Research in the Kawakami lab was supported by NIH grants: R01 AR064418-01A1, R01 HL124283-01, R21 AI 115534-01, and R41AI124734-01.

All animal experiments were approved by the Animal Care and Use Committee of the La Jolla Institute for Allergy and Immunology.
1. Mouse Body Temperature Measurement During Anesthetization
<ol>
	<li>Place the mouse in an anesthesia induction box. Anesthetize by using 1 L/min flow of oxygen with 5% isoflurane. 
	NOTE: Anesthetization is confirmed when the mouse ceases voluntary movement and has been immobile for over 30 s. Alternatively, monitor respiratory rate, and once mice are breathing at 1 breath per every 2 s or longer, anesthetization is confirmed.</li>
	<li>Hold the mouse by the nape of the neck with an index finger and thumb and hold the tail with a pinky finger in order to expose the lower abdomen.</li>
	<li>Place the infrared thermometer sensor below the lower abdomen while holding the mouse with its body parallel to the ground. 
	NOTE: The outer flat surface of the thermometer (not the surface of the sensor) should be approximately 2&#8211;5 mm away from the surface of the abdomen. This replicate results of temperatures measured while mice are not anesthetized (described in section 2). It is important to determine the target site of the abdomen. Aiming between two upper nipples allows for consistent results.</li>
	<li>Hold the trigger to measure the temperature. Ensure the stable holding of the mouse and thermometer.</li>
</ol>
2. Mouse Body Temperature Measurement Without Anesthetic
<ol>
	<li>Pick up the mouse by the middle of the tail.</li>
	<li>Expose the abdomen of the mouse.
	<ol>
		<li>Allow the mouse to hold on to a straight-edge surface, such as the lip of an open cage or cage-top with its forepaws. 
		NOTE: This allows the mouse to stretch its upper body and expose the abdomen.</li>
		<li>Alternatively, allow the mouse to hold on to the upper straight-edge of the thermometer, and make the mouse sit on the outer flat surface of the thermometer with its abdomen just over the infrared sensor. 
		NOTE: Any time the hind paws are resting on the surface of the thermometer, ensure that the feet are not obstructing the sensor from the abdominal surface. Thermometers with foot obstruction will measure a temperature lower than that of the abdomen.</li>
	</ol>
	</li>
	<li>Hold the trigger to measure the temperature. 
	NOTE: Mice tend to move; take care in measuring the same location of the body consistently by taking temperature measurements when mice are relatively less mobile.</li>
</ol>
3. Passive Systemic Anaphylaxis7
<ol>
	<li>Day 0: Sensitize with immunoglobulin E (IgE).
	<ol>
		<li>Prepare 200 &#181;L (per mouse) of anti-dinitrophenyl (DNP) IgE at a concentration of 100 &#181;g/mL in PBS.</li>
		<li>Intraperitoneally inject 200 &#181;L of anti-dinitrophenyl (DNP) IgE prepared in step 3.1.1 or PBS only (PBS-only is the negative control). Use a 26 G needle for injection. Perform the injections just lateral to the midline, approximately between the two most inferior nipples.</li>
	</ol>
	</li>
	<li>Day 1: Induce anaphylaxis with DNP-HSA.
	<ol>
		<li>Prepare 100 &#181;L (per mouse) of DNP-HSA at a concentration of 10 mg/mL in 0.9% NaCl.</li>
		<li>Anesthetize the mice as described in step 1.1.</li>
		<li>Measure the body temperature using the technique described in step 1.</li>
		<li>Intravenously inject 100 &#181;L of DNP-HSA prepared in step 3.2.1. Use a 30 G needle for injection. Perform the injections on the retroorbital venous sinus. Insert the needle on the medial side of the eye at a shallow angle, aiming behind the eye.</li>
		<li>After injection, place the mice in individual cages. Ensure that the mice recover from anesthesia. Observe that they awaken and become voluntarily mobile.</li>
		<li>Measure body temperatures using the infrared thermometer and observe their activity every 10 min for 70 min.</li>
	</ol>
	</li>
</ol>
4. Mouse Model of Food Allergy8,9
NOTE: The schematic is shown in Figure 2.
<ol>
	<li>Day 0: Sensitize the mice with OVA/alum.
	<ol>
		<li>Prepare 100 &#181;L (per mouse) of OVA (0.5 mg/mL) and alum (10 mg/mL) together in PBS. Vortex on the low setting for 30 min.</li>
		<li>Anesthetize the mice as described in step 1.1.</li>
		<li>Using a 26 G needle, intraperitoneally inject each mouse with 100 &#181;L of the OVA/alum mixture prepared in step 4.1.1. 
		&#8203;NOTE: Vortex the OVA/alum mixture again, briefly prior to each injection, to best ensure a homogenized mixture. See step 3.1.3 for details on the injection site.</li>
		<li>After injection, place mice back in their original cages. Ensure that the mice recover from anesthesia. Observe that they awaken and become voluntarily mobile.</li>
	</ol>
	</li>
	<li>Day 14: Perform a second sensitization with OVA/alum.
	<ol>
		<li>Repeat steps 4.1.1, 4.1.2, and 4.1.3.</li>
	</ol>
	</li>
	<li>Days 28-46: Challenge the mice with OVA every other day.
	<ol>
		<li>Prepare 100 &#181;L (per mouse) of OVA at a concentration of 250 mg/mL in PBS. 
		&#8203;NOTE: The mixture should be made by gently rocking back and forth by hand rather than with a vortex to minimize bubble formation.
		<ol>
			<li>If using food allergy inhibitors, prepare the inhibitors9. Prepare 100 &#181;L (per mouse) of inhibitor at a concentration of 1 mg/mL in PBS. This allows a delivery of 100 &#181;g of inhibitor for each mouse. 
			&#8203;NOTE: Do not exceed 100 &#181;L of inhibitor per mouse, as the mice are only able to handle 200 &#181;L of total gavage volume during one challenge day. As 100 &#181;L are necessary for the OVA challenge as well, 100 &#181;L is the maximum recommended volume for inhibitor usage.</li>
		</ol>
		</li>
		<li>Anesthetize the mice as described in step 1.1.</li>
		<li>Orally gavage each mouse with 100 &#181;L of the OVA solution (25 mg of OVA in 100 &#181;L of PBS gavaged per mouse) that was prepared in step 4.3.1.
		<ol>
			<li>If using inhibitors, utilize the same techniques described in the following steps for the gavage of inhibitor (or PBS control) prepared in step 4.3.1.1 30 minutes prior to the OVA challenge. After 30 min, move on to the next step.</li>
			<li>Use a gavage needle with a 1 mL syringe. Take up 100 &#181;L of OVA into the syringe.</li>
			<li>Insert the needle into the mouth; then, pointing the needle toward the left or right side of the throat, gently slide the needle through the esophagus. Inject 100 &#181;L of OVA. 
			NOTE: To ensure that the needle is not in the trachea, observe active breathing before injecting the OVA. The insertion of the needle will also be resisted early if it has entered the trachea rather than the esophagus.</li>
		</ol>
		</li>
		<li>Place the mice into their individual cages. For easy observation of stool quality, use cages without bedding.</li>
		<li>Ensure recovery from anesthesia. Observe that the mice awaken and become voluntarily mobile.</li>
		<li>Measure body temperatures of the mice at timepoints 10, 20, 40, and 60 min using the technique described in section 2.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Finkelman, F. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anaphylaxis:+lessons+from+mouse+models.">Anaphylaxis: lessons from mouse models.</a> The Journal of Allergy and Clinical Immunology. 120 (3), 506-515 (2007).</li><li>Lee, J. K., Vadas, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anaphylaxis:+mechanisms+and+management.">Anaphylaxis: mechanisms and management.</a> Clinical and Experimental Allergy: Journal of the British Society for Allergy and Clinical Immunology. 41 (7), 923-938 (2011).</li><li>Newsom, D. M., Bolgos, G. L., Colby, L., Nemzek, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+body+surface+temperature+measurement+and+conventional+methods+for+measuring+temperature+in+the+mouse.">Comparison of body surface temperature measurement and conventional methods for measuring temperature in the mouse.</a> Contemporary Topics in Laboratory Animal Science. 43 (5), 13-18 (2004).</li><li>Wong, J. P., Saravolac, E. G., Clement, J. G., Nagata, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+murine+hypothermia+model+for+study+of+respiratory+tract+influenza+virus+infection.">Development of a murine hypothermia model for study of respiratory tract influenza virus infection.</a> Laboratory Animal Science. 47 (2), 143-147 (1997).</li><li>Quimby, J. M., Olea-Popelka, F., Lappin, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+digital+rectal+and+microchip+transponder+thermometry+in+cats.">Comparison of digital rectal and microchip transponder thermometry in cats.</a> Journal of American Association for Laboratory Animal Science. 48 (4), 402-404 (2009).</li><li>Mei, J., Riedel, N., Grittner, U., Endres, M., Banneke, S., Emmrich, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Body+temperature+measurement+in+mice+during+acute+illness:+implantable+temperature+transponder+versus+surface+infrared+thermometry.">Body temperature measurement in mice during acute illness: implantable temperature transponder versus surface infrared thermometry.</a> Scientific Reports. 8 (1), 3526 (2018).</li><li>Doyle, E., Trosien, J., Metz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocols+for+the+induction+and+evaluation+of+systemic+anaphylaxis+in+mice.">Protocols for the induction and evaluation of systemic anaphylaxis in mice.</a> Methods in Molecular Biology. 1032, 133-138 (2013).</li><li>Brandt, E. B., 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=Mast+cells+are+required+for+experimental+oral+allergen-induced+diarrhea.">Mast cells are required for experimental oral allergen-induced diarrhea.</a> The Journal of Clinical Investigations. 112 (11), 1666-1677 (2003).</li><li>Ando, T., 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=Histamine-releasing+factor+enhances+food+allergy.">Histamine-releasing factor enhances food allergy.</a> The Journal of Clinical Investigations. 127 (12), 4541-4553 (2017).</li><li>Brandt, E. B., 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=Targeting+IL-4/IL-13+signaling+to+alleviate+oral+allergen-induced+diarrhea.">Targeting IL-4/IL-13 signaling to alleviate oral allergen-induced diarrhea.</a> The Journal of Allergy and Clinical Immunology. 123 (1), 53-58 (2009).</li></ol>

The authors have nothing to disclose.

The protocol described was established with the goal of measuring body temperature without the use of anesthesia. Despite its relative ease with which temperature readings can be obtained, there are several caveats that accommodate this technique, in addition to the more obvious effects such as handling stress and different ambient temperatures.
First, in order to maintain consistent temperature readings throughout the experiment, the location where the temperature is being measured must be predetermined using anatomic landmarks, and investigators must be able to replicate the measurement of said body location. By securing the mouse&#39;s forepaws on a straight-edge surface, such as the lip of a cage or the edge of the thermometer, the abdomen is readily exposed to the infrared sensor of the thermometer. Generally, the abdomen is a large surface area that allows some flexibility for consistent results. Furthermore, the abdomen has sparser hair than the dorsal surface of the mouse, and therefore allows for a more accurate body temperature reading.
Secondly, hind paws must always be monitored when taking a temperature measurement. When the thermometer is close to the abdomen, the mice tend to place their feet on the thermometer itself. When the feet or toes are within the circumference of the sensor, the temperature reading may be lower than that of the abdomen, as demonstrated in Video 4. It is important to allow the mouse to straddle with its feet on either side of the sensor, while keeping the circumference free of any body parts or objects besides the abdominal surface.
Thirdly, the abdominal surfaces of mice cannot be wet when taking the measurements. A wet surface will alter the thermometer&#39;s ability to measure temperature accurately; usually, the measured temperature will be lower than the actual value. This issue can be avoided by keeping the mice in cages with fresh bedding during the course of the observational period. This also ensures that the dryness of the abdominal surface is standardized each time the temperatures are measured.
Lastly, infrared thermometers have a larger margin of error than rectal probes, albeit only slightly. The margin of error for measurements with rectal probes is &#177; 0.1 &#176;C, whereas for infrared thermometers, it is &#177; 0.2 &#176;C. This margin of error was minimal compared to the overall differences in temperature drop between populations in the PSA experiment. More importantly, a project requiring a difference as subtle as &#177; 0.1 &#176;C may also foresee other margin of error issues, regardless of whether an infrared thermometer or rectal probe is used. During the preparation of this protocol, it had also been shown that there is a very good correlation between body core temperatures measured by implantable temperature transponders and body surface temperatures measured by infrared thermometers during lipopolysaccharide-induced hypothermia6.
Temperature drop in the food allergy model used had not been observed previously8,10. The standard for evaluation had previously been to determine the phenotype severity by diarrhea scoring; however, this method lacked objectivity. By using the infrared thermometer, we showed that subtle temperature drops could be detected. We were then able to correlate these with activity levels, as the mice were not anesthetized during the procedure. This method allows for a fast, accurate, easy-to-use, noninvasive, and inexpensive way to measure body temperature in mice while being able to observe other phenotypes that would otherwise be missed if mice are anesthetized&#160;for rectal probe measurements.

Measurement of body temperature has been an essential part of monitoring the effects of anaphylactic symptoms in animal models1,2. Temperature differences have been traditionally measured by rectal probe thermometers in mice3,4. With these measurements, investigators have reliably portrayed differences in temperature among variables; however, this method is a time-consuming procedure and causes distress to mice, which can increase the body core temperature. Rectal probing can also cause mucosal tearing and infection3. Moreover, the mice should be anesthetized in order to humanely insert the rectal probe to measure the temperature3. This is a slow process, and it prohibits the measurement of successive temperatures within a short period of time. Furthermore, mice activity phenotypes cannot be observed during this time until the anesthetic is completely worn off, which is another time-consuming process. More recently, other reliable methods to measure body temperature have used subcutaneously implanted passive infrared transponder tags or radio transmitters that include a temperature sensor3,5,6. Although they are accepted as the ideal practice by some researchers, these methods are not widely used because of high initial costs and distress to mice, due to the surgical implantation of a temperature sensor under the skin or another part of the body.
In order to demonstrate that a temperature difference is an accurate reflection of symptoms in a disease model1,2, mice must be awake during the temperature measurement and be able to return to their normal phenotypic activity immediately before and after the measurement. To this end, we sought a method by which this could be achieved.
Our goal was to accurately and inexpensively measure mouse body temperature, without the need for anesthesia and without restrictions on activity, to enable observation of behavioral phenotypes during and after the time of temperature measurement. To achieve this goal, it was apparent that a technique less invasive than the standard rectal temperature probes was required. Infrared thermometers have been used for decades in clinical medicine, especially in pediatrics, to obtain accurate temperature readings. It has been an alternative method that has allowed clinicians to quickly and accurately obtain temperature measurements in infants and fussy children that are actively mobile. We implemented this same technique in mice and have developed a successful method to obtain temperatures without anesthesia. Importantly, we show that this method is capable of replicating the well-established passive systemic anaphylaxis results regarding temperature changes, while also being able to observe the activity of the mouse throughout the measurement. Furthermore, we use the same method to evaluate body temperatures of food-allergic mice, while simultaneously investigating other symptoms, to demonstrate that body temperature is indeed an accurate reflection of the activity level and overall phenotype of the mouse.

<table><tbody><tr><td>Non-contact infrared thermometer</td><td>SinoPie</td><td>DT-8861</td><td></td></tr><tr><td>Anti-dinitrophenyl (DNP) IgE</td><td>Sigma Aldrich</td><td>D8406-.2MG</td><td></td></tr><tr><td>PrecisionGlide 30 G needle</td><td>BD</td><td>305128</td><td></td></tr><tr><td>PrecisionGlide 26 G needle&amp;nbsp;</td><td>BD</td><td>305111</td><td></td></tr><tr><td>1 mL syringe</td><td>BD</td><td>309659</td><td></td></tr><tr><td>Dinitrophenyl - human serum albumin</td><td>Biosearch Technologies</td><td>D-5059-10</td><td></td></tr><tr><td>Ovalbumin from chicken egg white</td><td>Sigma Aldrich</td><td>A5503-50G</td><td></td></tr><tr><td>Imject Alum</td><td>ThermoFisher Scientific</td><td>77161</td><td></td></tr><tr><td>Animal Feeding Needles, disposable</td><td>Fisher Scientific</td><td>01-208-87</td><td></td></tr></tbody></table>

mouse body temperature measurement using infrared thermometer during passive systemic anaphylaxis and food allergy evaluation

Mouse body temperature measurement is of paramount importance for investigating allergies and anaphylactic symptoms. Rectal probes for temperature readings is common, and they have been proven to be accurate and invaluable in this regard. However, this method of temperature measurement requires the mice to be anesthetized in order to insert the probe without injury to the animal. This limits the ability to observe other phenotypes of the mouse simultaneously. In order to investigate other phenotypes while measuring temperatures, rectal probes are not ideal, and another method is desired. Here, we introduce a noninvasive method of temperature measurement that foregoes the requirement for mouse anesthesia while maintaining equal reliability to rectal probes in measuring body temperature. We use an infrared thermometer that detects body surface temperatures at ranges between 2 and 150 mm. This method of body temperature measurement is successful in reliably replicating temperature change trends during passive system anaphylaxis experiments in mice. We show that body surface temperatures are about 2.0 &#176;C lower than rectal probe measurements, but the degree of temperature drop follows the same trend. Furthermore, we use the same technique to observe mice in a food allergy model to evaluate temperature and activity levels simultaneously.

Passive systemic anaphylaxis: For iv injection, 10 week old female BALB/c mice were anesthetized. Prior to the injection, we measured their body temperatures (Video 1) as described in step 1. Figure 1 shows the temperature trend of both populations after iv injection. The IgE-sensitized mouse showed a maximum temperature drop of 3.0 &#176;C at 20 min, while the PBS control mouse had a maximum drop of 1.1 &#176;C at 20 min7. Furthermore, throughout this process, activity scores were evaluated based on the mobility of each mouse. Mice were first observed without any agitation. Then, they were observed with agitation by introducing the evaluator&#39;s hand into the cage and making soft contact with the mouse. Based on their reactions, the mice are evaluated and scored on a scale of 1 to 3. A score of 1 indicates a mouse that is immobile or minimally mobile with and without agitation; a score of 2 indicates a mouse that is immobile without agitation and is mobile with agitation without making quick evasive movements; and a score of 3 indicates a mouse that is either mobile or immobile without agitation, and mobile with agitation and makes quick evasive movements. Videos 2 and 3 provide examples of the two groups that exemplify the temperature measurement methods as well as the corresponding activity level measurements. Usage of the infrared thermometer allowed for temperature measurements without anesthesia, which in turn allowed for behavioral phenotype evaluation before, during, and after the temperature measurement; in contrast, a traditional rectal probe would require anesthesia and therefore preclude any behavioral evaluation within the several minutes surrounding the temperature measurements.
Mouse model of food allergy8: The schematic of the mouse model is shown in Figure 2.&#160;Mice are female BALB/c mice of ages 8 to 10 weeks old at the time of sensitization. Mice were observed at 10, 20, 40, and 60 min after each challenge for body temperature and activity. A representative result is shown in Figure 3 of the temperature change during challenge 7. The mice showed a maximal temperature drop at 10 min; therefore, the 10 min temperatures were used to correlate with body temperatures at the 10 min activity levels (with scoring based on the criteria depicted above). Activity score and temperature drop showed a statistically significant correlation (Figure 4). As mentioned in the passive systemic anaphylaxis section, behavioral observations were performed due to the lack of anesthesia, and the infrared thermometer was utilized. In contrast, rectal probes would forestall any behavioral observations or evaluations during the time of temperature measurement. Any time that temperature and other activity phenotypes must be recorded, it is essential to utilize a temperature-measuring system that does not require anesthesia.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58391/58391fig1.jpg" /> 
Figure 1: Passive systemic anaphylaxis in BALB/c mice. Two mice were ip injected with anti-DNP IgE on day 0, then iv injected with either normal saline or DNP-HSA on day 1. Body temperature was measured using an infrared thermometer before injection and every 10 min after injection for 70 min. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58391/58391fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58391/58391fig2.jpg" /> 
Figure 2: Food allergy mouse model schematic. On day 0, BALB/c mice were sensitized ip with OVA and alum, and again on day 14. On day 28, 30 min prior to each challenge, mice were orally gavaged with either PBS control or a food allergy inhibitor (HRF-2CArecombinant histamine-releasing factor with its two cysteines exchanged with alanines)9. Mice were then challenged ig with 25 mg of OVA. Challenges were performed every other day for 9 challenges. Mice were observed for body temperature measurement and activity scoring at 10, 20, 40, and 60 min after the challenge. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58391/58391fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58391/58391fig3.jpg" /> 
Figure 3: Food allergy model in BALB/c mice. Temperature change after the 7th OVA challenge was monitored by infrared thermometer. Typical results are shown from at least four independent experiments (**, p &#60;0.01 between PBS and 2CA treatments by Student&#39;s t-test; ns: not significant). Error bars = SEM. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58391/58391fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58391/58391fig4.jpg" /> 
Figure 4: Correlation between activity and temperature at 10 min in food allergy experiments. The results reflect measurements taken from 4 separate sets of experiments. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58391/58391fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Movie Image 1" src="/files/ftp_upload/58391/58391movie1.jpg" /> 
Video 1: Body temperature measurement while mice are anesthetized. Mice body temperature measurements can be taken with or without anesthesia. For consistent measurements, it is essential to always aim the thermometer at the same body location. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58391/IMG_2416.mov" target="_blank">Please click here to view this video. (Right-click to download.)</a>
<img alt="Movie Image 2" src="/files/ftp_upload/58391/58391movie2.jpg" /> 
Video 2: Activity level at 40 min after DNP-HSA iv injection. Mice are observed without agitation initially, then with agitation. Based on their reactions, the mice were evaluated and scored on an activity scale of 1 to 3. The mouse on the left (anti-DNP sensitized mouse) is given a score of 1, while the mouse on the right (PBS control mouse) is given a score of 3. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58391/IMG_2442.mov" target="_blank">Please click here to view this video. (Right-click to download.)</a>
<img alt="Movie Image 3" src="/files/ftp_upload/58391/58391movie3.jpg" /> 
Video 3: Body temperature measurement at 40 min after DNP-HSA iv injection. This figure demonstrates the ability of the infrared thermometer to accurately and noninvasively measure body temperature without anesthetization. Moreover, the activity scores demonstrated in Video 2 reflect the degree of temperature drop measured in this video. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58391/IMG_2446.mov" target="_blank">Please click here to view this video. (Right-click to download.)</a>
<img alt="Movie Image 4" src="/files/ftp_upload/58391/58391movie4.jpg" /> 
Video 4: Errors in temperature reading by hind paw obstruction of the sensor. This video demonstrates the difference in temperatures with and without the foot within sensor circumference. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58391/IMG_2455.mov" target="_blank">Please click here to view this video. (Right-click to download.)</a>

Watch this Scientific Journal Video about Mouse Body Temperature Measurement Using Infrared Thermometer During Passive Systemic Anaphylaxis and Food Allergy Evaluation at JoVE.com

Mouse Body Temperature Measurement Using Infrared Thermometer During Passive Systemic Anaphylaxis and Food Allergy Evaluation

Here we present a new method to accurately measure body temperature differences in passive systemic anaphylaxis (PSA) and food allergy mouse models using an infrared thermometer. This procedure has been accurately duplicated in previous PSA results.

Mouse body temperature measurement is of paramount importance for investigating allergies and anaphylactic symptoms. Rectal probes for temperature ...

mouse-body-temperature-measurement-using-infrared-thermometer-during

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

25.4K Views.  La Jolla Institute for Allergy and Immunology. Here we present a new method to accurately measure body temperature differences in passive systemic anaphylaxis (PSA) and food allergy mouse models using an infrared thermometer. This procedure has been accurately duplicated in previous PSA results.

Video: Mouse Body Temperature Measurement Using Infrared Thermometer During Passive Systemic Anaphylaxis and Food Allergy Evaluation

Murine Model of Allergen Induced Asthma

Asthma is a major cause of morbidity and mortality, affecting some 300 million people throughout the world.1 More than 8% of the US population has asthma, with the prevalence increasing.2 As with other diseases, animal models of allergic airway disease greatly facilitate understanding of the underlying pathophysiology, help identify potential therapeutic targets, and allow preclinical testing of possible new therapies. Models of allergic airway disease have been developed in several animal species, but murine models are particularly attractive due to the low cost, ready availability, and well-characterized immune systems of these animals.3 Availability of a variety of transgenic strains further increases the attractiveness of these models.4 Here we describe two murine models of allergic airway disease, both employing ovalbumin as the antigen. Following initial sensitization by intraperitoneal injection, one model delivers the antigen challenge by nebulization, the other by intratracheal delivery. These two models offer complementary advantages, with each mimicking the major features of human asthma.5
The major features of acute asthma include an exaggerated airway response to stimuli such as methacholine (airway hyperresponsiveness; AHR) and eosinophil-rich airway inflammation. These are also prominent effects of allergen challenge in our murine models,5,6 and we describe techniques for measuring them and thus evaluating the effects of experimental manipulation. Specifically, we describe both invasive7 and non-invasive8 techniques for measuring airway hyperresponsiveness as well as methods for assessing infiltration of inflammatory cells into the airways and the lung. Airway inflammatory cells are collected by bronchoalveolar lavage while lung histopathology is used to assess markers of inflammation throughout the organ. These techniques provide powerful tools for studying asthma in ways that would not be possible in humans.

Experimental mouse models of allergic asthma offer new possibilities for studying disease pathogenesis and developing new therapeutics. These models are well suited to measuring factors governing the allergic immune response, airway inflammation, and pulmonary pathophysiology.

Asthma is a major cause of morbidity and mortality, affecting some 300 million people throughout the world.1 More than 8% of the US population has asthma, ...

Measuring Local Anaphylaxis in Mice

Allergic responses are the result of the activation of mast cells and basophils, and the subsequent release of vasoactive and proinflammatory mediators. Exposure to an allergen in a sensitized individual can result in clinical symptoms that vary from minor erythema to life threatening anaphylaxis. In the laboratory, various animal models have been developed to understand the mechanisms driving allergic responses. Herein, we describe a detailed method for measuring changes in vascular permeability to quantify localized allergic responses. The local anaphylaxis assay was first reported in the 1920s, and has been adapted from the technique published by Kojima et al. in 20071. In this assay, mice sensitized to OVA are challenged in the left ear with vehicle and in the right ear with OVA. This is followed by an intravenous injection of Evans Blue dye. Ten min after injecting Evans Blue, the animal is euthanized and the dye that has extravasated into the ears is extracted overnight in formamide. The absorbance of the extracted dye is then quantified with a spectrophotometer. This method reliably results in a visual and quantifiable manifestation of a local allergic response.

Allergic responses, characterized by the activation of mast cells and basophils, are driven by the cross-linking of IgE and release of proinflammatory mediators. A quantitative assessment of allergic responses can be achieved by using Evans Blue dye to monitor changes in vascular permeability after allergen challenge.

Allergic responses are the result of the activation of mast cells and basophils, and the subsequent release of vasoactive and proinflammatory mediators. ...

Measuring Changes in Tactile Sensitivity in the Hind Paw of Mice Using an Electronic von Frey Apparatus

Measuring inflammation-induced changes in thresholds of hind paw withdrawal from mechanical pressure is a useful technique to assess changes in pain perception in rodents. Withdrawal thresholds can be measured first at baseline and then following drug, venom, injury, allergen,&#160;or otherwise evoked inflammation by applying an accurate force on very specific areas of the skin. An electronic von Frey apparatus allows precise assessment of mouse hind paw withdrawal thresholds that are not limited by the available filament sizes in contrast to classical von Frey measurements. The ease and rapidity of measurements allow for incorporation of assessment of tactile sensitivity outcomes in diverse models of rapid-onset inflammatory and neuropathic pain as multiple measurements can be taken within a short time period. Experimental measurements for individual rodent subjects can be internally controlled against individual baseline responses and exclusion criteria easily established to standardize baseline responses within and across experimental groups. Thus, measurements using an electronic von Frey apparatus represent a useful modification of the well-established classical von Frey filament-based assays for rodent mechanical allodynia that may also be applied to other nonhuman mammalian models.

Electronic von Frey measurements are an effective and improved alternative to classical von Frey filaments, as they allow rapid and precise measurement of changes in rodent mechanical withdrawal thresholds to continuously applied pressure stimuli before and after an inflammatory event.

Measuring inflammation-induced changes in thresholds of hind paw withdrawal from mechanical pressure is a useful technique to assess changes in pain ...

Neuroscience

A Reversible, Non-invasive Method for Airway Resistance Measurements and Bronchoalveolar Lavage Fluid Sampling in Mice

Airway hyperreactivity (AHR) measurements and bronchoalveolar lavage (BAL) fluid sampling are essential to experimental asthma models, but repeated procedures to obtain such measurements in the same animal are generally not feasible.  Here, we demonstrate protocols for obtaining from mice repeated measurements of AHR and bronchoalveolar lavage fluid samples.  Mice were challenged intranasally seven times over 14 days with a potent allergen or sham treated.  Prior to the initial challenge, and within 24 hours following each intranasal challenge, the same animals were anesthetized, orally intubated and mechanically ventilated.  AHR, assessed by comparing dose response curves of respiratory system resistance (RRS) induced by increasing intravenous doses of acetylcholine (Ach) chloride between sham and allergen-challenged animals, were determined.  Afterwards, and via the same intubation, the left lung was lavaged so that differential enumeration of airway cells could be performed.  These studies reveal that repeated measurements of AHR and BAL fluid collection are possible from the same animals and that maximal airway hyperresponsiveness and airway eosinophilia are achieved within 7-10 days of initiating allergen challenge.  This novel technique significantly reduces the number of mice required for longitudinal experimentation and is applicable to diverse rodent species, disease models and airway physiology instruments.  

Repeated measurements of rodent respiratory physiology and sampling of airway inflammatory cells are desirable, but generally not feasible. Here we describe a repeatable method for orally intubating mice that permits repeated measurements of airway hyperreactivity and sampling of airway inflammatory cells.

Airway hyperreactivity (AHR) measurements and bronchoalveolar lavage (BAL) fluid sampling are essential to experimental asthma models, but repeated ...

Biology

Measuring Skeletal Muscle Thermogenesis in Mice and Rats

Skeletal muscle thermogenesis provides a potential avenue for better understanding metabolic homeostasis and the mechanisms underlying energy expenditure. Surprisingly little evidence is available to link the neural, myocellular, and molecular mechanisms of thermogenesis directly to measurable changes in muscle temperature. This paper describes a method in which temperature transponders are utilized to retrieve direct measurements of mouse and rat skeletal muscle temperature.
Remote transponders are surgically implanted within the muscle of mice and rats, and the animals are given time to recover. Mice and rats must then be repeatedly habituated to the testing environment and procedure. Changes in muscle temperature are measured in response to pharmacological or contextual stimuli in the home cage. Muscle temperature can also be measured during prescribed physical activity (i.e., treadmill walking at a constant speed) to factor out changes in activity as contributors to the changes in muscle temperature induced by these stimuli.
This method has been successfully used to elucidate mechanisms underlying muscle thermogenic control at the level of the brain, sympathetic nervous system, and skeletal muscle. Provided are demonstrations of this success using predator odor (PO; ferret odor) as a contextual stimulus and injections of oxytocin (Oxt) as a pharmacological stimulus, where predator odor induces muscle thermogenesis, and Oxt suppresses muscle temperature. Thus, these datasets display the efficacy of this method in detecting rapid changes in muscle temperature.

Mice and rats are surgically implanted with remote temperature transponders and then habituated to the testing environment and procedure. Changes in muscle temperature are measured in response to pharmacological or contextual stimuli in the home cage or during prescribed physical activity (i.e., treadmill walking at a constant speed).

Skeletal muscle thermogenesis provides a potential avenue for better understanding metabolic homeostasis and the mechanisms underlying energy expenditure. ...

Behavior

Thermal Imaging to Study Stress Non-invasively in Unrestrained Birds

Stress, a central concept in biology, describes a suite of emergency responses to challenges. Among other responses, stress leads to a change in blood flow that results in a net influx of blood to key organs and an increase in core temperature. This stress-induced hyperthermia is used to assess stress. However, measuring core temperature is invasive. As blood flow is redirected to the core, the periphery of the body can cool. This paper describes a protocol where peripheral body temperature is measured non-invasively in wild blue tits (Cyanistes caeruleus) using infrared thermography. In the field we created a set-up bringing the birds to an ideal position in front of the camera by using a baited box. The camera takes a short thermal video recording of the undisturbed bird before applying a mild stressor (closing the box and therefore capturing the bird), and the bird&#8217;s response to being trapped is recorded. The bare skin of the eye-region is the warmest area in the image. This allows an automated extraction of the maximum eye-region temperature from each image frame, followed by further steps of manual data filtering removing the most common sources of errors (motion blur, blinking). This protocol provides a time series of eye-region temperature with a fine temporal resolution that allows us to study the dynamics of the stress response non-invasively. Further work needs to demonstrate the usefulness of the method to assess stress, for instance to investigate whether eye-region temperature response is proportional to the strength of the stressor. If this can be confirmed, it will provide a valuable alternative method of stress assessment in animals and will be useful to a wide range of researchers from ecologists, conservation biologists, physiologists to animal welfare researchers.

There is a need for a non-invasive assessment of stress. This paper describes a simple protocol using thermal imaging to detect a significant response in eye-region temperature in wild blue tits to a mild acute stressor.

Stress, a central concept in biology, describes a suite of emergency responses to challenges. Among other responses, stress leads to a change in blood ...

Application of Biochip Microfluidic Technology to Detect Serum Allergen-specific Immunoglobulin E (sIgE)

Allergic disease is common in both adults and children. Identification of the causative allergens is significant in disease management and prevention. However, a specific immunoglobulin E (IgE) measurement system with a high price-performance ratio is lacking in mainland China, especially in the primary care hospitals. This paper describes the principle and operation procedures of using a microfluidic cartridge-based chemiluminescence system to detect allergen-specific IgE in serum. The results were compared with those from ImmunoCAP (System 1), the industrial standard, and the reproducibility of the system to detect patients sensitized to common allergens is evaluated. The results showed that in comparison with ImmunoCAP (System 1), the BioIC System (System 2) has good precision and sensitivity in detecting serum-specific IgE against various inhalant and food allergens but with a significantly lower cost. It can serve as a good alternative to System 1 in primary care hospitals in mainland China who have lower financial affordability.

Application of Biochip Microfluidic Technology to Detect Serum Allergen-specific Immunoglobulin E sIgE

This paper presents a protocol for detecting serum-specific immunoglobulin E with a microfluidic cartridge-based chemiluminescence system and the evaluation of its usefulness in allergy diagnoses.

Allergic disease is common in both adults and children. Identification of the causative allergens is significant in disease management and prevention. ...

Medicine

Basophil Activation Test for Allergy Diagnosis

The basophil activation test (BAT) is a complementary in vitro diagnostic test that can be used in addition to clinical history, skin test (ST), and specific IgE (sIgE) determination in the evaluation of IgE-mediated allergic reactions to food, insect venom, drugs, as well as some forms of chronic urticaria. However, the role of this technique in the diagnostic algorithms is highly variable and not well determined.
BAT is based on the determination of basophil response to allergen/drug cross-linking IgE activation through the measurement of activation markers (such as CD63, CD203c) by flow cytometry. This test can be a useful and complementary tool to avoid controlled challenge tests&#160;to confirm allergy diagnosis, especially in subjects experiencing severe life-threatening reactions. In general, the performance of BAT should be considered if i) the allergen/drug produces false positive results in ST; ii) there is no allergen/drug source to use for ST or sIgE determination; iii) there is discordance between patient history and ST or sIgE determination; iv) symptoms suggest that ST may result in systemic response; v) before considering a CCT to confirm the culprit allergen/drug. The main limitations of the test are related to non-optimal sensitivity, especially in drug allergy, the need to perform the test no longer than 24 h after sample extraction, and the lack of standardization between laboratories in terms of procedures, concentrations, and cell markers.

The basophil activation test is a complementary in vitro diagnostic test for the evaluation of IgE-mediated allergic reactions based on the detection of basophil activation in the presence of a specific stimulus through the measure of activation markers by flow cytometry.

The basophil activation test (BAT) is a complementary in vitro diagnostic test that can be used in addition to clinical history, skin test (ST), and ...

Infrared Thermography for the Detection of Changes in Brown Adipose Tissue Activity

Measuring brown adipose tissue (BAT) activity by positron emission tomography computed tomography (PET-CT) via the accumulation of 18F-fluorodeoxyglucose (FDG) after a meal or in obese or diabetic patients fails as the method of choice. The main reason is that 18F-FDG competes with the postprandial high glucose plasma concentration for the same glucose transporter on the membrane of BAT cells. In addition, BAT uses fatty acids as a source of energy as well, which is not visible with PET-CT and could be changed along with glucose concentration in obese and diabetic patients. Therefore, to estimate the physiological importance of BAT in animals and humans, a new infrared thermography method used in recent publications is applied.
After overnight fasting, BAT activity was measured by infrared thermography before and after a meal in human volunteers and&#160;female wild-type mice. The camera software calculates the object&#39;s temperature using distance from the object, skin emissivity, reflected room temperature, air temperature, and relative humidity. In mice, the shaved area above the BAT was a region of interest for which average and maximal temperatures were measured. The phase of the estrous cycle in female mice was determined after an experiment by vaginal smears stained with cresyl violet (0.1%) stain solution. In healthy volunteers, two skin areas of the neck were selected: the supraclavicular area (above the collarbone, where BAT cells are present) and the interclavicular area (between the collarbones, where there is no BAT tissue detected). BAT activity is determined by the subtraction of those two values. Also, the average and maximal temperatures of skin areas could be determined in animals and human participants.
Changes in BAT activity after a meal measured by infrared thermography, a non-invasive and more sensitive method, were shown to be sex, age, and phase of the estrous cycle dependent in laboratory animals. As part of diet-induced thermogenesis, BAT activation in humans was also proven to be sex, age, and body mass index dependent. Further determining the pathophysiological changes in BAT activity after a meal will be of great importance for participants with high glucose plasma concentrations (obesity and diabetes mellitus type 2), as well as in different laboratory animals (knock-out mice). This method is also a variable tool for determining possible activating drugs that could rejuvenate BAT activity.

Here, we present a protocol for measuring brown adipose tissue activity after a meal in humans and laboratory animals.

Measuring brown adipose tissue (BAT) activity by positron emission tomography computed tomography (PET-CT) via the accumulation of 18F-fluorodeoxyglucose ...

A Microfluidic Cartridge-Based Detection System for Allergen-Specific Immunoglobulin E

Source: Huang, Z., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/59100">Application of Biochip Microfluidic Technology to Detect Serum Allergen-specific Immunoglobulin E (sIgE).</a>&#160;J. Vis. Exp.&#160;(2019)
This video demonstrates a protocol for detecting serum-specific immunoglobulin E using a microfluidic cartridge-based chemiluminescence system. Serum IgEs interact with distinct allergens immobilized in the reaction zone of the cartridge, which are subsequently detected with peroxidase-conjugated anti-IgE antibodies and a chemiluminescent substrate, allowing simultaneous detection of multiple allergen-specific IgEs.

Oparty na kartridżu mikroprzepływowym system wykrywania swoistej dla alergenu E immunoglobuliny E

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Basic information of the study ...