Use of Environmental Enrichment as a Clinical Methodology During Acute Experimental Infection by Trypanosoma Cruzi

One critial point in the Laboratory Animal Science (CAL) is the non-invasive evaluation of Animal Welfare (BEA). The detection (predictive or diagnostic) of the presence of suffering in mice when used for scientific purposes is a gap. The manuscrip aim to create a technical-scientific form to preditive presence of suffering in mice lab, based on the principle of Refinement of the 3Rs and BEA monitoring. Our methodology was based in usual environmental enrichment the objects, the trapeze. From these observations, our study hypothesis emerged, which was, the creation of the #1 Trap prototype and the measurement through the daily counting of the number of trapeze use during 15 minutes. Experimental acute infection by Trypanosoma cruzi was experimental model in Swiss Webster mice. Our results described that, after division into four groups: [N] without infection and [Inf] mice infected and without treatment. The predictive statistical difference was observed on the 11 th day post infection (dpi) when compared ([N]: 72±18.5 Events number/15min) and ([Inf]: 32±13.1 Events number/15min) (p ≤ 0.05). The 21 st dpi was the most severe time point ([N]: 75±17.3 and [Inf]: 11±5.1 Events number/15min, respectively) (p ≤ 0.05). So, our results suggest the #1 Trap prototype has predictive and monitoring capacity for experimental disease course. It makes it possible to prevent suffering and preservation of mice welfare during acute experimental model.


Introduction
Despite detailed studies on the genus Mus, or Mus musculus species, their genealogy, phylogeny, and historical aspects of the lineage origin currently used in the laboratory, including classical inbred lineages, the lack of documentation and scientific reports become imprecise in explaining exactly which species (or subspecies). It is known that the first lineage developed in the laboratory was DBA isogenic [1]. Outbred stock mice (nonisogenic) Swiss and CD-1are the best known colonies. Interestingly, its origin is related to 200 mice from the Pasteur Institute (France) by Amédée Borrel provided by an unknown person. These animals were provided to André de Coulon of the Anticancereux Romand Center in Lausanne (Switzerland) [2].
In 1926, Clara J. Lynch, an oncologist researcher of the Rockefeller Institute (New York) imported two male and seven female mice from André de Coulon. In the laboratory all animals derived from these mice were known as the "swiss" a nickname used by Lynch in her laboratory. Lynch also collaborated and gave some of his new mouse, of the "swiss" to other researchers, both internally and to other institutions [3].
In conclusion, mice lab or Swiss Webster is a complex and extremely interesting animal. All knowledge about this animal, be it about your life in the wild or in animal facilities, is essential for Welfare condition and the presence discomfort and suffering with fast recognition. According to the Brazilian legislation, it is imperative to know the biology, physiology and behavior of these animals in order to simulate their natural conditions in animal facilities [4]. Moreover, the behavioral characteristics knowledge of these animals, promote empathy between the handler and the animals. Which increased maintenance the laboratory mice with high life quality [5].
The aim of our study was to evaluate the effectiveness of an environmental enrichment (EE) object, the Trapezium, for the assessment of animal health quality during, mainly in biomedical tests. So, having a large experience in experimental infection by Trypanosoma cruzi in Swiss Webster mice, we decided to use this experimental model to evaluate the effectiveness of the new methodology [6].
In resume, The experimental model formed by Swiss Webster mice (18 to 20 grams) and T. cruzi Y strain (inoculum of 1x10 4 parasites/ml via the i.p. route) is characterized by a relatively fast course of the experimental acute phase [7,8]. Infection by the infective blood form of T. cruzi, during the first week, starts its pathogenesis, however without blood parasites presence (parasitaemia). Standard model demonstrates the "parasitemia peak", that is, the maximum number of parasites in the bloodstream at the 8th days post infection (dpi). Around the 15th dpi, there is the so-called critical point of acute myocarditis, where there is the presence of parasitic replication and activated inflammatory response. So between the 15 th and 21 st dpi is the most clinically critical phase for the animal. Probaly due to the heart and cardiovascular involvement. Observed, change in body posture, prostration, piloerection and cachexia. In some cases, we can also observe other clinical signs, such digestive system alterations. The acute mortality expected arround 60% of animals occurs due to the severity of acute myocarditis and its cardiovascular consequences.
However, 40% of infected animals survive. Between at 28-30 dpi, we have the characterization of the acute phase, with the absence of parasites in the blood, remission clinical symptoms and the beginning of the chronic phase [9].
Although other EE objects, such as the formation of nests [6] can be used as objects for evaluating the BEA in several lineages, we decided use of the Trapezium. The main reason for choosing this object were: a) From the age of 5 weeks of life (relative to the body weight to be infected by T. cruzi) Swiss Webster mice make intensive motor and exploratory activity and high use of the Trapezium; b) Easy observation animal use of the object and fast to count of the events number in the Trapezium use; and c) Mice with discomfort and suffering, showed hipoactive behavior and decrease motor and exploratory activity.
Finally, the main objective for the achievement of this manuscript how apply a technical-scientific methodology (without personal subjectivities) and able demonstrates, through statistical results, impairment of clinical mice conditions [10]. Creation of a non-invasive parameter adequate of evaluating groups mice and without manipulation. This initiative is highly important to avoid murine model suffering and pain during trials and the reseacher decision of the animal end point. Photoperiod of (12h day/12h night). The floor used wood shavings with exchange for twice a week. In addition, animals were offered filtered water and autoclaved feed ad libitum. All procedures

Parasites and Infection
The Y strain of T. cruzi was maintained by passage in vivo, and the trypomastigote forms were isolated from the bloodstream as previously described [7]. The parasites were resuspended in phosphate buffered saline (PBS) and counted in a hemocytometer.

Parasitological Parameters
Parasitemia (parasites count in the bloodstream) was determined daily from 5 to 30 o dpi using the Pizzi-Brener method (Brener, 1962). Mortality was noted daily and the cumulative

Non-Invasive Parameters Food Consumption
Feed/water consumption was measured weekly (0, 7, 14, 21 and 30 o dpi). Food consumption was performed by the difference between the weight (grams)/volume (milliliters) offered (300 g/300 ml) and that removed and calculated after 07 days in each cage. The estimate of individual consumption was calculated using the formulas: Cons total = weight or added volume -weight or volume, after 7 days. Then the individual consumption estimate was: Cons ind = Cons total / number of animals per cage / number days.

Trap #1 Protocol use
The mice were received 5 days before the start of the tests. They were placed in their respective groups, in number 4 mice, after temporary individual marking (c1 to c4). We used a digital timer

Record Tape
Data collection was performed by filming outside the ventilated shelf by two portable cameras, with a frontal and lateral plane, using Sony HDR -PJ760® and Sony HDR -XR550® digital cameras.

Statistical Analysis
Initially, we will evaluate, using Excel and Prism, the sum, mean, percentage and standard deviation (±SD) of the data obtained. We defined it as a significance factor (p ≤ 0.05) under the Student T statistical test. These results will be confirmed by applying the Mann Whitney non-parametric statistical test with the same significance value.

Results
The results of the parasitological parameters (   Figure 3C).  The Trapezium is an object used for environmental enrichment in mouse cages. In our routine we observed that sick animals, or with some kind of compromise in their harmonious social relationship, there was a decrease in the use of the trapezium.
For this reason, we studied to structure a protocol that could be applied in experimental models and capable of predicting the onset of animal discomfort and suffering and/or diagnosing the animals that need to be removed from the trial and promoted end point ( Figure 5).  models, in our case, the acute chagasic. As already described, the group [N: blue circle] demonstrates that mice use the Trapezium constantly and continuously. However, the evolution of the disease, the presence of discomfort and suffering promotes hypoactivity to the animals, gradually losing interest in its use. Furthermore, it is directly related to health status, as animals surviving T. cruzi infection increase their interaction with the Trapezium [InF: red circle]. The asterisk illustrates a significant decreased between [Inf] and [N] (p ≤ 0.001). Statistical significance calculated using Student's T Test and Mann Withney test. We express the ±DP by the bars at each point.

Discussion
Our discussion will be based on the demonstration of the efficiency of the prototype #1Trap in monitoring the BEA commitment of mice used for didactic or scientific purposes (experimental models).
Our first point is that so far, several studies have sought to assess subordinate [11].
The second point we would like to question is the classic test of corticosterone dosage as a direct relationship to the animal's discomfort and suffering. We believe that the interpretation of results is often completely wrong, in addition to being invasive in itself. The Mus mus musculus species, or as we believe, the Mus mus laboratorius, has a fight and flight system intrinsic to its preservation [12,13]. Then the stress response and adaptation system will be activated quickly, depending on the time and intensity of the stressful stimulus. Therefore, we must be careful when interpreting a single dose of corticosterone is a diagnosis of pain and suffering. We need to analyze that corticosterone itself has the capacity for negative feedback in the reaction axis to stressors, in short they would be: a. Stressor factor; b. Activation of the HPA axis with the release of CRF; c. ACTH release and activation of the adrenal cortex and therefore the release of corticosterone that will act in various areas of the body, including the brain and neuromodulation in a stressful situation.
So everything depends on the relationship between three primary factors: III. If the stressor is within the animal's adaptive capacity, there will be a balance, which in the short medium term will tend to decrease serum corticosterone levels.
Therefore, the dosage of corticosterone, from our point of view, as a stress marker, will only be valid if: i. In a situation characterized as stressful, with weight loss, hyper or hypoactivity, corticosterone levels remain low; ii.
The continuous elevation of corticosterone, demonstrating that the animal is not able to adapt to the stressful situation, whether due to the intensity of the stressor or its resilience to any type of stress induction [14].
At this point, the question is right, which is the differential factor in our study. The evaluation of the experimental model, with pathogenicity with a cardiac focus, but profoundly systemic and of a relatively long course [15], to the point that we can assess the animals daily, where the pathogenesis begins to affect the animal's health, or better, to promote impairment of the BEA and of your health condition?
Second prototype #1 Trap at 11 th dpi. After the parsitaemia peak, we began to observe a decrease in the use of the trapezium, however at 11th dpi the difference between the animal without infection [N] and healthy and the animals infected with T. cruzi [InF] is statistically significant. Making a parallel between the pathogenesis (still not fully clarified) the pathogenesis of acute experimental infection by T. cruzi, the clinical symptoms of the animals and the use of the trapezium, we can make the following description: In all groups, the use of the trapezium is something that promotes BEA to the animal, being an efficient environmental enrichment. After infection, animals [InF] undergo an acute kidney injury, but mild and transient, which does not promote clinical signs, but activates RAAS that after the peak of parasitemia will act together, aggravating acute myocarditis [16]. This picture can be observed, starting at the 11 th dpi and a marked drop until the 15 th dpi, a period where the parasites are in their greatest multiplication activity in the cardiac muscle and the greatest response At this point, the question is right, which is the differential factor in our work [17]. The evaluation of the experimental model, with a pathogenesis with a cardiac focus, but profoundly systemic and with an immunological course, in an attempt to eradicate the infection. Then, from the association of cardiovascular compromise, with attempted compensation, and now the compromised heart, the mouse began to evolve into a clinical picture of cardiogenic shock, as observed by the absence of trapezium use between 18th and 23rd dpi and clinically cachectic effects [18]. of exacerbation of the immune response, mainly of TNF (called by Tarleton, 1988) as cachexin. So depending on the severity and susceptibility of the animal, mortality started at the 14th dpi, however between the 16th and 20th dpi are the periods of greatest severity, that is, exactly as demonstrated by the use of the trapezium [19]. close our discussion, convinced that, despite a simple methodology, it is highly efficient and can be better developed as a brazilian technology, affordable and most importantly capable of predicting during the course of a systemic pathology the moment at which the animal begins to present discomfort and pain and suggest based on data (values) the best time for a end point.

Conclusions
The mouse is an extremely interesting and complex species.
There are 11 to 14 million people live with human beings and have adapted to the most varied situations possible. Physiologically, and naturally, and accelerated, ready for the classic "Fight or Flight Response", then having a different stress threshold than ours. In short, we believe that the objective of the mouse is to live in a group, have a territory, offer food and reproduce. The most stressful factors for these animals are the abrupt change of environment, change in the group (mainly in adult males) and lack of food, especially water.
We describe this paragraph to support that we believe in the development of equipment, materials, inputs aimed at the refinement for the use of mice in the laboratory. We do not question the replacement of animals with methodologies that provide the same results. However, most of the time, reduction as an ethical concept is like saying we don't need a solution because there is no problem. Unfortunately, this is the result of the lack of knowledge about the animal model, in this case the mouse.
Our main conclusions were: a. The objective of the study was achieved with the development of prototype #1 Trap, which showed efficiency and sensitivity to determine that the decrease in the use of the trapezium as an indicator, including a predictor, of the presence of discomfort, suffering and pain, in different degrees; b. The prototype's simplicity does not interfere with its efficiency, and it can be used both in loco for observation of the researcher, as well as for filming. The difference between these two modes did not exceed 15% difference, only when the animals, in groups, had high activity. However, with the evolution of the disease and the fall in the use of the trapezium, there was no difference between counting in loco or by filming the group of animals; c. Trap#1 can be used daily, without handling the animals, totally non-invasively, without the need for individualization of the animals to perform the test and with the possibility of filming. Filming can provide further results such as: a) which individual in the group has clinical signs prior to the group; b) which individual is more severe during the course of the disease;