James H. Fitzpatrick

Effects of Nitrous Oxide on the Cerebrovascular Tone, Oxygen Metabolism, and Electroencephalogram of the Isolated Perfused Canine Brain

Nitrous oxide has been reported to act both as a stimulant and as a depressant of cerebral oxygen metabolism (CMRO2) and blood flow under a variety of experimental conditions in the intact animal. The isolated brain preparation is advantageous because it permits direct measurement of blood flow and allows the study of drug effects without interference from other organ systems or drugs. In this study, six isolated perfused canine brain preparations were used to compare the CMRO2, cerebral vascular resistance (CVR), and the EEG of brains perfused with normocapnic, normoxic blood equilibrated with either 70% N2O or 70% N2. There was no significant change in CMRO2. Cerebral vascular resistance fell [16.4% ± 3.4% SEM (P <0.015)] during exposure to N2O. The EEG pattern was reduced in amplitude, but showed an increase in both low-voltage β activity (14–40 Hz), and 3–5 Hz activity. In the isolated brain, N2O reduces cerebral vascular tone while exhibiting no effect on cerebral oxygen metabolism.

31P-MRS-Based Determination of Brain Intracellular and Interstitial pH: Its Application to In Vivo H+ Compartmentation and Cellular Regulation during Hypoxic/Ischemic Conditions

In the last decade, significant progress has been made in the characterization of pH regulation in nervous tissue in vitro. However, little work has been directed at understanding how pH regulatory mechanisms function in vivo. We are interested in how ischemic acidosis can effect pH regulation and modulate the extent of post-ischemic brain damage. We used 31P-MRS to determine normal in vivo pHi and pHe simultaneously in both the isolated canine brain and the intact rat brain. We observed that the 31Pi peak in the 31P-MRS spectrum is heterogeneous and can be deconvoluted into a number of discrete constituent peaks. In a series of experiments, we identified these peaks as arising from either extracellular or intracellular sources. In particular, we identified the peak representing the neurons and astrocytes and showed that they maintain different basal pH (6.95 and 7.05, respectively) and behave differently during hypoxic/ischemic episodes.

In vivo microdialysis of 2-deoxyglucose 6-phosphate into brain: a novel method for the measurement of interstitial pH using 31P-NMR.

Abstract : A unique method for simultaneously measuring interstitial (pHe) as well as intracellular (pHi) pH in the brains of lightly anesthetized rats is described. A 4‐mm microdialysis probe was inserted acutely into the right frontal lobe in the center of the area sampled by a surface coil tuned for the collection of 31P‐NMR spectra. 2‐Deoxyglucose 6‐phosphate (2‐DG‐6‐P) was microdialyzed into the rat until a single NMR peak was detected in the phosphomonoester region of the 31P spectrum. pHe and pHi values were calculated from the chemical shift of 2‐DG‐6‐P and inorganic phosphate, respectively, relative to the phosphocreatine peak. The average in vivo pHe was 7.24 ± 0.01, whereas the average pHi was 7.05 ± 0.01 (n = 7). The average pHe value and the average CSF bicarbonate value (23.5 ± 0.1 mEq/L) were used to calculate an interstitial Pco2 of 55 mm Hg. Rats were then subjected to a 15‐min period of either hypercapnia, by addition of CO2 (2.5, 5, or 10%) to the ventilator gases, or hypocapnia (Pco2 < 30 mm Hg), by increasing the ventilation rate and volume. pHe responded inversely to arterial Pco2 and was well described (r2 = 0.91) by the Henderson‐Hassel‐balch equation, assuming a pKa for the bicarbonate buffer system of 6.1 and a solubility coefficient for CO2 of 0.031. This confirms the view that the bicarbonate buffer system is dominant in the interstitial space. pHi responded inversely and linearly to arterial Pco2. The intracellular effect was muted as compared with pHe (slope = ‐0.0025, r2 = 0.60). pHe and pHi values were also monitored during the first 12 min of ischemia produced by cardiac arrest. pHe decreases more rapidly than pHi during the first 5 min of ischemia. After 12 min of ischemia, pHe and pHi values were not significantly different (6.44 ± 0.02 and 6.44 ± 0.03, respectively). The limitations, advantages, and future uses of the combined microdialysis/31P‐NMR method for measurement of pHe and pHi are discussed.

Hyperglycemic Damage to Mitochondrial Membranes During Cerebral Ischemia: Amelioration by Platelet‐Activating Factor Antagonist BN 50739

Abstract: The Pulsinelli‐Brierley four‐vessel occlusion model was used to study the consequences of hyperglycemic ischemia and reperfusion. Rats were subjected to either 30 min of normo‐ or hyperglycemic ischemia or 30 min of normo‐ or hyperglycemic ischemia followed by 60 min of reperfusion. In some animals, 2 mg/kg BN 50739, a platelet‐activating factor receptor antagonist, was administered intraarterially either before or after the ischemic insult. The changes in mitochondrial membrane free fatty acid levels, phosphatidylcholine fatty acyl composition, and thiobarbituric acid‐reactive material (TBAR) content plus the mitochondrial respiratory control ratio (RCR) were monitored. When the platelet‐activating factor antagonist was present during normoglycemia, (a) the mitochondrial free fatty acid release both during and after ischemia was slowed, (b) reacylation of phosphatidylcholine following ischemia was promoted, and (c) TBAR accumulation during and following ischemia was decreased. The detrimental effects of hyperglycemia were muted when BN 50739 was present during ischemia. The RCR was preserved and phosphatidylcholine hydrolysis during ischemia was decreased. TBAR levels were consistently higher in hyperglycemic brain mitochondria both during and after ischemia. The RCR correlated directly with mitochondrial phosphatidylcholine polyunsaturated fatty acid content during ischemia and reperfusion. BN 50739 protection of mitochondrial membranes in brain may be influenced by tissue pH.

NMR-Based Identification of Intra- and Extracellular Compartments of the Brain Pi Peak

Abstract: The Pi peak in a 31P NMR spectrum of the brain can be deconvoluted into six separate Lorentzian peaks with the same linewidth as that of the phosphocreatine peak in the spectrum. In an earlier communication we showed that the six Pi peaks in normal brain represent two extracellular and four intracellular compartments. In that report we have identified the first of the extracellular peaks by marking plasma with infused Pi, thereby substantially increasing the amplitude of the single peak at pH 7.35. 2‐Deoxyglucose‐6‐phosphate (2‐DG‐6‐P) was placed in the brain interstitial space by microdialysis. The resulting 2‐DG‐6‐P peak was deconvoluted into three separate peaks. The chemical shift of the principle 2‐DG‐6‐P peak gave a calculated pH of 7.24 ± 0.02 for interstitial fluid pH, a value that agreed well with the pH of the second extracellular Pi peak at pH 7.25 ± 0.01. We identified the intracellular compartments by selectively stressing cellular energy metabolism in three of the four intracellular spaces. A seizure‐producing chemical, flurothyl, was used to activate the neuron, thereby causing a demand for energy that could not be completely met by oxidative phosphorylation alone. The resulting loss of high‐energy phosphate reserves caused a significant increase in intracellular Pi only in those cells associated with the Pi peak at pH 6.95 ± 0.01. This suggests that this compartment represents the neuron. Ammonia is detoxified in the astrocyte (glutamine synthetase) by incorporating it into glutamine, a process that requires large amounts of glucose and ATP. The intraarterial infusion of ammonium acetate into the brain stressed astrocyte energy metabolism resulting in an increase in the Pi of the cells at pH of 7.05 ± 0.01 and 7.15 ± 0.02. This finding, coupled with our observation that these same cells take up infused Pi probably via the astrocyte end‐foot processes, lead us to conclude that these two compartments represent two different types of astrocytes, probably protoplasmic and fibrous, respectively. As a result of this study, we now believe the brain contains four extracellular and four intracellular compartments.

The cerebral origin of the alpha rhythm

The EEG alpha rhythm was recorded from 8 isolated canine brains in the absence of orbital contents, drug effect, or pulsatile cerebral blood flow. Abrupt shift to hypoxic perfusion, with maintenance of other perfusion variables, resulted in a loss of alpha coincident with a fall in CMRO2 and rise in oxygen deficit. It is concluded that the alpha rhythm reflects neural electrical activity, and that sources in the eye muscles or in cardiac-induced electromechanical properties of the brain may be rejected.

NMR studies of Pi-containing extracellular and cytoplasmic compartments in brain.

Abstract: The inorganic phosphate (Pi) NMR peak in brain has an irregular shape, which suggests that it represents more than a single homogeneous pool of Pi. To test the ability of the Marquardt‐Levenberg (M‐L) nonlinear curve fit algorithm software (Peak‐Fit) to separate multiple peaks, locate peak centers, and estimate peak heights, we studied simulated Pi spectra with defined peak centers, areas, and signal‐to‐noise (S/N) ratios ranging from ∞ to 5.8. As the S/N ratio decreased below 15, the M‐L algorithm located peak centers accurately when they were detected; however, small peaks tended to grow smaller and disappear, whereas the amplitudes of larger peaks increased. We developed an in vitro three‐compartment model containing a mixture of Pi buffer, phosphocreatine, phosphate diester, and phosphate monoester (PME), portions of which were adjusted to three different pHs before addition of agar. Weighed samples of each buffered gel together with phospholipid extract and bone chips were placed in an NMR tube and covered with mineral oil. Following baseline correction, it was possible to separate the Pi peaks arising from the three compartments with different pH values if each peak made up 10–35% of total Pi area. In vivo, we identified the plasma compartment by intraarterial infusion of Pi. It was assumed that intracellular compartments contained high‐energy phosphates and took up glucose. Based on these assumptions we subjected the brains to complete ischemia and observed that Pi compartments at pH 6.82, 6.92, 7.03, and 7.13 increased markedly in amplitude. If the brain cells took up and phosphorylated 2‐deoxyglucose (2‐DG), 2‐DG‐6‐phosphate (2‐DG‐6‐P) would appear in the PME portion of the spectrum ionized according to pHi. Four 2‐DG‐6‐P peaks with calculated pH values of 6.86, 6.94, 7.04, and 7.15 did appear in the spectrum, thereby confirming that the four larger Pi peaks represented intracellular spaces.

The effect of a free radical scavenger and platelet-activating factor antagonist on FFA accumulation in post-ischemic canine brain

The effects of the platelet-activating factor antagonist BN 50739 and a free radical scavenger dimethyl sulfoxide on the accumulation of free fatty acids in post-ischemic canine brain are reported. Following 14 min of complete normothermic ischemia and 60 min of reperfusion, the total brain FFAs were approximately 150% higher than in the control group (p<0.05). Perfusion with the platelet-activating factor antagonist BN50739 in its diluent dimethyl sulfoxide during 60 min of post-ischemic reoxygenation resulted in a 61.8% (p<0.01) reduction in the total brain free fatty acid accumulation. Palmitic, stearic, oleic, linoleic, and arachidonic acids decreased by 53.8%, 63.5%, 69.0%, 47.4%, and 57.2%, respectively. Although dimethyl sulfoxide alone caused stearic and arachidonic acids to return to the normal concentration range, BN 50739 had a significant influence on recovery of palmitic, oleic, and linoleic acids and was previously shown to provide significant therapeutic protection against damage to brain mitochondria following an ischemic episode. Because free fatty acid accumulation is one of the early phenomena in cerebral ischemia, this study provides evidence to support the hypothesis that both platelet-activating factor and free radicals are involved in initiating cerebral ischemic injury.

Inorganic Phosphate Compartmentation in the Normal Isolated Canine Brain

Abstract: In vivo 31P magnetic resonance spectra of 16 isolated dog brains were studied by using a 9.4‐T wide‐bore superconducting magnet. The observed Pi peak had an irregular shape, which implied that it represented more than one single homogeneous pool of Pi. To evaluate our ability to discriminate between single and multiple peaks and determine peak areas, we designed studies of simulated 31Pi spectra with the signal‐to‐noise (S/N) ratios ranging from ∞ to 4.4 with reference to the simulated Pi peak. For the analysis we used computer programs with a linear prediction algorithm (NMR‐Fit) and a Marquardt–Levenberg nonlinear curve‐fit algorithm (Peak‐Fit). When the simulated data had very high S/N levels, both methods located the peak centers precisely; however, the Marquardt‐Levenberg algorithm (M‐L algorithm) was the more reliable at low S/N levels. The linear prediction method was poor at determining peak areas; at comparable S/N levels, the M‐L algorithm determined all peak areas relatively accurately. Application of the M‐L algorithm to the individual experimental in vivo dog brain data resolved the Pi peak into seven or more separate components. A composite spectrum obtained by averaging all spectral data from six of the brains with normal O2 utilization was fitted using the M‐L algorithm. The results suggested that there were eight significant peaks with the following chemical shifts: 4.07, 4.29, 4.45, 4.62, 4.75, 4.84, 4.99, and 5.17 parts per million (ppm). Although linear prediction demonstrated the presence of only three peaks, all corresponded to values obtained using the M‐L algorithm. The peak indicating a compartment at 5.17 ppm (pH 7.34) was assigned to venous pH on the basis of direct simultaneous electrode‐based measurements. On the basis of earlier electrode studies of brain compartmental pH, the peaks at 4.99 ppm (pH 7.16) and 4.84 ppm (pH 7.04) were thought to represent interstitial fluid and the astrocyte cytoplasm, respectively.