Nitric oxide detection with intracerebral microdialysis: Important considerations in the application of the hemoglobin-trapping technique

Nitric oxide (NO') is involved in processes such as neurotransmission, memory, brain injury, vessel relaxation, etc. To study the functional and pathological roles of NO' in the brain, a reliable method to monitor NO' directly is needed. Since oxyhemoglobin (Hb) has a high affinity for NO' and upon binding is converted quantitatively to methemoglobin (MetHb), spectrophotometry of Hb conversion to MetHb can give a credible measurement of NO' concentration. Although this method is especially promising for in vivo microdialysis, factors can influence the reproducibility and stability, making it difficult to obtain reliable results at low NO' levels. Evaluation of the diffusion rates of NO' and sodium nitroprusside across the microdialysis membrane indicates that NO' readily diffuses through the membrane. By taking into account protein degradation and Hb autoxidation as well as integrating the difference spectra, this assay has a practical differential detection limit of about 7 nM (0.4 pmol) in vivo. We evaluated this method in anesthetized and awake rats by measuring the release of NO' induced by the excitotoxin kainic acid (13 mg/kg, i.p.). A protocol with detailed analytical parameters for NO' monitoring in neurobiological research is given.