Enhancing the Development and Growth of Infant Cerebral Palsy Rats Using Selective Spinal Manipulations

This study was approved by the Experimental Animal Ethics Committee of Yunnan University of Traditional Chinese Medicine. All experimental operations on animals followed the 3R principle of experimental animal reduction, optimization, and replacement (No. R-06202018). Healthy Sprague Dawley (SD) rats (14 males and 7 females) of Specific pathogen-free (SPF) grade with an average body weight of 250-300 g were used in this experiment. The rats were raised in the SPF animal room of Yunnan University of Traditional Chinese Medicine, Certificate number: SYXK(Yunnan)K2022-0004. All rats were housed under a 12-h light/dark cycle with a natural light-controlled environment at a room temperature of 22-26 °C and a relative humidity of 40%-50%. After 1 week of adaptive feeding, the male/female ratio was maintained at 1:2. After 1 week, the pregnant rats were housed in a single cage, fed normally, eating and drinking freely. After natural delivery, the healthy infant rats born to the pregnant rats were selected. The infant rats were divided into the Sham, Control, and Treatment groups to observe the effect of selective spinal manipulation on the growth and development of infant rats with cerebral palsy. From each group, 6 male pups were selected for the experiment. The Treatment group was treated with selective spinal manipulation from the 5^th day after birth, and the treatment was given for 6 days followed by rest for 1 day.

1. Establishing cerebral palsy model

1. Sterilize the microscope, ophthalmic scissors, forceps, suture, surgical plate, scissors, cotton swabs, and gloves required for surgery, and place them in the operating room.
2. Separate a pup temporarily from its mother on the third day of life, place it in a single frame, and transport it to the sterile operating room.
3. Measure the body weight. Include infant rats with body weight ranging from 7-9 g in the study, and exclude those who did not conform to body weight. Randomly divide the infant rats into Sham, Control, and Treatment groups.
4. Induce anesthesia with isoflurane at a concentration of 2% and maintain anesthesia at 1% using a small-animal inhalation gas anesthesia machine.
   1. Connect the small animal gas anesthetic to the power supply and open the valve connected to the induction box.
   2. Put the infant rat into the induction box, turn the concentration adjustment knob, and adjust the concentration of isoflurane to 2% to induce anesthesia.
   3. When the infant rat is unconscious, remove it from the induction box. Place it on the operation plate and close channel 1 of the anesthesia machine.
   4. Place the infant rat supine. Fix the anesthesia inhalation mask to maintain anesthesia.
   5. Open the channel connected to the anesthesia inhalation mask. Maintain anesthesia by turning the concentration adjustment knob and adjusting the concentration of isoflurane to 1%.
5. Disinfect the neck skin of the infant rat and cut a longitudinal 1 cm incision on the neck skin to expose the subcutaneous tissue.
6. Cut the skin of the left neck under a dissecting microscope, and use ophthalmic tweezers to bluntly separate the sternohyoid muscle and the left sternomastoid muscle.
   1. Find the left common carotid artery and vagus nerve, and separate the left common carotid artery and vagus nerve.
   2. In the Model group, cut off the left common carotid artery with an electric coagulation pen. Clean and suture the neck incision.
   3. In the Sham group, separate the left common carotid artery from the vagus nerve.
7. After the operation, place the infant rat in the thermostatic water bath in the prone position for resuscitation for 1 h.
   NOTE: After natural recovery, the skin color of the limbs and the whole body was ruddy. The infant rat could move its limbs autonomously and crawl independently.
8. Place the resuscitated rats of the Control and Treatment groups in a closed incubator at 37 °C in the prone position. Introduce a mixture of 5% oxygen and 95% nitrogen gas to induce hypoxia, and remove the rats after 2 h.
9. Remove the infant rat that completed the hypoxia process from the hypoxia box and place it in the prone position in a box with normal atmospheric oxygen content. Place them in a 37 °C thermostatic water bath for 1 h. After complete recovery, transfer the infant rat back to the mother's cage.
   NOTE: After recovering naturally, the skin color of the limbs and the whole body was red. Infant rat could move their limbs autonomously and could crawl independently.
10. Place the infant rat of the Sham group in one box with normal atmospheric oxygen content, and place the box in a 37 °C thermostatic water bath for 1 h. After complete recovery, put the infant rat back into the mother's cage for feeding.
    NOTE: After recovering naturally, the skin color of the limbs and the whole body was red. Infant rat could move their limbs autonomously and could crawl independently. The whole operation process was completed in the special operation room of the SPF animal room of Yunnan University of Traditional Chinese Medicine. During resuscitation and hypoxia, the operator observes the infant rat at all times to prevent death caused by improper posture.

2. Righting reflex experiment

1. On the 4^th day after birth (the 2^nd day after modeling), perform the righting reflex experiment to verify whether the model was successful (Figure 1 and Table 1).
   1. Lift the tail of the infant rat, rest its back on a horizontal board, and use the thumb and index finger to secure its belly and neck. Check if the rat can return to its normal position. The inability of the rat to return to the normal position from the abnormal position due to the loss of the righting reflex is recorded as a successful model^16,22.
   2. Record the time each group of pups takes to turn over from the supine to the prone position. Begin to record the time when the thumb and index fingers are released simultaneously in the supine position, allow the infant rat to turn to the prone position, and stop recording when the front and back paws are placed on the floor. If the infant rat could not return to the normal position for more than 20 s, record the righting time as 20 s.

3. Preparation before the operation

1. After modeling, separate the infant rat with cerebral palsy from its mother and bring it to an operating room with a constant room temperature of 22-26 °C for 2 min to adapt to the surrounding environment.
2. Wear disposable gloves to keep the palm mild and soft and the temperature between 36-37 °C.
3. Place the rat gently in the palm of the left hand. Bend the left thumb to cover the eyes of the rat for 2 min to form a dark field of vision so that the rat can adapt to the environment of the operator's palm. Use the right index and middle fingers for manipulation.
   NOTE: Covering the eyes of the infant rat can create a dark environment. This will keep the infant rat from becoming agitated because of the unsafe and uncertain environment affecting the experimental results.

4. Dividing the rats into the Control and the Treatment groups

1. On the second day after modeling (P5), verify the model by the righting reflex test and randomly divide the infant rats into the Control and Treatment groups. Perform selective spinal manipulation on the infant rats of the Treatment group.
2. Place the infant rat in the prone position. Ensure that the spine is always kept at a straight level (Figure 2A).
3. Massage the spine and both sides of the spine for 1 min using the rubbing method, and use the pressing and kneading method to the spine and the muscles on both sides of the spine for 5 min.
   1. Massage the spine and skin on both sides using the rubbing method to relax the infant rat and relieve its tension fully. Ensure that the frequency of rubbing is 100-120 times/min for 1 min after the infant rat is quiet and without agitation.
      1. Use the right index finger, middle finger, and ring finger as the contact surface, and make a circular rubbing motion on the surface skin of the cervical spine, thoracic spine, and lumbar spine from head to tail without disturbing the subcutaneous tissue.
      2. With the right index finger, middle finger, and ring finger as contact surfaces, perform circular motion on the skin surface of the trapezius muscle, superficial gluteus muscle, cervical rhomboid muscle, pectoral rhomboid muscle, latissimus dorsi muscle, and external oblique muscle in order from head to tail according to the animal anatomical standard²³, without disturbing the subcutaneous tissue.
   2. Treat the spinal column first with pressing and kneading for 5 min, followed by pressing and kneading the muscles on both sides of the spine for 5 min.
      1. Apply the kneading method to the spine at a frequency of 120 times/min for 5 min. Use the right index or middle fingers as the contact surface. From head to tail, perform circular kneading on the surface skin of the cervical, thoracic, and lumbar vertebrae, combined with downward pressing (1.77 ± 0.54 N). Ensure that the force reaches the cervical vertebrae, thoracic vertebrae, lumbar vertebrae, and supraspinous ligaments.
      2. Apply the pressing and kneading method to the muscles on both sides of the spine at a frequency of 120 times/min for 5 min. Use the right index finger or middle finger as the contact surface. According to the animal anatomical standard²³, press down the skin surface of the trapezius, superficial gluteus, rhomboid neck, rhomboid pectoralis, latissimus dorsi, and external oblique abdominal muscles in a round kneading motion from head to tail, with a force of 1.77 ± 0.54 N, so that the force reaches the trapezius, superficial gluteus, rhomboid neck, rhomboid pectoralis, latissimus dorsi, and external oblique abdominal muscles (Figure 2B)²³.
4. Press and knead the sensitive spinal acupoint points (such as Ganshu, Xinshu, Pishu, Shenshu, and Feishu²⁴) at a frequency of 100-120 times/min for 2 min. Using the right index or middle finger as the contact surface, perform circular kneading and downward pressing(1.77 ± 0.54 N) on the surface skin of local acupoints (such as Ganshu, Xinshu, Pishu, Shenshu, and Feishu) in infant rats with cerebral palsy.
   NOTE: The acupoints were selected according to the "Animal Acupuncture Point Map" formulated by the Experimental Acupuncture Research Branch of the Chinese Acupuncture Society) (Figure 3)²⁵.
5. Locally stimulate the affected limb by the twisting method at a frequency of 25 times/min for 1 min.
   1. With the right thumb and index finger, pinch the affected limb of the infant rat with cerebral palsy, exerting symmetrical force on the two fingers. Ensure that the force is gentle and the action is light and gentle. Rub the right extensor carpi radialis, extensor digitorum common, extensor carpi ulnaris, phalanx, interphalangeal joint, gastrocnemius muscle, semitendinosus muscle, metatarsal bone, and intermetatarsal joint of infant rat back and forth.
6. Apply the rubbing method to the midline of the forehead to the back of the brain at a frequency of 100-120 times/min for 1 min. Use the right index finger as the contact surface, and use the rubbing method on an imaginary line from the forehead to the middle of the posterior brain of the infant rat (namely, one line of Baihui, Fengfu, and Dazhui) (Figure 3).
   NOTE: Massage is mainly applied to the skin. It is a circular movement on the skin surface of the head of the infant rat with cerebral palsy. It does not need to drive the subcutaneous muscle movement of an infant rat with cerebral palsy.
7. At the end of the procedure, leave the infant rat alone for 30 min before returning it to the mother rat cage.
   NOTE: With age, the hair of the infant rats becomes thick, so it is necessary to remove the back hair of all groups of infant rats during the massage operation to avoid affecting the experimental effect.

5. Detecting the temperature of the local acupoints

1. Use an infrared thermal imager to detect the temperature of the local acupoint area before and after the operation.
   1. Before selective spinal massage, connect the Type-C interface of the mobile phone with the USB interface of the infrared imaging device with a data cable, and turn on the Power button.
   2. Click Analyze to enter the mobile phone terminal of the software, select Photo to enter the photo mode, and click the Picture in Picture Mode in the lower right corner.
   3. Focus the camera at the acupoint areas of Dazhui, Xinshu, and Shenshu acupoints (Figure 3) on the back of the infant rat to take temperature shots. Click Photo to complete the image capture and Save it in JPG format.
   4. Connect the computer with a data cable and upload the saved image to a computer installed with the Fotric software for analysis.
      1. Open the Fotric software on the PC, open the Local File, and find the location of the image.
      2. Select the Thermogram workspace, select the image to be analyzed, and enter the analysis interface.
      3. On the Analysis screen, select the picture in the Thermal image box on the right and open the Lock.
      4. Select the Zoom Function, adjust the size of the Thermal image and the left box Background image to coincide, and turn off the Lock on the right.
      5. Find the Set measurement rectangle in the toolbar on top and mark the acupoint to be detected in the figure.
      6. Click Report on the right to record the temperature value.

6. Detecting the motor balance function of infant rats with cerebral palsy

1. Use the Behavioral Rotarod test¹⁹ to detect the motor function of rats in the Sham, Control, and Treatment groups on the 61^st day after birth.
   1. Parameter settings: Start the power switch, select Do experiment to enter the next step, select Positive, Rats, Timing 5 min to enter the next step, and select Acc time:10s, Speed:10rpm.
   2. Then, place the rats on the running track and begin the test by clicking Run after they stand firm. Adjust the running state of each run to ON. Train each rat once for 5 min, and then begin to perform the test thrice.
   3. After the training, place the infant rats on the rotarod-running track. After it is stable, click Run, and adjust the running state to ON. At the end of 5 min, select Date to record the time and speed of each rat falling from the rotarod-running track for the first time, i.e., the Run time data.
2. Use the Foot-fault test^20,21 to evaluate the balance motor function of the right forelimb of the Sham, Control, and Treatment groups on the 61^st day after birth.
   1. Place the infant rats at the beginning end of the horizontal ladder and record a video of them crossing it to the other end (100 cm long, and the distance between each ladder is 2 cm). After one training session, perform the formal test thrice for each rat at a 5 min interval.
   2. Observe the right forelimb when the animal crosses the ladder using low-speed playback to make a score, as shown in the scoring standard (Table 2).

7. Western blotting

1. Perform western blotting analysis of the tissue samples as described previously²⁶.
   NOTE: The amount of lysate was determined according to the size of the tissue block. Antibodies used were as follows: primary anti-Growth Hormone antibody (0.5 µg [0.5 ng/lane]); anti-Growth Hormone receptor antibody (1/1000); internal reference anti-beta Actin antibody (1/2000) for primary antibodies; Goat anti-RabbitIgG H&L (HRP) (1/10000) for secondary antibody.

8. Detecting the strength of the hand

1. Wear the device used to detect the manipulation force on the hand used for massage. Fit the finger used to massage the infant rat with the sensor chip of the device, and turn the Power button on.
2. Connect the hard disk interface that stores the manual tester software to the computer, and enter the dedicated IP address of the manual tester in the URL input box.
3. Install the SpringVR client on the computer, click to enter the collection interface, and select the Finger Pattern in the lower right corner to test the manipulation force.
4. Select the Pressure option and click Start to start recording the manual force of the operator during operation. The software will automatically record the intensity of the manipulation and generate a data table on the local disk where the data in the file name PressureSensor will be found.

9. Data statistics

NOTE: SPSS26.0 software was used for statistical analysis, Graphpad Prism9.0.0 for bar chart production, and Image J for gray value analysis of protein bands.

1. Analyze and present all data as mean ± standard deviation (Mean ± SD).
   NOTE: The test standard was α = 0.05, and P ≤ 0.05 was considered statistically significant. Fisher's test was used because the data were analyzed by analysis of variance and normality and conformed to the normal distribution characteristics and variance homogeneity.