Effects of Ang-(1–7) and A-779 Into the LPA on Intravesical Pressure, Arterial Pressure, Heart Rate, and Renal Conductance in the Female Rats (n = 6/group)

As shown in Figure 1, first of all, the rats under ketamine + xylazine anesthesia underwent an implant of a guide cannula into the LPA, using a stereotaxic apparatus. Five days later, after recovery of the stereotaxic surgery, the animals were anesthetized with 2% isoflurane in 100% O₂ and submitted to the cannulation of the femoral artery and vein for PAP, MAP, and HR recordings, and infusion of drugs, respectively. A miniaturized Doppler flow probe was placed around the left renal artery for indirect blood flow measurement. Polyethylene tubing was also inserted into the urinary bladder for IP recordings. Rectal temperature was maintained between 37 and 38°C, using a heating pad. The animals were anesthetized with 2% isoflurane in 100% O₂ during the whole experiment and were unresponsive to a noxious toe pinch. This experimental approach was carried out as previously reported by Cafarchio et al. (2016, 2018, 2020) and Magaldi et al. (2020). A steady level of arterial pressure was maintained under anesthesia. The rats were kept in supine position in order to avoid pressure of abdominal organs on the urinary bladder, which could affect the IP values. After baseline PAP, MAP, HR, renal blood flow, and IP recordings for 15 min, Ang-(1–7) (5 nmol/μL, catalog # A9202, Sigma Aldrich, St, MO, United States) or saline (vehicle, 1 μL) or A-779 trifluoroacetate salt (50 nmol/μL, a Mas receptor antagonist, catalog # SML1370, Sigma Aldrich, St, MO, United States) was injected into the LPA unilaterally, and all the parameters were recorded for additional 30 min. In another group of the rats, after the baseline recordings, saline (vehicle, 1 μL) or A-779 (50 nmol/μL, 1 μL) was injected bilaterally into the LPA, and the effectiveness of Mas receptor blockade was evaluated by Ang-(1–7) injections (5 nmol/μL) bilaterally into the LPA at 15-min post-A-779 injections into the LPA, and all the parameters were recorded for at least 30 min. At the end of the experiment, a 4% Chicago Sky blue dye (1 μL) was administrated in the injection sites. An overdose of sodium thiopental (170 mg/kg, i.v.) was used to euthanize the animals. The brains were removed for posterior histological evaluation. Figure 2 shows the site of dye deposition in the LPA. Only the animals with histological confirmation of microinjection sites in the LPA were considered in this study.

A flowchart depicting the experimental design details.

Photomicrograph of histological sections (coronal), showing the drug injection site into the lateral preoptic area unilaterally (A) and bilaterally (B) marked with 4% Chicago Sky Blue dye (1 μL). A schematic representation of the lateral preoptic area at –0.84 mm from bregma (C) and at –0.96 mm from bregma (D) is shown in the brain sections according to Paxinos and Watson (2009). The arrows indicate the location of the LPA. CP, caudate nucleus/putamen; GP, globus pallidus; LPA, lateral preoptic area; LV, lateral ventricle; SFO, subfornical organ; 3V, third brain ventricle. Amplification: 160×.