Repeated administration of propofol upregulated the expression of c-Fos and cleaved-caspase-3 proteins in the developing mouse brain.

OBJECTIVES AND AIM: This study was designed to analyze the relationship between the expression of c-Fos protein and apoptosis in the hippocampus following propofol administration in infant mice. There are reports that certain drugs, including the general anesthetics applied in pediatrics and obstetrics, could block N-methyl-D-aspartate glutamate receptors and activate γ-aminobutyric acid type A receptors. Furthermore, some anesthetics could trigger neuroapoptosis and the expression of c-Fos in the developing rodent brain. Propofol is a general anesthetic increasingly used in pediatrics and obstetrics, and is reported to be able to interact with both γ-aminobutyric acid type A and N-methyl-D-aspartate glutamate receptors. No adequate evaluations have been available as to whether the dosage of propofol to maintain anaesthesia could trigger the expression of c-Fos and apoptosis.
MATERIALS AND METHODS: Intraperitoneal injections of propofol (50, 100 and 150 mg/kg) or vehicle were administered every 90 minutes (4 times) in infant mice (5-7 days old). 30 minutes after the final administration, the protein expressions of c-Fos and cleaved-caspase-3 in the hippocampus were determined by immunohistochemistry and Western blotting.
RESULTS: It was demonstrated that the expressions of cleaved-caspase-3 and c-Fos were upregulated in the hippocampal CA3 region in this study.
CONCLUSIONS: The upregulated c-Fos expression induced by repeated injections of propofol might evoke neuroapoptosis.

Introduction

There has been an increase in the number and duration of anesthetic procedures, as well as the surgical complexity of procedures carried out in the in pediatric and obstetric patients. Concerns have been raised for the effects or risks of anesthetics on the developing brain. Some anesthetics could block N-methyl-D-aspartate (NMDA) glutamate receptors or activate γ-amino butyric acid type A (GABAA) receptors, triggering widespread apoptotic neurodegeneration in the developing brain when administered to infant rodents.[1-3] The expression of c-Fos protein is modified under various physiological and pathological stimuli in the brain, e.g., some anesthetics, such as ketamine and phencyclidine, can upregulate the level of c-Fos protein.[4-6] Anesthetic-induced apoptotic neurodegeneration is age-dependent, and is closely correlated with the timing of synaptogenesis, e.g., the immature brain is most sensitive when synaptogenesis is at its peak.[7]

Propofol could interact with both GABAA receptors and NMDA glutamate receptors, and clinically serves as an intravenous anesthetic to induce and maintain the general anaesthesia and sedation, either by continuous infusion or intermittent bolus doses.[89] Irifune et al., showed that GABAA receptor agonist muscimol and NMDA receptor antagonist MK-801 could also increase the duration of the righting reflex loss, and the recovery period induced by propofol.[10] Many reports have documented that propofol has analgesic properties.[11]

Despite the controversy regarding the use of propofol in children, it is commonly administered to young children including neonates.[1213] Davide et al., have reported that propofol could trigger significant neuroapoptosis at a dose of 50 mg/kg, while the dosage required to induce a surgical plane of anaesthesia is 200 mg/kg.[14] However, it still remained obscure whether the anaesthesia-maintaining dosage of propofol could trigger the expression of c-Fos. This study was focused on the immunoreactivity of c-Fos and cleaved-caspase-3 so as to identify the biochemical changes of neurons after propofol administration.

Materials and Methods

Animals

All animal procedures were approved by the Experimental Animal Centre of Xuzhou Medical College. Kunming mice (5–7 days old), weighing 4.0 ± 0.5g, were used in all the experiments. Some studies have demonstrated that in young rodents, the growth spurt of the rodent brain occurs during the first 14 postnatal days (PNDs) and at 7th PND in particular, and this is the critical period of the brain development, when it is most vulnerable to the neuroapoptotic damage induced by some anesthetics.[15]

The Dose of Propofol for Inducing Anaesthesia

In this experiment, anaesthesia was induced by different doses of propofol (AstraZeneca) in infant mice. Sequential method was used to detect the doses of 50% effective dose (ED50) and median lethal dose (LD50) in mice (n = 15 each), and the behavioral responses, i.e., loss of righting reflex and response to pain, were tested at a 5-minute interval for the first 30 minutes; and then, every 20 minutes for the remainder of the experiment, as descried by Loepke et al.[16] In brief, juvenile mice underwent a single injection of propofol or vehicle (Intraperitoneal - i.p.) at doses of 50, 100 and 150 mg/kg (n = 8 each).

Immunohistochemistry

In this study, the expressions of c-Fos and cleaved-caspase-3 proteins were monitored at the neuroanatomical regions which serve to maintain anaesthesia, in order to study their effect on the activity of the developing neurons, and to further investigate the correlation between c-Fos and neuroapoptosis. Infant mice (n=8 each) underwent four injections of propofol (50, 100 and 150 mg/kg) or vehicle every 90 minutes. 30 minutes after the last administration, the animals were perfused with 10 ml normal saline and 30 ml 4% paraformaldehyde in 0.1 mol/L ice-cold sodium phosphate buffer (pH 7.4). Afterwards, murine brains were quickly removed and further post-fixed with the same fixation solution at 4°C overnight. Subsequently, the murine brains were cryoprotected in 30% sucrose with 0.1 mol/L sodium phosphate buffer (pH 7.4) for 48 hours at 4°C, and coronal sectioning (40 μm-thick) of the frozen brains on a cryostat (CM1900, Leica, Germany) was carried out. The sections were immersed in 1% hydrogen peroxide (H2O2) to block endogenous peroxidase activity at room temperature for 30 minutes. At the end of three rinses in Phosphate Buffered Saline (PBS) (0.01 mol/L), the sections were further blocked with 8% goat serum for 15 minutes, and then incubated with the primary antibody (c-Fos, Santa Cruz Biotechnology) (1:1000) and activated caspase-3 (Beijing Biosynthesis Biotechnology Co., LTD., China) (1:100) at 4°C overnight. No antibody was added to the negative control sections. Afterwards, the slides were rewashed with PBS, followed by addition of the secondary antibody SPN100 (anti-rabbit) for incubation overnight at 4°C. Finally, the sections were incubated with avidin-conjugated horseradish peroxidase for 4 hours at room temperature. The peroxidase reaction was visualized in phosphate buffer (0.1 M), 0.03% diaminobenzidine and 0.01% H2O2.

Western blotting

For brain tissue preparation, infant mice were sacrificed 30 minutes after the last administration of propofol dose. Whole brains were removed for dissection. The hippocampal CA3 regions were microdissected from both sides of the hippocampal fissure, and immediately frozen in liquid nitrogen. The samples were placed in lysis buffer containing protease inhibitor, homogenized and then centrifuged. The protein concentrations were determined by the Lowry's method, with bovine serum albumin as standard. Western blot analysis was performed on 10% Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Briefly, proteins were electrotransferred onto a nitrocellulose filter (NC, pore size: 0.45 μm). At the end of 2 hous of blockage in phosphate buffer with 0.1% Tween 20 (Phosphate-buffered saline with Tween 20 - PBST) and 3% bovine serum albumin (BSA), the membranes were incubated overnight with primary antibodies (1: 1000 dilution for actived caspase-3, 1: 500 for c-Fos). Detection was carried out by the use of proper alkaline phosphatase conjugated IgG (1: 500) and developed with Nitro blue tetrazolium /5-Bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) assay kit (Beyotime Institute of Biotechnology, China).

Results

The anaesthesia-inducing dose of propofol

The ED50 and LD50 of propofol in infant mice was 50 mg/kg and 200 mg/kg, i.p., respectively. Five to ten minutes after propofol administration, the righting reflex, consciousness and response to pain disappeared in mice. Animals recovered from administration of propofol at 90 minutes at 100 mg/kg and 110 minutes at 150 mg/kg. Since the duration of anaesthesia at the dose of 100 mg/kg was 90 minutes, the doses were administered synchronously, and repeatedly four times to maintain the simulated clinical sedation and anaesthesia.

Cleaved-Capase-3 and c-Fos Expression

In the control brains it was found that the immunoreactivity of c-Fos and activated caspase-3 was scarce in the hippocampal CA3 area [Figures 1a and d]. However, propofol triggered robust expressions of c-Fos, and activated-caspase-3 proteins at the doses of 100 mg/kg and 150 mg/kg every 90 minutes for four times, respectively [Figures 1b–f]. The histograms in Figure 2 showed the quantitative counts of CA3 [mean ± Standard error of mean (S.E.M)] under each treatment condition. We also detected the quantitative expressions of c-Fos and cleaved-caspase-3 proteins by Western blot [Figure 3a]. The upregulated expressions of c-Fos and cleaved-caspase-3 demonstrated that repeated administration of propofol (100 mg/kg and 150 mg/kg every 90 minutes for four times) could evoke the expression of c-Fos and apoptosis in the hippocampal CA3 region [Figures 3b and c].

Figure 1

Photomicrographs of cleaved-caspase-3 and c-Fos-positive profiles were shown in hippocampal CA3 region. (a, b, c) Positive cleaved-caspase-3 profi les in CA3. (d, e, f) Positive c-Fos profi les in CA3. (b, e) Propofol 100 mg/kg. (c, f) Propofol 150 mg/kg. (a, d) Control group. Scale: 20 μm

Figure 2

The histograms show the quantitative counts [mean ± Standard error of mean (S.E.M)] of positive caspase-3 and c-Fos profiles for each treatment condition in CA3 of hippocampal region. The counts were performed on serial sections. Signifi cant differences from the control group were determined when P < 0.01 (**)

Figure 3

Western blotting analyses of c-Fos and cleaved caspase-3 in hippocampal CA3 region in propofol group with four repeated intraperitoneal injections of propofol (50, 100 and 150 mg/kg) and vehicle group. The bands by Western blotting represent four experiments with similar results (a) Quantifi cation of the cleavedcaspase-3/caspase-3 ratio. (b) Quantifi cation of c-Fos expression (c). Results are represented by mean ± Standard Deviation (SD). *P< 0.05 vs. control; *P < 0.01 vs. control

Discussion

In the present study, we demonstrated that there was a significant increase in the populations of c-Fos-positive and cleaved-caspase-3-positive cells in the hippocampal CA3 region subsequent to propofol anaesthesia for 6 hours. The increase of neuroapoptosis induced by general anesthetics has been well documented, and c-Fos protein has a causative role to play in the initiation of apoptosis.[1718] However, other researches indicated that the c-Fos expression mediated by PI-3K signaling pathway could enhance the survival ability of the HaCaT cell line.[19]

It is unclear whether the expression of c-Fos could initiate or inhibit the neuroapoptosis under anaesthesia, and further studies are hence required. Regardless of the obscure intermediary mechanism for the injury of anaesthesia-induced developmental murine brain, our findings on the sensitivity of c-Fos expression to propofol stimulation might be of interest in humans as well. The neurodevelopment of rodent brain within the first two postnatal weeks corresponds to the last trimester of brain development in humans. Moreover, the responses of c-Fos to propofol in neonatal mice and the consequent apoptosis indicated that the sensitivity of developing human brain to adverse effects of anesthetics may also extend to the follow–up neurogenesis, the synaptic organization and the phases of terminal differentiation.[20-22] Furthermore, in obstetrics and pediatrics, the developing brain may remain vulnerable to general anesthetic well into neonatal life. Given the potential risks of anaesthesia-induced neurodegeneration in the perinatal period which might be attenuated with increasing age (as demonstrated in this animal study), it would be a prudent practice to subject infants to elective surgery.[7]