GRT-X Stimulates Dorsal Root Ganglia Axonal Growth in Culture via TSPO and Kv7.2/3 Potassium Channel Activation.

GRT-X, which targets both the mitochondrial translocator protein (TSPO) and the Kv7.2/3 (KCNQ2/3) potassium channels, has been shown to efficiently promote recovery from cervical spine injury. In the present work, we investigate the role of GRT-X and its two targets in the axonal growth of dorsal root ganglion (DRG) neurons. Neurite outgrowth was quantified in DRG explant cultures prepared from wild-type C57BL6/J and TSPO-KO mice. TSPO was pharmacologically targeted with the agonist XBD173 and the Kv7 channels with the activator ICA-27243 and the inhibitor XE991. GRT-X efficiently stimulated DRG axonal growth at 4 and 8 days after its single administration. XBD173 also promoted axonal elongation, but only after 8 days and its repeated administration. In contrast, both ICA27243 and XE991 tended to decrease axonal elongation. In dissociated DRG neuron/Schwann cell co-cultures, GRT-X upregulated the expression of genes associated with axonal growth and myelination. In the TSPO-KO DRG cultures, the stimulatory effect of GRT-X on axonal growth was completely lost. However, GRT-X and XBD173 activated neuronal and Schwann cell gene expression after TSPO knockout, indicating the presence of additional targets warranting further investigation. These findings uncover a key role of the dual mode of action of GRT-X in the axonal elongation of DRG neurons.

The Comprehensive Steroidome in Complete TSPO/PBR Knockout Mice under Basal Conditions.

The 18 kDa translocator protein (TSPO/PBR) is a multifunctional evolutionary highly conserved outer mitochondrial membrane protein. Decades of research has reported an obligatory role of TSPO/PBR in both mitochondrial cholesterol transport and, thus, steroid production. However, the strict dependency of steroidogenesis on TSPO/PBR has remained controversial. The aim of this study was to provide insight into the steroid profile in complete C57BL/6-Tspotm1GuWu(GuwiyangWurra)-knockout male mice (TSPO-KO) under basal conditions. The steroidome in the brain, adrenal glands, testes and plasma was measured by gas chromatography coupled to tandem mass spectrometry (GC-MS/MS). We found that steroids present in wild-type (WT) mice were also detected in TSPO-KO mice, including pregnenolone (PREG), progestogens, mineralo-glucocorticosteroids and androgens. The concentrations of PREG and most metabolites were similar between genotypes, except a significant decrease in the levels of the 5α-reduced metabolites of progesterone (PROG) in adrenal glands and plasma and of the 5α-reduced metabolites of corticosterone (B) in plasma in TSPO-KO compared to WT animals, suggesting other regulatory functions for the TSPO/PBR. The expression levels of the voltage-dependent anion-selective channel (VDAC-1), CYP11A1 and 5α-reductase were not significantly different between both groups. Thus, the complete deletion of the tspo gene in male mice does not impair de novo steroidogenesis in vivo.

Midazolam exhibits antitumour and anti-inflammatory effects in a mouse model of pancreatic ductal adenocarcinoma.

BACKGROUND: Anaesthesia and perioperative management contribute to long-term outcomes of patients with cancer, including pancreatic ductal adenocarcinoma. We assessed the antitumour, anti-inflammatory, and analgesic effects of midazolam on LSL-KrasG12D/+;Trp53flox/flox;Pdx-1cre/+ transgenic mice with pancreatic ductal adenocarcinoma. METHODS: Six-week-old transgenic mice were administered midazolam 30 mg kg-1 day-1 p.o. (n=13); midazolam 30 mg kg-1 day-1 with 1-(2-chlorophenyl)-N-methyl-N(1-methylpropyl)-3-isoquinoline carboxamide (PK11195) 3 mg kg-1 day-1 i.p., a peripheral benzodiazepine receptor antagonist (n=10); or vehicle (water; n=14) until the humane endpoint. Cancer-associated pain was evaluated using hunching score and mouse grimace scale. Tumour stage and immuno-inflammatory status were determined histopathologically. Anti-proliferative and apoptotic potentials of midazolam were investigated using mouse pancreatic ductal adenocarcinoma cell lines. RESULTS: Midazolam significantly inhibited tumour size and proliferative index of Ki-67 and cyclins in pancreatic ductal adenocarcinoma, which was blocked by administration of PK11195. Local myeloperoxidase+ tumour-associated neutrophils, arginase-1+ M2-like tumour-associated macrophages, and CD11b+Ly-6G+ polymorphonuclear myeloid-derived suppressor cells were reduced by midazolam, which was antagonised by administration of PK11195. Hunching and mouse grimace scale were improved by midazolam, whereas the scores increased with midazolam+PK11195 treatment. Plasma pro-inflammatory cytokines, such as interleukin-6 and CC chemokine ligand (CCL)2, CCL3, and CCL5, were reduced by midazolam, whereas these cytokines increased with PK11195. Midazolam inhibited pancreatic ductal adenocarcinoma proliferation through downregulation of cyclins and cyclin-dependent kinases and induced apoptosis in vitro. CONCLUSIONS: These results suggest that midazolam inhibits pancreatic ductal adenocarcinoma proliferation and local infiltration of tumour-associated neutrophils, tumour-associated macrophages, and polymorphonuclear myeloid-derived suppressor cells, thereby inhibiting pancreatic ductal adenocarcinoma progression.

Neuroprotective effect of mitochondrial translocator protein ligand in a mouse model of tauopathy.

BACKGROUND: The translocator protein (TSPO) has been identified as a positron emission tomography (PET)-visible biomarker of inflammation and promising immunotherapeutic target for the treatment of Alzheimer's disease (AD). While TSPO ligands have been shown to reduce the accumulation of the toxic Alzheimer's beta-amyloid peptide, their effect on tau pathology has not yet been investigated. To address this, we analyzed the effects of TSPO ligand, Ro5-4864, on the progression of neuropathology in rTg4510 tau transgenic mice (TauTg). METHODS: Brain atrophy, tau accumulation, and neuroinflammation were assessed longitudinally using volumetric magnetic resonance imaging, tau-PET, and TSPO-PET, respectively. In vivo neuroimaging results were confirmed by immunohistochemistry for markers of neuronal survival (NeuN), tauopathy (AT8), and inflammation (TSPO, ionized calcium-binding adaptor molecule 1 or IBA-1, and complement component 1q or C1q) in brain sections from scanned mice. RESULTS: TSPO ligand treatment attenuated brain atrophy and hippocampal neuronal loss in the absence of any detected effect on tau depositions. Atrophy and neuronal loss were strongly associated with in vivo inflammatory signals measured by TSPO-PET, IBA-1, and levels of C1q, a regulator of the complement cascade. In vitro studies confirmed that the TSPO ligand Ro5-4864 reduces C1q expression in a microglial cell line in response to inflammation, reduction of which has been shown in previous studies to protect synapses and neurons in models of tauopathy. CONCLUSIONS: These findings support a protective role for TSPO ligands in tauopathy, reducing neuroinflammation, neurodegeneration, and brain atrophy.

Osteopontin/secreted phosphoprotein-1 behaves as a molecular brake regulating the neuroinflammatory response to chronic viral infection.

BACKGROUND: Osteopontin (OPN) as a secreted signaling protein is dramatically induced in response to cellular injury and neurodegeneration. Microglial inflammatory responses in the brain are tightly associated with the neuropathologic hallmarks of neurodegenerative disease, but understanding of the molecular mechanisms remains in several contexts poorly understood. METHODS: Micro-positron emission tomography (PET) neuroimaging using radioligands to detect increased expression of the translocator protein (TSPO) receptor in the brain is a non-invasive tool used to track neuroinflammation in living mammals. RESULTS: In humanized, chronically HIV-infected female mice in which OPN expression was knocked down with functional aptamers, uptake of TSPO radioligand DPA-713 was markedly upregulated in the cortex, olfactory bulb, basal forebrain, hypothalamus, and central grey matter compared to controls. Microglia immunoreactive for Iba-1 were more abundant in some HIV-infected mice, but overall, the differences were not significant between groups. TSPO+ microglia were readily detected by immunolabeling of post-mortem brain tissue and unexpectedly, two types of neurons also selectively stained positive for TSPO. The reactive cells were the specialized neurons of the cerebellum, Purkinje cells, and a subset of tyrosine hydroxylase-positive neurons of the substantia nigra. CONCLUSIONS: In female mice with wild-type levels of osteopontin, increased levels of TSPO ligand uptake in the brain was seen in animals with the highest levels of persistent HIV replication. In contrast, in mice with lower levels of osteopontin, the highest levels of TSPO uptake was seen, in mice with relatively low levels of persistent infection. These findings suggest that osteopontin may act as a molecular brake regulating in the brain, the inflammatory response to HIV infection.

Steroidogenic abnormalities in translocator protein knockout mice and significance in the aging male.

The translocator protein (TSPO) has been proposed to act as a key component in a complex important for mitochondrial cholesterol importation, which is the rate-limiting step in steroid hormone synthesis. However, TSPO function in steroidogenesis has recently been challenged by the development of TSPO knockout (TSPO-KO) mice, as they exhibit normal baseline gonadal testosterone and adrenal corticosteroid production. Here, we demonstrate that despite normal androgen levels in young male TSPO-KO mice, TSPO deficiency alters steroidogenic flux and results in reduced total steroidogenic output. Specific reductions in the levels of progesterone and corticosterone as well as age-dependent androgen deficiency were observed in both young and aged male TSPO-KO mice. Collectively, these findings indicate that while TSPO is not critical for achieving baseline testicular and adrenal steroidogenesis, either indirect effects of TSPO on steroidogenic processes, or compensatory mechanisms and functional redundancy, lead to subtle steroidogenic abnormalities which become exacerbated with aging.

Midazolam inhibits chondrogenesis via peripheral benzodiazepine receptor in human mesenchymal stem cells.

Midazolam, a benzodiazepine derivative, is widely used for sedation and surgery. However, previous studies have demonstrated that Midazolam is associated with increased risks of congenital malformations, such as dwarfism, when used during early pregnancy. Recent studies have also demonstrated that Midazolam suppresses osteogenesis of mesenchymal stem cells (MSCs). Given that hypertrophic chondrocytes can differentiate into osteoblast and osteocytes and contribute to endochondral bone formation, the effect of Midazolam on chondrogenesis remains unclear. In this study, we applied a human MSC line, the KP cell, to serve as an in vitro model to study the effect of Midazolam on chondrogenesis. We first successfully established an in vitro chondrogenic model in a micromass culture or a 2D high-density culture performed with TGF-ß-driven chondrogenic induction medium. Treatment of the Midazolam dose-dependently inhibited chondrogenesis, examined using Alcian blue-stained glycosaminoglycans and the expression of chondrogenic markers, such as SOX9 and type II collagen. Inhibition of Midazolam by peripheral benzodiazepine receptor (PBR) antagonist PK11195 or small interfering RNA rescued the inhibitory effects of Midazolam on chondrogenesis. In addition, Midazolam suppressed transforming growth factor-ß-induced Smad3 phosphorylation, and this inhibitory effect could be rescued using PBR antagonist PK11195. This study provides a possible explanation for Midazolam-induced congenital malformations of the musculoskeletal system through PBR.

TSPO: kaleidoscopic 18-kDa amid biochemical pharmacology, control and targeting of mitochondria.

The 18-kDa translocator protein (TSPO) localizes in the outer mitochondrial membrane (OMM) of cells and is readily up-regulated under various pathological conditions such as cancer, inflammation, mechanical lesions and neurological diseases. Able to bind with high affinity synthetic and endogenous ligands, its core biochemical function resides in the translocation of cholesterol into the mitochondria influencing the subsequent steps of (neuro-)steroid synthesis and systemic endocrine regulation. Over the years, however, TSPO has also been linked to core cellular processes such as apoptosis and autophagy. It interacts and forms complexes with other mitochondrial proteins such as the voltage-dependent anion channel (VDAC) via which signalling and regulatory transduction of these core cellular events may be influenced. Despite nearly 40 years of study, the precise functional role of TSPO beyond cholesterol trafficking remains elusive even though the recent breakthroughs on its high-resolution crystal structure and contribution to quality-control signalling of mitochondria. All this along with a captivating pharmacological profile provides novel opportunities to investigate and understand the significance of this highly conserved protein as well as contribute the development of specific therapeutics as presented and discussed in the present review.

Does elevated peripheral benzodiazepine receptor gene expression relate to cognitive deficits in methamphetamine dependence?

BACKGROUND: Methamphetamine (METH) is a neurotoxin and psychostimulant drug with potent effects on the central nervous system. With chronic METH administration, an inflammatory glial response is observed as a result of METH-induced neurotoxicity. One inflammatory marker is the peripheral benzodiazepine receptor (PBR). OBJECTIVE: The purpose of the present study was to determine whether PBR expression is changed in METH dependence and whether the changes relate to cognitive deficits. METHODS: Reverse transcriptase-polymerase chain reaction was used to investigate PBR gene expression in blood samples taken from 14 male subjects with METH dependence and 14 controls. RESULTS: The results showed a significant increase in PBR gene expression in METH dependence, suggestive of a systemic inflammatory response. The increase remained elevated for more than 1 year following abstinence from METH use, but eventually returned to normal. Subjects with elevated PBR also exhibited a deficit in one domain of the Wisconsin Card Sorting Test. CONCLUSION: The results suggest that systemic inflammatory effects can be associated with chronic METH abuse, and this may relate to the cognitive deficits seen in METH dependence. Copyright © 2016 John Wiley & Sons, Ltd.

Brain inflammation in a chronic epilepsy model: Evolving pattern of the translocator protein during epileptogenesis.

AIMS: A hallmark in the neuropathology of temporal lobe epilepsy is brain inflammation which has been suggested as both a biomarker and a new mechanistic target for treatments. The translocator protein (TSPO), due to its high upregulation under neuroinflammatory conditions and the availability of selective PET tracers, is a candidate target. An important step to exploit this target is a thorough characterisation of the spatiotemporal profile of TSPO during epileptogenesis. METHODS: TSPO expression, microglial activation, astrocyte reactivity and cell loss in several brain regions were evaluated at five time points during epileptogenesis, including the chronic epilepsy phase in the kainic acid-induced status epilepticus (KASE) model (n = 52) and control Wistar Han rats (n = 33). Seizure burden was also determined in the chronic phase. Furthermore, ¹8F-PBR111 PET/MRI scans were acquired longitudinally in an additional four KASE animals. RESULTS: TSPO expression measured with in vitro and in vivo techniques was significantly increased at each time point and peaked two weeks post-SE in the limbic system. A prominent association between TSPO expression and activated microglia (p < 0.001; r = 0.7), as well as cell loss (p < 0.001; r = -0.8) could be demonstrated. There was a significant positive correlation between spontaneous seizures and TSPO upregulation in several brain regions with increased TSPO expression. CONCLUSIONS: TSPO expression was dynamically upregulated during epileptogenesis, persisted in the chronic phase and correlated with microglia activation rather than reactive astrocytes. TSPO expression was correlating with spontaneous seizures and its high expression during the latent phase might possibly suggest being an important switching point in disease ontogenesis which could be further investigated by PET imaging.

Differential influence of propofol and isoflurane anesthesia in a non-human primate on the brain kinetics and binding of [(18)F]DPA-714, a positron emission tomography imaging marker of glial activation.

Translocator protein 18 kDa (TSPO) expression at the mitochondrial membrane of glial cells is related to glial activation. TSPO radioligands such as [(18)F]DPA-714 are useful for the non-invasive study of neuroimmune processes using positron emission tomography (PET). Anesthetic agents were shown to impact mitochondrial function and may influence [(18)F]DPA-714 binding parameters and PET kinetics. [(18) F]DPA-714 PET imaging was performed in Papio anubis baboons anesthetized using either intravenous propofol (n = 3) or inhaled isoflurane (n = 3). Brain kinetics and metabolite-corrected input function were measured to estimate [(18) F]DPA-714 brain distribution (VT). Displacement experiments were performed using PK11195 (1.5 mg/kg). In vitro [(18)F]DPA-714 binding experiments were performed using baboon brain tissue in the absence and presence of tested anesthetics. Brain radioactivity peaked higher in isoflurane-anesthetized animals compared with propofol (SUVmax = 2.7 ± 0.5 vs. 1.3 ± 0.2, respectively) but was not different after 30 min. Brain VT was not different under propofol and isoflurane. Displacement resulted in a 35.8 ± 8.4% decrease of brain radioactivity under propofol but not under isoflurane (0.1 ± 7.0%). In vitro, the presence of propofol increased TSPO density and dramatically reduced its affinity for [(18)F]DPA-714 compared with control. This in vitro effect was not significant with isoflurane. Exposure to propofol and isoflurane differentially influences TSPO interaction with its specific radioligand [(18)F]DPA-714 with subsequent impact on its tissue kinetics and specific binding estimated in vivo using PET. Therefore, the choice of anesthetics and their potential influence on PET data should be considered for the design of imaging studies using TSPO radioligands, especially in a translational research context.

Sigma-2 receptor binding is decreased in female, but not male, APP/PS1 mice.

The sigma-2 receptor is a steroid-binding membrane-associated receptor which has been implicated in cell survival. Sigma-2 has recently been shown to bind amyloid-ß (Aß) oligomers in Alzheimer's disease (AD) brain. Furthermore, blocking this interaction was shown to prevent or reverse the effects of Aß to cause cognitive impairment in mouse models and synaptic loss in neuronal cultures. In the present work, the density of sigma-2 receptors was measured in a double transgenic mouse model of amyloid-ß deposition (APP/PS1). Comparisons were made between males and females and between transgenic and wt animals. Sigma-2 receptor density was assessed by quantitative autoradiography performed on coronal brain slices using [(3)H]N-[4-(3,4-dihydro-6,7-dimethoxyisoquinolin-2(1H)-yl)butyl]-2-methoxy-5-methyl-benzamide ([(3)H]RHM-1), which has a 300-fold selectivity for the sigma-2 receptor over the sigma-1 receptor. The translocator protein of 18 kDa (TSPO) is expressed on activated microglia and is a marker for neuroinflammation. TSPO has been found to be upregulated in neurodegenerative disorders, including AD. Therefore, in parallel with the sigma-2 autoradiography experiments, we measured TSPO expression using the selective radioligand, [(3)H]PBR28. We also quantified Aß plaque burden in the same animals using a monoclonal antibody raised against aggregated Aß. Sigma-2 receptor density was significantly decreased in piriform and motor cortices as well as striata of 16-month old female, but not male, APP/PS1 mice as compared to their wt counterparts. [(3)H]PBR28 binding and immunostaining for Aß plaques were significantly increased in piriform and motor cortices of both male and female transgenic mice. In striatum however, significant increases were observed only in females.

The methodology of TSPO imaging with positron emission tomography.

The 18-kDA translocator protein (TSPO) is consistently elevated in activated microglia of the central nervous system (CNS) in response to a variety of insults as well as neurodegenerative and psychiatric conditions. It is therefore a target of interest for molecular strategies aimed at imaging neuroinflammation in vivo. For more than 20 years, positron emission tomography (PET) has allowed the imaging of TSPO density in brain using [(11)C]-(R)-PK11195, a radiolabelled-specific antagonist of the TSPO that has demonstrated microglial activation in a large number pathological cohorts. The significant clinical interest in brain immunity as a primary or comorbid factor in illness has sparked great interest in the TSPO as a biomarker and a surprising number of second generation TSPO radiotracers have been developed aimed at improving the quality of TSPO imaging through novel radioligands with higher affinity. However, such major investment has not yet resulted in the expected improvement in image quality. We here review the main methodological aspects of TSPO PET imaging with particular attention to TSPO genetics, cellular heterogeneity of TSPO in brain tissue and TSPO distribution in blood and plasma that need to be considered in the quantification of PET data to avoid spurious results as well as ineffective development and use of these radiotracers.

Structure of the mammalian TSPO/PBR protein.

The 3D structure of the 18-kDa transmembrane (TM) protein TSPO (translocator protein)/PBR (peripheral benzodiazepine receptor), which contains a binding site for benzodiazepines, is important to better understand its function and regulation by endogenous and synthetic ligands. We have recently determined the structure of mammalian TSPO/PBR in complex with the diagnostic ligand PK11195 [1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide; Jaremko et al. (2014) Science 343: , 1363-1366], providing for the first time atomic-level insight into the conformation of this protein, which is up-regulated in various pathological conditions including Alzheimer's disease and Parkinson's disease. Here, we review the studies which have probed the structural properties of mammalian TSPO/PBR as well as the homologues bacterial tryptophan-rich sensory proteins (TspOs) over the years and provide detailed insight into the 3D structure of mouse TSPO (mTSPO)/PBR in complex with PK11195.

The development of an automated and GMP compliant FASTlab™ Synthesis of [(18) F]GE-180; a radiotracer for imaging translocator protein (TSPO).

The level of the translocator protein (TSPO) increases dramatically in microglial cells when the cells are activated in response to neuronal injury and insult. The radiotracer [(18) F]GE-180 binds selectively and with high affinity to TSPO and can therefore be used to measure neuroinflammation in a variety of disease states. An optimized, automated synthesis of [(18) F]GE-180 has been developed for the GE FASTlab™ synthesizer. The entire process takes place on the single-use cassette. The radiolabelling is performed by nucleophilic fluorination of the S- enantiomer mesylate precursor. The crude product is purified post-radiolabelling using two solid-phase extraction cartridges integrated on the cassette. Experimental design and multivariate data analysis were used to assess the robustness, and critical steps were optimized with respect to efficacy and quality. The average radiochemical yield is 48% (RSD 6%, non-decay corrected), and the synthesis time including purification is approximately 43 min. The radiochemical purity is ≥95% for radioactive concentration ≤1100 MBq/mL. The total amount of precursor-related chemical impurities is 1-2 µg/mL. The use of solid-phase extraction purification results in a robust GMP compliant process with a product of high chemical and radiochemical purity and consistent performance across positron emission tomography (PET) centers.

Targeting Mitochondria for Cancer Treatment.

There is an increasing accumulation of data on the exceptional importance of mitochondria in the occurrence and treatment of cancer, and in all lines of evidence for such participation, there are both energetic and non-bioenergetic functional features of mitochondria. This analytical review examines three specific features of adaptive mitochondrial changes in several malignant tumors. The first feature is characteristic of solid tumors, whose cells are forced to rebuild their energetics due to the absence of oxygen, namely, to activate the fumarate reductase pathway instead of the traditional succinate oxidase pathway that exists in aerobic conditions. For such a restructuring, the presence of a low-potential quinone is necessary, which cannot ensure the conventional conversion of succinate into fumarate but rather enables the reverse reaction, that is, the conversion of fumarate into succinate. In this scenario, complex I becomes the only generator of energy in mitochondria. The second feature is the increased proliferation in aggressive tumors of the so-called mitochondrial (peripheral) benzodiazepine receptor, also called translocator protein (TSPO) residing in the outer mitochondrial membrane, the function of which in oncogenic transformation stays mysterious. The third feature of tumor cells is the enhanced retention of certain molecules, in particular mitochondrially directed cations similar to rhodamine 123, which allows for the selective accumulation of anticancer drugs in mitochondria. These three features of mitochondria can be targets for the development of an anti-cancer strategy.

PET Imaging of Neuro-Inflammation with Tracers Targeting the Translocator Protein (TSPO), a Systematic Review: From Bench to Bedside.

Parkinson's disease is the second most common neurodegenerative disorder, affecting 2-3% of the population of patients >65 years. Although the standard diagnosis of PD is clinical, neuroimaging plays a key role in the evaluation of patients who present symptoms related to neurodegenerative disorders. MRI, DAT-SPECT, and PET with [18F]-FDG are routinely used in the diagnosis and focus on the investigation of morphological changes, nigrostriatal degeneration or shifts in glucose metabolism in patients with parkinsonian syndromes. The aim of this study is to review the current PET radiotracers targeting TSPO, a transmembrane protein that is overexpressed by microglia in another pathophysiological process associated with neurodegenerative disorders known as neuroinflammation. To the best of our knowledge, neuroinflammation is present not only in PD but in many other neurodegenerative disorders, including AD, DLB, and MSA, as well as atypical parkinsonian syndromes. Therefore, in this study, specific patterns of microglial activation in PD and the differences in distribution volumes of these radiotracers in patients with PD as compared to other neurodegenerative disorders are reviewed.

The preventative effect of of Ro5-4864 (peripheral benzodiazepine receptor agonist) on spinal epidural fibrosis after laminectomy in a rat model.

OBJECTIVE: To investigate the histopathological effects of a peripheral benzodiazepine receptor agonist (Ro5-4864) on epidural fibrosis (EF) in an experimental study model (post-laminectomy) in rats. METHODS: A total of 32 albino Wistar rats were randomly divided into four equal groups (n = 8). In Group 1, no treatment was applied after laminectomy (control group). In Group 2, hemostasis was achieved after Laminectomy, and the surgical procedure was terminated by placing a 2-mm absorbable gelatin sponge dipped in saline into the epidural space. In Group 3, low-dose (4 mg/kg) Ro5-4864 was administered 30 minutes before the surgery. In Group 4, high-dose (8 mg/kg) Ro5-4864 was administered 30 minutes before the surgery. A histopathological examination was performed to evaluate arachnoidal invasion and EF. RESULTS: Our data revealed the EF was significantly reduced in rats treated with high-dose Ro5-4864 (Group 4) compared to the control and saline-soaked Spongostan groups (p = 0.000 and p = 0.006, respectively). There was no significant difference between the groups treated with high- and low-dose Ro5-4864. Arachnoidal invasion was not seen in any of the rats in the high-dose R05-4864 group. However, the arachnoidal invasion results did not significantly differ between the study groups (p = 0.052 = 0.05). CONCLUSIONS: Our study showed that Ro5-4864 could be effective in reducing EF in rats after.

Anticancer Effects of Midazolam on Lung and Breast Cancers by Inhibiting Cell Proliferation and Epithelial-Mesenchymal Transition.

Despite improvements in cancer treatments resulting in higher survival rates, the proliferation and metastasis of tumors still raise new questions in cancer therapy. Therefore, new drugs and strategies are still needed. Midazolam (MDZ) is a common sedative drug acting through the γ-aminobutyric acid receptor in the central nervous system and also binds to the peripheral benzodiazepine receptor (PBR) in peripheral tissues. Previous studies have shown that MDZ inhibits cancer cell proliferation but increases cancer cell apoptosis through different mechanisms. In this study, we investigated the possible anticancer mechanisms of MDZ on different cancer cell types. MDZ inhibited transforming growth factor ß (TGF-ß)-induced cancer cell proliferation of both A549 and MCF-7 cells. MDZ also inhibited TGF-ß-induced cell migration, invasion, epithelial-mesenchymal-transition, and Smad phosphorylation in both cancer cell lines. Inhibition of PBR by PK11195 rescued the MDZ-inhibited cell proliferation, suggesting that MDZ worked through PBR to inhibit TGF-ß pathway. Furthermore, MDZ inhibited proliferation, migration, invasion and levels of mesenchymal proteins in MDA-MD-231 triple-negative breast cancer cells. Together, MDZ inhibits cancer cell proliferation both in epithelial and mesenchymal types and EMT, indicating an important role for MDZ as a candidate to treat lung and breast cancers.