This study introduced a surgical manipulation to expose the thoracic DRG in anesthetized mice for in vivo calcium imaging along with the synchronized electrocardiogram recording. By doing this, we try to answer whether acupuncture at PC6 on the foramen could activate DRG neuron and simultaneously regulate the electrocardiogram. Techniques currently used in acupuncture studies include in vivo or vitro electrophysiology, neural tracing, and various strategy combined with optogenetics and chemogenetics.
The in vivo calcium imaging introduced in this study helps to understand better the activities of DRG neuron populations induced by acupuncture, all in different animal models, via dynamic and real-time recordings. Due to the physiological curvature of the thoracic spine at thoracic one to five vertebral segments, thoracic DRGs are quite difficult to expose. Besides, the influences of cardiac and pulmonary rhythms cause the fixation of thoracic DRGs to be challenging.
Otherwise, respiratory anesthesia and condition maintenance of the mice for hours are not easy. In vivo calcium imaging reveals the activity of specific neuron populations by using genetic engineering mice. This is the first time the thoracic DRG neurons have been observed in vivo.
This approach realizes observation of neuronal activities produced by somatic or visceral stimulations as well as their crosstalk. Our laboratory has established methods for in vivo calcium imaging of thoracic and lumbar DRG, and in the future, we will review the receptors related to initiating factors of acupuncture, accupoints'hypersensitivity, interactions between somatic acupuncture stimulation and visceral inputs. After performing a tracheotomy on an anesthetized mouse, place the mouse in a prone position on a heated pad.
Make a two centimeter longitudinal incision in the center of the nape of the neck, extending from the cervical six to thoracic three vertebrae. Carefully separate the fat and hibernating glands at the anterior aspect of the mouse's thoracic vertebrae, avoiding the blood vessels beneath the glands. Use spring scissors to cut through the skin and muscle layers, including the trapezius muscle.
Insert a retractor between the muscles to assist with further exposure. Remove the muscles attaching to the head clamp and the straight portion of the long neck muscles, exposing the spinous processes of thoracic two. Displace the semispinalis and spinalis muscles to expose the vertebral arch from C6 to T3.Sever the connection between the vertebral arch plate and the articular process of the thoracic one.
Use fine forceps to meticulously remove the left and right articular processes and mammillary processes of the thoracic one. Clear away the overlying connective tissue. Carefully expose either the left or right thoracic one dorsal root ganglion, or DRG, ensuring the epineurium's integrity on the chosen side.
Place a small cotton ball soaked in warm saline over the exposed DRG to maintain moisture. Connect the ECG monitor with the negative pole on the right upper limb, the ground wire on the right lower limb, and the positive pole on the left lower limb. Place the mouse on the stage of the custom spinal clamp with a heating pad.
Secure the mouse using two clips attached to the articular processes of cervical six and thoracic three. Position the spinal clamp with the secured mouse under the confocal microscope. Place the 10x/0.32 long working distance air objective over the exposed thoracic 1 DRG.
Set the step size to 25 micrometers and a resolution of 512 by 512 or 1, 024 by 1, 024 pixels. Adjust the stage z-axis up and down and capture the entire thoracic 1 DRG. Apply brush stimulation to the upper limb of the mouse and assess the responsiveness of the imaged DRG neurons.
Perform peripheral nerve stimulations at PC6 stimulation using a stimulator. Under baseline conditions, most neurons in the thoracic 1 DRG did not exhibit GFP fluorescence. Somatic stimulation resulted in a rapid and transient increase in GCaMP fluorescence with an increase in the number and intensity of GFP.
Peripheral nerve stimulations at PC6 application resulted in similar changes in GCaMP fluorescence as somatic stimulation. The neurons were marked and numbered within a single thoracic 1 DRG after tracing with imaging software. Neurons showing fluorescence intensity changes exceeding 130%of the F0 threshold were considered positive responses.
The histogram displayed the different diameters of neurons responsive to peripheral nerve stimulations at PC6. The peripheral nerve stimulations at PC6 stimulation showed increased heart rate.
