Damian Garcia

Quantitative relationship between brain temperature and energy utilization rate measured in vivo using 31P and 1H magnetic resonance spectroscopy

ABSTRACT: In neonatal and adult animals, modest reduction in brain temperature (2–3°C) during ischemia and hypoxia-ischemia provides partial or complete neuroprotection. One potential mechanism for this effect is a decrease in brain energy utilization rate with consequent preservation of brain ATP, as occurs with profound hypothermia. To determine the extent to which modest hypothermia is associated with a decrease in brain energy utilization rate, in vivo 31P and 1H magnetic resonance spectroscopy (MRS) was used to measure the rate of change in brain concentration of phosphocreatine, nucleoside triphosphate, and lactate after complete ischemia induced by cardiac arrest in 11 piglets (8–16 d). Preischemia metabolite concentrations and MRS-determined rate constants were used to calculate the initial flux of high energy phosphate equivalents (d[∼P]/dt, brain energy utilization rate). Baseline physiologic and MRS measurements were obtained at 38.2°C and repeated after brain temperature was adjusted between 28 and 41°C. This was followed by measurement of d[∼P]/dt during complete ischemia at 1–2°C increments within this temperature range. Adjusting brain temperature did not alter any systemic variable except for heart rate which directly correlated with brain temperature (r = 0.95, p < 0.001). Before ischemia brain temperature inversely correlated with phosphocreatine (r = −0.89, p < 0.001), and reflected changes in the phosphocreatine-ATP equilibrium, because brain temperature inversely correlated with intracellular pH (r = −0.77, p = 0.005). Brain temperature and d[∼P]/dt were directly correlated and described by a linear relationship (slope = 0.61, intercept = −12, r = 0.92, p < 0.001). A reduction in brain temperature from normothermic values of 38.2°C was associated with a decline in d[∼P]/dt of 5.3% per 1°C, and therefore decreases in d[∼P]/dt during modest hypothermia represent a potential mechanism contributing to neuroprotection.

Modest Hypothermia Provides Partial Neuroprotection for Ischemic Neonatal Brain

ABSTRACT: Hypothermia is a frequent occurrence in newborns, and thermoregulatory management is a fundamental part of medical stabilization. Although modest reduction in brain temperature (2–3°C) before ischemia provides neuroprotection in adults, the effect of modest hypothermia on immature brain has not been examined. Nine-day-old swine were exposed to 15 min of incomplete global brain ischemia, with intraischemic rectal temperatures of either 38.3 ± 0.4°C (n = 10, normothermic) or 35.4 ± 0.5°C (n = 10, hypothermic). The relationship between rectal and brain temperature was delineated in preliminary experiments on four swine. Animals with intraischemic rectal temperatures maintained at either 39.5°C or 35.5°C were associated with a similar magnitude of difference in brain temperature. Therefore, rectal temperature was used to monitor brain temperature for 20 animals studied subsequently. Ischemia was induced by combining neck compression with hemorrhagic hypotension and resulted in similar group values for mean arterial pressure and changes in pH and blood gases at the completion of ischemia. A clinical overall performance score and brain tissue structure were evaluated after 72 h (or earlier if animals died prematurely). Hypothermic animals had less severe stages of impairment compared with the normothermic group (p = 0.023). Hypothermie piglets had less histologic damage in the neocortex at 0.5 cm beneath the brain surface (p = 0.048), the caudate nucleus (p = 0.038), and the pons/midbrain (p = 0.04) and the same direction of effect in neocortex at 1 cm beneath the surface (p = 0.07) and the cerebellum (p = 0.07) as compared with normothermic animals. The results demonstrate that a 2–3°C reduction in brain temperature during 15 min of incomplete ischemia provides partial neuroprotection in neonatal swine.

Modest hypothermia provides partial neuroprotection when used for immediate resuscitation after brain ischemia

Intraischemic reduction in temperature of 2-3 °C (modest hypothermia) has been demonstrated to provide partial neuroprotection in neonatal animals. This investigation determined if modest hypothermia initiated immediately after brain ischemia provides neuroprotection. Piglets were studied with rectal temperature maintained during the 1st h after 15 min of brain ischemia at either 38.3 ± 0.3 °C (normothermia, n = 11) or at 35.8± 0.5 °C (modest hypothermia, n = 11). The severity of brain ischemia was similar between groups as indicated by equivalent reduction in mean blood pressure (90 ± 15 to 24 ± 3 versus 92± 13 to 26 ± 3 mm Hg), and changes in cerebral metabolites and intracellular pH (pHi) measured by magnetic resonance spectroscopy(β-nucleoside triphosphate = 44 ± 9 versus 42 ± 18% of control, control = 100%, pHi: 6.25±.15 versus 6.24 ± 0.22 for normothermic and modestly hypothermic groups, respectively). In the first 90 min after ischemia, there were no differences between groups in the duration and extent of brain acidosis, and relative concentrations of phosphorylated metabolites. Categorical assessment of neurobehavior was evaluated at 72 h postischemia (n = 16), or earlier if an animals condition deteriorated (n = 6). Postischemic hypothermia was associated with less severe stages of encephalopathy compared with normothermia (p = 0.05). Histologic neuronal injury was assessed categorically in 16 brain regions, and postischemic hypothermia resulted in less neuronal injury in temporal (p = 0.024) and occipital (p = 0.044) cortex at 10 mm beneath the cortical surface, and in the basal ganglia (p = 0.038) compared with that in normothermia. Modest hypothermia for 1 h immediately after brain ischemia provides partial neuroprotection and may represent an adjunct to resuscitative strategies.

End-Tidal CO2 detection of an audible heart rate during neonatal cardiopulmonary resuscitation after asystole in asphyxiated piglets

Even brief interruption of cardiac compressions significantly reduces critical coronary perfusion pressure during cardiopulmonary resuscitation (CPR). End-tidal CO2 (ETCO2) monitoring may provide a continuous noninvasive method of assessing return of spontaneous circulation (ROSC) without stopping to auscultate for heart rate (HR). However, the ETCO2 value that correlates with an audible HR is unknown. Our objective was to determine the threshold ETCO2 that is associated with ROSC after asphyxia-induced asystole. Neonatal swine (n = 46) were progressively asphyxiated until asystole occurred. Resuscitation followed current neonatal guidelines with initial ventilation with 100% O2 followed by cardiac compressions followed by epinephrine for continued asystole. HR was auscultated every 30 s, and ETCO2 was continuously recorded. A receiver operator curve was generated using the calculated sensitivity and specificity for various ETCO2 values, where a positive test was defined as the presence of HR >60 bpm by auscultation. An ETCO2 cut-off value of 14 mm Hg is the most sensitive ETCO2 value with the least false positives. When using ETCO2 to guide uninterrupted CPR in this model of asphyxia-induced asystole, auscultative confirmation of return of an adequate HR should be performed when ETCO2 ≥14 mm Hg is achieved. Correlation during human neonatal CPR needs further investigation.

Randomized Trial of Volume Infusion During Resuscitation of Asphyxiated Neonatal Piglets

Despite its use, there is little evidence to support volume infusion (VI) during neonatal cardiopulmonary resuscitation (CPR). This study compares 5% albumin (ALB), normal saline (NS), and no VI (SHAM) on development of pulmonary edema and restoration of mean arterial pressure (MAP) during resuscitation of asphyxiated piglets. Mechanically ventilated swine (n = 37, age: 8 ± 4 d, weight: 2.2 ± 0.7 kg) were progressively asphyxiated until pH <7.0, Paco2 >100 mm Hg, heart rate (HR) <100 bpm, and MAP <20 mm Hg. After 5 min of ventilatory resuscitation, piglets were randomized blindly to ALB, NS, or SHAM infusion. Animals were recovered for 2 h before euthanasia and lung tissue sampled for wet-to-dry weight ratio (W/D) as a marker of pulmonary edema. SHAM MAP was similar to VI during resuscitation. At 2 h post-resuscitation, MAP of SHAM (48 ± 13 mm Hg) and ALB (43 ± 19 mm Hg) was higher than NS (29 ± 10 mm Hg; p = 0.003 and 0.023, respectively). After resuscitation, SHAM piglets had less pulmonary edema (W/D: 5.84 ± 0.12 versus 5.98 ± 0.19; p = 0.03) and better dynamic compliance (Cd) compared with ALB or NS (Cd: 1.43 ± 0.69 versus 0.97 ± 0.37 mL/cm H2O, p = 0.018). VI during resuscitation did not improve MAP, and acute recovery of MAP was poorer with NS compared with ALB. VI was associated with increased pulmonary edema. In the absence of hypovolemia, VI during neonatal resuscitation is not beneficial.

Energy Reserves and Utilization Rates in Developing Brain Measured in vivo by 31P and 1H Nuclear Magnetic Resonance Spectroscopy

Age-related changes in cerebral energy utilization were examined in swine, a species whose maximal rate of development is known to occur in the perinatal period. Interleaved in vivo 31P and 1H nuclear magnetic resonance spectroscopy was used to measure the rates of change in cerebral concentrations of phosphocreatine (PCr), nucleoside triphosphates, and lactate following complete ischemia, induced via cardiac arrest, in a total of 19 newborn, 10-day-old, and 1-month-old piglets. Preischemic concentrations of these three metabolites plus glucose and glycogen were determined in a separate experiment on 12 piglets whose brains were funnel-frozen in situ. The rate constants for the PCr and ATP decline and lactate increase were determined by nonlinear regression fits to the experimental data, assuming first-order kinetics. The rate constants and preischemic metabolite concentrations were used to calculate the initial flux of high-energy phosphate equivalents (∼P), which was used as an estimate of cerebral energy utilization at the point when ischemia was initiated. Cerebral energy utilization equaled 6.5 ± 1.9, 9.5 ± 3.2, and 15.1 ± 3.2 μmol ∼P/g/min in newborn, 10-day-old, and 1-month-old piglets, respectively. Within each age group the energy utilization rate was not altered by hyperglycemia-induced increases in cerebral energy reserves, but during hypoglycemia cerebral energy utilization rates decrease. The slope of ∼P versus time decreased with the duration of ischemia, indicating that cerebral energy utilization rates decrease after the first few minutes of ischemia. Newborn piglets had higher cerebral energy utilization rates compared with literature values for newborn rats and mice. This is consistent with the concept that newborns from a species with a perinatal stage of maximal growth and development will have higher cerebral energy demands compared with newborns from a species such as rodents, whose maximal growth occurs postnatally. However, this conclusion remains tentative because literature cerebral utilization rates estimated from the initial slope of ∼P-versus-time plots tend to underestimate the true rate, since the assumption of continued linearity may not be valid for the interval chosen.

Age‐Related Differences in the Effect of Dichloroacetate on Postischemic Lactate and Acid Clearance Measured In Vivo Using Magnetic Resonance Spectroscopy and Microdialysis

Abstract: Numerous studies using adult animal models suggest that dichloroacetate (DCA) may have neuroprotective properties by virtue of its ability to increase rates of metabolism and, therefore, clearance of brain lactic acidosis, which may accumulate during cerebral ischemia. We tested the hypothesis that postischemic DCA administration affects lactate and acid clearance to different extents in immature versus mature brain. 31P and 1H magnetic resonance spectroscopy were used to measure intracellular acid and lactate clearance rates in vivo in newborn and 1‐month‐old swine after a 14‐min episode of transient near‐complete global ischemia. Simultaneous monitoring of extracellular lactate efflux and clearance was measured in the same animals by in vivo microdialysis. Plasma glucose concentrations were elevated in order to study animals with severe cerebral lactic acidosis. Maximal levels of brain lactosis (16–20 µmol/g) and acidosis (pHintracellular 5.8–6.0) were reached during the first 10 min of recovery and were the same in age groups and in subgroups either acting as controls or treated with DCA (200 mg/kg) given from the last minute of ischemia to 5–7 min after ischemia. For newborns, DCA administration improved the postischemic clearance rate of cerebral acidosis and cerebral phosphocreatine, with similar trends for the clearance of lactosis and increased rates of recovery of nucleotide triphosphates, compared with controls. In contrast, DCA administration in 1‐month‐olds resulted in a modest trend for improvement of cerebral lactate clearance, but did not affect acid clearance or the recovery rate of phosphocreatine or nucleotide triphosphates. Extracellular brain lactate concentrations had similar relative increases and rates of decline for subgroups of either age treated with DCA versus controls. The results of this study indicate that postischemic DCA administration helps to resolve cerebral acidosis to a greater degree in immature than more mature brain, suggesting that DCA may have cerebroprotective properties for neonatal hypoxic‐ischemic encephalopathy.

Maturational changes in cerebral lactate and acid clearance following ischemia measured in vivo using magnetic resonance spectroscopy and microdialysis

Intraischemic hyperglycemia has different effects on neurologic outcome in mature vs. immature brain, and may reflect differences in the extent or duration of cerebral lactic acidosis. We examined the hypotheses that post-ischemic lactate and acid clearance rates depend on the severity of intraischemic cerebral acidosis, and that rates of clearance change as a function of brain maturation. In vivo 31P and 1H magnetic resonance spectroscopy (MRS) was used to compare intracellular acid and lactate clearance rates in newborn and 1-month old swine following a 14-min episode of transient near-complete global ischemia. In the same animals, in vivo microdialysis was used to determine if extracellular lactate clearance changed as a function of cerebral lactic acidosis or differed between age groups following ischemia. Plasma glucose concentration was altered in individual animals to study a range of intraischemic cerebral lactic acidosis. For both age-groups, maximal brain acidosis and lactosis occurred in the post-ischemia interval, indicating a delay in the re-establishment of oxidative metabolism following ischemia. Clearance half-lives of both cerebral acidosis and lactosis increase as a function of increased intraischemic cerebral acidosis. For either age group, the clearance half-life for acidosis was faster than the half-life for lactate. However, the subgroup of 1-month old swine who experienced severe cerebral acidosis (i.e., pH<6.1) had a longer cerebral lactate clearance half-life as compared to the subgroup of newborn animals with a similar severity of acidosis. In both age groups, there were comparable maximal increases in extracellular lactate concentrations in the post-ischemic period and similar rates of decline from the maximum. These results demonstrate that post-ischemic lactate and acid clearance are altered by the extent of intraischemic acidosis, and the extent of post-ischemic uncoupling between brain acid and lactate clearance increases with advancing age. The transmembrane clearance of lactate was not a prominent mechanism that differentiated lactate clearance rates between newborn and 1-month old swine.

Cerebral acid buffering capacity at different ages measured in vivo by 31P and 1H nuclear magnetic resonance spectroscopy.

Cerebral acidosis occurring during ischemia has been proposed as one determinant of tissue damage. Newborn animals appear to be less susceptible to ischemic tissue damage than adults. One possible component of ischemic tolerance could derive from maturational differences in the extent of acid production and buffering in newborns compared to adults. The purpose of this study was to measure the dependency of acid production on the blood plasma glucose concentrations and acid buffering capacity of piglets at different stages of development. Complete ischemia was induced in 29 piglets ranging in postconceptual age from 111 to 156 days (normal term conception, 115 days). Brain buffering capacity during the first 30 min of ischemia was quantified in vivo, via 31P and 1H nuclear magnetic resonance (NMR) spectroscopy, by measuring the change in intracellular brain pH for a given change in the concentration of compounds that contribute to the production of hydrogen ions. Animals from all four age groups showed a similar linear correlation between preischemia blood glucose concentration and intracellular pH after 30 min of ischemia. For each animal the slope of the plot of intracellular pH versus cerebral buffer base deficit was used to calculate the buffer capacity. Using data obtained over the entire 30 min of ischemia, there was no difference in the mean buffer capacity of the different age groups, nor was there a significant correlation between buffer capacity and age. However, there was a significant increase in buffer capacity for the intracellular pH range 6.6‐6.0, compared to 7.0‐6.6, for all age groups. No significant differences in buffer capacity for these two pH ranges were observed between any of the age groups. Acid buffering capacity was also measured by performing pH titrations on brain tissue homogenized in the presence of inhibitors of glycolysis and creatine kinase. Plots of homogenate pH versus buffer base deficit showed a nonlinear trend similar to that seen in vivo, indicating an increase in buffer capacity as intracellular pH decreases. A comparison of newborn and 1‐month‐old brain tissue frozen under control conditions or after 45 min of ischemia revealed no differences that could be attributed to age and a slight decrease in buffer capacity of ischemic brain compared to control brain tissue homogenates. There was no difference between the brain buffering capacity measured in vivo using 31P and 1H NMR and that measured in vitro using brain homogenates.

The effect of age on glucose-modulated cerebral agonal glycolytic rates measured in vivo by 1H NMR spectroscopy

ABSTRACT: The purpose of this study was to investigate the effect of plasma glucose concentration on cerebral agonal glycolytic rates in piglets of different ages. Twenty-four piglets were divided into four different age groups corresponding to 113, 121, 128, and 145 d postconception (normal gestation = 115 d). For each group the agonal glycolytic rate was measured by monitoring the rate of cerebral lactate accumulation after total ischemia. Ischemia was induced by cardiac arrest, and the rate of lactate formation was measured in vivo using proton nuclear magnetic resonance spectroscopy. Before cardiac arrest, the blood plasma glucose concentration for individual piglets was adjusted to a specific value in the range 1–30 mM. The dependence of agonal glycolytic rate upon blood glucose concentration was analyzed for each age group, using the Michaelis-Menten equation to evaluate Vmax, the maximal rate of glucose utilization, and Km the concentration of plasma glucose at which the half maximal rate of utilization occurs. Vmax for the two youngest age groups of piglets had significantly different (p < 0.05) values compared with each other (1.38 ± 0.17 and 1.92 ± 0.64 μmol·g-1·min-1, respectively) and with the two older groups of animals (2.99 ± 0.52 and 3.42 ± 0.65 μmol·g-1·min-1, respectively). The Km values determined for the two youngest age groups (0.79 ± 0.70 and 1.79 ± 0.33 mM, respectively) also were significantly lower than for the two older age groups (4.96 ± 2.90 and 4.82 ± 2.96 mM, respectively). We conclude that throughout the first 2 wk of life there are marked increases in the cerebral glycolytic capacity. It follows, therefore, that rates of lactate formation in newborns are not as strongly accelerated by increased blood glucose compared with older piglets. During combined hyperglycemia and severe ischemia, newborns will not be exposed to harmful levels of cerebral lactate as rapidly as 2− to 4-wk-old piglets. However, despite the differences in glycolytic rates, all four age groups showed similar potentials to generate high cerebral lactate concentrations. Furthermore, the final brain lactate concentration showed the same linear correlation with preischemia plasma glucose concentration for all four age groups. The implications of this study are that piglets of any age have the same potential to generate high concentrations of brain lactate, although newborns will reach this level more slowly than older animals.