Optical clearing method for monitoring cutaneous ...

Optical clearing method for monitoring cutaneous microcirculation response to vasoactive drugs with high sensitivity Rui Shi1, 2 , Min Chen3 , Ruilin Wang2 , Cong Ma2 , Junbo Jin2 , Yuhua Lu2 , Polina Timoshina4 , Valery V. Tuchin4 , Dan Zhu1, 2 , * 1 Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and Technology, Wuhan 430074, China 2 Department of Biomedical Engineering, Key Laboratory of Biomedical Photonics of Ministry of Education, Huazhong University of Science and Technology, Wuhan 430074, China 3 Affiliated Hospital, Huazhong University of Science and Technology, Wuhan 430074, China 4 Department of Optics and Biomedical Physics, Saratov State University, Saratov, 410012, Russia ABSTRACT Laser speckle contrast imag ing technique has been playing an important role in monitoring cutaneous microcirculat ion, but the strong scattering of skin restricts the imaging depth and contrast, and also makes it impossible to assess the cutaneous microcircu lation response dynamically with high sensitivity. The tissue optical clearing is helpful for opening a visible window on mouse dorsal skin. In this work, the cutaneous microcirculat ion response to vasoactive noradrenaline is monitored with the laser speckle contrast imag ing system before and after skin optical clearing. The results show that the optical clearing method can significantly enhance the contrast of laser speckle contrast imaging, and small blood vessels whose diameter less than 20μm can be distinguished with high resolution. The dynamic changes in cutaneous microvascular d iameter and blood flow velocity caused by drug can be monitored sensitively. In contrast, it is difficult to detect the cutaneous microcircu lation response that occurred in the blood vessels more than 100μm in the intact skin, and the signal-to-noise ratio is too low to monitor the dynamic changes caused by the same drug. Thus, skin optical clearing method can enhance the ability of laser speckle contrast imaging in accessing cutaneous microcircu lation response, including the imaging contrast, resolution and sensitivity. Keywords: Laser speckle contrast imaging, optical clearing method, noradrenaline, vascular diameter, flow velocity

1. INTRODUCTION The accessed vascular bed will be helpfu l fo r understanding the cutaneous microcircu lation function and dysfunction in a variety of diseases status [1, 2]. Thus, imaging the structure and function of dermal blood vessels is of considerable importance for investigating the cutaneous microcirculation . Two types of microvascular tests have been developed to study the cutaneous microcirculat ion function, one is the type without pharmacolog ical intervention, such as the local cooling [3], post-occlusive hyperemia [4] and pressure-induced vasodilation [5], and the other is the type with pharmacological intervention, including Acetylcholine (Ach)[6-8], Sodium n itroprusside (SNP)[7] and Noradrenaline (NA)[6]. These tests have been widely used to evaluate the difference in cutaneous microcirculat ion of the physiological or pathological status. So far, various imag ing techniques, such as Magnetic Resonance imaging (M RI), Co mputed Tomography (CT), Ultrasound, Laser Doppler flowmet ry (LDF), Angiography and Laser Speckle Contrast Imaging (LSCI), have been developed to evaluate the function of cutaneous microcirculat ion by monitoring the microcircu lation response to vasoactive drugs [6-9]. However, some techniques lack in temporal-spatial resolution (M RI, CT, LDF), and some need the exogenous marker (M RI, CT, Angiography). Moreover, most of these techniques cannot provide both the structural and functional information simultaneously. The above disadvantages reduce the sensitivity for monitoring the cutaneous *

Address all correspondence to Dan Zhu, Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and T echnology, Wuhan 430074, China; Tel: (86)27–87792033; Fax:(86) 27–87792034; E-mail: [email protected] Optical Diagnostics and Sensing XIV: Toward Point-of-Care Diagnostics, edited by Gerard L. Coté, Proc. of SPIE Vol. 8951, 89510W · © 2014 SPIE CCC code: 1605-7422/14/$18 · doi: 10.1117/12.2038446 Proc. of SPIE Vol. 8951 89510W-1 Downloaded From: http://proceedings.spiedigitallibrary.org/ on 07/12/2015 Terms of Use: http://spiedl.org/terms

microcirculat ion response to vasoactive drugs. In contrary, a fu ll-field laser speckle contrast imaging technique [10] can provide a two -dimensional (2-D) map of b lood flow with h igh spatiotemporal resolution, which has played an important role in studying cerebral b lood flow[11, 12], mesentery microcirculat ion [13], etc. Unfortunately, LSCI suffers fro m the limited penetration depth of light in turbid tissues, so the previous investigations were mainly restricted within the imaging of blood flow in transparent tissues. The strong scattering tremendously attenuates the penetration depth of the light in the tissue. Especially, the surface static speckle signals conceal the dynamic speckle signals derived from the moving red b lood cells in the dermal blood vessels, which results in poor image contrast and SNR. Thus, the LSCI technique has seldom been used to monitor the cutaneous microcircu lation response to vasoactive drugs in the intact skin. In order to monitor cutaneous microcirculation response to vasoactive drugs with high resolution and contrast, tissue optical clearing technique has attracted more and mo re attention [14]. Zhu’s group developed an in vivo skin optical clearing method, and enhanced the SNR and contrast of LSCI for dermal b lood flow imaging [15, 16]. In this work, based on the developed method, the cutaneous microcirculat ion response to vasoconstrictor NA was mon itored dynamically by the LSCI, and the enhancement of spatiotemporal resolution and sensitivity was evaluated.

2. MATERIALS AND METHODS 2.1 Animal preparation 30 Male Balb/c mice (8 weeks, 25-30g) were supplied by Wuhan University Center for Animal Experiment (Wuhan, China) and fed under specific pathogen free (SPF) level with free food and water, animal care and experimental procedures were approved by the Experimental Animal Management Ordinance of Hubei Province, P. R. China. M ice were anesthetized with the mixture of 2% α-chloralose and 10% urethane (0.8mL/ 100g) via intraperitoneal inject ion, the dorsal hair was shaved and the residual hair was removed with depilatory cream (sensitive hair removal cream, Veet, India). After that, Mice were rando mly div ided into two groups: 10 mice were used to study the effect of topical application of OCA on the cutaneous microcircu lation as the control group; 20 mice were used to monitor the cutaneous microcirculat ion response to vasoconstrictor NA (0.2 mg/ ml, Sig ma-Aldrich Co, USA) before and after OCA treat ment as the experimental group. 2.2 LSCI for monitoring the cutaneous microcirculation In this study, LSCI was used to monitor the cutaneous microcirculat ion response to vasoconstrictor NA. The schematic of LSCI system was shown in Figure 1. Briefly, a He -Ne laser (632.8 n m, 3 mW ) was used to illu minate the interested cutaneous areas through a beam expanded collimat ing lens, a sequence of raw speckle images were recorded by a charge coupled device (CCD) camera (Pixelfly USB, PCO Co mputer, Germany) mounted on a stereo microscopy (SZ61TR, Oly mpus, Japan). The CCD exposure time was set to 5 ms. The Laser Speckle Contrast Temporal Analysis ( LSTCA) method was used to obtain the 2-D blood flo w distribution maps of cutaneous blood vessels because of its highly effective for suppressing the influence of static scattering and non -uniform light distribution.

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Fig 1. The schematic of Laser Speckle Contrast Imaging system.

After animal model was prepared, the mice were placed on the experimental platform. For the control group, during the first 2 minutes, the initial raw speckle images of intact skin were recorded by LSCI, then optical clearing skin window model was prepared based on previous method, and next the effect of OCA on the cutaneous microcirculat ion was monitored for 35 minutes with an intravenous injection of saline. For the experimental g roup, after preparation of the animals, the intact skin was monitored for the first 2 minutes, and then 100u l of NA bitartrate was one-time injected intravenously into the mice, the intact cutaneous microcircu lation response to NA was monitored with LSCI for 30

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minutes with an interval of 1 minute. The next day, the intact skin microcircu lation in the same location as the previous day was monitored for the first 2 minutes, then optical clearing skin window model was prepared on the same cutaneous areas and the optical clearing cutaneous microcircu lation was recorded for 5 minutes, and then the same dosage of NA was injected into the mice with same method, the raw speckle images after NA in jection were measured for 30 minutes with an interval of 1 minute. 2.3 Data analysis LSTCA method was used to process the raw speckle images , wh ich can effectively suppress the static speckle signals and non-uniform distribution of light. Under the assumption of a random Gaussian or Lorentzian velocity distribution, the speckle contrast C can be linked with the decorrelation time  c with following equation [17, 18]:

C 

1 s   { c [1 - exp(-2T c )]}2  I  2T

(1)

Where C is the speckle contrast,  s is the standard deviation of the speckle intensity and  I  is the mean intensity. T is the exposure time of the CCD camera and  c is the decorrelation t ime. And then 2-D cutaneous blood flow distribution maps at different time-point after intravenous injection of vasoconstrictor NA were acquired. In order to calculate the relative changes in vascular diameter and the flow velocity, we first set a value in the region of interest (ROI) in the velocity images as the threshold, and then we identified the blood vessels as having pixels with the value above the threshold. The relative diameter was expressed as the ratio o f the measured diameter under condition of NA injection to that of the init ial condition. The b lood flow velocity was expressed as the mean value of a ROI in the blood vessels of the velocity images, and the relative flow velocity was also calculated as the rat io of the measured flow velocity under condition of NA in jection to that of the init ial condition. At the end, we co mpared the cutaneous microcirculation response to NA in intact skin and optical clearing skin based on mathematic statistical analysis.

3. RESULTS 3.1 Short-term effect of OCA on cutaneous microcirculation The whole time for mon itoring the cutaneous microcirculat ion response to vasoconstrictor NA based on optical clearing skin window model was about 35 minutes, so we monitored the cutaneous blood vessels with an intravenous injection of saline as control for 35 minutes with an interval of 1 minute to study the influence of OCA on the cutaneous microvasculature, from the aspects of cutaneous vascular diameter and blood flow velocity. Figure 2(a) demonstrated some time -points of white light photographs and speckle contrast images during topical application of OCA on the skin. The cutaneous vascular diameter and the blood flo w velocity were also calculated based on statistical analysis of 6-8 cutaneous blood vessels with almost the same size, respectively. The figure 2(b -c) showed the time -lapse changes in the cutaneous vascular diameter and blood flow velocity with the diameter in the range of 3080 μm and 100-200μm of the cutaneous blood vessels, and it indicated that the OCA almost has no influence on the cutaneous vascular diameter and blood flo w velocity in 35 minutes, no matter the s mall blood vessels or the big ones . It was consistent with previous studies , J. Wang et al[15] demonstrated that the topical application of OCA did not affect the microvasculature of dermal b lood vessels based on H&E stains and Masson stains . That is just to say, the optical clearing skin window model based on topical application of OCA on the skin is safe, and it does not influence the cutaneous vascular structure and blood flow distribution. So we can use this kind of method to estimate the cutaneous microcirculat ion response to some vasoactive drugs safely and accurately with high temporal-spatial resolution and contrast.


4. DISCUSSION AND CONCLUSION In this study, we used an endotheliu m-independent vasoconstrictor NA to study the cutaneous microvascular response to vasoactive substance based on the combination of the LSCI and optical clearing skin window model. Co mpared with the traditional methods, such as intravital microscopy (IVM) [19], orthogonal polarizat ion spectral imag ing (OPS)[20] as well as tissue viability imaging (TiVi)[21] and laser Doppler perfusion technique, our method has the advantages of high temporal-spatial resolution and better images quality in assessing the skin microcirculat ion. And our results indicated that OCA had no influence on cutaneous microvasculature, for the intact skin, we could not monitor the cutaneous microcirculat ion response to vasoactive drugs by LSCI with high temporal-spatial resolution, contrast and sensitivity. While the usage of optical clearing skin window makes it possible to monitor the time -lapse changes in cutaneous microcirculat ion response to NA in vascular diameter and blood flow velocity with high contrast and sensitivity. Meanwhile we found there were so me different vasoconstrictive responses to NA between the small blood vessels and the big ones. The much stronger cutaneous microvascular vasoconstriction in the big blood vessels induced the higher elevating blood flow velocity. On the contrary, the vasoconstrictor NA had a slight vasoconstrictive effect on the small cutaneous vascular microcirculat ion and it gave rise to a mild increase in cutaneous blood flow velocity in the end. But this different cutaneous microcircu lation response to vasoconstrictor NA between the small blood vessels and the big blood vessels could not be distinguished in the intact skin, that means that the skin optical clearing window has a significant value and importance in studying the cutaneous microcircu lation response to vasoactive drugs effectively and accurately by LSCI with high temporal-spatial resolution and contrast, no matter in the normal physiological status or in the pathological status.

ACKNOWLEDGMENTS This study was supported by the National Nature Science Foundation of China (Grant Nos. 81171376, 91232710, 812111313), the Research Fund for the Doctoral Program of Higher Education of Ch ina (Grant No. 20110142110073), and the Science Fund for Creative Research Group (Grant No. 61121004).

REFERENCE [1] [2] [3] [4] [5] [6] [7] [8] [9] [10]

Holowat z, L. A., Thompson-Torgerson, C. S., and Kenney, W. L., “The human cutaneous circulation as a model of generalized microvascular function,” J. Appl. Physiol. 105(1), 370-372 (2008). Cracowski, J. L., Minson, C. T., Salvat -Melis, M., and Halliwill, J. R., “Methodological issues in the assessment of skin microvascular endothelial function in humans,” Trends Pharmacol. Sci. 27(9), 503-508 (2006). Roustit, M., Maggi, F., Isnard, S., Hellmann, M., Bakken, B., and Cracowski, J. L., “Reproducibility of a local cooling test to assess microvascular function in human skin,” Microvasc. Res. 79(1), 34-39 (2010). Loren zo, S., and Minson, C. T., “Hu man cutaneous reactive hyperaemia: role of BK Ca channels and sensory nerves,” J. Physiol. 585(1), 295-303 (2007). Fro my, B., Sigaudo-Roussel, D., Gaubert-Dahan, M. L., Rousseau, P., Abraham, P., Ben zoni, D., Berrut, G., and Saumet, J. L., “Aging-associated sensory neuropathy alters pressure-induced vasodilation in hu mans,” J. Invest. Dermatol. 130(3), 849-855 (2010). Bro wn, H., Moppett, I. K., and Mahajan, R. P., “Transient hyperaemic response to assess vascular reactivity of skin: effect of locally iontophoresed acetycholine, bradykinin, epinephrine and phenylephrine,” Br. J. Anaesth. 90(4), 461-451 (2003). Morris, S. J., and Shore, A. C., “Skin b lood flo w responses to the iontophoresis of acetylcholine and sodium nitroprusside in man: possible mechanisms,” J. Physiol, 496(2), 531-542 (1996). Gaubert, M. L., Sigaudo-Roussel, D., Tartas, M., Berrut, G., Sau met, J. L., and Fro my, B., “Endotheliu mderived hyperpolarizing factor as an in vivo back-up mechanism in the cutaneous microcircu lation in old mice,” J. Physiol. 585(2), 617-626 (2007). Daly, S. M., and Leahy, M . J., “'Go with the flow ': a review of methods and advancements in blood flow imaging,” J. Biophotonics, 6(3), 217-255 (2013). Choi, B., Kang, N. M., and Nelson, J. S., “Laser speckle imaging for mon itoring b lood flow dynamics in the in vivo rodent dorsal skin fold model,” Microvasc. Res. 68(2), 143-146 (2004).


Figure 3(a) demonstrated that only big blood vessels could be recognized (with the diameter in the range of 100-200 μ m), and the changes in cutaneous vascular diameter and blood flo w velocity after NA injection in the intact skin could not be detected accurately and effectively. However, th is situation would be changed when OCA was topically applied on the skin, because that OCA can tremendously reduce the scattering of the skin and make the skin transparent. Thus, the penetration depth of the laser in the tissue can be deeper, and mo re dynamic speckle signals can be detected through the transparent skin. At the end, the better images qualities compared with the intact skin permit us to assess the cutaneous vascular response to vasoactive drugs with LSCI conveniently and accurately. After we verified that topical application of OCA had no effect on the cutaneous vascular diameter and blood flow velocity, we monitored the cutaneous microcirculation response to vas oconstrictive NA based on optical clearing skin window. Figure 3(b) showed the cutaneous vascular structure and blood flow distribution in the same cutaneous areas, it suggested that we can image the dermal blood flow with high contrast and SNR. Meanwhile we can monitor the time-lapse changes in the cutaneous vascular diameter and blood flow velocity after NA injection . Figure 4(a) demonstrated the time -lapse changes in the intact cutaneous vascular diameter and blood flow velocity of the big b lood vessels in the range of 100-200 μm. It suggested that there were very weak changes in the diameter and flow velocity after in jection of the NA, wh ich is mainly because of the strong scattering of the skin. The strong scattering decreased the dynamic speckle signal intensity from the red blood cells in the deep dermal blood vessels, and then leads to an inaccurate evaluation in cutaneous microcirculation response to NA. While t he time-lapse changes in cutaneous vascular d iameter and blood flow velocity to vasoconstrictor NA with the d iameter in the range of 100-200 μm and 30-80 μm based on skin optical clearing window were dynamically monitored in Figure 4(b) and Figure 4(c), respectively. It suggested that the different size of blood vessels had the different response to vasoconstrictor NA, the small b lood vessels ranging from 30 to 80 μm had no significant decrease in diameter, and just had increased about 24.3% ± 4.69% in blood flo w velocity, whereas the b lood vessels in the range of 100-200 μm have a decrease about 25.5 % ± 3.11% in the vascular diameter, and this much stronger vasoconstriction led to an increase about 61.8% ± 5.2% in the blood flow velocity. It demonstrated that the NA had a stronger vasoconstrictive effect on the big blood vessels compared with the small ones . And about 20 minutes after injection of NA, the cutaneous vascular diameter and blood flow velocity can return to the baseline. In verse, we cannot detect the changes in cutaneous vascular diameter and blood flow velocity in the intact skin effectively and accurately with high resolution and contrast. That means that the skin optical clearing window has a significant value and importance not only for studying the cutaneous microcircu lation response to vasoactive drugs with LSCI, but also for diagnosis and treatment of some peripheral vascular d iseases, including tumor research with various optical clearing techniques.

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Fig 4. Dynamically monitoring the cutaneous microcirculation response to NA for 30 minutes. (a): dynamic time-lapse monitoring the effect of NA on the intact cutaneous vascular diameter and blood flow velocity (in the range of 100-200μm) (b-c): dynamic time-lapse monitoring the effect of NA on cutaneous vascular diameter and blood flow velocity based on optical clearing skin window (with the size in the range of 100-200μm and 30-80μm, respectively). Values were expressed with mean ± SEM .


4. DISCUSSION AND CONCLUSION In this study, we used an endotheliu m-independent vasoconstrictor NA to study the cutaneous microvascular response to vasoactive substance based on the combination of the LSCI and optical clearing skin window model. Co mpared with the traditional methods, such as intravital microscopy (IVM) [19], orthogonal polarizat ion spectral imag ing (OPS)[20] as well as tissue viability imaging (TiVi)[21] and laser Doppler perfusion technique, our method has the advantages of high temporal-spatial resolution and better images quality in assessing the skin microcirculat ion. And our results indicated that OCA had no influence on cutaneous microvasculature, for the intact skin, we could not monitor the cutaneous microcirculat ion response to vasoactive drugs by LSCI with high temporal-spatial resolution, contrast and sensitivity. While the usage of optical clearing skin window makes it possible to monitor the time -lapse changes in cutaneous microcirculat ion response to NA in vascular diameter and blood flow velocity with high contrast and sensitivity. Meanwhile we found there were so me different vasoconstrictive responses to NA between the small blood vessels and the big ones. The much stronger cutaneous microvascular vasoconstriction in the big blood vessels induced the higher elevating blood flow velocity. On the contrary, the vasoconstrictor NA had a slight vasoconstrictive effect on the small cutaneous vascular microcirculat ion and it gave rise to a mild increase in cutaneous blood flow velocity in the end. But this different cutaneous microcircu lation response to vasoconstrictor NA between the small blood vessels and the big blood vessels could not be distinguished in the intact skin, that means that the skin optical clearing window has a significant value and importance in studying the cutaneous microcircu lation response to vasoactive drugs effectively and accurately by LSCI with high temporal-spatial resolution and contrast, no matter in the normal physiological status or in the pathological status.

ACKNOWLEDGMENTS This study was supported by the National Nature Science Foundation of China (Grant Nos. 81171376, 91232710, 812111313), the Research Fund for the Doctoral Program of Higher Education of Ch ina (Grant No. 20110142110073), and the Science Fund for Creative Research Group (Grant No. 61121004).

REFERENCE [1] [2] [3] [4] [5] [6] [7] [8] [9] [10]

Holowat z, L. A., Thompson-Torgerson, C. S., and Kenney, W. L., “The human cutaneous circulation as a model of generalized microvascular function,” J. Appl. Physiol. 105(1), 370-372 (2008). Cracowski, J. L., Minson, C. T., Salvat -Melis, M., and Halliwill, J. R., “Methodological issues in the assessment of skin microvascular endothelial function in humans,” Trends Pharmacol. Sci. 27(9), 503-508 (2006). Roustit, M., Maggi, F., Isnard, S., Hellmann, M., Bakken, B., and Cracowski, J. L., “Reproducibility of a local cooling test to assess microvascular function in human skin,” Microvasc. Res. 79(1), 34-39 (2010). Loren zo, S., and Minson, C. T., “Hu man cutaneous reactive hyperaemia: role of BK Ca channels and sensory nerves,” J. Physiol. 585(1), 295-303 (2007). Fro my, B., Sigaudo-Roussel, D., Gaubert-Dahan, M. L., Rousseau, P., Abraham, P., Ben zoni, D., Berrut, G., and Saumet, J. L., “Aging-associated sensory neuropathy alters pressure-induced vasodilation in hu mans,” J. Invest. Dermatol. 130(3), 849-855 (2010). Bro wn, H., Moppett, I. K., and Mahajan, R. P., “Transient hyperaemic response to assess vascular reactivity of skin: effect of locally iontophoresed acetycholine, bradykinin, epinephrine and phenylephrine,” Br. J. Anaesth. 90(4), 461-451 (2003). Morris, S. J., and Shore, A. C., “Skin b lood flo w responses to the iontophoresis of acetylcholine and sodium nitroprusside in man: possible mechanisms,” J. Physiol, 496(2), 531-542 (1996). Gaubert, M. L., Sigaudo-Roussel, D., Tartas, M., Berrut, G., Sau met, J. L., and Fro my, B., “Endotheliu mderived hyperpolarizing factor as an in vivo back-up mechanism in the cutaneous microcircu lation in old mice,” J. Physiol. 585(2), 617-626 (2007). Daly, S. M., and Leahy, M . J., “'Go with the flow ': a review of methods and advancements in blood flow imaging,” J. Biophotonics, 6(3), 217-255 (2013). Choi, B., Kang, N. M., and Nelson, J. S., “Laser speckle imaging for mon itoring b lood flow dynamics in the in vivo rodent dorsal skin fold model,” Microvasc. Res. 68(2), 143-146 (2004).


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