Physiological homeostasis during a prolonged exposure of propofol
To investigate whether the prolonged exposure of anesthesia cause disturbance of physical homeostasis in the PND 7 rats, arterial oxygen, respiratory rate, body temperature, and heart rate were continuously monitored and recorded. No significant differences in these physiological parameters had been discovered between the groups during a 5–h exposure of propofol (data not shown). Artery blood was collected for blood gas analysis at the immediate end of the 5–h exposure and there is no statistically significant difference between the groups (Table 1). These findings suggested that the administrated doses of propofol have no side effect on the physiological parameters in neonatal rats.
Table 1
Arterial blood physiological parameters between propofol exposure and intralipid control
Items | Control intralipid exposure rats | Propofol exposure rats |
pH (mmol•L−1) | 7.32±0.13 | 7.29±0.09 |
PO2 (mmHg) | 97.40±1.84 | 96.10±1.91 |
SO2 (%) | 98.16±2.72 | 96.22±3.53 |
PaCO2 (mmHg) | 43.41±3.66 | 47.06±4.42 |
HCO3 (mmol•L−1) | 27.30±3.30 | 29.45±3.58 |
Na+ (mmol•L−1) | 140.40±2.50 | 138.30±4.27 |
K+ (mmol•L−1) | 3.45±0.04 | 3.46±0.07 |
Ca2+ (mmol•L−1) | 1.38±0.03 | 1.36±0.06 |
Glucose (mg •dL−1) | 9.24±0.09 | 9.31±0.13 |
Data are presented as mean ± SD (n=10). pH= arterial hydrogen ion concentration; SO2= oxygen saturation; PaO2= arterial oxygen partial pressure; PaCO2= arterial carbon dioxide tension; HCO3= bicarbonate radical. Propofol exposure did not affect arterial blood gas values and blood glucose levels significantly. |
Long-term behavioral disorder after a prolonged exposure of propofol
MWM was used for testing spatial learning and memory in the PND 35–42 rats. Propofol treated rats had significant longer escape latency when compared with intralipid treated rats during PND 39–41 (all P < 0.01, two-way repeated measure ANOVA with Bonferroni post-hoc test; Fig. 2A). A probe trial was then used 24 h after the last training of MWM to evaluate the reference memory. Propofol treatment decreased platform crossing times as well as time spent in the target quadrant when compared with placebo (both P< 0.01, Mann-Whitney test; Fig. 2C and D), indicating that propofol treated rats had long-term spatial learning and memory deficits. There is no statistically significant difference in the swimming speed between the groups (Fig. 2B). Overall locomotor activity functions were also evaluated using open field tests and there is no statistically significant difference between the groups as well (Fig. 2E).
Acute neuronal injury after a prolonged exposure of propofol
Several anesthetic drugs including propofol have been linked to significant cell death in neonatal rat cerebral cortex [5, 9, 22]. As shown in Fig. 3, significant increases in number of TUNEL–positive cells and cleaved-caspase-3 expression in the neonatal prefrontal cortex were induced at 0, 4, 12, and 24 h during the recovery from the propofol anesthesia (quantitative values of number of TUNEL–positive cells were 5.00, 7.56, 11.25, and 5.06–fold induction, and those on cleaved–caspase-3 expression were 1.49, 1.21, 1.78, and 2.03–fold induction, when compared with the vehicle, respectively; P< 0.01-0.05), indicating an acute neuronal injury induced by a prolonged exposure of propofol. We thus chose the timepoint of 4 h after propofol anesthesia in the subsequent experiments to manifest the high acute neuronal injury.
TRPC6 contributes to acute neuronal injury after a prolonged exposure of propofol
To explore the potential role of TRPC6 in propofol-induced neuronal injury, we first examined expression levels of this protein in the neonatal prefrontal cortex. Immunoblotting analysis and the following densitometry showed that the level of TRPC6 was down-regulated at 0, 4, 12, and 24 h after the propofol anesthesia (densitometric values were 0.74, 0.68, 0.5, and 0.86–fold decreases compared with the control treatment, respectively; all P< 0.01, Bonferroni post-hoc test; Fig. 4A). To understand the role of TRPC6 in propofol-induced neuronal injury, we pretreated the rat pups with ICV injection of either TRPC6 agonist (hyperforin) or inhibitor (SKF96365) before the prolonged exposure. As expected, pretreatment with ICV injection of 5 µM and 10 µM hyperforin attenuated the down-regulated expression of TRPC6, whereas pretreatment with ICV injection of 20 µM SKF96365 exaggerated the down-regulation of this protein 4 h after the prolonged interventions (Fig. 4B). We then examined the pretreated effects on number of TUNEL-positive cells and cleaved-caspase-3 expression in the prefrontal cortex. With TRPC6 agonist, the number of TUNEL-positive cells and caspase-3 expression at the termination of propofol exposure were significantly reduced when compared with the propofol only treatment. In consistence, with TRPC6 antagonist, the number of TUNEL-positive cells and cleaved-caspase-3 expression were exaggerated as compared with the propofol only treatment (Fig. 4C-E). Taken together, these data indicate that propofol-induced acute neuronal damage involve a certain level of TRPC6 in the neonatal prefrontal cortex.
TRPC6 contributes to long-term behavioral disorder after a prolonged exposure of propofol
We next examined the effect of pharmacological manipulations of the TRPC6 on long-term spatial learning and memory deficits after the prolonged exposure of propofol. As expected, pretreatment with ICV injection of TRPC6 agonist rescued the propofol-induced increase in escape latency when compared with vehicle injection, and pretreatment with ICV injection of TRPC6 antagonist exaggerated the propofol-induced increase in escape latency (Fig. 5A). No significant changes in the swimming speed were observed among the ICV injected rats (Fig. 5B). The probe trial on PND 42 showed that pretreatment with hyperforin increased platform crossing times and time spent in the target quadrant when compared with vehicle treatment in the propofol-treated rats (Fig. 5C and D), indicating that TRPC6 contributes to the long-term spatial learning and memory deficits induced by prolonged propofol anesthesia. In open field tests, the rats given ICV injection of 5 µM, 10 µM hyperforin, or 20 µM SKF96365 did not produce any significant differences in locomotor distance when compared with the rats pre-treated with vehicle (Fig. 5E), indicating that TRPC6 intervention may not affect overall locomotor activity level.
Calpain as upstream of TRPC6 to suppress acute neuronal injury after a prolonged exposure of propofol
To explore the potential involvement of calpain in propofol-induced cortical injury, we first examined calpain activity using expression levels of SBDP145, a calpain specific spectrin breakdown product [24]. As compared with the control group, propofol induced a significant increase in the expression of SBDP145 from 0 to 24 h after the prolonged infusion (1.68, 1.53, 1.35, and 1.2 -fold induction when compared with the control vehicle, respectively; Fig. 6A). With the pretreatment of ICV injection of calpeptin, a calpain specific inhibitor, the immediate induction of SBDP145 in the prefrontal cortex was markedly suppressed in comparison to propofol anesthesia only (Fig. 6B). Similarly, the propofol-induced down-regulation of TRPC6 expression was reversed in the presence of calpeptin pretreatment (Fig. 6C). Western blot analysis showed that calpeptin pretreatment also alleviated the propofol-induced increase in levels of cleaved-caspase-3 in the prefrontal cortex (Fig. 6D). The TUNEL assay showed that the propofol-induced increase in number of TUNEL-positive cells, as seen at the termination of propofol exposure, was also significantly attenuated in the presence of calpeptin pretreatment (Fig. 6E and F). Collectively, our data indicate that calpain acts on the upstream of TRPC6 to suppress acute neuronal injury after propofol anesthesia.