Dp 304

A left flank incision was made and the left renal nerve was separated. The distal end of the nerve was severed to eliminate the nerve activity from the kidney. A pair of platinum electrodes were placed under the central end of the nerve and then immersed in warm mineral oil (37 °C). The RSNA was amplified 1000 times with a 100–3000 Hz bandpass filtration with a differential amplifier (DP-304, Warner Instruments, Hamden, CT, USA). The signals were integrated using LabChart 8 software (ADInstruments, Bella Vista, NSW, Australia) at 100 ms time constant. At the end of the recording, the nerve was cut at its central end to obtain the background noise. The RSNA value was equal to the raw RSNA minus the background noise [30 (link)].

A coated tungsten microelectrode (2 cm in length) with an exposed tip of 50 μm was inserted into the fat pad containing the ARGP, and an impedance of 9–12 MΩ at 1,000 Hz was mounted on a micromanipulator. Electrical signals generated by the ARGP were recorded using a PowerLab data acquisition system (8/35, ADInstruments, Bella Vista, New South Wales, Australia), and the signals were amplified using an amplifier (DP-304, Warner Instruments, Hamden, CT, United States) with bandpass filters set at 300 Hz to 1 kHz and an amplification range of 30–50 times (Yu et al., 2013 (link)). Neural activity was characterized using the recorded amplitude and frequency that was used to quantitate neural activity. Neural activity was defined as deflections with a signal-to-noise ratio greater than 3:1 (Yu et al., 2014 (link)).

Neural activities of the left vagal nerve (LVN) and left stellate ganglion (LSG) were recorded among the three groups at the 12th week, as described previously (Zhou et al., 2016 (link); Zhang et al., 2018 (link)). All the animals were anesthetized during the neural activity recordings. Briefly, the LVN and LSG were exposed via a left vertical incision in the supraclavicular fossa and bluntly dissected free from surrounding tissues with a glass dissecting needle. Neural activities were recorded through headstage electrodes into the LVN and SLGP, and nerve signals were analyzed with the Analysis Module of Lab Chart 8.0/proV7 software (Bio Amp; ADInstruments) of PowerLab (Bio Amp; ADInstruments). An amplifier (DP-304; Warner Instruments) was used to amplify nerve signals.

The left renal sympathetic nerve was isolated via a retroperitoneal incision. The renal nerve was cut distally to eliminate afferent activity and was placed on a pair of silver electrodes [34 (link),35 ]. To amplify the nerve signals, a four-channel AC/DC differential amplifier (DP-304, Warner Instruments, Hamden, CT, USA) was used. The data were collected using a high pass filter at 10 Hz and a low pass filter at 3,000 Hz, and RSNA was integrated at a time constant of 100 ms [34 (link),35 ]. At the end of each experiment, background noise was detected after section of the central end of the renal nerve at the end of the experiment and was subtracted from the integrated values of the recorded RSNA [21 (link),34 (link),35 ]. The change in RSNA was expressed as the percent change from baseline [21 (link)]. Baseline RSNA and MAP were determined by averaging 2 min of the maximal RSNA responses after microinjection into the PVN.

Left stellate ganglion (LSG) activity was recorded for 1 min in each group. The fascia of the LSG was inserted into a tungsten-coated microelectrode, and the chest wall was connected to a group lead. A Power Lab data acquisition system (8/35, AD Instruments, New South Wales, Australia) and an amplifier (DP-304, Warner Instruments, Hamden, CT, USA) were used to record LSG-generated electrical signals. Bandpass filters (from 300 Hz to 1 kHz) and amplification (from 30 to 50 times) were set up prior to each recording. The frequency and amplitude of neural activity, defined as deflections with a signal-to-noise ratio > 3:1, were recorded. LSG activity was measured at baseline and 15 min after AMI induction.

Renal sympathetic nerve activity (RSNA) were recorded as we previously reported [10 (link),22 (link)]. In brief, a left retroperitoneal incision was made, and the left renal nerve was isolated, and cut distally to eliminate its afferent activity. A pair of silver electrodes was placed on the central end of the nerve and immersed in warm mineral oil. The signals were amplified with a four-channel AC/DC differential amplifier (DP-304, Warner Instruments, Hamden, CT, USA) with a high pass filter at 10 Hz and a low pass filter at 3,000 Hz, and was integrated at a time constant of 100 ms. The RSNA and mean arterial pressure (MAP) were simultaneously recorded with a PowerLab data acquisition system (8/35, ADInstruments, Castle Hill, Australia). Background noise was determined after section of the central end of the renal sympathetic nerve at the end of the experiment, and was subtracted from the integrated values of the RSNA.