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 prevalency studies in various locations around the globe, followed by the search for specific biomarkers for a 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 a 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 host's clinical sites and behavior, as well as the parasite's behavior inside infected organism.

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 dilutions 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. 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 forepaw jumping. For visceral assessment, observe sudden abdominal retraction, immediate licking or scratching of the probed area, or forepaw 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. 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.

To begin, inject the experimental mice intraperitoneally with 0.1 milliliters of diluted blood solution infected with Trypanosoma evansi, or PBS for the control group. Before conducting a grimace assessment, ensure each mouse is clearly visible inside the chamber. Examine every mouse for the absence of lesions on their limbs, abdomen, and face, as well as any changes in their coat.

Observe the orbital area of the mouse. The open eyes of the mouse indicate the absence of pain, while closed eyes indicate noticeable pain. Then examine the mouse's nose area.

Categorize a normal nose as an absence of pain, while a bulge on the nose bridge signifies noticeable pain. Examine the mouse's cheeks and classify unaltered cheeks as painless. The presence of bulges on both cheeks indicates noticeable pain.

Next, observe the positioning of the mouse's ears. Round ears indicate no pain, while ears rotating outwards or backward forming a pointed shape away from the face, indicate obvious pain. Examine the mouse's whiskers.

Classify whiskers with the natural downward curve as the absence of pain, while whiskers either pulled back against the cheek or pulled forward signify noticeable pain. Infected mice displayed a notable reductions in mechanical threshold on the right hind paw by day three post infection, persisting lower than controls for the subsequent two days. Similarly, abdominal tactile sensitivity exhibited visceral allodynia in infected mice at day three post infection, which persisted for the following two days compared to controls.