Anesthesia and Sedation Exposure and Neurodevelopmental Outcomes in Infants Undergoing Congenital Cardiac Surgery: A Retrospective Cohort Study.

BACKGROUND: Children undergoing complex cardiac surgery are exposed to substantial cumulative doses of sedative medications and volatile anesthetics and are more frequently anesthetized with ketamine, compared with healthy children. This study hypothesized that greater exposure to sedation and anesthesia in this population is associated with lower neurodevelopmental scores at 18 months of age. METHODS: A secondary analysis was conducted of infants with congenital heart disease who participated in a prospective observational study of environmental exposures and neurodevelopmental outcomes to assess the impact of cumulative volatile anesthetic agents and sedative medications. Cumulative minimum alveolar concentration hours of exposure to volatile anesthetic agents and all operating room and intensive care unit exposures to sedative and anesthesia medications were collected before administration of Bayley Scales of Infant and Toddler Development, 3rd edition (Bayley III), at 18 months of age. RESULTS: The study cohort included 41 (37%) single-ventricle and 69 (63%) two-ventricle patients. Exposures to volatile anesthetic agents, opioids, benzodiazepines, and dexmedetomidine were not associated with abnormal Bayley III scores. At 18-month follow-up, after adjusting for confounders, each mg/kg increase in ketamine exposure was associated with a 0.34 (95% CI, -0.64 to -0.05) point decrease in Bayley III motor scores (P = 0.024). CONCLUSIONS: Total cumulative exposures to volatile anesthetic agents were not associated with neurodevelopmental impairment in infants with congenital heart disease undergoing various imaging studies and procedures, whereas higher ketamine doses were associated with poorer motor performance.

Effect of dexmedetomidine on sevoflurane-induced neurodegeneration in neonatal rats.

BACKGROUND: Structural brain abnormalities in newborn animals after prolonged exposure to all routinely used general anaesthetics have raised substantial concerns for similar effects occurring in millions of children undergoing surgeries annually. Combining a general anaesthetic with non-injurious sedatives may provide a safer anaesthetic technique. We tested dexmedetomidine as a mitigating therapy in a sevoflurane dose-sparing approach. METHODS: Neonatal rats were randomised to 6 h of sevoflurane 2.5%, sevoflurane 1% with or without three injections of dexmedetomidine every 2 h (resulting in 2.5, 5, 10, 25, 37.5, or 50 µg kg-1 h-1), or fasting in room air. Heart rate, oxygen saturation, level of hypnosis, and response to pain were measured during exposure. Neuronal cell death was quantified histologically after exposure. RESULTS: Sevoflurane at 2.5% was more injurious than at 1% in the hippocampal cornu ammonis (CA)1 and CA2/3 subfields; ventral posterior and lateral dorsal thalamic nuclei; prefrontal, retrosplenial, and somatosensory cortices; and subiculum. Although sevoflurane 1% did not provide complete anaesthesia, supplementation with dexmedetomidine dose dependently increased depth of anaesthesia and diminished responses to pain. The combination of sevoflurane 1% and dexmedetomidine did not reliably reduce neuronal apoptosis relative to an equianaesthetic dose of sevoflurane 2.5%. CONCLUSIONS: A sub-anaesthetic dose of sevoflurane combined with dexmedetomidine achieved a level of anaesthesia comparable with that of sevoflurane 2.5%. Similar levels of anaesthesia caused comparable programmed cell death in several developing brain regions. Depth of anaesthesia may be an important factor when comparing the neurotoxic effects of different anaesthetic regimens.

Standards for preclinical research and publications in developmental anaesthetic neurotoxicity: expert opinion statement from the SmartTots preclinical working group.

In March 2019, SmartTots, a public-private partnership between the US Food and Drug Administration and the International Anesthesia Research Society, hosted a meeting attended by research experts, anaesthesia journal editors, and government agency representatives to discuss the continued need for rigorous preclinical research and the importance of establishing reporting standards for the field of anaesthetic perinatal neurotoxicity. This group affirmed the importance of preclinical research in the field, and welcomed novel and mechanistic approaches to answer some of the field's largest questions. The attendees concluded that summarising the benefits and disadvantages of specific model systems, and providing guidance for reporting results, would be helpful for designing new experiments and interpreting results across laboratories. This expert opinion report is a summary of these discussions, and includes a focused review of current animal models and reporting standards for the field of perinatal anaesthetic neurotoxicity. This will serve as a practical guide and road map for novel and rigorous experimental work.

Immature murine hippocampal neurones do not develop long-term structural changes after a single isoflurane exposure.

BACKGROUND: Studies in developing animals show that a clinically relevant anaesthesia exposure increases neuronal death and alters brain structure. In the hippocampal dentate gyrus, the anaesthetic isoflurane induces selective apoptosis among roughly 10% of 2-week-old hippocampal granule cells in 21-day-old mice. In this work, we queried whether the 90% of granule cells surviving the exposure might be 'injured' and integrate abnormally into the brain. METHODS: The long-term impact of isoflurane exposure on granule cell structure was studied using a transgenic mouse model fate-mapping approach to identify and label immature granule cells. Male and female mice were exposed to isoflurane for 6 h when the fate-mapped granule cells were 2 weeks old. The morphology of the fate-mapped granule cells was quantified 2 months later. RESULTS: The gross structure of the dentate gyrus was not affected by isoflurane treatment, with granule cells present in the correct subregions. Individual isoflurane-exposed granule cells were structurally normal, exhibiting no changes in spine density, spine type, dendrite length, or presynaptic axon terminal structure (P>0.05). Granule cell axon terminals were 13% larger in female mice relative to males; however, this difference was evident regardless of treatment (difference of means=0.955; 95% confidence interval, 0.37-1.5; P=0.010). CONCLUSIONS: A single, prolonged isoflurane exposure did not impair integration of this age-specific cohort of granule cells, regardless of the animal's sex. Nonetheless, although 2-week-old cells were not affected, the results should not be extrapolated to other age cohorts, which may respond differently.

Comparison of Intranasal Dexmedetomidine and Oral Pentobarbital Sedation for Transthoracic Echocardiography in Infants and Toddlers: A Prospective, Randomized, Double-Blind Trial.

BACKGROUND: Acquisition of transthoracic echocardiographic (TTEcho) images in children often requires sedation. The optimal sedative for TTEcho has not been determined. Children with congenital heart disease are repeatedly exposed to sedatives and anesthetics that may affect brain development. Dexmedetomidine, which in animals alters brain structure to a lesser degree, may offer advantages in this vulnerable population. METHODS: A prospective, randomized, double-blind trial enrolled 280 children 3-24 months of age undergoing outpatient TTEcho, comparing 2.5 µg·kg intranasal dexmedetomidine to 5 mg·kg oral pentobarbital. Rescue sedation, for both groups, was intranasal dexmedetomidine 1 µg·kg. The primary outcome was adequate sedation within 30 minutes without rescue sedation, assessed by blinded personnel. Secondary outcomes included number of sonographer pauses, image quality in relation to motion artifacts, and parental satisfaction. RESULTS: Success rates with a single dose were not different between sedation techniques; 85% in the pentobarbital group and 84% in the dexmedetomidine group (P = .8697). Median onset of adequate sedation was marginally faster with pentobarbital (16.5 [interquartile range, 13-21] vs 18 [16-23] minutes for dexmedetomidine [P = .0095]). Time from drug administration to discharge was not different (P = .8238) at 70.5 (64-83) minutes with pentobarbital and 70 (63-82) minutes with dexmedetomidine. Ninety-five percent of sedation failures with pentobarbital and 100% of dexmedetomidine failures had successful rescue sedation with intranasal dexmedetomidine. CONCLUSIONS: Intranasal dexmedetomidine was comparable to oral pentobarbital sedation for TTEcho sedation in infants and did not increase the risk of clinically important adverse events. Intranasal dexmedetomidine appears to be an effective "rescue" sedative for both failed pentobarbital and dexmedetomidine sedation. Dexmedetomidine could be a safer option for repeated sedation in children, but further studies are needed to assess long-term consequence of repeated sedation in this high-risk population.

Anesthesia and the developing brain: A way forward for laboratory and clinical research.

All commonly used general anesthetics have been shown to cause neurotoxicity in animal models, including nonhuman primates. Opinion, however, remains divided over how cumulative evidence from preclinical and human studies in this field should be interpreted and its translation to current practices in pediatric anesthesia and surgery. A group of international experts in laboratory and clinical sciences recently convened in Genoa, Italy, to evaluate the current state of both laboratory and clinical research and discuss future directions for basic, translational, and clinical studies in this field. This paper describes those discussions and conclusions. A central goal identified was the importance of continuing to pursue laboratory research efforts to better understand the biological pathways underlying anesthesia neurotoxicity. The distinction between basic and translational experimental designs in this field was highlighted, and it was acknowledged that it will be important for future animal research to try to causally link structural changes with long-term cognitive abnormalities. While inherent limitations will continue to affect the ability of even large observational cohorts to determine if anesthesia impacts neurodevelopment or behavioral outcomes, the importance of conducting further large well-designed cohort studies was also emphasized. Adequately powered cohorts could clarify which populations are at increased risk, provide information on environmental and healthcare-related risk modifiers, and guide future interventional trials. If anesthetics cause structural or functional adverse neurological effects in young children, alternative or mitigating strategies need to be considered. While protective or mitigating strategies have been repeatedly studied in animals, there are currently no human data to support alternative anesthetic strategies in clinical practice. Lastly, it was noted that there is still considerable debate over the clinical relevance of anesthesia neurotoxicity, and the need to evaluate the impact of other aspects of perioperative care on neurodevelopment must also be considered.

Sedation methods for transthoracic echocardiography in children with Trisomy 21-a retrospective study.

BACKGROUND: Many children with Trisomy 21 have neurologic or behavioral problems that make it difficult for them to remain still during noninvasive imaging studies, such as transthoracic echocardiograms (TTEcho). Recently, intranasal dexmedetomidine sedation has been introduced for this purpose. However, dexmedetomidine has been associated with bradycardia. Children with Trisomy 21 have been reported to have a higher risk of bradycardia and airway obstruction with sedation or anesthesia compared to children without Trisomy 21. OBJECTIVE: Our aim was to quantify the incidence of age-defined bradycardia and other adverse effects in patients with Trisomy 21 under sedation for TTEcho using a variety of sedation and anesthesia techniques available and utilized at our institution in this challenging patient population, including intranasal dexmedetomidine, oral pentobarbital, general anesthesia with propofol, and general anesthesia with sevoflurane. Our primary hypothesis was that intranasal dexmedetomidine sedation would result in a significantly higher risk of bradycardia in patients with Trisomy 21, compared with other sedative or anesthetic regimens. METHODS: This is a retrospective, observational study of 147 consecutive patients with Trisomy 21 who were sedated or anesthetized for transthoracic echocardiography. Efficacy of sedation was defined as no need for rescue sedation or conversion to an alternate technique. Lowest and highest heart rate, systolic blood pressure, oxygen saturation, and PR interval from formal electrocardiograms were extracted from the electronic medical record. These data were compared to age-defined normal values to determine adverse events. RESULTS: Four methods of sedation or anesthesia were utilized to perform sedated transthoracic echocardiography: general anesthesia with sevoflurane by mask, general anesthesia with sevoflurane induction followed by intravenous propofol maintenance, oral pentobarbital, and intranasal dexmedetomidine. Intranasal dexmedetomidine 2.5 mcg·kg-1 was an effective sedative as a single dose for TTEcho in 37 of 41 (90%) cases. Oral pentobarbital 5 mg·kg-1 as a single dose for young children with Trisomy 21 was effective in 55 of 75 (73%) cases. Intranasal dexmedetomidine sedation was not associated with a significantly higher risk of bradycardia in patients with Trisomy 21, compared with other sedative or anesthetic regimens, when compared to oral pentobarbital for patients under 2 years of age and general anesthesia for children 3 years and older. The two general anesthesia groups showed lowest heart rates of 66.9 ± 15.9 min-1 for sevoflurane and 69.0 ± 11.5 min-1 for sevoflurane-propofol. Hypotension was present in all groups ranging between an incidence of 56% in the sevoflurane group to 11% in the oral pentobarbital group. Oxygen saturation and clinically significant desaturation occurred in 14% of the oral pentobarbital group. CONCLUSION: Intranasal dexmedetomidine sedation was not associated with a significantly higher risk of bradycardia in patients with Trisomy 21, compared with other sedative or anesthetic regimens.

Long-term Fate Mapping to Assess the Impact of Postnatal Isoflurane Exposure on Hippocampal Progenitor Cell Productivity.

BACKGROUND: Exposure to isoflurane increases apoptosis among postnatally generated hippocampal dentate granule cells. These neurons play important roles in cognition and behavior, so their permanent loss could explain deficits after surgical procedures. METHODS: To determine whether developmental anesthesia exposure leads to persistent deficits in granule cell numbers, a genetic fate-mapping approach to label a cohort of postnatally generated granule cells in Gli1-CreER::GFP bitransgenic mice was utilized. Green fluorescent protein (GFP) expression was induced on postnatal day 7 (P7) to fate map progenitor cells, and mice were exposed to 6 h of 1.5% isoflurane or room air 2 weeks later (P21). Brain structure was assessed immediately after anesthesia exposure (n = 7 controls and 8 anesthesia-treated mice) or after a 60-day recovery (n = 8 controls and 8 anesthesia-treated mice). A final group of C57BL/6 mice was exposed to isoflurane at P21 and examined using neurogenesis and cell death markers after a 14-day recovery (n = 10 controls and 16 anesthesia-treated mice). RESULTS: Isoflurane significantly increased apoptosis immediately after exposure, leading to cell death among 11% of GFP-labeled cells. Sixty days after isoflurane exposure, the number of GFP-expressing granule cells in treated animals was indistinguishable from control animals. Rates of neurogenesis were equivalent among groups at both 2 weeks and 2 months after treatment. CONCLUSIONS: These findings suggest that the dentate gyrus can restore normal neuron numbers after a single, developmental exposure to isoflurane. The authors' results do not preclude the possibility that the affected population may exhibit more subtle structural or functional deficits. Nonetheless, the dentate appears to exhibit greater resiliency relative to nonneurogenic brain regions, which exhibit permanent neuron loss after isoflurane exposure.

Dosing and efficacy of intranasal dexmedetomidine sedation for pediatric transthoracic echocardiography: a retrospective study.

PURPOSE: We designed this retrospective observational study on the use of α2-agonist dexmedetomidine to determine the optimum intranasal dose to achieve sedation for pediatric transthoracic echocardiography and to identify any dose-related adverse effects. METHODS: Outpatient children aged three months to three years with diverse diagnoses of congenital heart disease, including cyanotic cardiac defects, underwent transthoracic echocardiography under dexmedetomidine sedation. Aerosolized intranasal dexmedetomidine was administered with initial doses ranging from 1-3 µg·kg(-1). A rescue dose of 1 µg·kg(-1) was administered if adequate sedation was not achieved within 45 min following the first dose. The primary study outcome was the achievement of adequate sedation to allow transthoracic echocardiography (TTE) scanning, including subxiphoid and suprasternal probe manipulation. RESULTS: Sedation with intranasal dexmedetomidine for transthoracic echocardiography was successful in 62 of the 63 (98%) patients studied, with an intranasal rescue dose required in 13 (21%) patients. Intranasal doses of dexmedetomidine 2.5-3.0 µg·kg(-1) were required for tolerating TTE probe placement, including subxiphoid and suprasternal manipulation, with minimal response and a 90% success rate. Excluding patients who required a second dose of dexmedetomidine, the mean (standard deviation) time from administration to achieving such sedation (onset time) was 26 (8) min for low-dose (1-2 µg·kg(-1)) dexmedetomidine and 28 (8) min for moderate-dose (2.5-3.0 µg·kg(-1)) dexmedetomidine (P = 0.33). Time from administration of low-dose dexmedetomidine to discharge, including TTE scan time, was 80 (14) min, and it increased with moderate-dose dexmedetomidine to 91 (22) min (P = 0.05). Mild to moderate bradycardia and hypotension were observed, but no interventions were required. CONCLUSION: We found that aerosolized intranasal dexmedetomidine offers satisfactory conditions for TTE in children three months to three years of age with an optimal dose of 2.5-3.0 µg·kg(-1)administered under the supervision of a pediatric cardiac anesthesiologist.

Cell age-specific vulnerability of neurons to anesthetic toxicity.

OBJECTIVE: Anesthetics have been linked to widespread neuronal cell death in neonatal animals. Epidemiological human studies have associated early childhood anesthesia with long-term neurobehavioral abnormalities, raising substantial concerns that anesthetics may cause similar cell death in young children. However, key aspects of the phenomenon remain unclear, such as why certain neurons die, whereas immediately adjacent neurons are seemingly unaffected, and why the immature brain is exquisitely vulnerable, whereas the mature brain seems resistant. Elucidating these questions is critical for assessing the phenomenon's applicability to humans, defining the susceptible age, predicting vulnerable neuronal populations, and devising mitigating strategies. METHODS: This study examines the effects of anesthetic exposure on late- and adult-generated neurons in newborn, juvenile, and adult mice, and characterizes vulnerable cells using birth-dating and immunohistochemical techniques. RESULTS: We identify a critical period of cellular developmental during which neurons are susceptible to anesthesia-induced apoptosis. Importantly, we demonstrate that anesthetic neurotoxicity can extend into adulthood in brain regions with ongoing neurogenesis, such as dentate gyrus and olfactory bulb. INTERPRETATION: Our findings suggest that anesthetic vulnerability reflects the age of the neuron, not the age of the organism, and therefore may potentially not only be relevant to children but also to adults undergoing anesthesia. This observation further predicts differential heightened regional vulnerability to anesthetic neuroapoptosis to closely follow the distinct regional peaks in neurogenesis. This knowledge may help guide neurocognitive testing of specific neurological domains in humans following exposure to anesthesia, dependent on the individual's age during exposure.

A combination of mild hypothermia and sevoflurane affords long-term protection in a modified neonatal mouse model of cerebral hypoxia-ischemia.

BACKGROUND: Infant brain injury from hypoxia-ischemia (HI) can lead to life-long impairment, but protective strategies are lacking. Short-term but not long-term protection has been demonstrated in the Rice-Vannucci neonatal brain ischemia model (RVM) by volatile anesthetic administration before HI, while exposure during HI has not been tested. In the current study, we evaluated a combination of sevoflurane and mild hypothermia as a protective approach during HI, both short- and long-term, by introducing intubation and mechanical ventilation to the RVM. METHODS: The right common carotid artery was ligated in 10-day-old mice during brief sevoflurane anesthesia, followed by a 2-hour recovery with the dam. Littermates were then randomized to either: HI spontaneously breathing 10% oxygen for 60 minutes (the classical RVM); HI-Protect mild hypothermia and orotracheal intubation and mechanical ventilation with 3.5% sevoflurane in 10% oxygen for 60 minutes; or Room Air spontaneously breathing room air for 60 minutes. In a nonsurviving cohort, cerebral oxygenation was monitored in the area at risk and the contralateral hemisphere during HI or HI-Protect using visible-light spectroscopy (Spectros Corp). Mean arterial blood pressure and heart rate were measured. Arterial blood gases were obtained. Right/left brain hemispheric weight ratios and brain damage scores were determined 1 week after HI. In another group, learning and behavior were assessed in young adulthood (9 weeks) using spontaneous locomotion, Morris water maze, and apomorphine injection. RESULTS: During HI, ipsilateral and contralateral brain oxygenation, arterial blood pressures, blood gases, and glucose levels were similar in both ischemic groups, while heart rate was slower in the HI-Protect group. One week after ischemia, brain hemispheric weight ratios and injury scores in several brain regions were significantly worse after HI, compared with HI-Protect. Nine weeks after HI, Morris water maze hidden platform and reversal platform escape latencies, measures of spatial memory function, were superior after HI-Protect, compared with HI (P < 0.0001). HI-Protect animals demonstrated significantly less circling behavior after an apomorphine challenge (P < 0.0001), a measure of striatal integrity. CONCLUSIONS: To test the neuroprotective effects of volatile anesthetics during neonatal brain ischemia, we developed a modification of the RVM. By using mechanical ventilation and endotracheal intubation, sevoflurane administration during HI was survivable. The combination of sevoflurane administration and mild hypothermia during HI conferred not only short-term structural, but also long-term functional protection, compared with littermates treated according to the RVM. These findings warrant further studies to improve neurological outcome in critically ill infants.

Patient and procedural characteristics for successful and failed immediate tracheal extubation in the operating room following cardiac surgery in infancy.

BACKGROUND: Immediate extubation in the operating room after congenital heart surgery is practiced with rising frequency at many cardiac institutions to decrease costs and complications. Infants less than one year of age are also increasingly selected for this 'fast track'. However, factors for patient selection, success, or failure of this practice have not been well defined in this population, yet are critical for patient safety. OBJECTIVE: To identify selection criteria, patient and procedural characteristics for successful or failed very early endotracheal extubation in the operating room immediately following infant heart surgery. METHODS: A retrospective analysis was performed for 326 consecutive patients undergoing neonatal and infant heart surgery from 2009 to 2012. Extubation and reintubation data were taken from the institutional Society of Thoracic Surgeons database and patients' charts. Patient characteristics were derived using multivariable logistic regression models. RESULTS: Very early extubation in the operating room was performed for 130 of 326 neonates and infants (40%). Weight >4 kg, lesser procedural complexity, and absence of trisomy 21 were identified as significant predictors for attempted very early extubation. Of these patients, 12% required reintubation within 48 h following surgery, predominantly due to respiratory failure or for mediastinal re-exploration. Greater procedural complexity was associated with failed extubations. Reintubation was associated with prolonged hospitalization. CONCLUSIONS: Extubation immediately after infant heart surgery in the operating room can be safely achieved. However, our data suggest that patients undergoing more complex procedures should be selected more conservatively for immediate early extubation.

Association of preoperative coronavirus disease 2019 with mortality, respiratory morbidity and extrapulmonary complications after elective, noncardiac surgery: An observational cohort study.

STUDY OBJECTIVE: To assess the impact of preoperative infection with the contemporary strain of severe acute respiratory coronavirus 2 (SARS-CoV-2) on postoperative mortality, respiratory morbidity and extrapulmonary complications after elective, noncardiac surgery. DESIGN: An ambidirectional observational cohort study. SETTING: A tertiary and teaching hospital in Shanghai, China. PATIENTS: All adult patients (≥ 18 years of age) who underwent elective, noncardiac surgery under general anesthesia at Huashan Hospital of Fudan University from January until March 2023 were screened for eligibility. A total of 2907 patients were included. EXPOSURE: Preoperative coronavirus disease 2019 (COVID-19) positivity. MEASUREMENTS: The primary outcome was 30-day postoperative mortality. The secondary outcomes included postoperative pulmonary complications (PPCs), myocardial injury after noncardiac surgery (MINS), acute kidney injury (AKI), postoperative delirium (POD) and postoperative sleep quality. Multivariable logistic regression was used to assess the risk of postoperative mortality and morbidity imposed by preoperative COVID-19. MAIN RESULTS: The risk of 30-day postoperative mortality was not associated with preoperative COVID-19 [adjusted odds ratio (aOR), 95% confidence interval (CI): 0.40, 0.13-1.28, P = 0.123] or operation timing relative to diagnosis. Preoperative COVID-19 did not increase the risk of PPCs (aOR, 95% CI: 0.99, 0.71-1.38, P = 0.944), MINS (aOR, 95% CI: 0.54, 0.22-1.30; P = 0.168), or AKI (aOR, 95% CI: 0.34, 0.10-1.09; P = 0.070) or affect postoperative sleep quality. Patients who underwent surgery within 7 weeks after COVID-19 had increased odds of developing delirium (aOR, 95% CI: 2.26, 1.05-4.86, P = 0.036). CONCLUSIONS: Preoperative COVID-19 or timing of surgery relative to diagnosis did not confer any added risk of 30-day postoperative mortality, PPCs, MINS or AKI. However, recent COVID-19 increased the risk of POD. Perioperative brain health should be considered during preoperative risk assessment for COVID-19 survivors.

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.

Heterogeneous integration of adult-generated granule cells into the epileptic brain.

The functional impact of adult-generated granule cells in the epileptic brain is unclear, with data supporting both protective and maladaptive roles. These conflicting findings could be explained if new granule cells integrate heterogeneously, with some cells taking neutral or adaptive roles and others contributing to recurrent circuitry supporting seizures. Here, we tested this hypothesis by completing detailed morphological characterizations of age- and experience-defined cohorts of adult-generated granule cells from transgenic mice. The majority of newborn cells exposed to an epileptogenic insult exhibited reductions in dendritic spine number, suggesting reduced excitatory input to these cells. A significant subset, however, exhibited higher spine numbers. These latter cells tended to have enlarged cell bodies, long basal dendrites, or both. Moreover, cells with basal dendrites received significantly more recurrent mossy fiber input through their apical dendrites, indicating that these cells are robustly integrated into the pathological circuitry of the epileptic brain. These data imply that newborn cells play complex--and potentially conflicting--roles in epilepsy.

Abnormalities of granule cell dendritic structure are a prominent feature of the intrahippocampal kainic acid model of epilepsy despite reduced postinjury neurogenesis.

PURPOSE: Aberrant plastic changes among adult-generated hippocampal dentate granule cells are hypothesized to contribute to the development of temporal lobe epilepsy. Changes include formation of basal dendrites projecting into the dentate hilus. Innervation of these processes by granule cell mossy fiber axons leads to the creation of recurrent excitatory circuits within the dentate. The destabilizing effect of these recurrent circuits may contribute to hyperexcitability and seizures. Although basal dendrites have been identified in status epilepticus models of epilepsy associated with increased neurogenesis, we do not know whether similar changes are present in the intrahippocampal kainic acid model of epilepsy, which is associated with reduced neurogenesis. METHODS: In the present study, we used Thy1-YFP-expressing transgenic mice to determine whether hippocampal dentate granule cells develop hilar-projecting basal dendrites in the intrahippocampal kainic acid model. Brain sections were examined 2 weeks after treatment. Tissue was also examined using ZnT-3 immunostaining for granule cell mossy fiber terminals to assess recurrent connectivity. Adult neurogenesis was assessed using the proliferative marker Ki-67 and the immature granule cell marker calretinin. KEY FINDINGS: Significant numbers of cells with basal dendrites were found in this model, but their structure was distinct from basal dendrites seen in other epilepsy models, often ending in complex tufts of short branches and spines. Even more unusual, a subset of cells with basal dendrites had an inverted appearance; they completely lacked apical dendrites. Spines on basal dendrites were found to be apposed to ZnT-3 immunoreactive puncta, suggestive of recurrent mossy fiber input. Finally, YFP-expressing abnormal granule cells did not colocalize Ki-67 or calretinin, indicating that these cells were more than a few weeks old, but were found almost exclusively in proximity to the neurogenic subgranular zone, where the youngest granule cells are located. SIGNIFICANCE: Recent studies have demonstrated in other models of epilepsy that dentate pathology develops following the aberrant integration of immature, adult-generated granule cells. Given these findings, one might predict that the intrahippocampal kainic acid model of epilepsy, which is associated with a dramatic reduction in adult neurogenesis, would not exhibit these changes. Herein we demonstrate that hilar basal dendrites are a common feature of this model, with the abnormal cells likely resulting from the disruption of juvenile granule cell born in the weeks before the insult. These studies demonstrate that postinjury neurogenesis is not required for the accumulation of large numbers of abnormal granule cells.

Anesthetics and sedatives: toxic or protective for the developing brain?

Despite our insufficient understanding of the exact molecular mechanisms of general anesthetics and sedatives, every year millions of children are treated with these drugs in a seemingly safe manner. However, increasing evidence particularly from animal studies has suggested the possibility for deleterious effects in pediatric patients. All currently clinically utilized anesthetic drugs have been found to induce neuronal cell death in the developing brain and to potentially cause long-term neurological impairment. Conversely, painful stimuli without analgesia and anesthesia have also been shown to initiate a harmful stress response in young children and to trigger neurotoxic effects in the developing brain, which can be blunted by anesthetics. Moreover, anesthetic drugs may also confer neurological protection during hypoxic and ischemic insults. The mechanisms and human applicability of anesthetic neurotoxicity and neuroprotection remain under intense investigation and this Perspectives article summarizes the current state of research.

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.