ABSTRACT
OBJECTIVES: To study the regularity of central response to thermal needle stimulation of "Zusanli" (ST36) at different temperature, and to analyze the temperature difference of central responses. METHODS: Six male C57BL/6j adult mice were used in the present study. For observing activities of neurons in the hindlimb region of left primary somatosensory cortex (S1HL, A/P=0.46 mm, M/L=1.32 mm, D/V=-0.14 mm) by using a fast high-resolution miniature two-photon microscopy (FHIRM-TPM), the mice were anesthetized with 3% isoflurane (inhalation), with its head fixed in a stereotaxic apparatus, then, adeno-associated virus (AAV-hSyn-GCaMP6f-WPRE-hGHpA, for showing intracellular calcium transients in neurons transfected) was injected into the left S1HL region using a micro-syringe after scalp surgical operation. The mice's right ST36 were stimulated using internal thermal needles with the temperature being 43 â, or 45 â, or 47 â, separately. Image J software and MATLAB 2020b software were used to process the image data of neuronal calcium activity (Ca2+ signaling) in the left S1HL region, including the instant maximum calcium peak value (ΔF/F) in 2 s, instant calcium spike frequency in 2 s, short-term calcium peak value (ΔF/F) in 3.5 min, short-term calcium spike frequency in 3.5 min, calcium peak duration in 3.5 min, maximum calcium peak value (ΔF/F) at the 1st , 2nd and 3rd min, and calcium spike frequency at the 1st, 2nd and 3rd min after thermal needle stimulation. RESULTS: In comparison with the normal temperature needle stimulation, the instant intracellular maximum calcium peak value, instant calcium spike frequency, short-term maximum calcium peak value, short-term calcium spike frequency, and calcium peak duration of S1HL neurons in response to 43 â, 45 â and 47 â internal thermal needle stimulation of ST36 were significantly increased (P<0.001, P<0.01). Comparison among the 43 â, 45 â and 47 â thermal needle stimulation showed that the 45 â thermal needle stimulation was obviously superior to 43 â and 47 â thermal needle stimulation in increasing instant calcium spike frequency, short-term calcium spike frequency and calcium peak duration of S1HL neurons (P<0.001, P<0.01). The 47 â thermal needle stimulation was stronger than 43 â and 45 â thermal needle stimulation in increasing the instant maximum calcium peak value (P<0.001). The maximum calcium peak value was apparently higher (P<0.001) at the 2nd min than that at the 1st and 3rd min after 43 â, 45 â and 47 â thermal needle stimulation. No significant differences were found in the short-term maximum calcium peak value among the 3 thermal needle stimulation and in the calcium spike frequency among the 3 time points after 43 â, 45 â and 47 â thermal needle stimulation. CONCLUSIONS: S1HL neurons respond to all 43 â, 45 â and 47 â thermal needle stimulation of ST36 in mice, while more actively to 45 â thermal needle stimulation.