[TH-positive neurons in the rostral ventrolateral medulla mediating electroacupuncture to improve intestinal motility...

To investigate the role of tyrosine hydroxylase (TH)-positive neurons in the rostral ventrolateral medulla (RVLM) in the effects of electroacupuncture (EA) on intestinal dysmotility in mice with functional constipation (FC).

Firstly, transsynaptic tracing virus was employed to identify brain regions projecting to the proximal colon. Secondly, eighteen C57BL/6J mice were randomly assigned to the normal, model, and EA groups, with 6 mice in each group. FC model was established by oral administration of loperamide hydrochloride. Mice in the EA group received EA at "Tianshu" (ST25) and "Shangjuxu" (ST37), 5 days per treatment course for 2 courses. After intervention, the defecation status of mice in each group was observed, and the fecal water content was determined, the time for the first black stool excretion was recorded, the small-intestinal propulsion rate was measured. Serum and colonic TH levels were determined by ELISA. TH/c-Fos co-expression in the RVLM was assessed by immunofluorescence staining. Subsequently, 12 C57BL/6J mice were randomly divided into the control virus+clozapine-N-oxide (CNO) group and inhibitory-virus+CNO group, with 6 mice per group. Another 18 C57BL/6J mice were randomly assigned to the control virus+CNO group, EA group, and excitatory-virus+CNO+EA group, with 6 mice per group. The corresponding viral vectors were injected into the RVLM, followed by intraperitoneal administration of CNO (1 mg/kg) 21 d after viral expression. Mice in the EA and excitatory-virus+CNO+EA groups received EA treatment as described above 30 min after the administration of CNO. Following completion of EA treatment, fecal parameters and intestinal motility were assessed as described above. Finally, rAAV-hSyn-GCaMP6s virus was injected into the RVLM of another 18 C57BL/6J mice. After 21 days of viral transduction, mice were randomly assigned to normal control, model control, and model+EA groups (6 mice per group). Mice in the model+EA group received EA treatment as described above. Post-EA intervention, multichannel fiber photometry was employed to record calcium signal dynamics in RVLM neurons.

Neuronal fibers in the RVLM formed synaptic connections with the proximal colon. Compared with the normal group, the model group exhibited prolonged first black stool expulsion time, reduced total fecal pellet counts, decreased fecal water content and small intestinal transit rate (P<0.001, P<0.01), and elevated TH levels in serum and colon (P<0.01). TH and c-Fos, as well as TH/c-Fos co-expression positive cell counts in the RVLM, were all significantly increased (P<0.001). Compared with the model group, the EA group showed shortened first black stool expulsion time, increased total fecal pellet counts, elevated fecal water content and small intestinal transit rate (P<0.001, P<0.01), and reduced TH levels in serum and colon (P<0.01). TH and c-Fos expression, as well as TH/c-Fos positive cell counts in the RVLM, were all significantly decreased (P<0.001). Compared with the control virus+CNO group, the inhibitory-virus+CNO group exhibited shortened first black stool expulsion time, increased total fecal pellet counts, and elevated fecal water content and small intestinal transit rate (P<0.001, P<0.01). Compared with the control virus+CNO group, the EA group showed shortened first black stool expulsion time, increased total fecal pellet counts, elevated fecal water content and small intestinal transit rate (P<0.001, P<0.01). Compared with the EA group, the excitatory-virus+CNO+EA group showed prolonged first black stool expulsion time, reduced total fecal pellet counts, and decreased fecal water content and small intestinal transit rate (P<0.001, P<0.01). Compared with the normal control group, the model control group exhibited significantly increased Ca²⁺ activity in the RVLM (P<0.001), whereas EA significantly reduced RVLM Ca²⁺ activity compared with the model control group (P<0.001).

EA at ST25 and ST37 may improve intestinal dysmotility in FC mice by suppressing the activity of TH-positive neurons in the RVLM.