We would like to extend our gratitude to the Link&#246;ping staff of The Swedish Defense Agency (FOI) and the Center for disaster medicine and Traumatology (KMC) for their kind support.

The study described here followed the local ethic guidelines and was approved by the regional ethical review board in Link&#246;ping (permit number 2015/99-31).
1. Informed Consent and Screening
<ol>
	<li>Obtain informed consent from the subject.</li>
	<li>Screen according to inclusion/exclusion criteria. 
	NOTE: Inclusion criteria were: (i) Healthy adult &#62;18 years of age, (ii) Able to understand written and oral information, and (iii) Provides oral and written consent. Exclusion criteria were: (i) Unable or unwilling to provide informed consent, (ii) Ongoing skin conditions, (iii) Cardiovascular disease,&#160;(iv) Medication that can interfere with vascular function. Oral contraceptives are allowed. If the subject uses oral contraceptives this should be noted on the subject&#39;s protocol file,&#160;(v) Scar free and non-bruised skin of the forehead,&#160;(vi) No intake of caffeine or tobacco 2 h prior to the onset of the test, and&#160;(vii) No strenuous physical activity at least 2 h prior to onset of the test.</li>
</ol>
2. Acclimatization and Equipment Setup
<ol>
	<li>Let the test subject acclimatize for at least 20 min in a supine position prior to the onset of the test. 
	NOTE: Correct positioning of the subject and equipment is shown in supplemental figure 1.</li>
	<li>Set up the camera recording of the capillary refill tests using the designated software and digital camera (see Table of Materials).
	<ol>
		<li>Connect the USB cable to the camera and the computer and adjust camera settings to video mode.</li>
		<li>Turn on the camera and make sure that it connects to the computer.</li>
		<li>Start the remote-controlled camera software (see Table of Materials).</li>
		<li>Turn off ambient light and turn the computer screen away from the measurement area.</li>
	</ol>
	</li>
</ol>
3. Data Acquisition
<ol>
	<li>Click the &#34;life view&#34; (Sic) button and press &#34;Start&#34;.</li>
	<li>Switch between &#39;video&#39; mode and &#39;TiVi&#39; mode by clicking the radio buttons (this will not affect the recording, only the on-screen presentation). Ensure that a live video stream of the subject&#39;s forehead is visible on the display of the camera and on the computer screen.</li>
	<li>Adjust the camera to a height of approximately 15 cm directly above the subject&#39;s forehead.</li>
	<li>Make sure that the focus of the camera is set to &#34;AF&#34; (auto focus).</li>
	<li>Ask the subject to keep his/her head still for the duration of the test and to refrain from talking. Inform the subject that it is OK to keep his/her eyes closed during the test by saying: &#34;Please, place your head on this pillow and keep your head still for the duration of the test. Please, do not talk during the 20 seconds that the test will last. You may keep your eyes closed for the whole time if you find the light too bright&#34;.</li>
	<li>Do not move the camera while recording.</li>
	<li>In the &#34;life view&#34; window find and click &#34;HD record&#34; to start a continuous recording for 20 s. Note that the duration of the recording (seconds), can be seen on the camera display; the first 5 s of the recording is to acquire the normal capillary blood concentration of the forehead.</li>
	<li>At the end of the 5 s of baseline recording, apply a firm blanching pressure to the measurement area for 5 s. Use the index finger with a plastic teaspoon between the finger and the forehead for pressure equalization and temperature insulation.</li>
	<li>Release the pressure and quickly withdraw the examiner&#39;s finger and plastic teaspoon from the measurement area.</li>
	<li>Continue recording for another 10 s to capture the subsequent hyperemic response.</li>
	<li>Stop recording by clicking &#34;Stop&#34; in the &#34;life view&#34; window.</li>
	<li>Once the recording has stopped, a window will open for saving and naming the file (in .mov format); choose a folder and name for the video. Do not disconnect the camera from the computer while the video is downloading to the designated folder on the computer.</li>
</ol>
4. Data Analysis
<ol>
	<li>Proceed to performing image analysis and curve construction with a dedicated analysis software (see Table of Materials).</li>
	<li>Start the analysis software by double-clicking the desktop icon.</li>
	<li>Once the software has started, on the main screen, select &#34;Movie page.&#34;</li>
	<li>In the &#34;Movie page&#34; window, click &#34;Load movie.&#34;&#160;Wait for the software to upload and analyze the movie. This may take up to a couple of minutes depending on the size of the video file.</li>
	<li>Once the upload and analysis of the movie is finished, click &#34;Save as images&#34; and wait for the software to analyze the video. 
	NOTE: This will automatically create a new folder on the computer containing the video sequence divided into individual jpeg images.</li>
	<li>Once the division of the video into individual jpeg images is finished, find and select &#34;Crop images page&#34; in the main screen window.</li>
	<li>In the &#34;Crop images page&#34;, click &#34;First Photo&#34; and navigate to the folder that contains the jpeg photos from the video and select the first photo to be analyzed; the software will automatically select the following photos of that batch.</li>
	<li>Click the &#34;Edit&#34; menu and choose between circular or rectangular markers for the region of interest.</li>
	<li>In the &#34;Select Photos&#34; window, find the &#34;Actual photo&#34; box and type in the name of the first photo in the batch where the investigator&#39;s finger is fully retracted from the measurement area. To find this photo, look through the folder containing the individual photos from the video.</li>
	<li>Select a region of interest by clicking and holding the left mouse button and draw a circle or rectangle in the photo window that is within the borders of the blanched area. The selected region of interest is automatically applied to all photos of the batch.</li>
	<li>Click the &#34;File&#34; menu and choose &#34;Save ROI&#34; to save the location of the selected region of interest for future reference.</li>
	<li>Click the &#34;Curve tracker&#34; button and wait for the software to analyze the region of interest.</li>
	<li>Once the curve tracker window opens, the change in red blood cell concentration for the selected region of interest for the duration of the test is displayed as a curve. The on-screen presentation can be changed by clicking the different radio buttons. This will not affect the raw data.</li>
	<li>Click &#34;Export data&#34; and save the raw data transcript for further analysis.</li>
	<li>In the raw data transcript, navigate to the column named &#34;Mean intensity TiVi values.&#34;&#160;Generate three new columns next to this column for &#34;image number,&#34;&#160;&#34;total time,&#34;&#160;and &#34;capillary refill time&#34; starting at 1 s, 0 s, and 0 s and with an incrementation of 1 s, 0.02 s, and 0.02 s, respectively. 
	NOTE: &#34;Capillary refill time&#34; correlates to the time of the subsequent reaction to the release of the blanching pressure and starts at the mean value that represents the first photo where the investigator&#39;s finger is fully retracted from the measurement area.</li>
	<li>Delete the values between baseline and capillary refill time, as these only contain noise generated by the investigator&#39;s index finger and plastic teaspoon during the blanching maneuver.</li>
	<li>Calculate a mean value for the baseline measurement (first 5 s of the recording).</li>
	<li>Find the first value that equals or surpasses the calculated mean baseline value and note the time in the &#34;Capillary refill time&#34; column. This time point represents the &#34;time to return to baseline 1.&#34;</li>
	<li>Find the highest mean intensity value of the values generated after pressure release and note the time point in the &#34;Capillary refill time&#34; column. This time point represents the &#34;Time to peak.&#34;</li>
	<li>Find the second value that is less than or equal to the calculated mean value for the baseline measurement and note the time point in the &#34;Capillary refill time&#34; column. This time point represents the &#34;time to return to baseline 2.&#34;</li>
</ol>

<ol>
	<li>Strehlow, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+identification+of+shock+in+critically+ill+patients.">Early identification of shock in critically ill patients.</a> Emerg Med Clin North Am. 28 (1), 57-66 (2010).</li><li>Advanced Life Support, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Acute Medical Emergencies. , 67-84 (2010).</li><li>Brasel, K. J., Guse, C., Gentilello, L. M., Nirula, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+rate:+is+it+truly+a+vital+sign?.">Heart rate: is it truly a vital sign?.</a> J Trauma. 62 (4), 812-817 (2007).</li><li>De Backer, D., Ortiz, J. A., Salgado, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coupling+microcirculation+to+systemic+hemodynamics.">Coupling microcirculation to systemic hemodynamics.</a> Curr Opin Crit Care. 16 (3), 250-254 (2010).</li><li>Mrgan, M., Rytter, D., Brabrand, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capillary+refill+time+is+a+predictor+of+short-term+mortality+for+adult+patients+admitted+to+a+medical+department:+an+observational+cohort+study.">Capillary refill time is a predictor of short-term mortality for adult patients admitted to a medical department: an observational cohort study.</a> Emerg Med J. 31 (12), 954-958 (2014).</li><li>Advanced Life Support, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Acute Medical Emergencies. , 55-65 (2010).</li><li>Lewin, J., Maconochie, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capillary+refill+time+in+adults.">Capillary refill time in adults.</a> Emerg Med J. 25 (6), 325-326 (2008).</li><li>Lobos, A. T., Menon, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multidisciplinary+survey+on+capillary+refill+time:+Inconsistent+performance+and+interpretation+of+a+common+clinical+test.">A multidisciplinary survey on capillary refill time: Inconsistent performance and interpretation of a common clinical test.</a> Pediatr Crit Care Med. 9 (4), 386-391 (2008).</li><li>Otieno, H., 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=Are+bedside+features+of+shock+reproducible+between+different+observers?.">Are bedside features of shock reproducible between different observers?.</a> Arch Dis Child. 89 (10), 977-979 (2004).</li><li>Raju, N. V., Maisels, M. J., Kring, E., Schwarz-Warner, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capillary+refill+time+in+the+hands+and+feet+of+normal+newborn+infants.">Capillary refill time in the hands and feet of normal newborn infants.</a> Clin Pediatr (Phila). 38 (3), 139-144 (1999).</li><li>Klupp, N. L., Keenan, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+evaluation+of+the+reliability+and+validity+of+capillary+refill+time+test.">An evaluation of the reliability and validity of capillary refill time test.</a> The Foot. 17, 15-20 (2007).</li><li>Gorelick, M. H., Shaw, K. N., Baker, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+ambient+temperature+on+capillary+refill+in+healthy+children.">Effect of ambient temperature on capillary refill in healthy children.</a> Pediatrics. 92 (5), 699-702 (1993).</li><li>Schriger, D. L., Baraff, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+normal+capillary+refill:+variation+with+age,+sex,+and+temperature.">Defining normal capillary refill: variation with age, sex, and temperature.</a> Ann Emerg Med. 17 (9), 932-935 (1988).</li><li>Pandey, A., John, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capillary+refill+time.+Is+it+time+to+fill+the+gaps?.">Capillary refill time. Is it time to fill the gaps?.</a> Med J Armed Forces India. 69 (1), 97-98 (2013).</li><li>Strozik, K. S., Pieper, C. H., Roller, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capillary+refilling+time+in+newborn+babies:+normal+values.">Capillary refilling time in newborn babies: normal values.</a> Arch Dis Child Fetal Neonatal Ed. 76 (3), F193-F196 (1997).</li><li>Doherty, J., 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=Sub-epidermal+imaging+using+polarized+light+spectroscopy+for+assessment+of+skin+microcirculation.">Sub-epidermal imaging using polarized light spectroscopy for assessment of skin microcirculation.</a> Skin Res Technol. 13 (4), 472-484 (2007).</li><li>Noguchi, K., 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=Effect+of+caffeine+contained+in+a+cup+of+coffee+on+microvascular+function+in+healthy+subjects.">Effect of caffeine contained in a cup of coffee on microvascular function in healthy subjects.</a> J Pharmacol Sci. 127 (2), 217-222 (2015).</li><li>Reus, W. F., Robson, M. C., Zachary, L., Heggers, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+effects+of+tobacco+smoking+on+blood+flow+in+the+cutaneous+micro-circulation.">Acute effects of tobacco smoking on blood flow in the cutaneous micro-circulation.</a> Br J Plast Surg. 37 (2), 213-215 (1984).</li><li>Lenasi, H., Strucl, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+regular+physical+training+on+cutaneous+microvascular+reactivity.">Effect of regular physical training on cutaneous microvascular reactivity.</a> Med Sci Sports Exerc. 36 (4), 606-612 (2004).</li><li>Anderson, B., Kelly, A. M., Kerr, D., Clooney, M., Jolley, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+patient+and+environmental+factors+on+capillary+refill+time+in+adults.">Impact of patient and environmental factors on capillary refill time in adults.</a> Am J Emerg Med. 26 (1), 62-65 (2008).</li><li>John, R. T., Henricson, J., Nilsson, G. E., Wilhelms, D., Anderson, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reflectance+spectroscopy:+to+shed+new+light+on+the+capillary+refill+test.">Reflectance spectroscopy: to shed new light on the capillary refill test.</a> J Biophotonics. , (2017).</li><li>Blaxter, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+automated+quasi-continuous+capillary+refill+timing+device.">An automated quasi-continuous capillary refill timing device.</a> Physiological measurement. 37, 83-99 (2016).</li><li>Bordoley, A., Irwin, R., Kalyani, V., MacDonald, C., Standish, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> D1GIT: Automated, temperature-calibrated measurement of capillary refill time. , (2012).</li><li>Kviesis-Kipge, E., Curkste, E., Spigulis, J., Eihvalde, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+analysis+of+skin+capillary-refill+processes+using+blue+LED.">Real-time analysis of skin capillary-refill processes using blue LED.</a> Proc. of SPIE. 771523, (2010).</li><li>Shavit, I., Brant, R., Nijssen-Jordan, C., Galbraith, R., Johnson, D. 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+novel+imaging+technique+to+measure+capillary-refill+time:+improving+diagnostic+accuracy+for+dehydration+in+young+children+with+gastroenteritis.">A novel imaging technique to measure capillary-refill time: improving diagnostic accuracy for dehydration in young children with gastroenteritis.</a> Pediatrics. 118 (6), 2402-2408 (2006).</li><li>Cohen, J., 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=Monitor+to+Measure+Dermal+Blood+Flow+in+Critically+Ill+Patients:+Study.">Monitor to Measure Dermal Blood Flow in Critically Ill Patients: Study.</a> International Scholarly Research Notices. 578316, e578316 (2013).</li></ol>

No financial support from WheelsBridge AB was involved in the conduct of the study. The author JH is employed by the &#214;sterg&#246;tland County Council but has a royalty agreement with WheelsBridge AB. The senior author CDA has a full-time academic position but also limited involvement in WheelsBridge AB.

In order to get the best results with the system, variability caused by environmental factors must be controlled. All ambient light must be turned off. The camera must be positioned in vertical alignment with the measurement area. In order to ensure a constant measurement area, subjects should not move or talk during measurement. For the same reason, the camera is preferably mounted on a stand to avoid movement and to maintain a constant distance to the measurement area. Test subjects should avoid caffeine<sup class="xre...

Assessment and triage of the critically ill patient centers on the classical vital signs blood pressure (BP), heart rate (HR), respiratory rate (RR), oxygen saturation, and body temperature1. Changes in these parameters appear relatively late in the course of circulatory deterioration. For instance, in hemorrhage, a decrease in BP will not occur until blood loss becomes moderate to severe2, and HR increase can also be an insensitive and unspecific marker3.
The capillary refill test (CR test) may offer an earlier indication of incipient circulatory collapse, as the refill time is believed to change prior to the vital signs as well as clinical appearance of cold, clammy, and mottled skin1,4,5. The capillary refill test is typically performed by application and then release of a firm blanching pressure to the skin with timing (in seconds) of the return of blood to the blanched area. According to guidelines, refill should occur within 2 seconds after release of 5 seconds of firm pressure (e.g., by the physician&#39;s finger) in the normal healthy supine patient6. The rationale for the test is that a slower refill time would indicate poor skin perfusion, possible caused by one of a number of critical events such as sepsis, blood loss, acute heart failure, or hypothermia.
At present, there is no consensus on a state of the art method for performing the CR test6,7,8,9,10. Contentious issues include lack of standardization of the actual blanching maneuver and the dependence on subjective (i.e., naked eye) assessments of the refill endpoint7,9,11. Furthermore, there are indications that gender influences CR time12,13. The temperature, both ambient and skin, is known to affect the capillary refill time, but to what extent is not clear. Lastly, the use of different measurement sites, peripheral or central, is probably a further cause of variability in results with few studies in this area14,15.
In the present work, we used an optical bioengineering system to record the course of return of blood and the subsequent hyperemic response seen during the CR test. The system utilizes diffuse polarization spectroscopy to quantify and describe, in more detail than possible with the naked eye, the time and course of the capillary refill. The system comprises a standard digital camera, fitted with an external light ring with 92 white LEDs, and specially developed software. The lens and the two polarization filters, attached orthogonally in front of the LEDs, block light that has been directly reflected from the skin surface allowing only light that has become depolarized in the tissue to reach the camera. This generates a &#34;sub-epidermal&#34; image of the tissue to a depth of approximately 0.5 mm. The image is divided into its color planes and the red and green content for every pixel is calculated, generating a value which corresponds to the tissue concentration of red blood cells16. In video mode, the temporal resolution of the system is 0.02 s.

<table>
	<tbody>
		<tr>
			<td>TiVi701 Camera</td>
			<td>WheelsBridge AB</td>
			<td>TiVi701 Camera, version 1.5.1</td>
			<td>Software</td>
		</tr>
		<tr>
			<td>TiVi700</td>
			<td>WheelsBridge AB</td>
			<td>TiVi700 Analysis, version 1.2.9</td>
			<td>Software</td>
		</tr>
		<tr>
			<td>Canon EOS 700D</td>
			<td>Canon U.S.A., inc.</td>
			<td>Canon EOS 700D</td>
			<td>Digital SLR Camera</td>
		</tr>
		<tr>
			<td>Camera stand</td>
			<td>Manfrotto</td>
			<td>681B</td>
			<td>Modified camera stand to hold the digital camera in position</td>
		</tr>
		<tr>
			<td>Camera stand</td>
			<td>Disa Denmark</td>
			<td>9020B</td>
			<td>Modified camera stand to hold the digital camera in position</td>
		</tr>
	</tbody>
</table>

diffuse reflectance spectroscopy: getting the capillary refill test under one's thumb

The capillary refill test was introduced in 1947 to help estimate circulatory status in critically ill patients. Guidelines commonly state that refill should occur within 2 s after releasing 5 s of firm pressure (e.g., by the physician&#39;s finger) in the normal healthy supine patient. A slower refill time indicates poor skin perfusion, which can be caused by conditions including sepsis, blood loss, hypoperfusion, and hypothermia. Since its introduction, the clinical usefulness of the test has been debated. Advocates point out its feasibility and simplicity and claim that it can indicate changes in vascular status earlier than changes in vital signs such as heart rate. Critics, on the other hand, stress that the lack of standardization in how the test is performed and the highly subjective nature of the naked eye assessment, as well as the test&#39;s susceptibility to ambient factors, markedly lowers the clinical value. The aim of the present work is to describe in detail the course of the refill event and to suggest potentially more objective and exact endpoint values for the capillary refill test using diffuse polarization spectroscopy.

Filming the course of the capillary refilling generates vast amounts of data not possible to obtain by naked eye assessment. We suggest here new endpoints to further improve the usability of the CR test as an early indicator of deterioration in circulatory status. We call these endpoints: &#34;Baseline,&#34;&#160;&#34;Blood Zero&#34; (or &#34;BZ&#34;), &#34;Time to Return to Baseline 1&#34; (or &#34;tRtB1&#34;), &#34;Time to Peak&#34; or &#34;Tpk.&#34;&#160;The &#34;Baseline&#34; value is...

Watch this Scientific Journal Video about Diffuse Reflectance Spectroscopy: Getting the Capillary Refill Test Under One's Thumb at JoVE.com

Diffuse Reflectance Spectroscopy: Getting the Capillary Refill Test Under One's Thumb

This protocol describes how the use of diffuse polarization spectroscopy can improve the clinical usefulness of the capillary refill test. We suggest a more detailed analysis of the course of the capillary refill in healthy volunteers using diffuse reflectance spectroscopy videos and new informatic endpoints.

The capillary refill test was introduced in 1947 to help estimate circulatory status in critically ill patients. Guidelines commonly state that refill ...

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9.0K Views.  Region Östergötland. This protocol describes how the use of diffuse polarization spectroscopy can improve the clinical usefulness of the capillary refill test. We suggest a more detailed analysis of the course of the capillary refill in healthy volunteers using diffuse reflectance spectroscopy videos and new informatic endpoints.

Video: Diffuse Reflectance Spectroscopy: Getting the Capillary Refill Test Under One's Thumb

Imaging Odor-Evoked Activities in the Mouse Olfactory Bulb using Optical Reflectance and Autofluorescence Signals

In the brain, sensory stimulation activates distributed populations of neurons among functional modules which participate to the coding of the stimulus. Functional optical imaging techniques are advantageous to visualize the activation of these modules in sensory cortices with high spatial resolution. In this context, endogenous optical signals that arise from molecular mechanisms linked to neuroenergetics are valuable sources of contrast to record spatial maps of sensory stimuli over wide fields in the rodent brain.
Here, we present two techniques based on changes of endogenous optical properties of the brain tissue during activation. First the intrinsic optical signals (IOS) are produced by a local alteration in red light reflectance due to: (i) absorption by changes in blood oxygenation level and blood volume (ii) photon scattering. The use of in vivo IOS to record spatial maps started in the mid 1980's with the observation of optical maps of whisker barrels in the rat and the orientation columns in the cat visual cortex1. IOS imaging of the surface of the rodent main olfactory bulb (OB) in response to odorants was later demonstrated by Larry Katz's group2. The second approach relies on flavoprotein autofluorescence signals (FAS) due to changes in the redox state of these mitochondrial metabolic intermediates. More precisely, the technique is based on the green fluorescence due to oxidized state of flavoproteins when the tissue is excited with blue light. Although such signals were probably among the first fluorescent molecules recorded for the study of brain activity by the pioneer studies of Britton Chances and colleagues3, it was not until recently that they have been used for mapping of brain activation in vivo. FAS imaging was first applied to the somatosensory cortex in rodents in response to hindpaw stimulation by Katsuei Shibuki's group4.
The olfactory system is of central importance for the survival of the vast majority of living species because it allows efficient detection and identification of chemical substances in the environment (food, predators). The OB is the first relay of olfactory information processing in the brain. It receives afferent projections from the olfactory primary sensory neurons that detect volatile odorant molecules. Each sensory neuron expresses only one type of odorant receptor and neurons carrying the same type of receptor send their nerve processes to the same well-defined microregions of &tilde;100&mu;m3 constituted of discrete neuropil, the olfactory glomerulus (Fig. 1). In the last decade, IOS imaging has fostered the functional exploration of the OB5, 6, 7 which has become one of the most studied sensory structures. The mapping of OB activity with FAS imaging has not been performed yet.
Here, we show the successive steps of an efficient protocol for IOS and FAS imaging to map odor-evoked activities in the mouse OB.

This article presents the protocols of intrinsic optical signals and flavoproteins autofluorescence signals imaging to map odor-evoked activities at the surface of the olfactory bulb in mice.

In the brain, sensory stimulation activates distributed populations of neurons among functional modules which participate to the coding of the stimulus. ...

The Mouse Forced Swim Test

The forced swim test is a rodent behavioral test used for evaluation of antidepressant drugs, antidepressant efficacy of new compounds, and experimental manipulations that are aimed at rendering or preventing depressive-like states. Mice are placed in an inescapable transparent tank that is filled with water and their escape related mobility behavior is measured. The forced swim test is straightforward to conduct reliably and it requires minimal specialized equipment. Successful implementation of the forced swim test requires adherence to certain procedural details and minimization of unwarranted stress to the mice. In the protocol description and the accompanying video, we explain how to conduct the mouse version of this test with emphasis on potential pitfalls that may be detrimental to interpretation of results and how to avoid them. Additionally, we explain how the behaviors manifested in the test are assessed.

The forced swim test is validated as an experimental approach to assess potential antidepressant efficacy in rodents. Experimental animals are placed in a tank of water and escape-related mobility behavior is quantified. The common procedures for the mouse version of this test are described.

The forced swim test is a rodent behavioral test used for evaluation of antidepressant drugs, antidepressant efficacy of new compounds, and experimental ...

The Tail Suspension Test

The tail-suspension test is a mouse behavioral test useful in the screening of potential antidepressant drugs, and assessing of other manipulations that are expected to affect depression related behaviors. Mice are suspended by their tails with tape, in such a position that it cannot escape or hold on to nearby surfaces. During this test, typically six minutes in duration, the resulting escape oriented behaviors are quantified. The tail-suspension test is a valuable tool in drug discovery for high-throughput screening of prospective antidepressant compounds. Here, we describe the details required for implementation of this test with additional emphasis on potential problems that may occur and how to avoid them. We also offer a solution to the tail climbing behavior, a common problem that renders this test useless in some mouse strains, such as the widely used C57BL/6. Specifically, we prevent tail climbing behaviors by passing mouse tails through a small plastic cylinder prior to suspension. Finally, we detail how to manually score the behaviors that are manifested in this test.

The tail-suspension test is validated as an experimental procedure to assess antidepressant efficacy of drug treatments in mice. Mice are suspended by their tails for six minutes and escape-related behaviors are assessed. We describe procedures used in conducting the tail suspension test.

The tail-suspension test is a mouse behavioral test useful in the screening of potential antidepressant drugs, and assessing of other manipulations that ...

Laser Nanosurgery of Cerebellar Axons In Vivo

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to understand the mechanism that promotes neuronal regeneration, an in-depth analysis of the neuronal types that can remodel after injury is needed. Several studies showed that damaged climbing fibers are capable of regrowing also in adult animals1,2. The investigation of the time-lapse dynamics of degeneration and regeneration of these axons within their complex environment can be performed by time-lapse two-photon fluorescence (TPF) imaging in vivo3,4. This technique is here combined with laser surgery, which proved to be a highly selective tool to disrupt fluorescent structures in the intact mouse cortex5-9.
This protocol describes how to perform TPF time-lapse imaging and laser nanosurgery of single axonal branches in the cerebellum in vivo. Olivocerebellar neurons are labeled by anterograde tracing with a dextran-conjugated dye and then monitored by TPF imaging through a cranial window. The terminal portion of their axons are then dissected by irradiation with a Ti:Sapphire laser at high power. The degeneration and potential regrowth of the damaged neuron are monitored by TPF in vivo imaging during the days following the injury.

Laser Nanosurgery of Cerebellar Axons In Vivo

Two-photon imaging, coupled to laser nanodissection, are useful tools to study degenerative and regenerative processes in the central nervous system with subcellular resolution. This protocol shows how to label, image, and dissect single climbing fibers in the cerebellar cortex in vivo.

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to ...

The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster

For most animals, feeding is an essential behavior for securing survival, and it influences development, locomotion, health and reproduction. Ingestion of the right type and quantity of food therefore has a major influence on quality of life. Research on feeding behavior focuses on the underlying processes that ensure actual feeding and unravels the role of factors regulating internal energy homeostasis and the neuronal bases of decision-making. The model organism Drosophila melanogaster, with its great variety of genetically traceable tools for labeling and manipulating single neurons, allows mapping of neuronal networks and identification of molecular signaling cascades involved in the regulation of food intake. This report demonstrates the CApillary FEeder assay (CAFE) and shows how to measure food intake in a group of flies for time spans ranging from hours to days. This easy-to-use assay consists of glass capillaries filled with liquid food that flies can freely access and feed on. Food consumption in the assay is accurately determined using simple measurement tools. Herein we describe step-by-step the method from setup to successful execution of the CAFE assay, and provide practical examples to analyze the food intake of a group of flies under controlled conditions. The reader is guided through possible limitations of the assay, and advantages and disadvantages of the method compared to other feeding assays in D. melanogaster are evaluated.

The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster

The CApillary FEeder (CAFE) assay is a simple, budget-friendly, highly reliable method for investigating mechanisms underlying food intake. Used with the highly versatile genetic model organism Drosophila melanogaster, it provides a powerful means of gaining new insights into regulatory mechanisms of food intake.

For most animals, feeding is an essential behavior for securing survival, and it influences development, locomotion, health and reproduction. Ingestion of ...

Simultaneous Evaluation of Cerebral Hemodynamics and Light Scattering Properties of the In Vivo Rat Brain Using Multispectral Diffuse Reflectance Imaging

The simultaneous evaluation of cerebral hemodynamics and the light scattering properties of in vivo rat brain tissue is demonstrated using a conventional multispectral diffuse reflectance imaging system. This system is constructed from a broadband white light source, a motorized filter wheel with a set of narrowband interference filters, a light guide, a collecting lens, a video zoom lens, and a monochromatic charged-coupled device (CCD) camera. An ellipsoidal cranial window is made in the skull bone of a rat under isoflurane anesthesia to capture in vivo multispectral diffuse reflectance images of the cortical surface. Regulation of the fraction of inspired oxygen using a gas mixture device enables the induction of different respiratory states such as normoxia, hyperoxia, and anoxia. A Monte Carlo simulation-based multiple regression analysis for the measured multispectral diffuse reflectance images at nine wavelengths (500, 520, 540, 560, 570, 580, 600, 730, and 760 nm) is then performed to visualize the two-dimensional maps of hemodynamics and the light scattering properties of the in vivo rat brain.

Simultaneous Evaluation of Cerebral Hemodynamics and Light Scattering Properties of the In Vivo Rat Brain Using Multispectral Diffuse Reflectance Imaging

The simultaneous evaluation of cerebral hemodynamics and the light scattering properties of in vivo rat brain tissue is demonstrated using a conventional multispectral diffuse reflectance imaging system.

The simultaneous evaluation of cerebral hemodynamics and the light scattering properties of in vivo rat brain tissue is demonstrated using a conventional ...

Functional Magnetic Resonance Spectroscopy at 7 T in the Rat Barrel Cortex During Whisker Activation

Nuclear magnetic resonance (NMR) spectroscopy offers the opportunity to measure cerebral metabolite contents in vivo and noninvasively. Thanks to technological developments over the last decade and the increase in magnetic field strength, it is now possible to obtain good resolution spectra in vivo in the rat brain. Neuroenergetics (i.e., the study of brain metabolism) and, especially, metabolic interactions between the different cell types have attracted more and more interest in recent years. Among these metabolic interactions, the existence of a lactate shuttle between neurons and astrocytes is still debated. It is, thus, of great interest to perform functional proton magnetic resonance spectroscopy (1H-MRS) in a rat model of brain activation and monitor lactate. However, the methyl lactate peak overlaps lipid resonance peaks and is difficult to quantify. The protocol described below allows metabolic and lactate fluctuations to be monitored in an activated brain area. Cerebral activation is obtained by whisker stimulation and 1H-MRS is performed in the corresponding activated barrel cortex, whose area is detected using blood-oxygen-level-dependent functional magnetic resonance imaging (BOLD fMRI). All steps are fully described: the choice of anesthetics, coils, and sequences, achieving efficient whisker stimulation directly in the magnet, and data processing.

After checking by blood-oxygen-level-dependent functional magnetic resonance imaging (BOLD fMRI) that the corresponding somatosensory barrel field cortex area (called S1BF) is correctly activated, the main goal of this study is to quantify lactate content fluctuations in the activated rat brains by localized proton magnetic resonance spectroscopy (1H-MRS) at 7 T.

Nuclear magnetic resonance (NMR) spectroscopy offers the opportunity to measure cerebral metabolite contents in vivo and noninvasively. Thanks to ...

Ex Vivo Pressurized Hippocampal Capillary-Parenchymal Arteriole Preparation for Functional Study

From subtle behavioral alterations to late-stage dementia, vascular cognitive impairment typically develops following cerebral ischemia. Stroke and cardiac arrest are remarkably sexually dimorphic diseases, and both induce cerebral ischemia. However, progress in understanding the vascular cognitive impairment, and then developing sex-specific treatments, has been partly limited by challenges in investigating the brain microcirculation from mouse models in functional studies. Here, we present an approach to examine the capillary-to-arteriole signaling in an ex vivo hippocampal capillary-parenchymal arteriole (HiCaPA) preparation from mouse brain. We describe how to isolate, cannulate, and pressurize the microcirculation to measure arteriolar diameter in response to capillary stimulation. We show which appropriate functional controls can be used to validate the HiCaPA preparation integrity and display typical results, including testing potassium as a neurovascular coupling agent and the effect of the recently characterized inhibitor of the Kir2 inward rectifying potassium channel family, ML133. Further, we compare the responses in preparations obtained from male and female mice. While these data reflect functional investigations, our approach can also be used in molecular biology, immunochemistry, and electrophysiology studies.

The present manuscript details how to isolate hippocampal arterioles and capillaries from the mouse brain and how to pressurize them for pressure myography, immunofluorescence, biochemistry, and molecular studies.

From subtle behavioral alterations to late-stage dementia, vascular cognitive impairment typically develops following cerebral ischemia. Stroke and ...

Induction of Diffuse Axonal Brain Injury in Rats Based on Rotational Acceleration

Traumatic brain injury (TBI) is a major cause of death and disability. Diffuse axonal injury (DAI) is the predominant mechanism of injury in a large percentage of TBI patients requiring hospitalization. DAI involves widespread axonal damage from shaking, rotation or blast injury, leading to rapid axonal stretch injury and secondary axonal changes that are associated with a long-lasting impact on functional recovery. Historically, experimental models of DAI without focal injury have been difficult to design. Here we validate a simple, reproducible and reliable rodent model of DAI that causes widespread white matter damage without skull fractures or contusions.

This protocol validates a reliable, easy-to-perform and reproducible rodent model of brain diffuse axonal injury (DAI) that induces widespread white matter damage without skull fractures or contusions.

Traumatic brain injury (TBI) is a major cause of death and disability. Diffuse axonal injury (DAI) is the predominant mechanism of injury in a large ...

Culture of Brain Capillary Pericytes for Cytosolic Calcium Measurements and Calcium Imaging Studies

Pericytes are associated with endothelial cells and astrocytic endfeet in a structure known as the neurovascular unit (NVU). Brain capillary pericyte function is not fully known. Pericytes have been suggested to be involved in capillary development, regulation of endothelial barrier tightness and trancytosis activity, regulation of capillary tone and to play crucial roles in certain brain pathologies.
Pericytes are challenging to investigate in the intact brain due to the difficulties in visualizing processes in the brain parenchyma, as well as the close proximity to the other cells of the NVU. The present protocol describes a method for isolation and culture of primary bovine brain capillary pericytes and their following usage in calcium imaging studies, where effects of agonists involved in brain signaling and pathologies can be investigated. Cortical capillary fragments are allowed to attach to the bottom of culture flasks and, after 6 days, endothelial cells and pericytes have grown out from the capillary fragments. The endothelial cells are removed by gentle trypsinization and pericytes are cultured for 5 additional days before passaging.
Isolated pericytes are seeded in 96-well culture plates and loaded with the calcium indicator dye (Fura-2 acetoxymethyl (AM)) to allow for measurements of intracellular calcium levels in a plate reader setup. Alternatively, pericytes are seeded on coverslips and mounted in cell chambers. Following loading with the calcium indicator (Cal-520 AM), calcium live-imaging can be performed using confocal microscopy at an excitation wavelength of 488 nm and emission wavelength of 510-520 nm.
The method described here has been used to obtain the first intracellular calcium measurements from primary brain capillary pericytes, demonstrating that pericytes are stimulated via ATP and are able to contract in vitro.

Brain capillary pericytes are essential players in the regulation of blood-brain barrier properties and blood flow. This protocol describes how brain capillary pericytes can be isolated, cultured, characterized with respect to cell type and applied for investigations of intracellular calcium signaling with fluorescent probes.

Pericytes are associated with endothelial cells and astrocytic endfeet in a structure known as the neurovascular unit (NVU). Brain capillary pericyte ...