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23. A new adult rat brain slice preparation for ex vivo NMR spectroscopy studies of stroke
Abstracts/Journal of NeuroscienceMethods (1994)A 1-A28 reversibly eliminated the postsynaptic component of the field potential. A similar, although barely reversible, block occurred with the non-NMDA antagonists CNQX and DNQX (1-0.75 /zM). The specific NMDA antagonist, AP5 (75/zM), caused a much subtler reduction in response amplitude. The GABA antagonist, bicuculline (100/zM), caused a large enhancement of field potential amplitudes in retinorecipient layers. (Supported by NS 24560 and EY 06890 to H.J.K.) 23. A NEW ADULT RAT BRAIN SLICE PREPARATION FOR EX VIVO N M R SPECTROSCOPY STUDIES OF STROKE
M.T. Espanol, L. Litt, L.-H. Chang, T.L. James, P.R. Weinstein, P.H. Chan The University of California, San Francisco, CA 94143, USA NMR spectroscopy manifestations of intracellular energy failure were studied during ischemia and normocapnic ischemia (50% CO 2) in live, perfused ex vivo adult rat cerebrocortical slice preparations. This model allowed us to study cellular phenomena without the following confounding variables that characterize in vivo studies: cerebral blood flow changes, unknown •extracellular concentrations, blood-brain barrier limits to drug delivery, hepatic metabolism of drugs, limits to or requirements for anesthesia. The protocol was approved by the UCSF Committee on Animal Research. Hypothermia was used to extend the revival time of brain tissue, and time needed to harvest adult brains. Spontaneously breathing adult rats (350 g) were anesthetized in a chamber using an isoflurane/oxygen mixture that was reduced from approximately 3 to 1% as rectal temperatures decreased to about 28°C. A midline thoracotomy was then performed and, through a ventriculostomy, 70 ml of heparinized saline at 4°C was injected into the proximal aorta, cooling the brain to about 19°C (as measured after excision). Forty cortical slices (350/zm thin, about 3 g. total tissue weight) were obtained and transferred to a cylindrical NMR tube and perfused, as described previously. Interleaved 31p/~H NMR spectra were obtained in 5 min epochs at 4.7 Tesla before, during, and after 20 min of no-flow ischemia. Stable high-energy phosphorus metabolite levels were obtained within 2-3 h post-decapitation perfusion. Tissue slices were viable for more than 15 h. Slices were removed for histology (Nissl stain) at different times in the protocol. Parallel studies of wet/dry weights were used to quantitate brain edema. Three experiments were done with normocapnic ACSF (5% CO2), and three with hypercapnic ACSF (50% CO2).
A 11
NMR spectra from adult rats had exceptional spectral resolution, exceeding that seen in vivo. PCr and ATP completely disappeared, and lactate/NAA increased about 3-fold. All metabolites recovered to control values within 2 h of the restoration of oxygen. There was no statistical difference between hypercapnic and normocapnic groups. The methods produce brain slices of very high metabolic integrity. From a cellular perspective, pure hypercapnia does not add to metabolic toxicity during the limited ischemia time studied. By correlating NMR energy failure with cell-specific histological measures of injury one can use the model for important studies of cellular stroke mechanisms and the design of therapeutic and protective regimens. 24. A NOVEL APPROACH TO STUDY THE REGULATED RELEASE OF SECRETORY PROTEINS FROM BRAIN SLICES
S.A. Farber a, R.M. Nitsch b, J.H. Growdon b, R.J. Wurtman a a Department of Brain and Cognitive Sciences, MIT, Cambridge, MA 02139, USA; o Department of Neurology, Massachusetts General Hospital, Boston, MA 02114, USA We have developed a brain slice superfusion system to measure protein secretion from mammalian brain tissue. Brain slices derived from individual regions are rapidly prepared on a Mcllwain tissue slicer and loaded into 0.7 ml chambers that are continuously superfused (0.8 ml/min) with oxygenated Krebs-Ringer buffer supplemented with glucose and maintained at 37°C. Slices are equilibrated for 60-90 min after which superfusates are collected and processed for protein purification, separation and quantitation. Five methods with high protein yield will be discussed: (1) ultrafiltration through cellulose against water and subsequent Western blotting; (2) affinity chromatography followed by size exclusion chromatography and Western blotting; (3) acid precipitation and Western blotting; (4) vacuum blotting onto PVDF membranes and Western blotting; (5) vacuum blotting onto PVDF membranes and direct immunostaining. As an exemplar, we measured the secreted ectodomain (APP s) of the amyloid beta-protein precursor (APP), which is present at high levels in cell culture media and human cerebrospinal fluid, by using antibodies against various APP epitopes. We found time-dependent release of substantial amounts of APP s which lacked the cytoplasmic Cterminal domain suggesting ectodomain cleavage of the membrane-associated full-length APP prior to secretion. APP s was modulated by electrical depolarization and tetrodotoxin. The effects of other pharmacological
M.T. Espanol, L. Litt, L.-H. Chang, T.L. James, P.R. Weinstein, P.H. Chan The University of California, San Francisco, CA 94143, USA NMR spectroscopy manifestations of intracellular energy failure were studied during ischemia and normocapnic ischemia (50% CO 2) in live, perfused ex vivo adult rat cerebrocortical slice preparations. This model allowed us to study cellular phenomena without the following confounding variables that characterize in vivo studies: cerebral blood flow changes, unknown •extracellular concentrations, blood-brain barrier limits to drug delivery, hepatic metabolism of drugs, limits to or requirements for anesthesia. The protocol was approved by the UCSF Committee on Animal Research. Hypothermia was used to extend the revival time of brain tissue, and time needed to harvest adult brains. Spontaneously breathing adult rats (350 g) were anesthetized in a chamber using an isoflurane/oxygen mixture that was reduced from approximately 3 to 1% as rectal temperatures decreased to about 28°C. A midline thoracotomy was then performed and, through a ventriculostomy, 70 ml of heparinized saline at 4°C was injected into the proximal aorta, cooling the brain to about 19°C (as measured after excision). Forty cortical slices (350/zm thin, about 3 g. total tissue weight) were obtained and transferred to a cylindrical NMR tube and perfused, as described previously. Interleaved 31p/~H NMR spectra were obtained in 5 min epochs at 4.7 Tesla before, during, and after 20 min of no-flow ischemia. Stable high-energy phosphorus metabolite levels were obtained within 2-3 h post-decapitation perfusion. Tissue slices were viable for more than 15 h. Slices were removed for histology (Nissl stain) at different times in the protocol. Parallel studies of wet/dry weights were used to quantitate brain edema. Three experiments were done with normocapnic ACSF (5% CO2), and three with hypercapnic ACSF (50% CO2).
A 11
NMR spectra from adult rats had exceptional spectral resolution, exceeding that seen in vivo. PCr and ATP completely disappeared, and lactate/NAA increased about 3-fold. All metabolites recovered to control values within 2 h of the restoration of oxygen. There was no statistical difference between hypercapnic and normocapnic groups. The methods produce brain slices of very high metabolic integrity. From a cellular perspective, pure hypercapnia does not add to metabolic toxicity during the limited ischemia time studied. By correlating NMR energy failure with cell-specific histological measures of injury one can use the model for important studies of cellular stroke mechanisms and the design of therapeutic and protective regimens. 24. A NOVEL APPROACH TO STUDY THE REGULATED RELEASE OF SECRETORY PROTEINS FROM BRAIN SLICES
S.A. Farber a, R.M. Nitsch b, J.H. Growdon b, R.J. Wurtman a a Department of Brain and Cognitive Sciences, MIT, Cambridge, MA 02139, USA; o Department of Neurology, Massachusetts General Hospital, Boston, MA 02114, USA We have developed a brain slice superfusion system to measure protein secretion from mammalian brain tissue. Brain slices derived from individual regions are rapidly prepared on a Mcllwain tissue slicer and loaded into 0.7 ml chambers that are continuously superfused (0.8 ml/min) with oxygenated Krebs-Ringer buffer supplemented with glucose and maintained at 37°C. Slices are equilibrated for 60-90 min after which superfusates are collected and processed for protein purification, separation and quantitation. Five methods with high protein yield will be discussed: (1) ultrafiltration through cellulose against water and subsequent Western blotting; (2) affinity chromatography followed by size exclusion chromatography and Western blotting; (3) acid precipitation and Western blotting; (4) vacuum blotting onto PVDF membranes and Western blotting; (5) vacuum blotting onto PVDF membranes and direct immunostaining. As an exemplar, we measured the secreted ectodomain (APP s) of the amyloid beta-protein precursor (APP), which is present at high levels in cell culture media and human cerebrospinal fluid, by using antibodies against various APP epitopes. We found time-dependent release of substantial amounts of APP s which lacked the cytoplasmic Cterminal domain suggesting ectodomain cleavage of the membrane-associated full-length APP prior to secretion. APP s was modulated by electrical depolarization and tetrodotoxin. The effects of other pharmacological