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 ℃, or 45 ℃, or 47 ℃, 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 ℃, 45 ℃ and 47 ℃ internal thermal needle stimulation of ST36 were significantly increased (P<0.001, P<0.01). Comparison among the 43 ℃, 45 ℃ and 47 ℃ thermal needle stimulation showed that the 45 ℃ thermal needle stimulation was obviously superior to 43 ℃ and 47 ℃ 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 ℃ thermal needle stimulation was stronger than 43 ℃ and 45 ℃ 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 ℃, 45 ℃ and 47 ℃ 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 ℃, 45 ℃ and 47 ℃ thermal needle stimulation.
Conclusions: S1HL neurons respond to all 43 ℃, 45 ℃ and 47 ℃ thermal needle stimulation of ST36 in mice, while more actively to 45 ℃ thermal needle stimulation.
目的: 研究不同温度热刺激“足三里”的中枢响应规律，分析中枢响应的温度差异性。方法: 健康成年C57BL/6j小鼠6只，采用动物脑立体定位仪定位小鼠大脑左侧初级体感皮层后肢区（S1HL），注射0.3 μL腺相关病毒，通过双光子显微镜观察其神经元钙活动荧光成像，以荧光强度显示神经元中的钙离子浓度。分别以43、45、47 ℃热刺激小鼠右侧“足三里”，使用双光子显微镜直接记录小鼠大脑左侧S1HL神经元活动，观察并比较S1HL神经元对3种温度热刺激“足三里”的响应差异性，包括热刺激后2 s内的即时最大钙尖峰值和即时钙尖峰频率，3.5 min内的短时最大钙尖峰值、短时钙尖峰频率和钙尖峰持续时间，以及3 min内的分时段最大钙尖峰值和分时段钙尖峰频率。结果: 与常温比较，43、45、47 ℃热刺激“足三里”均可使小鼠左侧S1HL神经元的即时最大钙尖峰值、即时钙尖峰频率、短时最大钙尖峰值、短时钙尖峰频率和钙尖峰持续时间升高（P<0.001，P<0.01）。不同温度热刺激比较，从即时最大钙尖峰值来看，小鼠S1HL神经元对47 ℃热刺激响应程度最高（P<0.001）；从即时钙尖峰频率来看，其对45 ℃热刺激响应最频繁（P<0.001）。从短时最大钙尖峰值来看，不同温度热刺激对其影响差异无统计学意义；从短时最大钙尖峰频率来看，小鼠S1HL神经元对45 ℃热刺激响应最频繁（P<0.001）。从钙尖峰持续时间来看，小鼠S1HL神经元对45 ℃热刺激响应更为明显（P<0.01）。从神经元响应分时段分析来看，在3 min内，43、45、47 ℃热刺激下，小鼠S1HL神经元最大钙尖峰值都在第2分钟达到最高（P<0.001）；钙尖峰频率与热刺激时段无显著联系。结论: S1HL神经元对43、45、47 ℃热刺激“足三里”均有响应，且响应存在特定温度差异性，小鼠S1HL神经元对45 ℃热刺激“足三里”响应较为活跃。.
Keywords: Neuron response; Primary somatosensory cortex; Thermal needle stimulation; Two-photon technology; Zusanli (ST36).