| Abstract No.: | A-C1101 |
| Country: | Canada |
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| Title: | THE TRIPEPTIDE FEG REDUCES INTRASPINAL INFILTRATION OF LEUKOCYTES AND OXIDATIVE DAMAGE AFTER SPINAL CORD INJURY
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| Authors/Affiliations: | 1 Feng Bao*; 1 Lynne Weaver;
1 Robarts Research Institute, Schulich School of Medicine & Dentistry, The University of Western Ontario, London, ON, Canada
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| Content: | Objectives: Traumatic insult to the spinal cord is followed by progressive, secondary damage at the injury site. This can be attributed in part, to inflammatory processes. Salivary gland peptides such as feG (D-isomeric form of the tripeptide phenylalanine glutamate glycine) can modulate activation states and migratory potential of circulating leukocytes. We have shown that intravenous treatment with feG has anti-inflammatory, neuroprotective effects, improving motor and sensory outcomes. We concluded that this treatment has potential as a clinical therapeutic agent. The objective of this study was to determine the dose-response relationship and optimal dose for effects of feG on intraspinal inflammation and oxidative damage and to expand the behavioural outcomes previously examined.
Materials and Methods: SCI was induced in female Wistar rats using clip compression injury at the fourth thoracic (T4) spinal cord segment. Five feG doses were tested (range: 20µg/kg - 1mg/kg) and delivered at 2hr,12hr and 24hr post-injury by bolus injections (i.v.). Control rats received equal volumes of saline. Leukocyte infiltration, free radical formation, oxidative damage, and cell death were evaluated at 72hr post-SCI by immunohistochemistry, western blotting, biochemical analyses and fluorescent probes in tissue sections or homogenates. After determining the optimal dose, motor function and pain were assessed for 8 weeks.
Results: Antibodies to neutrophils and to ED1 (for macrophages) identified leukocyte infiltration in spinal cord sections. feG treatment (200µg/kg) reduced the density of neutrophils and macrophages. Myeloperoxidase (MPO) assays and western blot analysis of ED1 also estimated the neutrophil and macrophage influx and activation. feG treatment significantly decreased MPO activity at all doses, especially at 100µg/kg (-34%) and 200µg/kg (-38%); ED1 expression was significantly reduced at 200µg/kg (-49%) and 100µg/kg (-40%).
Expression of the gp91Phox subunit of NADPH oxidase (western blot analysis) was significantly reduced by the 200µg/kg and 100µg/kg doses (-42% and -39%). Free radical formation was detected using oxidation of 2’-7’-dichlorofluorescin diacetate to the fluorescent 2’-7’-dichlorofluorescein (DCF) in vivo or ex vivo. DCF was monitored by fluorescence intensity in tissue sections (microscopy) or tissue homogenates (spectrophotometry). feG treatment clearly reduced DCF fluorescence, particularly at the 200µg/kg and 100µg/kg doses: -54% and -41%,in tissue sections and -30% and -26% in homogenates.
Oxidative damage was estimated by the amount of malondiadehyde (MDA, by a thiobarbituric acid reactive substances (TBARS) assay and western blot analysis of 4-hydroxynonenal (HNE)-bound protein in tissue homogenates. feG treatment significantly decreased the TBARS concentration (-32% at 200µg/kg) and HNE-bound protein (-58% and -57% at 200µg/kg and 100µg/kg).
Cell death was estimated activated caspase-3 protein expression (western blotting) in injured spinal cord. Activated caspase-3 expression significantly increased by approximately 6-11 fold in injured compared to uninjured cords. The feG treatment at 200µg/kg and 100µg/kg significantly reduced the activated caspase-3 protein (-45% and -37%) after SCI. |
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