Study on the regulation and effect of neurobud protein after botulinum toxin injection in rats with hemicerebral palsy

This study aims to assess the capacity to prolong the efficacy of BoNT-A in hemiplegic CP(HCP) by analyzing its impact on muscle morphology and neurogenesis in a rat model. It seeks to identify neurogenesis-related proteins for potential targeting to extend BoNT-A's benefits, overcoming the challenges of repeated injections and resistance.

Seven-day-old Wistar rats were employed to establish an ischemic brain injury model for HCP, and spasticity was confirmed at 18 days using behavioral tests. At 21 days, BoNT-A was injected into the gastrocnemius muscle, and rats were divided into five groups for evaluations at 4 and 12 weeks post-injection. Outcomes included TTC-stained brain injury, Nissl-stained motor cortex neurons, motor function, spasticity, and protein expression of IGF-1, GAP43, and S100 via immunofluorescence and Western blot. NMJ morphology and muscle pathology were analyzed using staining and microscopy. Protein expression changes were quantified label-free and validated by PCR and Western blot, with statistical analysis performed using ANOVA and non-parametric tests.

Model Validation:

• Damaged rats showed cortical neuronal loss and myelin disruption.
•  Post-surgery, HCP rats had reduced grip strength and shorter rotarod times.
• Ashworth scores and neurophysiological measures indicated higher spasticity.
• Histological analysis confirmed muscle atrophy and disorder in HCP rats.

Motor Ability:

•  HCP+BoNT-A rats had improved motor function at 4 weeks but not at 12 weeks.

Muscle Morphology and NMJ Morphology:

• BoNT-A injection led to muscle atrophy and altered NMJ staining at 4 weeks.
•  At 12 weeks, mixed muscle fiber changes were observed.
• Muscle weight was significantly reduced post-BoNT-A injection.

Spasticity Evaluation:

• BoNT-A reduced spasticity measures at 4 weeks, with no difference at 12 weeks.
•  Neurophysiological tests showed temporary spasticity reduction post-BoNT-A.

Protein Expression:

• Post-BoNT-A, levels of IGF-1, GAP43, and S100 increased at 4 weeks, returning to baseline by 12 weeks.

Proteomics:

• Proteomics identified 534 proteins, with 25 differentially expressed. KEGG and GO analyses tied these to processes like mitochondrial function, protein transport, and antioxidation.

This study established a hemiplegic CP rat model showing significant symptoms and dysfunction. BoNT-A improved motor function and elevated neurogenesis-related proteins like IGF-1 and GAP43 at 4 weeks, but effects waned by 12 weeks. Spasticity measures improved 4 weeks post-injection but were not sustained. Proteomics revealed key proteins for potentially extending BoNT-A's benefits.

The significance of this project lies in its potential to enhance the treatment of CP by investigating the use of BoNT-A and its effects on muscle morphology and neurogenesis. The study addresses the clinical challenges associated with the transient efficacy of BoNT-A and the need for repeated injections, which can lead to resistance and reduced patient quality of life.