Abstract
Recovery from severe spinal injury in adults is limited, compared to immature animals who demonstrate some capacity for repair. Using laboratory opossums (Monodelphis domestica), the aim was to compare proteomic responses to injury at two ages: one when there is axonal growth across the lesion and substantial behavioural recovery and one when no axonal growth occurs. Anaesthetized pups at postnatal day (P) 7 or P28 were subjected to complete transection of the spinal cord at thoracic level T10. Cords were collected 1 or 7 days after injury and from age-matched controls. Proteins were separated based on isoelectric point and subunit molecular weight; those whose expression levels changed following injury were identified by densitometry and analysed by mass spectrometry. Fifty-six unique proteins were identified as differentially regulated in response to spinal transection at both ages combined. More than 50% were cytoplasmic and 70% belonged to families of proteins with characteristic binding properties. Proteins were assigned to groups by biological function including regulation (40%), metabolism (26%), inflammation (19%) and structure (15%). More changes were detected at one than seven days after injury at both ages. Seven identified proteins: 14-3-3 epsilon, 14-3-3 gamma, cofilin, alpha enolase, heart fatty acid binding protein (FABP3), brain fatty acid binding protein (FABP7) and ubiquitin demonstrated age-related differential expression and were analysed by qRT-PCR. Changes in mRNA levels for FABP3 at P7+1day and ubiquitin at P28+1day were statistically significant. Immunocytochemical staining showed differences in ubiquitin localization in younger compared to older cords and an increase in oligodendrocyte and neuroglia immunostaining following injury at P28. Western blot analysis supported proteomic results for ubiquitin and 14-3-3 proteins. Data obtained at the two ages demonstrated changes in response to injury, compared to controls, that were different for different functional protein classes. Some may provide targets for novel drug or gene therapies.
Highlights
Several mammalian organ systems, such as the heart [1] and the central nervous system (CNS) have an increased capacity to regenerate following injury when immature compared to adult
Conclusions and future directions Results obtained in this study clearly indicate that proteomic analysis, western blotting, Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunocytochemistry are approaches that yield complementary information to the study of age-related responses of the spinal cord to injury
Comparison of proteomic data with results obtained from qRT-PCR for the seven candidate genes indicated that there was a possibility that an injury could influence biological response at three levels of molecular function: (1) gene expression levels, (2) protein synthesis levels and (3) post-translational modifications of proteins
Summary
Several mammalian organ systems, such as the heart [1] and the central nervous system (CNS) have an increased capacity to regenerate following injury when immature compared to adult. If the spinal cord in an opossum Monodelphis domestica [3,4] or Didelphis virginiana [5,6] is transected in the mid thoracic region in the first week of life, many axons regenerate, reaching in Monodelphis domestica nearly 50% [7]. At the same time a substantial growth of new undamaged axons occurs as part of normal development. These animals demonstrate near normal locomotor ability when adult [4]. Following transection at about one month of age, growth of axons across the lesion site cannot be detected [6] and the animals have substantially impaired locomotion [8]
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