Characterization and quantification of isoflurane-induced developmental apoptotic cell death in mouse cerebral cortex.

BACKGROUND: Accumulating evidence indicates that isoflurane and other, similarly acting anesthetics exert neurotoxic effects in neonatal animals. However, neither the identity of dying cortical cells nor the extent of cortical cell loss has been sufficiently characterized. We conducted the present study to immunohistochemically identify the dying cells and to quantify the fraction of cells undergoing apoptotic death in neonatal mouse cortex, a substantially affected brain region. METHODS: Seven-day-old littermates (n = 36) were randomly assigned to a 6-hour exposure to either 1.5% isoflurane or fasting in room air. Animals were euthanized immediately after exposure and brain sections were double-stained for activated caspase 3 and one of the following cellular markers: Neuronal Nuclei (NeuN) for neurons, glutamic acid decarboxylase (GAD)65 and GAD67 for GABAergic cells, as well as GFAP (glial fibrillary acidic protein) and S100ß for astrocytes. RESULTS: In 7-day-old mice, isoflurane exposure led to widespread increases in apoptotic cell death relative to controls, as measured by activated caspase 3 immunolabeling. Confocal analyses of caspase 3-labeled cells in cortical layers II and III revealed that the overwhelming majority of cells were postmitotic neurons, but some were astrocytes. We then quantified isoflurane-induced neuronal apoptosis in visual cortex, an area of substantial injury. In unanesthetized control animals, 0.08% ± 0.001% of NeuN-positive layer II/III cortical neurons were immunoreactive for caspase 3. By contrast, the rate of apoptotic NeuN-positive neurons increased at least 11-fold (lower end of the 95% confidence interval [CI]) to 2.0% ± 0.004% of neurons immediately after isoflurane exposure (P = 0.0017 isoflurane versus control). In isoflurane-treated animals, 2.9% ± 0.02% of all caspase 3-positive neurons in superficial cortex also coexpressed GAD67, indicating that inhibitory neurons may also be affected. Analysis of GABAergic neurons, however, proved unexpectedly complex. In addition to inducing apoptosis among some GAD67-immunoreactive neurons, anesthesia also coincided with a dramatic decrease in both GAD67 (0.98 vs 1.84 ng/mg protein, P < 0.00001, anesthesia versus control) and GAD65 (2.25 ± 0.74 vs 23.03 ± 8.47 ng/mg protein, P = 0.0008, anesthesia versus control) protein levels. CONCLUSIONS: Prolonged exposure to isoflurane increased neuronal apoptotic cell death in 7-day-old mice, eliminating approximately 2% of cortical neurons, of which some were identified as GABAergic interneurons. Moreover, isoflurane exposure interfered with the inhibitory nervous system by downregulating the central enzymes GAD65 and GAD67. Conversely, at this age, only a minority of degenerating cells were identified as astrocytes. The clinical relevance of these findings in animals remains to be determined.

Comparison of the neuroapoptotic properties of equipotent anesthetic concentrations of desflurane, isoflurane, or sevoflurane in neonatal mice.

BACKGROUND: Volatile anesthetics facilitate surgical procedures and imaging studies in millions of children every year. Neuronal cell death after prolonged exposure to isoflurane in developing animals has raised serious concerns regarding its safe use in children. Although sevoflurane and desflurane are becoming more popular for pediatric anesthesia, their cytotoxic effects have not been compared with those of isoflurane. Accordingly, using newborn mice, the current study established the respective potencies of desflurane, isoflurane, and sevoflurane and then compared equipotent doses of these anesthetics regarding their effects on cortical neuroapoptosis. METHODS: Minimum alveolar concentrations were determined in littermates (aged 7-8 days, n = 42) using tail-clamp stimulation in a bracketing study design. By using equipotent doses of approximately 0.6 minimum alveolar concentration, another group of littermates was randomly assigned to receive desflurane, isoflurane, or sevoflurane or to fast in room air for 6 h. After exposure, animals (n = 47) were euthanized, neocortical apoptotic neuronal cell death was quantified, and caspase 3 activity was compared between the four groups. RESULTS: The minimum alveolar concentration was determined to be 12.2% for desflurane, 2.7% for isoflurane, and 5.4% for sevoflurane. After a 6-h exposure to approximately 0.6 minimum alveolar concentration of desflurane, isoflurane, or sevoflurane, neuronal cell death and apoptotic activity were significantly increased, irrespective of the specific anesthetic used. CONCLUSIONS: In neonatal mice, equipotent doses of the three commonly used inhaled anesthetics demonstrated similar neurotoxic profiles, suggesting that developmental neurotoxicity is a common feature of all three drugs and cannot be avoided by switching to newer agents.

Red blood cell transfusion in critically ill children: a narrative review.

OBJECTIVE: To review the pathophysiology of anemia, as well as transfusion-related complications and indications for red blood cell (RBC) transfusion, in critically ill children. Although allogeneic blood has become increasingly safer from infectious agents, mounting evidence indicates that RBC transfusions are associated with complications and unfavorable outcomes. As a result, there has been growing interest and efforts to limit RBC transfusion, and indications are being revisited and revamped. Although a so-called restrictive RBC transfusion strategy has been shown to improve morbidity and mortality in critically ill adults, there have been relatively few studies on RBC transfusion performed in critically ill children. DATA SOURCES: Published literature on transfusion medicine and outcomes of RBC transfusion. STUDY SELECTION, DATA EXTRACTION, AND SYNTHESIS: After a brief overview of physiology of oxygen transportation, anemia compensation, and current transfusion guidelines based on available literature, risks and outcomes of transfusion in general and in critically ill children are summarized in conjunction with studies investigating the safety of restrictive transfusion strategies in this patient population. CONCLUSIONS: The available evidence does not support the extensive use of RBC transfusions in general or critically ill patients. Transfusions are still associated with risks, and although their benefits are established in limited situations, the associated negative outcomes in many more patients must be closely addressed. Given the frequency of anemia and its proven negative outcomes, transfusion decisions in the critically ill children should be based on individual patient's characteristics rather than generalized triggers, with consideration of potential risks and benefits, and available blood conservation strategies that can reduce transfusion needs.

The effects of neonatal isoflurane exposure in mice on brain cell viability, adult behavior, learning, and memory.

BACKGROUND: Volatile anesthetics, such as isoflurane, are widely used in infants and neonates. Neurodegeneration and neurocognitive impairment after exposure to isoflurane, midazolam, and nitrous oxide in neonatal rats have raised concerns regarding the safety of pediatric anesthesia. In neonatal mice, prolonged isoflurane exposure triggers hypoglycemia, which could be responsible for the neurocognitive impairment. We examined the effects of neonatal isoflurane exposure and blood glucose on brain cell viability, spontaneous locomotor activity, as well as spatial learning and memory in mice. METHODS: Seven-day-old mice were randomly assigned to 6 h of 1.5% isoflurane with or without injections of dextrose or normal saline, or to 6 h of room air without injections (no anesthesia). Arterial blood gases and glucose were measured. After 2 h, 18 h, or 11 wk postexposure, cellular viability was assessed in brain sections stained with Fluoro-Jade B, caspase 3, or NeuN. Nine weeks postexposure, spontaneous locomotor activity was assessed, and spatial learning and memory were evaluated in the Morris water maze using hidden and reduced platform trials. RESULTS: Apoptotic cellular degeneration increased in several brain regions early after isoflurane exposure, compared with no anesthesia. Despite neonatal cell loss, however, adult neuronal density was unaltered in two brain regions significantly affected by the neonatal degeneration. In adulthood, spontaneous locomotor activity and spatial learning and memory performance were similar in all groups, regardless of neonatal isoflurane exposure. Neonatal isoflurane exposure led to an 18% mortality, and transiently increased Paco(2), lactate, and base deficit, and decreased blood glucose levels. However, hypoglycemia did not seem responsible for the neurodegeneration, as dextrose supplementation failed to prevent neuronal loss. CONCLUSIONS: Prolonged isoflurane exposure in neonatal mice led to increased immediate brain cell degeneration, however, no significant reductions in adult neuronal density or deficits in spontaneous locomotion, spatial learning, or memory function were observed.

General anesthetics and the developing brain.

PURPOSE OF REVIEW: General anesthetics and sedatives are used in millions of children every year to facilitate surgical procedures, imaging studies, and sedation in operating rooms, radiology suites, emergency departments, and ICUs. Mounting evidence from animal studies suggests that prolonged exposure to these compounds may induce widespread neuronal cell death and neurological sequelae, seriously questioning the safety of pediatric anesthesia. This review presents recent developments in this rapidly emerging field. RECENT FINDINGS: In animals, all currently available anesthetics and sedatives that have been studied, such as ketamine, midazolam, diazepam, clonazepam, propofol, pentobarbital, chloral hydrate, halothane, isoflurane, sevoflurane, enflurane, nitrous oxide, and xenon, have been demonstrated to trigger widespread neurodegeneration in the immature brain. In humans, recent preliminary findings from epidemiological studies suggest an association between surgery and anesthesia early in life and subsequent learning abnormalities. SUMMARY: Neurodegeneration following exposure to anesthetics and sedatives has been clearly established in developing animals. However, while some of the biochemical pathways have been revealed, the phenomenon's particular molecular mechanisms remain unclear. As the phenomenon is difficult to study in humans, clinical evidence is still scarce and amounts to associative and not causal relationships. Owing to the lack of alternative anesthetics, further animal studies into the mechanism as well as clinical studies defining human susceptibility are both urgently needed.

The impact of the perioperative period on neurocognitive development, with a focus on pharmacological concerns.

Mounting evidence from animal studies has implicated that all commonly used anaesthetics and sedatives may induce widespread neuronal cell death and result in long-term neurological abnormalities. These findings have led to serious questions regarding the safe use of these drugs in young children. In humans, recent findings from retrospective, epidemiological studies do not exclude the possibility of an association between surgery with anaesthesia early in life and subsequent learning abnormalities. These results have sparked discussions regarding the appropriate timing of paediatric surgery and the safe management of paediatric anaesthesia. However, important questions need to be addressed before findings from laboratory studies and retrospective clinical surveys can be used to guide clinical practice. This article summarises the currently available preclinical and clinical information regarding the impact of anaesthetics, sedatives, opioids, pain and stress, inflammation, hypoxia-ischaemia, co-morbidities and genetic predisposition on brain structure and long-term neurological function. Moreover, this article outlines the putative mechanisms of anaesthetic neurotoxicity, and the phenomenon's implications for clinical practice in this rapidly emerging field.

Management of traumatic tracheobronchial separation in a teenager using a fabricated extra-long endotracheal tube.

Tracheobronchial separation (TBS) due to blunt chest trauma in children is extremely rare. We report the case of a 14-year-old boy who fell 20 feet and developed respiratory distress, bilateral pneumothoraces, pneumomediastinum, and subcutaneous emphysema. Computed tomography imaging at the initial institution failed to detect tracheobronchial disruption, and the patient was managed conservatively. The patient's worsening condition prompted bronchoscopic examination which revealed complete separation of the right main bronchus from the trachea. Single-lung ventilation was instituted using a fabricated extra-long nasotracheal tube due to the patient's large size and mandibular fracture, and the airway was primarily anastamosed with an open thoracotomy approach. The clinical features of tracheobronchial separation as well as anesthetic, clinical and surgical management of this rare but life-threatening injury are described.