preparation and inoculation of trypanosoma evansi in mouse to evaluate allodynia and pain

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

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.

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.

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.

preparation-inoculation-trypanosoma-evansi-mouse-to-evaluate

Research

JoVE Journal

Immunology and Infection

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.

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 (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.