Name: Author Spotlight: Assessing Pain and Promoting Animal Welfare in Laboratory Animals Infected with Trypanosoma evansi
Uploaded: 2024-07-19T12:30:00
Description: 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 ...

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) (protoc...

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>

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 Trypanosoma evansi

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

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 ...