Hemodynamic and metabolic effects of a new pediatric dobutamine formulation in hypoxic newborn pigs

Background: The aim of our study was to measure drug-related changes in hemodynamics and oxygen metabolism in response to different doses of an age-appropriate dobutamine formulation in hypoxic pigs. A secondary aim was to validate superior vena cava flow (SVCF) as a marker of cardiac index (CI) for subsequent clinical trials of this formulation in humans. Methods: Newborn pigs (n=18) were exposed to 2h-hypoxia (10-15% oxygen) followed by reoxygenation (21-30% oxygen 4h). After 1h-reoxygenation, pigs were randomized to: control group (no treatment), dobutamine infusion at a rate of 10-15μg/kg/min or 15-20μg/kg/min. Dobutamine groups received two dobutamine doses during 30min with a 60min washout period between doses. Cardiovascular profile and oxygen metabolism were monitored. In four animals an ultrasonic perivascular flow probe was placed around superior vena cava to measure SVCF. Results: Hypoxia significantly decreased CI, systemic-vascular-resistance and mean-arterial-bloodpressure (MABP). Dobutamine doses significantly increased heart-rate, CI and oxygen-delivery without changes in stroke-volume and MABP. Only 10-15μg/kg/min increased oxygen consumption and peripheral tissue oxygenation measured by Near-infrared-spectroscopy. A positive correlation was observed between SVCF and CI. Conclusion: The new pediatric dobutamine formulation improved hemodynamic status, with dose-specific differences in metabolic response. SVCF may be a useful surrogate for CI in subsequent clinical trials

Early Cerebral Hemodynamic, Metabolic, and Histological Changes in Hypoxic–Ischemic Fetal Lambs during Postnatal Life

The hemodynamic, metabolic, and biochemical changes produced during the transition from fetal to neonatal life may be aggravated if an episode of asphyxia occurs during fetal life. The aim of the study was to examine regional cerebral blood flow (RCBF), histological changes, and cerebral brain metabolism in preterm lambs, and to analyze the role of oxidative stress in the first hours of postnatal life following severe fetal asphyxia. Eighteen chronically instrumented newborn lambs were randomly assigned to either a control group or the hypoxic–ischemic (HI) group, in which case fetal asphyxia was induced just before delivery. All the animals were maintained on intermittent positive pressure ventilation for 3 h after delivery. During the HI insult, the injured group developed acidosis, hypoxia, hypercapnia, lactic acidosis, and tachycardia (relative to the control group), without hypotension. The intermittent positive pressure ventilation transiently improved gas exchange and cardiovascular parameters. After HI injury and during ventilatory support, there continued to be an increased RCBF in inner regions among the HI group, but no significant differences were detected in cortical flow compared to the control group. Also, the magnitude of the increase in TUNEL positive cells (apoptosis) and antioxidant enzymes, and decrease of ATP reserves was significantly greater in the brain regions where the RCBF was not higher. In conclusion, our findings identify early metabolic, histological, and hemodynamic changes involved in brain damage in premature asphyxiated lambs. Such changes have been described in human neonates, so our model could be useful to test the safety and the effectiveness of different neuroprotective or ventilation strategies applied in the first hours after fetal HI injury

MgSO\u3csub\u3e4\u3c/sub\u3e Treatment Preserves the Ischemia-Induced Reduction in S-100 Protein Without Modification of the Expression of Endothelial Tight Junction Molecules

The aim of this work was to evaluate the effect of magnesium sulphate (MgSO4) administration on blood-brain barrier (BBB) permeabilization after cerebral hypoxia-ischemia (HI) induced by partial occlusion of the umbilical cord of premature fetal lambs. We also characterized BBB dysfunction in terms of the levels of expression of a panel of BBB proteins; Occludin, Claudin, Zona Occludens-1, Zonula Occludens-2, VE-cadherin and beta-catenin. Lambs were assigned to: Control group: non-injured animals, 0 h post-partial cord occlusion (0h-PCO) group: animals subjected to 60 min HI and sacrificed just after the insult, 3h-PCO group: HI injured animals resuscitated and managed on ventilation for 3 hours and MgSO4 group: animals which received a dose of 400 mg/kg MgSO4 after the HI event and managed on ventilation for 3 hours. Brains were fixed and blocks processed for S-100 protein immunohistochemistry. Other brains were dissociated and processed for S-100 and BBB protein immunochemistry for analysis by flow cytometry. The percentage of S-100 positive cells was found to be dramatically reduced in all studied brain tissues in the 3h-PCO group with respect to the other groups. No differences were found in the percentage or mean intensity of BBB protein immunolabeled cells among the groups. In the MgSO4 group, the percentage of S-100 positive cells 3 h after the HI event was similar to the control group. These results suggest that MgSO4 treatment preserves the ischemia-induced reduction in S-100 protein without modification in the expression of endothelial tight junction molecules. We speculate that MgSO4 treatment confers neuroprotection by restoration of blood brain permeability in hypoxia-ischemia

MgSO\u3csub\u3e4\u3c/sub\u3e Treatment Preserves the Ischemia-Induced Reduction in S-100 Protein Without Modification of the Expression of Endothelial Tight Junction Molecules

The aim of this work was to evaluate the effect of magnesium sulphate (MgSO4) administration on blood-brain barrier (BBB) permeabilization after cerebral hypoxia-ischemia (HI) induced by partial occlusion of the umbilical cord of premature fetal lambs. We also characterized BBB dysfunction in terms of the levels of expression of a panel of BBB proteins; Occludin, Claudin, Zona Occludens-1, Zonula Occludens-2, VE-cadherin and beta-catenin. Lambs were assigned to: Control group: non-injured animals, 0 h post-partial cord occlusion (0h-PCO) group: animals subjected to 60 min HI and sacrificed just after the insult, 3h-PCO group: HI injured animals resuscitated and managed on ventilation for 3 hours and MgSO4 group: animals which received a dose of 400 mg/kg MgSO4 after the HI event and managed on ventilation for 3 hours. Brains were fixed and blocks processed for S-100 protein immunohistochemistry. Other brains were dissociated and processed for S-100 and BBB protein immunochemistry for analysis by flow cytometry. The percentage of S-100 positive cells was found to be dramatically reduced in all studied brain tissues in the 3h-PCO group with respect to the other groups. No differences were found in the percentage or mean intensity of BBB protein immunolabeled cells among the groups. In the MgSO4 group, the percentage of S-100 positive cells 3 h after the HI event was similar to the control group. These results suggest that MgSO4 treatment preserves the ischemia-induced reduction in S-100 protein without modification in the expression of endothelial tight junction molecules. We speculate that MgSO4 treatment confers neuroprotection by restoration of blood brain permeability in hypoxia-ischemia

MgSO4 treatment preserves the ischemia-induced reduction in S-100 protein without modification of the expression of endothelial tight junction molecules

The aim of this work was to evaluate the effect of magnesium sulphate (MgSO4 ) administration on blood-brain barrier (BBB) permeabilization after cerebral hypoxia-ischemia (HI) induced by partial occlusion of the umbilical cord of premature fetal lambs. We also characterized BBB dysfunction in terms of the levels of expression of a panel of BBB proteins; Occludin, Claudin, Zona Occludens-1, Zonula Occludens-2, VE-cadherin and ß-catenin. Lambs were assigned to: Control group: non-injured animals, 0 h post-partial cord occlusion (0h-PCO) group: animals subjected to 60 min HI and sacrificed just after the insult, 3h-PCO group: HI injured animals resuscitated and managed on ventilation for 3 hours and MgSO4 group: animals which received a dose of 400 mg/kg MgSO4 after the HI event and managed on ventilation for 3 hours. Brains were fixed and blocks processed for S-100 protein immunohistochemistry. Other brains were dissociated and processed for S-100 and BBB protein immunochemistry for analysis by flow cytometry. The percentage of S-100 positive cells was found to be dramatically reduced in all studied brain tissues in the 3h-PCO group with respect to the other groups. No differences were found in the percentage or mean intensity of BBB protein immunolabeled cells among the groups. In the MgSO4 group, the percentage of S-100 positive cells 3 h after the HI event was similar to the control group. These results suggest that MgSO4 treatment preserves the ischemia-induced reduction in S- 100 protein without modification in the expression of endothelial tight junction molecules. We speculate that MgSO4 treatment confers neuroprotection by restoration of blood brain permeability in hypoxia-ischemia

The Cannabinoid Receptor Agonist Win 55,212-2 Reduces the Initial Cerebral Damage After Hypoxic-Ischemic Injury in Fetal Lambs

The aim of the present work was to evaluate in an early time point the effect of the cannabinoid agonist WIN 55,212-2 after hypoxic–ischemic (HI) brain injury induced by partial occlusion of the umbilical cord of premature fetal lambs. Lambs were assigned to three experimental groups: one SHAM group: non-injured animals, and two hypoxic–ischemic groups that received a dose of 0.01 μg/kg WIN 55,212-2 (HI + WIN group) or not (HI +VEH) after 60 min of a hypoxic–ischemic event. All animals were managed on mechanical ventilation for 3 h and then sacrificed. Brains were perfusion-fixed and different regions separated for regional cerebral blood flow measurement, apoptosis quantification by TUNEL method and S-100 protein analysis by flow cytometry. The number of apoptotic cells was lower in the HI + WIN group in all regions studied. Moreover, animals treated with the cannabinoid agonist showed higher values in the percentage of S-100 positive cells in all regions, except in the cortex. In both studies we obtained similar values between SHAM group and HI + WIN group. Our results suggest that the administration of the cannabinoid agonist WIN 55,212-2 after hypoxic–ischemic brain injury in preterm lambs decreases brain injury reducing the delayed cell death and glial damage

The Cannabinoid Receptor Agonist Win 55,212-2 Reduces the Initial Cerebral Damage After Hypoxic-Ischemic Injury in Fetal Lambs

The aim of the present work was to evaluate in an early time point the effect of the cannabinoid agonist WIN 55,212-2 after hypoxic–ischemic (HI) brain injury induced by partial occlusion of the umbilical cord of premature fetal lambs. Lambs were assigned to three experimental groups: one SHAM group: non-injured animals, and two hypoxic–ischemic groups that received a dose of 0.01 μg/kg WIN 55,212-2 (HI + WIN group) or not (HI +VEH) after 60 min of a hypoxic–ischemic event. All animals were managed on mechanical ventilation for 3 h and then sacrificed. Brains were perfusion-fixed and different regions separated for regional cerebral blood flow measurement, apoptosis quantification by TUNEL method and S-100 protein analysis by flow cytometry. The number of apoptotic cells was lower in the HI + WIN group in all regions studied. Moreover, animals treated with the cannabinoid agonist showed higher values in the percentage of S-100 positive cells in all regions, except in the cortex. In both studies we obtained similar values between SHAM group and HI + WIN group. Our results suggest that the administration of the cannabinoid agonist WIN 55,212-2 after hypoxic–ischemic brain injury in preterm lambs decreases brain injury reducing the delayed cell death and glial damage

Early Cerebral Hemodynamic, Metabolic, and Histological Changes in Hypoxic-Ischemic Fetal Lambs During Postnatal Life

The hemodynamic, metabolic, and biochemical changes produced during the transition from fetal to neonatal life may be aggravated if an episode of asphyxia occurs during fetal life. The aim of the study was to examine regional cerebral blood flow (RCBF), histological changes, and cerebral brain metabolism in preterm lambs, and to analyze the role of oxidative stress in the first hours of postnatal life following severe fetal asphyxia. Eighteen chronically instrumented newborn lambs were randomly assigned to either a control group or the hypoxic–ischemic (HI) group, in which case fetal asphyxia was induced just before delivery. All the animals were maintained on intermittent positive pressure ventilation for 3h after delivery. During the HI insult, the injured group developed acidosis, hypoxia, hypercapnia, lactic acidosis, and tachycardia (relative to the control group), without hypotension. The intermittent positive pressure ventilation transiently improved gas exchange and cardiovascular parameters. After HI injury and during ventilatory support, there continued to be an increased RCBF in inner regions among the HI group, but no significant differences were detected in cortical flow compared to the control group. Also, the magnitude of the increase in TUNEL positive cells (apoptosis) and antioxidant enzymes, and decrease of ATP reserves was significantly greater in the brain regions where the RCBF was not higher. In conclusion, our findings identify early metabolic, histological, and hemodynamic changes involved in brain damage in premature asphyxiated lambs. Such changes have been described in human neonates, so our model could be useful to test the safety and the effectiveness of different neuroprotective or ventilation strategies applied in the first hours after fetal HI injury

Early Cell Death in the Brain of Fetal Preterm Lambs After Hypoxic-Ischemic Injury

The objective of the present study was to evaluate using premature fetal lambs the effect of cerebral hypoxia–ischemia induced by partial occlusion of the umbilical cord on the type of cell death which occurs in different brain regions and to ascertain some of the neural pathways which may underlie the associated pathologies. Lambs were sacrificed either immediately after a 1 h hypoxic–ischemic insult or 3 h later. Brains were fixed by perfusion and blocks of the different brain territories were processed for light microscopy (hematoxylin–eosin, Nissl staining), electron transmission microscopy and quantification of apoptosis by the TUNEL method. Other fixed brains were dissociated and labeled by nonyl acridine orange to determine mitochondrial integrity. Non-fixed brains were also used for membrane asymmetry studies, in which cell suspensions were analyzed by flow cytometry to quantify apoptosis. In both hypoxic–ischemic groups, necrotic-like neurons were observed mainly in the mesencephalon, pons, deep cerebellar nuclei and basal nuclei, whereas apoptotic cells were extensively found both in white and gray matter and were not limited to regions where necrotic neurons were present. The 3 h post-partial cord occlusion group, but not the 0 h group, showed a generalized alteration of cell membrane asymmetry and mitochondrial integrity as revealed by Annexin V/PI flow cytometry and nonyl acridine orange studies, respectively. Our results show that the apoptotic/necrotic patterns of cell death occurring early after hypoxic–ischemic injury are brain-region-specific and have distinct dynamics and suggest that therapeutic strategies aimed at rescuing cells from the effects of hypoxia/ischemia should be aimed at blocking the apoptotic components of brain damage

Early Cell Death in the Brain of Fetal Preterm Lambs After Hypoxic-Ischemic Injury

The objective of the present study was to evaluate using premature fetal lambs the effect of cerebral hypoxia–ischemia induced by partial occlusion of the umbilical cord on the type of cell death which occurs in different brain regions and to ascertain some of the neural pathways which may underlie the associated pathologies. Lambs were sacrificed either immediately after a 1 h hypoxic–ischemic insult or 3 h later. Brains were fixed by perfusion and blocks of the different brain territories were processed for light microscopy (hematoxylin–eosin, Nissl staining), electron transmission microscopy and quantification of apoptosis by the TUNEL method. Other fixed brains were dissociated and labeled by nonyl acridine orange to determine mitochondrial integrity. Non-fixed brains were also used for membrane asymmetry studies, in which cell suspensions were analyzed by flow cytometry to quantify apoptosis. In both hypoxic–ischemic groups, necrotic-like neurons were observed mainly in the mesencephalon, pons, deep cerebellar nuclei and basal nuclei, whereas apoptotic cells were extensively found both in white and gray matter and were not limited to regions where necrotic neurons were present. The 3 h post-partial cord occlusion group, but not the 0 h group, showed a generalized alteration of cell membrane asymmetry and mitochondrial integrity as revealed by Annexin V/PI flow cytometry and nonyl acridine orange studies, respectively. Our results show that the apoptotic/necrotic patterns of cell death occurring early after hypoxic–ischemic injury are brain-region-specific and have distinct dynamics and suggest that therapeutic strategies aimed at rescuing cells from the effects of hypoxia/ischemia should be aimed at blocking the apoptotic components of brain damage