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 T. evansi-Infected Animals

<ol>
	<li>Desquesnes, M., 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+and+surra:+a+review+and+perspectives+on+origin,+history,+distribution,+taxonomy,+morphology,+hosts,+and+pathogenic+effects.">Trypanosoma evansi and surra: a review and perspectives on origin, history, distribution, taxonomy, morphology, hosts, and pathogenic effects.</a> BioMed Research International. 2013, 194176 (2013).</li><li>Cipriani, D. 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=Experimental+Trypanosoma+evansi+infection+induces+pain+along+with+oxidative+stress,+prevented+by+COX-2+inhibition.">Experimental Trypanosoma evansi infection induces pain along with oxidative stress, prevented by COX-2 inhibition.</a> Experimental Parasitology. 247, 108477 (2023).</li><li>Paim, F. 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. 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=Murine+liver+response+to+Allium+sativum+treatment+during+infection+induced-trypanosomiasis.">Murine liver response to Allium sativum treatment during infection induced-trypanosomiasis.</a> Saudi Journal of Biological Sciences. 28 (6), 3270-3274 (2021).</li><li>Martins de Moraes, 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=Infection+by+Trypanosoma+evansi+in+horses+from+Brazil.">Infection by Trypanosoma evansi in horses from Brazil.</a> Revista Portuguesa de Ci&#234;ncias Veterin&#225;rias. 102 (561-562), 159-163 (2007).</li><li>Martinov, 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=Measuring+changes+in+tactile+sensitivity+in+the+hind+paw+of+mice+using+an+electronic+von+Frey+apparatus.">Measuring changes in tactile sensitivity in the hind paw of mice using an electronic von Frey apparatus.</a> Journal of Visualized Experiments. 82, e51212 (2013).</li><li>Rodr&#237;guez-Angulo, 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=Role+of+TNF+in+sickness+behavior+and+allodynia+during+the+acute+phase+of+Chagas'+disease.">Role of TNF in sickness behavior and allodynia during the acute phase of Chagas' disease.</a> Experimental Parasitology. 134 (4), 422-429 (2013).</li><li>Langford, D. 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. M., 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=Differential+virulence+of+camel+Trypanosoma+evansi+isolates+in+mice.">Differential virulence of camel Trypanosoma evansi isolates in mice.</a> Parasitology. 145 (9), 1235-1242 (2018).</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=Isolation,+cloning,+and+pathologic+analysis+of+Trypanosoma+evansi+field+isolates.">Isolation, cloning, and pathologic analysis of Trypanosoma evansi field isolates.</a> Parasitology Research. 112, 1513-1521 (2013).</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=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 determine the correct period of the experiment and prevent frustration with unexpected mouse death events15,16. Additionally, this data may determine humane endpoints for the experiment, if applicable.
In addition, the same isolate must be used to infect all mice in the experiment to respect an equal number of blood passages, as explained in the protocol section. In the same way, cryopreserved or fresh infected blood can be used to infect the mice, as some cryopreserved samples may be inactivated after defrosting in a water bath. However, fresh infected blood may produce faster parasitemia and, therefore, earlier death events17,18.
Modifications in the experiment, such as the addition of trypanocidal or analgesic drugs, are viable. New groups mean more animals and it is important to remember that even control groups for the chosen drugs must be submitted to baseline measurements. In our experience, approximately 20-25% of mice are excluded from the experiment after the second baseline measurement, which is consistent with previous data8. This means that the initial number of animals must be higher than the experimental number of animals, which can be an issue when more groups are evaluated and a consequently higher number of mice is estimated.
Pharmacokinetics and pharmacodynamics must be taken into consideration for this model. Some drugs take extended periods to exert their pharmacological action, which may impact the experimental design of a model where mice usually die in an average of 4 to 5 days2,6. In addition, if animals are fasting for the chosen treatment, the acclimation period may be an important factor to consider, as it will affect both drug dynamics and experimental schedule and procedure, as reported in the protocol section.
A great improvement in the present method is that a single well-trained researcher may perform the entire EvF reading procedure (both baseline and experimental measurements) while remaining blinded to the infection or treatments. Another investigator must perform the infection at the beginning of the experiment. This is not necessary after the infection procedure, as the EvF apparatus has a specific von Frey Wi-Fi measurement program that allows only one person to fully operate the equipment. Furthermore, this method is faster than the ordinary Filament von Frey equipment and easier to perform8,19.
However, fatigue may be a complication, as the same researcher must perform all animal readings and may develop exhaustion due to the repetitive motion after some time8. Taking into consideration the survival expectation for mice infected by T. evansi, a high number of groups in the experimental design is not encouraged. In our experience, an average of 10 mice at a time (depending on the experimental design) can be measured in less than 30 min. Furthermore, only one target tissue must be chosen for each mouse in every experiment so the animals do not get used to being probed, as explained in the protocol section, which also diminishes the chance of development of fatigue by the investigator.
In addition, both EvF (right hind paw and visceral measurements) and Mouse Grimace Scale assessments need well-trained researchers. Before performing the experiments, the investigator must spend long periods practicing. To correctly evaluate the facial expression changes in mice, the researcher must know not only the expected alterations but also the normal facial expression of an ordinary mouse and its variations of normality and behaviors10,20. Furthermore, to correctly evaluate mice&#39;s mechanical threshold using the EvF apparatus, the researcher must repeat several baseline measurements until the mouse response to the probe stimuli is easily recognized and consistency is achieved2,8.
Future applications of the protocol involve the evaluation of allodynia and pain, as well as its treatment, on mice infected by T. evansi. The present model enables scientific investigators to evaluate the pain-related pathogenesis of a disease commonly found in livestock, in mice under a controlled environment in the laboratory.

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><tbody><tr><td>26 G 1/2&quot; 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 &amp;micro;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 (&lt;em&gt;Mus musculus&lt;/em&gt;) from both gender</td><td>UFSC</td><td>Swiss Webster</td><td>Laboratory animals used for controlled experiments</td></tr><tr><td>&lt;em&gt;Trypanosoma evansi &lt;/em&gt;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 &amp;micro;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.

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 next 2 days post infection (Figure 1A) compared to uninfected mice. Similar results for abdominal tactile sensitivity showed visceral allodynia in infected mice starting at day 3 post infection and remaining significantly lower than the control group during the next 2 days post&#160;infection (Figure 1B).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65743/65743fig01.jpg" /> 
Figure 1: Allodynia evaluation of mice experimentally infected by Trypanosoma evansi. (A) Right hind paw allodynia and (B) visceral allodynia. Data expressed as the mean &#177; SD of ten animals for each experimental group. Statistical analysis: Student&#39;s t-test, one unpaired analysis per time point. Asterisks show significant differences between experimental groups considering the same analyzed time point (p &#60; 0.05). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65743/65743fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Induction of mouse pain-related facial features by T. evansi infection and numerical interpretation by Mouse Grimace Scale pain assessment 
Infected mice started to manifest significant signs of pain-related facial features on day 3 post infection, showing a mean score of 0.4 [0-1] at the referred time point. The same tendency was observed during the experiment, in which the infected group showed significant differences when compared to the control group, presenting mean scores of 0.6 [0-1] and 1.3 [0-2] for days 4 and 5 post infection, respectively. The control group did not score on the Mouse Grimace Scale, as expected. The statistical analysis was performed using Student&#39;s t-test, one unpaired analysis per time point, considering as significant a value of p &#60; 0.05. Furthermore, the Pearson correlation coefficient (r) identified interaction patterns between the Mouse Grimace Scale pain assessment and right hind paw allodynia or between the same scale and visceral allodynia, which values were -96.35% and -84.08%, respectively. In addition, the coefficients of determination (R2) for the Mouse Grimace Scale pain assessment and right hind paw allodynia or visceral allodynia were 0.9283 and 0.7070, respectively.
The present study agrees with previous data, which confirmed the presence of inflammation and pain through the course of T. evansi infection2,3,7,12. Moreover, the described method provides an accurate methodology to identify and measure allodynia and pain in mice. Furthermore, the use of Mouse Grimace Scale pain assessment in infected mice indicated a very high negative r (90 to 100%) when compared to the results expressed by the right hind paw allodynia evaluation and high negative r (70 to 89%) for visceral allodynia evaluation of the respective animals13. Similarly, the use of Mouse Grimace Scale pain assessment in infected mice indicated a very strong R2 (0.90 to 1.00) when compared to the results expressed by the right hind paw allodynia evaluation and strong R2 (0.70 to 0.89) for visceral allodynia evaluation of the same animals14.

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

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.

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

951 Views.  Santa Catarina State University. De vraag die we met deze studie willen beantwoorden, is het evalueren van nociceptie bij proefdieren die experimenteel zijn geïnfecteerd met Trypanosoma evansi, zoals waargenomen in eerdere studies door onze groep. Met betrekking tot Trypanosoma evansi hebben de meest recente ontwikkelingen betrekking op prevalentiestudies op verschillende locaties in de wereld, gevolgd door de zoektocht naar specifieke biomarkers voor differentiële diagnose. Sommige experimentele uitdagingen hebben vooral ...

Video: Auteur in de schijnwerpers: Pijn beoordelen en dierenwelzijn bevorderen bij proefdieren die besmet zijn met Trypanosoma evansi

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

An Improved Assay and Tools for Measuring Mechanical Nociception in Drosophila Larvae

Published assays for mechanical nociception in Drosophila have led to variable assessments of behavior. Here, we fabricated, for use with Drosophila larvae, customized metal nickel-titanium alloy (nitinol) filaments. These mechanical probes are similar to the von Frey filaments used in vertebrates to measure mechanical nociception. Here, we demonstrate how to make and calibrate these mechanical probes and how to generate a full behavioral dose-response from subthreshold (innocuous or non-noxious range) to suprathreshold (low to high noxious range) stimuli. To demonstrate the utility of the probes, we investigated tissue damage-induced hypersensitivity in Drosophila larvae. Mechanical allodynia (hypersensitivity to a normally innocuous mechanical stimulus) and hyperalgesia (exaggerated responsiveness to a noxious mechanical stimulus) have not yet been established in Drosophila larvae. Using mechanical probes that are normally innocuous or probes that typically elicit an aversive behavior, we found that Drosophila larvae develop mechanical hypersensitization (both allodynia and hyperalgesia) after tissue damage. Thus, the mechanical probes and assay that we illustrate here will likely be important tools to dissect the fundamental molecular/genetic mechanisms of mechanical hypersensitivity.

An Improved Assay and Tools for Measuring Mechanical Nociception in Drosophila Larvae

The goal of this protocol is to show how to perform an improved assay for mechanical nociception in Drosophila larvae. We use the assay here to demonstrate that mechanical hypersensitivity (allodynia and hyperalgesia) exists in Drosophila larvae.

Published assays for mechanical nociception in Drosophila have led to variable assessments of behavior. Here, we fabricated, for use with Drosophila ...

Behavior

Back Mechanical Sensitivity Assessment in the Rat for Mechanistic Investigation of Chronic Back Pain

Low back pain is the leading cause of disability worldwide, with dramatic personal, economic, and social consequences. To develop novel therapeutics, animal models are needed to examine the mechanisms and effectiveness of novel therapies from a translational perspective. Several rodent models of back pain are used in current investigations. Surprisingly, however, no standardized behavioral test was validated to assess mechanical sensitivity in back pain models. This is critical to confirm that animals with presumed back pain present local hypersensitivity to nociceptive stimuli, and to monitor sensitivity during interventions designed to relieve back pain. The objective of this study is to lay down a simple and accessible test to assess mechanical sensitivity in the back of rats. A test cage was fabricated specifically for this method; length x width x height: 50 x 20 x 7 cm, having a stainless-steel mesh on the top. This test cage allows the application of mechanical stimuli to the back. To perform the test, the back of the animal is shaved in the region of interest, and the test area is marked to repeat the test on different days, as needed. The mechanical threshold is determined with Von Frey filaments applied to the paraspinal muscles, utilizing the up-down method described previously. The positive responses include (1) muscle twitching, (2) arching (back extension), (3) rotation of the neck (4) scratching or licking the back, and (5) escaping. This behavioral test (Back Mechanical Sensitivity (BMS) test) is useful for mechanistic research with rodent models of back pain for the development of therapeutic interventions for the prevention and management of back pain.

To develop novel therapeutic interventions for the prevention and management of back pain, animal models are required to examine the mechanisms and effectiveness of these therapies from a translational perspective. The present protocol describes the BMS test, a standardized method to assess back mechanical sensitivity in the rat.

Low back pain is the leading cause of disability worldwide, with dramatic personal, economic, and social consequences. To develop novel therapeutics, ...

Inflammatory TMJ Pain Mouse Model: Bite Force and Von Frey Filament Assessments

Temporomandibular Joint Pain Measurement by Bite Force and Von Frey Filament Assays in Mice

Temporomandibular joint (TMJ) pain and osteoarthritis (OA) are common and debilitating disorders that impair patients' quality of life. The mechanisms driving diseases-related pain are poorly understood, and current treatments fail to provide effective and long-term therapeutic effects. Additionally, pain assessment in research, particularly orofacial pain, poses several challenges that complicate studies in both clinical and basic science settings. Therefore, we have established an inflammatory TMJ pain mouse model via intra-articular injection of CFA (Complete Freund's Adjuvant) and evaluated pain behaviors by bite force measurement and the von Frey filament test. Mice with CFA injection exhibited orofacial pain behaviors compared to PBS injection, including reduced bite force and head withdrawal threshold in the von Frey filament test. These methods are relatively easy to execute to have reproducible results and can be potentially extended to pain studies for other disease models related to TMJ disorders. Together, bite force, and the von Frey filament tests are reliable in measuring orofacial pain, as demonstrated in CFA injection-induced painful TMJOA mouse models.

This protocol describes how measuring bite force and head withdrawal threshold can capture pain behavior in Complete Freund's Adjuvant (CFA)-induced inflammatory temporomandibular joint (TMJ) pain.

Temporomandibular joint (TMJ) pain and osteoarthritis (OA) are common and debilitating disorders that impair patients' quality of life. The mechanisms ...

The Monoiodoacetate Model of Osteoarthritis Pain in the Mouse

A major symptom of patients with osteoarthritis (OA) is pain that&#160;is triggered by peripheral as well as central changes within the pain pathways. The current treatments for OA pain such as NSAIDS or opiates are neither sufficiently effective nor devoid of detrimental side effects. Animal models of OA are being developed to improve our understanding of OA-related pain mechanisms and define novel pharmacological targets for therapy. Currently available models of OA in rodents include surgical and chemical interventions into one knee joint. The monoiodoacetate (MIA) model has become a standard for modelling joint disruption in OA in both rats and mice. The model, which is easier to perform in the rat, involves injection of MIA into a knee joint that induces rapid pain-like responses in the ipsilateral limb, the level of which can be controlled by injection of different doses. Intra-articular injection of MIA disrupts chondrocyte glycolysis by inhibiting glyceraldehyde-3-phosphatase dehydrogenase and results in chondrocyte death, neovascularization, subchondral bone necrosis and collapse, as well as inflammation. The morphological changes of the articular cartilage and bone disruption are reflective of some aspects of patient pathology. Along with joint damage, MIA injection induces referred mechanical sensitivity in the ipsilateral hind paw and weight bearing deficits that are measurable and quantifiable. These behavioral changes resemble some of the symptoms reported by the patient population, thereby validating the MIA injection in the knee as a useful and relevant pre-clinical model of OA pain.
The aim of this article is to describe the methodology of intra-articular injections of MIA and the behavioral recordings of the associated development of hypersensitivity with a mind to highlight the necessary steps to give consistent and reliable recordings.

Osteoarthritis (OA), or degenerative joint disease, is a debilitating condition associated with pain that remains only partially controlled by available analgesics. Animal models are being developed to improve our understanding of OA-related pain mechanisms. Here we describe the methodology for the monoiodoacetate model of OA pain in the mouse.

A major symptom of patients with osteoarthritis (OA) is pain that&#160;is triggered by peripheral as well as central changes within the pain pathways. The ...

Medicine

Automated Gait Analysis in Mice with Chronic Constriction Injury

The von Frey test is a classical method that has been widely used to examine the sensory function of neuropathic pain animals. However, it has some disadvantages such as subjective data and the requirement of a skilled, experienced experimenter. To date, a variety of modifications have improved the von Frey method, but it still has a few limitations. Recent reports have suggested that gait analysis produces more accurate and objective data from the neuropathic animals. This protocol demonstrates how to perform the automated gait analysis to determine the degree of neuropathic pain in mice. After several days of acclimation, the mice were allowed to walk freely on the glass floor to illuminate footprints. Then, quantification of the footprints and gait were performed through video clips with automatic analysis of various walking parameters, such as area of paw print, swing time, angle of paw, etc.
The main purpose of this study is to describe the methodology of automated gait analysis and briefly compare it with data from the classical sensory test using von Frey filament.

The precise assessment of pain response in a neuropathic animal model is critical to investigate the pathophysiology of pain diseases and develop new analgesics. We present a sensitive and objective method to determine the sensory function of the rodent hind paw by an automated gait analysis system.

The von Frey test is a classical method that has been widely used to examine the sensory function of neuropathic pain animals. However, it has some ...

Establishing a Mouse Model of a Pure Small Fiber Neuropathy with the Ultrapotent Agonist of Transient Receptor Potential Vanilloid Type 1

Patients with diabetes mellitus (DM) or those experiencing the neurotoxic effects of chemotherapeutic agents may develop sensation disorders due to degeneration and injury of small-diameter sensory neurons, referred to as small fiber neuropathy. Present animal models of small fiber neuropathy affect both large- and small-diameter sensory fibers and thus create a neuropathology too complex to properly assess the effects of injured small-diameter sensory fibers. Therefore, it is necessary to develop an experimental model of pure small fiber neuropathy to adequately examine these issues. This protocol describes an experimental model of small fiber neuropathy specifically affecting small-diameter sensory nerves with resiniferatoxin (RTX), an ultrapotent agonist of transient receptor potential vanilloid type 1 (TRPV1), through a single dose of intraperitoneal injection, referred to as RTX neuropathy. This RTX neuropathy showed pathological manifestations and behavioral abnormalities that mimic the clinical characteristics of patients with small fiber neuropathy, including intraepidermal nerve fiber (IENF) degeneration, specifically injury in small-diameter neurons, and induction of thermal hypoalgesia and mechanical allodynia. This protocol tested three doses of RTX (200, 50, and 10 &#181;g/kg, respectively) and concluded that a critical dose of RTX (50 &#181;g/kg) is required for the development of typical small fiber neuropathy manifestations, and prepared a modified immunostaining procedure to investigate IENF degeneration and neuronal soma injury. The modified procedure is fast, systematic, and economic. Behavioral evaluation of neuropathic pain is critical to reveal the function of small-diameter sensory nerves. The evaluation of mechanical thresholds in experimental rodents is particularly challenging and this protocol describes a customized metal mesh that is suitable for this type of assessment in rodents. In summary, RTX neuropathy is a new and easily established experimental model to evaluate the molecular significance and intervention underlying neuropathic pain for the development of therapeutic agents.

This study establishes an experimental model of pure small fiber neuropathy with resiniferatoxin (RTX). A unique dose of RTX (50 &#181;g/kg) is optimal for developing a small fiber neuropathy model that mimics patient characteristics and could help investigate the nociceptive molecular significance underlying neuropathic pain.

Patients with diabetes mellitus (DM) or those experiencing the neurotoxic effects of chemotherapeutic agents may develop sensation disorders due to ...

Development of Recombinant Proteins to Treat Chronic Pain

Chronic pain is difficult to treat and new approaches to resolve persistent pain are urgently needed. Anti-inflammatory cytokines are promising candidates for treating debilitating pain conditions due to their capacity to regulate aberrant neuro-immune interactions. However, physiologically they work in a network of various cytokines, and therefore their therapeutic effect may not be optimal when used as stand-alone drugs. To overcome this limitation, we developed a fusion protein of the anti-inflammatory cytokines IL4 and IL10. Here, we describe the methods for production and quality control of IL4-10 recombinant fusion protein and we test the effectiveness of the IL4-10 fusion protein to resolve pain in a mouse model of persistent inflammatory pain.

In this paper we provide details for the methods of production and quality control of an IL4-10 recombinant fusion protein. We also show how to test the effectiveness of this protein to resolve pain in a mouse model of inflammatory pain.

Chronic pain is difficult to treat and new approaches to resolve persistent pain are urgently needed. Anti-inflammatory cytokines are promising candidates ...

Cheek Injection Model for Simultaneous Measurement of Pain and Itch-related Behaviors

Itch was defined as &#34;an unpleasant cutaneous sensation that provokes a desire to scratch&#34;&#160;by Rothman in 1941. In mouse models, scratch bouts are typically counted to evaluate itch induced by pruritogens. However, previous reports have shown that algesic substances also induce scratching behaviors in a mouse neck injection model, which is the most common test used for scratching behaviors. This finding makes it difficult to study itch in mice. &#160;In contrast, capsaicin, a common algogen, reduced scratching behaviors in some neck injection experiments. Therefore, the effect of pain on scratching behaviors remains unclear. It is thus necessary to develop a method to concurrently investigate itch and pain sensation using behavioral tests. Here, a cheek injection model is introduced which can be used to simultaneously measure pain- and itch-related behaviors. In this model, pruritogens induce scratching behaviors while algesic substances induce wiping behaviors. Using this model, lysophosphatidic acid (LPA), an itch mediator found in cholestatic patients with itch, is shown to exclusively induce itch but not pain. However, in mouse models, LPA has been reported to be both a pruritogen and an algogen. Investigation into the effects of LPA in a mouse cheek injection model showed that LPA only induced scratching, but not wiping behaviors. This indicates that LPA acts as a pruritogen similarly in mice and humans, and demonstrates the utility of a cheek injection model for itch research.

Typically, the mouse neck injection model is used for evaluate pruritogen-induced scratch behaviors. However, the model provides information only on itch, not pain. Here, a cheek injection model is introduced in mice which can be used to simultaneously measure pain and itch-related behaviors.

Itch was defined as &#34;an unpleasant cutaneous sensation that provokes a desire to scratch&#34;&#160;by Rothman in 1941. In mouse models, scratch bouts ...

Dural Stimulation and Periorbital von Frey Testing in Mice As a Preclinical Model of Headache

The cranial meninges, comprised of the dura mater, arachnoid, and pia mater, are thought to primarily serve structural functions for the nervous system. For example, they protect the brain from the skull and anchor/organize the vascular and neuronal supply of the cortex. However, the meninges are also implicated in nervous system disorders such as migraine, where the pain experienced during a migraine is attributed to local sterile inflammation and subsequent activation of local nociceptive afferents. Of the layers in the meninges, the dura mater is of particular interest in the pathophysiology of migraines. It is highly vascularized, harbors local nociceptive neurons, and is home to a diverse array of resident cells such as immune cells. Subtle changes in the local meningeal microenvironment may lead to activation and sensitization of dural perivascular nociceptors, thus leading to migraine pain. Studies have sought to address how dural afferents become activated/sensitized by using either in vivo electrophysiology, imaging techniques, or behavioral models, but these commonly require very invasive surgeries. This protocol presents a method for comparatively non-invasive application of compounds on the dura mater in mice and a suitable method for measuring headache-like tactile sensitivity using periorbital von Frey testing following dural stimulation. This method maintains the integrity of the dura and skull and reduces confounding effects from invasive techniques by injecting substances through a 0.65 mm modified cannula at the junction of unfused sagittal and lambdoid sutures. This preclinical model will allow researchers to investigate a wide range of dural stimuli and their role in the pathological progression of migraine, such as nociceptor activation, immune cell activation, vascular changes, and pain behaviors, all while maintaining injury-free conditions to the skull and meninges.

The most notable symptom of migraine is severe head pain, and it is hypothesized that this is mediated by sensory neurons innervating the meninges. Here, we present a method to locally apply substances to the dura in a minimally invasive manner while using facial hypersensitivity as an output.

The cranial meninges, comprised of the dura mater, arachnoid, and pia mater, are thought to primarily serve structural functions for the nervous system.  ...

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

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