ADULT-RAT BRAIN-SLICE PREPARATION FOR NUCLEAR-MAGNETIC-RESONANCE SPECTROSCOPY STUDIES OF HYPOXIA

Background: When perfused neonatal brain slices are studied ex vivo wi th nuclear magnetic resonance (NMR) spectroscopy, it is possible to us e P-31 detection to monitor levels of intracellular adenosine triphosp hate (ATP), cytosolic pH, and other high-energy phosphates and H-1 det ection to monitor lactate and glutamate. Adult brain slices of high me tabolic integrity are more difficult to obtain for such studies, becau se the adult cranium is thicker, and postdecapitation revival time is shorter. A common-clinical anesthesia phenomenon-loss of temperature r egulation during anesthesia, with surface cooling and deep hypothermia , was used to obtain high-quality adult rat cerebrocortical slices for NMR studies. Methods: Spontaneously breathing adult rats (350 g), ane sthetized with isoflurane in a chamber, were packed in ice and cooled until rectal temperatures decreased to approximate to 30 degrees C. An intraaortic injection of heparinized saline at 4 degrees C further co oled the brain to approximate to 18 degrees C. Slices were obtained an d then recovered at 37 degrees C in oxygenated medium. Interleaved P-3 1/H-1 NMR spectra were acquired continually before, during, and after 20 min of no-now hypoxia (PO2 approximate to 0 mmHg). Histologic (Niss l stain) measurements were made from random slices removed at differen t times in the protocol. Three types of pretreatment were compared in no-flow hypoxia studies. The treatments were: (1) hyperoxia; (2) hyper capnia (50% CO2); and (3) hypoxia, which was accomplished by washing t he slices with perfusate equilibrated with 100% N-2 and maintaining a 100% N-2 gas flow in the air space above the perfusate. Results: Durin g hyperoxia, P-31 NMR metabolite ratios were identical to those seen i n vivo in adult brains, except that, in vitro, the P-1 peak was slight ly larger than in vivo. A lactate peak was seen in in vitro H-1 spectr a of slices after metabolic recovery from decapitation, although lacta te is barely detectable in vivo in healthy brains. The in vitro lactat e 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 ex periments. Phosphocreatine and ATP became undetectable during oxygen d eprivation, which also caused a three- to sixfold increase in the rati o of lactate to N-acetyl-aspartate, Within experimental error, all met abolite concentrations except pH(1) recovered to control values within 2 h after oxygen restoration. Nissl-stained sections suggested that p retreatment with hypercapnia protected neurons from cell swelling duri ng the brief period of no-flow oxygen deprivation. Conclusions: Perfus ed, respiring adult brain slices having intact metabolic function can be obtained for NMR spectroscopy studies, Such studies have higher spe ctral resolution than can be obtained in vivo. During such NMR experim ents, one can deliver drugs or molecular probes to brain cells and obt ain brain tissue specimens for histologic and immunochemical measures of injury:Important ex vivo NMR spectroscopy studies that are difficul t or impossible to perform in vivo are feasible in this model.