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

Published: February 02, 2024

doi:

Tianlei Gao*¹, Yuanwang Wang*¹, Min Li, Liuying Yang, Suyu Chen, Lin Gao, Yinghua Shi, XingHe Zhang, XianTao Tai

¹Yunnan University of Traditional Chinese Medicine

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-α 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 Me…

Representative 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 ± 0.035 g, and the body weight of the Control group was 3.15 ± 0.43 g. The body weight of the Treatment group was 4.42 ± 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…

Declarações

The authors have nothing to disclose.

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 Science 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

96-well plates	Beijing Lanjieke Biotechnology Co., LTD	11510	Determination of protein concentration
Anti-beta Actin antibody	Abacm	Ab8227	Dilution: 1/2000
Anti-Growth Hormone antibody	Abacm	Ab126882	0.5 µg (0.5 ng/lane)
Anti-Growth Hormone receptor antibody	Abacm	Ab202964	Dilution: 1/1000
Basic operating microscope	Shanghai YuYAN Scientific Instrument Co. LTD	SM-101	The common carotid artery was isolated under microscope during modeling
BCA developer	Biyuntian Biological Engineering Co., LTD	P0010	Determination of protein concentration
Chemiluminescence imaging system	Shanghai Qinxiang Scientific Instrument Co., LTD	100240073	Protein banding imaging
Direct-load Color Prestained Marker	Beijing Kangrunchengye Biotechnology Co., LTD (GenStar)	M221	Western Blot
DK-30Automatic snow ice maker	Henan Brothers instrument equipment Co., LTD	SHDX0023	Ice-making
ECL luminescent substrate kit	Beijing Lanjieke Biotechnology Co., LTD	BL520B	Convert latent images in exposed film into visible images
Electric-heated thermostatic water bath	TAISITE INSTRUMENT	DK-98-II	The young rats were resuscitated after modeling
Electronic scales	Kunshan YoukeWEI ELECTRONIC Technology Co. LTD	CN-LQC10002	The body weight of the young rats was measured
German small white electric coagulation pen hemostat	Haohang	L55×W125×H37	It was used to coagulate the left common carotid artery
Glove	Jiangsu YANGzi LiDE Medical Device Co. LTD	Q/320684 YZYL001-2017	For massage operation
Glycine	Beijing Soleibao Technology Co., Ltd.	Cat#G8200	Electrophoretic solution, Configure the transfer fluid
Goat Anti-RabbitIgG H&L (HRP)	Abacm	Ab6721	Dilution: 1/10000
Intelligent laboratory ultra-pure water machine	Chongqing huachuag water treatment engineering co.,LTD	N/A	Filtration (15 L)
Isoflurane	Shandong Ante Animal Husbandry Technology Co. LTD	15198	Anesthesia was maintained by induction in young rats
LinkIR	FOTIRC	V1.3.2.134	Infrared image analysis software
Low temperature high speed tissue grinder	Wuhan Servicebio technology CO.,LTD	SKZ3F20200191	Tissue grinding
Methanol	Guangdong Guanghua Sci-Tech Co., Ltd	20220519	Configure the transfer fluid
Mini-PROTEAN Tetra	Bole Life Medical Products (Shanghai) Co., Ltd	552BR 233193	Electrophoresis
Multiskan Spectrum Microplate Spectrophotometer	TECAN	Spark	The absorbance and concentration of tissue protein were detected
Pressure-sensing smart gloves	Jinan Super Sense Intelligent Technology Co. LTD	Miiglove	It is used to measure the manipulative strength of the operator
PVDF membrane	MerckMillipore Corporation	IPVH00010	Western Blot
Refrigerated centrifuge	Hettich Precision Technology (Zhuhai) Co., LTD	MIKRO 220R	Centrifuge
Research three-in-one thermal imager	FOTIRC	226S (384 x 288)	Temperature measurement
RIPA lysate	Beijing Solaibao Technology Co., LTD (Solarbio)	R0010	Lytic tissue
SHA-CA digital display water bath thermostatic oscillator	Changzhou Aohua Instrument Co. LTD	SHA-CA	Young rats were used in hypoxia
Six-rat fatigue rotarod apparatus	Shanghai Duoyi Industry Co., LTD	DO01104RT703	CP motor function was detected
Skim milk powder	Guangzhou Saiguo Biotechnology Co., LTD (BIOFROXX)	1172GR500	Confining liquid
Surgical plate	Shanghai YuYAN Scientific Instrument Co. LTD	51002	The model operating table was established in young rats
TS-200 Orbital shaker	Haimen Qilin Bell Manufacturing Co., Ltd.	TS-8S	Gel fixation
Tween 80	MedChemExpress	HY-Y1819	Configure TBST
ZS-MV Portable anesthesia machine	ZHONGSHI SCIENCE &TECHNOLOGY	ZS-MV-I	Anesthesia was induced and maintained in experimental animals

Referências

Paul, S., Nahar, A., Bhagawati, M., Kunwar, A. J. A review on recent advances of cerebral palsy. Oxidative Medicine and Cellular Longevity. , 2622310 (2022).
Şimşek, T. T., Tuç, G. Examination of the relation between body mass index, functional level and health-related quality of life in children with cerebral palsy. Turk Pediatri Arsivi. 49 (2), 130-137 (2014).
Dahlseng, M. O., et al. Feeding problems, growth and nutritional status in children with cerebral palsy. Acta Paediatrica. 101 (1), 92-98 (2012).
Fogarasi, A., et al. The purple n study: Objective and perceived nutritional status in children and adolescents with cerebral palsy. Disability and Rehabilitation. 44 (22), 6668-6675 (2022).
Xiaojie, L., et al. Epidemiological characteristics of cerebral palsy in twelve province in China. Chinese Journal of Practical Pediatrics Clinical. 33 (5), 378-383 (2018).
Kakooza-Mwesige, A., et al. Prevalence of cerebral palsy in Uganda: A population-based study. The Lancet. Global Health. 5 (12), e1275-1282 (2017).
Korzeniewski, S. J., Slaughter, J., Lenski, M., Haak, P., Paneth, N. The complex aetiology of cerebral palsy. Nature Reviews. Neurology. 14 (9), 528-543 (2018).
Vargus-Adams, J. N., Martin, L. K. Domains of importance for parents, medical professionals and youth with cerebral palsy considering treatment outcomes. Child: Care, Health and Development. 37 (2), 276-281 (2011).
Koy, A., et al. Quality of life after deep brain stimulation of pediatric patients with dyskinetic cerebral palsy: A prospective, single-arm, multicenter study with a subsequent randomized double-blind crossover (stim-cp). Movement Disorders. 37 (4), 799-811 (2022).
Pool, D., Valentine, J., Taylor, N. F., Bear, N., Elliott, C. Locomotor and robotic assistive gait training for children with cerebral palsy. Developmental Medicine and Child Neurology. 63 (3), 328-335 (2021).
Li, J., et al. Evaluating the effects of 5-hz repetitive transcranial magnetic stimulation with and without wrist-ankle acupuncture on improving spasticity and motor function in children with cerebral palsy: A randomized controlled trial. Frontiers In Neuroscience. 15, 771064 (2021).
Chen, K., Shu, S., Yang, M., Zhong, S., Xu, F. Meridian acupuncture plus massage for children with spastic cerebral palsy. American Journal of Translational Research. 13 (6), 6415-6422 (2021).
Gao, J., et al. Rehabilitation with a combination of scalp acupuncture and exercise therapy in spastic cerebral palsy. Complementary Therapies in Clinical Practice. 35, 296-300 (2019).
Chen, Z., et al. Effects of traditional Chinese medicine combined with modern rehabilitation therapies on motor function in children with cerebral palsy: A systematic review and meta-analysis. Frontiers In Neuroscience. 17, 1097477 (2023).
Zhang, Y., et al. Tuina massage improves cognitive functions of hypoxic-ischemic neonatal rats by regulating genome-wide DNA hydroxymethylation levels. Evidence-Based Complementary and Alternative Medicine: ECAM. 2019, 1282085 (2019).
Zhang, P., et al. Chinese tuina protects against neonatal hypoxia-ischemia through inhibiting the neuroinflammatory reaction. Neural Plasticity. 2020, 8828826 (2020).
Niu, F., et al. Spinal tuina improves cognitive impairment in cerebral palsy rats through inhibiting pyroptosis induced by nlrp3 and caspase-1. Evidence-Based Complementary and Alternative Medicine: ECAM. 2021, 1028909 (2021).
Bowen, Z., Guangyi, X., Qian, Z., Xueping, H., Xiantao, T. Time-dose effect of spinal manipulation on growth and motor function in infant rat with cerebral palsy. Shi Zhen National Medicine and National Medicine. 28 (09), 2274-2277 (2017).
Yang, L. J., Cui, H. Olig2 knockdown alleviates hypoxic-ischemic brain damage in newborn rats. Histology and Histopathology. 36 (6), 675-684 (2021).
Martins, L. A., Schiavo, A., Xavier, L. L., Mestriner, R. G. The foot fault scoring system to assess skilled walking in rodents: A reliability study. Frontiers in Behavioral Neuroscience. 16, 892010 (2022).
Li, S., et al. Plxna2 knockdown promotes m2 microglia polarization through mtor/stat3 signaling to improve functional recovery in rats after cerebral ischemia/reperfusion injury. Experimental Neurology. 346, 113854 (2021).
Wei, W., et al. Neuroprotective effect of verbascoside on hypoxic-ischemic brain damage in neonatal rat. Neuroscience Letters. 711, 134415 (2019).
Taiyi, W., Ziyu, H. . Anatomical Atlas of Experimental Animals in Chinese and English. , (2000).
Na, X. . Study on the sensitization rule of dorsal acupoints in children with spastic cerebral palsy based on infrared thermal imaging technology and the effect of massage intervention. , (2021).
China Association for Acupuncture and Moxibustion. Names and localization of commonly used acupoints in laboratory animals Part 2: Rats. Acupuncture Research. 46 (04), 351-352 (2021).
Yinghua, S., Xiantao, T. Effect of spinal manipulation on the expression of growth hormone and its receptor protein in hypothalamus of infant rat with cerebral palsy. Sichuan Traditional Chinese Medicine. 39 (02), 55-59 (2021).
Qi, H., et al. Effect of spinal manipulation on learning and memory in infant rat with cerebral palsy and its mechanism. Guide to Traditional Chinese Medicine. 24 (11), 36-39 (2018).
Tai, W. -. C., Burke, K. A., Dominguez, J. F., Gundamraj, L., Turman, J. E. Growth deficits in a postnatal day 3 rat model of hypoxic-ischemic brain injury. Behavioural Brain Research. 202 (1), 40-49 (2009).
Huang, L., et al. Animal models of hypoxic-ischemic encephalopathy: Optimal choices for the best outcomes. Reviews In the Neurosciences. 28 (1), 31-43 (2017).
Lyu, H., et al. A new hypoxic-ischemic encephalopathy model in neonatal rats. Heliyon. 7 (12), e08646 (2021).
Hegazi, M. A., et al. Growth hormone/insulin-like growth factor-1 axis: A possible non-nutritional factor for growth retardation in children with cerebral palsy. Jornal de Pediatria. 88 (3), 267-274 (2012).
Boboc, I. K. S., et al. A preclinical systematic review and meta-analysis of behavior testing in rat models of ischemic stroke. Life. 13 (2), 567 (2023).
Lubrich, C., Giesler, P., Kipp, M. Motor behavioral deficits in the cuprizone model: Validity of the rotarod test paradigm. International Journal of Molecular Sciences. 23 (19), 11342 (2022).
Tan, Y., et al. Vof-16 knockout improves the recovery from hypoxic-ischemic brain damage of neonatal rats. Brain Research. 1748, 147070 (2020).
Xiantao, T., Pengyue, Z., Xinghe, Z. Construction and application of selective spinal manipulation in the treatment of children with cerebral palsy. China’s Scientific and Technological Achievements. 23 (9), 1-2 (2022).
Matos, L. C., Machado, J., Greten, H. J., Monteiro, F. J. Changes of skin electrical potential in acupoints from ren mai and du mai conduits during qigong practice: Documentation of a clinical phenomenon. Journal of Bodywork and Movement Therapies. 23 (4), 713-720 (2019).
Levkovets, I. L., Kiryanova, V. V. Systemic and pathogenetic approach: A new look at traditional Chinese medicine. Voprosy Kurortologii, Fizioterapii, I Lechebnoi Fizicheskoi Kultury. 99 (1), 80-88 (2022).
Efferth, T., Xu, A. -. L., Lee, D. Y. W. Combining the wisdoms of traditional medicine with cutting-edge science and technology at the forefront of medical sciences. Phytomedicine: International Journal of Phytotherapy and Phytopharmacology. 64, 153078 (2019).
Zhang, Z., et al. Correlated sensory and sympathetic innervation between the acupoint bl23 and kidney in the rat. Frontiers In Integrative Neuroscience. 14, 616778 (2020).
Zhu, X., et al. Effects of electroacupuncture at st25 and bl25 in a sennae-induced rat model of diarrhoea-predominant irritable bowel syndrome. Acupuncture In Medicine: Journal of the British Medical Acupuncture Society. 35 (3), 216-223 (2017).
Gimarc, K., Yandow, S., Browd, S., Leibow, C., Pham, K. Combined selective dorsal rhizotomy and single-event multilevel surgery in a child with spastic diplegic cerebral palsy: A case report. Pediatric Neurosurgery. 56 (6), 578-583 (2021).
Zhang, W. -. B., Wang, Y. -. P., Li, H. -. Y. Analysis on correlation between meridians and viscera in book the yellow emperor’s internal classic. Acupuncture Research. 43 (7), 424-429 (2018).
Wang, Y. -. P., Hou, X. -. S. Discussion on the classification of acupoints. Chinese Acupuncture & Moxibustion. 39 (10), 1069-1072 (2019).
Chen, R. -. X., Kang, M. -. F. Clinical application of acupoint heat-sensitization. Chinese Acupuncture & Moxibustion. 27 (3), 199-202 (2007).