Supported by NIH awards RO1 HL073087, GM 077185, and GM 069589 to CKS. The work is also supported by a grant from the Regenerative Medicine program of the DHLRI. The authors are grateful to the technical support and clinical inputs from the following personnel at the Ohio State University: Dr. Sabyasachi.Biswas (Comprehensive Wound Center), Dr. Allison Spiwak (Circulation Technology Division), Joseph Agoston (Circulation Division), Thoma Shives (Circulation Division), Joseph Ewing (Mechanical Engineering), Scott Killinger (Mechanical Engineering), and Xiaoyin Ge (Electrical Engineering).

1. Mapping tissue oxygenation by multispectral imaging
A Comprehensive Wound Center (CWC) dual-mode imaging system, henceforth referred to as the CWC system, was developed at the Ohio State University for multispectral imaging of cutaneous tissue oxygenation and thermographic imaging of vascular function. The wide gap second derivative spectroscopic technique was used to reconstruct tissue oxygenation maps based on multispectral images 1. In this study, a healthy subject sat with the left forearm resting on a countertop. A portion of the forearm within the field of view of the CWC system was painted with India ink (1% dissolved in ethanol) to mimic different skin colors. Multispectral images were acquired and an oxygenation map was reconstructed based on the wide gap second derivative spectrum. The reconstructed oxygenation map was compared with that acquired by a commercial Hypermed OxyVu hyperspectral tissue oxygenation measurement system.
Tissue oxygen responses to vascular occlusion were studied on the same subject following a protocol of post-occlusive reactive hyperemia (PORH) 2. Before the PORH test, the subject's systolic and diastolic blood pressures were recorded by a pressure cuff placed on the left upper arm. The PORH protocol consisted of a pre-occlusive baseline period of two minutes, a suprasystolic occlusion (systolic + 50 mm Hg) period of two minutes, and a reactive hyperemia period of two minutes. Multispectral images were acquired at four wavelengths (i.e., 530nm, 550nm, 570nm, and 590nm) during the PORH test at the sampling rate of 0.75 seconds per wavelength. Deep tissue oxygen saturation and cutaneous tissue oxygen tension on the same arm were simultaneously recorded by an OxiplexTS tissue spectrophotometer (ISS Inc., Urbana Champaign, IL) and a TCM transcutaneous oxygen monitor (Radiometer, Denmark) respectively.
2. Mapping vascular function by dynamic thermographic imaging
Dynamic thermographic imaging was demonstrated on a healthy subject using the same CWC system. The subject comfortably lied on a table in supine position, with the left arm resting on a countertop and the dorsum of the left hand facing up toward the infrared camera unit of the CWC system. A laser Doppler probe was placed on the finger tip of the same hand for continuous monitoring of finger skin perfusion. A pressure cuff was placed on the left upper arm to produce different levels of occlusion. Before the experiment, the subject was asked to rest for at least 10 minutes, with the systolic and diastolic pressures recorded by the pressure cuff. Dynamic thermographic images were captured at the following cuff pressure levels: no occlusion, 0.5 x diastolic blood pressure, 0.5 x (diastolic blood pressure + systolic blood pressure), and 1.5 x systolic blood pressure. At each cuff pressure level, a thermal stimulation was introduced by placing a water bag of room temperature (25&deg;C) on the left hand for 30 seconds. Immediately after the removal of the thermal stimulation, the left hand thermographic images were acquired at a rate of 2 frames/second and the finger skin perfusion was recorded by the laser Doppler probe at the sampling rate of 10 Hz. The time interval between tests was 10 min. The position of the left hand was marked in advance so that the subsequent measurements were at the same position. Tissue vascular functions at different occlusion levels were evaluated by calculating both the temperature response and the vascular function index. The vascular function index was defined as the ratio between the square root of the time after thermal stimulation and the corresponding temperature change.
3. Co-registration between tissue oxygenation and vascular function maps
The map of cutaneous tissue vascular function acquired by dynamic thermographic imaging and the map of cutaneous tissue oxygenation acquired by multispectral imaging were co-registered by an advanced imaging algorithm. Four fiduciary markers, with simultaneous thermal and optical contrasts, were placed on the biological tissue to facilitate image co-registration. Currently the protocol for co-registering the optical image (and the oxygenation map) with the thermal image (and the vascular function map) includes the following key steps: 1) transform all the images to gray scale and normalize the pixel intensity values between 0 and 1; 2) for the optical photo, identify the foreground (skin) region which has high pixel intensity values than the an empirical global threshold (0.8 times the mean intensity value of the whole image); the small holes in the foreground region were filled using the morphological close operation; 3) identify the fiduciary marker regions as darker regions in the foreground whose intensity values are below both the global threshold and the adaptive local threshold defined by the mean pixel values in the 20-by-20 pixel neighborhood. The adaptive local threshold allowed us to accommodate the illumination variation; 4) refine the fiduciary marker regions using morphological operations to remove noise and spikes; the centroids of the four regions were selected as control points in the optical photo; 5) repeat similar steps to identify marker regions in the thermal image; 6) match the two sets of control points based on proximity (given that the two cameras were closely positioned); 7) compute an affine transformation between the two images with the thermal image as the reference and transform the optical photo and the oxygen map (which is obtained from the same camera as the optical photo) correspondingly; 8) finally generate the overlaid images for visualization.
4. Representative Results:
The representative results for the oxygenation protocol (i.e., #1) are cutaneous tissue oxygenation maps reconstructed based on wide gap second derivative spectroscopy. The method of wide gap second derivative spectroscopy effectively reduced the measurement artifacts caused by tissue background absorption so that the cutaneous oxygenation measurement was less affected by the simulated skin color changes. We also demonstrated a PORH protocol where the cutaneous tissue oxygenation map, the deep tissue oxygenation, and the cutaneous tissue oxygen tension were simultaneously recorded.
The representative results for the vascular function protocol (i.e., #2) include the cutaneous tissue temperature distributions, the cutaneous tissue temperature changes in response to external thermal stimulation, and the cutaneous tissue vascular index maps derived from dynamic thermographic imaging. Correlations were observed between the thermographic imaging of cutaneous tissue vascular index and the laser Doppler measurement of skin tissue perfusion.
The representative results for the co-registration protocol (i.e., #3) include the tissue oxygenation map, the vascular index map, and the image fusion among photographic, oxygenation, and vascular index images co-registered by multi-contrast fiduciary markers.
<img src="/files/ftp_upload/2095/2095fig1.jpg" alt="Figure 1" /> 
 Figure 1. CWC dual-mode system setup for non-contact imaging of tissue oxygenation and vascular function. The system was installed on a mobile cart. It consisted of an infrared thermal camera, a CCD camera, a liquid crystal tunable filter, and a broadband light source. A computer was used to synchronize the tasks of data collection, analysis, and display.
<img src="/files/ftp_upload/2095/2095fig2.jpg" alt="Figure 2" /> 
 Figure 2. Software interface for the CWC system. The software interface was programmed in the Labview environment. On the top left of the interface is the control panel for hardware configuration and system calibration. On the right of the interface is the real-time display of the tissue temperature map acquired by the infrared camera. On the bottom of the interface are laser Doppler measurements of skin tissue perfusion.
<img src="/files/ftp_upload/2095/2095fig3.jpg" alt="Figure 3" /> 
 Figure 3. Software interface for the CWC system (Cont.). This is a pop-up window to show tissue oxygenation parameters. On the left is the tissue oxygenation map reconstructed from the multispectral images. The right side of the interface displays the following tissue oxygenation parameters from top down: (1) cutaneous tissue oxygenation averaged from a selected region of interest (ROI) in the tissue oxygenation map; (2) deep tissue oxygenation monitored by an OxplexTS tissue oximeter; (3) cutaneous tissue oxygen tension monitored by a TCM transcutaneous oxygen monitor.
<img src="/files/ftp_upload/2095/2095fig4.jpg" alt="Figure 4" /> 
 Figure 4. Cutaneous tissue oxygenation imaging by an OxyVu imaging system. The left figure is a grayscale image of the skin tissue. A layer of ink was painted on the skin to simulate the skin color. The map of cutaneous tissue oxygenation acquired by the OxyVu system is shown on the right. Two square-shaped regions of interest (ROIs) were selected within and outside the ink-painted area respectively. For the ROI within the ink-painted area, the averaged cutaneous tissue oxygenation was 62.8&plusmn;11.0%. For the ROI outside the ink-painted area, the averaged cutaneous tissue oxygenation was 44.0&plusmn;11.0%. A difference of 18.8% was observed for the cutaneous tissue oxygenation measurements within and outside the ink-painted skin area.
<img src="/files/ftp_upload/2095/2095fig5.jpg" alt="Figure 5" /> 
 Figure 5. Cutaneous tissue oxygenation imaging by the CWC system. The multispectral images were captured by the CWC system at the same skin location on the same subject as that in Figure 4. The left figure is the single wavelength image of the skin tissue and the right figure is the reconstructed cutaneous tissue oxygenation map. A layer of ink was painted on the skin with the ink concentration the same as that in Figure 4. . Two square-shaped regions of interest (ROIs) were selected within and outside the ink-painted area respectively. For the ROI within the ink-painted area, the averaged cutaneous tissue oxygenation was 60.9&plusmn;6.9%. For the ROI outside the ink-painted area, the averaged cutaneous tissue oxygenation was 65.8&plusmn;5.5%. A difference of 4.9% was observed for the cutaneous tissue oxygenation measurements within and outside the ink-painted skin area.
<img src="/files/ftp_upload/2095/2095fig6.jpg" alt="Figure 6" /> 
 Figure 6. The effect of simulated skin color changes on the reliability of cutaneous tissue oxygenation measurements. Statistic analysis was carried out to determine the significance of the simulated skin color changes to oxygenation measurements for both the OxyVu and the CWC imaging systems. For each imaging system, the averaged cutaneous tissue oxygenations were calculated within and outside the ink-painted skin areas by randomly selecting 10 regions of interest (ROIs) in each area. Our null hypothesis is that the change of skin color will not affect the cutaneous tissue oxygenation measurement. This null hypothesis was tested using oxygenation maps in Figure 4 (i.e., the OxyVu measurements) and Figure 5 (i.e., the CWC measurements) respectively. Student's t-tests show that the p value for the OxyVu measurements is much less than 0.001, implying that the null hypothesis is rejected. Therefore, the change of skin color does affect the cutaneous tissue oxygenation measurements in an OxyVu imaging system. In contrast, the p value for the CWC measurements is 0.728, implying the likelihood that the change of skin color does not affect the cutaneous tissue oxygenation measurements in a CWC imaging system.
<img src="/files/ftp_upload/2095/2095fig7.jpg" alt="Figure 7" /> 
 Figure 7. Cutaneous tissue oxygenation images obtained from the CWC system during post-occlusive reactive hyperemia (PORH). The PORH test was carried out on the forearm of a healthy subject following #1. (a). Single wavelength gray scale image of the arm with four fiduciary markers placed for image co-registration. (b) The baseline cutaneous tissue oxygenation map acquired before vascular occlusion. (c) The cutaneous tissue oxygenation map after vascular occlusion (systolic pressure + 50mmHg) for 2 minutes. (d) The cutaneous tissue oxygenation map after reactive hyperemia. Significant changes in cutaneous tissue oxygenation were observed before, during, and after vascular occlusion.
<img src="/files/ftp_upload/2095/2095fig8.jpg" alt="Figure 8" /> 
 Figure 8. Tissue oxygen parameters continuously monitored during a PORH test. The test protocol in (a) shows a period of pre-occlusion baseline, followed by a suprasystolic occlusion period, and end with a reactive hyperemia period. The history of cutaneous tissue oxygenation duration the PORH procedure is plotted in (b). It was obtained by averaging a selected region of interest (ROI) in the CWC cutaneous oxygenation map. We also monitored the deep tissue oxygenation by an OxiplexTS tissue oximeter, as plotted in (c). The transcutaneous tissue oxygen tension was also monitored by a TCM device and plotted in (d). The CWC measurements of cutaneous tissue oxygenation coincide well with other oxygen parameters during the PORH procedure.
<img src="/files/ftp_upload/2095/2095fig9.jpg" alt="Figure 9" /> 
 Figure 9. Cutaneous tissue responses at different vascular occlusion pressures. Thermographic images were acquired immediately after the thermal stimulation (i.e., a water bag at room temperature 25&deg;C) was removed from the subject's left hand. The horizontal axis corresponds to the different time spots after the thermal stimulation was removed. The vertical axis corresponds to the following 4 levels of vascular occlusion pressures: 0 (No occlusion), 0.5DBP, 0.5(DBP+SBP), 1.5SBP, where DBP is the diastolic blood pressure and SBP is the systolic blood pressure. For this specific subject, the DBP is 69mmHg and the SBP is 123mmHg. The test results indicate that the tissue temperature response to the external thermal stimulation is correlated with the level of vascular occlusion. Increasing the occlusion pressure reduces the thermal response rate.
<img src="/files/ftp_upload/2095/2095fig10.jpg" alt="Figure 10" /> 
 Figure 10. Reconstructed vascular index maps at different vascular occlusion pressures. Vascular function index (v) at each pixel of the thermographic images was derived by regressing the cutaneous tissue temperature change (&Delta;T) against the square root of the time after the thermal stimulation (&radic;t): &Delta; &radic;t + K + &#949; , where &#949; is the random error and K is a constant. Vascular function index maps from left to right correspond to the following occlusion conditions: (a) no occlusion, (b) 0.5DBP, (c) 0.5 (DBP+SBP), (d) 1.5SBP. For this specific subject, the diastolic blood pressure (DBP) is 69mmHg and the systolic blood pressure (SBP) is 123mmHg.
<img src="/files/ftp_upload/2095/2095fig11.jpg" alt="Figure 11" /> 
 Figure 11. Correlation between the vascular function index and the skin tissue perfusion. The finger tip vascular function index at each occlusion pressure in #2 was calculated by averaging five regions of interest (ROIs) in the tissue vascular index map. The skin tissue perfusion was measured by a laser Doppler device at one of the finger tip. The finger tip vascular index correlated with the laser Doppler measurement, indicating the potential of using the dynamic thermographic method for quantitative assessment of tissue vascular function.
<img src="/files/ftp_upload/2095/2095fig12.jpg" alt="Figure 12" /> 
 Figure 12. The image co-registration process. In the first row, we show: (a) the normalized photo, (b) the segmented foreground region, and (c) the segmented markers in the photo image. In the second row, we show: (d) the normalized perfusion map, (e) the segmented foreground region, and (f) the segmented markers in the perfusion map. The affine transformation between the two sets of marker positions was calculated. The co-registered images (g and h) were obtained by transforming the photo image and the oxygen map using this recovered affine transformation.
<img src="/files/ftp_upload/2095/2095fig13.jpg" alt="Figure 13" /> 
 Figure 13. The co-registration results. The transformed photo and oxygen map, together with the perfusion map were co-registered and displayed in as a heat map. The heat map in (a) was presented with 100% of transformed photo image in the red channel, 100% of transformed oxygen image in the green channel and 50% of the perfusion map in the blue channel. In order to visualize the vessels better, we present another version of the co-registration result as in (b). The heat map was composed of 100% of transformed photo image in the red channel, 50% of transformed oxygen image in green channel, and 50% of changed perfusion map in the blue channel, where the vascular index map was inverted and only the information within the foreground region was kept.

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	<li>Myers, D. E., Anderson, L. D., Seifert, R. P., Ortner, J. P., Cooper, C. E., Beilman, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+method+for+measuring+local+hemoglobin+oxygen+saturation+in+tissue+using+wide+gap+second+derivative+near-infrared+spectroscopy.">Noninvasive method for measuring local hemoglobin oxygen saturation in tissue using wide gap second derivative near-infrared spectroscopy.</a> J Biomed Opt. 10 (3), 034017-034017 (2005).</li><li>Jarm, T., Kragelj, R., Liebert, A., Lukasiewitz, P., Erjavec, T., Preseren-Strukelj, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postocclusive+reactive+hyperemia+in+healthy+volunteers+and+patients+with+peripheral+vascular+disease+measured+by+three+noninvasive+methods.">Postocclusive reactive hyperemia in healthy volunteers and patients with peripheral vascular disease measured by three noninvasive methods.</a> Adv Exp Med Biol. 530, 661-669 (2003).</li><li>Treacher, D. F., Leach, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygen+transport-1.+Basic+principles.">Oxygen transport-1. Basic principles.</a> Bmj. 317 (7168), 1302-1306 (1998).</li><li>Leach, R. M., Treacher, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygen+transport-2.+Tissue+hypoxia.">Oxygen transport-2. Tissue hypoxia.</a> BMJ. 317 (7169), 1370-1373 (1998).</li><li>Sen, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound+healing+essentials:+let+there+be+oxygen.">Wound healing essentials: let there be oxygen.</a> Wound Repair Regen. 17 (1), 1-18 (2009).</li><li>RL, G. r. e. e. n. m. a. n., Panasyuk, S., Wang, X., TE, L. y. o. n. s., Dinh, T., Longoria, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+changes+in+the+skin+microcirculation+and+muscle+metabolism+of+the+diabetic+foot.">Early changes in the skin microcirculation and muscle metabolism of the diabetic foot.</a> Lancet. 366 (9498), 1711-1717 (2005).</li><li>Valvano, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The use of thermal diffusivity to quantify tissue perfusion [dissertation]. , (1981).</li><li>Hassan, M., Togawa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Observation+of+skin+thermal+inertia+distribution+during+reactive+hyperaemia+using+a+single-hood+measurement+system.">Observation of skin thermal inertia distribution during reactive hyperaemia using a single-hood measurement system.</a> Physiol Meas. 22 (1), 187-200 (2001).</li><li>Ley, O., Deshpande, C., Prapamcham, B., Naghavi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lumped+parameter+thermal+model+for+the+study+of+vascular+reactivity+in+the+fingertip.">Lumped parameter thermal model for the study of vascular reactivity in the fingertip.</a> J Biomech Eng. 130 (3), 031012-031012 (2008).</li><li>Wilson, S. B., Spence, V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+thermographic+imaging+method+for+quantifying+dermal+perfusion:+potential+and+limitations.">Dynamic thermographic imaging method for quantifying dermal perfusion: potential and limitations.</a> Med Biol Eng Comput. 27 (5), 496-501 (1989).</li></ol>

No conflicts of interest declared.

Oxygen exists in biological tissue in multiple forms such as hemoglobin or myoglobin bound oxygen, dissolved oxygen, and reactive oxygen species. Oxygen transport plays a critical role in maintaining tissue viability and normal metabolic processes 3. Acute mild to moderate hypoxia will initiate metabolic adaptation, vascular regulation, and angiogenic responses 4. Extreme hypoxia and anoxia will lead to insufficient angiogenesis and cell death. The imbalance between limited oxygen supply and increased oxygen demand is one of major causative factors for many disorders such as chronic wounds 5. In the case of ischemic wounds, oxygen supply is limited by lack of perfusion and cannot meet the metabolic need of the healing process and compromising respiratory burst function aimed at fighting infection. Simultaneous assessment of cutaneous tissue oxygenation and vascular function has clinical significance in chronic wound management. The hyperspectral imaging technique estimates cutaneous tissue oxygenation by illuminating tissue and detect tissue reflectance at different wavelengths 6. One major advantage of hyperspectral imaging is non-invasive and non-contact detection of tissue functional properties. However, the oxygen measurement reliability for many hyperspectral imaging systems is affected by skin color and other background absorption variations. Wide gap second derivative spectroscopy was developed previously for tissue oxygenation measurement with minimal effect of scattering and skin pigmentation 1. We applied the principle of the second derivative spectroscopy to multispectral imaging and demonstrated the consistent measurement of cutaneous tissue oxygenation independent with the simulated skin color changes. Tissue perfusion was previously studied by measuring tissue thermal diffusivity 7. A single hood method was developed to introduce thermal stimulation and image tissue dynamic responses in order to estimate the skin tissue thermal inertia distribution 8. Experiments on human forearm skin subjected to arterial cuff occlusion demonstrated linear relationship between thermal inertia and blood perfusion measured by a laser Doppler imager before and during blood flow occlusion 8. A lumped bioheat model was also used to estimate the finger tip vascular reactivity during venous occlusion plethysmography 9. Despite the above efforts, quantifying skin blood perfusion from skin temperature measurements is challenging because of the lack of sensitivity, the dependence on subcutaneous fat thickness, and other contributing factors such as vasoconstriction, vasodilation, and motion artifacts 10. In this protocol, we used an infrared camera to capture tissue temperature dynamics in response to a thermal stimulation at room temperature. The thermal stimulation was selected so that the effect of vasoconstriction and vasodilation was minimized. Further modeling and measurement efforts are necessary in order to delineate a reliable quantitative correlation between the vascular function index and the skin tissue perfusion. In #3, we used the affine transformation for the co-registration tasks. However, given that the two cameras are positioned with different angles in the 3D space, it is potentially more accurate to apply a transformation related to the 3D transformation between the two cameras. Currently we are exploring in that direction which involve extrinsic calibration of the cameras in 3D space using epipolar geometry.

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<td>Higgins Calligraphy waterproof black ink</td>
<td>&nbsp;</td>
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<td>diluted to 1%</td>
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<td>Hamamatsu ORCA ER deep cooling CCD camera</td>
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dual-mode imaging of cutaneous tissue oxygenation and vascular function

Accurate assessment of cutaneous tissue oxygenation and vascular function is important for appropriate detection, staging, and treatment of many health disorders such as chronic wounds. We report the development of a dual-mode imaging system for non-invasive and non-contact imaging of cutaneous tissue oxygenation and vascular function. The imaging system integrated an infrared camera, a CCD camera, a liquid crystal tunable filter and a high intensity fiber light source. A Labview interface was programmed for equipment control, synchronization, image acquisition, processing, and visualization. Multispectral images captured by the CCD camera were used to reconstruct the tissue oxygenation map. Dynamic thermographic images captured by the infrared camera were used to reconstruct the vascular function map. Cutaneous tissue oxygenation and vascular function images were co-registered through fiduciary markers. The performance characteristics of the dual-mode image system were tested in humans.

Watch this Scientific Journal Video about Dual-mode Imaging of Cutaneous Tissue Oxygenation and Vascular Function at JoVE.com

Dual-mode Imaging of Cutaneous Tissue Oxygenation and Vascular Function

A dual-mode imaging system was developed for non-contact assessment of cutaneous tissue oxygenation and vascular function.

Accurate assessment of cutaneous tissue oxygenation and vascular function is important for appropriate detection, staging, and treatment of many health ...

dual-mode-imaging-cutaneous-tissue-oxygenation-vascular

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JoVE Journal

Medicine

16.4K vues.  The Ohio State University. Un système bi-mode d'imagerie a été développé pour les non-contact avec l'évaluation de l'oxygénation des tissus cutanés et la fonction vasculaire.

Vidéo: Dual-mode Imaging of Cutaneous Tissue Oxygenation and Vascular Function

Diagnostic Ultrasound Imaging of Mouse Diaphragm Function

Function analysis of rodent respiratory skeletal muscles, particularly the diaphragm, is commonly performed by isolating muscle strips using invasive surgical procedures. Although this is an effective method of assessing in vitro diaphragm activity, it involves non-survival surgery. The application of non-invasive ultrasound imaging as an in vivo procedure is beneficial since it not only reduces the number of animals sacrificed, but is also suitable for monitoring disease progression in live mice. Thus, our ultrasound imaging method may likely assist in the development of novel therapies that alleviate muscle injury induced by various respiratory diseases. Particularly, in clinical diagnoses of obstructive lung diseases, ultrasound imaging has the potential to be used in conjunction with other standard tests to detect the early onset of diaphragm muscle fatigue. In the current protocol, we describe how to accurately evaluate diaphragm contractility in a mouse model using a diagnostic ultrasound imaging technique.

Diagnostic ultrasound imaging has proven to be effective in diagnosing various respiratory diseases in human and animal subjects. We demonstrate a comprehensive ultrasound protocol utilized by Dr. Zuo's lab to analyze diaphragm kinetics specifically in mouse models. This is also a non-invasive research technique which can provide quantitative information on mouse respiratory muscle function.

Function analysis of rodent respiratory skeletal muscles, particularly the diaphragm, is commonly performed by isolating muscle strips using invasive ...

Assessment of Vascular Function in Patients With Chronic Kidney Disease

Patients with chronic kidney disease (CKD) have significantly increased risk of cardiovascular disease (CVD) compared to the general population, and this is only partially explained by traditional CVD risk factors. Vascular dysfunction is an important non-traditional risk factor, characterized by vascular endothelial dysfunction (most commonly assessed as impaired endothelium-dependent dilation [EDD]) and stiffening of the large elastic arteries. While various techniques exist to assess EDD and large elastic artery stiffness, the most commonly used are brachial artery flow-mediated dilation (FMDBA) and aortic pulse-wave velocity (aPWV), respectively. Both of these noninvasive measures of vascular dysfunction are independent predictors of future cardiovascular events in patients with and without kidney disease. Patients with CKD demonstrate both impaired FMDBA, and increased aPWV. While the exact mechanisms by which vascular dysfunction develops in CKD are incompletely understood, increased oxidative stress and a subsequent reduction in nitric oxide (NO) bioavailability are important contributors. Cellular changes in oxidative stress can be assessed by collecting vascular endothelial cells from the antecubital vein and measuring protein expression of markers of oxidative stress using immunofluorescence. We provide here a discussion of these methods to measure FMDBA, aPWV, and vascular endothelial cell protein expression.

The degree of vascular dysfunction and contributing physiological mechanisms can be assessed in patients with chronic kidney disease by measuring brachial artery flow-mediated dilation, aortic pulse-wave velocity, and vascular endothelial cell protein expression.

Patients with chronic kidney disease (CKD) have significantly increased risk of cardiovascular disease (CVD) compared to the general population, and this ...

A Methodological Approach to Non-invasive Assessments of Vascular Function and Morphology

The endothelium is the innermost lining of the vasculature and is involved in the maintenance of vascular homeostasis. Damage to the endothelium may predispose the vessel to atherosclerosis and increase the risk for cardiovascular disease. Assessments of peripheral endothelial function are good indicators of early abnormalities in the vascular wall and correlate well with assessments of coronary endothelial function. The present manuscript details the important methodological steps necessary for the assessment of microvascular endothelial function using laser Doppler imaging with iontophoresis, large vessel endothelial function using flow-mediated dilatation, and carotid atherosclerosis using carotid artery ultrasound. A discussion on the methodological considerations for each of the techniques is also presented, and recommendations are made for future research.

The present article describes the methodological considerations for several non-invasive assessments of vascular function and morphology that are commonly used in medical research to assess different stages of atherosclerosis.

The endothelium is the innermost lining of the vasculature and is involved in the maintenance of vascular homeostasis.  Damage to the endothelium may ...

Chronic Thromboembolic Pulmonary Hypertension and Assessment of Right Ventricular Function in the Piglet

An original piglet model of Chronic Thromboembolic Pulmonary Hypertension (CTEPH) associated with chronic Right Ventricular (RV) dysfunction is described. Pulmonary Hypertension (PH) was induced in 3-week-old piglets by a progressive obstruction of the pulmonary vascular bed. A ligation of the left Pulmonary Artery (PA) was performed first through a mini-thoracotomy. Second, weekly embolizations of the right lower pulmonary lobe were done under fluoroscopic guidance with n-butyl-2-cyanoacrylate during 5 weeks. Mean Pulmonary Arterial Pressure (mPAP) measured by ritght heart catheterism, increased progressively, as well as Right Atrial pressure and Pulmonary Vascular Resistances (PVR) after 5 weeks compared to sham animals. Right Ventricular (RV) structural and functional remodeling were assessed by transthoracic echocardiography (RV diameters, RV wall thickness, RV systolic function). RV elastance and RV-pulmonary coupling were assessed by Pressure-Volume Loops (PVL) analysis with conductance method. Histologic study of the lung and the right ventricle were also performed. Molecular analyses on RV fresh tissues could be performed through repeated transcutaneous endomyocardial biopsies. Pulmonary microvascular disease in obstructed and unobstructed territories was studied from lung biopsies using molecular analyses and pathology. Furthermore, the reliability and the reproducibility was associated with a range of PH severity in animals. Most aspects of the human CTEPH disease were reproduced in this model, which allows new perspectives for the understanding of the underlying mechanisms (mitochondria, inflammation) and new therapeutic approaches (targeted, cellular or gene therapies) of the overloaded right ventricle but also pulmonary microvascular disease.

Chronic Thromboembolic Pulmonary Hypertension (CTEPH) and Right Ventricular (RV) dysfunction were induced in piglets by progressive obstruction of the pulmonary arteries. Consequences were remarkably similar to those observed in CTEPH patients. This animal model would be a very useful tool for pathophysiology and therapeutic experiments on CTEPH and RV failure.

An original piglet model of Chronic Thromboembolic Pulmonary Hypertension (CTEPH) associated with chronic Right Ventricular (RV) dysfunction is described. ...

Visualization of Vascular and Parenchymal Regeneration after 70% Partial Hepatectomy in Normal Mice

A modified silicone injection procedure was used for visualization of the hepatic vascular tree. This procedure consisted of in-vivo injection of the silicone compound, via a 26 G catheter, into the portal or hepatic vein. After silicone injection, organs were explanted and prepared for ex-vivo micro-CT (&#181;CT) scanning. The silicone injection procedure is technically challenging. Achieving a successful outcome requires extensive microsurgical experience from the surgeon. One of the challenges of this procedure involves determining the adequate perfusion rate for the silicone compound. The perfusion rate for the silicone compound needs to be defined based on the hemodynamic of the vascular system of interest. Inappropriate perfusion rate can lead to an incomplete perfusion, artificial dilation and rupturing of vascular trees.
The 3D reconstruction of the vascular system was based on CT scans and was achieved using preclinical software such as HepaVision. The quality of the reconstructed vascular tree was directly related to the quality of silicone perfusion. Subsequently computed vascular parameters indicative of vascular growth, such as total vascular volume, were calculated based on the vascular reconstructions. Contrasting the vascular tree with silicone allowed for subsequent histological work-up of the specimen after &#181;CT scanning. The specimen can be subjected to serial sectioning, histological analysis and whole slide scanning, and thereafter to 3D reconstruction of the vascular trees based on histological images. This is the prerequisite for the detection of molecular events and their distribution with respect to the vascular tree. This modified silicone injection procedure can also be used to visualize and reconstruct the vascular systems of other organs. This technique has the potential to be extensively applied to studies concerning vascular anatomy and growth in various animal and disease models.

Tools used for visualizing vascular regeneration require methods for contrasting the vascular trees. This film demonstrated a delicate injection technique used to achieve optimal contrasting of the vascular trees and illustrate the potential benefits resulting from a detailed analysis of the resulting specimen using &#181;CT and histological serial sections.

A modified silicone injection procedure was used for visualization of the hepatic vascular tree. This procedure consisted of in-vivo injection of the ...

Calcification of Vascular Smooth Muscle Cells and Imaging of Aortic Calcification and Inflammation

Cardiovascular disease is the leading cause of morbidity and mortality in the world. Atherosclerotic plaques, consisting of lipid-laden macrophages and calcification, develop in the coronary arteries, aortic valve, aorta, and peripheral conduit arteries and are the hallmark of cardiovascular disease. In humans, imaging with computed tomography allows for the quantification of vascular calcification; the presence of vascular calcification is a strong predictor of future cardiovascular events. Development of novel therapies in cardiovascular disease relies critically on improving our understanding of the underlying molecular mechanisms of atherosclerosis. Advancing our knowledge of atherosclerotic mechanisms relies on murine and cell-based models. Here, a method for imaging aortic calcification and macrophage infiltration using two spectrally distinct near-infrared fluorescent imaging probes is detailed. Near-infrared fluorescent imaging allows for the ex vivo quantification of calcification and macrophage accumulation in the entire aorta and can be used to further our understanding of the mechanistic relationship between inflammation and calcification in atherosclerosis. Additionally, a method for isolating and culturing animal aortic vascular smooth muscle cells and a protocol for inducing calcification in cultured smooth muscle cells from either murine aortas or from human coronary arteries is described. This in vitro method of modeling vascular calcification can be used to identify and characterize the signaling pathways likely important for the development of vascular disease, in the hopes of discovering novel targets for therapy.

Vascular calcification is an important predictor of and contributor to human cardiovascular disease. This protocol describes methods for inducing calcification of cultured primary vascular smooth muscle cells and for quantifying calcification and macrophage burden in animal aortas using near-infrared fluorescence imaging.

Cardiovascular disease is the leading cause of morbidity and mortality in the world.  Atherosclerotic plaques, consisting of lipid-laden macrophages and ...

Assessment of Human Adipose Tissue Microvascular Function Using Videomicroscopy

While obesity is closely linked to the development of metabolic and cardiovascular disease, little is known about mechanisms that govern these processes. It is hypothesized that pro-atherogenic mediators released from fat tissues particularly in association with central/visceral adiposity may promote pathogenic vascular changes locally and systemically, and the notion that cardiovascular disease may be the consequence of adipose tissue dysfunction continues to evolve. Here, we describe a unique method of videomicroscopy that involves analysis of vasodilator and vasoconstrictor responses of intact small human arterioles removed from the adipose depot of living human subjects. Videomicroscopy is used to examine functional properties of isolated microvessels in response to pharmacological or physiological stimuli using a pressured system that mimics in vivo conditions. The technique is a useful approach to gain understanding of the pathophysiology and molecular mechanisms that contribute to vascular dysfunction locally within the adipose tissue milieu. Moreover, abnormalities in the adipose tissue microvasculature have also been linked with systemic diseases. We applied this technique to examine depot-specific vascular responses in obese subjects. We assessed endothelium-dependent vasodilation to both increased flow and acetylcholine in adipose arterioles (50 - 350 &#181;m internal diameter, 2 - 3 mm in length) isolated from two different adipose depots during bariatric surgery from the same individual. We demonstrated that arterioles from visceral fat exhibit impaired endothelium-dependent vasodilation compared to vessels isolated from the subcutaneous depot. The findings suggest that the visceral microenvironment is associated with vascular endothelial dysfunction which may be relevant to clinical observation linking increased visceral adiposity to systemic disease mechanisms. The videomicroscopy technique can be used to examine vascular phenotypes from different fat depots as well as compare findings across individuals with different degrees of obesity and metabolic dysfunction. The method can also be used to examine vascular responses longitudinally in response to clinical interventions.

Videomicroscopy systems are used to examine functional properties of isolated adipose tissue arterioles in response to physiological and pharmacological stimuli. This technique can be used to examine microvascular phenotypes in different adipose tissue domains in obese humans.

While obesity is closely linked to the development of metabolic and cardiovascular disease, little is known about mechanisms that govern these processes. ...

Skeletal Muscle Neurovascular Coupling, Oxidative Capacity, and Microvascular Function with 'One Stop Shop' Near-infrared Spectroscopy

Exercise represents a major hemodynamic stress that demands a highly coordinated neurovascular response in order to match oxygen delivery to metabolic demand. Reactive hyperemia (in response to a brief period of tissue ischemia) is an independent predictor of cardiovascular events and provides important insight into vascular health and vasodilatory capacity. Skeletal muscle oxidative capacity is equally important in health and disease, as it determines the energy supply for myocellular processes. Here, we describe a simple, non-invasive approach using near-infrared spectroscopy to assess each of these major clinical endpoints (reactive hyperemia, neurovascular coupling, and muscle oxidative capacity) during a single clinic or laboratory visit. Unlike Doppler ultrasound, magnetic resonance images/spectroscopy, or invasive catheter-based flow measurements or muscle biopsies, our approach is less operator-dependent, low-cost, and completely non-invasive. Representative data from our lab taken together with summary data from previously published literature illustrate the utility of each of these end-points. Once this technique is mastered, application to clinical populations will provide important mechanistic insight into exercise intolerance and cardiovascular dysfunction.

Skeletal Muscle Neurovascular Coupling, Oxidative Capacity, and Microvascular Function with &#039;One Stop Shop&#039; Near-infrared Spectroscopy

Here, we describe a simple, non-invasive approach using near-infrared spectroscopy to assess reactive hyperemia, neurovascular coupling and skeletal muscle oxidative capacity in a single clinic or laboratory visit.

Exercise represents a major hemodynamic stress that demands a highly coordinated neurovascular response in order to match oxygen delivery to metabolic ...

Veno-Venous Extracorporeal Membrane Oxygenation in a Mouse

The use of extracorporeal membrane oxygenation (ECMO) has increased substantially in recent years. ECMO has become a reliable and effective therapy for acute as well as end-stage lung diseases. With the increase in clinical demand and prolonged use of ECMO, procedural optimization and prevention of multi-organ damage are of critical importance. The aim of this protocol is to present a detailed technique of veno-venous ECMO in a non-intubated, spontaneously breathing mouse. This protocol demonstrates the technical design of the ECMO and surgical steps. This murine ECMO model will facilitate the study of pathophysiology related to ECMO (e.g., inflammation,bleeding and thromboembolic events). Due to the abundance of genetically modified mice, the molecular mechanisms involved in ECMO-related complications can also be dissected.

Here we present a protocol describing the technique of veno-venous extracorporeal membrane oxygenation (ECMO) in a non-intubated, spontaneously breathing mouse. This murine model of ECMO can be effectively implemented in experimental studies of acute and end-stage lung diseases.

The use of extracorporeal membrane oxygenation (ECMO) has increased substantially in recent years. ECMO has become a reliable and effective therapy for ...

Measurement of Tissue Oxygenation Using Near-Infrared Spectroscopy in Patients Undergoing Hemodialysis

Near-infrared spectroscopy (NIRS) has recently been applied as a tool to measure regional oxygen saturation (rSO2), a marker of tissue oxygenation, in clinical settings including cardiovascular and brain surgery, neonatal monitoring and prehospital medicine. The NIRS monitoring devices are real-time and noninvasive, and have mainly been used for evaluating cerebral oxygenation in critically ill patients during an operation or intensive care. Thus far, the use of NIRS monitoring in patients with chronic kidney disease (CKD) including hemodialysis (HD) has been limited; therefore, we investigated rSO2 values in some organs during HD. We monitored rSO2 values using a NIRS device transmitting near-infrared light at 2 wavelengths of attachment. The HD patients were placed in a supine position, with rSO2 measurement sensors attached to the foreheads, the right hypochondrium and the lower legs to evaluate rSO2 in the brain, liver and lower leg muscles, respectively. NIRS monitoring could be a new approach to clarify changes in organ oxygenation during HD or factors affecting tissue oxygenation in CKD patients. This article describes a protocol to measure tissue oxygenation represented by rSO2 as applied in HD patients.

We present a protocol to measure regional oxygen saturation (rSO2) in hemodialysis (HD) patients by using a near-infrared spectroscopy monitor. The rSO2 value is an index of tissue oxygenation. This noninvasive and real-time monitoring could be useful for confirming changes in organ oxygenation during HD.

Near-infrared spectroscopy (NIRS) has recently been applied as a tool to measure regional oxygen saturation (rSO2), a marker of tissue oxygenation, in ...