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,letc. To study the functional and pathological roles of NO. in the brain, a reliable method to moni tor NO. directly is needed. Since oxyhemoglobin (Hb) has a high affini ty for NO. and upon binding is converted quantitatively to methemoglob in (MetHb), spectrophotometry of Hb conversion to MetHb can give a cre dible measurement of NO. concentration. Although this method is especi ally promising for in vivo microdialysis, factors can influence the re producibility and stability, making it difficult to obtain reliable re sults 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 prot ein degradation and Hb autoxidation as well as integrating the differe nce spectra, this assay has a practical differential detection limit o f about 7 nM (0.4 pmol) in vivo. We evaluated this method in anestheti zed and awake rats by measuring the release of NO. induced by the exci totoxin kainic acid (13 mg/kg, i.p.). A protocol with detailed analyti cal parameters for NO. monitoring in neurobiological research is given .