Tumor Necrosis Factor Alpha in the Cerebrospinal Fluid of Dogs with Central Nervous System Disease
Tumor necrosis factor alpha (TNF-α) is a pro-inflammatory cytokine essential in the inflammatory phase of the innate immune response. Cells that express TNF-α include, but are not limited to, B cells, macrophages, neutrophils, plasma cells and astrocytes. Most of the veterinary research on cytokine expression in canine central nervous system (CNS) disease has been performed using reverse transcription-polymerase chain reaction (RT-PCR) on biopsies of CNS tissue or cells from cerebrospinal fluid (CSF). Production of pro-inflammatory cytokines in situ in CSF of canine patients with CNS disease has not been investigated to date. The purpose of this prospective study was to evaluate whether TNF-α could be detected in measurable quantities in CSF samples in situ in dogs with CNS disease and to compare levels to those of normal, healthy dogs. Our hypothesis was that TNF-α would be measured in appreciable quantities in CSF and that the levels would be statistically different between various CNS disease processes in dogs.
Thirty-seven client owned dogs that presented to the Iowa State University Veterinary Teaching Hospital with signs of CNS disease underwent collection of CSF as part of their diagnostic work up. These samples were routinely analyzed; cell count, cytology and protein level. A small aliquot of CSF from each patient (approximately 100µL) was reserved for TNF-α production analysis. The samples were centrifuged at high speed for clarification and frozen at -80 degrees centigrade until time of assay. CSF samples from 8 normal, healthy dogs that presented to the hospital from the local humane society for routine spay or castration were used as the negative control group. Samples were analyzed via quantitative sandwich enzyme immunoassay technique using the Quantikine® Canine TNF-α Immunoassay (R&D Systems, Minneapolis, MN, USA). Study dogs were allotted into one of 6 groups based on disease process. These included intervertebral disk disease (9), vascular disease (2), inflammatory disease (5), idiopathic epilepsy (9), neoplasia (6) and other CNS disease such as congenital disease and trauma (4). Ages of study dogs ranged from 1 to 12 years. Statistical analysis was performed with SAS® version 9.1 using Wilcoxon rank sum and Kruskal-Wallis tests.
TNF-α could be consistently measured in the CSF samples of both normal and diseased dogs via ELISA kit with a median of 20 pg/ml and a 95% confidence interval of 15-27 pg/ml. A dog with acute intervertebral disk disease had the highest single TNF-α level at 47 pg/ml. There was no statistically significant difference in TNF-α levels among the groups of diseased dogs or between normal and diseased groups.
In conclusion, levels of TNF-α could be consistently measured in the CSF in both diseased and normal dogs using a commercially available ELISA kit. Levels of TNF-α were not significantly different between diseased and normal groups or among dogs in diseased groups.