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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

This study illustrates the effect of selective spinal manipulation on the growth and development of infant rats with cerebral palsy, emphasizing the specific procedure and standardized protocol. Body weight measurement, Rotarod test, Foot-fault score, other behavioral tests, and growth hormone detection were performed to evaluate the protocol.

Abstract

Cerebral palsy (CP) is a refractory pediatric disease with a high prevalence, high disability rate, and difficult treatment. A variety of treatments are currently used for CP. The treatment involves drug and non-drug therapy. Traditional Chinese medicine external therapy is a very distinctive treatment method in non-drug therapy. As one of the external therapies of traditional Chinese medicine, massage is used in treating cerebral palsy and has good efficacy, small side effects, and strong operability. As a part of TCM external therapy, selective spinal manipulation can effectively promote the growth and development of infant rats with cerebral palsy.The operation was mainly divided into four steps: first, the rubbing method was applied to the spine and both sides of the spine for 1 min. The pressing and kneading method was applied to the spine for 5 min, and the muscles on both sides of the spine for 5 min. Second, pressing and kneading the sensitive local acupoints in the spine for 2 min were performed. Thirdly, the affected limb was treated by twisting method for 1 min. Fourth, the rubbing method was applied to a midline from the forehead to the back of the brain for 1 min. This study aimed to use selective spinal manipulation to treat infant rats with cerebral palsy. The weight, Rotarod test, Foot-fault score, and growth hormone of infant rats with cerebral palsy were detected to understand the effect of selective spinal manipulation on the growth and development of infant rats with cerebral palsy. The results showed that it can promote weight gain, improve balance ability and motor function, promote growth and development of infant cerebral palsy rats, promote growth hormone secretion, and increase the temperature of sensitive parts of the back.

Introduction

Cerebral Palsy (CP), caused by non-progressive damage to the brain in the fetus or infancy, is a group of disorders characterized by abnormal motor and postural development1, developmental disorders including slow weight gain2,3,4, and motor dysfunction. The incidence of cerebral palsy in China is 2.48%, and the prevalence is 2.46‰ (1-6 years old)5, respectively. Foreign studies reported a prevalence of 2.4%-3.6%6. Cerebral palsy is a major cause of physical injury and disability in children, among which balance and movement disorders significantly impact daily living activities1. Cerebral palsy can be caused by premature birth, infection, genetics, neonatal ischemia and hypoxia, neonatal jaundice, and other complex and diverse pathogenic factors7. The goal of treatment for cerebral palsy is to improve physical function and quality of life 8. Currently, the treatment methods for cerebral palsy include deep brain stimulation9, robot-assisted gait training10, wrist and ankle acupuncture11, and meridian acupuncture combined with massage12.

Traditional Chinese medicine (TCM) has been increasingly used as an effective treatment for CP13,14. Massage, as a part of it, also plays a certain role. For example, selective spinal manipulation can regulate the state of DNA hydroxymethylation to regulate neural development and improve learning and memory function15. In the treatment of infant rat with cerebral palsy, selective spinal manipulation may improve the inflammatory homeostasis of the cortex and hippocampus by regulating the methylation level of inflammatory cytokines TNF-Ξ± and IL-10 gene promoter region16 and play a positive therapeutic role in the balance ability of infant rat with cerebral palsy16. Selective spinal manipulation can improve the cognitive function of infant rats with cerebral palsy17. Selective spinal manipulation was used in the treatment of infant rats with cerebral palsy, and it was observed that it also had therapeutic effects on their growth and development18.

The purpose of this study is to illustrate the effect of selective spinal manipulation on the growth and development of infant rats with cerebral palsy by measuring the body weight, Rotarod test19, Foot-fault score20,21, and other behavioral tests and the detection of growth hormone, and to provide research ideas for relevant personnel.

Protocol

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 Β°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 5th 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.
    NOTE: This experiment is mainly to observe the effects of selective spinal manipulation on the growth and development of infant rats with cerebral palsy. Anesthesia has been carried out in accordance with the general requirements during the modeling process, and additional analgesia will affect the observation results.
    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%.
      NOTE: After anesthesia, assess the anesthetic depth by forcibly lifting and pinching the toes and tail of the infant rats. A no response when the toes and tail were lifted and pinched confirms the depth of anesthesia has reached.
  5. Disinfect the neck skin of the infant rat three times with 75% alcohol and cut a longitudinal 1 cm incision on the neck skin to expose the subcutaneous tissue.
    NOTE: Longitudinal incision of neck skin does not cause skin defect.
  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 Β°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 Β°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 Β°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. Lactating female rats were fed with Specific Pathogen Free (SPF) breeding feed, and when the pups were able to self-feed, the pups were weaned (postnatal day 21) and fed with SPF maintenance feed.

2. Righting reflex experiment

  1. On the 4th day after birth (the 2nd 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 model16,22.
    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 Β°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 Β°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 Β± 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 Β± 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 Β± 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 61st 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 test20,21 to evaluate the balance motor function of the right forelimb of the Sham, Control, and Treatment groups on the 61st 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 Β΅g [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 Β± standard deviation (Mean Β± SD).
    NOTE: The test standard was Ξ± = 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.

Results

Selective spinal manipulation can promote body weight gain in infant rats with cerebral palsy.
During the treatment, body weight was measured on postnatal days 3, 14, 28, 42, and 61 (Figure 4, Table 3). On the third day after birth, the body weight of the Sham group was 5.53 Β± 0.035 g, and the body weight of the Control group was 3.15 Β± 0.43 g. The body weight of the Treatment group was 4.42 Β± 0.13 g, and it was not significantly differe...

Discussion

Because ischemia and hypoxia are important pathogenic factors of cerebral palsy, the internationally recognized method of establishing the cerebral palsy model is combined with hypoxia to prepare the cerebral palsy model16,28,29,30. When cerebral palsy develops, it causes global developmental delay, including weight and motor function, which may be related to the level of growth hormone secreti...

Disclosures

The authors have no competing financial or other conflicts of interest under this work.

Acknowledgements

This work has been supported by the General Program of National Natural Science Foundation of China (82374614), the Major Biomedical Project of the Science and Technology Department of Yunnan Province (202102AA100016), the Joint Major Project of Applied Basic Research of the Department of ScienceΒ and Technology of Yunnan Province -- Yunnan University of Traditional Chinese Medicine (201901AI070004), Supported by the Key Laboratory of Acupuncture, Moxibustion, and Massage for Prevention and Treatment of Encephalopathy in Colleges and Universities of Yunnan Province (2019YGZ04), Department of Science and Technology of Yunnan Province -- Youth Project of Basic Research Program of Yunnan Province (202101AU070002), Graduate Program of Science Research Fund of Education Department of Yunnan Province, (2023Y0433); Scientific Research Foundation of Education Department of Yunnan Province, (2023Y0462).

Materials

NameCompanyCatalog NumberComments
96-well platesBeijing Lanjieke Biotechnology Co., LTD11510Determination of protein concentration
Anti-beta Actin antibodyAbacmAb8227Dilution: 1/2000
Anti-Growth Hormone antibodyAbacmAb1268820.5 Β΅g (0.5 ng/lane)
Anti-Growth Hormone receptor antibodyAbacmAb202964Dilution: 1/1000
Basic operating microscopeShanghai YuYAN Scientific Instrument Co. LTDSM-101The common carotid artery was isolated under microscope during modeling
BCA developerBiyuntian Biological Engineering Co., LTDP0010Determination of protein concentration
Chemiluminescence imaging systemShanghai Qinxiang Scientific Instrument Co., LTD100240073Protein banding imaging
Direct-load Color Prestained MarkerBeijing Kangrunchengye Biotechnology Co., LTD (GenStar)M221Western Blot
DK-30Automatic snow ice makerHenan Brothers instrument equipment Co., LTDSHDX0023Ice-making
ECL luminescent substrate kitBeijing Lanjieke Biotechnology Co., LTDBL520BConvert latent images in exposed film into visible images
Electric-heated thermostatic water bathTAISITE INSTRUMENTDK-98-IIThe young rats were resuscitated after modeling
Electronic scalesKunshan YoukeWEI ELECTRONIC Technology Co. LTDCN-LQC10002The body weight of the young rats was measured
German small white electric coagulation pen hemostatHaohangL55Γ—W125Γ—H37It was used to coagulate the left common carotid artery
GloveJiangsu YANGzi LiDE Medical Device Co. LTDQ/320684 YZYL001-2017For massage operation
GlycineBeijing Soleibao Technology Co., Ltd.Cat#G8200Electrophoretic solution, Configure the transfer fluid
Goat Anti-RabbitIgG H&L (HRP)Β AbacmAb6721Dilution: 1/10000
Intelligent laboratory ultra-pure water machineChongqing huachuag water treatment engineering co.,LTDN/AFiltration (15 L)
IsofluraneShandong Ante Animal Husbandry Technology Co. LTD15198Anesthesia was maintained by induction in young rats
LinkIRFOTIRCV1.3.2.134Infrared image analysis software
Low temperature high speed tissue grinderWuhan Servicebio technology CO.,LTDSKZ3F20200191Tissue grinding
MethanolGuangdong Guanghua Sci-Tech Co., Ltd20220519Configure the transfer fluid
Mini-PROTEAN TetraBole Life Medical Products (Shanghai) Co., Ltd552BR 233193Electrophoresis
Multiskan Spectrum Microplate SpectrophotometerTECANSparkThe absorbance and concentration of tissue protein were detected
Pressure-sensing smart glovesJinan Super Sense Intelligent Technology Co. LTDMiigloveIt is used to measure the manipulative strength of the operator
PVDF membraneMerckMillipore CorporationΒ IPVH00010Β Western Blot
Refrigerated centrifugeHettich Precision Technology (Zhuhai) Co., LTDMIKRO 220RCentrifuge
Research three-in-one thermal imagerFOTIRC226S (384 x 288)Temperature measurement
RIPA lysateBeijing Solaibao Technology Co., LTD (Solarbio)R0010Lytic tissue
SHA-CA digital display water bath thermostatic oscillatorChangzhou Aohua Instrument Co. LTDSHA-CAYoung rats were used in hypoxia
Six-rat fatigue rotarod apparatusShanghai Duoyi Industry Co., LTDDO01104RT703CP motor function was detected
Skim milk powderGuangzhou Saiguo Biotechnology Co., LTD (BIOFROXX)1172GR500Confining liquid
SPF breeding feedSPF(Beijing)biotechnology co.,Ltd.A1EC30005A1S4285266Lactating female rats were fed
SPF maintenance feedSPF(Beijing)biotechnology co.,Ltd.A1EC30005A1S4285267The pups were fed after weaning
Surgical plateShanghai YuYAN Scientific Instrument Co. LTD51002The model operating table was established in young rats
TS-200 Orbital shakerHaimen Qilin Bell Manufacturing Co., Ltd.TS-8SGel fixation
Tween 80MedChemExpressHY-Y1819Configure TBSTΒ 
ZS-MV Portable anesthesia machineZHONGSHI SCIENCE &TECHNOLOGYZS-MV-IAnesthesia was induced and maintained in experimental animals

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Cerebral PalsyInfant RatsSelective Spinal ManipulationTraditional Chinese MedicineTCM External TherapyMassage TherapyWeight GainMotor FunctionGrowth HormoneRotarod TestFoot fault ScoreTreatment Efficacy

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