This work was supported by research grants awarded to SKL from Abbott CARO (Canadian Association of Radiation Oncologists) Uro-Oncologic Radiation Awards and the Alan E. Tiffin Foundation. EK was supported by the Frederick Banting and Charles Best Canada Graduate Scholarship, the Scace Graduate Fellowship in Prostate Cancer Research and Paul Starita Graduate Student Fellowship.

The following methods are in accordance with the guidelines of the Sunnybrook Research Institute Animal Care Ethics Committee.
1. Diffuse Reflectance Spectroscopy System
<ol>
	<li>Collect diffuse reflectance spectra using a handheld, fiber-optic probe and portable spectroscopic acquisition system that has been previously described (Kim et al. 2010) and is briefly reviewed in Figure 1 (and related captions) for completeness1,2.</li>
</ol>
2. Preparation of Mouse Model of Acute Radiation Skin Damage
<ol>
	<li>Order 6-week old mice (preferably hairless, such as athymic or SKH-1) and allow them to acclimate in the animal facility for a week before starting experiments. Reserve at least 3 mice for a non-irradiated control group and 5 mice for an irradiated group.</li>
	<li>Before baseline DOS measurements and irradiation, label the mice using ear punches or permanent marker markings on the tail. If mice are not nude, remove the hair on a 2 cm by 2 cm patch of flank skin, but this may cause skin irritation.</li>
</ol>
3. Diffuse Optical Spectroscopy Data Acquisition
<ol>
	<li>Turn on the power supply to the electronics.</li>
	<li>For mouse skin, set the signal parameters for the acquisition software by typing in 25 msec for collection time, 25 for signal averages and 1 for boxcar filter width. These parameters offer a reasonable balance between acquisition time and signal to noise.</li>
	<li>Using custom programmed acquisition software, automatically acquire a background reading, Rbg (LED off) and diffuse reflectance at two source-detector separation distances, Rmeas (260 &#956;m, 520 &#956;m) by clicking the &#34;Acquire&#34; button. The total acquisition time is ~ 2 sec.</li>
	<li>Switch off all fluorescent room lights by pressing on the room light switch before performing measurements. 
	NOTE: Fluorescent room light interfere with the detected signal (these lights produce a time-varying light intensity and thus it is difficult to subtract as a background signal). Although incandescent bulbs may be employed keep the lights at a distance from the DOS probe to avoid high background levels (and poor signal to noise).</li>
</ol>
4. Animal Anesthesia and Baseline DOS Measurements
<ol>
	<li>Prepare the anesthesia machine by ensuring that all connections are intact and liquid isoflurane level is adequate. Use an anesthesia induction chamber with an attached tube and nose cone that can be taped down to a sterilized, softly padded surface within comfortable reach of the DOS probe.</li>
	<li>Anaesthetize one cage of mice at a time in the induction chamber by inducing with 4% isoflurane for 30 sec. Lower the isoflurane amount to 2% for the next 2 min. Verify that the mouse is anaesthetized by observing no response from pinching a toe of the hind limb.</li>
	<li>Quickly move one mouse onto the sterilized DOS probing area, place it on its side, fasten its snout into the nose cone and open the nose cone tubing to the flow of anesthesia (2% isoflurane). 
	NOTE: If the procedure takes longer than 1 - 2 min, apply vet ointment on the eyes to prevent dryness.</li>
	<li>Before acquiring mouse skin measurements, sterilize the probe by wiping with 70% ethanol. Do not sterilize the skin.</li>
	<li>Place the probe gently on the flank skin making sure to avoid dispersing the local vasculature. Hold the probe by hand for the duration of the measurement.</li>
	<li>Acquire reflectance data by probing a flank skin area of about 2 cm by 2 cm (the area to be irradiated) by following the 5-dot formation on a die. Keep this probing pattern, area, probe pressure and body side (left or right) consistent for all subsequent measurements. 
	NOTE: The complete scan takes approximately 60 sec. Probe pressure should be just enough to obtain a scan without dispersing local vasculature.</li>
	<li>Move the mouse into a recovery cage, and move the next mouse over to the DOS probing area. Repeat steps 4.2 - 4.6 until all mice have been measured. Do not leave an animal unattended until it has regained sufficient consciousness to maintain sternal recumbency.</li>
</ol>
5. Animal irradiation
NOTE: This protocol requires the use of an irradiator, and animal preparation may need to be adjusted to meet the needs of the irradiator device. During irradiation, only the small area of flank skin should be exposed to the radiation beam. The irradiator should be located in a sterile facility and appropriate cage sterilization should be observed when returning mice to their sterile housing area.
<ol>
	<li>Prepare the anesthesia machine (as in steps 4.1 - 4.2) and anaesthetize one mouse at a time in the induction chamber before preparing it for irradiation.</li>
	<li>Remove the mouse from the induction chamber, gently pinch the flank skin and place tape over and below the stretched skin, forming a flap.</li>
	<li>Place the mouse onto a plexiglass stage and cover the body with a custom lead jig (a working design is a rectangular box with the bottom and at least one end open, along with a side window to allow flank skin to be pulled through). Pull the skin flap through the jig window and gently tape the flap onto the stage. 
	NOTE: The custom lead jig is small enough to immobilize the mouse. If the custom jig does not completely immobilize the mouse, then use additional restrainers and/or administer ketamine (80 - 100 mg/kg) and xylazine (10 - 12.5 mg/kg) via intraperitoneal injection to keep the mouse immobilized throughout the entire irradiation procedure.</li>
	<li>Place the plexiglass stage with the jig and mouse into the irradiator. Determine the settings (skin distance from x-ray source, voltage, duration and amperage) and deliver the desired dose (e.g., 11 cm from a 160 kVp x-ray source for 2.5 min with 6.3 mA). 
	NOTE: Use CAUTION with the x-ray source by following machine use guidelines to avoid burns and DNA damage. 
	NOTE: Athymic nude mice develop moist desquamation around 14 days post irradiation in response to 35 Gy, but only minor patchy desquamation with 17 Gy.</li>
	<li>Take the apparatus and mouse out of the irradiator, remove the shielding, remove the tape and place it into an individual recovery cage. Return the mouse to its normal shared cage after it has recovered from the anesthesia. Repeat steps 5.2 - 5.4 for all mice, and perform a sham operation on control mice.</li>
	<li>After irradiation, house the animals in their regular conditions. If abnormal behavior develops (e.g., hunched posture, which may signify pain), consult a veterinarian to diagnose the problem. Pain alleviation may include administering 0.1 mg/kg buprenorphine subcutaneously or as directed by the veterinarian.&#160;If weight loss exceeds 20% of normal body mass, house it separately in its own cage and provide high-nutrient food.</li>
</ol>
6. Follow-up DOS Measurements
<ol>
	<li>Monitor and measure skin reaction intensity using the quantitative DOS technique.&#160;Visual inspection of skin changes and previous work suggest that large changes in DOS parameters can be expected (relative to baseline) around 6 - 12 days following irradiation3,4. However, since appreciable changes may take place even earlier or later depending on the model, other measurement time points may be useful to investigate.</li>
	<li>Set up DOS equipment and calibrations as described in section 3.Prepare the anesthesia machine and acquire DOS measurements as described in section 4.</li>
</ol>
7. Post-acquisition Processing
NOTE: All steps in the following section are performed using a custom program created in a high performance software environment. Standardized naming conventions for each spectral acquisition file are employed to allow for batch processing. All steps are illustrated in Figure 2.
<ol>
	<li>Subtract the baseline (noise floor) from all the measured spectra including the background reading.</li>
	<li>Subtract the background reading, Rbg (LED off), obtained in Step 3.3 from the measurement spectrum, Rmeas. 
	NOTE: For the remainder of this article all spectra are assumed to be noise floor and background subtracted and referred to as Rcorr.</li>
	<li>Convert Rcorr to absolute reflectance, Rabs, as described in references 1,2 in Section 1.
	<ol>
		<li>Obtain relative reflectance measurements, Rrel, in Intralipid-20% phantoms (Fresenius Kabi, Sweden) phantoms with increasing 3% aliquot fractions up to 48% (i.e., 3%,6%,9%, ..., 48%) and create of plot of Rrel versus Intralipid concentration.</li>
		<li>Generate an absolute plot of Rabs versus&#160;&#956;s&#39; using the diffusion equation for reflectance.14</li>
		<li>Match the peak of both curves and adjust the Rrel x-axis to match the Rabs x-axis.</li>
		<li>At a given wavelength and source-detector separation, scale the y-axis using: 
		<img alt="Figure 1" src="/files/ftp_upload/53573/53573eq2.jpg" /> 
		NOTE: In the following section, all fitting of measurements will refer to Rabs.</li>
	</ol>
	</li>
</ol>
8. Spectral Data Fitting
NOTE: The following section outlines the theory and fitting algorithm utilized for extracting functional parameters of mice skin. For all theory employed, refer to the following articles14-18 and references therein. All equations are assumed to be programmed in a high end scientific software environment (containing pre-programmed modules) commonly used in physics or engineering labs.
<ol>
	<li>Program a function that describes the absorption spectrum, &#956;a(&#955;) of skin as the sum of relevant individual chromophores in the spectral range of interest using the equation: 
	<img alt="Figure 1" src="/files/ftp_upload/53573/53573eq4.jpg" /> 
	Here, Hb is the total hemoglobin concentration (g/L) , while StO2 is the unitless oxygen saturation ranging from 0 to 1.</li>
	<li>Obtain oxy, <img alt="Figure 1" src="/files/ftp_upload/53573/53573eq5.jpg" />, and deoxy, <img alt="Figure 1" src="/files/ftp_upload/53573/53573eq6.jpg" />, hemoglobin spectra (stored as text files) from the on-line collection of Prahl19.</li>
	<li>Program a function that describes the scattering spectrum of skin, <img alt="Figure 1" src="/files/ftp_upload/53573/53573eq7.jpg" />, using a power law dependence, where A (cm-1) is the value of &#956;s&#39; at &#955;o = 1 nm and k is a medium dependent power factor16.</li>
	<li>Program a mathematical function for the forward model of diffuse reflectance based on equations from reference 14 that incorporate the spectral equations from Steps 8.2 - 8.3 into the forward model function (i.e., R(r,&#160;&#956;a(&#955;),&#160;&#956;s&#39;(&#955;)) = R(r, Hb, StO2, A, k). 
	NOTE: While various models exist, the steady-state diffusion theory equation provides a simple and accurate description of the light distribution in tissue.</li>
	<li>Program a function that squares the difference between the forward modeled reflectance spectra from Section 8.4 and the measured reflectance spectra.</li>
	<li>Iteratively change Hb, StO2, A, and k until the least squares difference function in Section 8.5 is smallest. MatLab&#39;s lsqcurvefit can be used to automatically perform this step.</li>
	<li>Repeat steps 8.5 - 8.6 to obtain DOS parameters (Hb, StO2, A, and k) for all measured reflectance data sets.</li>
	<li>Plot the relative change in DOS parameter with the corresponding unique baseline measurement using the average of each mouse&#39;s set of 3 - 5 normalized probe spot measurements. These plots are created using MatLab&#39;s plot command.</li>
</ol>
9. Visual Radiation Dermatitis Scoring Period
<ol>
	<li>Monitor and score skin reaction intensity using a qualitative grading scale (see Douglas and Fowler grading scale20) after irradiation every 48 hr (one may also observe changes 3 - 24 hr following irradiation). Two blinded investigators are ideal. Acquiring photographs with a hand-held camera and reference scale (i.e., ruler) may help with evaluations.&#160;</li>
	<li>NOTE: Scoring the skin every two days after irradiation may help determine optimal DOS measurement times for the model. More frequent scoring may yield important data depending on the model and research question.</li>
	<li>Plot the median of each group at each time point. Compare groups at specific time points or the median overall areas under each curve.</li>
	<li>After mice have been followed to the point of skin healing that is desired (e.g., 4 weeks), euthanize the mice by an appropriate (approved) method.</li>
</ol>

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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin+perfusion+and+oxygenation+changes+in+radiation+fibrosis.">Skin perfusion and oxygenation changes in radiation fibrosis.</a> Plast. Reconstr. Surg. 131 (4), 707-716 (2013).</li><li>Yohan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+monitoring+of+radiation+induced+skin+toxicities+in+nude+mice+using+optical+biomarkers+measured+from+diffuse+optical+reflectance+spectroscopy.">Quantitative monitoring of radiation induced skin toxicities in nude mice using optical biomarkers measured from diffuse optical reflectance spectroscopy.</a> Biomed. Opt. 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The authors have nothing to disclose.

A DOS approach for quantitatively assessing radiation skin toxicities using optical biomarkers has been presented. Visual skin toxicity scoring systems require expert training and even then are prone to inter-observer variability and subjectivity. The DOS system and analysis software is simple to use, requires minimal training and returns objective functional parameters for interpreting physiological changes in skin. Furthermore, instead of describing the appearance of a skin lesion as a single parameter, DOS provides a wealth of information in spectral shape, optical properties and functional / microstructural parameters that offer an added degree of sensitivity and specificity not available in current qualitative scoring methods. Sections 1 and 7 highlight the main processing steps for obtaining absolute spectral data that can be utilized for quantitative fitting of optical biomarkers. Background and baseline subtraction are vital to allow the user to perform the DOS measurements under normal lighting conditions. Section 8 provides the necessary models and equations needed to describe athymic mice before and after x-ray irradiation. Here, the choice of appropriate absorbers is vital for an accurate description of the measured spectra. It is advised that the user thoroughly investigate in the literature the key absorbers that dominate the wavelength range and tissue of interest used in a given study prior to constructing an optical biomarker fitting model. Finally, Sections 3-5 describe the handling of the athymic mice during DOS acquisition. To avoid disrupting the local vasculature, use gentle force to place the DOS probe on the mouse skin surface.
While relatively inexpensive compared to hyperspectral camera systems3,4, a clear limitation of the described DOS approach is the use of a point probe for measuring diffuse reflectance. This reflectance geometry necessities gentle contact with the skin and has the potential to introduce measurement uncertainty by dispersing the vasculature if consistent probe-skin pressure is not employed. Future designs of the DOS probe may incorporate a pressure sensor to maintain consistent results. Further, while the use of close source-detector separation (&#60; 2-3 mm) allows for optical probing depths specific to the skin surface, the improved specificity comes at a loss of spatial resolution compared to 2D hyperspectral imaging. To minimize this limitation, a 5 point quadrant scan that captures the overall irradiated volume was employed. Despite the lack of spatial resolution, previous work in mice5 has shown the ability of optical biomarkers averaged over a sparse area to differentiate not only irradiated and non-irradiated skin but also the impact of skin sparing interventional drugs such as Vasculotide6.
It should be noted that while the overall system design can be modified for different skin models, the underlying basis spectra and scattering shape may need to be optimized. Specifically, while oxy- and deoxy-Hb well describe an athymic mouse model, the application of the same model to darker skin may require the addition of melanin for optimal fitting. In addition, extension of the DOS bandwidth to higher wavelengths &#62; 950 nm would necessitate the addition of water, which dominates at higher wavelengths. Furthermore, animal models with different skin thicknesses may require a different source-detector separation to optimize depth sensitivity. Lastly, the hairless feature makes algorithms simpler. Although non-hairless models may be optimal for certain research questions, they will require hair removal before DOS measurements, and skin irritation from this process may affect results. For research where total immune function is crucial, an immunocompetent hairless mouse (e.g., SKH-1) may serve as a better model due to its euthymic nature.
Important considerations for DOS probe measurements are consistent RT and estimation of the irradiated area. Temperature fluctuations can affect tissue Hb and StO2 levels. Measuring a group of 3 non-irradiated animals at each data collection time may serve as a baseline to which unintended environmental fluctuations in parameter values can be normalized. Additionally, the irradiated area may be difficult to estimate (if skin flap preparations were not consistent) before damage begins to manifest visually around day 5 (40 Gy). If using black permanent marker to dot the boundaries of the radiation-exposed skin, avoid excessive ink use to prevent ink smudging, which can compromise readings.
An added feature of the system is the ability to separate absorption from scattering properties. While alternative hyperspectral imaging systems also provide the ability to monitor oxyHb and Hb concentration, the free-space geometry of hyperspectral imaging is unable to resolve scattering changes. This limitation may result in inaccuracies in the returned oxyHb, Hb and StO2 parameters if significant changes in scattering occur due to erythema (redness). Further, monitoring of scattering changes using DOS may provide additional optical biomarkers for erythema evaluation. As shown in Figure 6, the initial results from Yohan et al. (2014) indicate that A and k demonstrate a temporal trend following ionizing radiation that does not correlate with trends observed from other alternative methods such as visual scoring systems. This indicates that scattering changes do not manifest in a visually descriptive manner and may in fact be describing a separate biological process. Therefore, compared to alternative methods, DOS provides a high resolution for superficial scattering changes, an avenue for investigating novel skin damage biomarkers that may be separate from the usual Hb-based measurements.
Although our model employs a large single radiation dose (rather than multiple small fractionated doses that are used in the clinical setting), this mimics the pathophysiology of acute human skin radiotoxicity21. It is envisioned that with further optimization, DOS may provide a quantitative approach for automated and standardized scoring of radiation induced skin reactions. After mastering this technique, future applications may include monitoring differences between skin sparing therapeutics (e.g., comparing oxyHb levels between a control and experimental treatment for skin radioprotection, or for wound healing promotion). While ideal for high-throughput drug screening in animal models, the DOS system is potentially adaptable to the clinical environment due to ease of usability and the ability to measure in normal lighting conditions. In this case, the probe design may require minor modifications with slightly larger optode separations to account for the increased thickness of human skin. A clinical DOS system would allow for on-line evaluation of interventional therapies that could minimize painful skin reactions and improve patient comfort and compliance. In the future, it may be interesting to expand DOS-based quantification to the features of chronic radiation induced skin damage (e.g., fibrosis).

Technological improvements in radiation therapy (RT) planning and delivery now allow for highly conformal therapeutic doses to be delivered to the tumor region, while simultaneously sparing normal surrounding structures. Yet, acute and sometimes severe toxicities are unavoidable when the high dose target is in close proximity to the skin. If severe enough, the resulting normal tissue damage can negatively affect the RT treatment outcome and patient quality of life1,2.
Despite the detrimental consequences, present management of radiation skin erythema remains nonspecific, employing creams or ointments that ignore the underlying biological mechanisms leading to damage. These approaches are based upon minimizing symptoms rather than the cause. Moreover, the timing and administration of interventional therapies is complicated by the qualitative and subjective nature of radiation skin injury assessment. While several recognized organizations (RTOG, EORTC) provide visual grading recommendations, institutions vary in their choice of preferred scoring, thereby obscuring comparisons of normal tissue toxicities for the purposes of meta-analyses. Further, such grading systems are crude and prone to inter-observer variability, such that differences in radiation injury severity may be indiscernible in studies evaluating toxicity reduction strategies.
Rather than visually describing the degree of erythema in irradiated skin, an alternative approach is to measure parameters that quantitatively describe the underlying physiological changes that occur in the organ. Blood hemoglobin (Hb), tissue oxygen saturation (StO2) or oxygenated hemoglobin (oxyHb) levels have been used as proxies for irradiation-induced erythema in mice3-6. Following irradiation, total Hb levels undergo fluctuations, but oxyHb or StO2 undergo a characteristic early sharp rise, followed by a fall and another more persistent rise3,6. When irritants are used to induce skin erythema, vascular oxyHb levels directly correlate with the severity of the local erythema and inflammation7.
Diffuse optical spectroscopy (DOS) employs near-infrared light to provide functional information on the biochemical and microstructural components of vital tissues components. This quantitative, non-invasive optical technology offers a method to measure the cytokine-induced vasodilation in blood vessels that occur during erythema via functional surrogates of Hb concentration and StO2. Recent studies comparing DOS measured parameters with controlled clinical scoring methods8-11 indicate the potential of the technique for overcoming the limitations inherent to current grading systems.
Here we describe an in-house, portable, DOS system that employs functional surrogates for quantitatively detecting differences in radiation-induced skin toxicity in a pre-clinical mouse model5. The described platform may provide a means of standardized erythema scoring with high sensitivity for early detection and subtle differentiation of interventional drug response. Moreover, with only minor adaptations, the instrumentation may eventually be employed clinically for real-time bedside monitoring.

<table border="0" cellpadding="0" cellspacing="0" width="1146">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Nude mice</td>
			<td style="width:347px;">e.g. Charles River</td>
			<td colspan="2" style="width:555px;">Athymic nude Crl:NU(NCr)-Foxn1nu, or immunocompetent nude Crl:SKH1-Hrhr</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Small animal irradiator&#160;</td>
			<td>e.g. Faxitron X-Ray Corp.</td>
			<td>Faxitron CP160</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Animal anaesthesia&#160;</td>
			<td></td>
			<td></td>
			<td>If using isoflurane vaporizer machine with induction chamber, need tube and nose cone</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lead jig and plexiglass stage</td>
			<td>Custom made</td>
			<td></td>
			<td>If irradiator device exposes whole animal body to radiation, lead shielding must be used to expose only the skin flap</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Medical tape&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Permanent marker/ear puncher</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Matlab</td>
			<td>Mathworks Inc., Natick, MA</td>
			<td></td>
			<td>With StatisticsToolbox&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Labview</td>
			<td>National Instruments, Vaudreuil-Dorian, QB</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DOS system</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Optical multiplexer</td>
			<td>Ocean Optics, Dunedin, FL</td>
			<td>Model MPM-2000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spectrometer</td>
			<td>Ocean Optics, Dunedin, FL</td>
			<td>Model S200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">White light source</td>
			<td>Ocean Optics, Dunedin, FL</td>
			<td>Model LS-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Intralipid-20%</td>
			<td>Kabi Pharmacia, New York, NY</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Reflectance standard</td>
			<td>INO, Quebec City, QB</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

diffuse optical spectroscopy for the quantitative assessment of acute ionizing radiation induced skin toxicity using a mouse model

Acute skin toxicities from ionizing radiation (IR) are a common side effect from therapeutic courses of external beam radiation therapy (RT) and negatively impact patient quality of life and long term survival. Advances in the understanding of the biological pathways associated with normal tissue toxicities have allowed for the development of interventional drugs, however, current response studies are limited by a lack of quantitative metrics for assessing the severity of skin reactions. Here we present a diffuse optical spectroscopic (DOS) approach that provides quantitative optical biomarkers of skin response to radiation. We describe the instrumentation design of the DOS system as well as the inversion algorithm for extracting the optical parameters. Finally, to demonstrate clinical utility, we present representative data from a pre-clinical mouse model of radiation induced erythema and compare the results with a commonly employed visual scoring. The described DOS method offers an objective, high through-put evaluation of skin toxicity via functional response that is translatable to the clinical setting.

The DOS reflectance technique provides an objective alternative to traditional qualitative methods of evaluating radiation induced skin toxicity. Visual changes in skin appearance following toxic doses of radiation present as alterations in both the magnitude and shape of the measured reflectance spectra. Both are related to functional changes in the underlying cellular microstructure and physiological tissue state. In this section, representative results from previously published work by Yohan et al. 20145 are reviewed.
Figure 3 (left) show representative spectra (thin blue lines) measured at a 260 &#956;m source-separation in an athymic mouse model of skin erythema 6 days after 40 Gy irradiation. Compared to pre-irradiation (Figure 3, right panel), differences in the spectral shape at ~ 550-650 nm are observed, likely due to an increase in oxygenated hemoglobin. A small rise in absolute reflectance is also seen that is correlated to an increase in tissue scattering power. The observed spectra on day 6 following irradiation correlated to a visual skin score of 0.75.
An evaluation of post irradiation reflectance changes at select wavelengths does not make use of the complete reflectance spectrum and also carries the potential issue of noise sensitivity. However, fitting the complete spectrum allows the entire data set to be converted into intuitive optical biomarkers (Hb, StO2). Figure 3 show the resulting fits (solid green line) of the measured data (thin noisy line) using the equations presented in Section 4. Excellent agreement is observed, confirming that the choice of basis chromophores and scattering shape adequately describe the mouse skin model.
Due to the non-invasive and self-calibrating nature of the DOS system, measurements can be conveniently performed over multiple days in different lighting conditions. Figure 4 shows relative changes in skin StO2 for various time-points (6, 9, 12 days) in an irradiated mouse cohort (n=8) while Figure 5 shows the corresponding qualitative skin reaction scores. A progressive increase in StO2 is observed that is statistically different compared to pre-irradiation values over all 3 days (p &#60; 0.05). These trends mirror the visually observed increases in skin damage severity that peak at day 12 (average score of ~ 3) demonstrating the potential of StO2 as a visual scoring surrogate (Figure 5).
It should be noted that no statistically significant changes were seen for any of the returned optical biomarkers for the non-irradiated control group (n=3) over the 12 days measured (data not shown). Changes in A and k can also be monitored over time (Figure 6), and these indicate that the scattering properties of the skin are changing in response to the radiation.
<img alt="Figure 1" src="/files/ftp_upload/53573/53573fig1.jpg" /> 
Figure 1. DOS instrumentation. (A) Schematic of the diffuse reflectance measurement geometry. (B) Fiber-optic probe: The optical probe consists of a linear array of 200 &#956;m core optical fibers that are bundled into an 18 G metal needle and spaced 260 &#956;m apart. Two source fibers are coupled to two broadband light emitting diodes while a detection fiber is connected to an optical spectrometer. By sequentially turning on each of the sources, the spectrometer can collect diffuse reflectance at distances of 260 &#956;m and 520 &#956;m from each of the source fibers. (C) Complete DOS system including laptop, attached fiber-optic probe and optic box: An automated data acquisition program is used to drive the sequential collection of spectra. The electronics are housed in an acquisition box that connects to the fiber-optic probe via SMA connectors. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53573/53573fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53573/53573fig2.jpg" /> 
Figure 2. Spectral processing. All x-axis scales are in nm: (A) Raw relative spectra, the baseline is the reading approximately between 900 - 1,000 nm and approximately equal to the background signal. (B) Relative background reading. (C) Background and baseline subtracted relative spectra. (D) Absolutely calibrated spectrum following scaling of processed spectra shown in (C). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53573/53573fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53573/53573fig3.jpg" /> 
Figure 3. Typical white light reflectance spectra of non-irradiated (left) and irradiated (right) mouse skin 6 days post irradiation. Excellent agreement between measurement (noisy blue) and fits (solid green) were typically observed. Two key differences were seen between the two groups: 1) an overall increase in absolute reflectance and 2) a distinct change in spectral shape between 550 - 600 nm. With permission from Yohan et al. 20145. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53573/53573fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/53573/53573fig4.jpg" /> 
Figure 4. Change in the oxygenation fraction of mouse skin following 40 Gy irradiation. The baseline-normalized mean difference between the two groups (per mouse) is significant for Days 6 (Box 1), 9 (Box 2) and 12 (Box 3). With permission from Yohan et al. 20145. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53573/53573fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/53573/53573fig5.jpg" /> 
Figure 5. Average qualitative skin reactions scores (n=8) as a function of days following 40 Gy irradiated mice skin. Adapted from Yohan et al. 20145. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53573/53573fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/53573/53573fig6.jpg" /> 
Figure 6. Relative changes in A and k of mouse skin following 40 Gy irradiation on Days 6 (Box 1), 9 (Box 2) and 12 (Box 3). The change in A (left side) and k (right side) on Day 6 (Box 1, left and right sides) was found to be significant (p &#60; 0.026). With permission from Yohan et al. 20145. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53573/53573fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Diffuse Optical Spectroscopy for the Quantitative Assessment of Acute Ionizing Radiation Induced Skin Toxicity Using a Mouse Model at JoVE.com

Diffuse Optical Spectroscopy for the Quantitative Assessment of Acute Ionizing Radiation Induced Skin Toxicity Using a Mouse Model

We present a diffuse optical spectroscopic (DOS) approach that provides quantitative optical biomarkers of skin response to radiation. We describe DOS instrumentation design, optical parameters extraction algorithms and the animal handling procedures required to yield representative data from a pre-clinical mouse model of radiation induced erythema.

Acute skin toxicities from ionizing radiation (IR) are a common side effect from therapeutic courses of external beam radiation therapy (RT) and ...

diffuse-optical-spectroscopy-for-quantitative-assessment-acute

Nanyang Technological University

Research

JoVE Journal

Medicine

8.0K Views.  University of Toronto. We present a diffuse optical spectroscopic (DOS) approach that provides quantitative optical biomarkers of skin response to radiation. We describe DOS instrumentation design, optical parameters extraction algorithms and the animal handling procedures required to yield representative data from a pre-clinical mouse model of radiation induced erythema.

Video: Diffuse Optical Spectroscopy for the Quantitative Assessment of Acute Ionizing Radiation Induced Skin Toxicity Using a Mouse Model

Quantitative Visualization and Detection of Skin Cancer Using Dynamic Thermal Imaging

In 2010 approximately 68,720 melanomas will be diagnosed in the US alone, with around 8,650 resulting in death 1. To date, the only effective treatment for melanoma remains surgical excision, therefore, the key to extended survival is early detection 2,3. Considering the large numbers of patients diagnosed every year and the limitations in accessing specialized care quickly, the development of objective in vivo diagnostic instruments to aid the diagnosis is essential. New techniques to detect skin cancer, especially non-invasive diagnostic tools, are being explored in numerous laboratories. Along with the surgical methods, techniques such as digital photography, dermoscopy, multispectral imaging systems (MelaFind), laser-based systems (confocal scanning laser microscopy, laser doppler perfusion imaging, optical coherence tomography), ultrasound, magnetic resonance imaging, are being tested. Each technique offers unique advantages and disadvantages, many of which pose a compromise between effectiveness and accuracy versus ease of use and cost considerations. Details about these techniques and comparisons are available in the literature 4.
Infrared (IR) imaging was shown to be a useful method to diagnose the signs of certain diseases by measuring the local skin temperature. There is a large body of evidence showing that disease or deviation from normal functioning are accompanied by changes of the temperature of the body, which again affect the temperature of the skin 5,6. Accurate data about the temperature of the human body and skin can provide a wealth of information on the processes responsible for heat generation and thermoregulation, in particular the deviation from normal conditions, often caused by disease. However, IR imaging has not been widely recognized in medicine due to the premature use of the technology 7,8 several decades ago, when temperature measurement accuracy and the spatial resolution were inadequate and sophisticated image processing tools were unavailable. This situation changed dramatically in the late 1990s-2000s. Advances in IR instrumentation, implementation of digital image processing algorithms and dynamic IR imaging, which enables scientists to analyze not only the spatial, but also the temporal thermal behavior of the skin 9, allowed breakthroughs in the field.
In our research, we explore the feasibility of IR imaging, combined with theoretical and experimental studies, as a cost effective, non-invasive, in vivo optical measurement technique for tumor detection, with emphasis on the screening and early detection of melanoma 10-13. In this study, we show data obtained in a patient study in which patients that possess a pigmented lesion with a clinical indication for biopsy are selected for imaging. We compared the difference in thermal responses between healthy and malignant tissue and compared our data with biopsy results. We concluded that the increased metabolic activity of the melanoma lesion can be detected by dynamic infrared imaging.

We demonstrated that malignant pigmented lesions with increased metabolic activity generate quantifiable amounts of heat and the measurement of the transient thermal response of the skin to a cooling excitation allows quantitative identification of melanoma and other skin cancers (vs. non-proliferative nevi) at an early stage of the disease.

In 2010 approximately 68,720 melanomas will be diagnosed in the US alone, with around 8,650 resulting in death 1. To date, the only effective treatment ...

Ischemic Tissue Injury in the Dorsal Skinfold Chamber of the Mouse: A Skin Flap Model to Investigate Acute Persistent Ischemia

Despite profound expertise and advanced surgical techniques, ischemia-induced complications ranging from wound breakdown to extensive tissue necrosis are still occurring, particularly in reconstructive flap surgery. Multiple experimental flap models have been developed to analyze underlying causes and mechanisms and to investigate treatment strategies to prevent ischemic complications. The limiting factor of most models is the lacking possibility to directly and repetitively visualize microvascular architecture and hemodynamics. The goal of the protocol was to present a well-established mouse model affiliating these before mentioned lacking elements. Harder et al. have developed a model of a musculocutaneous flap with a random perfusion pattern that undergoes acute persistent ischemia and results in ~50% necrosis after 10 days if kept untreated. With the aid of intravital epi-fluorescence microscopy, this chamber model allows repetitive visualization of morphology and hemodynamics in different regions of interest over time. Associated processes such as apoptosis, inflammation, microvascular leakage and angiogenesis can be investigated and correlated to immunohistochemical and molecular protein assays. To date, the model has proven feasibility and reproducibility in several published experimental studies investigating the effect of pre-, peri- and postconditioning of ischemically challenged tissue.

The window of the murine dorsal skinfold chamber presented visualizes a zone of acute persistent ischemia of a musculocutaneous flap. Intravital epi-fluorescence microscopy permits for direct and repetitive assessment of the microvasculature and quantification of hemodynamics. Morphologic and hemodynamic results can further be correlated with histological and molecular analyses.

Despite profound expertise and advanced surgical techniques, ischemia-induced complications ranging from wound breakdown to extensive tissue necrosis are ...

A Mouse Fetal Skin Model of Scarless Wound Repair

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs across species but is age dependent. The transition from a scarless to scarring phenotype occurs in the third trimester of pregnancy in humans and around embryonic day 18 (E18) in mice. However, this varies with the size of the wound with larger defects generating a scar at an earlier gestational age. The emergence of lineage tracing and other genetic tools in the mouse has opened promising new avenues for investigation of fetal scarless wound healing. However, given the inherently high rates of morbidity and premature uterine contraction associated with fetal surgery, investigations of fetal scarless wound healing in vivo require a precise and reproducible surgical model. Here we detail a reliable model of fetal scarless wound healing in the dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

During mammalian development, early gestational skin wounds heal without a scar. Here we detail a reliable and reproducible model of fetal scarless wound healing in the cutaneous dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs ...

Scleral Cross-linking Using Riboflavin and Ultraviolet-A Radiation for Prevention of Axial Myopia in a Rabbit Model

Myopic individuals, especially those with severe myopia, are at higher-than-normal risk of cataract, glaucoma, retinal detachment and chorioretinal abnormalities. In addition, pathological myopia is a common irreversible cause of visual impairment and blindness1-3. Our study demonstrates the effect of scleral crosslinking using riboflavin and ultraviolet-A radiation on the development of axial myopia in a rabbit model. The axial length of the eyeball was measured by A-scan ultrasound in New Zealand white rabbits aged 13 days (male and female). The eye then underwent 360&#176; conjunctival peritomy with scleral crosslinking, followed by tarsorrhaphy. Axial elongation was induced in 13 day-old New Zealand rabbits by suturing their right eye eyelids (tarsorrhaphy). The eyes were divided into quadrants, and every quadrant had two scleral irradiation zones, each with an area of 0.2 cm&#178; and a radius of 4 mm. Crosslinking was performed by dropping 0.1% dextran-free riboflavin-5-phosphate onto the irradiation zones 20 sec before ultraviolet-A irradiation and every 20 sec during the 200 sec irradiation time. UVA radiation (370 nm) was applied perpendicular to the sclera at 57 mW/cm&#178; (total UVA light dose, 57 J/cm&#178;). Tarsorrhaphies were removed on day 55, followed by repeated axial length measurements. This study demonstrates that scleral crosslinking with riboflavin and ultraviolet-A radiation effectively prevents occlusion-induced axial elongation in a rabbit model.

We demonstrate the effect of scleral crosslinking with riboflavin and UVA on an axial elongation rabbit eye. Axial elongation was induced in 13 day-old New Zealand rabbits (male and female) by suturing their right eye eyelids (tarsorrhaphy).

Myopic individuals, especially those with severe myopia, are at higher-than-normal risk of cataract, glaucoma, retinal detachment and chorioretinal ...

A Neonatal Mouse Spinal Cord Compression Injury Model

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal cord. A major current area of research is focused on the mechanisms of adaptive plasticity that underlie spontaneous or induced functional recovery following SCI. Spontaneous functional recovery is reported to be greater early in life, raising interesting questions about how adaptive plasticity changes as the spinal cord develops. To facilitate investigation of this dynamic, we have developed a SCI model in the neonatal mouse. The model has relevance for pediatric SCI, which is too little studied. Because neural plasticity in the adult involves some of the same mechanisms as neural plasticity in early life1, this model may potentially have some relevance also for adult SCI. Here we describe the entire procedure for generating a reproducible spinal cord compression (SCC) injury in the neonatal mouse as early as postnatal (P) day 1. SCC is achieved by performing a laminectomy at a given spinal level (here described at thoracic levels 9-11) and then using a modified Yasargil aneurysm mini-clip to rapidly compress and decompress the spinal cord. As previously described, the injured neonatal mice can be tested for behavioral deficits or sacrificed for ex vivo physiological analysis of synaptic connectivity using electrophysiological and high-throughput optical recording techniques1. Earlier and ongoing studies using behavioral and physiological assessment have demonstrated a dramatic, acute impairment of hindlimb motility followed by a complete functional recovery within 2 weeks, and the first evidence of changes in functional circuitry at the level of identified descending synaptic connections1.

This article describes a method for generating a reproducible spinal cord compression injury (SCI) in the neonatal mouse. The model provides an advantageous platform for studying mechanisms of adaptive plasticity that underlie spontaneous functional recovery.

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal ...

A Mouse Model for Laser-induced Choroidal Neovascularization

The mouse laser-induced choroidal neovascularization (CNV) model has been a crucial mainstay model for neovascular age-related macular degeneration (AMD) research. By administering targeted laser injury to the RPE and Bruch&#8217;s membrane, the procedure induces angiogenesis, modeling the hallmark pathology observed in neovascular AMD. 
First developed in non-human primates, the laser-induced CNV model has come to be implemented into many other species, the most recent of which being the mouse. Mouse experiments are advantageously more cost-effective, experiments can be executed on a much faster timeline, and they allow the use of various transgenic models. The miniature size of the mouse eye, however, poses a particular challenge when performing the procedure. Manipulation of the eye to visualize the retina requires practice of fine dexterity skills as well as simultaneous hand-eye-foot coordination to operate the laser. However, once mastered, the model can be applied to study many aspects of neovascular AMD such as molecular mechanisms, the effect of genetic manipulations, and drug treatment effects. 
The laser-induced CNV model, though useful, is not a perfect model of the disease. The wild-type mouse eye is otherwise healthy, and the chorio-retinal environment does not mimic the pathologic changes in human AMD. Furthermore, injury-induced angiogenesis does not reflect the same pathways as angiogenesis occurring in an age-related and chronic disease state as in AMD.
Despite its shortcomings, the laser-induced CNV model is one of the best methods currently available to study the debilitating pathology of neovascular AMD. Its implementation has led to a deeper understanding of the pathogenesis of AMD, as well as contributing to the development of many of the AMD therapies currently available.

Here, we present the mouse laser-induced choroidal neovascularization (CNV) protocol, an experimental model that re-creates the vascular hallmarks of neovascular age-related macular degeneration (AMD). Once mastered, it can reliably and effectively induce CNV as a model system to test various experimental measures.

The mouse laser-induced choroidal neovascularization (CNV) model has been a crucial mainstay model for neovascular age-related macular degeneration (AMD) ...

Primary Outcome Assessment in a Pig Model of Acute Myocardial Infarction

Mortality after acute myocardial infarction remains substantial and is associated with significant morbidity, like heart failure. Novel therapeutics are therefore required to confine cardiac damage, promote survival and reduce the disease burden of heart failure. Large animal experiments are an essential part in the translational process from experimental to clinical therapies. To optimize clinical translation, robust and representative outcome measures are mandatory. The present manuscript aims to address this need by describing the assessment of three clinically relevant outcome modalities in a pig acute myocardial infarction (AMI) model: infarct size in relation to area at risk (IS/AAR) staining, 3-dimensional transesophageal echocardiography (TEE) and admittance-based pressure-volume (PV) loops. Infarct size is the main determinant driving the transition from AMI to heart failure and can be quantified by IS/AAR staining. Echocardiography is a reliable and robust tool in the assessment of global and regional cardiac function in clinical cardiology. Here, a method for three-dimensional transesophageal echocardiography (3D-TEE) in pigs is provided. Extensive insight into cardiac performance can be obtained by admittance-based pressure-volume (PV) loops, including intrinsic parameters of myocardial function that are pre- and afterload independent. Combined with a clinically feasible experimental study protocol, these outcome measures provide researchers with essential information to determine whether novel therapeutic strategies could yield promising targets for future testing in clinical studies.

Reliable and accurate outcome assessment is the key for translation of preclinical therapies into clinical treatment. The current paper describes how to assess three clinically relevant primary outcome parameters of cardiac performance and damage in a pig acute myocardial infarction model.

Mortality after acute myocardial infarction remains substantial and is associated with significant morbidity, like heart failure. Novel therapeutics are ...

Quantitative Visualization of Leukocyte Infiltrate in a Murine Model of Fulminant Myocarditis by Light Sheet Microscopy

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently labelled structures in large biological specimens, and is down to cellular resolution. Meanwhile, the constant refinement of underlying protocols and the enhanced availability of specialized commercial systems enable us to investigate the microstructure of whole mouse organs and even allow for the characterization of cellular behavior in various live-cell imaging approaches. Here, we describe a protocol for the spatial whole-mount visualization and quantification of the CD45+ leukocyte population in inflamed mouse hearts. The method employs a transgenic mouse strain (CD11c.DTR)that has recently been shown to serve as a robust, inducible model for the study of the development of fulminant fatal myocarditis, characterized by lethal cardiac arrhythmias. This protocol includes myocarditis induction, intravital antibody-mediated cell staining, organ preparation, and LSFM with subsequent computer-assisted image post-processing. Although presented as a highly-adapted method for our particular scientific question, the protocol represents the blueprint of an easily adjustable system that can also target completely different fluorescent structures in other organs and even in other species.

Here, we describe a light-sheet microscopy approach to visualize the cardiac CD45+ leukocyte infiltrate in a murine model of aseptic fulminant myocarditis, which is induced by the intratracheal diphtheria toxin treatment of CD11c.DTR mice.

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently ...

Quantitative Micro-CT Analysis of Aortopathy in a Mouse Model of &#946;-aminopropionitrile-induced Aortic Aneurysm and Dissection

Aortic aneurysm and dissection is associated with significant morbidity and mortality in the population and can be highly lethal. While animal models of aortic disease exist, in vivo imaging of the vasculature has been limited. In recent years, micro-computerized tomography (micro-CT) has emerged as a preferred modality for imaging both large and small vessels both in vivo and ex vivo. In conjunction with a method of vascular casting, we have successfully used micro-CT to characterize the frequency and distribution of aortic pathology in &#946;-aminopropionitrile-treated C57/Bl6 mice. Technical limitations of this method include variations in the quality of the perfusion introduced by poor animal preparation, the application of proper methodologies for vessel size quantification, and the non-survivability of this procedure. This article details a methodology for the intravascular perfusion of a lead-based radiopaque silicone rubber for the quantitative characterization of aortopathy in a mouse model of aneurysm and dissection. In addition to visualizing aortic pathology, this method may be used for examining other vascular beds in vivo or vascular beds removed post-mortem.

This article describes a detailed methodology of using a radiopaque lead-based silicone rubber to perfuse the murine vasculature for aortic diameter quantification in a mouse model of aortic aneurysm and dissection.

Aortic aneurysm and dissection is associated with significant morbidity and mortality in the population and can be highly lethal. While animal models of ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...