Repetitive Electroacupuncture Attenuates Cold-Induced Hypertension through Enkephalin in the Rostral Ventral Lateral Medulla

Abstract

Acupuncture lowers blood pressure (BP) in hypertension, but mechanisms underlying its action are unclear. To simulate clinical studies, we performed electroacupuncture (EA) in unanesthetized rats with cold-induced hypertension (CIH) induced by six weeks of cold exposure (6 °C). EA (0.1 - 0.4 mA, 2 Hz) was applied at ST36-37 acupoints overlying the deep peroneal nerve for 30 min twice weekly for five weeks while sham-EA was conducted with the same procedures as EA except for no electrical stimulation. Elevated BP was reduced after six sessions of EA treatment and remained low 72 hrs after EA in 18 CIH rats, but not in sham-EA (n = 12) and untreated (n = 6) CIH ones. The mRNA level of preproenkephalin in the rostral ventrolateral medulla (rVLM) 72 hr after EA was increased (n = 9), compared to the sham-EA (n = 6), untreated CIH rats (n = 6) and normotensive control animals (n = 6). Microinjection of ICI 174,864, a δ-opioid receptor antagonist, into the rVLM of EA-treated CIH rats partially reversed EA's effect on elevated BP (n = 4). Stimulation of rVLM of CIH rats treated with sham-EA using a δ-opioid agonist, DADLE, decreased BP (n = 6). These data suggest that increased enkephalin in the rVLM induced by repetitive EA contributes to BP lowering action of EA.

Figure 1. Timeline of experiments for four groups of rats.

Cold-induced hypertension (CIH) rats were randomly divided into EA, sham-EA or untreated hypertension groups. They were treated with repetitive EA or sham-EA at the ST36-37 acupoints, or restraint only, for 30 min twice weekly for another five weeks following staying in the cold room for six weeks. Rats kept at room temperature were restrained for 30 min twice weekly for eleven weeks. Blood pressures (BPs) in all groups of rats were evaluated weekly using tail-cuff. Twenty-four or 72 hrs after five weeks of treatment, rats were decapitated or microinjected to respectively examine the preproenkephalin mRNA levels and role of δ-opioid receptors in the rVLM.

Figure 2. Repetitive EA treatment in cold-induced hypertension (CIH).

Cold exposure increased systolic blood pressure (SBP; Panel A), diastolic blood pressure (DBP; Panel B) and heart rate (HR; Panel C) after four weeks and induced sustained hypertension by six weeks. BPs and HRs of rats in the hypertension and sham-EA groups remained elevated at 11 weeks. In contrast, elevated SBP and DBP, but not HR in the EA group was reduced after six sessions of EA and remained low throughout EA treatment. BPs and HR of normotensive rats remained stable. SBP and DBP of EA-treated CIH rats, although beginning to return to pre-treatment levels were still significantly reduced for three days following termination of EA compared to sham-EA. Note that the numbers of sham-EA, EA and normotensive rats respectively were reduced to 6, 9, and 6 rats at 72 h after termination of treatments. Values represent means ± SEM. ^, * and # indicate P < 0.05 compared with sham-EA, CIH and normotensive controls, respectively.

Figure 3. Expression of preproenkephalin (PPE) mRNA in the rVLM of rats.

Panel A: Real time PCR traces display levels of fluorescence in a logarithmic scale from four rats, one from each group. The trace crosses the arbitrary line of threshold shown as a dashed horizontal line indicated by the perpendicular arrow. The tissue sample obtained from the EA-treated rat with cold-induced hypertension (CIH) expressed a higher concentration (i.e., at lower number of cycles) of PPE mRNA, represented by the blue curve indicated by (b), compared to control curves (c), which included CIH treated with sham-EA, CIH and normotension (red, blue and light blue curves, respectively). Expression of housekeeping gene 18s (arrow a) was similar among the four groups. They had very similar cycle thresholds. Panel B: Group data show that PPE mRNA, 72 hr after termination of EA treatment, was increased relative to controls. ^, * and # indicate significant differences (P < 0.05) compared respectively with CIH treated with sham-EA, CIH and the normotensive control.

Figure 4. Involvement of rVLM δ-opioid receptors in EA modulation of blood pressure (BP) in rats with cold-induced hypertension (CIH).

Panels A,B: δ-opioid receptor blockade in the rVLM reversed EA’s action in lowering BP in CIH rats. Panels C,D: decrease in BP after activation of δ-opioid receptors in the rVLM of CIH rats treated with sham-EA. Panels A,C show original BP tracings before and after bilateral microinjection of a δ-opioid receptor antagonist (ICI 174,864; 1 mM in 50 nl) and agonist (DADLE; 10 nM in 50 nl) into the rVLM. Arrows in Panels A,C indicate sequential bilateral microinjections into the rVLM. In Panels B,D, *indicates significant differences compared to before microinjection, *P < 0.05; **P < 0.01.

Figure 5. Anatomical locations of microinjection sites in the rat.

Left panel: An original slide of the medulla oblongata (+1.44 mm from the obex) shows blue-dyed track of a microdialysis probe insertion used for injections. Ventral aspect of blue dye represents site of microinjection in the rVLM, as indicated by arrow. Middle and right panels: Composite maps displaying histologically verified sites of microinjections in the rVLM of rats. The middle panel displays a magnified area in the right panel, indicated by the box. Brain section shows composite of planes of brain stem rostral to the obex (Paxinos and Watson’s atlas). Symbols represent microinjection of ICI 174,864 (★), DADLE (◾), naloxone (▴) and injections outside the rVLM (⚪). All injections were bilateral, although for ease of display, sites are superimposed on the left. rVLM, rostral ventrolateral medulla; Py, pyramidal tract; Sp5, spinal trigeminal nucleus; NA, nucleus ambiguus; 4V, 4th ventricle.