Saulius Satas

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°C; n = 7) or hypothermia (35°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.

Head cooling with mild systemic hypothermia in anesthetized piglets is neuroprotective

Hypothermia is potentially therapeutic in the management of neonatal hypoxic‐ischemic brain injury. However, not all studies have shown a neuroprotective effect. It is suggested that the stress of unsedated hypothermia may interfere with neuroprotection. We propose that selective head cooling (SHC) combined with mild total‐body hypothermia during anesthesia enhances local neuroprotection while minimizing the occurrence of systemic side effects and stress associated with unsedated whole‐body cooling. Our objective was to determine whether SHC combined with mild total‐body hypothermia while anesthetized for a period of 24 hours reduces cerebral damage in our piglet survival model of global hypoxia‐ischemia. Eighteen anesthetized piglets received a 45‐minute global hypoxic‐ischemic insult. The pigs were randomized either to remain normothermic or to receive SHC. We found that the severity of the hypoxic‐ischemic insult was similar in the SHC versus the normothermic group, and that the mean neurology scores at 30 and 48 hours and neuropathology scores were significantly better in the SHC group versus the normothermic group. We conclude that selective head cooling combined with mild systemic hypothermia and anesthesia is neuroprotective when started immediately after the insult in our piglet model of hypoxic‐ischemic encephalopathy. Ann Neurol 2003;53:000–000

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 (Fio2 ∼ 6%) global insult (n = 36) or sham hypoxia (n = 3). On reoxygenation, 18 were maintained normothermic (NT, 39.0°C) for 72 h, and 21 were cooled from 39 (NT) to 35°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 < 7μV) was similar in the NT and HT groups, mean ± SD (28 ± 7.2 versus 27 ± 8.6 min), as was the mean Fio2 (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 μV). 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 μM 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°C) to hypothermia (33.5–33.8°C) using a head cap perfused with cold (6–24°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°C; range, 4.2–8.5°C versus 3.0°C; 1.7-7.4°C;p = 0.008). During rewarming to normothermia, the gradient was maintained at 4.5°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.

Delayed Hypothermia as Selective Head Cooling or Whole Body Cooling Does Not Protect Brain or Body in Newborn Pig Subjected to Hypoxia-Ischemia

The neuroprotective efficacy of hypothermia (HT) after hypoxia-ischemia (HI) falls dramatically the longer the delay in initiating HT. Knowledge is scarce regarding protective or adverse effects of HT in organs beyond the brain. In addition, the relative effectiveness of selective head cooling (SHC) and whole body cooling (WBC) has not been studied. We aimed to examine whether 24 h HT, initiated 3 h after global HI is brain- and/or organ-protective using pathology, neurology, and biochemical markers. Fifty, ≤1-d-old pigs were subjected to global HI causing permanent brain injury. Animals were randomized to normothermia (NT), (Trectal) 39.0°C, SHCTrectal 34.5°C, or WBCTrectal 34.5°C for 24 h, all followed by 48 h NT. There was no difference in injury to the brain or organs between groups. There was no gender difference in brain injury but females had significantly more organs injured [2.3 (± 1.3) [mean ± SD] vs. 1.4 ± (1.0)]. The postinsult decline in lactate was temperature independent. However, HT animals normalized their plasma-calcium, magnesium, and potassium significantly faster than NT. Delayed SHC or WBC, initiated 3 h after HI, does not reduce pathology in the brain nor in organs. Delayed HT improves postinsult recovery of plasma-calcium, magnesium, and potassium. There were no differences in adverse effects across groups.

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°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 (Tdeep brain) to 7 mm (Tsuperficial 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°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) Tdeep brain during the seven hour cooling period was 31.1 (4.9)°C while Trectal remained stable at 38.8 (0.4)°C. The mean Trectal−Tdeep brain difference throughout the cooling period was 9.8 (6.1)°C. The mean Tskin required was 40.8 (1.1)°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.

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 ∼6% O2. On reoxygenation, pigs were randomised to NT or HT. The 18 NT piglets were maintained at rectal temperature 39.0°C for 72 h. Twenty-three HT pigs (20 experimental HT and 3 sham controls) were cooled to rectal temperature 35°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] AUCHT 4.4 mmol/l vs. AUCNT 3.9 mmol/l, p = 0.04 and for [Ca] AUCHT 2.7 mmol/l vs. AUCNT 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 (AUCHT 11.1 vs. AUCNT 15.3 103/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.

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°C) for 72 h (n = 8) or mild hypothermia (35°C) for 24 h followed by normothermia (39°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°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.

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 µV. 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.

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°C) or hypothermia (35°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.