Effect of Hypercarbia and Isoflurane on Brain Cell Death and Neurocognitive Dysfunction in 7-day-old Rats

MILLIONS of neonates undergo anesthesia for surgical or diagnostic procedures each year.1,2Anesthesia kills neonatal brain cells of several animal species, including primates,3–6and causes long-term neurocognitive dysfunction.3,5,6Consequently, anesthesia-induced apoptotic brain cell death is a growing concern within the anesthesia community, the public, and regulatory authorities alike.7However, a causal link between anesthesia-induced brain cell death and anesthesia-induced long-term neurocognitive dysfunction is lacking. If such a causal relationship existed, the degree of anesthesia-induced brain cell death should predict the severity of anesthesia-induced neurocognitive dysfunction. We therefore studied if 0, 1, 2, or 4 h of isoflurane at 1 minimum alveolar concentration (MAC) kills brain cells of postnatal day (P)7 rats and causes long-term neurocognitive dysfunction. We previously observed that the arterial carbon dioxide tension of spontaneously ventilating isoflurane-treated P7 rats increases shortly after induction of general anesthesia; therefore, we also tested if hypercarbia alone affects cell death in the P7 rat brain and if it alters long-term neurocognitive function.Pavlovian fear conditioning is a well-established method of studying learning and memory.8,9In a typical fear-conditioning experiment, a rodent is taught to associate a neutral cue (e.g. , a tone) with an aversive event (e.g. , a shock). After rodents have been trained with one or more pairings of tone and shock, they learn that tone predicts shock. Rodents will exhibit their innate fear response (i.e. freezing, immobility) even when the tone is presented without the shock. Memory of the association between the tone and shock is assessed by quantifying freezing behavior in response to the tone. In addition to learning that tone predicts shock, rodents also learn that the surrounding environment made up of visual, auditory, olfactory, and tactile cues (the context) also predicts the shock. When rodents are given multiple pairings of tone and shock, not only will they freeze in response to the tone, they will also freeze in response to the context in which the training took place.The basolateral amygdala, located within the medial temporal lobe, is essential for the acquisition of conditional fear. The basolateral amygdala is the site at which convergence of information about the shock and its predictive cues occurs.10,11The hippocampus assembles the representation of the context and communicates it to the amygdala. The roles of these two structures have been well characterized. Lesions of the amygdala block fear to tone and context, whereas lesions of the hippocampus block only fear to context and leave fear to tone intact.12,13The differential contribution of the amygdala and hippocampus allows us to perform two different tests of memory after training: (1) measurement of freezing in response to tone in a second context distinct from the training context, providing an assessment of the effect of anesthesia on an amygdala-dependent, hippocampal-independent task; (2) measurement of freezing in response to the training context in the absence of tone or shock, providing an assessment of the effect of anesthetics on a hippocampal-dependent task.In the spatial reference memory task of the Morris water maze, rats are trained to find a submerged platform to escape from the maze. Due to the absence of local visual or olfactory cues that would indicate platform position, rats have to use distal cues. This requires formation of an allocentric spatial representation of the maze, which depends on the hippocampus and the neocortex.14,15Both memory acquisition (learning) and memory retention can be tested in the Morris water maze. The spatial working memory task of the Morris water maze, which tests executive function, uses a submerged platform placed in a different location each day. This task requires integration of information from the hippocampus, the cortex and the thalamus, among others.16–19Here we show that three interventions cause significant cell death in multiple brain regions, namely 2 h and 4 h of isoflurane as well as 4 h of carbon dioxide. Of these, only 4 h of isoflurane caused a neurocognitive deficit.All experiments were conducted with approval from the Institutional Animal Care and Use Committee at University of California, San Francisco. Rats (n = 17) with litters containing male (n = 162) and female (n = 2) Sprague-Dawley rats (from Simonsen Laboratories, Gilroy, CA, and Charles River Laboratories, Davis, CA) were cross-fostered before starting the experiment. When pups were 7 days old, they were given anesthesia or sham anesthesia. After treatment, the three anesthesia groups and two control groups (4 h of sham anesthesia, 4 h of carbon dioxide) were distributed equally among litters.Anesthesia was conducted as described elsewhere.20Briefly, isoflurane was administered to at least 10 rats anesthetized simultaneously. MAC was determined by tail clamping every 15 min. Physiologic variables were assessed, and temperature was controlled at 36.5°C.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. Thirty rats were kept in a carbon dioxide–enriched atmosphere for 4 h to rule out hypercarbia as the cause of the effects observed in the anesthesia group. The fraction of inspired carbon dioxide was varied between 15 and 30% to mimic the carbon dioxide tension in the anesthesia groups. The assumption was made that as long as breathing did not appear depressed, arterial carbon dioxide tension would be less than 10 mmHg higher than inspired first carbon dioxide tension.21Cardiac puncture for blood draws in these rats was performed under local anesthesia (0.05 mm of 1% lidocaine) injected subcutaneously.Tissue preparation and FluoroJade (FJ) staining was performed as described in the accompanying article in this issue of Anesthesiology.20FJ stains were scanned and photographed at 4× before a tile scan at 20× of areas with appreciable cell death. The brain was mapped according to Paxinos’ atlas of the rat brain.22Data are presented as fold increase from control. Density of FJ-positive (FJ+) cells in control animals, representing physiologic cell death, is provided for reference. All FJ+ cells were counted in three slices of the structure of interest, and the results were averaged. The individually assessed thalamic nuclei combined in the analysis of P7 rats were the paracentral, centromedial, paraventricular, mediodorsal (central and lateral), intermediodorsal, rhomboid, reuniens (including ventral reuniens), and the lateral posterior (laterocaudal, laterorostral, mediocaudal, and mediorostral). Cortical layers 2-6 were combined and the hippocampal subregions analyzed were the cornu ammonis (CA)-1, CA-2, CA-3, pyramidal cell layer, and dentate gyrus.Eight weeks after anesthesia, rats underwent fear conditioning. 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 shock was composed of 19 stainless steel bars, each 4 mm in diameter and spaced 16 mm on center. These floors were connected to a shock delivery system (Med Associates, St. Albans, VT). The chambers were wiped with a pine-scented cleaner (5% Pine Scented Disinfectant; Midland, Inc., Sweetwater, TN) before and after each session. The room in which training took place was illuminated with overhead fluorescent bulbs, and a ventilation fan provided background noise (65 db). The appearance, odor, and texture of the chambers and room comprised the training context.After a 3-min baseline exploratory period in the chambers, rats received three tone (2000 Hz, 90 db)–shock (1 mA, 2 s) pairings separated by 1 min. Freezing, the absence of all movement except that necessary for respiration, is an innate defensive fear response in rodents and a reliable measure of learned fear.12Each animal’s behavior was scored every 8 s during the observation period, and a percentage was calculated using the formula 100 × f/n, where f is the number of freezing events per rat and n is the total number of observations per rat.The next day, rats were tested for fear to the training context and fear to tone. For the context test, each rat was once again placed in the chamber in which it was trained for a period of 8 min (in the absence of tone and shock). For the tone test, groups of rats were transported in separate plastic pots (height, 14 cm; diameter, 15.5 cm) to a distinct context in a different room. The test chambers were triangular in shape with an acrylic floor (length, 28 cm; width, 25 cm) and two acrylic sidewalls (length, 28 cm; width, 22 cm) at a 45-degree angle. The chambers were equipped with a speaker and were wiped down with acetic acid (1%; Fisher Scientific, St. Louis, MO) before and after each session. The room appeared dark to the rats, being lit by a single 30-Watt red bulb. A different kind of white noise (65 db) was used for background noise. Rats were given a 3-min exploratory period before three 30-s tones (2000 Hz, 90 db) separated by 60 s. Rats were removed from the chamber after an additional 30 s. The order of the context and tone tests was counterbalanced such that half of each treatment group was tested to context first and tone second and vice versa . Freezing was scored by three observers blinded to group assignment during the 3-min exploratory period, the training, and both tests.A platform (diameter, 10.3 cm) was submerged in a circular pool (diameter, 180 cm; depth, 50 cm) filled with warm (24°C) opaque water. Two training were administered each day h the of its first a rat was placed the platform for 60 s and given three 15 in which it was from one of the rats were not placed the platform for 60 s before the three were given 60 s to the If the rat did not the platform in the it was by the In the rat was removed from the platform after 15 s. with platform of rats in each group were one of platform for the of training in were administered the rats were to the platform in less than 15 s a after or more training to the platform and to the platform were analyzed using a system to per retention of the platform a was administered with the platform removed from the days after the training the learning was the the of in the each of the was determined for each of two 30-s and the number of platform was and the the of the room was by both the and a of spatial cues. the of the escape platform was made by it 1 the water and its with red that be from the water but not by the Rats were tested in three platform per in which the platform was each variables and analyzed during the platform were also and analyzed during platform this the room was again by the and a of spatial cues. The platform was submerged 1 the water in one of platform in that were used with each on every session. This to the use of hippocampal-dependent place than was administered per day with a during which the rat was to the maze. If the platform was the rat was to on it for 15 s. If the platform was not located within 60 the animal was to it and to on it for 15 s. during the was not After the three were administered in which the rat was from one of the of the The platform location was identical for all animals in a but the location was varied to one one and one long This did not when the platform was located in the of the in which all animals were placed in the pool at the three were administered the rats were to the platform in less than 15 s a or more training to the platform and were with a system to per the rats that they the of the a was between the and the to increase task day a between the and the three scored was on day the was to 4 to the to and were again with the system as described reference memory training, working memory on the first after the on day day and day 14 was used as a measure of working are as and for the control and as a fold increase from control for cell death the if isoflurane increases cell death between the isoflurane groups and the control group were using a test with of and by a for multiple to the at which isoflurane causes a significant increase in cell death. this the test each isoflurane group to the control group. the if hypercarbia causes cell death, between the hypercarbia and the control group were using the the if the degree of cell death in the hypercarbia group from the degree of cell death in the isoflurane a test was the of the between the from in Paxinos’ rat brain atlas and FJ+ cell the was death were analyzed using for Inc., San conditioning freeze were as and were analyzed both and for the tone or context of the test for to the in the variables freezing, freezing, freezing, and context were using a test with of and with a for multiple conditioning were analyzed using for were as In the water of and to the platform were all within therefore, to platform are presented for and in the variables to freezing were using a analysis of and using When the significant of tests were were analyzed using or were at cell death a group of animals was to a between of with an at a of the to a 30% between groups at and a of was animals per group. A group of than 14 was to a of was used for physiologic variables during this of anesthetic are described in elsewhere.20Briefly, or during anesthesia in group. and and after induction of general anesthesia. The in rats anesthetized for 4 h and 2 h was with animals during the of anesthesia and two animals in the group after of the The arterial carbon dioxide and of the carbon animals of animals all areas with anesthesia-induced cell death, the thalamic nuclei were the After 4 h of the cell of FJ+ cells was to than control in the lateral posterior and thalamic The effect of carbon dioxide on the varied by In the lateral and thalamic the of cell death caused by carbon dioxide was and and was not different from that of the isoflurane group. In these regions, carbon dioxide caused more cell death than 1 h and 2 h of the in these was not In the rhomboid, and thalamic carbon dioxide did not cause cell death, but 4 h of isoflurane did cell death, the and thalamic nuclei were not in the to of for these between from in Paxinos’ atlas of the rat brain and FJ+ cell in the lateral thalamic nuclei of both the isoflurane and carbon dioxide control groups was significant in isoflurane and in carbon dioxide the group the isoflurane group a of cell the hippocampus and dentate the of FJ+ cells was on the of isoflurane exposure The of cell death in the control group did not from the cell death in the carbon dioxide control group of isoflurane significant effect on cell death, whereas 2 h of isoflurane caused a increase in the of FJ+ cells from and 4 h caused a This that but not carbon causes hippocampal cell death, in a if administered for at least 2 layers of the isoflurane treatment caused significant cell death, but 4 h of isoflurane did The of cell death was an increase from control caused by 4 h of the was significant increase in cell death in the carbon or the group. both the and isoflurane cell death in the of observed such as the or or to cell death in of the freezing before the of the first during training was and less in the and isoflurane groups than in or carbon dioxide of the shock and freezing, the formation of memory the next day, the baseline freezing were again baseline freezing before training the day, were between groups freezing and were not different between groups and was effect of freezing were again and was effect of treatment was effect of order of test, , tone test first context test first a significant treatment × effect = on to the A test all groups the platform at about the the isoflurane group did perform and the carbon dioxide group than the control group 2 and in the first was a treatment effect on number of to learning s three = Rats to isoflurane for 4 h more training to learning than all groups did was also a treatment effect on rats to carbon dioxide than the groups the first in that these animals performed as well as or than their than The show that neonatal exposure (4 h but not 2 h or 1 to isoflurane in the Morris water neonatal carbon dioxide exposure not but to in this The groups all performed equally well on platform not that and to the task was not by neonatal exposure to anesthesia. days after the spatial reference memory the platform was removed from the pool and was assessed for 60 s. All groups about the of the and made of platform These indicate that neonatal exposure to isoflurane did not memory for the platform location once it was show that all groups on well on working memory task training the rats to carbon dioxide appeared to the control group on training an that was significant treatment × effect on in this task on to s on all groups about the number of to carbon which this the between groups that were observed in the spatial reference memory task on training in this task such that all groups as as the carbon dioxide group did in the spatial reference memory This that the carbon group than the groups in the first task but that the groups with When we task by the rats to the platform location for 1 h and 4 h group in to the platform location learned on the that at the rats to carbon dioxide performed = whereas rats to isoflurane for 4 h to perform = group when a was between the and next between groups = with rats previously to carbon dioxide more than the groups and on the in which a was between the and the next = are several of this isoflurane at 1 MAC causes brain cell death in P7 rats when administered for at least 2 this brain cell death in areas is with being as MAC × cell death is caused by 4 h of in the thalamic This of anesthesia-induced brain cell a single agent used at a concentration of 1 4 h of carbon dioxide alone causes cell death in thalamic the degree and of thalamic cell death was to the degree and of cell death caused by 4 h of that of the cell death be caused by The brain as a and structures within it from to and from to that cells in are of of this is where are located in layers and more If the of to anesthetic were a of of for the at which are from the brain for to with would of anesthesia-induced cell death in areas that more cells at this the of FJ+ cell in the lateral thalamic nuclei of both the and carbon dioxide treatment groups that the two the cell this not of the of the cells by carbon dioxide or is that a different kind of cell is degree of cell death and of cell death caused by 4 h of isoflurane or 4 h of of the three interventions cell death (4 h of 2 h of 4 h of carbon only 4 h of isoflurane caused a long-term neurocognitive which was in spatial reference memory and spatial working memory but not in fear conditioning We the neurocognitive test on the of the well-established about the in these on their different for at the of different of behavior in these carbon which caused cell death in areas of the spatial reference memory and spatial working and we can for this in the thalamus, 2 h of isoflurane caused significant cell death in the and hippocampal cell death but neurocognitive of long-term neurocognitive dysfunction after neonatal anesthesia in between brain cell death and long-term neurocognitive in this that cell death alone not be to cause neurocognitive dysfunction. a to cell death should be after cell with in the plastic neonatal in an accompanying article in this we the of the to be the with a and a to at least one of a the areas by cell death, the hippocampus, and to the of tested 8 weeks after as thalamic cell death not the long-term it is that the hippocampal cell death to the neurocognitive observed is also that a of cell death, which was only by 4 h of is to the of the results is that the degree of cell death that was caused only by 4 h of isoflurane is for the neurocognitive these be additional or even that anesthesia-induced long-term two in this a anesthetic effect on or on and These two of brain namely and In of these are the that the of a in spatial reference memory but in fear conditioning been 4 weeks after of in weeks after that only the more water be at this time. a single exposure of P7 to which anesthetic dentate in is with of a in for at least 4 days after isoflurane anesthesia in P7 have been to these to anesthetic effects on long-term are two by which anesthesia the brain for a that the of the of this are For of cell death as not for apoptotic or cell of anesthesia used in this is more a of anesthesia clamping causes and which be a This is both a from a and a from a anesthesia without is administered to only In a anesthetic was used in this which within the of the by between rats and to a by the of this of is the of the presented to that anesthesia-induced cell death in the rat brain to anesthesia-induced long-term dysfunction is on the of from to which they are a period, to to the period of between of and in the brain several after cell death caused during the period of causes neurocognitive dysfunction in if the were in anesthesia cause brain cell death and neurocognitive dysfunction several after the assumption that cell death causes neurocognitive dysfunction. in the of also be namely the that the period of is by the of and that the cells are been that to anesthetic which at P7 in to the period of contribution in of this is a of brain during different that to the of the brain it not to per . The of in rodents not and to at least period of to anesthesia-induced cell death not therefore, the between and to anesthesia-induced cell death is of anesthetic have caused brain cell death in including are these cells Due to the period of rats, the in the structures by cell death of P7 rats are days of with the being even in P7 rats from in that they are to have total acid and they are by not the should be that the of anesthetic be an than a The of cells by anesthesia be by than or of of which a temporal or of the of cells to anesthetic would a more of the in anesthesia-induced cell a of the the of a for anesthesia-induced neurocognitive in it be to if and when the brain is to anesthetic if anesthesia cells in the it is if neurocognitive dysfunction would The results that the two are less than previously and that at least to the long-term of neonatal anesthesia. This is not to the The of anesthesia-induced long-term neurocognitive dysfunction in animals both concern and to if the in In the in in and in are also to us of the of if that anesthesia cause in as well as the or that the that hypercarbia a in anesthesia-induced brain cell death and that cell death alone is to for the long-term neurocognitive after neonatal anesthesia in The of anesthesia-induced neurocognitive dysfunction after neonatal anesthesia and its to neonatal anesthesia and of University of California, San and of University of California, San for on anesthesia and carbon dioxide in The also of and University of for on the