Significant head cooling can be achieved while maintaining normothermia in the newborn piglet.

BACKGROUND: Hypothermia has been shown to be neuroprotective in animal models of hypoxia-ischaemia. It is currently being evaluated as a potentially therapeutic option in the management of neonatal hypoxic-ischaemic encephalopathy. However, significant hypothermia has adverse systemic effects. It has also recently been found that the stress of being cold can abolish the neuroprotective effects of hypothermia. It is hypothesised that selective head cooling (SHC) while maintaining normal core temperature would enable local hypothermic neuroprotection while limiting the stress and side effects of hypothermia. OBJECTIVE: To determine whether it is possible to induce moderate cerebral hypothermia in the deep brain of the piglet while maintaining the body at normothermia (39 degrees C). METHODS: Six piglets (<48 hours old) were anaesthetised, and temperature probes inserted into the brain. Temperature was measured at different depths from the brain surface (21 mm (T(deep brain)) to 7 mm (T(superficial brain))). After a 45 minute global hypoxic-ischaemic insult, each piglet was head cooled for seven hours using a cap circulated with cold water (median 8.9 degrees C (interquartile range 7.5-14)) wrapped around the head. Radiant overhead heating was used to warm the body during cooling. RESULTS: During SHC it was possible to cool the brain while maintaining a normal core temperature. The mean (SD) T(deep brain) during the seven hour cooling period was 31.1 (4.9) degrees C while T(rectal) remained stable at 38.8 (0.4) degrees C. The mean T(rectal)-T(deep brain) difference throughout the cooling period was 9.8 (6.1) degrees C. The mean T(skin) required was 40.8 (1.1) degrees C. There was no evidence of skin damage secondary to these skin temperatures. During cooling only one piglet shivered. CONCLUSIONS: It is possible to maintain systemic normothermia in piglets while significantly cooling the deeper structures of the brain. This method of cooling may further limit the side effects associated with systemic hypothermia and be feasible for premature infants.

Cardiac output, pulmonary artery pressure, and patent ductus arteriosus during therapeutic cooling after global hypoxia-ischaemia.

OBJECTIVE: To assess by Doppler echocardiography the effects of 24 hours of whole body mild hypothermia compared with normothermia on cardiac output (CO), pulmonary artery pressure (PAP), and the presence of a persistent ductus arteriosus (PDA) after a global hypoxic-ischaemic insult in unsedated newborn animals. DESIGN: Thirty five pigs (mean (SD) age 26.6 (12.1) hours and weight 1.6 (0.3) kg) were anaesthetised with halothane, mechanically ventilated, and subjected to a 45 minute global hypoxic-ischaemic insult. At the end of hypoxia, halothane was stopped; the pigs were randomised to either normathermia (39 degrees C) or hypothermia (35 degrees C) for 24 hours. Rewarming was carried out for 24-30 hours followed by 42 hours of normothermia. Unanaesthetised pigs were examined with a VingMed CFM 750 ultrasound scanner before and 3, 24, 30, and 48 hours after the hypoxic-ischaemic insult. Aortic valve diameter, forward peak flow velocities across the four valves, and the occurrence of a PDA were measured. Tricuspid regurgitation (TR) velocity was used to estimate the PAP. Stroke volume was calculated from the aortic flow. RESULTS: Twelve animals (seven normothermic, five hypothermic) had a PDA on one or more examinations, which showed no association with cooling or severity of insult. There were no differences in stroke volume or TR velocity between the hypothermic and normothermic animals at any time point after the insult. CO was, however, 45% lower at the end of cooling in the subgroup of hypothermic pigs that had received a severe insult compared with the pigs with mild and moderate insults. CO and TR velocity were transiently increased three hours after the insult: 0.38 (0.08) v 0.42 (0.08) litres/min/kg (p = 0.007) for CO; 3.0 (0.42) v 3.4 (0.43) m/s (p < 0.0001) for TR velocity (values are mean (SD)). CONCLUSIONS: The introduction of mild hypothermia while the pigs were unsedated did not affect the incidence of PDA nor did it lead to any changes in MABP or PAP. Stroke volume was also unaffected by temperature, but hypothermic piglets subjected to a severe hypoxic-ischaemic insult had reduced CO because the heart rate was lower. Global hypoxia-ischaemia leads to similar transient increases in CO and estimated PAP in unsedated normothermic and hypothermic pigs. There were no signs of metabolic compromise in any subgroup, suggesting that 24 hours of mild hypothermia had no adverse cardiovascular effect.

Effect of global hypoxia-ischaemia followed by 24 h of mild hypothermia on organ pathology and biochemistry in a newborn pig survival model.

BACKGROUND: Perinatal asphyxia may lead to multiorgan damage as well as brain injury. Posthypoxic hypothermia (HT) may protect other organs in addition to the brain. The aim of this study was to assess the systemic effects of our global hypoxic-ischaemic (HI) insult and compare the effect of mild 24-hour HT with normothermia (NT) during unsedated recovery. METHOD: Thirty-eight newborn pigs were subjected to 45 min of global HI by ventilating them with approximately 6% O2. On reoxygenation, pigs were randomised to NT or HT. The 18 NT piglets were maintained at rectal temperature 39.0 degrees C for 72 h. Twenty-three HT pigs (20 experimental HT and 3 sham controls) were cooled to rectal temperature 35 degrees C for 24 h before NT was resumed and the animals then survived a further 48 h. RESULTS: All lesions were small with no apparent clinical effect. The incidence of any damage to the heart (6 HT vs. 9 NT), liver (9 HT vs. 7 NT), kidney (6 HT vs. 9 NT) or intestinal injury (8 HT vs. 2 NT, p = 0.07) was not different in the two groups. More HT piglets developed lung injury, 10 HT and 3 NT. Plasma [Na], [K], [Ca] and [Mg] increased significantly after the HI insult as compared to baseline values. For the 24-hour period plasma [K] and [Ca] were significantly higher in the HT group, the mean area under the curve (AUC) being for [K] AUC(HT) 4.4 mmol/l vs. AUC(NT) 3.9 mmol/l, p = 0.04 and for [Ca] AUC(HT) 2.7 mmol/l vs. AUC(NT) 2.5 mmol/l, p = 0.01, respectively. Aspartate aminotransferase peaked at 48 h in the HT group and at 24 h in the NT group. Creatinine peaked at >72 h in the HT pigs and at 48 h in the NT pigs. White blood cells (WBC) peaked at 12 h for the HT pigs and at 6 h for the NT animals. AUC of the WBC during the cooling was significantly lower in the HT pig (AUC(HT) 11.1 vs. AUC(NT) 15.3 10(3)/mm3, p = 0.04). The HT pigs needed more glucose to maintain normal glucose during the last 12 h of HT. Also HT animals needed more oxygen during cooling to maintain PaO2. CONCLUSION: Twenty-four hours of mild HT did not reduce damage in any organ. There was a slight increase in lung damage in the HT group. None of the biochemical or pathological changes were of clinical significance. We conclude that mild HT for 24 h does not affect the organ systems adversely when compared to NT. Additional glucose and oxygen is needed during cooling to maintain normal values.

Twenty-four hours of mild hypothermia in unsedated newborn pigs starting after a severe global hypoxic-ischemic insult is not neuroprotective.

Three to 12 h of mild hypothermia (HT) starting after hypoxia-ischemia is neuroprotective in piglets that are anesthetized during HT. Newborn infants suffering from neonatal encephalopathy often ventilate spontaneously and are not necessarily sedated. We aimed to test whether mild posthypoxic HT lasting 24 h was neuroprotective if the animals were not sedated. Thirty-nine piglets (median weight 1.6 kg, range 0.8-2.2 kg; median age 24 h, range 7-48 h) were anesthetized and ventilated and subjected to a 45-min hypoxic (FiO(2) approximately 6%) global insult (n = 36) or sham hypoxia (n = 3). On reoxygenation, 18 were maintained normothermic (NT, 39.0 degrees C) for 72 h, and 21 were cooled from 39 (NT) to 35 degrees C (HT) for the first 24 h before NT was resumed (18 experimental, three sham hypoxia). Cardiovascular parameters and intermittent EEG were documented throughout. The brain was perfusion fixed for neuropathology and five main areas examined using light microscopy. The insult severity (duration in minutes of EEG amplitude < 7 microV) was similar in the NT and HT groups, mean +/- SD (28 +/- 7.2 versus 27 +/- 8.6 min), as was the mean FiO(2) (5.9 +/- 0.7 versus 5.8 +/- 0.8%) during the insult. Six NT and seven HT piglets developed posthypoxic seizures that lasted 29 and 30% of the time, respectively. The distribution and degree of injury (0.0-4.0, normal-maximal damage) within the brain (hippocampus, cortex/white matter, cerebellum, basal ganglia, thalamus) were similar in the NT and HT groups (overall score, mean +/- SD, 2.3 +/- 1.5 versus 2.4 +/- 1.3) as was the EEG background amplitude at 3 h (13 +/- 3.5 versus 10 +/- 3.3 microV). The HT animals shivered and were more active. The sham control group (n = 3) shivered but had normal physiology and neuropathology. Plasma cortisol was significantly higher in the HT group during the HT period, 766 +/- 277 versus 244 +/- 144 microM at 24 h. Mild postinsult HT for 24 h was not neuroprotective in unsedated piglets and did not reduce the number of animals that developed posthypoxic seizures. Cortisol reached 3 times the NT value at the end of HT. We speculate that the stress of shivering and feeling cold interfered with the previously shown neuroprotective effect of HT. Research on the appropriateness of sedation during clinical HT is urgent.

Effective selective head cooling during posthypoxic hypothermia in newborn piglets.

Selective head cooling has been proposed as a neuroprotective intervention after hypoxia-ischemia in which the brain is cooled without subjecting the rest of the body to significant hypothermia, thus minimizing adverse systemic effects. There are little data showing it is possible to cool the brain more than the body. We have therefore applied selective head cooling to our hypoxia-ischemia piglet model to establish whether it is possible. Nine piglets were anesthetized, and brain temperature was measured at the surface and in the superficial (0.2 cm) and deep (1.7-2.0 cm) gray matter. Rectal (6-cm depth), skin, and scalp temperatures (T) were recorded continuously. Lowering T-rectal from normothermia (39 degrees C) to hypothermia (33.5-33.8 degrees C) using a head cap perfused with cold (6-24 degrees C) water was undertaken for up to 6 h. To assess the impact of the 45-min hypoxia-ischemia insult on the effectiveness of selective head cooling, four piglets were cooled both before and after the insult, and four, only afterward. During selective head cooling, it was possible to achieve a lower T-deep brain than T-rectal in all animals both before and after hypoxia. However, this was only possible when overhead body heating was used. The T-rectal to T-deep brain gradient was significantly smaller after the insult (median, 5.3 degrees C; range, 4.2-8.5 degrees C versus 3.0 degrees C; 1.7-7.4 degrees C; p = 0.008). During rewarming to normothermia, the gradient was maintained at 4.5 degrees C. We report for the first time a study, which by direct measurement of deep intracerebral temperatures, validates the cooling cap as an effective method of selective brain cooling in a newborn animal hypoxia-ischemia model.

Influence of mild hypothermia after hypoxia-ischemia on the pharmacokinetics of gentamicin in newborn pigs.

The renal function is often affected in asphyxiated newborn infants. The pharmacokinetics of drugs like aminoglycosides eliminated through the kidneys may be impaired and require a different than usual dosage regimen. A decrease in body temperature is associated with a decrease in glomerular filtration rate and may, therefore, impair the elimination of aminoglycosides. When hypothermia is applied as neuronal rescue therapy after birth asphyxia, the pharmacokinetics of kidney-eliminated drugs may be impaired even more. We used our well-established global hypoxia-asphyxia newborn pig model to evaluate the effect of mild hypothermia after hypoxia-ischemia on gentamicin pharmacokinetics. Newborn pigs underwent global hypoxia-ischemia followed by normothermia (39 degrees C) for 72 h (n = 8) or mild hypothermia (35 degrees C) for 24 h followed by normothermia (39 degrees C) for 48 h (n = 8). Gentamicin pharmacokinetics was studied after three gentamicin doses: before hypoxia-ischemia, after hypoxia-ischemia during mild hypothermia or normothermia, and during normothermia 48 h after the first dose. The gentamicin pharmacokinetics variables were calculated using a SAAM II program. Hypoxia-ischemia altered renal function and gentamicin pharmacokinetics. The gentamicin clearance correlated with the creatinine plasma concentration (r = 0.89) and with the kidney pathology score (r = 0.55). There was no significant difference in gentamicin pharmacokinetics at 35 and 39 degrees C in newborn pigs after hypoxia-ischemia. The gentamicin pharmacokinetics variables were not different in the hypothermic or normothermic pigs after all three studied doses. Mild hypothermia for 24 h after hypoxia-ischemia does not affect gentamicin pharmacokinetics.

Cardiac function and morphology studied by two-dimensional Doppler echocardiography in unsedated newborn pigs.

The newborn pig is currently the most used species in animal neonatal research. Valid non-invasive monitoring is important in particular for long-term survival of unsedated animals. In the unsedated newborn pig (n = 35, median age 24 h, range 7-48 h) we standardized two-dimensional Doppler echocardiography and determined the normal ranges for cardiac function. Probe positioning had to be adjusted to the V-shaped thorax and the mid-line position of the heart. Six out of the sixteen animals < 20 h had a patent ductus arteriosus compared with one of the twenty animals > 20 h old. One atrial septal defect (5 mm) and one small ventricular septal defect were diagnosed. The average heart size was 0.7-0.9% of body weight which is similar to human infants of the same size. The mean aortic diameter was 6.0 +/- 0.5 mm (mean +/- S.D.) and cardiac output was 0.38 +/- 0.08 l min-1; both correlate with body weight (r = 0.80 and 0.73, respectively). Tricuspid regurgitation velocity was 3.0 +/- 0.4 m s-1 (mean +/- S.D.), giving an estimated pressure gradient across the tricuspid valve of 37 +/- 9.7 mmHg. The aortic diameter and the heart weight per kg body weight are comparable to those reported for preterm neonates. The cardiac output and velocities across the four valves are more comparable with term neonates.

Lactate and pyruvate changes in the cerebral gray and white matter during posthypoxic seizures in newborn pigs.

Cerebral lactate rises after chemically induced seizures, but it is not known if this occurs with posthypoxic seizures. We examined changes in lactate and pyruvate in gray and white matter in the newborn pig brain after a hypoxic insult known to produce seizures and permanent brain damage. Fourteen halothane-anesthetized piglets aged 24-49 h, were instrumented with a two-channel scalp EEG and microdialysis probes positioned in white and gray matter. Forty-five minutes of hypoxia were induced by reducing the fraction of inspired O2 to the maximum concentration at which EEG amplitude was < 7 microV. Postinsult EEG was classified as electroconvulsive activity (ECA) (n = 4) or burst suppression (n = 2), persistently low amplitude (n = 2), or intermittent spikes on normal background activity (n = 6). Six hours after the insult the brains were perfusion fixed for histologic probe localization. Plasma lactate and brain lactate had different time courses with brain having a persistently elevated lactate/pyruvate (L/P) ratio. The highest L/P ratios in gray and white matter were in the two pigs with persistently low amplitude EEG. There was no association between onset of electroconvulsive activity and an increase in lactate or L/P ratio. Posthypoxic energy metabolism is disturbed in both gray and white matter probably because of mitochondrial dysfunction. Seizure activity does not increase cerebral lactate or L/P ratio above the already raised levels found in posthypoxic encephalopathy. These findings cast further doubt on the hypothesis that such seizures are, in themselves, damaging.

Post-hypoxic hypothermia reduces cerebrocortical release of NO and excitotoxins.

Hypothermia applied after hypoxia offers neuroprotection in neonatal animals, but the mechanisms involved remain unknown. Hypoxia was induced in newborn piglets and changes in excitatory amino acids (EAAs) and the citrulline:arginine ratio (CAR) were followed by microdialysis for 5 h. After the 45 min hypoxic insult, the animals were randomized to receive normothermia (39 degrees C; n=7) or hypothermia (35 degrees C; n = 7). After reoxygenation, extracellular glutamate, aspartate and the excitotoxic index were significantly lower in the cerebral cortex of hypothermic animals than in normothermic animals. A progressive rise of the CAR occurred during reoxygenation in the normothermic group whereas the ratio tended to decrease in the hypothermic group. In conclusion, post-hypoxic hypothermia attenuated NO production and overflow of EAAs.

Lidocaine pharmacokinetics and toxicity in newborn pigs.

In newborn infants suffering from perinatal asphyxia seizures, lidocaine (LD) has proved to be an effective anticonvulsant. At high concentrations, however, LD can itself cause convulsions. The convulsive concentration of LD (LD(conv)) varies among species. The aim of this study was to describe LD pharmacokinetics and to define the LD(conv) in awake newborn pigs. Eighteen Land race newborn pigs aged 12-60 h, weight 1.0-2.5 kg, were enrolled. LD, 2 mg/kg intravenous (IV) bolus, (n = 11) was given to estimate pharmacokinetic variables. Continuous LD infusion 2 mg x kg(-1) x min(-1) IV (n = 5) and repeated bolus doses of 15 mg/kg (n = 4) were given until electroencephalogram-confirmed seizures appeared. After the bolus injection, the elimination half-life for LD was 0.87-5.44 h. Increasing plasma concentration (LD(pl)) during infusion resulted in sedation after 5-10 min and in shivering, nystagmus, neck extension, tonic-clonic seizures at LD(conv) of 40.6 +/- 12.7 mg/L (mean +/- SD). The unbound LD(pl) at seizures was 4.4 +/- 2.4 mg/L. Younger animals convulsed at higher LD(conv) (r2 = 0.85). LD pharmacokinetics in newborn pigs were found to be dose-dependent at high plasma concentrations. At lower plasma concentrations, LD pharmacokinetics appeared to be linear. The central nervous system is the primary target for the toxic effect of LD in awake newborn pigs. LD neurotoxicity is age-dependent, and younger pigs convulse at a higher LD(conv).

MAC for halothane and isoflurane during normothermia and hypothermia in the newborn piglet.

BACKGROUND: Halothane and isoflurane are frequently used in studies of perinatal hypoxia and ischemia. Little information exists on the minimum alveolar concentration (MAC) necessary to prevent movement to a painful stimulus in newborn pigs and no information on the effects of hypothermia on MAC in pigs. Hypothermia is currently investigated as a posthypoxic neuroprotective intervention. METHODS: The MAC of halothane and isoflurane necessary to prevent movement when a 25 cm hemostatic clamp was applied to the tail were determined in six 20-48-hour-old piglets, and when the same stimulus was applied to the hoof. MAC for halothane was first determined at 39 degrees C, then at 35 degrees C, whereafter halothane was discontinued and MAC for isoflurane determined first at 35 degrees C and then at 39 degrees C. RESULTS: In all six piglets MAC was lower at 35 degrees C than at 39 degrees C for both anesthetics with both tail and hoof determination, lower for halothane than isoflurane for both stimuli at both temperatures, and lower for tail than hoof determination for both anesthetics at both temperatures. For halothane at 39 degrees C, mean (SD) MAC hoof was 0.82 (0.05)% vs tail 0.60 (0.12)%, and at 35 degrees C, hoof 0.65 (0.06)% vs tail 0.42 (0.10)%. For isoflurane at 39 degrees C, MAC hoof was 2.47 (0.28)% vs tail 1.83 (0.28)%, and at 35 degrees C, hoof was 1.83 (0.18)% vs tail 0.85 (0.25)%. CONCLUSION: In the newborn piglet, MAC should be determined by hoof clamp, MAC of isoflurane is approximately three times that of halothane, and both are reduced during hypothermia.