and provide an optical view for recording vessel images.
Make a one centimeter incision along the right saphenous vein to expose the vessel. Successively infuse 100 microliters each of the FITC-Dextran solution and the blood cell suspension into the animal through the saphenous vein to enable visualization of the blood vessels and tracking of blood flow. Observe the blood flow in real time on a video monitor five minutes after the injection.
Image the blood vessels using a fluorescence microscope equipped with a long working distance objective and a lamp housing containing a multiple band excitation filter and compatible emission filter. Record the video using a high resolution digital black and white charge coupled device camera acquiring more than 350 images per video. To ensure successful analysis of the flow velocity.
Measure the vessel diameter using appropriate software and determine the distance between two fixed points across the vessel in the acquired images. Open the video of blood flow in the software and set the scale of the images. Track the selected DiO stained blood cells using the tracking function.
Then 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. Calculate the volumetric flow using the equation given in the text manuscript. To create the noise exposure model before blood flow recording, place the animals in wire mesh cages and expose them to broadband noise at 120 decibels sound pressure level in a sound exposure booth for three hours.
Then for an additional three hours on the next day. Record blood flow two weeks after noise exposure. After surgical exposure of the cochlear capillaries in the lateral wall, intravital high resolution fluorescence microscopic observation of DiL labeled blood cells in FITC Dextran labeled vessels was performed through an open vessel window.
DiL labeled blood cells in Stria vessels labeled with FITC Dextran are shown here. Dio labeled blood cells labeled with FITC Dextran show that the vessel density of the spiral ligament is sparser than that of the denser Stria vascularis. High resolution IVM shows the blood flow patterns in control and noise exposed groups.
The disturbed pattern of blood circulation can be seen in the noise exposed groups. Anomalies included reduced vessel diameter and increased variation in vessel diameter. The blood flow velocities in the control and noise exposed groups were calculated by tracking the roots of the DiO labeled blood cells.
The blood velocity and volume in the noise exposed group were significantly lower than in the control group. The successful establishment of the open vessel window requires a high degree of surgical skill. Care must be exercised in removing the bone of the cochlear lateral wall.
