CIMT combined with BoNT-A regenerates skeletal muscle and improves upper limb function through activating IGF-1/FGFR2...

Hemiplegic cerebral palsy (HCP) is a prevalent cause of pediatric motor disability. Constraint-induced movement therapy (CIMT), when combined with botulinum neurotoxin type A (BoNT-A), improves upper limb function and social participation in individuals with HCP. However, the mechanisms underlying the combined interventions remain unclear.

In this study, sixty children with CP were initially recruited and assigned to receive either CIMT or CIMT combined with BoNT-A intervention. The differences between the two therapeutic regimens were subsequently assessed using the Melbourne Assessment 2 (MA2) and the Modified Ashworth Scale (MAS). Subsequently, forty-six additional children with CP were enrolled. Fifteen children received CIMT and other thirty-one received CIMT combined with BoNT-A intervention. The serum samples were collected from these patients for proteomic analysis. Proteomic analysis was conducted to identify potential therapeutic targets of the CIMT and BoNT-A combined intervention in the serum of children with CP. HCP rats were treated with CIMT alone, the CIMT and BoNT-A combined intervention, or the combined intervention with the insulin-like growth factor-1 receptor (IGF-1R) inhibitor PQ401, respectively. ELISA was utilized to quantify IGF-1 levels in serum and skeletal muscle. Hematoxylin-eosin (HE) staining and transmission electron microscopy (TEM) were employed to observe muscle morphology. The expression of muscle satellite cells (MSCs) quiescence marker PAX7, early activation marker MyoD, and late activation marker MyoG were detected by fluorescent in situ hybridization and immunofluorescence double staining (FISH-IF). RNA sequencing (RNA-seq) was performed to identify down- stream targets of IGF-1. The expression of Fibroblast Growth Factor Receptor 2 (Fgfr2) was validated by quantitative real-time PCR (qRT-PCR).

The combined intervention group exhibited significantly greater improvements in the MA2 and MAS test. Serum IGF-1 levels were significantly increased following the CIMT combined with BoNT-A intervention in children with CP. The combined intervention significantly improved forelimb function in rats, which was positively correlated with elevated IGF-1 levels in serum and skeletal muscle. Muscle fibers exhibited a more orderly arrangement, and the expression of PAX7, MyoD, and MyoG was higher in the combined intervention group than in the HCP group. In contrast, when treated with PQ401, a decline in forelimb function and a reversal of the therapeutic effects of CIMT combined with BoNT-A intervention was observed in HCP rats. RNA-seq identified Fgfr2 as a downstream target of IGF-1, and the combined intervention significantly upregulated Fgfr2 mRNA expression in the skeletal muscle of HCP rats. In contrast, the expression of Fgfr2 was downregulated when the combination intervention was administered alongside PQ401.

CIMT combined with BoNT-A may enhance MSCs differentiation into skeletal muscle in HCP by upregulating the IGF-1/Fgfr2 pathway, providing new evidence for the mechanism of improving HCP by the combination intervention.