Victoria Mielgo

Neuroprotective Effects of the Nonpsychoactive Cannabinoid Cannabidiol in Hypoxic-Ischemic Newborn Piglets

To test the neuroprotective effects of the nonpsychoactive cannabinoid cannabidiol (CBD), piglets received i.v. CBD or vehicle after hypoxia-ischemia (HI: temporary occlusion of both carotid arteries plus hypoxia). Nonhypoxic-ischemic sham-operated piglets remained as controls. Brain damage was studied by near-infrared spectroscopy (NIRS) and amplitude-integrated electroencephalography (aEEG) and by histologic assessment (Nissl and FluoroJadeB staining). In HI+vehicle, HI led to severe cerebral hemodynamic and metabolic impairment, as reflected in NIRS by an increase in total Hb index (THI) and a decrease in the fractional tissue oxygenation extraction (FTOE); in HI+CBD the increase of THI was blunted and FTOE remained similar to SHAM. HI profoundly decreased EEG amplitude, which was not recovered in HI+vehicle, indicating cerebral hypofunction; seizures were observed in all HI+vehicle. In HI+CBD, however, EEG amplitude recovered to 46.4 ± 7.8% baseline and seizures appeared only in 4/8 piglets (both p < 0.05). The number of viable neurons decreased and that of degenerating neurons increased in HI+vehicle; CBD reduced both effects by more than 50%. CBD administration was free from side effects; moreover, CBD administration was associated with cardiac, hemodynamic, and ventilatory beneficial effects. In conclusion, administration of CBD after HI reduced short-term brain damage and was associated with extracerebral benefits.

Cannabidiol reduces brain damage and improves functional recovery after acute hypoxia-ischemia in newborn pigs.

Newborn piglets exposed to acute hypoxia-ischemia (HI) received i.v. cannabidiol (HI + CBD) or vehicle (HI + VEH). In HI + VEH, 72 h post-HI brain activity as assessed by amplitude-integrated EEG (aEEG) had only recovered to 42 ± 9% of baseline, near-infrared spectroscopy (NIRS) parameters remained lower than normal, and neurobehavioral performance was abnormal (27.8 ± 2.3 points, normal 36). In the brain, there were fewer normal and more pyknotic neurons, while astrocytes were less numerous and swollen. Cerebrospinal fluid concentration of neuronal-specific enolase (NSE) and S100β protein and brain tissue percentage of TNFα(+) cells were all higher. In contrast, in HI + CBD, aEEG had recovered to 86 ± 5%, NIRS parameters increased, and the neurobehavioral score normalized (34.3 ± 1.4 points). HI induced histological changes, and NSE and S100β concentration and TNFα(+) cell increases were suppressed by CBD. In conclusion, post-HI administration of CBD protects neurons and astrocytes, leading to histological, functional, biochemical, and neurobehavioral improvements.

Cerebral blood flow and morphological changes after hypoxic-ischaemic injury in preterm lambs

AIM To evaluate the effect of cerebral hypoxia-ischaemia induced by partial occlusion of the umbilical cord on the relationship of the regional cerebral blood flow and the cerebral cell death in near-term fetal lambs. METHODS Fifteen near-term lambs were assigned to two hypoxic-ischaemic groups with or without life support (3 h), and a healthy one. Hypoxia-ischaemia was induced by partial occlusion of the umbilical cord (60 min). Routine light and electron microscopy, and the TUNEL method for apoptosis were performed. Regional cerebral blood flow was measured by coloured microspheres. Cardiovascular, gas exchange and pH parameters were also evaluated. RESULTS Both hypoxic-ischaemic groups produced a transient acidosis and a decrease of base excess in comparison to the healthy group. Cortical and cerebellar zones, where the regional cerebral blood flow values were similar to baseline, showed an increased number of oligodendrocyte-like apoptotic cells. In contrast, in the inner zones, where regional cerebral blood flow was increased, the number of apoptotic cells did not increase. Necrotic neurons were observed in the basal nuclei, mesencephalon, pons and deep cerebellar nuclei. CONCLUSION Our results suggest that regional cerebral blood flow and the presence of apoptotic cells, 3 h after hypoxic-ischemic injury, are correlated.

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.

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 0.01μg/kg WIN 55,212-2 (HI+WIN group) or not (HI+VEH) after 60min of a hypoxic-ischemic event. All animals were managed on mechanical ventilation for 3h 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.

Acute and sustained effects of aerosolized vs. bolus surfactant therapy in premature lambs with respiratory distress syndrome

Background:Surfactant (SF) instillation may produce acute deleterious effects on gas exchange and both systemic and cerebral hemodynamics. Our aim was to compare the effects of aerosolized SF (SF-aero) with those of bolus SF (SF-bolus) administration on gas exchange, lung mechanics, and cardiovascular function in premature lambs with respiratory distress syndrome (RDS).Methods:Fourteen preterm lambs (85% gestation) were randomly assigned to receive SF-aero or SF-bolus. Oxygenation index (OI), PaCO2, cardiovascular parameters, carotid blood flow (CBF), lung compliance (mean dynamic compliance), and tidal volume (VT) were measured every 30 min for 6 h. Biochemical and histological analyses were performed.Results:After delivery, lambs developed severe RDS (inspiratory fraction of oxygen: 1; pH < 7.15; PaCO2 > 80 mm Hg; PaO2 < 30 mm Hg, mean dynamic compliance < 0.08 ml/cm H2O/kg). By 60 min after treatment, both groups showed an improvement in OI, PaCO2, mean dynamic compliance, and VT that was maintained until the end of the experiment. PaCO2 and CBF increased significantly in the SF-bolus group during the first 15–30 min, without concomitant changes in cardiovascular parameters, whereas in the SF-aero group, PaCO2 and CBF decreased gradually. SF-aero induced less alveolar hemorrhage and inflammation.Conclusion:SF-aero produced improvements in gas exchange and lung mechanics similar to those produced by bolus administration but with less lung injury and fewer cerebral hemodynamic changes.

Bronchoalveolar lavage versus bolus administration of lucinactant, a synthetic surfactant in meconium aspiration in newborn lambs

This study was designed to study effects of lung lavage versus the classical bolus instillation with a peptide‐based synthetic surfactant (lucinactant) in a model of Meconium Aspiration Syndrome (MAS). Eighteen newborn lambs received meconium and were randomized to: the experimental meconium installation (eMAS) group—lambs with eMAS kept on conventional mechanical ventilation (control); the SF‐Bolus group—eMAS receiving a lucinactant bolus (30 mg/ml); or the D‐SF‐Lavage group—eMAS treated with dilute lucinactant bronchoalveolar lavage (10 mg/ml). Systemic and pulmonary arterial pressures, blood gases, and pulmonary mechanics were recorded for 180 min. In addition, the intrapulmonary distribution of the lucinactant was determined using dye‐labeled microspheres. Following meconium instillation, severe hypoxia, hypercapnia, acidosis, and pulmonary hypertension developed, and dynamic compliance decreased (50% from baseline). After lung lavage with dilute lucinactant, gas exchange significantly improved versus bolus instillation (P < 0.05). Further, only in the lavage group did pulmonary arterial pressure return to basal values and dynamic compliance significantly increased. Both lung lavage and bolus techniques for the administration of lucinactant resulted in a non‐uniform lung distribution. In conclusion, in newborn lambs with respiratory failure and pulmonary hypertension induced by meconium, lung lavage with dilute lucinactant seems to be an effective and safe alternative for treatment for MAS. Pediatr. Pulmonol. 2011; 46:991–999.

Surfactant and Perfluorocarbon Aerosolization by Means of Inhalation Catheters for the Treatment of Respiratory Distress Syndrome: An In Vitro Study

BACKGROUND The aerosolization of perfluorocarbons or surfactant has emerged as a feasible alternative to instillation, for the treatment of experimental respiratory distress syndrome. However, the biophysical properties that make these compounds useful in such therapies, significantly affect the performance of nebulizers. Therefore, in vitro studies are required to assess the suitability of new aerosolization technologies for use with these compounds. METHODS The aim of the present in vitro study was to investigate the influence of the biophysical properties of perfluorocarbons (PFD, FC75, and PFOB) and a natural porcine surfactant, Curosurf®; on aerosolization and to assess the suitability of three intratracheal inhalation catheters (IC) with different air flow rates (IC-1.23, IC-1.1, IC-1.4) coupled to a jet nebulizer, for aerosol delivery of these compounds. RESULTS With IC-1.23 significantly higher aerosol production rates were achieved (p < 0.0001), ranging between 6.05 ± 0.17 mL/min (FC75) and 1.94 ± 0.09 mL/min (Curosurf®), and lower percentage losses of the compound (5-21%), compared to IC-1.1 and IC-1.4 catheters. The lowest aerosolization rates were produced with IC-1.4 ranging from 0.58 ± 0.02 mL/min (FC75) to 0.14 ± 0.01 mL/min (Curosurf®), and this catheter also resulted in the highest percentage losses (25-60%). The mass median aerodynamic diameter (MMAD) ranged between 0.77 μm (PFD) and 8.29 μm (Curosurf®) with IC-1.1, whereas higher MMAD values, of between 4.84 μm (FC75) and 13.42 μm (PFOB), were observed with IC-1.23. Regardless of the catheter used during aerosolization, the perfluorocarbon with the highest kinematic viscosity showed the lowest aerosolization and emission rates and vice versa, which reveals the substantial contribution of this parameter that should accordingly be considered in the design of perfluorocarbon aerosol drug delivery systems. CONCLUSIONS Jet aerosolization of perfluorocarbons or surfactant with the intratracheal inhalation catheters seems to be a suitable method for treating experimental respiratory distress syndrome, because it delivers relatively high doses of perfluorocarbons and surfactant to the lungs in a respirable size droplets.

Reference values of respiratory and peripheral muscle function in rats

Skeletal muscle dysfunction is a common systemic manifestation in several prevalent diseases. Predictive values are useful tools for the diagnosis and prognosis of diseases. In experimental animals, no reference values of muscle function evaluation have been so far reported. The objective was to obtain predictive values of maximal inspiratory pressure (MIP) and grip strength measurements in healthy rats. In 70 healthy rats, MIP and grip strength were measured in vivo weekly for five consecutive weeks using non-invasive methodologies. Three ranges of rat body weights (250-299, 300-349 and 350-399 g) and lengths (37.0-41.0, 41.1-42.0 and 42.1-44.0 cm) were established. MIP and grip strength measurements falling within the ranges of weight 350-399 and 300-349 g and length 42.1-44.0 cm were significantly greater than values falling within 250-299 g and 37.0-41.0 cm ranges respectively. Specific weight- and length-percentile distributions for MIP and grip strength measurements were calculated. As significant direct correlations were observed between rat weights and lengths and either MIP or grip strength measurements, regression equations relating all these variables were also determined. Skeletal muscle dysfunction is frequently associated with highly prevalent conditions. The significant predictive equations described for both MIP and grip strength measurements will enable scientists to better estimate the respiratory and peripheral muscle dysfunctions of laboratory animals, especially when conducting follow-up and/or intervention investigations.

Comparative effects of bronchoalveolar lavage with saline, surfactant, or perfluorocarbon in experimental meconium aspiration syndrome.

Objective: Today, in meconium aspiration syndrome, treatment focuses on bronchoalveolar lavage, because it removes meconium and proinflammatory factors from airways. This technique might be more effective if different solutions were used such as saline solution, a protein-free surfactant, or a perfluorocarbon, because these would be less inhibited by meconium proteins. Setting: Pulmonary physiology research unit, Cruces Hospital. Design: Prospective, randomized study. Subjects: We studied 24 lambs (<6 days) on mechanical ventilation for 180 mins. Catheters were placed and femoral and pulmonary arteries pressures registered (systemic and pulmonary arterial pressures). Interventions: Lambs were instilled with 20% meconium (3–5 mL/Kg) and were randomly assigned to one of the following groups (n = 6): control: only continuous mechanical ventilation; saline bronchoalveolar lavage: bronchoalveolar lavage with 30 mL/kg of saline solution; dilute surfactant bronchoalveolar lavage: bronchoalveolar lavage with 32 mL/kg of diluted surfactant (lucinactant, 10 mg/mL); or perfluorocarbon bronchoalveolar lavage: bronchoalveolar lavage with 30 mL/kg of perfluorocarbon. Measurements and Main Results: Blood gases, cardiovascular parameters, and pulmonary mechanics were assessed. Meconium instillation produced severe hypoxia, hypercapnia, acidosis, and pulmonary hypertension with impairment of pulmonary mechanics (p < .05). Lung lavage with dilute surfactant resulted in the resolution of pulmonary hypertension as well as better gas exchange and pulmonary mechanics than the control group (p < .05). Bronchoalveolar lavage with perfluorocarbon produced a transient improvement in gas exchange and ventilatory indices in comparison with control and saline bronchoalveolar lavage groups. Conclusions: In lambs with meconium aspiration syndrome, bronchoalveolar lavage with diluted lucinactant is an effective therapy producing significant improvements in gas exchange, pulmonary hypertension, and pulmonary mechanics. In addition, bronchoalveolar lavage with perfluorocarbon appears to confer some advantages over lavage with equal volumes of saline or no lavage.