ß2-Adrenergic Receptor Stimulation Upregulates Cx43 Expression on Glioblastoma Multiforme and Olfactory Ensheathing Cells.

Glioblastoma multiforme (GBM) is described as an invasive astrocytic tumor in adults. Despite current standard treatment approaches, the outcome of GBM remains unfavorable. The downregulation of connexin 43 (Cx43) expression is one of the molecular transformations in GBM cells. The Cx43 levels and subsequently gap junctional intercellular communication (GJIC) have an important role in the efficient transfer of cytotoxic drugs to whole tumor cells. As shown in our previous study, the stimulation of the ß2-adrenergic receptor (ß2-AR) leads to the modulation of Cx43 expression level in the GBM cell line. Here we further examine the effect of clenbuterol hydrochloride as a selective ß2-AR agonist on the Cx43 expression in human GBM-derived astrocyte cells and human olfactory ensheathing cells (OECs) as a potent vector for future gene therapy. In this experiment, first we established a primary culture of astrocytes from GBM samples and verified the purity using immunocytofluorescent staining. Western blot analysis was performed to evaluate the Cx43 protein level. Our western blot findings reveal that clenbuterol hydrochloride upregulates the Cx43 protein level in both primary human astrocyte cells and human OECs. Conversely, ICI 118551 as a ß2-AR antagonist inhibits these effects. Moreover, clenbuterol hydrochloride increases the Cx43 expression in primary human astrocyte cells and OECs co-culture systems, and ICI 118551 reverses these effects. To confirm the western blot results, immunocytofluorescent staining was performed to evaluate the ß2-AR agonist effect on Cx43 expression. Our immunocytofluorescent results supported western blot analysis in primary human astrocyte cells and the OECs co-culture system. The results of this study suggest that the activation of ß2-AR with regard to Cx43 protein levels enhancement in GBM cells and OECs might be a promising approach for GBM treatment in the future.

Longitudinal Effects of Bumetanide on Neuro-Cognitive Functioning in Drug-Resistant Epilepsy.

Antiepileptic drugs (AEDs) have repeatedly shown inconsistent and almost contradictory effects on the neurocognitive system, from substantial impairments in processing speed to the noticeable improvement in working memory and executive functioning. Previous studies have provided a novel insight into the cognitive improvement by bumetanide as a potential antiepileptic drug. Through the current investigation, we evaluated the longitudinal effects of bumetanide, an NKCC1 co-transporter antagonist, on the brain microstructural organization as a probable underlying component for cognitive performance. Microstructure assessment was completed using SPM for the whole brain assay and Freesurfer/TRACULA for the automatic probabilistic tractography analysis. Primary cognitive operations including selective attention and processing speed, working memory capacity and spatial memory were evaluated in 12 patients with a confirmed diagnosis of refractory epilepsy. Participants treated with bumetanide (2 mg/ day) in two divided doses as an adjuvant therapy to their regular AEDs for 6 months, which followed by the re-assessment of their cognitive functions and microstructural organizations. Seizure frequency reduced in eight patients which accompanied by white matter reconstruction; fractional anisotropy (FA) increased in the cingulum-cingulate gyrus (CCG), anterior thalamic radiation (ATR), and temporal part of the superior longitudinal fasciculus (SLFt) in correlation with the clinical response. The voxel-based analysis in responder patients revealed increased FA in the left hippocampus, right cerebellum, and right medial temporal lobe, while mean diffusivity (MD) values reduced in the right occipital lobe and cerebellum. Microstructural changes in SLFt and ATR accompanied by a reduction in the error rate in the spatial memory test. These primary results have provided preliminary evidence for the effect of bumetanide on cognitive functioning through microstructural changes in patients with drug-resistant epilepsy.