Comparison of immunohistochemical and serological evaluation of axonal degeneration after acute through sub-acute cervical spinal cord injury (SCI)

Understanding the etiology of spinal cord injury (SCI) is fundamentally important for the development and optimization of therapies aimed at minimizing injury induced neurodegeneration. Here we evaluated the time course of neurodegeneration after SCI in the adult rat visualized immunohistochemically (IHC) with a recently developed antibody, MCA-6H63 (EnCor Biotechnology Inc.). MCA-6H63 binds to an epitope on neurofilament light chain (NF-L) only accessible following neurofilament degradation (PMID: 37091583), as occurs during axonal degeneration. Our goal was to compare the pattern of axonal degeneration in the spinal cord using the MCA-6H63 antibody for IHC and serum NF-L levels using the Quanterix ® Simoa TM NF-L serum assay, a promising biomarker under investigation for the detection of neurodegeneration in a variety of diseases. Adult Sprague Dawley rats received a unilateral 150 kydne contusion at C4. Brainstem and spinal cord were harvested and serum was collected at 1-hr (n=9; 4 F), 6-hr (n=8; 3 F), 1-day (n=9; 5 F), 3-days (n=10, 6 F), and 10-days (n=9; 4 F) post-injury. A group of spinally intact (uninjured) rats (n=8; 4F) were included as a control. Tissue sections spanning the caudal medulla to C8 were stained with the MCA-6H63 antibody. MCA-6H63 staining was absent in control sections. MCA-6H63 positive axons were observed at all time points post SCI. At 1-hr post SCI very few MCA-6H63 positive axons were seen only in the immediate vicinity of the lesion. The number of MCA-6H63 positive axons dramatically increased by 6 hours post injury but were still confined to the vicinity of lesion epicenter. At 1- and 3- days post injury there was a peak in the number of MCA-6H63 positive fibers and staining spanned the cervical cord and caudal medulla. By 10 days post injury, there was an obvious decline in the number of MCA-6H63 positive axons. Serum NF-L levels, determined with the Simoa TM assay, were increased by 6-hrs post injury and peaked at 1-3 days after SCI (all p<0.05 vs. non-injured). The timing of serum NF-L elevation correlated with the time course of axonal degeneration seen with MCA-6H63 staining. The MCA-6H63 immunohistological data establish the time course of axonal degeneration following cervical spinal contusion in the adult rat. The close matching of immunohistological data with serum NF-L measurements shows for the first time the morphological correlate of the Simoa TM NF-L assay, suggesting that serum NF-L measurements are indicative of the extent of ongoing axonal degeneration within the injured spinal cord. Funding: 1R01NS139422-01 (DDF); 1K99NS133388-01A (SR); Frank M Davis Chairman Emeritus Grant (AFF). AFF supported by NIH training grant T32HD043730-19 This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.