The Effect And Mechanism Of Propofol In Adult Neurogenesis

1. BackgroundAdult neurogenesis in mammals is among the most important discoveries to date. The process of adult neurogenesis contains consecutive steps, including the initial proliferation of neural stem cells. Accumulating evidences have shown that adult neurogenesis is involved in various physiological functions, such as learning and memory, which is strongly related to the functions of propofol. This makes us believe that propofol is a hopefully potential agent to regulate neurogenesis. Several studies focused on the in vivo effects of propofol on adult neurogenesis. The number of BrdU-positive cells was shown to be significantly reduced in propofol treated young male Fisher 344 rats. Tung’s group, however, found no effect of prolonged anesthesia with propofol on neural cell proliferation in adult male Sprague-Dawley rats. Engelhard’s group found neurogenesis significantly increased in male Sprague-Dawley rats with a low-dose (36 mg/kg/h for 3 hours) of propofol, administered during cerebral ischemia compared with naive animals. Unfortunately, the significant results become negative in another test. Contrast to the above results of contradiction, effects of another anesthetics sevoflurane is highly consistent. The contradiction may be due to different choices of rodent species and unknown in vivo factors that conceal the infirm effects of propofol.Adult neurogenesis in hippocampus has been strongly implicated in spatial learning and memory. During the adult neurogenesis, neuronal progenitors proliferate constantly; however, undergo a mass of (even up to 50%) death after proliferation. Thereafter, live progenitors can differentiate into newborn neurons in the dentate gyrus of the hippocampus before several stages of morphological and physiological development, when dendritic complexity and neuronal excitability serve as reliable markers accompanying the function of memory. The newly matured neurons are able to contribute to spatial learning about 4 weeks after birth. Up to now, the effect and mechanisms of propofol on learning and memory remains unclear. There were reports that propofol blocks working memory and even induces profound amnesia in human. Similarly, the deleterious effects were found on several animal models. However, some other studies have shown that propofol anesthesia does not cause memory retardation in adult and aged rats after a single bolus injection or intravenous injection. It is worth nothing that the practical administration of propofol is usually repeated, even for several days. Therefore, the controversial results in experimental animals may be due to the different susceptibility between species and distinct doses of medication.On the ground of above research, the present research hypothesized that propofol shows clear effects on neural stem cells in vitro and took the advantage to reveal the direct effects of propofol on adult neural stem cells and the underlying molecular mechanisms. Then the present research hypothesized that repeated propofol sedation may show beneficial effect to the spatial learning and memory ability after propofol sedation in rodents. We administered propofol repeatedly in mice and checked the performance of spatial tasks and status of adult hippocampal neurogenesis later, to test the effects of propofol and its relationship with adult neurogenesis.2. Methods:In vitro study, cultured rat adult hippocampal neural stem cells (SCR022) were treated with propofol at concentrations of 10,50 or 100μg/mL, vehicle, or without anything respectively, after administration of 5-bromo-2-deoxyuridine (BrdU). After 24 hours of treatment, BrdU incorporation was shown by immunostaining, cell nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI). The cell viability was checked by cell counting and 3-(4,5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. The proportions of BrdU-positive cells and pyknotic nuclei were also checked. Caspase activity was measured by a colorimetric assay. Cytoplasmic calcium concentration and the expression of CREB and phospho-CREB in cells were measured before and after KN93, a specific inhibitor of cAMP-response binding protein phosphorylation, was used. Cytoplasmic calcium concentrations were determined with Fura 2-AM. The expression of CREB and phospho-CREB in cells was shown by immunostaining. Phosphorylation level of CREB was analyzed by western blot. Quantitative data were analyzed by ANOVA or student t-test. P values less than 0.05 were considered significant.In vivo study, Enhanced green fluorescence protein (EGFP) male transgenic mice receive intraperitoneal injection of propofol or Intralipid twice a day for 7 days, respectively. Spatial learning and memory was assessed by Morris water maze test 28 days after treatment.5-Bromo-2’-deoxyuridine (BrdU) was injected after treatment; numbers of BrdU positive cell in Subgranular zone (SGZ) were checked 1,14 or 28 days later treatment. Dendritic complexity of newborn neurons in dentate gyrus were assessed in EGFP transgenic mice 14 days after treatment by confocal microscopy.3. Results:In vitro study, propofol did not promote the proliferation of ANSCs at a low concentration of 10μg/mL, while it did so at a moderate concentration of 50μg/mL and a high concentration of 100μg/mL in a dose-dependent manner. Propofol increased the percentages of BrdU-positive cells. Percentages of cell death maintained at a low level (<2%). Inhibition of cell death caspase-3 activity with propofol was slight without significance. Propofol elevated the cytoplasmic free calcium concentrations in ANSCs. CREB and phospho-CREB were generally expressed in ANSCs with or without application of propofol. Propofol up-regulated the phosphorylation level of CREB in ANSCs. The cytoplasmic free calcium concentrations and phospho-CREB expressive level were significant decrease after using KN93, the specific inhibitor of CREB phosphorylation.In vivo study, propofol sedation exerts significant delay in mice to find hidden objects 28 days after treatment. Numbers of BrdU positive cell in SGZ did not change one day after treatment, while decreased 14 and 28 days after propofol injection. Total dendritic length and branch number of newborn neurons in dentate gyrus decreased 14 days after propofol injection in transgenic mice; ratios of EGFP cells to DCX positive cells remained consistent.4. Discussion:According to the vitro study, we found that the anesthetics propofol can enhance the proliferation of ANSCs at moderate (50μg/mL) and high (100μg/mL) doses in a dose-dependent manner. The vehicle and propofol at low dose (10μg/mL), however, did not show any significant effect. Most of the cultured adult neural stem cell maintained undifferentiated according to being marked with anti-nestin antibody, which indicates the contribution of proliferation but not differentiation to increased cell numbers.Next, we detected the changed percentage of proliferating cells after application of propofol. BrdU, a thymidine analog, is able to be incorporated into newly synthesized DNA strands of actively proliferating cells. In this work, we found remarkably increased percentage of BrdU-positive cells after treatment of propofol (more than 50μg/mL), which confirms proliferation contributing to increased viability. Because the growth of cells may attribute to suppression of cell death, we measured the percentage of dead cell by counting pyknotic nuclei and measured the activity of activated caspase-3, an indicator of apoptotic cell death, of cells in whole plate-wells. The results of cell counting and activity assay show consistently that cell death was not affected significantly after propofol treatment. That helped us exclude the possibility of decreased apoptosis as the major reason for proliferation.Calcium is vital for neocortex development and proliferation of neural stem cells. In the vitro study, we found cytoplasmic calcium increased in propofol treated ANSCs, which indicates a role of calcium in the effect of propofol on ANSCs. In addition, the induced less than two-fold fluctuation of intracellular calcium is in the physiological range, which suggests the physiological effect of propofol in our experiments. The following results of the upregulation of phosphorylated CREB proteins increased the possibility of calcium signaling. Recent studies suggest that CREB activation via phosphorylation is important for neurogenesis in the adult rodent brain. In fact, CREB phosphorylation has been implicated in the proliferation of ANSCs conservatively during evolution. Having stated the above, we can deduce that propofol might improve the activity of CREB through cytoplasmic sensors, e.g. calcium, to promote the proliferation. In addition, the induced less than two-fold fluctuation of intracellular calcium is in the physiological range, which suggests the physiological effect of propofol in our experiments.In the vitro study, we chose an adult hippocampal neural stem cell line (SCR022) from a Fisher344 rat for its high homogeneity. The abundant nutrient in commercial culture medium and simple micro-environment around cultured cells ensured fast growth with limited fluctuation. In the other hand, the superior conditions may conceal some effect of propofol at a low concentration, which can also be reflected from the low apoptotic index. It is difficult yet to infer the effect in vivo from that in vitro, since the niche in the central nervous system, including microvessels, is strongly implicated in neurogenesis. Propofol has the ability to regulate the hemodynamics within the brain and modify the functional status of endothelial cells, which may interfere the final in vivo effects of propofol.Enhanced green fluorescent protein (EGFP) mice have granule cells which express EGFP in a couple of days, about two weeks after birth. We used these transgenic mice to investigate the cognitive and neurogenetic effect of repeated propofol sedation in vivo. On the contrary of our hypothesis, we found the deleterious effect of repeated propofol sedation on spatial learning ability in mice through Morris water maze. Previous studies focused on either the early effects (within minutes to hours) of propofol anesthesia or the long-term effects (14 days) of a single dose of propofol anesthesia. However, our study concerned about the long-term effects of repeated propofol sedation on cognition, which is closer to some clinical practice in intensive care units.It should be noted that the behavioral deficits appeared 4 weeks after treatment when new granule cells were able to participate in the existed circuitry. To quickly assess the possible role and extent of adult neurogenesis which might contribute to the impairment, we counted the BrdU-positive cell numbers in the subgranular zone of mice in both groups after treatment. The numbers equaled statistically on the next day after treatment, which indicated no influence on the proliferation of adult neural stem cells in hippocampus. The diminished numbers in the propofol sedation group revealed the injured viability of proliferating cells in the subgranular zone during the first month after sedation. These results have clearly shown that repeated propofol impairs adult neurogenesis, especially the survival of neural progenitors, which has been strongly associated to learning. The survived neural progenitors would thereafter differentiate into several cell lineages, among which neurons might be the mostly important for cognition.To elucidate the exact effects of repeated propofol sedation on the newborn neurons, we determined to trace the developmental status of newborn neurons that derived from the decreased progenitors. We also took the advantage of the EGFP transgenic mice to portray the newborn neurons two weeks after treatment. We confirmed the intrinsic character of EGFP-positive cells by colocalizing them with DCX immunostaining, since DCX is a reliable marker of newly generated neurons in dentate gyrus of adult hippocampus. EGFP-positive cells in our experiments were almost stained as DCX-positive cells, which was consistent to other’s results and served as the basis for further exploration of the newborn neurons.The numbers of EGFP-positive cells were reduced days after propofol sedation, which was consistent with those of BrdU-positive cell. This indicated a consequence of less survival of neuronal progenitors. Moreover, the reductive newborn neurons endured delayed maturation in morphologically. Our results showed that two indices of dendritic complexity, total length and numbers of branches, were weakened by propofol sedation. Studies have demonstrated that even minor alterations in dendritic structure can have a marked impact on the biophysical properties of neurons and dramatically affect the manner in which neurons transmit information. Actually, dendritic complexity has been strong related to hippocampus-dependent learning in several models, including enriched environment, voluntary exercises and short photoperiods. During neuronal development, dendritic complexity is a marker indicating the extent of maturation, and is able to suggest future ability of learning. Subsequently, the propofol sedation-induced decline of adult neurogenesis, both in quantity and morphology, can be inferred to render the behavioral deficits later. The vivo study revealed toxic effects of propofol on learning and adult neurogenesis. These findings suggest anesthetists and physicians pay attention to an adverse impact when using propofol as sedation repeatedly. The vivo study also presents a model of anesthetic toxicity in mice, which links spatial learning to adult neurogenesis in hippocampus.5. Conclusion:In vitro, propofol might improve the activity of CREB through cytoplasmic sensors, e.g. calcium, to promote the proliferation. However, in vivo, we found the deleterious effect of repeated propofol sedation on spatial learning ability in EGFP transgenic mice. Furthermore, the propofol sedation-induced decreased the numbers and dendritic complexity of the newborn neurons in the subgranular zone of the dentate gyrus of mice hippocampus.