Adult Rat Brain-Slice Preparation for Nuclear Magnetic Resonance Spectroscopy Studies of Hypoxia


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Abstract

BackgroundWhen perfused neonatal brain slices are studied ex vivo with nuclear magnetic resonance (NMR) spectroscopy, it is possible to use sup 31 Phosphorus detection to monitor levels of intracellular adenosine triphosphate (ATP), cytosolic pH, and other high-energy phosphates and1 Hydrogen detection to monitor lactate and glutamate. Adult brain slices of high metabolic integrity are more difficult to obtain for such studies, because the adult cranium is thicker, and postdecapitation revival time is shorter. A common clinical anesthesia phenomenon--loss of temperature regulation during anesthesia, with surface cooling and deep hypothermia, was used to obtain high-quality adult rat cerebrocortical slices for NMR studies.MethodsSpontaneously breathing adult rats (350 g), anesthetized with isoflurane in a chamber, were packed in ice and cooled until rectal temperatures decreased to [nearly equal] 30 degrees C. An intraaortic injection of heparinized saline at 4 degrees C further cooled the brain to [nearly equal] 18 degrees C. Slices were obtained and then recovered at 37 degrees C in oxygenated medium. Interleaved31 Phosphorous/sup 1 Hydrogen NMR spectra were acquired continually before, during, and after 20 min of no-flow hypoxia (PO2 [nearly equal] 0 mmHg). Histologic (Nissl stain) measurements were made from random slices removed at different times in the protocol. Three types of pretreatment were compared in no-flow hypoxia studies. The treatments were: (1) hyperoxia; (2) hypercapnia (50% CO2); and (3) hypoxia, which was accomplished by washing the slices with perfusate equilibrated with 100% Nitrogen2 and maintaining a 100% Nitrogen2 gas flow in the air space above the perfusate.ResultsDuring hyperoxia,31 Phosphorus NMR metabolite ratios were identical to those seen in vivo in adult brains, except that, in vitro, the Phosphorusi peak was slightly larger than in vivo. A lactate peak was seen in in vitro1 Hydrogen spectra of slices after metabolic recovery from decapitation, although lactate is barely detectable in vivo in healthy brains. The in vitro lactate peak was attributed to a small population of metabolically impaired cells in an injury layer at the cut edge. NMR spectral resolution from the solenoidal coil exceeded that obtained in vivo in surface coil experiments. Phosphocreatine and ATP became undetectable during oxygen deprivation, which also caused a three- to sixfold increase in the ratio of lactate to N-acetyl-aspartate. Within experimental error, all metabolite concentrations except pHi recovered to control values within 2 h after oxygen restoration. Nissl-stained sections suggested that pretreatment with hypercapnia protected neurons from cell swelling during the brief period of no-flow oxygen deprivation.ConclusionsPerfused, respiring adult brain slices having intact metabolic function can be obtained for NMR spectroscopy studies. Such studies have higher spectral resolution than can be obtained in vivo. During such NMR experiments, one can deliver drugs or molecular probes to brain cells and obtain brain tissue specimens for histologic and immunochemical measures of injury: Important ex vivo NMR spectroscopy studies that are difficult or impossible to perform in vivo are feasible in this model.

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