| Background:There is great concern about the safety of currently used anesthetics in the young. Indeed, preclinical data suggests that commonly used anesthetics including ketamine, sevoflurane, and isoflurane are neurotoxic to the developing brain and caused long-term cognitive deficits in rodents and even primates. It is clear from animal studies that commonly used anesthetic agents affect early brain development both histologically and functionally. Features of anesthetic-related developmental neurotoxicity including abnormal intra-neuronal calcium homeostasis, neuronal mitochondrial dysfunction, apoptotic neuronal death during synaptogenesis, suppression of neurogenesis, deformation of neuronal and astroglial cytoskeletal, and impairment of hippocampal long-term potentiation. This concept is also supported by retrospective clinical data in which there was an association between anesthesia and/or surgery before the age of4leading to late-onset reading, learning and mathematics disabilities. So public pays more and more attention to the safety of anesthetics in developmental brains. However, most studies of this kind only concentrated on acute effect of anesthetics on neuroapoposis without further investigation on neuronal development and neurogenesis. In particular, long-term learning and spatial memory impairments and other neurological disorders including depression, anxiety, emotional blunting and apathy were largely neglected. Memory impairment and mood disorders have a significant negative impact to people’s daily lives and have raised great concerns. The relationship between mood disorders and cognitive impairment is very complex, as well as the development process of the behavioral disturbances. The two aspects of which can influence each other, also can exist separately. Cognitive impairments (such as memory impairment) may be just a symptom of mood disorders. Further more, most investigators have focused their attention on anesthetic neurotoxicity of neonatal. Unfortunately, only a few previous reports looked into the effects of general anesthesia on the neurodevelopmental consequences for the fetus before birth. Most obstetric anesthesia studies, however, have focused on either the teratogenic and abortive effects of anesthetic agents in the first trimester or on the neonatal status immediately after delivery, and ignored the second trimester. Traditionally, most elective surgical procedures during the first trimester (<12gestational weeks) are delayed to a later gestational age, which makes a larger proportion of surgery in the second trimester. In fact, both neurogenesis and neuronal migration in human accelerate and reach a peak during and after the second trimester, and vulnerable phase of synaptogenesis extends from the third trimester to the first few years of life in humans. Further more, most general anesthetic agents are lipophilic and can cross the placenta easily to blood circulation system of the fetal, and then adversely affect on the fetal’s brain development. Between0.75%and2%of pregnant women require non-obstetric surgery every year (such as acute inflammation of appendix and gallbladder, ovarian tumor and trauma), and most of non-obstetric surgery during pregnancy is unavoidable. This number is increasing, partly because of laparoscopic procedures and fetal surgery becoming more widely performed. Accordingly, a question arises whether maternally-administered anesthetic agents causes adverse effects during fetal neurodevelopment.Ketamine, classical NMDA receptor antagonist, is a commonly used clinical general anesthesia drugs, and is no longer in use in developed countries. But in developing countries, it is widely used in clinical anesthesia, analgesia and sedation and still plays a significant role. In recent years, use of ketamine as recreational drug in Asia Pacific area especially China and India increases sharply. The complex pharmacological efficacy and side effects such as induced schizophrenia, hallucinogenic and cognition dysfunction also drew great concerns of researchers.In this study, taking many factors into consideration, such as maternal-fetal factors, differences in gestational period, and operational stimulation, we simulate general anesthesia with ketamine for non-obstetric surgery in the second trimester, to observe behavioral disorders (cognitive impairment and mood disorders) in adult period and study its effects on hippocampal development, which help to explore the possible mechanism under neurotoxicity of ketamine. We believe our work have great significance for anesthesiologists to seek for relatively safe anesthetics, anesthesia protocols and prevention measures for non-obstetric surgery in the second trimester.Objective:Through a series of behavioral tests, such as sucrose preference test (SPT), openfield test (OFT), water maze test (WMT), and forced swimming test (FST), to observe the effects of second trimester of maternal intravenous administration of ketamine anesthesia on cognitive and emotional state of their offsprings. The hippocampus of postnatal rats morphology were studied using Nissl stain for neuronal cell count and Golgi-Cox staining method for revealing morphological changes of dendrites of hippocampal pyramidal neurons. Indication of cell proliferation in subventricular zone (SVZ) and dentate gyrus (DG) were investigated with Bromodeoxyuridine (BrdU) labeling. In addition, the expression of several proteins, including NR1and NR2subunits of N-methyl-D-aspartate (NMDA) receptor, brain-derived neurotrophic factor (BDNF) and postsynaptic density protein95(PSD-95) in the hippocampus, was studied and compared to provide insight into possible cellular and molecular mechanisms underlying any observable morphological and behavioral changes.Methods:The dams,12adult female virgin Sprague-Dawley rats, weighing between180-220g, were acclimated to the approved housing facility for7days. Then they were mated at7:00pm for the experiment, and vaginal plug tests were carried out in the next morning, a positive vaginal plug was defined as gestational day0, GO. On G14, dams were randomly divided into control (C) and ketamine groups (K). Controls were left undisturbed in their home cages while the ketamine group received a bolus dose (40mg/kg) of ketamine via lower back intramuscular injection, followed by continuous intravenous infusion with a pump via a tail vein at a rate of40-60mg/kg/hr (10mg/ml diluted with saline) for2hrs. The infusion rate was varied to induce a sedative state between a deep sedation and light anesthesia level that was evidenced by the lack of voluntary movement, decreased muscle tone, and minimal reaction to painful stimulation with the maintenance of an intact palpebral reflex. The core body temperature was measured with a rectal probe and maintained between36.5-37.5℃by a servo-controlled infrared lamp and heating pad throughout experiments. At the same time, we monitored the skin color, respiratory amplitude, respiratory frequency and oral mucosa of the anesthetized dams. At the end of infusion, dams were returned to their home cages after the righting reflex was recovered. On gestational day22, six neonatals (2/dam/group) by cesarean section after2h of intraperitoneal injection of bromodeoxyuridine of dams (50mg/kg) were used for the neurogenesis study whilst another three in each group were allowed to give birth naturally (the date of birth was designed as PO) Two pups from each dam (total6pups in each group) were killed about6hrs after birth for Nissl staining. The remaining pups were allowed to grow up with their mothers until P21, at which time pups were weaned for behavioral tests. Two groups (n=10or11) of rats were randomly selected from each group (3-4from each dam) at the P25to30for open field activity test (OFT) and forced swimming test (FST) or Morris water maze test (MWM) and sucrose preference test (SPT) for3consecutive days respectively. Subsequently, their ex vivo brain samples were harvested by decapitation at the P30for Nissl staining (n=6) to test cell density of hippocampal, immunostaining to test neurogenesis (n=5), and Golgi stain to determine the characteristics of dendrites; hippocampal fragments (n=3) were also isolated on ice to determine the expression NR1and NR2subunits of N-methyl-D-aspartate (NMDA) receptor, brain-derived neurotrophic factor (BDNF) and postsynaptic density protein95(PSD-95) used Western Blot. Another cohort including3age-matched pregnant and unpregnant rats conducted same ketamine administration protocol, arterial blood samples were sampled from the left cardiac ventricle immediately at the end of the2-hr ketamine adminstration. The dams of this group were not involved in subsequent experiment.Result:1. All the anesthetized dams recovered fully without any respiratory depression and other complications. All measurements were within the normal physiological ranges and no statistical significance was found between the two groups (P>0.05). At P28, the body weight of rats whose mother received ketamine was significantly lower than that of controls (P<0.01). There were two offspring with extreme low body weight (36g and44g) excluded from further studies.2. At P25-30, comparison with control group, ketamine offspring showed depression-and anxiety-like behaviours and impaired memory up to young adult age, which was demonstrated by reduction in sucrose intake in SPT (P<0.05), an increase in immobility time in FST (P<0.05), less time of lingering the center area of the environment in OFT (P<0.05), and more time to escape onto the platform in WMT (P<0.01). These data indicate that ketamine treated offspring showed depression-and anxiety-like behavior without memory impairment.3. There was no significant differences of cell density in the CA1region at the PND0and PND30between the control and ketamine treated group (P>0.05), In contrast cell density of ketamine treated offspring was decreased by23%at the PND0and21%at the PND30in the CA3region(p0, P<0.01; P30, P<0.05). At P30, Pyramidal dendrites in the ketamine treated pups were less branched (P<0.01) and the total branch length was shorter (P<0.01), Furthermore, spine density was significantly decreased in the ketamine treated offspring compared with that of the controls in the CA3region (P<0.01). These data indicate neurons in the CA3are more susceptible to the ketamine treatment. All these also show us that ketamine administrated in second trimester induce neuronal cell loss, and cause less maturation of pyramidal neuron in the hippocampal of CA3region in offspring.4.Both at P0and P30, ketamine-treated pups via mother resulted in a significant decrease in BrdU+cells in both the DG and SVZ regions compared with control pups (P0-DG, P<0.05, P0-SVZ, P<0.01; P30-DG, P<0.05, P30-SVZ, P<0.05). When compared to the controls, ketamine exposure caused a decrease of co-labelled BrdU/DCX cells relative to total BrdU+cells in both the DG and SVZ regions but this only reached to a statistical significance in the DG area (P<0.01). All these indicated that the proliferating function of brain cells including neurons was impaired.5. At P30, there were no significant differences of NR1protein levels between the two groups (P>0.05).At the same time, the expression level of NR2A was significantly higher in the ketamine treated rats than in the controls (P<0.05). In contrast, NR2B was decreased nearly to a quarter of controls (P<0.01). The expression levels of BDNF and PSD-95levels were significantly lower in the ketamine treated offspring than the controls (BDNF, P<0.05; PSD-95, P<0.01) All these results indicate that ketamine administrated in the second trimester perturbed NMDA receptor subunits and down-regulated BDNF and PSD-95in the hippocampus.Conclusion:The present study has provided provides preclinical evidence that pregnant rats receiving ketamine during the second trimester causes memory impairment, depression-like and anxiety-like behavioral disorders together with lower body weight in their litters. These neurological abnormalities found in the offspring were associated with neuronal loss, pyramidal neuronal abnormality, a reduction in and neurogenesis, reduction together with mismatched expression of NMDA receptor subunits and a reduction of BDNF and PSD-95in the hippocampus. |
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