ANESTHESIA kills neonatal brain cells of several animal species, including primates.1A combination of γ-aminobutyric-acid (GABA)–ergic and N -methyl-d-aspartate antagonist agents is particularly neurotoxic; however propofol, isoflurane, ketamine, and midazolam have all caused apoptosis individually.1We have shown that 4 h of hypercapnia, which caused a similar degree and distribution of cell death as 4 h of isoflurane, did not cause a neurocognitive deficit.2Likewise, 2 h of isoflurane caused cell death in many areas of the brain relevant to the behavioral outcome tested, but no neurocognitive deficit was observed.2The only intervention that caused both cell death and neurocognitive dysfunction was 4 h of isoflurane. This raises suspicion about whether anesthesia-related brain cell death in postnatal day (P)7 rats can really account for the cognitive deficit observed after anesthesia.2–5The developing brain has a high degree of plasticity. In P7 rats, cell birth is a very common event. Additional division(s) of progenitors in the P7 rat brain should be able to compensate for the loss of cells due to anesthesia-induced cell death unless anesthesia also impairs neurogenesis. Neurogenesis occurs during development and persists in the subventricular zone and the hippocampal dentate gyrus (DG) in the adult brain, where it is important for learning and memory.6–10Neurogenesis in the subventricular zone, and the DG has been shown to increase when the brain loses neurons, such as in stroke (for review see Sharp et al. 11). This begs the question of whether isoflurane-induced brain cell death might also increase neurogenesis. Neurogenesis is the composite outcome of progenitor proliferation, neuronal differentiation, migration, and survival through activity-dependent integration into existing neural networks. We hypothesized that isoflurane, in addition to causing cell death in the developing rat brain, impairs one or more events required for DG neurogenesis such as progenitor proliferation, neuronal differentiation, and new neuronal survival.Curiously, young adult rats do not develop neurocognitive decline after anesthesia. In fact, Culley et al. 12have shown an unexplained improvement in neurocognitive function of young adult rats after anesthesia. Neurogenesis occurs both during development and in the adult brain and can by itself affect cognitive outcome6–10; we therefore hypothesized that anesthesia enhances some aspect of neurogenesis and improves neurocognitive outcome in young adult rats.Here we show that 4 h of isoflurane does not cause cell death in P60 rats and increases neuronal differentiation, causing a temporary decrease and subsequent compensatory increase in progenitor proliferation followed by an improvement in neurocognitive function 8 weeks later. In P7 rats on the other hand, isoflurane decreases progenitor proliferation without increasing neuronal differentiation and causes a neurocognitive deficit that is progressive and persistent.All experiments were approved by the Institutional Animal Care and Use Committee of the University California, San Francisco, California. Male P60 rats (n = 40) and P7 rats (n = 141) were anesthetized in groups of 10–20 at 1 minimum alveolar concentration (MAC) as determined by tail clamping. MAC was initially assumed to be 3.5% for P7 and 2.4% for P60 rats. A supramaximal pain stimulus was generated by application of an alligator clamp to each rat's tail for 30 s or until the rat moved. Movement was defined as any movement except breathing. Tail clamping was repeated every 15 min, starting 15 min after induction of general anesthesia. The anesthetic concentration estimated as MAC and alterations in the delivered concentration were obtained from the determined concentration and the empirically derived algorithm in table 1(see legend to table 1). The algorithm takes into account the tendency for anesthetic requirements to decrease over time in P7 rats. Custom-made temperature probes were inserted subcutaneously over the skull to facilitate control of temperature at 36.5 ± 1°C using computer-controlled Peltier heater/cooler plates integrated into the floor of the anesthetizing chamber. Hemoglobin oxygen saturation and heart rate were measured by application of a rodent transreflectance sensor (Nonin 2000T; Nonin Medical, Minneapolis, MN) to the ventral thoracic chest wall. The probe was coupled to a Nonin V8600 pulse oximeter (Nonin Medical). At various intervals throughout the anesthetic, blood (0.25 ml) was withdrawn either blindly through a 30-gauge needle from the heart of designated homeostatic P7 rats or from a P10 polyethylene catheter inserted into the tail artery of P60 cardiorespiratory control rats (n = 6). PH, arterial oxygen, and carbon dioxide tensions, base excess, blood hemoglobin, and blood glucose was analyzed by a blood gas analyzer (ABL 520; Radiometer, Copenhagen, Denmark). Although a total of 4 blood draws were performed in P60 rats, blood was withdrawn from only 1 rat per timepoint in P7 rats.Control rats were placed in the anesthesia glove box for 4 h without exposure to anesthetic agent but otherwise identical conditions as animals in the isoflurane group.Bromodeoxyuridine (BrdU; Sigma, St. Louis, MO; 15 mg/ml) was injected intraperitoneally as listed in table 2to separately target the processes of progenitor proliferation, neuronal differentiation, and new neuronal survival. We were specifically interested in how isoflurane affects proliferation of progenitors that are in the S-phase of the cell cycle during the time of the anesthetic (labeling during anesthesia), how it affects proliferation and early lineage selection of progenitors that were labeled just before 4 h of anesthesia, how isoflurane affects progenitor proliferation 4 days after anesthesia, and if survival of these progenitor populations differently labeled with respect to the anesthetic is affected.Animals were deeply anesthetized with isoflurane and then transcardially perfused with 0.9% saline followed by 4% paraformaldehyde in 0.1 m phosphate buffered saline (PBS), pH 7.4. The brains were removed, postfixed overnight in 4% paraformaldehyde/PBS, and placed in 20% sucrose until sunk. Coronal sections (40 μm) were cut on a microtome and stored in PBS. For immunocytochemical detection of BrdU-labeled nuclei, DNA was denatured to expose the antigen. Incubation with 50% formamide in PBS for 2 h at 65°C preceded 2-normal hydrochloric acid incubation for 30 min at 37°C and neutralization with 0.5 m boric acid pH 8.5 for 10 min at room temperature (RT) and 3 washes with PBS for 10 min in between each of these steps. Blocking of nonspecific epitopes with 3% serum and 0.1% bovine serum albumin in PBS with 0.3% Triton-X (Fisher Scientific, Pittsburg, PA) for 30 min at RT preceded incubation overnight at 4°C with the primary antibody listed in table 3in PBS and bovine serum albumin 0.2%.On day 2 of the single BrdU stain, incubation with a biotinylated antibody (sheep anti-mouse IgG, 1:200; Amersham [GE Healthcare], Piscattaway, NJ) for 2 h at RT was performed. Streptavidin-biotin treatment for 2 h at RT (Vectastain, ABC kit, Catalogue # PK 6100; Vector Laboratories, Burlingame, CA) was followed by three thorough washes to eliminate residual peroxidase activity. This was followed by incubation with diaminobenzidine + urea (Fast DAB tablets, Sigma) with nickel chloride augmentation (7.5 μl of 8% stock in 25 ml) for 5 min followed by mounting and coverslipping with Depex mounting medium (Electronmicroscopy Sciences, Fort Washington, PA).Double immunofluorescent stains were performed using an identical protocol for day 1 as the above single stain except for incubation with the Serotec (Serotec, Raleigh, NC) anti-BrdU antibody (table 2). On day 2, incubation with the appropriate secondary fluorescent antibodies Alexafluor 488 and Alexafluor 594 (1:500; Invitrogen, Carlsbad, CA) occurred for 2 h at RT. Nuclear counterstaining was performed with 4′,6-diamidino-2-phenylindole (DAPI, 1:1000; Sigma) followed by mounting and coverslipping with an acqueous mounting medium.Slices of P60 rat brain tissue were mounted on glass slides and covered in 100% ethanol for 3 min, then 70% ethanol for 1 min, and then deionized water (dH2O) for 1 min. Each slide contained one positive control slice of brain tissue from a P60 rat treated with 10 mg/kg kainic acid intraperioneally. Tissue was incubated with 0.06% potassium permanganate for 20 min on a shaker at RT before a 1-min wash with dH2O. Slides were then incubated with FluoroJade staining solution (4 ml of 0.01% FluoroJade [in dH2O] stock solution, 36 ml of 0.1% acetic acid in dH2O, 40 μl DAPI) for 30 min at RT before 3 washes with dH2O for 1 min each. After air-drying, slides were briefly rinsed with xylene and coverslipped with Depex mounting solution. All areas of the brain were inspected for FluoroJade staining in every twelfth section.Single-stained BrdU+ cells were detected using brightfield microscopy with a 4×, 20×, and 100× objective lenses if required to distinguish cells within tightly packed cell conglomerates. The granule cell layer and the subgranular zone of each DG was traced using StereoInvestigator® software (MicroBrightField, Williston, VT). An observer blinded to group assignment determined the number of BrdU+ cells per DG in every twelfth slice for each animal.Using double immunofluorescent staining, the proportion of BrdU+ cells that coexpressed the immature neuronal marker NeuroD or the mature neuronal marker NeuN was assessed by an observer blinded to group assignment. Colocalization was confirmed using image stacks on a laser scanning confocal microscope (Zeiss LSM 510, 63× NA 1.3; Carl Zeiss, Peabody, MA) or a grid confocal microscope (Optigrid; Improvision, Waltham, MA) using a Volocity® image acquisition suite (Improvision). The number of new neurons in the DG cell layer was assessed in every twelfth slice.Four rats, counterbalanced for group assignment, were trained at a time. The chambers (length, 32 cm; width, 25 cm; height, 25 cm) were constructed of clear acrylic. The grid floor used to deliver was of each 4 in and to were to a St. VT). The chambers were with a before and after each The room in which was with fluorescent and a The and of the chambers and room the a in the rats 3 2 by 1 min. the of all movement except that for is an in and a of was every 8 s during the and a was using the 100× where is the number of events per rat and is the total number of per rats were for to the and to For the each rat was placed in the in which it was trained for a of 8 min the of and For the groups of rats were in cm; cm) to a in a The chambers were in with an floor (length, cm; width, 25 cm) and (length, cm; width, cm) at a The chambers were with a and were with acetic acid Scientific, St. Louis, before and after each The room to the rats, by a single A of was used for were a followed by three by were from the after an 30 The of the and was such that of each treatment group was to and and was by blinded to group assignment during the the and both of rats a total of three of in the water was performed on animals (n = after a (n = = 8 was anesthetized and in the water to the timepoint at which a neurocognitive deficit is In the water the of rats to a in a in with water was The rats were trained to a and then to a In a subsequent the was per day for Each of three with a The was to be over one of in the of each The the for each rats. The animals were placed in the at one of to The various in a and for each The between the was 5 the rats the were to on it for 20 to the and were with an to 10 per for in the to the during the we as a primary outcome the to the the probe the proportion of time in the target each of the other was determined separately for each of three were as and except blood glucose and survival A was performed to were analyzed by using NC) each of a was used from which were starting with and then all in the were or required to be in for the was a which and a for were used to the and was used to for were analyzed by using the was a group of animals was required to a between of with an at a of the water the required to a between groups at and a of was animals per A group of was to a of For a water in the P60 the required to a in to of with an of 10 as observed in the with at a of was animals per animals were per group to a of NC) was used for all P60 rats, the MAC of isoflurane a of after min. MAC of isoflurane in P7 rats, on the other hand, from 3.5% isoflurane at 15 min to at 4 h or occurred during anesthesia in either and in P7 but not in P60 rats after induction of general anesthesia, with a arterial carbon dioxide of at 1 h and a decrease to at 4 P60 rat during the anesthetic, and the in the P7 group was was during the of anesthesia. The of death is of these occurred during anesthesia. The is a control group of P7 rats by of carbon dioxide no were to by of 1 mg/kg at 3 h not or ml of saline with or without 1 of at 2 h of anesthesia not or by tail clamping until the not at with a throughout the anesthetic and did not with arterial not In P60 cardiorespiratory control the blood concentration to 10 by the blood the of anesthesia. In P7 rats, the blood concentration from a of = just after induction of general anesthesia to a of = at 4 h of anesthesia. to the blood gas in P60 rats, the rate of P60 rats was not The rate of P7 rats from to 20 from after induction of general anesthesia and until 3 h of anesthesia, when it to at h of P60 rats, were any cells in the brain of anesthetized or was cell death in both anesthetized and P7 the of which is in in an in of BrdU was injected 4 h before anesthesia in P60 rats, the proportion of BrdU+ cells the early neuronal marker NeuroD at the of the anesthetic was to a of in animals the protocol in P7 rats, the proportion of BrdU+ cells NeuroD was at the of 4 h of isoflurane anesthesia isoflurane The proportion of BrdU+ cells NeuroD was in anesthetized P60 in anesthetized P7 rats differentiation of progenitor cells cell cycle and isoflurane neuronal differentiation only in P60 isoflurane should decrease progenitor proliferation in P60 but not in P7 rats. We that BrdU during anesthesia the number of BrdU+ cells per DG in both groups by and to a of in P60 and in P60 rats and in P7 in P7 rats a control mg/kg BrdU was injected 4 h before anesthesia in both the of of BrdU is 4 number of BrdU+ cells after anesthesia should not be we that isoflurane no on the number of cells labeled with BrdU in P60 control isoflurane or P7 rats control isoflurane if the isoflurane-induced decrease in progenitor proliferation observed in both groups over we labeled progenitors with BrdU 4 days after anesthesia. after the of BrdU on day 4 after anesthesia, the number of BrdU+ cells was in the P60 animals to P60 by In P7 rats, the number of BrdU+ cells was by at the time to of isoflurane on the number of new granule cell neurons was assessed 4 weeks after BrdU before anesthesia or on the day after anesthesia in both the increase in the proportion of BrdU+ cells that assumed a neuronal lineage in P60 rats the decrease in progenitor proliferation in P7 rats in a in the number of BrdU+ cells the mature neuronal marker NeuN days not At time of BrdU+ cells in coexpressed the mature neuronal marker NeuN in the isoflurane not if the in progenitor proliferation in P60 rats to new granule cell neurons, BrdU was injected on days after anesthesia. days after the the proportion of BrdU+ cells NeuN was in P60 animals in the total number of new granule cells at time was not between P60 and control animals was observed in 4 after 4 h of isoflurane to P60 rats (n = to (n = not After 4 h of isoflurane at was 15 and days after anesthesia and in a of rats at 5 after anesthesia. deficit was at the early time A and but the of rats were of at 5 in the but not the of the rats that 4 h of isoflurane on P60 were in the water the isoflurane group a in the and a proportion of time for the in the target the isoflurane group and the control group at a but rate when was in the water on postnatal days after 4 h of isoflurane on P7 a total of of for learning to the be from the was no between groups in of for which the is or the probe for which the is from the and The animals were weeks after the anesthetic, at which was was no in to the in or between groups rats that isoflurane at days of time for the in the target during the probe a of animals was 8 after of 4 h of isoflurane on was in the animals probe was not of is an deficit of but not learning and in P7 rats. In isoflurane in P60 rats causes a improvement in In P7 rats, isoflurane caused a decrease in progenitor proliferation for at 4 and it neuronal differentiation in P60 in the DG of the for in animals and (for see et al. 6). The of a in the subgranular zone is of a of The by neurogenesis the DG to new neurons that it to a DG and DG neurogenesis is in in neurogenesis hippocampal learning (for review see of neurogenesis impairs hippocampal progenitors are to the of by et al. and brain causes hippocampal dysfunction in brain causes a progressive hippocampal on the at treatment and of it can also affect general to a degree by between decrease in neurogenesis and the cognitive decline in has not been the deficit caused by 4 h of isoflurane in P7 rats as progressive in in that we were to a deficit until weeks after anesthesia, but we it from that on in both the and a deficit is initially and can be by increases of 5 the as used in the hippocampal This is with that is not to in progenitor between the and in these were 4 weeks and and it is that the deficit in a is not at with we did not any on 8 weeks after 4 h of isoflurane, when and were in rats that neonatal isoflurane deficit observed to but not 5 after the does which was weeks and 8 after isoflurane, an other brain which was by isoflurane, does not the neurogenesis is to the at in P60 rats. that an anesthetic on neurogenesis affect DG function as to general hippocampal For DG neurogenesis to be a for anesthesia-induced neurocognitive it is required that the DG dysfunction a This to be et al. into the causing a DG and other hippocampal This in and but not In other a DG caused a was not assessed in that and the hippocampal deficit caused by cell death in the DG over the hippocampal deficit caused by isoflurane to both the of deficit caused by isoflurane in P7 rats and time to be with neurogenesis as a of a have to be in P60 rats, we that isoflurane-induced increase in neuronal differentiation was with an decrease in progenitor proliferation, which we as a secondary decrease in proliferation due to cell cycle with neuronal lineage This is the was not to if isoflurane causes cell cycle or cell cycle The subsequent increase in progenitor proliferation be a compensatory to the progenitor We base on the that only a very proportion of progenitors labeled from days after anesthesia assumed a mature neuronal days later. The of the BrdU+ to be in P7 rats we were to an of isoflurane on neuronal This is that both adult cell cycle and neuronal lineage the P7 rat brain, the DG has only just the from the to the has not occurred by et al. we isoflurane, a anesthetic, to cause cell cycle and neuronal differentiation of progenitors in P7 rats. is that protocol the but we repeated the experiments with a at the of the anesthetic followed by survival and were to show an of isoflurane on neuronal differentiation This is the that isoflurane has been shown to neuronal differentiation and cell cycle of neural progenitors from rats in therefore the decrease in progenitor proliferation in P7 rats as a primary one that is not secondary to an increase in neuronal for a primary decrease in progenitor proliferation be isoflurane in neural as has been to after isoflurane is not to neural progenitors in is if the isoflurane-induced decrease in progenitor proliferation at P7 cell cycle or cell cycle and if progenitor proliferation is also if the isoflurane-induced on progenitor proliferation occurs in other areas of the developing brain, such as the subventricular zone of adult animals or the or of immature rats, where a decrease in progenitor proliferation isoflurane-induced cell anesthetic used a relevant isoflurane concentration with a supramaximal pain stimulus causing tissue in fact, anesthesia which is a from a and a from a The of anesthesia have to be from of caused by tissue of the of is a decrease in progenitor proliferation caused by isoflurane in rats. We do not the time of the on proliferation in neonatal animals for more 4 days after anesthesia. This be important for if and how an isoflurane on neurogenesis affects cognitive outcome of anesthesia. For if the degree of progenitor proliferation at the time of with in neurocognitive a be for a between aspect of neurogenesis and that no in the number of new neurons was observed after a decrease in progenitor proliferation in both groups and an increase in neuronal differentiation in P60 rats be as the of an of isoflurane on neurogenesis is a is that the number of new neurons 4 weeks after anesthesia is not a primary of This is by of a between cell proliferation and cell to tightly control the granule cell the rate of cell death with the rate of cell also be is neuronal in the DG a of through of neurons that a The total number of granule cells not be as important for hippocampal function as neuronal as by in hippocampal function by of progenitor also by of cell death in the an et al. that cell death of immature neuronal is for learning and and that in with the of cell death of BrdU-labeled cells and with the number of new granule of a between granule cell number and function the that a decrease in progenitor proliferation causing did not affect the total number of dentate granule that of the decrease in hippocampal by with a increase in the rate of the of hippocampal these that not all affect function by increasing or the number of new dentate granule more an between an isoflurane on of neurogenesis and anesthesia-related neurocognitive is for other of anesthesia-induced neurocognitive the and of the between target and anesthesia-induced neurocognitive dysfunction of the to anesthesia is is if anesthesia-induced cognitive occurs in to the with which of learning and after anesthesia in immature rats have been is and that isoflurane a neurocognitive improvement in P60 rats and a deficit in hippocampal function in P7 rats. The of isoflurane-induced increase in neuronal differentiation in P60 rats and decrease in progenitor proliferation in P7 rats a in the cognitive outcome of anesthesia.
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Stratmann et al. (2009) studied this question.
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