| Background The developmental neurotoxicity of general anesthetics has aroused extensive concern in the field of anesthesiologists, even the society. Animal studies has demonstrated that exposure of postnatal day 6 or 7 rats to isoflurane causes widespread neuroapoptosis, synaptogenesis impairment and decline of learning and memory in the adulthood. However, the underlying mechanisms remain unclear. Insulin-like growth factor 1(IGF-1) activated ribosomal protein S6 through PI3K/Akt and MEK/ERK pathways. The consequence is to promote the protein systhesis, cell growth and cell survival. Also, the IGF-1/PI3K/Akt/S6 and IGF-1/MEK/ERK/S6 pathways play a pivotal role in the process of dendritic arborization, spinogenesis and synaptogenesis. Therefore, this reasearch program was to investigate the effect of these two pathways on the developmental neurotoxicity of isoflurane. Specifically, we planed to reveal their roles in the neuroapoptosis, synapogenesis impairment and decline of learning and memory in the adulthood caused by postnatal exposure to isoflurane.Methods 1. To reveal the activities of IGF-1/PI3K/Akt/S6 and IGF-1/MEK/ERK/S6 pathways after postnatal isoflurane exposure. Primary cultured day 7 hippocampal neurons or postnatal day 7 rats were treated with or without 1.5% isoflurane for different time. After that, neuronal cultures or hippocampi in both sides were harvested to detect the expression levels of phosphorylated S6 ribosomal protein or activities of IGF-1 and IGF-1 receptor. Then, we investigated the role of PI3K/Akt and MEK/ERK pathways in the IGF-1 induced S6 activity performance using different inhibitors of these two pathways above.2. To investigate the role of IGF-1/PI3K/Akt/S6 and IGF-1/MEK/ERK/S6 pathways in the isoflurane induced neuroapoptosis Primary cultured day 7 hippocampal neurons were treated with or without 1.5% isoflurane for different time. The apoptotic cells were assayed by FITC Annexin V detection and the expression level of cleaved caspase-3 was evaluated by Western blot. To investigate the role of IGF-1/PI3K/Akt/S6 and IGF-1/MEK/ERK/S6 pathways in the isoflurane induced neuroapoptosis, the neuronal cultures were pre-treated with 200 ng/ml IGF-1 or 5 u M PF-4708671 for 30 min following by treatment with 1.5% isoflurane for 6 h. The level of apoptotic cells were tested by FITC Annexin V detection. Also, the expression levels of Bad, Bcl-x L and cleaved caspase-3 were detected by Western blot. 3. To investigate the role of IGF-1/PI3K/Akt/S6 and IGF-1/MEK/ERK/S6 pathways in the isoflurane induced synaptogenesis impairment Primary cultured day 10 hippocampal neurons or postnatal day 7 rats were exposed with or without 1.5% isoflurane for different time. The cells or hippocampi in both sides were collected to detect the level of PSD95 and Gephyrin, which were makers of excitatory synapses and inhibitory synapses respectively. Then, primary cultured day 10 hippocampal neurons were treated with 200 ng/ml IGF-1 or 5 u M PF-4708671 for 30 min before exposure to 1.5% isoflurane for 6 h. The levels of PSD95 and Gephyrin were detected to investigate the role of IGF-1/PI3K/Akt/S6 and IGF-1/MEK/ERK/S6 pathways in the isoflurane induced synaptogenesis impairment. 4. To investigate the role of IGF-1/PI3K/Akt/S6 and IGF-1/MEK/ERK/S6 pathways in the isoflurane induced decline of learning and memory Postnatal day 7 rats were pre-exposed with 1.8 ug/g IGF-1 or 75 mg/kg PF-4708671 or 0.9% saline for 30 min prior to treatment with 1.5% isoflurane for 6 h. After treatment, the rats were returned to cage. On the second and third day, the postnatal rats received the same doses of IGF-1or PF-4708671 or saline. Until postnatal day 47, the locomotor activities of rats were measured by Open-field test. Two days later, Morris water maze wasused to detect the spatial learning and memory of rats.Results 1. Isoflurane inhibited the activity of ribosomal protein S6 in a dose-dependent manner and this effect lasted almost 24 hours. Further study showed that the S6 depression occurred in the neurons, especially in the CA3 area. Meanwhile, we found that isoflurane suppressed the activation of IGF-1 and IGF-1 receptor, which led to S6 inhibition through PI3K/Akt and MEK/ERK pathways. 2. FITC-Annexin V and cleaved caspase-3 detection all showed that exposure to 1.5% isoflurane for 3 h did not trigger neuroapoptosis, while treatment for 6 h or 12 h significantly increased the level of neuroapoptosis. We further showed that isoflurane decreased the ratio of Bcl-x L/Bad, and PF-4708671 worsened this situation, however, it could be reversed by IGF-1. 3. Isoflurane inhibited the expression of PSD95 in a dose-dependent manner and this effect lasted almost for 24 hours. The level of PSD95 further decreased when co-treated with PF-4708671, but returned to normal level after adding IGF-1. The present study also showed that isoflurane, IGF-1or PF-4708671 did not influence the Gephyrin expression. 4. Postnatal isoflurane exposure caused decline of spatial learning and memory in the adulthood. PF-4708671 aggravated this trend, but it could be rescued by IGF-1.Conclusions 1. Postnatal isoflurane exposure suppressed the activities of IGF-1/PI3K/Akt/S6 and IGF-1/MEK/ERK/S6 pathways. 2. Isoflurane decreased the ratio of Bcl-x L/Bad and triggered neuroapoptosis via inhibiting IGF-1/PI3K/Akt/S6 and IGF-1/MEK/ERK/S6 pathways. 3. Inhibition of IGF-1/PI3K/Akt/S6 and IGF-1/MEK/ERK/S6 pathways were involved in the isoflurane induced impairment of excitatory synapses formation. 4. Inhibition of IGF-1/PI3K/Akt/S6 and IGF-1/MEK/ERK/S6 pathways were associated with isoflurane induced decline of spatial learning and memory when grown up. |
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