Corneal lesions related to an anesthetic mixture of medetomidine, midazolam, and butorphanol treatment in rats.

An anesthetic mixture of medetomidine, midazolam and butorphanol (MMB) has been recently used in laboratory animals. We observed corneal opacity in nephrectomized rats that had undergone two operations under MMB anesthesia at 4 and 5 weeks of age. To evaluate the features of this corneal opacity, ophthalmic examinations were conducted in 83 nephrectomized rats, and 8 representative animals with corneal opacity were evaluated histopathologically 4 weeks after operation. The ophthalmic examinations revealed that 66/83 animals had corneal opacity, which was characterized histopathologically by mineralization with or without inflammation in the corneal stroma. In addition, to examine the possible causes of this corneal opacity, we investigated whether similar corneal changes were induced by the MMB anesthetic treatment in normal rats. The MMB anesthetic was administered twice to 4- and 5-week-old normal SD rats (5 animals/age) in the same manner as for the nephrectomized rats. Ophthalmic examinations were conducted in all the animals once a week, and the animals were necropsied 4 weeks after the first administration. In normal rats, similar corneal opacity was observed after the first administration, and increases in the severity and size of the corneal opacity were noted after the second administration. In conclusion, this study revealed the features of corneal opacity in rats undergoing nephrectomy under MMB anesthesia and the occurrence of similar corneal opacity in normal rats treated with MMB anesthetic. To the best of our knowledge, this is the first report of corneal opacity related to MMB anesthetic treatment in rats.

Testing for midazolam and oxycodone in blood after formalin-embalmment: About a complex medico-legal case.

The stability of compounds in formalin solution is an important factor for drug analysis in a toxicological investigation. In this article, the authors report a complex medico-legal case involving midazolam and oxycodone. The complexity of this case comes from the fact that the body was embalmed with formalin solution before the autopsy. This technique, called thanatopraxy, allows the preservation of corpses from decomposition, the destruction of a maximal number of micro-organisms, and the presentation of the body with a natural appearance to the family. Unfortunately, when thanatopraxy is performed before the collection of biological specimens, the toxicological results are not representative of the time of the death. In addition, the interpretation of the results is difficult, because formalin can cause oxidation of xenobiotics present in the body at the time of the death, alter the pH of the tissues and dilute the compounds. To document the chemical stability of midazolam and oxycodone in formalin solution and interpret the results, a stability study was conducted for 21 days. Blood containing midazolam and oxycodone was spiked with formalin, kept at 4°C and regularly tested for both drugs. This study showed a rapid degradation of midazolam and oxycodone (85% during the first 24 hours for oxycodone). In the peripheral blood of the victim, methanol (1.31 g/L), midazolam (74ng/mL) and oxycodone (152 ng/mL) were identified. According to the stability study, the measured concentrations in formalin fixed-tissues are to be interpreted very carefully, knowing that significant degradation has occurred.

Early Developmental Exposure to Repetitive Long Duration of Midazolam Sedation Causes Behavioral and Synaptic Alterations in a Rodent Model of Neurodevelopment.

There is a large body of preclinical literature suggesting that exposure to general anesthetic agents during early life may have harmful effects on brain development. Patients in intensive care settings are often treated for prolonged periods with sedative medications, many of which have mechanisms of action that are similar to general anesthetics. Using in vivo studies of the mouse hippocampus and an in vitro rat cortical neuron model we asked whether there is evidence that repeated, long duration exposure to midazolam, a commonly used sedative in pediatric intensive care practice, has the potential to cause lasting harm to the developing brain. We found that mice that underwent midazolam sedation in early postnatal life exhibited deficits in the performance on Y-maze and fear-conditioning testing at young adult ages. Labeling with a nucleoside analog revealed a reduction in the rate of adult neurogenesis in the hippocampal dentate gyrus, a brain region that has been shown to be vulnerable to developmental anesthetic neurotoxicity. In addition, using immunohistochemistry for synaptic markers we found that the number of presynaptic terminals in the dentate gyrus was reduced, while the number of excitatory postsynaptic terminals was increased. These findings were replicated in a midazolam sedation exposure model in neurons in culture. We conclude that repeated, long duration exposure to midazolam during early development has the potential to result in persistent alterations in the structure and function of the brain.

Disruption of brain MEK-ERK sequential phosphorylation and activation during midazolam-induced hypnosis in mice: Roles of GABAA receptor, MEK1 inactivation, and phosphatase MKP-3.

Midazolam is a positive allosteric modulator at GABAA receptor that induces a short hypnosis and neuroplasticity, in which the sequential phosphorylation of MEK1/2 and ERK1/2 was shown to play a role. This study investigated the parallel activation of p-MEK and p-ERK and regulatory mechanisms induced by midazolam through the stimulation of GABAA receptors in the mouse brain. During the time course of midazolam (60mg/kg)-induced sleep in mice (lasting for about 2h) p-Ser217/221 MEK1/2 was increased (+146% to +258%) whereas, unexpectedly, p-Tyr204/Thr202 ERK1/2 was found decreased (-16% to -38%), revealing uncoupling of MEK to ERK signals in various brain regions. Midazolam-induced p-MEK1/2 upregulation was prevented by pretreatment (30min) with flumazenil (10mg/kg), indicating the involvement of GABAA receptors. Also unexpectedly, midazolam-induced p-ERK1/2 downregulation was not prevented by flumazenil (10 or 30mg/kg). Notably, during midazolam-induced sleep the content of inactivated p-Thr286 MEK1, which can dampen ERK1/2 activation, was increased (+33% to +149%) through a mechanism sensitive to flumazenil (10mg/kg). Midazolam also increased MKP-3 (+13% to +73%) content and this upregulation was prevented by flumazenil (10mg/kg); an effect suggesting ERK inactivation because MKP-3 is the phosphatase selective for ERK1/2 dephosphorylation. The results indicate that during midazolam-induced sleep in mice there is an uncoupling of p-MEK (increased) to p-ERK (decreased) signals. p-ERK1/2 downregulation (not involving GABAA receptors) is the result of increased inactivated MEK1 and phosphatase MKP-3 (both effects involving GABAA receptors). These findings are relevant for the neurobiology and clinical use of benzodiazepines.

Effects of pentobarbital, isoflurane, or medetomidine-midazolam-butorphanol anesthesia on bronchoalveolar lavage fluid and blood chemistry in rats.

Bronchoalveolar lavage fluid (BALF) is commonly examined for pulmonary toxicity in animal studies. Two common means of anesthesia before euthanasia and bronchoalveolar lavage in rats are intraperitoneal injection of pentobarbital and inhalation of isoflurane. Medetomidine-midazolam-butorphanol is an alternative anesthesia to pentobarbital for animal welfare; however, the effect of this combination on BALF and blood chemistry is unknown. Here, we compared the effects of anesthesia by intraperitoneal injection of pentobarbital or one of two combinations of medetomidine-midazolam-butorphanol (dose, 0.375-2.0-2.5 or 0.15-2.0-2.5 mg/kg) or by inhalation of isoflurane on BALF and blood chemistry in rats with or without pulmonary inflammation. In BALF, we determined total protein, albumin, lactate dehydrogenase, total cell count and neutrophil count. In serum, we conducted a general chemistry screen. After anesthesia with pentobarbital or isoflurane, there were no significant differences between any of the BALF or blood chemistry parameters with or without inflammation. After anesthesia with either of the combinations of medetomidine-midazolam-butorphanol, lactate dehydrogenase, total cell count, neutrophil count, and almost all of the blood chemistry parameters were comparable with those observed after pentobarbital or isoflurane; however, BALF albumin and serum glucose were significantly increased in rats without inflammation. After the combination of low-dose medetomidine in rats with inflammation, BALF parameters were comparable with those observed after pentobarbital or isoflurane. Our results show that, of the anesthetics examined, inhalation of isoflurane is the most appropriate means of anesthesia when examining BALF or serum for toxicity studies in rats.

Cytotoxic Effects of Intranasal Midazolam on Nasal Mucosal Tissue.

The aim of this experimental study was to investigate the cytotoxic effects of intranasal midazolam on nasal mucosal tissue in rats. Forty healthy rats were randomly divided into 5 groups. Group 1 (n = 8) was the control group, group 2 (n = 8) received intranasal saline, group 3 (n = 8) received intranasal midazolam, group 4 (n = 8) received intraperitoneal saline, and group 5 received intraperitoneal midazolam (n = 8). Midazolam and saline were administered via intraperitoneal and intranasal routes at doses of 200 µg/kg. Nasal septal mucosal stripe tissues were removed at the 6th hour. All materials were evaluated according to Ki67 and p53 staining to evaluate proliferation and apoptosis, respectively, and hemotoxylin and eosin staining was performed for histopathology evaluation. Ki67 values and inflammation in group 3 were statistically higher compared to group 1, group 2, and group 4. P53 values in group 3 were statistically higher compared to group 1. Assessment of subepithelial edema between group 3 and the other groups revealed no statistically significant differences. Assessment of cilia loss between group 3 and group 1, group 2, and group 4 revealed no statistically significant difference. The evaluation of goblet cell loss between group 3 and group 1 revealed a statistically significant difference. Intranasal midazolam had adverse effects on nasal mucosa. However, intranasal midazolam is as safe as systemic midazolam administration with respect to nasal mucosa.

Early Exposure to General Anesthesia Disrupts Spatial Organization of Presynaptic Vesicles in Nerve Terminals of the Developing Rat Subiculum.

Exposure to general anesthesia (GA) during critical stages of brain development induces widespread neuronal apoptosis and causes long-lasting behavioral deficits in numerous animal species. Although several studies have focused on the morphological fate of neurons dying acutely by GA-induced developmental neuroapoptosis, the effects of an early exposure to GA on the surviving synapses remain unclear. The aim of this study is to study whether exposure to GA disrupts the fine regulation of the dynamic spatial organization and trafficking of synaptic vesicles in presynaptic terminals. We exposed postnatal day 7 (PND7) rat pups to a clinically relevant anesthetic combination of midazolam, nitrous oxide, and isoflurane and performed a detailed ultrastructural analysis of the synaptic vesicle architecture at presynaptic terminals in the subiculum of rats at PND 12. In addition to a significant decrease in the density of presynaptic vesicles, we observed a reduction of docked vesicles, as well as a reduction of vesicles located within 100 nm from the active zone, in animals 5 days after an initial exposure to GA. We also found that the synaptic vesicles of animals exposed to GA are located more distally with respect to the plasma membrane than those of sham control animals and that the distance between presynaptic vesicles is increased in GA-exposed animals compared to sham controls. We report that exposure of immature rats to GA during critical stages of brain development causes significant disruption of the strategic topography of presynaptic vesicles within the nerve terminals of the subiculum.

Early Exposure to General Anesthesia with Isoflurane Downregulates Inhibitory Synaptic Neurotransmission in the Rat Thalamus.

Recent evidence supports the idea that common general anesthetics (GAs) such as isoflurane (Iso) and nitrous oxide (N2O; laughing gas) are neurotoxic and may harm the developing mammalian brain, including the thalamus; however, to date very little is known about how developmental exposure to GAs may affect synaptic transmission in the thalamus which, in turn, controls the function of thalamocortical circuitry. To address this issue we used in vitro patch-clamp recordings of evoked inhibitory postsynaptic currents (eIPSCs) from intact neurons of the nucleus reticularis thalami (nRT) in brain slices from rat pups (postnatal age P10-P18) exposed at age of P7 to clinically relevant GA combinations of Iso and N2O. We found that rats exposed to a combination of 0.75 % Iso and 75 % N2O display lasting reduction in the amplitude and faster decays of eIPSCs. Exposure to sub-anesthetic concentrations of 75 % N2O alone or 0.75 % Iso alone at P7 did not affect the amplitude of eIPSCs; however, Iso alone, but not N2O, significantly accelerated decay of eIPSCs. Anesthesia with 1.5 % Iso alone decreased amplitudes, caused faster decay and decreased the paired-pulse ratio of eIPSCs. We conclude that anesthesia at P7 with Iso alone or in combination with N2O causes plasticity of eIPSCs in nRT neurons by both presynaptic and postsynaptic mechanisms. We hypothesize that changes in inhibitory synaptic transmission in the thalamus induced by GAs may contribute to altered neuronal excitability and consequently abnormal thalamocortical oscillations later in life.

Reducing risk of overdose with midazolam injection in adults: an evaluation of change in clinical practice to improve patient safety in England.

RATIONALE AIMS AND OBJECTIVES: This study sought to evaluate potential reductions in risk associated with midazolam injection, a sedating medication, following a UK National Patient Safety Alert. This alert, 'Reducing risk of overdose with midazolam injection in adults', was sent to all National Health Service organizations as a Rapid Response Report detailing actions services should take to minimize risks. METHOD: To evaluate any potential changes arising from this alert, a number of data sources were explored including reported incidents to a national reporting system for health care error, clinician survey and audit data, pharmaceutical purchasing patterns and feedback from National Health Service managers. RESULTS: Prior to the Rapid Response Report, 498 incidents were received by the National Patient Safety Agency including three deaths. Post-implementation of the Rapid Response Report (June 2009), no incidents resulting in death or severe harm had been received. All organizations reported having completed the Rapid Response Report actions. Purchase and use of risk-prone, high-strength sedating midazolam by health care organizations decreased significantly as did the increased use of safer, lower strength doses (as recommended in the Rapid Response Report). CONCLUSIONS: Organizations can achieve safer medication practices, better knowledge, awareness and implementation of national safer practice recommendations. Risks from inadvertent overdose of midazolam injection were reduced post-implementation of national recommendations. Ongoing monitoring of this particular adverse event will be required with a sustained patient safety message to health services to maintain awareness of the issue and reduction in the number of midazolam-related errors.

Mechanisms underlying midazolam-induced peripheral nerve block and neurotoxicity.

BACKGROUND AND OBJECTIVES: The benzodiazepine midazolam has been reported to facilitate the actions of spinally administrated local anesthetics. Interestingly, despite the lack of convincing evidence for the presence of γ-aminobutyric acid type A (GABAA) receptors along peripheral nerve axons, midazolam also has been shown to have analgesic efficacy when applied alone to peripheral nerves.These observations suggest midazolam-induced nerve block is due to another site of action. Furthermore, because of evidence indicating that midazolam has equal potency at the benzodiazepine site on the GABAA receptor and the 18-kd translocator protein (TSPO), it is possible that at least the nerve-blocking actions of midazolam are mediated by this alternative site of action. METHODS: We used the benzodiazepine receptor antagonist flumazenil, and the TSPO antagonist PK11195, with midazolam on rat sciatic nerves and isolated sensory neurons to determine if either receptor mediates midazolam-induced nerve block and/or neurotoxicity. RESULTS: Midazolam (300 µM)-induced block of nerve conduction was reversed by PK11195 (3 µM), but not flumazenil (30 µM). Midazolam-induced neurotoxicity was blocked by neither PK11195 nor flumazenil. Midazolam also causes the release of Ca from internal stores in sensory neurons, and there was a small but significant attenuation of midazolam-induced neurotoxicity by the Ca chelator, BAPTA. BAPTA (30 µM) significantly attenuated midazolam-induced nerve block. CONCLUSIONS: Our results indicate that processes underlying midazolam-induced nerve block and neurotoxicity are separable, and suggest that selective activation of TSPO may facilitate modality-selective nerve block while minimizing the potential for neurotoxicity.

Midazolam suppresses osteogenic differentiation of human bone marrow-derived mesenchymal stem cells.

OBJECTIVE: Previous study showed that peripheral-type benzodiazepine receptors (PBRs) are expressed in human mesenchymal stem cells (hMSCs) and diazepam was found to inhibit hMCSs viability in high concentration. Midazolam, a benzodiazepine derivative, is widely used as an intravenous sedative in hospital. Peripheral-type benzodiazepine receptors (PBRs) affect a broad spectrum of cellular functions. We tested the cell viability and osteogenic differentiation of hMSCs. PATIENTS AND METHODS: Bone marrow was collected from 12 patients during the operation of spine internal fixation. Cultivated with basal medium, the hBMSCs were incubated with or without midazolam (0.1, 1, 5, 10, 15, 20 µM, respectively). Cell viability were tested with MTS assay after 2, 4, 6 hours respectively. Cell morphology was observed and recorded at 6 hour. After cultivated with osteogentic medium, the hBMSCs were incubated with or without midazolam (5, 10, 15, 20 µM, respectively). Alkaline phosphatase (ALP) activity and alizarin red S staining were measured. Cultivated with osteogentic medium with or without treatment of 15 µM midazolam, the mRNA expression of ALP, type 1 collagen (COL1), Runx2 and PPARγ was analyzed by real-time RT-PCR. RESULTS: The treatments of midazolam inhibited cell viability to 85%-16% respectively (p < 0.05). Rounded up phenomenon with floating cells, Membrane-blebbed cells and cytoplasmic contraction were observed after 10, 15 or 20 µM midazolam treatment. The ALP activity and Calcium deposition of hBMSCs exposed to 15 and 20 µM midazolam was significantly inhibited at 7, 14 and 21 days (p < 0.05). And the mRNA expression of ALP, COL1 and PPARγ was significantly suppressed in the hBMSCs cultured with 15 µM midazolam (p < 0.05). CONCLUSIONS: Midazolam exert negative effect on cell viability and osteogenic differentiation of cultured hBMSCs. During sedation in critical care, the use of midazolam may suppress activity of hBMSCs.

Midazolam exacerbates morphine tolerance and morphine-induced hyperactive behaviors in young rats with burn injury.

Midazolam and morphine are often used in pediatric intensive care unit (ICU) for analgesia and sedation. However, how these two drugs interact behaviorally remains unclear. Here, we examined whether (1) co-administration of midazolam with morphine would exacerbate morphine tolerance and morphine-induced hyperactive behaviors, and (2) protein kinase C (PKC) would contribute to these behavioral changes. Male rats of 3-4 weeks old were exposed to a hindpaw burn injury. In Experiment 1, burn-injured young rats received once daily saline or morphine (10mg/kg, subcutaneous, s.c.), followed 30min later by either saline or midazolam (2mg/kg, intraperitoneal, i.p.), for 14 days beginning 3 days after burn injury. In Experiment 2, young rats with burn injury were administered with morphine (10mg/kg, s.c.), midazolam (2mg/kg, i.p.), and chelerythrine chloride (a non-specific PKC inhibitor, 10nmol, intrathecal) for 14 days. For both experiments, cumulative morphine anti-nociceptive dose-response (ED50) was tested and hyperactive behaviors such as jumping and scratching were recorded. Following 2 weeks of each treatment, ED50 dose was significantly increased in rats receiving morphine alone as compared with rats receiving saline or midazolam alone. The ED50 dose was further increased in rats receiving both morphine and midazolam. Co-administration of morphine and midazolam also exacerbated morphine-induced hyperactive behaviors. Expression of the NR1 subunit of the N-methyl-d-aspartate (NMDA) receptor and PKCγ in the spinal cord dorsal horn (immunohistochemistry; Western blot) was upregulated in burn-injured young rats receiving morphine alone or in combination with midazolam, and chelerythrine prevented the development of morphine tolerance. These results indicate that midazolam exacerbated morphine tolerance through a spinal NMDA/PKC-mediated mechanism.

[Comparison of dexmedetomidine and midazolam on neurotoxicity in neonatal mice].

Dexmedetomidine and midazolam have been widely used in clinical anesthesia and intensive care unit sedation. These two drugs differ in sedative mechanism. We hypothesized that the neurotoxicity of repeated exposure to dexmedetomidine or midazolam for neonatal mice might be different. Twenty four mice of postnatal day 8 were randomly divided into control (n=8), dexmedetomidine (n=8) and midazolam group (n=8) respectively. In the three groups, saline(10mL/kg), dexmedetomidine(10microg/kg) or midazolam(40mg/kg) was injected intraperitoneally once a day, in the next five days, respectively. Then the brains of the mice in the three qroups were removed and cryosectioned. Apoptotic neural cell in hippocampus region was detected with terminal deoxynucleotydyl transferase -mediated dUTP nick end labeling(TUNEL). Bcl2 and Bax protein expression level in the hippocampus were determined by immunofluorescent staining. In the present study, the number of TUNEL-positive cells from midazolam group ((20 +/-3) /mm2) was larger than that from dexmedetomidine group ((15+/-2) /mm2, P<0. 05) and control group((13+/-3) /mm2, P<0. 05); Bcl-2 protein quantity in hippocampus from control group((790+/-103)/mm2)was significantly lower than that in midazolam group((1187+/- 162)/mm2, P<0.05)and dexmedetomidine group((890+/-125)/mm2, P<0. 05). Bax protein level in control group((942+/-104)/mm2) was also significantly lower than that in midazolam group((1839+/-160)/mm2, P<0. 05)and dexmedetomidine group((1143+/-125)/mm2, P<0. 05); Bax/Bcl-2 ratio in midazolam group(0. 64+/-0. 13) was significantly lower than that in dexmedetomidine group(0. 78 +/-0. 14, P<0. 05) and control group(0. 84+/-0. 15, P<0. 05). Our results suggest that dexmedetomidine has lower neurotoxicity than midazolam for neonatal mice.

Cytotoxicity and type of cell death induced by midazolam in human oral normal and tumor cells.

BACKGROUND: Intravenous anesthetics have been used during the treatment of various malignant tumors, however, their effects on oral tissues is not well-understood. In the present study, the cytotoxicity of five intravenous anesthetics towards oral tumor and normal cells was compared. MATERIALS AND METHODS: Tumor specificity index was determined by the ratio of the mean 50% cytotoxic concentration for normal cells to that for tumor cells. Apoptosis induction was monitored by internucleosomal DNA fragmentation and caspase-3, -8, and -9 activation. Fine cell structure was observed under transmission electron microscopy. RESULTS: Benzodiazepines (midazolam and diazepam) exhibited higher cytotoxicity than barbiturates (thiopental and thiamylal), whereas propofol had the intermediate range of cytotoxicity. Midazolam showed the highest cytotoxicity. HL-60 cells were the most sensitive to midazolam, followed by epidermal keratinocytes, oral squamous cell carcinoma (OSCC), glioblastoma and then oral normal cells. Midazolam did not induce the production of apoptosis markers such as internucleosomal DNA fragmentation and activation of caspase-3, -8 and -9, but did induce the appearance of many vacuoles, mitochondrial swelling and cell membrane rupture in OSCC cell lines (HSC-2 and HSC-4) cells. The cytotoxicity of midazolam was not reduced by pre-treatment with autophagy inhibitors (3-methyladenine and bafilomycin A1). CONCLUSION: These results suggest that midazolam may induce necrotic cell death, rather than apoptosis or autophagy, in OSCC cell lines.

Role of state-dependency in memory impairment induced by acute administration of midazolam in mice.

Although the memory deficits produced by pre-training benzodiazepines administration have been extensively demonstrated both in humans and in animal studies, there is considerable controversy about the involvement of the state-dependency phenomenon on benzodiazepines-induced anterograde amnesia. The present study aimed to characterize the role of state-dependency on memory deficits induced by the benzodiazepine midazolam (MID) in mice submitted to the plus-maze discriminative avoidance task (PM-DAT). This animal model concomitantly evaluates learning and retention of discriminative avoidance task, exploratory habituation as well as anxiety-like behavior and motor activity. Mice received 2mg/kg MID before training and/or before testing in the PM-DAT. Pre-training (but not pre-test) MID administration impaired the retention of the discriminative avoidance task, which was not counteracted by a subsequent pre-test administration of this drug, thus refuting the role of state-dependency. Conversely, the pre-training administration of MID also led to an impairment of the habituation of exploration in the PM-DAT (an animal model of non-associative memory). This habituation deficit was state-dependent since it was absent in pre-training plus pre-test MID treated mice. Concomitantly, MID pre-training administration induced anxiolytic effects and diminished the aversive effectiveness of the aversive stimuli of the task, leading to an impairment of the acquisition of the discriminative avoidance task. Our findings suggest that pre-training benzodiazepine administration can impair the retention of different types of memory by producing specific deleterious effects on learning or by inducing state-dependent memory deficits.

Toxic effects of midazolam on differentiating neurons in vitro as a consequence of suppressed neuronal Ca2+-oscillations.

BACKGROUND: In immature neurons anesthetics induce apoptosis and influence neuronal differentiation. Neuronal Ca(2+)-oscillations regulate differentiation and synaptogenesis. We examined the effects of the long-term blockade of hippocampal Ca(2+)-oscillations with midazolam on neuronal synapsin expression. MATERIAL AND METHODS: Hippocampal neurons were incubated at day 15 in culture with the specific GABA(A) receptor agonist muscimol (50µM) or with midazolam (100 and 300nM), respectively, for 24h. TUNEL and activated-Caspase-3 staining were used to detect apoptotic neurons. Ca(2+)-oscillations were detected using the Ca(2+)-sensitive dye FURA-2 and dual wavelength excitation fluorescence microscopy. Synapsin was identified with confocal anti-synapsin immunofluorescence microscopy. RESULTS: Muscimol, when applied for 24h, decreased the amplitude and frequency Ca(2+)-oscillations significantly. Midazolam concentration-dependently suppressed the amplitude and frequency of the Ca(2+)-oscillations. This was associated by a downregulation of the synapsin expression 24h after washout. CONCLUSION: Neuronal Ca(2+)-oscillations mediate neuronal differentiation and are involved in synaptogenesis. By acting via the GABA(A) receptor, midazolam exerts its toxic effect through the suppression of neuronal Ca(2+)-oscillations, a reduction in synapsin expression and consecutively reduced synaptic integrity.

Benzodiazepines medazepam and midazolam are activators of pregnane X receptor and weak inducers of CYP3A4: investigation in primary cultures of human hepatocytes and hepatocarcinoma cell lines.

Benzodiazepines have wide-spread used in pharmacotherapy for their anxiolytic, myorelaxant, hypnotic, amnesic and anticonvulsive properties. Despite benzodiazepines are used in clinics over 50 years, they have not been surprisingly tested for capability to induce major drug-metabolizing cytochromes P450. In the current study, we have examined the potency of Alprazolam, Bromazepam, Chlordiazepoxide, Clonazepam, Diazepam, Lorazepam, Medazepam, Midazolam, Nitrazepam, Oxazepam, Tetrazepam and Triazolam to induce CYP1A2 and CYP3A4 in primary cultures of human hepatocytes. Benzodiazepines were tested in therapeutic concentrations and in concentrations corresponding to their plasma levels in intoxicated patients. We found weak but significant induction of CYP3A4 mRNA by Midazolam and Medazepam, while other benzodiazepines did not induce CYP3A4 expression. None of the tested compounds induced CYP1A2 mRNA in three independent human hepatocytes cultures. In addition, employing gene reporter assays with transiently transfected hepatocarcinoma cells, we found that tested benzodiazepines did not activate aryl hydrocarbon receptor (AhR), whereas Midazolam and Medazepam slightly activated pregnane X receptor (PXR). Consistently, two-hybrid mammalian assay using hybrid fusion plasmids GAL4-PXR ligand-binding domain (LBD) and VP16-SRC-1-receptor-interacting domain (RID) confirmed PXR activation by Midazolam and Medazepam. In conclusion, Alprazolam, Bromazepam, Chlordiazepoxide, Clonazepam, Diazepam, Lorazepam, Nitrazepam, Oxazepam, Tetrazepam and Triazolam can be considered as safe drugs in term of their inability to induce PXR- and AhR-dependent cytochrome P450 enzymes CYP1A2 and CYP3A4. Medazepam and Midazolam slightly activated pregnane X receptor and displayed weak potency to induce CYP3A4 mRNA in human hepatocytes.

The effect of midazolam on mouse Leydig cell steroidogenesis and apoptosis.

The peripheral-type benzodiazepine receptor (PBR), a putative receptor in Leydig cells, modulates steroidogenesis. Since benzodiazepines are commonly used in regional anesthesia, their peripheral effects need to be defined. Therefore, this study set out to investigate in vitro effects of the benzodiazepine midazolam (MDZ) on Leydig cell steroidogenesis, and the possible underlying mechanisms. The effects of MDZ on steroidogenesis in primary mouse Leydig cells and MA-10 Leydig tumor cells were determined by radioimmunoassay. PBR, P450scc, 3beta-HSD and StAR protein expression induced by MDZ was determined by Western blotting. Inhibitors of the signal transduction pathway and a MDZ antagonist were used to investigate the intracellular cascades activated by MDZ. In both cell types, MDZ-stimulated steroidogenesis in dose- and time-dependent manners, and induced the expression of PBR and StAR proteins, but had no effect on P450scc and 3beta-HSD expressions. Moreover, H89 (PKA inhibitor) and GF109203X (PKC inhibitor) attenuated MDZ-stimulated steroid production. Interestingly, the MDZ antagonist (flumazenil) did not decrease MDZ-induced steroid production in both cell types. These results highly indicated that MDZ-induced steroidogenesis in mouse Leydig cells via PKA and PKC pathways, along with the expression of PBR and StAR proteins. In addition, MDZ at high dosages induced rounding-up, membrane blebbing, and then death in MA-10 cells. In conclusion, midazolam could induce Leydig tumor cell steroidogenesis, and high dose of midazolam could induce apoptosis in Leydig tumor cells.

Additives in intravenous anesthesia modulate pulmonary inflammation in a model of LPS-induced respiratory distress.

BACKGROUND: It has been suggested that propofol with ethylenediaminetetraacetic acid (EDTA) can modulate the systemic inflammatory response. Prolonged higher levels of pulmonary inflammation are associated with poor outcome of patients with acute lung injury. In the present study, we hypothesized that pulmonary inflammation could be modulated by propofol with EDTA compared with propofol with sulfite. METHODS: Respiratory distress was induced in rats (n=25) by intratracheal nebulization of lipopolysaccharide (LPS). After 24 h, animals were randomized to either propofol with EDTA (Propofol(EDTA)), propofol with sulfite (Propofol(sulfite)) or ketamine/midazolam (Ket/Mid); control animals received saline (n=30). Animals were ventilated for 4 h and blood gases were measured hourly. Bronchoalveolar lavage (BAL) was performed for cytokine analysis of: tumor necrosis factor (TNF), interleukin (IL)-6 and macrophage inflammatory protein (MIP)-2. RESULTS: LPS led to increased pulmonary inflammation in all groups compared with the control groups. Gas exchange deteriorated over time only in the LPS Propofol(sulfite) group and was significantly lower than the Ket/Mid group. Only IL-6 was significantly higher in the LPS Propofol(sulfite) group compared with both the Ket/Mid group and the Propofol(EDTA) group. CONCLUSION: Pulmonary IL-6 can be modulated by additives in systemic anesthesia. IMPLICATION STATEMENT: This study demonstrates that pulmonary inflammation caused by direct lung injury can be modulated by intravenous anesthesia used in critically ill patients.

Midazolam administered to 8-day-old mouse pups for three weeks induces cerebellar cortex alterations.

Midazolam (MDZ) administered during the neonatal period induces histological changes in the cerebellar cortex of mouse pups. We investigated whether these changes are similar to those induced by prenatal exposure to diazepam (DZ). Two neonatal ICR-strain mouse groups were injected daily from day 8 to 29. The first group (MDZ) was treated with a single daily MDZ dose (2.0 mg/kg/body weight) and the second with saline solution. The mice (10 MDZ and 10 control), were sacrificed in a CO2 atmosphere at 30 days; the cerebellum was fixed in 2.5% glutaraldehyde, post-fixed in 1% OsO4 and embedded in epoxy resin. Semifine sections were stained with toluidine blue and observed under a light microscope. The cerebellar cortex of the MDZ group was thinner than that of controls. The cortex showed a reduced number of Purkinje cells, some with a shrunken cytoplasm and degenerative signs. The dendrite tree was swollen with disoriented branches. Numerous granular cells remained in the molecular layer with few in the granular layer (p<0.05). The nucleus of Purkinje cells and granular cells showed atypical heterochromatin distribution, irregular nuclear surface, and voluminous nucleoli. The neuropile and myelin fibers showed delayed differentiation. Postnatal exposure to MDZ produces similar histological changes in the cerebellar cortex of the 30-day-old mouse treated for 21 days, to those induced by prenatal exposure to DZ.