The authors give special thanks to the Santa Catarina State University for financial support, to the Pharmacology Laboratory for the animals and space, and to the Hemoparasites and Vectors Biochemistry Laboratory for the cryopreserved blood sample infected with T. evansi used in these experiments.

All experiments were performed using 10-week-old adult female Swiss mice (35-55 g). Animals were housed in polysulfone cages (3-5 animals per cage) in a room with controlled temperature (21 &#177; 1 &#176;C), 12 h light/12 h dark cycle, and standard lab chow and water ad libitum. Ten animals were assigned to each group in every test to demonstrate the consistent effects of drug treatments. The experimental design was submitted and approved by the Ethics Committee of Santa Catarina State University (CEUA) (protocol number: 6019201123). All animal experiments complied with the ARRIVE guidelines (Animal Research: Reporting of in vivo Experiments) and were carried out in accordance with the National Research Council Guide for the Care and Use of Laboratory Animals.
1. Trypanosoma evansi preparation and inoculation
CAUTION: Wear a disposable lab coat, gloves, mask, and eye protection when handling infected blood samples, reagents, and other chemical compounds.
<ol>
	<li>Turn on the water bath equipment and adjust it at a constant temperature of 37 &#176;C.</li>
	<li>Take a microcentrifuge tube containing the preserved blood sample out of the ultrafreezer (-80 &#176;C). Place it in the water bath until the blood thaws. 
	NOTE: The same isolate should be used to infect all mice in every group of different experiments to maintain an equal number of blood passages.</li>
	<li>Use phosphate-buffered saline (PBS, 0.1 M) containing 60% D-(+)-glucose (pH 7.4) to perform serial dilution (10:1 v v) until 1 &#215; 104 trypanosomes per 0.1 mL are achieved. Determine the number of parasite cells by manual cell counting using a Neubauer chamber on a microscope with a 100x objective lens2.</li>
	<li>Inoculate 0.1 mL of the diluted blood solution (infected group), or the same volume in 0.9% saline vehicle (control group), intraperitoneally, using a 26 G &#189;&#34; needle coupled to an insulin syringe.</li>
</ol>
2. Electronic von Frey apparatus setup and operation
NOTE: Be careful about how the obtained data are recorded. Some EvF equipment has specific programs to transfer the obtained data from the apparatus to other electronic devices, such as computers, tablets, or smartphones.
<ol>
	<li>Turn the EvF apparatus on. Ensure that the equipment is well charged before starting the experiment, observing the battery level displayed on the equipment screen and charging it if necessary.</li>
	<li>Connect the EvF apparatus to the chosen electronic device via Wi-Fi to transfer and save the obtained data.</li>
	<li>Insert a 10 &#181;L polypropylene tip into the EvF apparatus cone and set the readout to zero (0.00 g) by pressing the button with a paw symbol.</li>
	<li>Observe that once the evaluated tissue is probed; the maximum applied pressure is shown on the EvF apparatus display. After that, transfer it to the chosen electronic device by pressing the button with an antenna symbol for safe recording.</li>
	<li>Reset the readout to zero and get ready to take a new measurement by simply pressing the button with the paw symbol again.</li>
</ol>
3. General considerations for mechanical threshold assessments
<ol>
	<li>Perform all experiments in a quiet room with controlled temperature at 21 &#176;C and a 12 h light/12 h dark cycle, and be sure to respect the mouse circadian cycle by performing all evaluations at the same time of the day2.</li>
	<li>Have the same person perform both baseline and experimental mechanical threshold assessments to reduce bias and ensure measurement consistency; make sure the experiments are blinded.</li>
	<li>Place each mouse gently inside an individual chamber made of polymethyl methacrylate (PMMA) with a perforated top placed on a 5 mm&#178; mesh floor stand2,8.</li>
	<li>Allow the mice to acclimate for 30 min in the PMMA chambers prior to assessing both baseline and experimental mechanical thresholds2.</li>
	<li>Place the mesh floor stand at a comfortable height (upon a steady surface) so the animals are accessible from all sides. Ensure that the abdomen and all four paws of the mice are easily accessible through the mesh floor openings.</li>
	<li>Cover the steady surface used to allocate the chambers with absorbent material to absorb or collect micturition and defecation. Make sure that the arms of the investigator can move freely below the chambers while operating the EvF apparatus.</li>
</ol>
4. Baseline mechanical threshold assessment and mouse response to stimuli
<ol>
	<li>For abdomen measurements, divide the abdomen into three virtual parts (cranial, medium, and caudal areas). Choose the cranial area (anatomically containing the liver) as the standard target tissue for visceral allodynia mechanical threshold assessment. For paw measurements, choose the right hind paw for standardization.</li>
	<li>Take a baseline measurement from each mouse 48 h prior to the infection. To do so, place the mice in the containing chambers, prepare the EvF apparatus, and use both hands to raise the probe slowly to stimulate the targeted tissue.</li>
	<li>Gradually increase the pressure on the tissue until the mouse expresses a nociceptive behavior. For right hind paw evaluation, look for paw retraction, paw licking, or four-paw jumping2,8,9. For visceral evaluation, look for a sharp retraction of the abdomen, immediate licking or scratching of the probed site, or four-paw jumping2,11.</li>
	<li>Save the obtained value by transferring the data to the chosen electronic device containing the specific EvF program or writing it in a grade book. Reset the display to zero and repeat the process four times to obtain five measurements8. 
	NOTE: Consider only three similar measurements to estimate the mean value2</li>
	<li>Measure the mechanical threshold of the mouse present in the adjacent chamber until each mouse is probed five times and a mean value is obtained for each animal.</li>
	<li>Repeat the baseline measurements 24 h later and exclude mice with mean values below 3.00 g or with differences between basal measurements higher than 2.00 g2,8. 
	&#8203;NOTE: Only one target tissue must be chosen for each mouse in every experiment so the animals do not get used to being probed, as the number of evaluations would be considerably higher in a short period.</li>
</ol>
5. Experimental right hind paw and visceral allodynia mechanical threshold assessment
<ol>
	<li>Assess the right hind paw or visceral allodynia mechanical threshold 1 h after the infection for day 0 evaluation.</li>
	<li>Repeat the procedure every 24 h over 5 days post infection. 
	NOTE: if there is a treatment to be administered, choose an appropriate time point within the experimental design.</li>
	<li>Return the mice to their original cages to prevent animals from fighting each other, with access to standard lab chow and water ad libitum after measurements are completed.</li>
	<li>Perform a normality test to check the normal distribution of the data and further appropriate statistical analysis of the collected data.</li>
</ol>
6. Mouse grimace scale assessment
<ol>
	<li>Ensure that each mouse is seen easily while inside the containing chamber and examine each mouse prior to the experiment to ensure there are no lesions on the limbs, abdomen, or face and no coat changes.</li>
	<li>Observe the mouse orbital area. Classify open eyes as an absence of pain (score 0) while an obvious pain may be represented when the mouse closes its eyes (score 2).</li>
	<li>Observe the mouse&#39;s nose. Classify a normal nose as the absence of pain while the presence of a bulge on the bridge of the nose represents obvious pain.</li>
	<li>Observe the mouse&#39;s cheeks. Classify normal cheeks as the absence of pain while the presence of a bulge on both cheeks represents obvious pain.</li>
	<li>Observe the position of the mouse&#39;s ears. Classify rounded ears as the absence of pain while ears rotating outwards or backwards, away from the face, in a pointed shape, represent obvious pain. Space between the ears increases with the pain score.</li>
	<li>Observe the mouse&#39;s whiskers. Classify whiskers with their natural downward curve as the absence of pain while whiskers that are either pulled back against the cheek or pulled forward represent obvious pain.</li>
	<li>As innate behaviors, such as self-grooming, may interfere with the facial expression, do not consider these to be pain-related behaviors8. Wait for the mouse to stop this behavior before every action unit evaluation.</li>
	<li>Perform a normality test to check the normal distribution of the data and further appropriate statistical analysis of the collected data. 
	NOTE: At the end of all the experiments, mice were humanely euthanized using ketamine (90 mg/kg) and xylazine (7.5 mg/kg), injected intraperitoneally. After reaching a confirmed deep anesthesia plan, they were submitted to cervical dislocation.</li>
</ol>

Assessing Pain and Allodynia in &lt;em&gt;T. evansi&lt;/em&gt;-Infected Animals

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C., 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=Cytokines+in+rats+experimentally+infected+with+Trypanosoma+evansi.">Cytokines in rats experimentally infected with Trypanosoma evansi.</a> Experimental Parasitology. 128 (4), 365-370 (2011).</li><li>Gillingwater, 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=In+vivo+investigations+of+selected+diamidine+compounds+against+Trypanosoma+evansi+using+a+mouse+model.">In vivo investigations of selected diamidine compounds against Trypanosoma evansi using a mouse model.</a> Antimicrobial Agents and Chemotherapy. 53 (12), 5074-5079 (2009).</li><li>Dkhil, M. A., 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=Treatment+of+Trypanosoma+evansi-infected+mice+with+Eucalyptus+camaldulensis+led+to+a+change+in+brain+response+and+spleen+immunomodulation.">Treatment of Trypanosoma evansi-infected mice with Eucalyptus camaldulensis led to a change in brain response and spleen immunomodulation.</a> Frontiers in Microbiology. 13, 833520 (2022).</li><li>Dkhil, M. 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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=Coding+of+facial+expressions+of+pain+in+the+laboratory+mouse.">Coding of facial expressions of pain in the laboratory mouse.</a> Nature Methods. 7, 447-449 (2010).</li><li>Eijkelkamp, N., 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=Increased+visceral+sensitivity+to+capsaicin+after+DSS-induced+colitis+in+mice:+spinal+cord+c-Fos+expression+and+behavior.">Increased visceral sensitivity to capsaicin after DSS-induced colitis in mice: spinal cord c-Fos expression and behavior.</a> American Journal of Physiology-Gastrointestinal and Liver Physiology. 293 (4), 749-757 (2007).</li><li>Mekata, 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=Expression+of+regulatory+dendritic+cell-related+cytokines+in+cattle+experimentally+infected+with+Trypanosoma+evansi.">Expression of regulatory dendritic cell-related cytokines in cattle experimentally infected with Trypanosoma evansi.</a> Journal of Veterinary Medical Science. 77 (8), 1017-1019 (2015).</li><li>Mukaka, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Statistics+corner:+a+guide+to+appropriate+use+of+correlation+coefficient+in+medical+research.">Statistics corner: a guide to appropriate use of correlation coefficient in medical research.</a> Malawi Medical Journal. 24 (3), 69-71 (2012).</li><li>Schober, P., 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=Correlation+coefficients:+appropriate+use+and+interpretation.">Correlation coefficients: appropriate use and interpretation.</a> Anesthesia and Analgesia. 126 (5), 1763-1768 (2018).</li><li>Kamidi, C. 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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trypanosoma+evansi+pathogenicity+strain+in+rats+inoculated+with+parasite+in+fresh+and+cryopreserved+blood.">Trypanosoma evansi pathogenicity strain in rats inoculated with parasite in fresh and cryopreserved blood.</a> Ci&#234;ncia Rural. 39 (6), 1842-1846 (2009).</li><li>Silva, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acetylcholinesterase+activity+and+lipid+peroxidation+in+the+brain+and+spinal+cord+of+rats+infected+with+Trypanosoma+evansi.">Acetylcholinesterase activity and lipid peroxidation in the brain and spinal cord of rats infected with Trypanosoma evansi.</a> Veterinary Parasitology. 175, 237-244 (2011).</li><li>Di&#243;genes, A. K. L., 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=Concurrent+validity+of+electronic+von+Frey+as+an+assessment+tool+for+burn+associated+pain.">Concurrent validity of electronic von Frey as an assessment tool for burn associated pain.</a> Burns. 46 (6), 1328-1336 (2020).</li><li>Kalueff, A. V., 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=Neurobiology+of+rodent+self-grooming+and+its+value+for+translational+neuroscience.">Neurobiology of rodent self-grooming and its value for translational neuroscience.</a> Nature Reviews Neuroscience. 17, 45-59 (2016).</li></ol>

The authors have no conflicts of interest to declare.

A critical step in experiments involving infected animals is the control of parasitemia levels. Different strains of T. evansi may behave in divergent ways in mice, leading from acute to chronic infections2,4,5,6. Furthermore, changes in the infection dose may decrease or extend the survival time of mice. Therefore, obtaining a survival curve prior to the experiments is recommended to ...

Trypanosoma evansi is the etiological agent of the disease called &#34;surra&#34; or &#34;mal-das-cadeiras&#34; in South America1,2. It commonly affects equines and cattle, but also wild fauna, being transmitted by hematophagous bats, tabanids, and stomoxes fly bites1,3. Surra is a major disease in domestic animals, which can be fatal in the absence of the correct treatment, exhibiting nonspecific clinical signs such as anemia, loss of appetite and weight, muscle weakness, and abortion, which may vary according to the host and geographic region2,4,5,6.
The expression of allodynia and pain in infected animals during the course of the disease is still a new topic2,7. The urge to understand the pathogenesis behind these signs is an important step to improve and refine the currently used treatment by adding effective analgesic drugs to the classic trypanocidal protocol5,6. In this scenario, the possibility of replicating the disease using a murine model represents an advantage, as mice can be easily kept under controlled environments in the laboratory and therefore, yield results that are more consistent than field experiments using livestock.
A mechanical threshold is commonly obtained using an electronic von Frey (EvF) apparatus for the evaluation of allodynia in a vast number of experiments2,8,9. This device is used to assess tissue sensitivity to mechanical stimulation: once the apparatus touches the paw of the animal, through a 20-200 &#181;L tip attached to the device, the force with which the animal withdraws the paw is recorded.
Rodents are usually used as standard animals in these experiments, and most results are extrapolated to other species, as most researched diseases come from other species in which it would be difficult to conduct a controlled study. Furthermore, allodynia and pain are intimately related. The use of a specific facial scale to evaluate pain in infected mice plays an important role as a confirming adjuvant to affirm the presence of pain during the course of T. evansi infection2,10.
In this protocol, we demonstrate a novel model to evaluate allodynia and pain in mice experimentally infected with T. evansi, showing a high correlation between variables, thus proving to be a strong model. Furthermore, it requires a small number of researchers to carry out the entire procedure, decreasing the chance of human interference during the experiment. It also enables the investigator to replicate the target disease during an acute course and add several different treatment drugs in the experimental design, thereby obtaining consistent results in a short period.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>26 G 1/2&#34; needle coupled to insulin syringe</td>
			<td>TKL</td>
			<td>80288090100</td>
			<td>Used to infund solutions on laboratory animals</td>
		</tr>
		<tr>
			<td>Accessories for von Frey analgesimeter</td>
			<td>INSIGHT</td>
			<td>EFF 303</td>
			<td>Containment box with support for digital analgesimeter assessment</td>
		</tr>
		<tr>
			<td>D-(+)-Glucose</td>
			<td>SIGMA-ALDRICH</td>
			<td>G7021</td>
			<td>A monosaccharide which is the main source of energy in the form of ATP for living organisms</td>
		</tr>
		<tr>
			<td>Digital analgesimeter</td>
			<td>INSIGHT</td>
			<td>von Frey Wi-Fi</td>
			<td>The von Frey Wi-Fi is a portable device used to assess tissue sensitivity to mechanical stimuli</td>
		</tr>
		<tr>
			<td>Gilson type 10 &#181;L polypropylene tip</td>
			<td>CRALPLAST</td>
			<td>18261</td>
			<td>Polypropylene to be used on eletronic von Frey apparatus, recommended for hind paw allodynia assessment</td>
		</tr>
		<tr>
			<td>Laboratory water bath</td>
			<td>BEING INSTRUMENT</td>
			<td>BW-22P</td>
			<td>Used to heat liquid and semi-solid substances contained in appropriate recipients to specific temperature</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>SIGMA-ALDRICH</td>
			<td>806552</td>
			<td>A balanced salt solution buffer used for a variety of cell culture applications</td>
		</tr>
		<tr>
			<td>Swiss mice (Mus musculus) from both gender</td>
			<td>UFSC</td>
			<td>Swiss Webster</td>
			<td>Laboratory animals used for controlled experiments</td>
		</tr>
		<tr>
			<td>Trypanosoma evansi cryiopreserved sample</td>
			<td>UDESC</td>
			<td>-</td>
			<td>Sample used to infect all mice, ceded by the Hemoparasites and Vectors Biochemistry Laboratory</td>
		</tr>
		<tr>
			<td>Universal type 10 &#181;L polypropylene tip</td>
			<td>CRALPLAST</td>
			<td>18171</td>
			<td>Polypropylene to be used on eletronic von Frey apparatus, recommended for visceral allodynia assessment</td>
		</tr>
	</tbody>
</table>

electronic von frey associated with the mouse grimace scale to assess allodynia and pain in trypanosoma evansi-infected mice

Infectious disease pathogenesis is still a complex field to study. The course of several clinical signs, such as allodynia and pain, may be observed in domestic animals. However, the knowledge of their pathways and correct treatment need controlled experiments, many of them using laboratory animals. Measuring changes in mechanical thresholds of the hind paw and viscera is a useful technique to observe changes in pain perception in rodents. Withdrawal response can be measured first in baseline tests, which creates better control of experimental groups. Subsequent tests can be performed after inducing infection and adding drugs to the protocol. The use of an electronic von Frey apparatus associated with the use of a facial scale to observe pain-like changes allows a simple, precise, and consistent assessment to evaluate allodynia and pain in mice. Thus, experiments using the present methodology for Trypanosoma evansi infection represent a useful method to evaluate allodynia and pain in laboratory-infected animals, which can be applied to the conventional treatment for livestock animals.

Author Spotlight: Assessing Pain and Promoting Animal Welfare in Laboratory Animals Infected with &lt;em&gt;Trypanosoma evansi&lt;/em&gt;

Electronic von Frey associated with the Mouse Grimace Scale to Assess Allodynia and Pain in Trypanosoma evansi-Infected Mice

The question we intend to answer with this study is to evaluate nociception in laboratory animals experimentally-infected with Trypanosoma evansi, as observed in previous studies by our group. In relation to Trypanosoma evansi, the most recent developments are regarding prevalence studies in various locations around the globe, followed by the search for specific biomarkers for differential diagnosis. Some experimental challenges are mainly related to animal welfare.All the care related to the three Rs must be reviewed and encouraged. We believe that an article with video instruction, as we show in this study, is a good incentive to discuss reduction in experimental models. Our experiments enable the necessity of analgesics during Trypanosoma evansi infection, since this infection was able to diminish the nociception threshold in our model.We can now ask ourselves how the inflammation, allodynia and pain caused by Trypanosoma evansi infection affects the disease's progression. We also need to understand how it molecularly impacts the hosts'clinical sites and behavior, as well as the parasites'behavior inside infected organism.

Decrease in the mechanical threshold due to T. evansi infection as evaluated by electronic von Frey apparatus on both right hind paw and visceral tissues 
The experiment was conducted over 5 days, according to previous data related to the available T. evansi sample2. Infected mice started to show a significant difference in mechanical threshold on the right hind paw at day 3 post infection and remained significantly lower than the control group during the ne...

Here, we present a protocol to evaluate nociception in mice infected with Trypanosoma evansi, using the electronic von Frey apparatus as a tool, measuring mechanical thresholds of the hind paw and viscera.

Infectious disease pathogenesis is still a complex field to study. The course of several clinical signs, such as allodynia and pain, may be observed in ...

electronic-von-frey-associated-with-mouse-grimace-scale-to-assess

Research

JoVE Journal

Immunology and Infection

453 Views.  Santa Catarina State University. The question we intend to answer with this study is to evaluate nociception in laboratory animals experimentally-infected with Trypanosoma evansi, as observed in previous studies by our group. In relation to Trypanosoma evansi, the most recent developments are regarding prevalence studies in various locations around the globe, followed by the search for specific biomarkers for differential diagnosis. Some experimental challenges are mainly related to animal welfare.All the care related...

Video: Author Spotlight: Assessing Pain and Promoting Animal Welfare in Laboratory Animals Infected with Trypanosoma evansi

Preparation and Inoculation of Trypanosoma evansi in Mouse to Evaluate Allodynia and Pain

To begin, turn on the water bath equipment and set it at 37 degrees Celsius, remove a micro centrifuge tube containing the preserved blood sample from the ultra freezer and place it in the water bath until the blood thaws. Perform serial dilution using PBS with 60%glucose to dilute the trypanosomes. Count the number of parasite cells using a neubauer chamber under a microscope with a 100 times objective lens.

Electronic Von Frey (EvF) Apparatus Set up for the Evaluation of Allodynia and Pain in Trypanosoma evansi Infected Animals

Set up a quiet room with a controlled temperature of 21 degrees Celsius in a 12-hour light and 12-hour dark cycle. Gently place each mouse into a chamber made of PMMA placed on a five square millimeter mesh floor stand. Allow the mice to acclimate for 30 minutes before assessing baseline and experimental mechanical thresholds.Then adjust the mesh floor stand to a comfortable height so that the mice can be assessed from all sides, and their abdomen and all four paws are easily reachable through the mesh floor openings. Cover the surface beneath the chambers with absorbent material to manage micturition and defecation. Ensure that there is ample arm space below the chambers while using the EVF apparatus.After setting up the EVF apparatus for mechanical threshold assessments, conduct abdomen measurements by dividing the mouse abdomen into three virtual sections. To obtain baseline measurements, 48 hours before infection, place the mice in the chambers, and half an hour after setting up the EVF apparatus, stimulate the designated tissue by gradually elevating the probe with both hands. Now, apply gradual pressure to the tissue until the mouse displays nociceptive behavior.To evaluate the right hind paw, watch for actions such as retraction, paw licking, or four-paw jumping. For visceral assessment, observe sudden abdominal retraction, immediate licking or scratching of the probed area, or four-paw jumping. Then save the recorded value into the designated electronic device with the EVF program or document it in a notebook.Reset the display to zero. Probe each mouse in the adjacent chamber five times, calculate their individual average values and reassess the baseline measurements after 24 hours.

Mechanical Threshold Assessment of Mice Infected with Trypanosoma evansi

Inject the experimental mice intraperitoneally with 0.1 Milliliters of diluted blood solution infected with Trypanosoma evansi. For the control group, inject 0.1 milliliters of PBS vehicle intraperitoneally into the mice. One hour later, set up the EVF apparatus for mechanical threshold assessments.For the day zero assessment, after one hour of infection, and 30 minutes of acclimatization of the mice, evaluate the mechanical threshold for the right hind paw or visceral allodynia. After taking the measurements, return the mice to their original cages to avoid conflicts among them and provide standard lab chow and water ad libitum.