Spinal Cord Lesion Study

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  • Potential Regenerative Strategies within the CNS with Attention to the Hippocampus

Contents

Project Background

Researchers have found that neurons in the central nervous system fail to regenerate after having been lesioned. This issue is relevant to countless neurological disorders including: traumatic brain injury (TBI), spinal cord injury, amyotrophic lateral sclerosis (ALS) and multiple sclerosis (MS). When considering neural regeneration it is critical to examine the differences between peripheral nervous system (PNS) and central nervous system (CNS) damage. After a lesion to the PNS, axons can re-grow and re-innervate targets. From the site of the lesion, the portion of the axon proximal to the soma regenerates a growth cone, which has the potential to grow back to the target. The portion of the axon distal to the soma from the site of the lesion degrades away into the extracellular space. In the PNS after a lesion, Schwann cells revert to an embryonic state producing axon stimulating factors including laminin and Nerve Growth Factor (NGF) which promote axonal re-grow. PNS axonal re-growth limitations include misdirected routing and atrophied targets. After a neuron is lesioned in the CNS, the distal nerve terminal and distal segment of the axon degenerates. Myelinating cells shed myelin and dedifferentiate while immune cells, macrophages and microglia, infiltrate and phagocyte debris. The Proximal portion of axon survives; the cut end of axon re-heals and reforms growth cone. After the CNS lesion, fibroblast reactive gliosis occurs which causes increased production of glia, resulting in a glial scar. The proximal axon tip reforms but fails to grow due to the glial scar and turns into a retraction bulb. Therefore, lesioned CNS neurons fail to regenerate because of reactive gliosis, misguided axonal growth, atrophied post-synaptic targets and CNS inhibition by oligodendrocytes, the myelin producing cell within the CNS. There are at least 3 factors secreted by oligodendrocytes that inhibit axonal re-growth, these factors include: Myelin Associated Glycoprotein (MAG), OMGP and Nogo. Nogo is a protein secreted by oligodendrocytes that inhibits axonal growth cones from re-growing. Research has shown that the use of Nogo knockouts or Nogo antibodies may stimulate re-growth with a CNS lesion such as a stroke.

Ideas for Neuronal Re-growth after CNS Lesions

Insertion of developmental CNS growth factors:

  • NGF
  • Brain Derived Neurotrophic Factor (BDNF)
  • Embryonic Epithelial Progenitor cells
  • Noggin

Inhibit adult CNS axonal growth inhibition

  • Nogo Inhibition
  • Repulsive guidance molecule (RGMa) antibody

Inhibition of reactive gliosis
Transplantation of Stem cells
Combinatorial Therapy

Proposed Experimental Design

Animals and Surgery

30 male and 30 female Sprague–Dawley rats will be anesthetized using sodium pentobarbital (60 mg/kg administered intraperitoneally) and surgically prepared for lateral fluid-percussion brain injury. A craniotomy will performed over the left parietal cortex. Animals will be attached to the fluid percussion device and brain injury of moderate severity will be induced in 20 male and 20 female rats by injecting a bolus of saline at high pressure into the closed cranial cavity. After the injury the skin will be sutured. The 20 sham-injured control animals will be anesthetized and surgically prepared as previously described but will not be subjected to brain injury.

  • Animals will be divided into three groups:
    • GROUP 1 will undergo fluid percussion brain injury and will be subsequently treated with anti–Nogo-A monoclonal antibody (mAb) 7B12. [n=20]
    • GROUP 2 will undergo fluid percussion brain injury and will be subsequently treated with a combinatorial therapy including administration of anti–Nogo-A monoclonal antibody (mAb) 7B12 as well as implantation of a fibroblast embedded into a collagen matrix, genetically altered to secrete NGF, into the lesion site. [n=20]
    • GROUP 3 will undergo sham brain injury and receive a control IgG (Immunoglobulin) monoclonal antibody. [n=20]

Behavioral and Motor Testing

  • MORRIS WATERMAZE TEST OF COGNITIVE FUNCTION
    • The MWM will evaluate the animals' visuospatial learning ability
    • The test is sensitive to damage in the hippocampus
    • Animals will be introduced into circular tank filled with water; the animal's ability to find a platform using visuospatial cues will be assessed
  • ROTAROD
    • The purpose of the Rotarod test is to assess the rodent’s sensorimotor coordination
    • The test is sensitive to damage in the basal ganglia and cerebellum and to drugs that effect motor function
    • The animals will be placed on a rotating rod; the speed of rotation is gradually increased and the rodent’s ability to remain on the rotating rod is recorded
  • COMPOSITE NEUROSCORE
    • The animals in all test groups will be subjected to this battery of neurological motor function tests at 24 hours post-injury and at 1,2,3 and 4 weeks post-injury
    • The tests administered will include a forelimb reflex, hindlimb flexion, lateral pulsion, and an angle board test
    • Tests will assess muscular strength and capacity to balance

Post- Mortem Neurological Evaluations

Loss of Hemispheric Tissue

  • Tissue loss will be evaluated by calculating hemispheric volume

Expression of Hippocampal GAP-43

  • Immunohistochemical analyses for GAP-43 will be performed in the CA1 region of the hippocampus

Sprouting of Axon Collaterals

  • The efficacy of axon collaterals to sprout will be evaluated in the area proximal to the damage

Additional Considerations

This study will seek to examine whether treatment with the Nogo-A neutralizing antibody 7B12, beginning at a clinically relevant time point postinjury, will increase regional expression of GAP-43 protein, believed to be associated with synaptic plasticity, and significantly improve the cognitive outcome following experimental brain trauma. The basis for this study is based on the observations reported in research that suggest that Nogo-A may be an important contributor to the pathophysiology of TBI, and treatment strategies targeting Nogo-A may be a possible treatment strategy for neurological damage and disease.






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