To explore the relation between acupoint sensitization and alterations of electrical signals of dorsal root ganglion (DRG) neurons from the perspective of ion channel dynamics in myocardial ischemia (MI) model mice.

Sixty-four male C57BL/6J mice were randomly assigned to control and model groups (n=32/group). The MI model was established by ligation of the left anterior descending (LAD) of the coronary artery. The MI was monitored by observing changes of ST segment of the standard limb-lead Ⅱ of electrocardiogram (ECG-ST_Ⅱ). 2, 3, 5-triphenyltetrazolium chloride (TTC) and H.E. staining were used to evaluate the infarct area and histopathological changes in the myocardial tissue. Evans blue (EB) staining was administered via tail vein injection to observe the location, distribution, and quantity of exudate points on the body surface in both groups. Nociceptive changes were evaluated by measuring mechanical pain and thermal pain thresholds. Subsequently, electrophysiological assessments were conducted in vitro to evaluate whole-cell membrane currents, intrinsic excitability, and Na⁺ channel current variations in different types of DRG neurons.

Compared with the control group, the ECG-ST_Ⅱ segment in the model group was significantly elevated (P＜0.0001), suggesting an induction of MI. TTC and H.E. staining showed a significant increase in the MI area (P＜0.001), accompanied by evident histopathological features such as myocardial fiber damage and inflammatory cell infiltration. The number of EB exudate points was significantly increased (P＜0.01), mainly distributing in the skin innervated by the T1—T5 spinal cord segments, as well as in the acupoints of “Feishu” (BL13), “Jueyinshu” (BL14) and“Xinshu” (BL15). Mechanical retraction reflex thresholds of the left upper and lower limbs of the model group significantly reduced (P＜0.0001), and the thermal retraction latency of the left upper and lower limbs were notably shortened in the model group (P＜0.01, P＜0.0001), and the mechanical pressure pain threshold on the left dorsal skin significantly reduced (P＜0.001). The DRG medium-sized neurons displayed an obvious decrease in rheobase (P＜0.05), along with a notable increase in whole-cell membrane current, spike number, and average instantaneous frequency (P＜0.05), reflecting enhanced intrinsic excitability. The activation curves of TTX-R/TTX-S sodium channels shifted towards hyperpolarization (P＜0.001), while the inactivation curves moved towards depolarization (P＜0.01).

DRG medium-sized neurons in the T1—T5 spinal cord segments may influence the acupoint sensitization of the body surface by modifying their Na⁺ channel kinetic properties.

To explore the relationship between sensitization points of the body surface and the expression of pituitary adenylate cyclase activating polypeptides (PACAP) in myocardial ischemia (MI) mice, so as to reveal the underlying mechanisms of acupoint sensitization from the perspective of molecular biology.

Male C57BL/6J mice were randomly divided into control and model groups (28 mice/group). The MI-induced visceral pain model was established by intraperitoneal injection of isoprenaline (ISO, 160 mg/kg). The mice of the control group received intraperitoneal injection of the same dose of normal saline. Six days after modeling, the Evans blue (EB) dye was injected into the tail vein of mice to observe the distribution and quantity of the plasma extravasated EB points at the body surface. Meanwhile, the mechanical pain threshold (MPT) was measured to evaluate the level of pain sensitivity in the activated area on their body surface and left forelimb and hindlimb, respectively. Hematoxylin-eosin (H. E.) staining was used to evaluate the morphologic and pathological changes of the heart tissue in the two groups. Then, the expressions of PACAP in the thoracic (T)1—T5 dorsal root ganglia (DRGs), spinal cord and skin in the dominant area of body surface were detected by Western blot and immunofluorescence staining, respectively.

Compared with the control group, the heart tissue of the model group was hypertrophic and the myocardial tissue showed obvious inflammatory cell infiltration and fibrosis. In addition to these pathologic changes, the number of EB exudation points on the body surface was significantly increased (P<0.01), and was mainly distributed in the innervated region of T1—T5 segments of the spinal cord, and the MPT of these EB exudation points was lower than that of non-exudation points (P<0.01). At the same time, the MPTs of left forelimb and hindlimb were significantly decreased in the model group (P<0.001). More importantly, the level of protein and positive expression of PACAP were significantly higher in the model group than those in the control group, which was observed in the innervated body surface, spinal cord and its DRG neurons of T1—T5 segments (P<0.01, P<0.001, P<0.05).

ISO injection resulted in histological lesions and cardiogenic referred pain on the body surface after the formation of MI in mice. The expression of PACAP in the body surface of the sensitization points, the corresponding T1—T5 segments of spinal cord and DRG neurons were significantly increased, which may partly explain the reason for acupoint sensitization in the animal model of visceral pain.

To elucidate the relationship between the auricular point stimulation and the activity of glutamatergic neurons in the anterior cingulate cortex (ACC) from the perspective of intrinsic excitability plasticity of neurons in mice with posttraumatic stress disorder (PTSD), so as to explore the underlying mechanisms of acupuncture at auricular points in improving emotional diseases induced by PTSD.

C57BL/6J male mice were randomly divided into control, PTSD model, sham electroacupuncture (EA), and EA groups, with 5 mice in each group. The glutamatergic neurons were labelled by injection of AAV2/9-CaMK Ⅱ α-EGFP viral fluid into the bilateral ACC. Fourteen days after the injection, the PTSD model was established by single prolonged stress (restraint stress, forced swimming, ether exposure) and plantar electrical shock. EA (2 Hz/15 Hz, 1 mA) was applied to bilateral "Xin" points and the center of auriculae of auricular concha area for 30 min, once daily for 7 days. Mice of the sham EA group were anesthetized for 30 min per day for 7 days, but no EA treatments were given. The anxiety-like behavior of mice was evaluated by open field (OF) and elevated plus maze (EPM) tests after 2 weeks of modeling. The whole-cell patchclamp method was used to record the intrinsic excitability level of ACC glutamatergic neurons.

Compared with the control group, the dwell time and locomotor distance in the central area of the OF, the dwell time and entry times percentage in the open arms of the EPM in the model group were significantly reduced (P < 0.001, P < 0.01). At the same time, the intrinsic excitability of ACC glutamatergic neurons in model mice was suppressed, presenting as enhanced rheobase currents (P < 0.01) and decreased spike number (P < 0.05, P < 0.01, P < 0.001). Compared with the model group, the dwell time and locomotor distance in the central area of the OF, the dwell time and entry times percentage in the open arms of the elevated plus maze were significantly increased (P < 0.001, P < 0.01), and the intrinsic excitability of ACC glutamatergic neurons was significantly improved, presenting as reduced rheobase currents (P < 0.05) and an increased spike number (P < 0.05) in the EA group. Compared with the sham EA group, the dwell time and locomotor distance in the central area of the OF, the dwell time and entry times percentage in the open arms of the EPM were significantly increased (P < 0.001, P < 0.01) in the EA group, and the intrinsic excitability of ACC glutamatergic neurons was significantly improved, presenting as reduced rheobase currents (P < 0.01) and an increased spike number (P < 0.05).

EA of auricular points can effectively alleviate the anxiety-like behavior of PTSD mice and increase the intrinsic excitability level of glutamatergic neurons in ACC, which may be one of the neural mechanisms of auricular point stimulation in the treatment of emotional diseases.