*2.4. Animal Model*

The animal model chosen was 30 male Wistar rats (*Rattus novergicus*) aged 38 ± 2 days old and with a mean weight of 65 ± 10 g, provided by the Universidad Industrial de Santander (UIS) Health Faculty's Bioterium in Bucaramanga, Colombia. A period of eight days of acclimatization to their new environment in the animal testing laboratory at the University of Santander (UDES) was required before their usage in research. In the lab, they were housed with two animals per cage (of dimensions 24 × 37 × 24 cm), in an individually ventilated (IVC) Easy Flow cage system, with a bed of patula pine shavings, food (commercial rodent diet, Lab-Diet®) and sterilized water ad libitum. Variables such as humidity, temperature (21 ± 22 ◦C), ammonia and CO2 concentrations, and standardized dark/light cycles (lights on at 06:00 and off at 18:00) were controlled. For the experiments, animals were randomly assigned using the standard function = RAND in Microsoft Excel into six groups of six animals each. Three of these groups were controls: negative (not infected, not treated); hLIMOX-Control (not infected, treated with higher doses of LIMOX (EOs enriched in limonene and with added caryophyllene oxide)); and positive (infected, not treated). The three other groups were experimental: LIMOX (infected, treated with LIMOX); LIMOXBNZ (infected, treated with LIMOX + BNZ); and BNZ (infected, treated with BNZ). The positive control and experimental groups (LIMOX, LIMOXBNZ, and BNZ) were made up of animals infected with *T. cruzi*. The therapeutic schemes were administrated as described in Table 1 via daily, oral doses for 31 days continuously following the onset of the chronic disease phase as established through echocardiography and parasitemia absence. All experiments were carried out according to the NIH Guide for the Care and Use of Laboratory Animals to minimize animal pain and distress. The protocol used

was approved by the Bioethics Committee of the Universidad Industrial de Santander and the Ethics Committee of the Universidad de Santander (Agreement Number 010-VII of 15 and 16 May 2017).


**Table 1.** Therapeutic schemes administrated in *Trypanosoma cruzi*-infected Wistar rats.

Tryp: metacyclic trypomastigote of *Trypanosoma cruzi* clone 338Cl2 TcI; EO: Essential oil; *q. s. p*: quantity sufficient provided.

#### *2.5. Experimental Animal Model of Chronic Chagas Disease*

Animals from the positive control and experimental groups were infected using an inoculum prepared as described in the literature [10]. Briefly, 2.5 × <sup>10</sup><sup>5</sup> metacyclic trypomastigotes of *T. cruzi* clone 338Cl2 (isolated from a confirmed CCC patient), characterized as Discrete Typing Unit (DTU) TcI, were obtained from TAU3AAG media, resuspended in a final volume of 100 μL of phosphate buffer saline-glucose (PBS-G), and injected into the intraperitoneal cavity [10]. After infection, the rats' behavior and clinical parameters (weight, ocular perimeter, position of the vibrissae, physical condition, stool moisture, and parasitemia) were monitored every three days. Parasitemia was evaluated by observing parasites in peripheral blood obtained through puncture of the ventral coccygeal vein with a 27-gauge needle. The blood was collected in a microhematocrit tube and observed between a microscope slide and coverslip under a light optical microscope at one hundred microscopic fields (100× magnification objective) as described by Brener [12].

To determine the development and progression of CCC, the rats' heart silhouettes were evaluated via Two-Dimensional Ultrasound, measuring the Maximum Length (ML) and Maximum Diameter (MD) along the long axis of the heart with a convex transducer and a DP20 (Mindray, Madrid, Spain) ultrasound machine. This analysis was performed at three specific points during the experiment: (i) one day prior to infection to establish baseline measurements; (ii) between 60 and 70 days after infection (d.a.i) to determine the onset of the chronic phase of the infection (presence of cardiomegaly) and to define the start date for therapy; and (iii) at the end of the treatments to evaluate the effect of the therapies applied. Animals in the experimental groups (LIMOX, LIMOXBNZ, and BNZ) received doses of the assigned treatments (Table 1) in a daily, oral scheme for 31 consecutive days, and at a specific hour (7:00 a.m.).

The test subjects were euthanized one day after the end of treatment (99 d.a.i) using an anesthetic mixture of ketamine at 90 mg/kg and xylazine at 7.5 mg/kg intraperitoneally, providing hypnosis, analgesia, and muscle relaxation (the anesthetic triad). Once reflexes such as patellar, palpebral, and corneal had ceased, an additional anesthetic dose was applied for the induction of euthanasia by anesthetic overdose. Before any further procedures were effected, death was verified by establishing the absence of vital signs and reflexes (corneal, eyelash, and rhythmic breathing). Blood was then collected by cardiac puncture to measure biomarkers of liver (ALT and AST) and kidney (BUN and creatinine) function and to conduct a hemogram. Transaminases, BUN, and creatinine assays were carried out from serum samples using commercial Ccromatest kits purchased from Linear Chemicals S.L.U. (Montgat, Spain), in accordance with the manufacturer's instructions; and measurements were made using a URIT-8030 Automated Chemistry Analyzer. EDTA

whole blood samples were used for hematological analysis. Total blood cell counts were performed using established URIT-3000Vet hematology equipment. Blood cell morphology and differential leukocyte counts were made on slides stained with Wright.

During the animals' necropsies, photographic and weight records were collected from solid organs (spleen, liver, heart, and kidney), which were sectioned for subsequent immunohistochemical (heart tissue) and histopathological (kidney, liver, and spleen) analysis. These sections were placed into tubes containing 10% neutral formalin stabilized for 72 h, and then embedded in paraffin blocks. A fragment of cardiac tissue from the apical region of the left ventricle was collected in RNAlaterTM storage reagent (Sigma-Aldrich, St. Louis, MO, USA), for parasitemia determination by real-time polymerase chain reaction (qPCR). The remainder of the heart tissue was then divided by cross sectional cuts, with each half being randomly assigned for histologic or immunohistochemical analysis. To guarantee data blindness, all procedures (control of parasitemia, echocardiography, euthanasia, and sampling) were performed by independent veterinary personal unaware of which group the animal had been assigned to. The microscopic study was also performed blind by an expert pathologist. The animal remains were properly handled in accordance with the safety and environmental responsibility protocols established by the Universidad de Santander, and disposed of by incineration in accordance with the same.
