Pharmacology and Therapeutics - Research Publications
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ItemPathological Changes in the White Matter after Spinal Contusion Injury in the Rat(PUBLIC LIBRARY SCIENCE, 2012-08-29)It has been shown previously that after spinal cord injury, the loss of grey matter is relatively faster than loss of white matter suggesting interventions to save white matter tracts offer better therapeutic possibilities. Loss of white matter in and around the injury site is believed to be the main underlying cause for the subsequent loss of neurological functions. In this study we used a series of techniques, including estimations of the number of axons with pathology, immunohistochemistry and mapping of distribution of pathological axons, to better understand the temporal and spatial pathological events in white matter following contusion injury to the rat spinal cord. There was an initial rapid loss of axons with no detectable further loss beyond 1 week after injury. Immunoreactivity for CNPase indicated that changes to oligodendrocytes are rapid, extending to several millimetres away from injury site and preceding much of the axonal loss, giving early prediction of the final volume of white matter that survived. It seems that in juvenile rats the myelination of axons in white matter tracts continues for some time, which has an important bearing on interpretation of our, and previous, studies. The amount of myelin debris and axon pathology progressively decreased with time but could still be observed at 10 weeks after injury, especially at more distant rostral and caudal levels from the injury site. This study provides new methods to assess injuries to spinal cord and indicates that early interventions are needed for the successful sparing of white matter tracts following injury.
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ItemSpontaneous Development of Full Weight-Supported Stepping after Complete Spinal Cord Transection in the Neonatal Opossum, Monodelphis domestica(PUBLIC LIBRARY SCIENCE, 2011-11-02)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.
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ItemAge-Dependent Changes in the Proteome Following Complete Spinal Cord Transection in a Postnatal South American Opossum (Monodelphis domestica)(PUBLIC LIBRARY SCIENCE, 2011-11-16)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.
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ItemEffects of neonatal systemic inflammation on blood-brain barrier permeability and behaviour in juvenile and adult rats.(Hindawi Limited, 2011)Several neurological disorders have been linked to inflammatory insults suffered during development. We investigated the effects of neonatal systemic inflammation, induced by LPS injections, on blood-brain barrier permeability, endothelial tight junctions and behaviour of juvenile (P20) and adult rats. LPS-treatment resulted in altered cellular localisation of claudin-5 and changes in ultrastructural morphology of a few cerebral blood vessels. Barrier permeability to sucrose was significantly increased in LPS treated animals when adult but not at P20 or earlier. Behavioural tests showed that LPS treated animals at P20 exhibited altered behaviour using prepulse inhibition (PPI) analysis, whereas adults demonstrated altered behaviour in the dark/light test. These data indicate that an inflammatory insult during brain development can change blood-brain barrier permeability and behaviour in later life. It also suggests that the impact of inflammation can occur in several phases (short- and long-term) and that each phase might lead to different behavioural modifications.
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ItemSpatio-Temporal Progression of Grey and White Matter Damage Following Contusion Injury in Rat Spinal Cord(PUBLIC LIBRARY SCIENCE, 2010-08-09)Cellular mechanisms of secondary damage progression following spinal cord injury remain unclear. We have studied the extent of tissue damage from 15 min to 10 weeks after injury using morphological and biochemical estimates of lesion volume and surviving grey and white matter. This has been achieved by semi-quantitative immunocytochemical methods for a range of cellular markers, quantitative counts of white matter axonal profiles in semi-thin sections and semi-quantitative Western blot analysis, together with behavioural tests (BBB scores, ledged beam, random rung horizontal ladder and DigiGait analysis). We have developed a new computer-controlled electronic impactor based on a linear motor that allows specification of the precise nature, extent and timing of the impact. Initial (15 min) lesion volumes showed very low variance (1.92+/-0.23 mm3, mean+/-SD, n=5). Although substantial tissue clearance continued for weeks after injury, loss of grey matter was rapid and complete by 24 hours, whereas loss of white matter extended up to one week. No change was found between one and 10 weeks after injury for almost all morphological and biochemical estimates of lesion size or behavioural methods. These results suggest that previously reported apparent ongoing injury progression is likely to be due, to a large extent, to clearance of tissue damaged by the primary impact rather than continuing cell death. The low variance of the impactor and the comprehensive assessment methods described in this paper provide an improved basis on which the effects of potential treatment regimes for spinal cord injury can be assessed.