This research was supported by NIH/NIDCD R21 DC016157 (X.Shi), NIH/NIDCD R01 DC015781 (X.Shi), NIH/NIDCD R01-DC010844 (X.Shi),&#160;and Medical Research Foundation from Oregon Health and Science University (OHSU) (X.Shi).

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 &#38; Science University (IACUC approval number: TR01_IP00000968).
1. Preparation of the fluorescent-labeled blood cells
<ol>
	<li>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). 
	&#8203;NOTE: This anesthesia protocol is very reliable and maintains systemic blood pressure.</li>
	<li>Lay the mouse on its back, open&#160;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.&#160;Collect 1 mL of blood in heparin (15 IU/mL blood) by cardiac puncture, and centrifuge at 3,000 &#215; g for 3 min at 4 &#176;C. Euthanize the mouse by cervical dislocation after blood collection.</li>
	<li>Remove the plasma, wash the blood cell pellet with 1 mL of phosphate-buffered saline (PBS), and centrifuge 3x at 3000 &#215; g for 3 min at 4 &#176;C.</li>
	<li>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 temperature23,24.</li>
	<li>Centrifuge and wash the labeled blood cells with 1 mL of PBS, centrifuge 3x at 3000 &#215; g for 3 min at 4 &#176;C, and resuspend the cell pellet in 30% hematocrit with ~0.9 mL of PBS (final volume ~1.3 mL) before injection.</li>
</ol>
2. Surgery to create an open window 25
<ol>
	<li>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 &#176;C.</li>
	<li>Place the animal tail in the CODATM monitor system for monitoring blood pressure and heartbeat (see the Table of Materials). Record the animal&#39;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 &#177; 11 mmHg vs. 97 &#177; 7 mmHg). The animal heart rate is stable under anesthetization, though lower (still within normal range) than in the non-anesthetized condition (357 &#177; 12 bmp vs. 709 &#177; 3 bmp). A slightly increased heart beat should be expected in animals placed in restraint26.</li>
	<li>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.
	<ol>
		<li>Make an incision along the midline of the animal&#39;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.</li>
		<li>Locate and expose the bony bulla by identifying the sternocleidomastoid muscle and facial nerve extending anterior toward the bulla.</li>
		<li>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&#160;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&#160;not be used for any comparison.</li>
	</ol>
	</li>
	<li>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).</li>
	<li>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&#39;s vital signs, including blood pressure and heartbeat, should also be monitored throughout the surgery.</li>
</ol>
3. Imaging of CoBF under FIVM
<ol>
	<li>Make an ~1 cm incision along the right saphenous vein to expose the vessel (Figure 1F).</li>
	<li>Infuse 100 &#956;L of the FITC-dextran solution (2000 kDa, 40 mg/mL in PBS) and 100 &#956;L 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.</li>
	<li>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).</li>
</ol>
4. Video analysis
<ol>
	<li>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.</li>
	<li>Calculate the blood flow velocity from captured video frames by tracing the movement of labeled blood cellsin the spatial distance between image locations27.
	<ol>
		<li>Open the video of blood flow in the software (Fiji [ImageJ] was used in this protocol), and set the scale of the images.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Calculate the volumetric flow (F) per the following equation: F = V &#215; A. (V: velocity; A: cross-sectional area of the vessel).</li>
</ol>
5. Noise Exposure
<ol>
	<li>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.</li>
</ol>

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The authors have nothing to disclose.

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&#160; 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&#160;3 &#38; Figure&#160;4). This approach can be used for several different applications, such as the assessment of vascular permeability and the examination&#160; of pericyte contractility, and&#160; 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-time25,29.&#160; 
&#160; 
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&#160; 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&#160; blood flow in the stria vascularis.&#160; However, blood circulation in the stria vascularis&#160; has&#160; 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&#160; &#160;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&#8217;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,&#160; 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&#160; 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&#160; to prevent loss of perilymphatic fluid and micro-injury to the outer layer of the cochlear spiral ligament. These could&#160; adversely affect cochlear homeostasis and compromise the imaging.

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.&#160;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 laboratories14,21,22.
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&#8242;-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.

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

measurement of strial blood flow in mouse cochlea utilizing an open vessel-window and intravital fluorescence microscopy

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.

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.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61857/61857fig01.jpg" /> 
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.&#160;(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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61857/61857fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61857/61857fig2v2.jpg" /> 
Figure 2:&#160;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 &#181;m and 100 &#181;m. Abbreviations: Dil = 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate; DiO = 3, 3&#8242;-dioctadecyloxacarbocyanine perchlorate; FITC = fluorescein isothiocyanate. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61857/61857fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61857/61857fig3v2.jpg" /> 
Figure 3:&#160;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)&#160;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&#39;s t-test, mean &#177; standard deviation [SD]). (D)&#160;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 &#60; 0.0001, Student&#39;s t-test, mean &#177; SD). Scale bars: 100 &#181;m. Abbreviations: DiO = 3, 3&#8242;-dioctadecyloxacarbocyanine perchlorate; FITC = fluorescein isothiocyanate; IVM = intravital microscopy. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61857/61857fig3v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61857/61857fig4v2.jpg" /> 
Figure 4:&#160;Blood flow changes in the stria two weeks after noise exposure. (A and B)&#160;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 (&#181;m/s) and volumetric flow rate (&#181;m3/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, ****pvelocity &#60; 0.0001; tvolume (106) = 15.342, ****pvolume &#60; 0.0001, Student&#39;s t-test, mean &#177; standard deviation). Scale bars: 100 &#181;m. Abbreviations: DiO = 3, 3&#8242;-dioctadecyloxacarbocyanine perchlorate1; FITC = fluorescein isothiocyanate; NE 2W = two-week noise exposure. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61857/61857fig4v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental video 1: Blood vessels of both the spiral ligament and stria vascularis.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61857/strial-spiral ligament-supplymental video 1 for revision.mp4" target="_blank">Please click here to download this video.</a>
Supplemental video 2: Tracking the routes of DiO-labeled blood cellsfor calculation of the blood flow velocities in a&#160;control animal. Abbreviations: DiO = 3, 3&#8242;-dioctadecyloxacarbocyanine perchlorate.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61857/sup_video_2-072321.mp4" target="_blank">Please click here to download this video.</a>

Watch this Scientific Journal Video about Measurement of Strial Blood Flow in Mouse Cochlea Utilizing an Open Vessel-Window and Intravital Fluorescence Microscopy at JoVE.com

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

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.

Transduction of sound is metabolically demanding, and the normal function of the microvasculature in the lateral wall is critical for maintaining ...

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Nanyang Technological University

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2.3K Views.  Oregon Health & Science University. 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.

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

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

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 &amp; 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.

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.

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of ...

Biology

Intravital Microscopy of the Mouse Brain Microcirculation using a Closed Cranial Window

This experimental model was designed to assess the mouse pial microcirculation during acute and chronic, physiological and pathophysiological hemodynamic, inflammatory and metabolic conditions, using in vivo fluorescence microscopy. A closed cranial window is placed over the left parieto-occipital cortex of the mice. Local microcirculation is recorded in real time through the window using epi and fluorescence illumination, and measurements of vessels diameters and red blood cell (RBC) velocities are performed. RBC velocity is measured using real-time cross-correlation and/or fluorescent-labeled erythrocytes. Leukocyte and platelet adherence to pial vessels and assessment of perfusion and vascular leakage are made with the help of fluorescence-labeled markers such as Albumin-FITC and anti-CD45-TxR antibodies. Microcirculation can be repeatedly video-recorded over several days. We used for the first time the close window brain intravital microscopy to study the pial microcirculation to follow dynamic changes during the course of Plasmodium berghei ANKA infection in mice and show that expression of CM is associated with microcirculatory dysfunctions characterized by vasoconstriction, profound decrease in blood flow and eventually vascular collapse.

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Neuroscience

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Alterations in retinal blood flow can contribute to, or be a consequence of, ocular disease and visual dysfunction. Therefore, quantitation of altered perfusion can aid research into the mechanisms of retinal pathologies. Intravital video microscopy of fluorescent tracers can be used to measure vascular diameters and bloodstream velocities of the retinal vasculature, specifically the arterioles branching from the central retinal artery and of the venules leading into the central retinal vein. Blood flow rates can be calculated from the diameters and velocities, with the summation of arteriolar flow, and separately venular flow, providing values of total retinal blood flow. This paper and associated video describe the methods for applying this technique to mice, which includes 1) the preparation of the eye for intravital microscopy of the anesthetized animal, 2) the intravenous infusion of fluorescent microspheres to measure bloodstream velocity, 3) the intravenous infusion of a high molecular weight fluorescent dextran, to aid the microscopic visualization of the retinal microvasculature, 4) the use of a digital microscope camera to obtain videos of the perfused retina, and 5) the use of image processing software to analyze the video. The same techniques can be used for measuring retinal blood flow rates in rats.

Intravital microscopy can be used in animals to visualize and measure retinal vascular diameters, bloodstream velocities, and total retinal blood flow.

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Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics

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In this study, IVM is applied to the mouse liver and protocols are designed to image in vivo the circulatory system of the liver and measure red blood cell (RBC) velocity in individual hepatic vessels. To visualize the different vessel subtypes that characterize the hepatic organ and perform blood flow speed measurements, C57Bl/6 mice are intravenously injected with a fluorescent plasma reagent that labels the liver-associated vasculature. IVM enables in vivo, real time, measurement of RBC velocity in a specific vessel of interest. Establishing this methodology will make it possible to investigate liver hemodynamics under physiological and pathological conditions. Ultimately, this imaging-based methodology will be important for studying the influence of L. donovani infection&#160;on hepatic hemodynamics.
This method can be applied to other infectious models and mouse organs and might be further extended to pre-clinical testing of a drug&#8217;s effect on inflammation by quantifying its effect on blood flow.

Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics

This article reports on a detailed method for the dynamic measurement and quantification of blood flow velocity within individual blood vessels of the mouse liver vasculature using intravital microscopy imaging in combination with a specific methodology for image acquisition and analysis.

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Immunology and Infection

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Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo

Many vision-threatening ocular diseases are associated with dysfunctional retinal microvessels. Therefore, the measurement of retinal arteriole responses is important to investigate the underlying pathophysiological mechanisms. This article describes a detailed protocol for mouse retinal arteriole isolation and preparation to assess the effects of vasoactive substances on vascular diameter.

Vascular insufficiency and alterations in normal retinal perfusion are among the major factors for the pathogenesis of various sight-threatening ocular ...

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Intravital microscopy of the mouse M. cremaster microcirculation offers a unique and well-standardized in vivo model for the analysis of peripheral bone marrow stem cell migration.

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to ...

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Presented here is a protocol to study the coronary microcirculation in living murine heart tissue by ex vivo monitoring of the arterial perfusion pressure and flow that maintains the pressure, as well as vascular tree components including the capillary beds and pericytes, as the septal artery is cannulated and pressurized.

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Saliva plays an important role in oral and overall health. The intact endothelial barrier function of blood vessels enables saliva secretion, whereas the ...

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

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Chapters in this video

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

Intravital Microscopy of the Mouse Brain Microcirculation using a Closed Cranial Window

Intravital Video Microscopy Measurements of Retinal Blood Flow in Mice

Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics

Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo

Intravital Microscopy of the Microcirculation in the Mouse Cremaster Muscle for the Analysis of Peripheral Stem Cell Migration

Dynamic Measurement and Imaging of Capillaries, Arterioles, and Pericytes in Mouse Heart

Intravital Imaging of Fluorescent Protein Expression in Mice with a Closed-Skull Traumatic Brain Injury and Cranial Window Using a Two-Photon Microscope

Measuring Retinal Vessel Diameter from Mouse Fluorescent Angiography Images

In Vivo Vascular Permeability Detection in Mouse Submandibular Gland