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Age-dependent changes in the proteome following complete spinal cord transection in a postnatal South American opossum (Monodelphis domestica).

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.

SUBMITTER: Noor NM

PROVIDER: S-EPMC3217969 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Spinal cord trauma in the adult nervous system usually results in permanent loss of function below the injury level. The immature spinal cord has greater capacity for repair and can develop considerable functionality by adulthood. This study used the marsupial laboratory opossum Monodelphis domestica, which is born at a very early stage of neural development. Complete spinal cord transection was made in the lower-thoracic region of pups at postnatal-day 7 (P7) or P28, and the animals grew to adulthood. Injury at P7 resulted in a dense neuronal tissue bridge that connected the two ends of the cord; retrograde neuronal labelling indicated that supraspinal and propriospinal innervation spanned the injury site. This repair was associated with pronounced behavioural recovery, coordinated gait and an ability to use hindlimbs when swimming. Injury at P28 resulted in a cyst-like cavity encased in scar tissue forming at the injury site. Using retrograde labelling, no labelled brainstem or propriospinal neurons were found above the lesion, indicating that detectable neuronal connectivity had not spanned the injury site. However, these animals could use their hindlimbs to take weight-supporting steps but could not use their hindlimbs when swimming. White matter, demonstrated by Luxol Fast Blue staining, was present in the injury site of P7- but not P28-injured animals. Overall, these studies demonstrated that provided spinal injury occurs early in development, regrowth of supraspinal innervation is possible. This repair appears to lead to improved functional outcomes. At older ages, even without detectable axonal growth spanning the injury site, substantial development of locomotion was still possible. This outcome is discussed in conjunction with preliminary findings of differences in the local propriospinal circuits following spinal cord injury (demonstrated with fluororuby labelling), which may underlie the weight bearing locomotion observed in the apparent absence of axons bridging the lesion site in P28-injured Monodelphis.

Project description:Complete spinal transection in the mature nervous system is typically followed by minimal axonal repair, extensive motor paralysis and loss of sensory functions caudal to the injury. In contrast, the immature nervous system has greater capacity for repair, a phenomenon sometimes called the infant lesion effect. This study investigates spinal injuries early in development using the marsupial opossum Monodelphis domestica whose young are born very immature, allowing access to developmental stages only accessible in utero in eutherian mammals. Spinal cords of Monodelphis pups were completely transected in the lower thoracic region, T10, on postnatal-day (P)7 or P28 and the animals grew to adulthood. In P7-injured animals regrown supraspinal and propriospinal axons through the injury site were demonstrated using retrograde axonal labelling. These animals recovered near-normal coordinated overground locomotion, but with altered gait characteristics including foot placement phase lags. In P28-injured animals no axonal regrowth through the injury site could be demonstrated yet they were able to perform weight-supporting hindlimb stepping overground and on the treadmill. When placed in an environment of reduced sensory feedback (swimming) P7-injured animals swam using their hindlimbs, suggesting that the axons that grew across the lesion made functional connections; P28-injured animals swam using their forelimbs only, suggesting that their overground hindlimb movements were reflex-dependent and thus likely to be generated locally in the lumbar spinal cord. Modifications to propriospinal circuitry in P7- and P28-injured opossums were demonstrated by changes in the number of fluorescently labelled neurons detected in the lumbar cord following tracer studies and changes in the balance of excitatory, inhibitory and neuromodulatory neurotransmitter receptors' gene expression shown by qRT-PCR. These results are discussed in the context of studies indicating that although following injury the isolated segment of the spinal cord retains some capability of rhythmic movement the mechanisms involved in weight-bearing locomotion are distinct.

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Age-dependent changes in the proteome following complete spinal cord transection in a postnatal South American opossum (Monodelphis domestica).

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