| Over the past decade, numerous preclinical and retrospective human studies have demonstrated that general anesthesia in infants may be associated with adverse neurodevelopmental outcomes, including the adverse effect on learning and cognitive impairment. These data have gained widespread attention from regulatory agencies and professional, including the public at large. With the increasing number of non-obstetric surgeries occurring in pregnant women (0.15% to 2%), whether anesthesia in pregnancy is neurotoxic to the developing fetus is becoming a question of concern, as is the possible association between anesthetic exposure in children and altered neurocognitive outcomes.Propofol is a widely used anesthetic with the advantage of short onset, rapid drug metabolism and lack of untoward effect. It is also an anesthetic for pediatric surgery, as well as for other procedures such as central line catheterization, angiography, and endoscopy. Several experimental studies have demonstrated that propofol causes widespread apoptosis in the developing brain and leads to long-term behavioral deficits. Furthermore, recent studies suggest that propofol induces alterations in synaptogenesis, including changes in dendritic growth, dendritic spine density and synapse number. Synaptogenesis correlates with several types of genes and proteins including mbp and synapsin Ⅱa (syn2a). Mbp code a special protein Myelin basic protein (Mbp), which is an important component of the myelin sheath. Mbp ensheaths axons and enables neurones to function effectively and uniaxially. Damage or deficiency to the myelin sheath is known to cause axonal loss and severe demyelinating diseases, such as multiple sclerosis (MS). Synapsin Ⅱa (syn2a) is a neuronal phosphoprotein that binds small synaptic vesicles to induce further synaptogenesis in mammals. This plays an important role in both synaptogenesis and neurotransmitter release. However, little is known about the adverse effects of exposure to propofol on the transcripts and protein expression of mbp and syn2a, especially during embryonic development. More studys are needed to investigated.It is repoted that the neurotoxicity of propofol is most sensitive in the time of synaptogenesis formatting. In rodents, it is 7-15 days after postnatal, while it is 5days after postnatal in non-humanprimates. However, due to the limit of mammal, there is a need for research and new in vivo experimental models to verify the link between anesthetic administration during pregnancy and possible neurotoxicity in the developing brain. As a model species, zebrafish have numerous strengths including high genetic homology, rapid embryonic development and high fecundity, compared to rodents or non-human primates. Moreover, the transparent eggs that undergo external fertilization make it possible for researchers to microscopically observe embryos from the very beginning of development. Because of these advantages, zebrafish have been widely used in the field of neurotoxicity to research the neurological effects of early-life exposures to various chemicals.Here, we used zebrafish to verify the effects of embryonic exposure to propofol on the developing brain. We hypothesized that anesthetic doses of propofol cause apoptosis in the developing embryo and suppress mbp expression. To test this hypothesis, zebrafish embryos were exposed to propofol at 6-48 hours post-fertilization (hpf) by immersion. Our results suggest that embryonic propofol exposure is neurotoxic, and we introduce the zebrafish as a new model to study the developmental toxicity of propofol.Materials and Methods1. Buliding zebrafish model of embryonic exposure to propofolOur first objective was to determine what dose of propofol and when to expose to propofol is best for zebrafsh embryos.1) Wild-type AB strain zebrafish (Danio rerio) were studied and raised according to the protocol described by Westerfield. Zebrafish embryos (50 per well) were randomly dosed with 1,2,3,4,6, or 9 μg/ml propofol in 5 ml fish water or control treatment by immersion from 6-48 hpf in six-well plates. There were two control groups in which embryos received equal volumes of fish water or DMSO (the vehicle for propofol,0.013%). Half of the fish water in all groups was changed to fresh water (containing propofol and DMSO) at 24 hpf. All embryos were incubated with system fish water at 48 hpf. Embryos/larvae were then collected at numerous developmental stages ranging from 24-120 hpf for experimental procedures.2) The effect of propofol on embryonic survival rate, aberration rate and hatchability was assessed. To measure the survival rate, propofol-exposed live embryos and larvae were counted at 24,48,72,96, and 120 hpf. Dead embryos were removed from the plates immediately. The embryonic hatching rate was counted at 48 and 72 hpf. The aberration rate was determined by examining 5 days post fertilization (dpf) larvae under a stereo microscope.3) The effect of propofol on brain structure of 7dpf larvae were assessed. The changes were analysed trough coronal plane of brains HE stainng.4) To measure the anesthetic effect of propofol on zebrafish,6 dpf larvae were exposed to 1,2, or 3 μg/ml propofol as described above. The larvae that had no response to poking with a syringe needle were regard as anesthetized. After 30 min of anesthesia, larvae were placed in fresh fish water to measure the time taken for anesthetized larvae to return to normal. The number of dead samples during anesthesia and 24 h later was also recorded.2. The adverse effect of embryonic exposure to propofol on developing brainSeveral studies have reported that propofol causes apoptosis in the developing brain. Accordingly, we evaluated the effect of propofol on anesthesia-induced apoptosis. Moreover, we detected the adverse effect of propofol on the transcription and protein expression of mbp and syn2a.1) The effect of propofol exposure on embryonic apoptosis was determined using the vital dye, acridine orange (AO).36 hpf embryos and 3 dpf larvae from each group were staining with AO. After staining, apoptotic cells were identified with a stereo fluorescence microscope and laser scanning confocal microscope, respectively. Apoptotic cells appeared as bright green fluorescence spots.2) To detect the mRNA expression of caspase-3,-8, and -9, total RNA was extracted from 36 hpf embryos (50 per group) and 3 dpf larvae (30 per group). A SYBR green PCR core reagent kit (TaKaRa, Dalian, China) was used to amplify caspase-3,-8, and -9, transcripts, as well as mbp, syn2a and the endogenous control, ef1a in a LightCycler Nano.3) To assess the expression pattern of mbp and syn2a mRNA, qRT-PCR (described previously) and whole-mount in situ hybridization (WISH) was performed. Mbp and syn2a cRNA probes labeled with digoxigenin (Roche, Basel, Switzerland) were synthesized from linearized template DNA by T7 RNA Polymerase using in vitro Transcription Systems.4) To assess the protein expression of Mbp,3dpf larvae from each group were homogenized. The supernatant was then stored at -80℃ until western blot analysis. Protein lysates were subjected to separation on a 12% or 8% SDS-PAGE gel, then electrotransferred to nitrocellulose membranes. Signals were detected with rabbit anti-Mbp.Results1.1,2,3μg/ml of propofol is reasonable concentrations for zebrafish embryosOur first objective was to determine the effect of propofol anesthesia on embryo survival, hatching and aberration rate. The average cumulative survival rates in the groups treated with 1,2 and 3 μg/ml propofol were higher than 30% in the first 120 hpf, which was similar to the control and DMSO groups, whereas the survival rate in the groups treated with 4,6 and 9 μg/ml propofol were significantly reduced, and reached zero in 96 and 48 hpf, respectively. The hatchability rate significantly decreased in the groups treated with 1,2 and 3 μg/ml propofol at 48hpf (P< 0.05) and 72 hpf (P< 0.05), compared to both control and DMSO groups. The rate aberration rate of larvae at 5 dpf was significantly increased in the groups treated with 1,2 and 3 μg/ml propofol (P< 0.05). But the hatchability and rate aberration rate are no significant difference (P>0.05) in DMSO and control groups. The observed malformations included pericardial cysts, an unconsumed yolk sac, small eyes, bleeding and a curly spinal cord. These results show that doses of 1,2, and 3 μg/ml propofol were reasonable concentrations for embryos, inducing alterations in hatchability and aberration rate, but not survival.2.1,2,3 μg/ml of propofol has an anesthetic effect in zebrafish larvaeTo determine the anesthetic effect of propofol in zebrafish,6dpf larvae were exposed to 1,2, or 3 μg/ml propofol as described above. We found that 6 dpf larvae were totally anaesthetized within 90s when they were immersed into fish water containing 1,2, and 3μg/ml propofol. The average of onset time (s) were 60 (45,75), 45(30,60),30(30,45), respectively. The loss of larval locomotor activity and stress responses were regarded as anesthetic samples. The larvae returned to normal in 60-150 min after 30 min of anesthesia. The average of awake time (min) were 75 (75, 90),120 (105,120),120 (120,135), respectively. No larvae died during anesthesia, or within 24 h after anesthesia. These results suggest that larvae are anesthetized by immersion into water containing 1,2, or 3 μg/ml of propofol, although the onset time and duration differ with varying concentrations of propofol.3. The adverse effect of embryonic exposure to propofol on 7dpf larvaeTo detect the adverse effect of embryonic exposure to propofol on developing brain is transient or permanent,7dpf larvae were collected. We found that, compared to the control larvae, the cavity of brain in the groups treated with 1,2 and 3 μg/ml propofol was extended and the amont of the cells were declined. These results indicate that the adverse effect of embryonic exposure to propofol is lasting.4. Propofol induces cell apoptosis at 36 hpf and upregulates caspases gene expressionSeveral studies have reported that propofol causes apoptosis in the developing brain. Accordingly, we evaluated the effect of propofol on anesthesia-induced apoptosis. No obvious apoptotic cells were observed in the control group at 36 hpf, whereas considerable numbers of apoptotic cells appeared in the head of propofol treated embryos. Some apoptotic cells were also observed in the body of embryos exposed to propofol. Next, we assessed the gene expression level of caspase-3,-8, and -9 in propofol exposed embryos, because the caspase pathway is important in the induction of apoptosis. Caspase-3,-8 and -9 expression were significantly upregulated in the groups of zebrafish exposed to 1,2 and 3μg/ml propofol at 36 hpf (P< 0.05). Moreover, the upregulation of caspase-8 and -9 were dose-dependent (P < 0.05). These results strongly suggest that propofol anesthesia at embryonic stages induces acute cell apoptosis, and that the caspase pathway is important in mediating this effect.5. Propofol induces cell apoptosis at 3dpfTo evaluate the long-term effect of propofol on the developing brain, we assessed the number of apoptotic cells at 3dpf when was 24 h after anesthesia. Consistently, an increased number of apoptotic cells was observed in the brain of 3dpf larvae in the propofol treatment groups (P< 0.05). Furthermore, there were more apoptotic cells in the group of zebrafish exposed to 3 μg/ml vs. the 1 μg/ml group (P< 0.05), indicating that the lasting adverse effect of propofol is associated with the initial dose.6. Propofol significantly inhibit the transcripts and protein expression of mbpMbp and syn2a gene play an important role in myelination and synapse development. However, little is known about the effect of propofol on mbp and syn2a expression. Here, we assessed the expression of mbp and syn2a gene by western blotting, WISH and qRT-PCR. The gene expression level of mbp was significantly decreased in the propofol treated group at 3 dpf (P< 0.05), as measured by qRT-PCR. Indeed, we were able to categorize the extent of downregulation into three groups, including those zebrafish that were unaffected, slightly affected and severely affected. Importantly, the expression of mbp decreased primarily in the central nervous system, whereas there were no significant difference in the peripheral nervous system. The expression of syn2a gene was not different in the control and treatment groups at 3 dpf. Consistent with the data presented above, the protein expression level of Mbp was significantly inhibited in a dose-dependent manner by 1,2, and 3 μg/ml propofol, compared to the control group (P< 0.05). Taken together, embryonic propofol exposure cause a decrease in mbp at the gene and protein level.ConclusionsThis is the first study to examine the effect of embryonic propofol exposure on zebrafish development. After exposure to 1,2 and 3 μg/ml propofol, Propofol caused delayed development of embryos and developmental defects. In agreement with the previous studies, propofol induced apoptosis and significantly upregulates the expression of caspases gene (caspase-3,-8,-9). More importantly, propofol decreased the transcripts and protein expression of mbp. These results strengthen the view that propofol induces neurotoxicity in the developing brain, and provide a new model system through which the mechanism of propofol neurotoxicity can be studied. |
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