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BACKGROUND Volatile anesthetic exposure during development leads to long-term cognitive deficits in rats which are dependent on age and sex. Female rats are protected relative to male rats for the same exposure on postnatal day 7. Here we test our hypothesis that androgens can modulate chloride cotransporter expression to alter the susceptibility to neurotoxicity from GABAergic drugs using female rats with exogenous testosterone exposure. METHODS Female rats were injected with testosterone (100 μg/animal) or vehicle on postnatal days 1 to 6. On postnatal day 7, the animals were randomized to either isoflurane exposure or sham. Spatial memory was assessed with the Barnes maze starting on postnatal day 41. Western blots were run from testosterone treated postnatal day 7 animals to measure levels of chloride cotransporters sodium-potassium-chloride symporter (NKCC1) and chloride-potassium symporter 5 (KCC2). RESULTS Exogenous testosterone modulated isoflurane anesthetic neurotoxicity in female rats based on poor performance in the probe trial of the Barnes Maze. By contrast, females with vehicle and isoflurane exposure were able to differentiate the goal position. These behavioral differences corresponded to differences in the protein levels of NKCC1 and KCC2 after exogenous testosterone exposure, with NKCC1 increasing (P

Volatile anesthetic exposure during development leads to long-term cognitive deficits in rats which are dependent on age and sex. Female rats are protected relative to male rats for the same exposure on postnatal day 7. Here we test our hypothesis that androgens can modulate chloride cotransporter expression to alter the susceptibility to neurotoxicity from GABAergic drugs using female rats with exogenous testosterone exposure.Female rats were injected with testosterone (100 μg/animal) or vehicle on postnatal days 1 to 6. On postnatal day 7, the animals were randomized to either isoflurane exposure or sham. Spatial memory was assessed with the Barnes maze starting on postnatal day 41. Western blots were run from testosterone treated postnatal day 7 animals to measure levels of chloride cotransporters sodium-potassium-chloride symporter (NKCC1) and chloride-potassium symporter 5 (KCC2).Exogenous testosterone modulated isoflurane anesthetic neurotoxicity in female rats based on poor performance in the probe trial of the Barnes Maze. By contrast, females with vehicle and isoflurane exposure were able to differentiate the goal position. These behavioral differences corresponded to differences in the protein levels of NKCC1 and KCC2 after exogenous testosterone exposure, with NKCC1 increasing ( P0.001) and KCC2 decreasing ( P =0.003) relative to female controls.The expression of chloride cotransporters, NKCC1 and KCC2, is altered by testosterone in female rats and corresponds to a cognitive deficit after isoflurane exposure. This confirms the role of androgens in perinatal anesthetic neurotoxicity and supports our hypothesis that the developing GABAergic system plays a critical role in the underlying mechanism.

Direct electrical motor cortex stimulation with short-train high-frequency stimulation (HFS) for motor evoked potentials (MEPs) has been used intraoperatively during supratentorial surgeries, but the safety threshold is poorly defined. The goal of this study is to establish a rat model for the investigation of neural damage in the cerebral cortex caused by high current HFS to aid in defining safety thresholds. We performed bilateral craniotomy on 12 rats. Cerebral sensory-motor cortex was stimulated with a high-frequency current for 100 times. The rats were sacrificed and the brains were sliced for Nissl, DAPI, and IBA-1 staining. Severe neural damage of the cerebral cortex was found in all cases, including markedly shrunken and pyknotic cells. IBA-1 staining revealed reactive microglia morphology in the lesion area. DAPI staining showed nucleus degeneration and deformation. The cell density were significantly lower within the lesion area compared to the contralateral side. This study has established a brain lesion model caused by HFS on rats. These results suggest HFS may carry a risk of serious neural damage if repeatedly applied to the same brain site. More experiments are needed to fully understand the safety threshold of direct cortical stimulation with HFS for clinical use.

Background Cognitive deficits after perinatal anesthetic exposure are well established outcomes in animal models. This vulnerability is sex-dependent and associated with expression levels of the chloride transporters NKCC1 and KCC2. The hypothesis was that androgen signaling, NKCC1 function, and the age of isoflurane exposure are critical for the manifestation of anesthetic neurotoxicity in male rats. Methods Flutamide, an androgen receptor antagonist, was administered to male rats on postnatal days 2, 4, and 6 before 6 h of isoflurane on postnatal day 7 (ntotal = 26). Spatial and recognition memory were subsequently tested in adulthood. NKCC1 and KCC2 protein levels were measured from cortical lysates by Western blot on postnatal day 7 (ntotal = 20). Bumetanide, an NKCC1 antagonist, was injected immediately before isoflurane exposure (postnatal day 7) to study the effect of NKCC1 inhibition (ntotal = 48). To determine whether male rats remain vulnerable to anesthetic neurotoxicity as juveniles, postnatal day 14 animals were exposed to isoflurane and assessed as adults (ntotal = 30). Results Flutamide-treated male rats exposed to isoflurane successfully navigated the spatial (Barnes maze probe trial F[1, 151] = 78; P < 0.001; mean goal exploration ± SD, 6.4 ± 3.9 s) and recognition memory tasks (mean discrimination index ± SD, 0.09 ± 0.14; P = 0.003), unlike isoflurane-exposed controls. Flutamide changed expression patterns of NKCC1 (mean density ± SD: control, 1.49 ± 0.69; flutamide, 0.47 ± 0.11; P < 0.001) and KCC2 (median density [25th percentile, 75th percentile]: control, 0.23 [0.13, 0.49]; flutamide, 1.47 [1.18,1.62]; P < 0.001). Inhibiting NKCC1 with bumetanide was protective for spatial memory (probe trial F[1, 162] = 6.6; P = 0.011; mean goal time, 4.6 [7.4] s). Delaying isoflurane exposure until postnatal day 14 in males preserved spatial memory (probe trial F[1, 140] = 28; P < 0.001; mean goal time, 6.1 [7.0] s). Conclusions Vulnerability to isoflurane neurotoxicity is abolished by blocking the androgen receptor, disrupting the function of NKCC1, or delaying the time of exposure to at least 2 weeks of age in male rats. These results support a dynamic role for androgens and chloride transporter proteins in perinatal anesthetic neurotoxicity. Editor’s Perspective What We Already Know about This Topic What This Article Tells Us That Is New

OBJECTIVEMaximal safe resection is a primary objective in the management of gliomas. Despite this objective, surgeons and referring physicians may, on the basis of radiological studies alone, assume a glioma to be unresectable. Because imaging studies, including functional MRI, may not localize brain functions (such as language) with high fidelity, this simplistic approach may exclude some patients from what could be a safe resection. Intraoperative direct electrical stimulation (DES) allows for the accurate localization of functional areas, thereby enabling maximal resection of tumors, including those that may appear inoperable based solely on radiological studies. In this paper the authors describe the extent of resection (EOR) and functional outcomes following resections of tumors deemed inoperable by referring physicians and neurosurgeons.METHODSThe authors retrospectively examined the cases of 58 adult patients who underwent glioma resection within 6 months of undergoing a brain biopsy of the same lesion at an outside hospital. All patients exhibited unifocal supratentorial disease and preoperative Karnofsky Performance Scale scores ≥ 70. The EOR and 6-month functional outcomes for this population were characterized.RESULTSIntraoperative DES mapping was performed on 96.6% (56 of 58) of patients. Nearly half of the patients (46.6%, 27 of 58) underwent an awake surgical procedure with DES. Overall, the mean EOR was 87.6% ± 13.6% (range 39.0%–100%). Gross-total resection (resection of more than 99% of the preoperative tumor volume) was achieved in 29.3% (17 of 58) of patients. Subtotal resection (95%–99% resection) and partial resection (PR; < 95% resection) were achieved in 12.1% (7 of 58) and 58.6% (34 of 58) of patients, respectively. Of the cases that involved PR, the mean EOR was 79.4% ± 12.2%. Six months after surgery, no patient was found to have a new postoperative neurological deficit. The majority of patients (89.7%, 52 of 58) were free of neurological deficits both pre- and postoperatively. The remainder of patients exhibited either residual but stable deficits (5.2%, 3 of 58) or complete correction of preoperative deficits (5.2%, 3 of 58).CONCLUSIONSThe use of DES enabled maximal safe resections of gliomas deemed inoperable by referring neurosurgeons. With rare exceptions, tumor resectability cannot be determined solely by radiological studies.

BackgroundEarly life anesthesia exposure results in long-term cognitive deficits in rats. Environmental enrichment consisting of social housing, a stimulating environment, and voluntary exercise can rescue this deficit. We hypothesized that exercise alone is sufficient to rescue the cognitive deficit associated with perinatal anesthesia.MethodsPostnatal day 7 male rats (P7) underwent isoflurane (Iso) or sham exposure and were subsequently weaned at P21. They were then singly housed in a cage with a running wheel or a fixed wheel. After 3 weeks of exercise, animals underwent behavioral testing for spatial and recognition memory assessments. Animals were killed at various time points to accomplish either bromodeoxyuridine (BrdU) labeling or quantitative real-time polymerase chain reaction (qRT-PCR) to quantify brain-derived neurotrophic factor (BDNF) messenger ribonucleic acid (mRNA) levels.ResultsPostweaning voluntary exercise rescued the long-term spatial memory deficit associated with perinatal Iso exposure. Iso-sedentary animals did not discriminate the goal quadrant, spending no more time than chance during the Barnes maze probe trial (1-sample t test, P = .524) while all other groups did (1-sample t test, PIso-exercise = .033; Pcontrol [Con]-sedentary = .004). We did not find a deficit in recognition memory tasks after Iso exposure as we observed previously. BrdU incorporation in the adult hippocampus of Iso-sedentary animals was decreased compared to sedentary controls (Tukey P = .005). Exercise prevented this decrease, with Iso-exercise animals having more proliferation than Iso-sedentary (Tukey P < .001). There was no effect of exercise or Iso on BDNF mRNA in either the cortex or hippocampus (cortex: FExercise[1,32] = 0.236, P = .631; FIso [1,32] = 0.038, P = .847; FInteraction [1,32] = 1.543, P = .223; and hippocampus: FExercise[1,33] = 1.186, P = .284; FIso [1,33] = 1.46, P = .236; FInteraction[1,33] = 1.78, P = .191).ConclusionsExercise restores BrdU incorporation and rescues a spatial memory deficit after early life anesthesia exposure. This demonstrates sufficiency of exercise alone in the context of environmental enrichment to recover a behavioral phenotype after a perinatal insult.

Summary 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.

Background Preclinical investigations of the effects of general anesthesia on the young brain show differences in vulnerability of males and females to anesthetic exposure at different times during development. However, the mechanism underlying this sex difference is poorly understood. Perinatal testosterone is the primary determinant of sexual differentiation and likely plays an important role in defining the period of susceptibility to anesthetic injury. We investigated whether the removal of testosterone through gonadectomy shortly after birth would improve cognitive outcomes in male rodents after early anesthesia exposure. Methods Male Sprague Dawley rats underwent gonadectomy at postnatal day 2 (P2), followed by exposure to 6 hours of isoflurane at P7. A control cohort of gonad-intact male littermates was simultaneously exposed. All rats were subjected to a series of object recognition and association tasks beginning at P42. Cell death in the thalamus and hippocampus was assessed in a separate cohort. Results All groups performed similarly on the Novel Object Recognition task; however, the gonad-intact isoflurane group exhibited decreased performance in the more difficult tasks. This deficit was ameliorated in the gonadectomized group. Cell death was similar between both isoflurane-exposed groups, regardless of gonadectomy. Conclusions The absence of testosterone does not block cell death after anesthesia in specific brain regions of interest; however, does provide some neuroprotection as evidenced by the improved cognitive test performance during adulthood. These findings suggest that testosterone may be mechanistically involved in the sex-specific effects of anesthetic injury on the developing brain by extending the vulnerable period in male rats.

Background The factors determining peak susceptibility of the developing brain to anaesthetics are unclear. It is unknown why postnatal day 7 (P7) male rats are more vulnerable to anaesthesia-induced memory deficits than littermate females. Given the precocious development of certain regions in the female brain during the neonatal critical period, we hypothesised that females are susceptible to anaesthetic brain injury at an earlier time point than previously tested. Methods Female rats were exposed to isoflurane (Iso) 1 minimum alveolar concentration or sham anaesthesia at P4 or P7. Starting at P35, rats underwent a series of behavioural tasks to test their spatial and recognition memory. Cell death immediately after anaesthesia was quantified by Fluoro-Jade C staining in select brain regions, and developmental expression of the chloride transporters KCC2 and NKCC1 was analysed by immunoblotting in male and female rats at P4 and P7. Results Female rats exposed to Iso at P4 displayed impaired spatial, object-place, -context, and social recognition memory, and increased cell death in the hippocampus and laterodorsal thalamus. Female rats exposed at P7 exhibited only decreased performance in object-context compared with control. The ratio of NKCC1/KCC2 expression in cerebral cortex was higher in P4 females than in P7 females, and similar to that in P7 males. Conclusions Female rats exposed to Iso at P4 are sensitive to anaesthetic injury historically observed in P7 males. This is consistent with a comparably immature developmental state in P4 females and P7 males. The window of anaesthetic vulnerability correlates with sex-specific cortical expression of chloride transporters NKCC1 and KCC2. These findings suggest that both sex and developmental age play important roles in determining the outcome after early anaesthesia exposure.

Volatile anesthetics are used widely for achieving a state of unconsciousness, yet these agents are incompletely understood in their mechanisms of action and effects on neural development. There is mounting evidence that children exposed to anesthetic agents sustain lasting effects on learning and memory. The explanation for these behavioral changes remains elusive, although acute neuronal death after anesthesia is commonly believed to be a principal cause. Rodent models have shown that isoflurane exposure in newborns induces acute neuroapoptosis and long-term cognitive impairment. However, the assessment of predisposing factors is lacking. We investigated the role of sex by delivering isoflurane to postnatal day (P)7 male and female Sprague Dawley rats for 4 h. Brain cell death was assessed 12 h later using FluoroJade C staining in the thalamus, CA1–3 regions of hippocampus, and dentate gyrus. Behavior was assessed separately using a series of object recognition tasks and a test of social memory beginning at P38. We found that isoflurane exposure significantly increased neuronal death in each brain region with no difference between sexes. Behavioral outcome was also equivalent in simple novel object recognition. However, only males were impaired in the recognition of objects in different locations and contexts. Males also exhibited deficient social memory while females were intact. The profound behavioral impairment in males relative to females, in spite of comparable cell death, suggests that males are more susceptible to long-term cognitive effects and this outcome may not be exclusively attributed to neuronal death.

BackgroundWith growing evidence that anesthesia exposure in infancy affects cognitive development, it is important to understand how distinct anesthetic agents and combinations can alter long-term memory. Investigations of neuronal death suggest that combining anesthetic agents increases the extent of neuronal injury. However, it is unclear how the use of simultaneously combined anesthetics affects cognitive outcome relative to the use of a single agent.MethodsPostnatal day 7 (P7) male rats were administered either sevoflurane as a single agent or the combined delivery of sevoflurane with nitrous oxide at 1 Minimum Alveolar Concentration for 4 h. Behavior was assessed in adulthood using the forced alternating T-maze, social recognition, and context-specific object recognition tasks.ResultsAnimals exposed to either anesthetic were unimpaired in the forced alternating T-maze test and had intact social recognition. Subjects treated with the combined anesthetic displayed a deficit, however, in the object recognition task, while those treated with sevoflurane alone were unaffected.ConclusionA combined sevoflurane and nitrous oxide anesthetic led to a distinct behavioral outcome compared with sevoflurane alone, suggesting that the simultaneous use of multiple agents may uniquely influence early neural and cognitive development and potentially impacts associative memory.

Anesthesia in infancy impairs performance in recognition memory tasks in mammalian animals, but it is unknown if this occurs in humans. Successful recognition can be based on stimulus familiarity or recollection of event details. Several brain structures involved in recollection are affected by anesthesia-induced neurodegeneration in animals. Therefore, we hypothesized that anesthesia in infancy impairs recollection later in life in humans and rats. Twenty eight children ages 6-11 who had undergone a procedure requiring general anesthesia before age 1 were compared with 28 age- and gender-matched children who had not undergone anesthesia. Recollection and familiarity were assessed in an object recognition memory test using receiver operator characteristic analysis. In addition, IQ and Child Behavior Checklist scores were assessed. In parallel, thirty three 7-day-old rats were randomized to receive anesthesia or sham anesthesia. Over 10 months, recollection and familiarity were assessed using an odor recognition test. We found that anesthetized children had significantly lower recollection scores and were impaired at recollecting associative information compared with controls. Familiarity, IQ, and Child Behavior Checklist scores were not different between groups. In rats, anesthetized subjects had significantly lower recollection scores than controls while familiarity was unaffected. Rats that had undergone tissue injury during anesthesia had similar recollection indices as rats that had been anesthetized without tissue injury. These findings suggest that general anesthesia in infancy impairs recollection later in life in humans and rats. In rats, this effect is independent of underlying disease or tissue injury.

BackgroundIsoflurane exposure causes improvement in long-term neurocognitive function in young adult rats; this is associated with an increase in dentate gyrus (DG) progenitor proliferation 4 days after anesthesia. However, the number of new neurons that were born from cells that incorporated bromodeoxyuridine (BrdU) 4 days after anesthesia is not affected by anesthesia. We tested the hypothesis that progenitor proliferation continues to increase past 4 days, which would imply the possibility that the number of new neurons after anesthesia could be increased if BrdU labeling occurred at a later time point.MethodsBrdU was injected at 0, 1, 2, 4, 9, 16 days after 4 hours of isoflurane exposure to 60-day old rats. Brains were harvested 2 hours later, immunohistochemically stained, and the number of BrdU+ cells in the DG was assessed microscopically.ResultsAfter 4 hours of exposure to isoflurane in 60-day old rats, the number of BrdU+ cells decreased on days 0 to 2, then increased on day 4 significantly, and regressed toward the control level on days 9 and 16.ConclusionsAnesthesia-induced progenitor proliferation in the DG was not sustained 9 days after anesthesia. We interpret these results to signify that an anesthetic effect on neurogenesis likely does not play a critical role in the previously observed isoflurane-induced long-term improvement in neurocognitive function in 60-day old rats and that the transient increase in progenitor proliferation serves to replenish the pool of neural stem cells. The mechanism of anesthesia-induced improvement in cognition of young adult rats remains elusive.

Background Many anaesthetics when given to young animals cause cell death and learning deficits that persist until much later in life. Recent attempts to compare the relative safety or toxicity between different agents have not adequately controlled for the relative dose of anaesthetic given, thereby making direct comparisons difficult. Methods Isoflurane or sevoflurane were given at 1 minimum alveolar concentration (MAC) for 4 h to postnatal day 7 (P7) rat pups. Beginning at P75 these animals underwent fear conditioning and at P83 Morris water maze testing to assess working memory, short-term memory and early long-term memory using delays of 1 min, 1 h, and 4 h. Results No difference between groups was seen in fear conditioning experiments. Morris water maze learning was equivalent between groups, and no difference was seen in working memory. Sevoflurane-treated animals had a deficit in early long-term memory, and isoflurane-treated animals had a deficit in both short-term and early long-term memory. Conclusions Both isoflurane and sevoflurane delivered at 1 MAC for 4 h to immature rats caused a deficit in long-term memory. Isoflurane also caused a deficit in short-term memory. Isoflurane might be more detrimental than sevoflurane in very young animals.

Evidence suggests that diabetic ketoacidosis (DKA) may cause subtle cognitive alterations in children but the mechanisms are poorly understood. Acute DKA is associated with reactive astrogliosis and microglial activation in a rat model. Whether these inflammatory changes permanently alter brain histology is unknown. We aimed to determine whether DKA results in permanent alterations in brain histology and whether these changes are associated with cognitive deficits in a rat model. We induced diabetes in juvenile rats with streptozotocin at 4 weeks of age. We induced DKA in one group (n=21) at 5 weeks of age and compared this group to rats with diabetes without DKA episodes (n=13). Beginning at 7 weeks, rats underwent a series of cognitive tests to evaluate memory. At 15 weeks, rat brains were harvested and examined using immunohistochemistry (IHC). In tests of novel object recognition and social recognition, both groups performed similarly, however, the DKA group performed more poorly in object-place recognition tests, suggesting alterations in hippocampal function. IHC studies demonstrated increased glial fibrillary acidic protein staining intensity in the hippocampus of DKA rats suggesting astrogliosis, and decreased NeuN positive cell counts in the cortex suggesting neuron loss. These studies demonstrate that DKA results in permanent alterations in brain microstructure in a rat diabetes model. These structural changes are associated with deficits in hippocampal function.

Preclinical data suggest that general anaesthetics affect brain development. There is mixed evidence from cohort studies that young children exposed to anaesthesia can have an increased risk of poor neurodevelopmental outcome. We aimed to establish whether general anaesthesia in infancy has any effect on neurodevelopmental outcome. Here we report the secondary outcome of neurodevelopmental outcome at 2 years of age in the General Anaesthesia compared to Spinal anaesthesia (GAS) trial.In this international assessor-masked randomised controlled equivalence trial, we recruited infants younger than 60 weeks postmenstrual age, born at greater than 26 weeks' gestation, and who had inguinal herniorrhaphy, from 28 hospitals in Australia, Italy, the USA, the UK, Canada, the Netherlands, and New Zealand. Infants were randomly assigned (1:1) to receive either awake-regional anaesthesia or sevoflurane-based general anaesthesia. Web-based randomisation was done in blocks of two or four and stratified by site and gestational age at birth. Infants were excluded if they had existing risk factors for neurological injury. The primary outcome of the trial will be the Wechsler Preschool and Primary Scale of Intelligence Third Edition (WPPSI-III) Full Scale Intelligence Quotient score at age 5 years. The secondary outcome, reported here, is the composite cognitive score of the Bayley Scales of Infant and Toddler Development III, assessed at 2 years. The analysis was as per protocol adjusted for gestational age at birth. A difference in means of five points (1/3 SD) was predefined as the clinical equivalence margin. This trial is registered with ANZCTR, number ACTRN12606000441516 and ClinicalTrials.gov, number NCT00756600.Between Feb 9, 2007, and Jan 31, 2013, 363 infants were randomly assigned to receive awake-regional anaesthesia and 359 to general anaesthesia. Outcome data were available for 238 children in the awake-regional group and 294 in the general anaesthesia group. In the as-per-protocol analysis, the cognitive composite score (mean [SD]) was 98.6 (14.2) in the awake-regional group and 98.2 (14.7) in the general anaesthesia group. There was equivalence in mean between groups (awake-regional minus general anaesthesia 0.169, 95% CI -2.30 to 2.64). The median duration of anaesthesia in the general anaesthesia group was 54 min.For this secondary outcome, we found no evidence that just less than 1 h of sevoflurane anaesthesia in infancy increases the risk of adverse neurodevelopmental outcome at 2 years of age compared with awake-regional anaesthesia.Australia National Health and Medical Research Council (NHMRC), Health Technologies Assessment-National Institute for Health Research UK, National Institutes of Health, Food and Drug Administration, Australian and New Zealand College of Anaesthetists, Murdoch Childrens Research Institute, Canadian Institute of Health Research, Canadian Anesthesiologists' Society, Pfizer Canada, Italian Ministry of Heath, Fonds NutsOhra, and UK Clinical Research Network (UKCRN).

During the Fifth Pediatric Anesthesia Neurodevelopmental Assessment (PANDA) Symposium, experts and stakeholders met to present and discuss recent advances made in the study of neurodevelopmental outcomes following exposure to anesthetic drugs in infants and children. This article summarizes the update of five ongoing clinical studies: General Anesthesia compared to Spinal Anesthesia (GAS), Toxicity of Remifentanil and Dexmedetomidine (T-Rex), Mayo Anesthesia Safety in Kids (MASK), the UCSF (University of California San Francisco) human cohort study, and Columbia University Medical Center (CUMC) Neonatal Magnetic Resonance Imaging (MRI) study. The purpose of this summary is to discuss the contributions and limitations of these studies, how they fit into the published literature, and what questions remain to be answered.

Background Anesthesia given to immature rodents causes cognitive decline, raising the possibility that the same might be true for millions of children undergoing surgical procedures under general anesthesia each year. We tested the hypothesis that anesthesia-induced cognitive decline in rats is treatable. We also tested if anesthesia-induced cognitive decline is aggravated by tissue injury. Methods Seven-day old rats underwent sevoflurane anesthesia (1 minimum alveolar concentration, 4 h) with or without tail clamping. At 4 weeks, rats were randomized to environmental enrichment or normal housing. At 8 weeks rats underwent neurocognitive testing, which consisted of fear conditioning, spatial reference memory, and water maze-based memory consolidation tests, and interrogated working memory, short-term memory, and early long-term memory. Results Sevoflurane-treated rats had a greater escape latency when the delay between memory acquisition and memory retrieval was increased from 1 min to 1 h, indicating that short-term memory was impaired. Delayed environmental enrichment reversed the effects of sevoflurane on short-term memory and generally improved many tested aspects of cognitive function, both in sevoflurane-treated and control animals. The performance of tail-clamped rats did not differ from those rats receiving anesthesia alone. Conclusion Sevoflurane-induced cognitive decline in rats is treatable. Delayed environmental enrichment rescued the sevoflurane-induced impairment in short-term memory. Tissue injury did not worsen the anesthesia-induced memory impairment. These findings may have relevance to neonatal and pediatric anesthesia.

The hippocampus is often injured in neonatal stroke. We have investigated the effect of erythropoietin (EPO) on oxygen-glucose deprived hippocampal slices and hypoxic progenitor cells. EPO improved survival of the organotypic hippocampal slices with significantly less cell death in the dentate gyrus and an increased number of proliferating cells 4-5 days after insult. Significantly fewer markers of neurogenesis were seen after the insult but when EPO was added to the culture medium, neurogenesis was sustained. When hippocampal progenitor cultures were stimulated into differentiation, more cells chose a neuronal cell fate when treated with EPO. These findings support the hypothesis that EPO not only prevents ischemia induced cell death but promotes neuronal cell fate committment in in-vitro models of neonatal stroke.

Background Roughly, 10% of elderly patients develop postoperative cognitive dysfunction. General anesthesia impairs spatial memory in aged rats, but the mechanism is not known. Hippocampal neurogenesis affects spatial learning and memory in rats, and isoflurane affects neurogenesis in neonatal and young adult rats. We tested the hypothesis that isoflurane impairs neurogenesis and hippocampal function in aged rats. Methods Isoflurane was administered to 16-month-old rats at one minimum alveolar concentration for 4 h. FluoroJade staining was performed to assess brain cell death 16 h after isoflurane administration. Dentate gyrus progenitor proliferation was assessed by bromodeoxyuridine injection 4 days after anesthesia and quantification of bromodeoxyuridine+ cells 12 h later. Neuronal differentiation was studied by determining colocalization of bromodeoxyuridine with the immature neuronal marker NeuroD 5 days after anesthesia. New neuronal survival was assessed by quantifying cells coexpressing bromodeoxyuridine and the mature neuronal marker NeuN 5 weeks after anesthesia. Four months after anesthesia, associative learning was assessed by fear conditioning. Spatial reference memory acquisition and retention was tested in the Morris Water Maze. Results Cell death was sporadic and not different between groups. We did not detect any differences in hippocampal progenitor proliferation, neuronal differentiation, new neuronal survival, or in any of the tests of long-term hippocampal function. Conclusion In aged rats, isoflurane does not affect brain cell death, hippocampal neurogenesis, or long-term neurocognitive outcome.

Anesthetic drugs cause brain cell death and long-term neurocognitive dysfunction in neonatal rats. Recently, human data also suggest that anesthesia early in life may cause cognitive impairment. The connection between cell death and neurocognitive decline is uncertain. It is conceivable that mechanisms other than brain cell death contribute to neurocognitive outcome of neonatal anesthesia. In a series of experiments, we demonstrate that isoflurane exposure causes significant hypercarbia in postnatal day 7 rats and that exposure to isoflurane or carbon dioxide for 4 h provoked brain cell death. However, 1 h of isoflurane exposure was not sufficient to cause brain cell death. Moreover, only 4 h of isoflurane exposure, but not 1 or 2 h of exposure or 4 h of carbon dioxide, led to impaired hippocampal function,questioning the association between anesthesia-induced brain cell death and neurocognitive dysfunction. Neurogenesis both in the developing and adult dentate gyrus is important for hippocampal function, specifically learning and memory. γ-Amino-butyric-acid regulates proliferation and neuronal differentiation both in the developing and the adult brain. Inhaled anesthetics are γ-amino-butyric-acid-ergic and may therefore affect neurogenesis, which could be an alternative mechanism mediating anesthesia-induced neurocognitive decline in immature rats. Understanding the mechanism will help guide clinical trials aiming to define the scope of the problem in humans and may lead to preventive and therapeutic strategies.

Background Anesthetic exposure early in life affects neural development and long-term cognitive function, but our understanding of the types of memory that are altered is incomplete. Specific cognitive tests in rodents that isolate different memory processes provide a useful approach for gaining insight into this issue. Methods Postnatal day 7 (P7) rats were exposed to either desflurane or isoflurane at 1 Minimum Alveolar Concentration for 4 h. Acute neuronal death was assessed 12 h later in the thalamus, CA1-3 regions of hippocampus, and dentate gyrus. In separate behavioral experiments, beginning at P48, subjects were evaluated in a series of object recognition tests relying on associative learning, as well as social recognition. Results Exposure to either anesthetic led to a significant increase in neuroapoptosis in each brain region. The extent of neuronal death did not differ between groups. Subjects were unaffected in simple tasks of novel object and object-location recognition. However, anesthetized animals from both groups were impaired in allocentric object-location memory and a more complex task requiring subjects to associate an object with its location and contextual setting. Isoflurane exposure led to additional impairment in object-context association and social memory. Conclusion Isoflurane and desflurane exposure during development result in deficits in tasks relying on associative learning and recognition memory. Isoflurane may potentially cause worse impairment than desflurane.

BACKGROUND:: Carbon monoxide poisoning is a significant problem in most countries, and a reliable method of quick diagnosis would greatly improve patient care. Until the recent introduction of a multiwavelength "pulse CO-oximeter" (Masimo Rainbow SET Radical-7), obtaining carboxyhemoglobin (COHb) levels in blood required blood sampling and laboratory analysis. In this study, we sought to determine whether hypoxemia, which can accompany carbon monoxide poisoning, interferes with the accurate detection of COHb. METHODS:: Twelve healthy, nonsmoking, adult volunteers were fitted with 2 standard pulse-oximeter finger probes and 2 Rainbow probes for COHb detection. A radial arterial catheter was placed for blood sampling during 3 interventions: (1) increasing hypoxemia in incremental steps with arterial oxygen saturations (SaO2) of 100% to 80%; (2) normoxia with incremental increases in %COHb to 12%; and (3) elevated COHb combined with hypoxemia with SaO2 of 100% to 80%. Pulse-oximeter (SpCO) readings were compared with simultaneous arterial blood values at the various increments of hypoxemia and carboxyhemoglobinemia (≈25 samples per subject). Pulse CO-oximeter performance was analyzed by calculating the mean bias (SpCO - %COHb), standard deviation of the bias (precision), and the root-mean-square error (Arms). RESULTS:: The Radical-7 accurately detected hypoxemia with both normal and elevated levels of COHb (bias mean ± SD: 0.44% ± 1.69% at %COHb

Pulse oximetry is a standard of care during anaesthesia in high-income countries. However, 70% of operating environments in low- and middle-income countries have no pulse oximeter. The 'Lifebox' oximetry project set out to bridge this gap with an inexpensive oximeter meeting CE (European Conformity) and ISO (International Organization for Standardization) standards. To date, there are no performance-specific accuracy data on this instrument. The aim of this study was to establish whether the Lifebox pulse oximeter provides clinically reliable haemoglobin oxygen saturation (Sp O2 ) readings meeting USA Food and Drug Administration 510(k) standards. Using healthy volunteers, inspired oxygen fraction was adjusted to produce arterial haemoglobin oxygen saturation (Sa O2 ) readings between 71% and 100% measured with a multi-wavelength oximeter. Lifebox accuracy was expressed using bias (Sp O2 - Sa O2 ), precision (SD of the bias) and the root mean square error (Arms). Simultaneous readings of Sa O2 and Sp O2 in 57 subjects showed a mean (SD) bias of -0.41% (2.28%) and Arms 2.31%. The Lifebox pulse oximeter meets current USA Food and Drug Administration standards for accuracy, thus representing an inexpensive solution for patient monitoring without compromising standards.

Propofol has been reported to have high stability in glass and relatively high stability up to 24 hours in polyvinyl chloride-based medical plastics. Recent publications have observed the effects of propofol on cells and tissues grown in culture. Many cell culture plastics are formulated from polystyrene but we could find little information on the stability of propofol exposed to these products. We observed very little change in the concentration of propofol diluted in cell culture medium over 24 hours when exposed to glass, but substantial loss of the drug when exposed to 96-well polystyrene cell culture plates. This decrease was most rapid in the first hour but continued until 24 hours. The type of plastic used in cell and tissue culture experiments with propofol may influence the results by increasing the apparent dose required to see an effect.

Genetic lines of the cricket Gryllus assimilis selected for elevated vs. decreased activity of hemolymph juvenile hormone esterase (JHE) exhibited a 6.8-fold difference in enzyme activity by the seventh generation of selection. This documents that hemolymph JHE activity has the capability for rapid evolutionary change. This is the only insect endocrine trait for which such data are currently available. The difference in hemolymph JHE activity between the lines was due, to an equivalent degree, to variation in whole-cricket enzyme activity and allocation of JHE activity to the hemolymph compartment. No differences in kinetic or thermostability characteristics were observed between JHEs from the high- vs. low-selected lines. This suggests that the variation in JHE activity between the lines resulted from selection of variable regulatory genetic factors affecting the synthesis, degradation, or activation of enzyme activity. JHE and JHbinding activity in the hemolymph are genetically correlated, possibly due to co-regulation by the same genetic factors. Finally, in vivo JH degradation was significantly elevated in lines selected for elevated JHE activity. This documents that evolutionary changes in JHE activity alter in vivo JH metabolism.

Background Propofol in the early postnatal period has been shown to cause brain cell death. One proposed mechanism for cognitive dysfunction after anesthesia is alteration of neural stem cell function and neurogenesis. We examined the effect of propofol on neural precursor or stem cells (NPCs) grown in vitro. Methods Hippocampal-derived NPCs from postnatal day 2 rats were exposed to propofol or Diprivan. NPCs were then analyzed for bromodeoxyuridine incorporation to measure proliferation. Cell death was measured by lactate dehydrogenase release. Immunocytochemistry was used to evaluate the expression of neuronal and glial markers in differentiating NPCs exposed to propofol. Results Propofol dose dependently increases the release of lactate dehydrogenase from NPCs under both proliferating and differentiating conditions at supraclinical concentrations (more than 7.1 µM). Both Diprivan and propofol had the same effect on NPCs. Propofol-mediated release of lactate dehydrogenase is not inhibited by blocking the γ-aminobutyric acid type A receptor or extracellular calcium influx and is not mediated by caspase-3/7. Direct γ-aminobutyric acid type A receptor activation did not have the same effect. In differentiating NPCs, 6 h of propofol at 2.1 µM increased the number neurons but not glial cells 4 days later. Increased neuronal differentiation was not blocked by bicuculline. Conclusions Only supraclinical concentrations of propofol or Diprivan kill NPCs in culture by a non-γ-aminobutyric acid type A, noncaspase-3 mechanism. Clinically relevant doses of propofol increase neuronal fate choice by a non-γ-aminobutyric acid type A mechanism.

Background While studying neurotoxicity in rats, we observed that the anesthetic minimum alveolar anesthetic concentration (MAC) of isoflurane decreases with increasing duration of anesthesia in 7-day-old but not in 60-day-old rats. After 15 min of anesthesia in 7-day-old rats, MAC was 3.5% compared with 1.3% at 4 h. We investigated whether kinetic or dynamic factors mediated this decrease. Methods In 7-day-old rats, we measured inspired and cerebral partial pressures of isoflurane at MAC as a function of duration of anesthesia. In 60-day-old rats, we measured inspired partial pressures of isoflurane at MAC as a function of duration of anesthesia. Finally, we determined the effect of administering 1 mg/kg naloxone and of delaying the initiation of the MAC determination (pinching the tail) on MAC in 7-day-old rats. Results In 7-day-old rats, both inspired and cerebral measures of MAC decreased from 1 to 4 h. The inspired MAC decreased 56%, whereas the cerebral MAC decreased 33%. At 4 h, the inspired MAC approximated the cerebral MAC (i.e., the partial pressures did not differ appreciably). Neither administration of 1 mg/kg naloxone nor delaying tail clamping until 3 h reversed the decrease in MAC. In 60-day-old rats, inspired MAC of isoflurane was stable from 1 to 4 h of anesthesia. Conclusions MAC of isoflurane decreases over 1-4 h of anesthesia in 7-day-old but not in 60-day-old rats. Both pharmacodynamic and a pharmacokinetic components contribute to the decrease in MAC in 7-day-old rats. Neither endorphins nor sensory desensitization mediate the pharmacodynamic component.

Background: Isoflurane causes long-term hippocampal-dependent learning deficits in rats despite limited isoflurane-induced hippocampal cell death, raising questions about the causality between isoflurane-induced cell death and isoflurane-induced cognitive function. Neurogenesis in the dentate gyrus is required for hippocampal-dependent learning and thus constitutes a potential alternative mechanism by which cognition can be altered after neonatal anesthesia. The authors tested the hypothesis that isoflurane alters proliferation and differentiation of hippocampal neural progenitor cells. Methods: Multipotent neural progenitor cells were isolated from pooled rat hippocampi (postnatal day 2) and grown in culture. These cells were exposed to isoflurane and evaluated for cell death using lactate dehydrogenase release, caspase activity, and immunocytochemistry for nuclear localization of cleaved caspase 3. Growth was assessed by cell counting and BrdU incorporation. Expression of markers of stemness (Sox2) and cell division (Ki67) were determined by quantitative polymerase chain reaction. Cell fate selection was assessed using immunocytochemistry to stain for neuronal and glial markers. Results: Isoflurane did not change lactate dehydrogenase release, activity of caspase 3/7, or the amount of nuclear cleaved caspase 3. Isoflurane decreased caspase 9 activity, inhibited proliferation, and decreased the proportion of cells in s-phase. messenger ribonucleic acid expression of Sox2 (stem cells) and Ki67 (proliferation) were decreased. Differentiating neural progenitor cells more often select a neuronal fate after isoflurane exposure. Conclusions: The authors conclude that isoflurane does not cause cell death, but it does act directly on neural progenitor cells independently of effects on the surrounding brain to decrease proliferation and increase neuronal fate selection. These changes could adversely affect cognition after isoflurane anesthesia.

Background: Anesthetic agents cause cell death in the developing rodent brain and long-term, mostly hippocampal-dependent, neurocognitive dysfunction. However, a causal link between thesefindings has not been shown. Postnatal hippocampal neurogenesis affects hippocampal function into adulthood; therefore, the authors tested the hypothesis that isoflurane affects long-term neurocognitive function via an effect on dentate gyrus neurogenesis. Methods: The S-phase marker 5-bromodeoxyuridine was administered at various times before, during, and afte r4ho f isoflurane given to postnatal day (P)60 and P7 rats to assess dentate gyrus progenitor proliferation, early neuronal lineage selection, and long-term survival of new granule cell neurons. Fear conditioning and spatial reference memory was tested at various intervals from 2 weeks until 8 months after anesthesia. Results: In P60 rats, isoflurane increased early neuronal differentiation as assessed by BrdU/NeuroD costaining, decreased progenitor proliferation for 1 day, and subsequently increased progenitor proliferation 5‐10 days after anesthesia. In P7 rats, isoflurane did not induce neuronal lineage selection but decreased progenitor proliferation until at least 5 days after anesthesia. Isoflurane improved spatial reference memory of P60 rats long-term, but it caused a delayed-onset, progressive, persistent hippocampal deficit in P7 rats in fear conditioning and spatial reference memory tasks. Conclusion: The authors conclude that isoflurane differentially affects both neurogenesis and long-term neurocognitive function in P60 and P7 rats. Neurogenesis might mediate the long-term neurocognitive outcome after isoflurane at different ages.

Background: Millions of neonates undergo anesthesia each year. Certain anesthetic agents cause brain cell death and longterm neurocognitive dysfunction in postnatal day (P)7 rats. Despite its intuitive appeal, a causal link between cell death and neurocognitive decline after anesthesia has not been established. If one existed, the degree of cell death would be expected to correlate with the degree of neurocognitive dysfunction caused by anesthesia. The authors therefore tested if cell death caused by various durations of isoflurane at 1 minimum alveolar concentration causes duration-dependent long-term neurocognitive dysfunction. Methods: Isoflurane was administered to P7 rats at 1 minimum alveolar concentration for 0, 1, 2, or 4 h. To control for the respiratory depressant effects of anesthesia, a group of rats was treated wit h4ho fcarbon dioxide. Cell death was assessed by FluoroJade staining 12 h after the end of each intervention, and neurocognitive outcome was assessed 8 weeks later by using fear conditioning, spatial reference memory, and spatial working memory tasks. Results: Widespread brain cell death was caused b y2ha nd 4 h of isoflurane and b y4ho fcarbon dioxide. The degree and distribution of thalamic cell death was similar in 4 h isofluranetreated and 4-h carbon dioxide‐treated rats. Onl y4ho fisoflurane caused a long-term neurocognitive deficit affecting both spatial reference memory and spatial working memory. Working memory was improved in carbon dioxide‐treated rats. Conclusion: Isoflurane-induced brain cell death may be partly caused by hypercarbia. The inconsistencies between cell death and neurocognitive outcome suggest that additional or alternative mechanisms may mediate anesthesia-induced longterm neurocognitive dysfunction.

Neovascularization stimulated by IGF-1 mediated induction of vascular endothelial growth factor (VEGF) is one of the leading causes of blindness in humans. It plays a central role in the pathogenesis of proliferative diabetic retinopathy (DR), neovascular glaucoma, exudative age-related macular degeneration (AMD) and retinopathy of prematurity. Neovascularization is a multi-step process that involves complex interactions of a variety of mitogenic factors such as VEGF and IGF-I which are produced locally in the human eye by a variety of cells including retinal pigment epithelial (RPE) cells, retinal capillary pericytes, endothelial cells, Mueller cells and ganglion cells. We hypothesized that somatostatin would inhibit the IGF-1 signal transduction pathway in RPE cells, resulting in decreased VEGF production. We have observed expression of somatostatin receptor protein in retinal pigment epithelial (RPE) cells of the human eye using immunohistochemistry and have confirmed expression of somatostatin receptors in cultured human RPE cells using reverse transcriptase-PCR. IGF-1 induced a dose dependent increase in IGF-1R phosphorylation and in VEGF mRNA levels in cultured human RPE cells. Somatostatin and octreotide, a somatostatin analogue, inhibited IGF-1 receptor (IGF-1R) phosphorylation and decreased VEGF production. Both IGF-1R phosphorylation and accumulation of VEGF mRNA were inhibited by physiological levels of somatostatin and octreotide (1 nM). These results demonstrate somatostatin and octreotide mediated attenuation of both IGF-1R signal transduction and VEGF mRNA accumulation via somatostatin receptor type 2 (sst2). Furthermore, these data suggest a rationale for the use of octreotide as a prophylactic and therapeutic option in disease states that cause ocular neovascularization.