Abstract
Objective—To measure 15F2t isoprostane concentrations in the urine of dogs undergoing ovariohysterectomy (OHE) and dogs undergoing surgery because of intervertebral disk disease (IVDD) and to assess relationships between urinary concentrations of 15F2t isoprostanes and neurologic score in dogs with IVDD.
Animals—11 dogs undergoing OHE and 32 dogs with IVDD undergoing hemilaminectomy.
Procedures—Paired urine samples were obtained at induction of anesthesia and approximately 1 hour after OHE (controls) and were collected from dogs with IVDD at induction of anesthesia (28 samples) and approximately 1 hour after hemilaminectomy (31 samples); 26 paired urine samples were obtained from dogs with IVDD. Urinary isoprostane concentrations were measured by use of a commercial ELISA, and results were adjusted on the basis of urinary creatinine concentrations. Differences in the mean isoprostane-to-creatinine ratio were analyzed. Neurologic score was determined in dogs with IVDD by use of the modified Frankel scoring system.
Results—Urinary isoprostane-to-creatinine ratios were significantly higher in dogs with IVDD than in control dogs before and after surgery. There was no significant difference between values before and after surgery for either group. There was a significant correlation of neurologic score and urinary isoprostane-to-creatinine ratio because dogs that had higher neurologic scores (ie, less severely affected) generally had higher isoprostane-to-creatinine ratios.
Conclusions and Clinical Relevance—Urinary isoprostane-to-creatinine ratios were higher in dogs with IVDD before and after surgery. Analysis of these data suggests that dogs with IVDD are in a state of oxidative stress and that preemptive treatment with antioxidants warrants further investigation.
Intervertebral disk disease is characterized by intervertebral disk degeneration and protrusion or extrusion of disk material into the vertebral canal. It is the most common cause of paresis and paralysis in dogs.1,2 It has been estimated1,2 that IVDD is the reason for admission for 2% of dogs admitted to veterinary teaching hospitals. Damage to the spinal cord is dependent on the duration and location of the lesion, volume of the extruded disk material, dynamic considerations (eg, peracute massive extrusion vs chronic progressive protrusion), and secondary injury. Biochemical and vascular events that start at the time of injury and peak 4 days later result in local hypoxia, demyelination, axonal degeneration, and malacia.3 Severe injury or delayed surgical intervention may result in permanent neurologic deficits and, in many cases, result in the owner's decision to euthanize a paralyzed dog. Although clinicians can do little to control the severity of the primary insult to the spinal cord, treatments aimed at minimizing secondary injury may improve outcome.4
Neuronal intracellular concentrations of ATP are reduced during severe ischemia resulting from spinal cord injury, which leads to release of the excitatory neurotransmitter glutamate.5 Glutamate initiates a pathogenic cascade that results in cellular overload of calcium and increased production of ROS.5 In addition, an inflammatory response is initiated, with recruitment of neutrophils and macrophages and upregulation of cellular adhesion molecules.6 This inflammatory response is more pronounced in the spinal cord than in the brain during CNS injury in mice.6 Recruitment of inflammatory cells leads to the release of inflammatory mediators, including ROS. Release of ROS can lead to a number of secondary effects, which includes recruitment of additional inflammatory cells and thus perpetuates the cycle of ROS-mediated tissue damage. The ROS play a critical role in pathogenesis of several processes, including cardiac disease, ischemia-reperfusion injury, and neurologic disease.7
Isosprostanes are considered an accurate marker of ROS activity in vivo in humans and several other animal species. Urinary or plasma concentration of isoprostanes correlate with disease severity in humans with asthma, heart failure, and pulmonary disease.8,9 Isoprostanes are produced when ROS react with arachidonic acid in cell membranes. Formation of isoprostanes is independent of cyclooxygenase and therefore not affected by nonsteroidal anti-inflammatory drugs.10 Isoprostane concentrations can be assessed in any body fluid, but lipid-containing fluids (ie, plasma or serum) auto-oxidize ex vivo, which is a major concern for confounding of results for analyses of lipid-containing solutions.8 For this reason, urine, which usually does not contain appreciable amounts of lipid and can be collected noninvasively, is the preferred biological sample for assessment of isoprostanes in vivo.
The purpose of the study reported here was to compare concentrations of 15F2t isoprostane in the urine of dogs with IVDD at the time of anesthetic induction and approximately 1 hour after hemilaminectomy with those at similar time points for healthy control dogs undergoing an OHE. In addition, relationships between concentrations of 15F2t isoprostane and neurologic score for dogs with IVDD were evaluated.
Materials and Methods
Animals—Two groups of client-owned dogs were used in the study. Eleven healthy client-owned dogs undergoing OHE served as a control group, and there were 32 client-owned dogs with newly diagnosed IVDD. Inclusion criteria for dogs with IVDD included an intervertebral disk rupture in the region of T3 to L3 that required surgical decompression, was characterized by a decrease in neurologic score (increased severity of neurologic deficits), occurred within the preceding 7 days before our initial examination, and was accompanied by a lack of other documented inflammatory disease (eg, aspiration pneumonia). Exclusion criteria included extradural compressive lesions caused by something other than IVDD (eg, tumors, fractures, or abscesses), intradural or intramedullary lesions, or chronic neurologic disease with little change during the preceding 7 days. The study was conducted in a manner consistent with principles established by the US National Institutes of Health11 and the Animal Welfare Act.
Neurologic assessment—For each dog with IVDD, a neurologic score was assigned at the time of admission. Neurologic scores were determined on the basis of the modified Frankel scoring system12 and assigned on a scale of 0 to 2 (0, no deep pain sensation evident; 1, deep pain evident but no motor function; and 2, deep pain and motor function are evident).
Sample collection—In all dogs, urine samples were obtained by use of midstream catch or aseptic catheterization of the bladder. Expression of urine from the bladder, as is clinically indicated and in accordance with the standard of care for our facility, was used on dogs before surgery. When necessary to obtain a urine sample, an indwelling urinary catheter was aseptically inserted into the bladder, and urine was collected from the closed urinary collection system by use of aseptic techniques. Urine samples were frozen immediately and stored frozen at −80°C until analyzed.
Samples were obtained from all dogs before surgery for use in urinalysis and after surgery for use in assessing hydration status. A urine sample was obtained from each of the 11 healthy dogs undergoing OHE before induction of anesthesia and again approximately 1 hour after surgery. Similarly, a urine sample was obtained at induction of anesthesia from 28 dogs with IVDD and approximately 1 hour after hemilaminectomy from 31 dogs with IVDD.
Analytic methods—Urinary concentrations of isoprostane were determined by use of a commercially available competitive enzyme immunoassay kit.a Samples were analyzed in duplicate and in batches to reduce interassay variation. This assay has undergone extensive analytic validation by the manufacturers, as indicated in results provided in the package insert.
Because 15F2t isoprostane is not a species-specific compound, our in-house validation of this assay was intended to confirm the optimal use of this assay for urine samples obtained from dogs. In-house validation was performed with urine samples previously collected from healthy dogs as part of an unrelated project. Our validation of the assay included assessment of dilutional parallelism of canine urine samples (5 samples at 3 dilutions in which we determined the correct dilution for samples such that they would be within the working range of the assay), assessment of the correlation between direct analysis of canine urine samples and analysis after affinity purification of isoprostane (by use of commercially available isoprostane affinity purification columnsb) from canine urine samples (9 samples in which we assessed evidence of cross-reactive substances in canine urine), and determination of interassay variability (5 samples assayed on 5 separate days). Urinary creatinine concentrations were measured in all samples by personnel at another laboratoryc by use of an automated dry chemistry system.d To account for variation in glomerular filtration rate and urinary concentrating capacity among dogs, urinary isoprostane concentrations were adjusted on the basis of the urinary creatinine concentration, and the isoprostane-to-creatinine ratio was calculated.
Statistical analysis—All statistical analyses were performed by use of a commercial software package.e Mean ± SE of untransformed data was reported.
Transformed data were analyzed for an effect of group (IVDD or control group) and time (before or after surgery) by use of a mixed linear modelf that accounted for the random variance of dog nested within each group and repeated measurements on each dog. When the group-by-time interaction was significant, predetermined comparisons were made within and between groups by use of least squares means with a Scheffé adjustment to maintain the type I error rate at 0.05.
The association between neurologic score and iso-prostane-to-creatinine ratio was explored by use of Cochran-Mantel-Haenszel methods, with stratification and control for time (before or after surgery). The isoprostane-to-creatinine ratio was categorized on the basis of obvious breaks in the distribution observed on scatterplots and through quartile assessment. Exploration was performed by use of categorization of the isoprostane-to-creatinine ratio (< 10, 10 to < 25, 25 to < 40, and ≥ 40; with a simple dichotomized categorization of < 25 or ≥ 25). Because this was an exploratory investigation, associations were evaluated by use of the χ2 statistic and considered significant at a conservative type I error rate of 0.1.
For all statistical analyses, the null hypothesis was that there was no difference between groups. This null hypothesis was rejected and statistical significance assigned for values of P < 0.05.
Results
Animals—Mean ± SE age of the OHE dogs was 2.63 ± 1.26 years. The group comprised 3 Labrador Retrievers, 2 Dachshunds, 1 Boxer, 1 Australian Shepherd, and 2 mixed-breed dogs; breed was not known for 2 dogs. There were 11 paired urine samples collected from these dogs.
Age of the dogs with IVDD was not normally distributed. Median age of the 32 dogs with IVDD was 5.0 years. This group comprised 24 Dachshunds, 5 Pekingese, 1 Golden Retriever, 1 Cocker Spaniel, and 1 mixed-breed dog. There were 16 females (13 spayed) and 16 males (11 castrated). Urine samples were collected from 28 dogs before surgery and 31 dogs after surgery. However, there were only 26 paired samples collected from these dogs. For the dogs with IVDD, 6 had an initial neurologic score of 0, 12 had a neurologic score of 1, and 14 had a neurologic score of 2.
Assay validation—Mean variation between concentrations determined by direct dilution of urine samples in enzyme immunoassay buffer and concentrations determined after affinity chromatography to remove competing substances from the urine was 43.0% for 9 samples. As per the manufacturer's instructions for the enzyme immunoassay kit, all subsequent analyses were performed on affinity-purified samples.
Dilutional parallelism evaluations revealed acceptable linearity for 3 dilutions. The optimum dilution for initial analysis of a sample was determined to be 1:10 (urine:buffer). Samples with concentrations outside the linear range of the assay on initial analysis were reanalyzed at higher or lower dilutions (as necessary) to obtain valid results.
Interassay variability was good; mean interassay coefficient of variation for 5 samples analyzed in 5 separate assays was 9.3%. Other investigators who used this kit have reported13 interassay variability of 7.5% for analysis of exhaled breath condensates.
Analysis of urine concentrations of isoprostane and creatinine—The isoprostane-to-creatinine ratio was considered to be a continuous variable. Data were transformed (ie, 1 divided by the isoprostane-to-creatinine ratio), which resulted in a normal distribution (Kolmogorov-Smirnov statistic).
Mean ± SE urinary concentration of 15F2t isoprostane for OHE dogs was 7.14 ± 0.94 pg/mL before OHE and 7.56 ± 1.18 pg/mL after OHE (Figure 1).
Mean ± SE concentrations of 15F2t isoprostane in urine samples collected before and approximately 1 hour after surgery for dogs undergoing an OHE and dogs with IVDD under-going hemilaminectomy. There were 11 paired samples for the OHE dogs and 26 paired samples for the dogs with IVDD. *Value differs significantly (P = 0.002) from the value before surgery for the OHE dogs. †Value differs significantly (P = 0.003) from the value after surgery for the OHE dogs.
Citation: American Journal of Veterinary Research 67, 7; 10.2460/ajvr.67.7.1226
Mean urinary concentration of 15F2t isoprostane for dogs with IVDD was 23.65 ± 5.87 pg/mL before hemilaminectomy and 30.47 ± 5.41 pg/mL after hemilaminectomy.
We detected a significant effect for the group-by-time interaction. There was not a significant difference between values before and after surgery for either group. However, the isoprostane-to-creatinine ratio for dogs with IVDD differed significantly from that of the control dogs both before (P = 0.002) and after (P = 0.003) surgery.
We detected a significant (P = 0.01) association between neurologic score and isoprostane-to-creatinine ratio. In that analysis, we controlled for the effect of time. The distribution among neurologic scores and isoprostane-to-creatinine ratios was not homogenous because higher neurologic scores tended to have higher (≥ 25) isoprostane-to-creatinine ratios.
Discussion
Analysis of results from dilutional parallelism and interassay variation evaluations revealed that the commercial immunoassay kit used for the study reported here was stable and appropriate for the measurement of 15F2t isoprostane in canine urine samples. The weak correlation between results obtained after solid-phase purification of 15F2t isoprostane and values obtained by direct dilution of the urine samples in enzyme immunoassay buffer indicated that there may be substances in canine urine that cross-react with the anti-isoprostane antibodies used in the kit; thus, solid-phase purification of 15F2t isoprostane from canine urine samples is necessary before analysis with this kit.
Age, sex, and breeds of the dogs with IVDD reported here are similar to those reported elesewhere.14 In our population of dogs, 15F2t isoprostane concentrations were higher for the IVDD group, compared with concentrations for the control OHE dogs. There was no significant difference observed in the urinary iso-prostane-to-creatinine concentrations before and after surgery for either of the 2 groups.
Analysis of the results of the study reported here suggests that IVDD is associated with substantial oxidative stress, most likely through ischemia of the spinal cord during disk extrusion. Resulting ischemia and inflammation likely contributes to the ROS-associated injury. The half-life of 15F2t isoprostane reportedly is 16 minutes in rats,15 and urinary concentrations of 15F2t isoprostane peak 20 minutes after IV injection in rabbits.16 In several studies,17–19 isoprostane concentrations increased immediately after reperfusion, peaked at 1 hour after reperfusion, and then decreased to base-line values by 2 to 3 hours after reperfusion.
Dogs with higher neurologic scores (less affected clinically) tended to have higher isoprostane-to-creatinine ratios, whereas dogs with lower neurologic scores (more severely affected) tended to have lower iso-prostane-to-creatinine ratios. It is intriguing that the more severely affected dogs had a lower isoprostane-to-creatinine ratio. One possible explanation could be that with greater spinal cord damage, there may be reduced perfusion of the damaged cord and an inability to mobilize isoprostane formed after partial reperfusion. However, many factors may influence the interpretation of neurologic score, including variability among clinicians who perform the evaluation and administration of medications. Any of these factors could have contributed to these results. Additional studies with multiple urine samples, particularly after improvement in neurologic score, may be warranted.
Evidence points to a central role of ROS in the pathophysiologic cascade of secondary damage that results after initial spinal cord injury.20–23 In 1 study,21 investigators detected ROS formation within 4 hours after spinal cord injury and ROS continued to be formed up to 24 hours later. An increase in ROS concentrations has also been detected in the semen of men with spinal cord injury.24 Furthermore, increases in ROS concentrations can contribute to death of motor neuron cells in mice with experimentally induced spinal cord injuries.25
Once formed, ROS may attack nucleic acids, proteins, and phospholipids that are critical to cellular function.26 Lipid peroxidation, which tends to be a self-propagating event, is one of the most damaging results that ROS has on spinal cord tissue, which results in the spread of deterioration to regions outside of the original zone of trauma and causes extensive tissue damage proximal and distal to the original trauma zone.27 Products of lipid peroxidation have been detected as early as 1 hour after spinal cord injury, which suggests that lipid peroxidation is one of the earliest biochemical changes after damage to the spinal cord.22,28,29
The CNS is particularly vulnerable to injury from ROS. Although the CNS comprises only 2% of the body mass, it uses 20% of the available oxygen, and high oxygen consumption correlates positively with ROS formation.30 The CNS also has a relatively low amount of glutathione, a critical component of the endogenous antioxidant defense mechanisms that protect cells from ROS-induced damage, which makes the spinal cord extremely susceptible to unchecked ROS-induced damage resulting from primary trauma.27,31 Finally, the CNS contains a high concentration of polyunsaturated fatty acids in the membranes of neurons and oligoden-drocytes, which make it exquisitely sensitive to lipid peroxidation.32
It has been suggested clinically and in 1 study27 that improving the defenses of spinal cord cells against lipid peroxidation is a promising strategy for minimizing damage associated with spinal cord injury. Methylprednisolone, the most widely used drug for humans and other animals with spinal cord injuries, is administered specifically because of its antioxidant activity.33,34 Unfortunately, the dose of methylprednisolone necessary to exert an antioxidant effect exceeds the dose necessary for activation of glucocorticoid receptors by a factor of approximately 1,000; thus, such a dose would contribute to the many adverse effects reported with use of this drug.33 Bleeding in the gastrointestinal tract, an increase in the number of wound infections, and pneumonia are the most commonly reported adverse effects associated with high doses of methylprednisolone in humans.35 In veterinary medicine, 36 of 40 (90%) dogs receiving a high dose of methylprednisolone in 1 study36 developed occult hemorrhage of the gastrointestinal tract, where-as in another study,37 dogs receiving high doses of methylprednisolone had a significant increase in the incidence of gastrointestinal complications, increased use of gastrointestinal tract protectants, and increased cost for each hospital stay, compared with results for dogs receiving other corticosteroids. Although reports38–43 revealed a slight benefit in neurologic score with the use of methylprednisolone, an evidence-based meta-analysis failed to detect benefits of methylpred-nisolone in people with acute spinal cord injury.35 The authors are unaware of similar studies in dogs.
Treatment of patients with spinal cord injury with anti-inflammatory doses of corticosteroids in combination with an effective antioxidant may optimize healing while minimizing adverse effects. Unfortunately, a major limitation in the assessment of clinical efficacy of antioxidants is the difficulty of quantifying their effect. Testing for markers of oxidative stress is not standardized and is fraught with inaccuracies.44,45 Results vary depending on the test used, the biological fluid or tissue tested, and the laboratory method used. Because testing is not standardized, results of treatment trials with antioxidants vary dramatically, and it is difficult to discern the role, if any, antioxidants play in treatment.44,45
The F2 isoprostanes are a relatively recently recognized class of free-radical–catalyzed products of the arachidonic acid pathway. They are produced in vivo independent of cyclooxygenase enzymes by free-radical–catalyzed peroxidation of arachidonic acid.16,45 These chemically stable products of ROS activity are now used extensively in the quantification of lipid peroxidation in vivo and have a high sensitivity and specificity for assessing oxidative stress in several diseases in humans and laboratory animals.46,47 In addition to acting as a marker for oxidant-mediated damage, F2 isoprostanes possess potent vasoconstrictive activity and may be important mediators for the adverse effects of oxidant injury.48,49
The F2 isoprostanes have been studied most extensively.48 There is disagreement as to the correct nomen-clature of the isoprostanes, and 15F2t isoprostane may also be referred to as 8-isoprostaglandin F2α.
Measurement of urinary F2 isoprostanes as a bio-marker of lipid peroxidation is now considered to be the criterion-referenced standard for assessment of oxidative damage in vivo.50 Approximately 90% of F2 isoprostanes are carried in plasma as lipid esters, with the remainder circulating in the free form.50 Free F2 isoprostanes are formed via oxidation of plasma or tissue lipids, and quantification of the free form is believed to provide an index of total body F2 isoprostane production.50
Isoprostanes have been detected in all types of biological fluids and tissue. However, urine may be the ideal biological fluid for measurement of isoprostanes because of the lack of artifactual lipid peroxidation in most species and the noninvasive method of collection.16,45
In pigs with experimentally induced spinal cord ischemia,51 investigators found that 15F2t isoprostane was significantly increased during ischemia and reperfusion, with plasma concentrations increasing almost instantaneously, which was followed by increased urine concentrations within 60 minutes. An excellent correlation was found between plasma and urinary concentrations of isoprostane in that study. Increased urinary concentrations of isoprostane have also been found in humans with hyperreflexic neurogenic bladder resulting from spinal cord injury.52 In dogs used to evaluate ischemia-reperfusion injury,17 15F2t isoprostane concentrations were increased significantly after reperfusion, compared with baseline concentrations.
In the study reported here, we found a significant increase in ROS-generated isoprostane concentrations in dogs with IVDD unrelated to general anesthesia or surgical trauma (surgical control dogs and dogs with IVDD both were subjected to general anesthesia and major surgical procedures), which suggests that oxidative stress is increased in dogs with spinal cord injury. Additional studies will be needed to determine whether increased urinary isoprostane-to-creatinine ratios affect the degree of function and time to return of function in dogs with IVDD undergoing decompressive surgery. Studies to determine whether oxidative damage to the spinal cord affects neurologic recovery and prognosis following spinal cord surgery are also warranted.
Several factors may have affected the results in the study reported here and prompt us to urge caution in interpretation of the results. This study was designed to detect a difference between control dogs and affected dogs. Therefore, the sample size may have been too small to find a difference between the measurements obtained before and after surgery in dogs with IVDD.
Major limitations of the study reported here include a lack of matching on the basis of age and breed for the OHE and IVDD dogs. Most studies53–55 do not support a connection between isoprostane concentration and age. Although the timing for collection of urine samples before surgery varied in the dogs of our report, a study56 in humans revealed that spot urine samples adequately represent daily isoprostane excretion, compared with results for 24-hour urine samples. Because the urinary bladders of the dogs were not completely emptied before surgery, it is possible that there was a dilutional effect (ie, urine remained in the bladder before reperfusion) in the samples obtained after surgery. This effect may have contributed to the lack of significant differences between isoprostane concentrations measured before and after surgery for the dogs with IVDD.
Dogs in the OHE group did not receive any drugs prior to collection of the first urine sample. Some dogs with IVDD had received treatment with nonsteroidal anti-inflammatory drugs or corticosteroids before collection of the first urine sample; however, those data were incomplete because of a lack of information from the referring veterinarians. No effect of nonsteroidal anti-inflammatory drugs on isoprostane excretion has been found in a study57 in humans. In asthmatic humans, corticosteroids depress isoprostane formation after challenge exposure with an allergen.58 Corticosteroids would be expected to depress iso-prostane concentrations, thus acting to minimize any significant differences between the OHE and IVDD groups. Treatment with these drugs before inclusion of dogs in the study may have decreased the difference between concentrations in the samples obtained before and after surgery. Other limitations of this study include the small sample size, a single neurologic score for dogs with IVDD, and a small number (ie, 2) of urine samples collected from each dog. Whether the increased concentration of isoprostane in the urine of dogs with IVDD reflects increased amounts of oxidative stress in the CNS or is a reflection of more generalized systemic oxidative stress remains to be determined.
An additional limitation to the study reported here is that a complete urinalysis was not performed in all dogs, and it is not known whether urinary tract infection or inflammation (ie, resulting from the urinary catheter) could have contributed to isoprostane formation. However, all urine samples before surgery were collected by use of manual bladder expression, and when urine samples were collected after surgery by use of a catheter, they were collected at the time of insertion of the urinary catheter.
In the future, measurement of urinary concentrations of F2 isoprostanes may be used as a noninvasive biomarker to determine the role of ROS in dogs with IVDD. It may also be used to guide dosing regimens and for use in evaluating effectiveness of antioxidants in clinical trials.
ABBREVIATIONS
| IVDD | Intervertebral disk disease |
| ROS | Reactive oxygen species |
| OHE | Ovariohysterectomy |
8-isoprostane enzyme immunoassay kit, Cayman Chemical Co, Ann Arbor, Mich.
8-isoprostane affinity purification kit, Cayman Chemical Co, Ann Arbor, Mich.
Texas Veterinary Medical Diagnostic Laboratory, College Station, Tex.
Roche Hitachi 911, Diagnostic Chemicals Ltd, Oxford, Conn.
SAS, version 9.0, SAS Institute Inc, Cary, NC.
Proc MIXED, SAS, version 9.0, SAS Institute Inc, Cary, NC.
References
- 1
Hoerlin BF. Intervertebral disc disease. In: Oliver JE, Hoerlin BF, Mayhew IG, eds. Veterinary neurology. Philadelphia: WB Saunders Co, 1987;321–341.
- 2
Toombs JP, Waters DJ. Intervertebral disc disease. In: Slatter D, ed. Textbook of small animal surgery. Philadelphia: WB Saunders Co, 2003;1193–1209.
- 3↑
Olby N. Current concepts in the management of acute spinal cord injury. J Vet Intern Med 1999;13:399–407.
- 4↑
Jerram RM, Dewey CW. Acute thoracolumbar disc extrusion in dogs—Part I. Compend Contin Educ Pract Vet 1999;21:922–930.
- 5↑
Juurlink BH, Paterson PG. Review of oxidative stress in brain and spinal cord injury: suggestions for pharmacological and nutritional management strategies. J Spinal Cord Med 1998;21:309–334.
- 6↑
Schnell L, Fearn S & Klassen H, et al. Acute inflammatory responses to mechanical lesions in the CNS: differences between brain and spinal cord. Eur J Neurosci 1999;11:3648–3658.
- 7↑
Cracowski JL, Durand T, Bessard G. Isoprostanes as a biomarker of lipid peroxidation in humans: physiology, pharmacology and clinical implications. Trends Pharmacol Sci 2002;23:360–366.
- 8↑
Morrow JD, Roberts LJ. The isoprostanes: their role as an index of oxidant stress status in human pulmonary disease. Am J Respir Crit Care Med 2002;166:S25–S30.
- 9
Mallat Z, Philip I & Lebret M, et al. Elevated levels of 8-isoprostaglandin F2alpha in pericardial fluid of patients with heart failure: a potential role for in vivo oxidant stress in ventricular dilatation and progression to heart failure. Circulation 1998;97:1536–1539.
- 10↑
Pilacik B, Nofer TW, Wasowicz W. F2-isoprostanes biomarkers of lipid peroxidation: their utility in evaluation of oxidative stress induced by toxic agents. Int J Occup Med Environ Health 2002;15:19–27.
- 11↑
Institute of Laboratory Animal Resources, National Research Council. Guide to the care and use of laboratory animals. Washington, DC: National Academy Press, 1996.
- 12↑
Frankel HL, Hancock DO & Hyslop G, et al. The value of postural reduction in the initial management of closed injuries of the spine with paraplegia and tetraplegia. I. Paraplegia 1969;7:179–192.
- 13↑
Van Hoydonck PG, Wuyts WA & Vanaudenaerde BM, et al. Quantitative analysis of 8-isoprostane and hydrogen peroxide in exhaled breath condensate. Eur Respir J 2004;23:189–192.
- 14↑
Priester WA. Canine intervertebral disc disease—occurrence by age, breed, and sex among 8,117 cases. Theriogenology 1976;6:293–303.
- 16↑
Morrow JD, Roberts LJ. The isoprostanes: unique bioactive products of lipid peroxidation. Prog Lipid Res 1997;36:1–21.
- 17↑
Delanty N, Reilly MP & Pratico D, et al. 8-epi PGF2 alpha generation during coronary reperfusion. A potential quantitative marker of oxidant stress in vivo. Circulation 1997;95:2492–2499.
- 18
Harry D, Anand R & Holt S, et al. Increased sensitivity to endotoxemia in the bile duct-ligated cirrhotic rat. Hepatology 1999;30:1198–1205.
- 19
Favreau F, Petit-Paris I & Hauet T, et al. Cyclooxygenase 1dependent production of F2-isoprostane and changes in redox status during warm renal ischemia-reperfusion. Free Radic Biol Med 2004;36:1034–1042.
- 20
Hall ED, Braughler JM. Free radicals in CNS injury. Res Publ Assoc Res Nerv Ment Dis 1993;71:81–105.
- 21↑
Azbill RD, Mu X, Bruce-Keller A. Impaired mitochondrial function, oxidative stress and altered antioxidant enzyme activities following traumatic spinal cord injury. Brain Res 1997;765:283–290.
- 22
Bondy S, LeBel C. The relationship between excitotoxicity and oxidative stress in the central nervous system. Free Radic Biol Med 1993;14:633–642.
- 23
Coyle JT, Puttfarcken P. Oxidative stress, glutamate, and neurodegenerative disorders. Science 1993;262:689–695.
- 24↑
Monga M, Dunn K, Rajasekaran M. Characterization of ultrastructural and metabolic abnormalities in semen from men with spinal cord injury. J Spinal Cord Med 2001;24:41–46.
- 25↑
Xu W, Chi L & Xu R, et al. Increased production of reactive oxygen species contributes to motor neuron death in a compression mouse model of spinal cord injury. Spinal Cord 2005;43:204–213.
- 26↑
Hall ED. Lipid antioxidants in acute central nervous system injury. Ann Emerg Med 1993;22:1022–1027.
- 27↑
Lucas JH, Wheeler DG & Guan Z, et al. Effect of glutathione augmentation on lipid peroxidation after spinal cord injury. J Neurotrauma 2002;19:763–775.
- 28
Barut S, Canbolat A, Bilge T. Lipid peroxidation in experimental spinal cord injury: time-level relationship. Neurosurg Rev 1993;16:53–59.
- 29
Ildan F, Oner A, Polat S. Correlation of alterations on Na(+)-K+/Mg ATPase activity, lipid peroxidation and ultrastructural findings following experimental spinal cord injury with and without prednisolone treatment. Neurosurg Rev 1995;18:35–44.
- 30↑
Sokoloff L. Circulation and energy metabolism of the brain. In: Sigel J, Agranoff B, Albers R, et al, eds. Basic neurochemistry. New York: Raven Press, 1989;565–590.
- 31
Romero FJ, Monsalve E & Hermenegildo C, et al. Oxygen toxicity in the nervous tissue: comparison of the antioxidant defense of rat brain and sciatic nerve. Neurochem Res 1991;16:157–161.
- 32↑
Agranoff B. Lipids. In: Sigel J, Agranoff B, Albers R, et al, eds. Basic neurochemistry. New York: Raven Press, 1989;91–107.
- 33↑
Amar AP, Levy ML. Pathogenesis and pharmacological strategies for mitigating secondary damage in acute spinal cord injury. Neurosurgery 1999;44:1027–1039.
- 34
Blight AR, Zimber MP. Acute spinal cord injury: pharmacotherapy and drug development perspectives. Curr Opin Investig Drugs 2001;2:801–808.
- 35↑
Hurlbert R. The role of steroids in acute spinal cord injury: an evidence-based analysis. Spine 2001;26(suppl 24):S39–S46.
- 36↑
Hanson SM, Bostwick DR & Twedt DC, et al. Clinical evaluation of cimetidine, sucralfate, and misoprostol for prevention of gastrointestinal tract bleeding in dogs undergoing spinal surgery. Am J Vet Res 1997;58:1320–1323.
- 37↑
Boag A, Otto C, Drobatz K. Complications of methylprednisolone sodium succinate therapy in Dachshunds with surgically treated intervertebral disc disease. J Vet Emerg Crit Care 2001;11:105–110.
- 38
Bracken MB, Collins WF & Freeman DF, et al. Efficacy of methylprednisolone in acute spinal cord injury. JAMA 1984;251:45–52.
- 39
Bracken MB, Shepard MJ & Hellenbrand KG, et al. Methylprednisolone and neurological function 1 year after spinal cord injury. Results of the National Acute Spinal Cord Injury Study. J Neurosurg 1985;63:704–713.
- 40
Bracken MB, Shepard MJ & Collins WF, et al. A randomized, controlled trial of methylprednisolone or naloxone in the treatment of acute spinal-cord injury. Results of the Second National Acute Spinal Cord Injury Study. N Engl J Med 1990;322:1405–1411.
- 41
Bracken MB, Shepard MJ & Collins WF Jr, et al. Methylprednisolone or naloxone treatment after acute spinal cord injury: 1-year follow-up data. Results of the second National Acute Spinal Cord Injury Study. J Neurosurg 1992;76:23–31.
- 42
Bracken MB, Shepard MJ & Holford TR, et al. Administration of methylprednisolone for 24 or 48 hours or tirilazad mesylate for 48 hours in the treatment of acute spinal cord injury. Results of the Third National Acute Spinal Cord Injury Randomized Controlled Trial. National Acute Spinal Cord Injury Study. JAMA 1997;277:1597–1604.
- 43
Bracken MB, Shepard MJ & Holford TR, et al. Methylprednisolone or tirilazad mesylate administration after acute spinal cord injury: 1-year follow up. Results of the third National Acute Spinal Cord Injury randomized controlled trial. J Neurosurg 1998;89:699–706.
- 44
Yin H, Porter NA. New insights regarding the autoxidation of polyunsaturated fatty acids. Antioxid Redox Signal 2005;7:170–184.
- 45
Moore K, Roberts LJ. Measurement of lipid peroxidation. Free Radic Res 1998;28:659–671.
- 46
Pratico D, Barry OP & Lawson JA, et al. IPF2alpha-I: an index of lipid peroxidation in humans. Proc Natl Acad Sci U S A 1998;95:3449–3454.
- 47
Pratico D, Lawson JA, Fitzgerald GA. Cyclooxygenasedependent formation of the isoprostane, 8-epi prostaglandin F2alpha. J Biol Chem 1995;270:9800–9808.
- 48↑
Cracowski JL. Isoprostanes: an emerging role in vascular physiology and disease? Chem Phys Lipids 2004;128:75–83.
- 49
Morrow JD, Minton TA & Mukundan CR, et al. Free radicalinduced generation of isoprostanes in vivo. Evidence for the formation of D-ring and E-ring isoprostanes. J Biol Chem 1994;269:4317–4326.
- 51↑
Basu S, Hellberg A & Ulus AT, et al. Biomarkers of free radical injury during spinal cord ischemia. FEBS Lett 2001;508:36–38.
- 52↑
Oner-Iyidogan Y, Kocak H & Gurdol F, et al. Urine 8-isoprostane F2alpha concentrations in patients with neurogenic bladder due to spinal cord injury. Clin Chim Acta 2004;339:43–47.
- 53
Block G, Dietrich M & Norkus EP, et al. Factors associated with oxidative stress in human populations. Am J Epidemiol 2002;156:274–285.
- 54
Feillet-Coudray C, Tourtauchaux R & Niculescu M, et al. Plasma levels of 8-epiPGF2alpha, an in vivo marker of oxidative stress, are not affected by aging or Alzheimer's disease. Free Radic Biol Med 1999;27:463–469.
- 55
Meghdadi S, Rodrigues M & Oguogho A, et al. 8-EpiPGF2alpha and 6-oxo-PGF1alpha in human (varicose) veins: influence of age, sex, and risk factors. Angiology 2003;54:317–324.
- 56↑
Basu S, Helmersson J. Factors regulating isoprostane formation in vivo. Antioxid Redox Signal 2005;7:221–235.
- 57↑
Catella F, Reilly MP & Delanty N, et al. Physiological formation of 8-epi-PGF2 alpha in vivo is not affected by cyclooxygenase inhibition. Adv Prostaglandin Thromboxane Leukot Res 1995;23:233–236.
- 58↑
Dworski R, Murray JJ & Roberts LJ, et al. Allergen-induced synthesis of F(2)-isoprostanes in atopic asthmatics. Evidence for oxidant stress. Am J Respir Crit Care Med 1999;160:1947–1951.
