Midazolam Activates Rat Cerebral Neuron Caspase-3 And Its Mechanism

Background and objectiveAmnesia, one of the important reasons for postoperative cognitive dysfunction, can be made by sedative and anesthetic agents in addition to hypnosis, sedation and anesthesia. As a notable feature of midazolam, anterograde amnesia can have effects on learning and memory even blow the dose of sedation (3mg/kg), and its mechanisms remain unclear [1].γ-aminobutyric acid-A (GABAA) receptor, one of inhibitory ionotropic receptor of amino acids, takes part in the establishment and regulation of potential in long-term potentiation (LTP) and long-term depression (LTD). So it plays important roles in the development of the central nervous system and learning and memory abilities. While regulating neuronal Ca²⁺ flow and excitement, it has complex regulation on synapse transmission, neuronal plasticity and growth. Recently, it was reported that infant rats exposure to sedative and anesthetic agents type of GABAA agonist, caused widespread apoptotic neurodegeneration in their developing brain. The human brain growth spurt period starts at the beginning of the trimester and ends several years after birth, thus the development of central nervous system of fetal and infant is in a sensitive period and peak time. Whether it will have impacts on human infants when exposure to sedative and anesthetic agents in this neurodevelopmental age? Midazolam, a GABAA agonist, is commonly used in pediatric and obstetric operation as sedative and anesthetic agents. The aim of our study was to observe effect of midazolam on the activation of caspase-3 and expressions of bax and bcl-2 in cerebral cortex neurons of infant rat, and explore the possible mechanisms, so as to found a basis for the use of clinical sedative and anesthetic agents.MethodsThe experiment consisted of part A and B, and totally 104 postnatal day 7 Sprague Dawley rats of either sex were enrolled. In part A, 24 rats were randomly divided into 4 groups according to the time points after intraperitoneal injection of 18.0 mg/kg midazolam (n=6 each group), i.e., group A0 without midazolam treatments as control, group A2 (15 min), group A3 (30 min), and group A4 (60 min). In part B, 80 rats were randomly divided into groups B45, B90, B135 and B180 (n=16 each group): according to the different dose of midazolam (4.5, 9.0, 14.5, 18.0 mg/kg) and group B0 as control received 18.0 mg/kg normal saline. Both control and experimental rats after treatment were maintained in a warm chamber (28℃) for a 6-hour observation period. The rats of each group were divided into 3 subgroups, then anesthetized to take brain specimen. After the rats were perfused transcardially with 4% paraformaldehyde PBS buffer, the brain tissue sections were made for C3A expression with immunohistochemistry. Other cerebral cortex tissues were fixed with 2.5% glutaraldehyde for electron microscopy. The last part was for bax and bcl-2 expressions with RT-PCR and Western blot.Results1. Blood gas values in 15, 30 and 60 min after single interaperitoneal injection of 18.0 mg/kg midazolam revealed there is no evidence of hypoxia/ischemia. Oxygen saturation, PaO2, PaCO2, pH, HCO3-, Lac and Anion Gap had no significant difference with those of normal rats (P>0.05). Single intraperitoneal injection of 18 mg/kg midazolam does not induce hypoxic in P7 rats, and there is no sign of respiratory and metabolic acid-base imbalance.2. Histological evaluation showed a significantly enhanced caspase-3 activation in the cerebral cortex of rats treated with 9.0, 13.5 and 18.0 mg/kg midazolam in these rats (P<0.01).But there was no such significant change in rats with 4.5mg/kg midazolam treatment (P>0.05).3. Transmission electron microscopy demonstrated that in 6 h post-treatment, the typical apoptotic neurons appeared in experimental rats. Ultrastrctural changes met classical criteria for neuroapoptosis, including budding, dense flocculent compacts in the cytoplasm, condensation and marginataion of chromatin, deformed cell shape, and membrane shrinkage.4. Bax and bcl-2 were both expressed at mRNA and protein levels in the cerebral cortex neurons in the normal juvenile rats. Their mRNA and protein expressions were increased after the rat treated with a single does of midazolam (4.5, 9.0, 13.5 and 18.0 mg/kg) (P<0.05). The bax mRNA and protein, and bcl-2 mRNA was up-regulated in a positively dose-dependent manner, while bcl-2 protein demonstrated in a negative way (P>0.05).Conclusions1. Exposure to the range of midazolam 9-18mg/kg as a single one-time injection may cause a short-term increase in caspase-3 activation and neuroapoptosis in cerebral cortex neurons in a dose dependent manner.2. Bax and bcl-2 expressions at mRNA and protein levels in the cerebral cortex neurons are short-term elevated after treatment of midazolam in the range of 4.5-18mg/kg. The bax mRNA and its protein expressions are relevent to durg does, the same as bcl-2 mRNA.3. Upregulate the expressions of mRNA and protein of bax, and activate Cyt c- dependent pathway would be one of the mechanisms of short-term increase in caspase-3 activation and neuroapoptosis in cerebral cortex neurons of infant rats treated with midazolam.