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

Summary

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.

Abstract

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.

Introduction

Trypanosoma evansi is the etiological agent of the disease called "surra" or "mal-das-cadeiras" in South America^1,2. It commonly affects equines and cattle, but also wild fauna, being transmitted by hematophagous bats, tabanids, and stomoxes fly bites^1,3. 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 region^2,4,5,6.

The expression of allodynia and pain in infected animals during the course of the disease is still a new topic^2,7. 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 protocol^5,6. 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 experiments^2,8,9. This device is used to assess tissue sensitivity to mechanical stimulation: once the apparatus touches the paw of the animal, through a 20-200 µL 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 infection^2,10.

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.

Protocol

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 ± 1 °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.

1. Turn on the water bath equipment and adjust it at a constant temperature of 37 °C.
2. Take a microcentrifuge tube containing the preserved blood sample out of the ultrafreezer (-80 °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.
3. 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 × 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.
4. 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 ½" needle coupled to an insulin syringe.

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.

1. 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.
2. Connect the EvF apparatus to the chosen electronic device via Wi-Fi to transfer and save the obtained data.
3. Insert a 10 µL polypropylene tip into the EvF apparatus cone and set the readout to zero (0.00 g) by pressing the button with a paw symbol.
4. 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.
5. Reset the readout to zero and get ready to take a new measurement by simply pressing the button with the paw symbol again.

3. General considerations for mechanical threshold assessments

1. Perform all experiments in a quiet room with controlled temperature at 21 °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.
2. 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.
3. Place each mouse gently inside an individual chamber made of polymethyl methacrylate (PMMA) with a perforated top placed on a 5 mm² mesh floor stand^2,8.
4. Allow the mice to acclimate for 30 min in the PMMA chambers prior to assessing both baseline and experimental mechanical thresholds².
5. 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.
6. 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.

4. Baseline mechanical threshold assessment and mouse response to stimuli

1. 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.
2. 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.
3. 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 jumping^2,8,9. For visceral evaluation, look for a sharp retraction of the abdomen, immediate licking or scratching of the probed site, or four-paw jumping^2,11.
4. 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
5. 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.
6. 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 g^2,8.
   ​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.

5. Experimental right hind paw and visceral allodynia mechanical threshold assessment

1. Assess the right hind paw or visceral allodynia mechanical threshold 1 h after the infection for day 0 evaluation.
2. 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.
3. 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.
4. Perform a normality test to check the normal distribution of the data and further appropriate statistical analysis of the collected data.

6. Mouse grimace scale assessment

1. 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.
2. 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).
3. Observe the mouse'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.
4. Observe the mouse's cheeks. Classify normal cheeks as the absence of pain while the presence of a bulge on both cheeks represents obvious pain.
5. Observe the position of the mouse'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.
6. Observe the mouse'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.
7. 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.
8. 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.

Representative Results

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 infection (Figure 1B).

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 ± SD of ten animals for each experimental group. Statistical analysis: Student's t-test, one unpaired analysis per time point. Asterisks show significant differences between experimental groups considering the same analyzed time point (p < 0.05). Please click here to view a larger version of this figure.

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's t-test, one unpaired analysis per time point, considering as significant a value of p < 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 infection^2,3,7,12. 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.

Discussion

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 infections^2,4,5,6. 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 events^15,16. 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 events^17,18.

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 days^2,6. 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 perform^8,19.

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 behaviors^10,20. Furthermore, to correctly evaluate mice'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 achieved^2,8.

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.

Materials

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