The presence of 4-hydroxynonenal/protein complex as an indicator of oxidative stress after experimental spinal cord contusion in a rat model

Abstract

The authors tested the hypothesis that breach of the blood-spinal cord barrier (BSCB) will produce evidence of oxidative stress and that a similar staining pattern will be seen between 4-hydroxynonenal (HNE)/protein complexes and extravasated immunoglobulin G (IgG). Adult female Fischer 344 rats, each weighing 200 to 225 g, were subjected to a spinal cord contusion at T-10 by means of a weight-drop device. Spinal cord tissue was assessed for oxidative stress by localizing extravasated plasma contents with a monoclonal antibody for rat IgG and protein conjugation with HNE, which is an aldehyde byproduct of lipid peroxidation. The animals were killed at 1 and 6 hours, and 1, 2, and 7 days after surgery. Maximum HNE/protein staining was observed at 2 days postinjury, and HNE/protein and IgG manifested similar staining patterns. Analysis revealed a graduated but asymmetrical rostral-caudal response relative to the T-10 injury site. Both HNE/protein complex and IgG staining revealed that the caudal levels T-11 and T-12 stained significantly more intensely than the rostral levels T-9 and T-8, respectively. A higher percentage of neurons positive for HNE/protein immunostaining was observed in spinal cord levels caudal to the injury site compared with equidistant rostral regions. Protein dot-blot assays also revealed a similar asymmetrical rostral-caudal HNE/protein content. To analyze the timing of the BSCB breach, another group of animals received identical contusions, and horseradish peroxidase (HRP) was injected 10 minutes before or at various times after injury (1, 3, and 6 hours, and 1, 2, and 7 days). Maximum HRP permeability was seen immediately after injury, with a significant decrease occurring by 1 hour and a return to control levels by 2 days posttrauma. Data from this study indicate possible compromise of neuronal, axonal, glial, and synaptic function after trauma, which may be a factor in motor deficits seen in animals after spinal cord contusion. The colocalization of the IgG stain with the HNE/protein stain is consistent with the hypothesis of a mutual cause-effect relationship between BSCB and oxidative stress in central nervous system trauma.