Biomarker Expressions of Spinal Cord Injury in Avulsed Neonatal Brachial Plexus

Introduction: Diagnostic methods play a crucial role in determining the severity and progression of injury. Neonatal Brachial Plexus Palsy (NBPP) occurs in infants when their shoulder is stuck behind the pelvis of mother. While most cases of NBPP recover spontaneously, 25-30% of cases remain unrecovered. Studies have reported concomitant spinal cord injury (SCI) in avulsion BP injury cases. The diagnosis of this condition is severely limited due to the complex anatomy of BP and the age of the neonate. Qualitative clinical examination, the current care standard, will significantly improve when coupled with a supporting diagnostic technique. Detecting injury biomarkers released in the serum and cerebral spinal fluid post NBPP can lead to accurate diagnosis such as occurrence of concomitant SCI and can help in determining future treatment. This study aims to measure the biomarkers expressed in the body fluids post neonatal brachial plexus injury and allow for a better understanding of injury progression.

Materials and

Methods: All procedures were approved by the institutional animal care and use committee. Two piglets (3-5 days old) were included in the study. The BP complex of anesthetized piglets was exposed using an axillary approach. The BP terminal nerves (Musculocutaneous, Median, Ulnar and Radial BP nerves were subjected to failure tensile testing using a custom-built biomechanical testing set-up ​(1)​. The nerves were stretched until complete failure occurred.
Sample collection
Serum and Cerebral Spinal Fluid (CSF) were collected at two different time points, pre-surgery, and post 3hour post injury. Blood was collected from the arterial line established in the piglet and CSF was collected from the lumbar region (Fig 1). Samples were centrifuged at 2000 rpm for 10 mins at 40C, flash-frozen, and stored at –80 degrees until further analysis. Total RNA was extracted from serum and CSF using the Qiagen miRNesy serum plasma kit and the RNA was then reverse transcribed into cDNA. The following biomarkers were assessed in the study: Myelin Basic Protein (MBP), Glial Fibrillary Acidic Protein (GFAP) (Structural biomarker), Interleukin 6 (IL6) (Inflammatory biomarker), and their expressions were determined by qPCR using TaqMan assays. The expression data was normalized to the expression of β-Actin and mRNA expression levels were analyzed using the ΔΔCt method.

Results, Conclusions, and Discussions: As shown in Figure 2, higher expressions of MBP and IL6 were observed in the serum after 3 hours post injury, while the expressions of GFAP, MBP, and IL6 were increased in CSF when compared to pre-surgery values. The higher expressions of MBP and IL6 were observed in both the serum and CSF post BP avulsion injuries, when compared to pre-injury validating the evidence of SCI in these animals. The reported elevated level of GFAP in the CSF only indicates its specificity as a biomarker during spinal cord injury. Previous studies in humans have reported an increase in the concentrations of GFAP and IL6 in the CSF when compared to serum ​(2)​. This supports our findings of elevated levels of GFAP found only in the CSF. ​​In conclusion, this study is the first to report increased expressions of SCI biomarkers, GFAP, MBP and IL6 in BP avulsion injury animal model.

Acknowledgements (Optional): Acknowledgements
This project was supported by the Eunice Kennedy Shriver National Institute of Child Health and Human Development of the National Institutes of Health (R15HD093024 and #1R01HD104910-01A1) and NSF CAREER grant Award #1752513.

References

​​1. Singh A, Orozco V, Balasubramanian S. In vivo biomechanical responses of neonatal brachial plexus when subjected to stretch. PLoS One [Internet]. 2023 Aug 1 [cited 2024 May 12];18(8). Available from: /pmc/articles/PMC10468090/

​2. Kwon BK, Stammers AMT, Belanger LM, Bernardo A, Chan D, Bishop CM, et al. Cerebrospinal Fluid Inflammatory Cytokines and Biomarkers of Injury Severity in Acute Human Spinal Cord Injury. https://home.liebertpub.com/neu [Internet]. 2010 Apr 15 [cited 2024 May 10];27(4):669–82. Available from: https://www.liebertpub.com/doi/10.1089/neu.2009.1080

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