Alginate-based 3D cancer cell culture for therapeutic response modeling.

Two-dimensional (2D) culture of tumor cells fails to recapitulate some important aspects of cellular organization seen in in vivo experiments. In addition, cell cultures traditionally use non-physiological concentration of nutrients. Here, we describe a protocol for a facile three-dimensional (3D) culture format for cancer cells. This 3D platform helps overcome the 2D culture limitations. In addition, it allows for longitudinal modeling of responses to cancer therapeutics. For complete details on the use and execution of this protocol, please refer to Lhuissier et al. (2017), Lehmann et al. (2016), Liu et al. (2016), and Duval et al. (2011).

Testosterone or Estradiol When Implanted in the Medial Preoptic Nucleus Trigger Short Low-Amplitude Songs in Female Canaries.

In male songbirds, the motivation to sing is largely regulated by testosterone (T) action in the medial preoptic area, whereas T acts on song control nuclei to modulate aspects of song quality. Stereotaxic implantation of T in the medial preoptic nucleus (POM) of castrated male canaries activates a high rate of singing activity, albeit with a longer latency than after systemic T treatment. Systemic T also increases the occurrence of male-like song in female canaries. We hypothesized that this effect is also mediated by T action in the POM. Females were stereotaxically implanted with either T or with 17ß-estradiol (E2) targeted at the POM and their singing activity was recorded daily during 2 h for 28 d until brains were collected for histological analyses. Following identification of implant localizations, three groups of subjects were constituted that had either T or E2 implanted in the POM or had an implant that had missed the POM (Out). T and E2 in POM significantly increased the number of songs produced and the percentage of time spent singing as compared with the Out group. The songs produced were in general of a short duration and of poor quality. This effect was not associated with an increase in HVC volume as observed in males, but T in POM enhanced neurogenesis in HVC, as reflected by an increased density of doublecortin-immunoreactive (DCX-ir) multipolar neurons. These data indicate that, in female canaries, T acting in the POM plays a significant role in hormone-induced increases in the motivation to sing.

Dehydroepiandrosterone protects male and female hippocampal neurons and neuroblastoma cells from glucose deprivation.

Dehydroepiandrosterone (DHEA) modulates neurogenesis, neuronal function, neuronal survival and metabolism, enhancing mitochondrial oxidative capacity. Glucose deprivation and hypometabolism have been implicated in the mechanisms that mediate neuronal damage in neurological disorders, and some studies have shown that these mechanisms are sexually dimorphic. It was also demonstrated that DHEA is able to attenuate the hypometabolism that is related to some neurodegenerative diseases, eliciting neuroprotective effects in different experimental models of neurodegeneration. The aim of this study was to evaluate the effect of DHEA on the viability of male and female hippocampal neurons and SH-SY5Y neuroblastoma cells exposed to glucose deprivation. It was observed that after 12h of pre-treatment, DHEA was able to protect SH-SY5Y cells from glucose deprivation for 6h (DHEA 10(-12), 10(-8) and 10(-6)M) and 8h (DHEA 10(-8)M). In contrast, DHEA was not neuroprotective against glucose deprivation for 12 or 24h. DHEA (10(-8)M) also protected SH-SY5Y cells when added together or even 1h after the beginning of glucose deprivation (6h). Furthermore, DHEA (10(-8)M) also protected primary neurons from both sexes against glucose deprivation. In summary, our findings indicate that DHEA is neuroprotective against glucose deprivation in human neuroblastoma cells and in male and female mouse hippocampal neurons. These results suggest that DHEA could be a promising candidate to be used in clinical studies aiming to reduce neuronal damage in people from both sexes.

The Selective Estrogen Receptor Modulator Raloxifene Regulates Arginine-Vasopressin Gene Expression in Human Female Neuroblastoma Cells Through G Protein-Coupled Estrogen Receptor and ERK Signaling.

The selective estrogen receptor modulator raloxifene reduces blood pressure in hypertensive postmenopausal women. In the present study we have explored whether raloxifene regulates gene expression of arginine vasopressin (AVP), which is involved in the pathogenesis of hypertension. The effect of raloxifene was assessed in human female SH-SY5Y neuroblastoma cells, which have been recently identified as a suitable cellular model to study the estrogenic regulation of AVP. Raloxifene, within a concentration ranging from 10(-10) M to 10(-6) M, decreased the mRNA levels of AVP in SH-SY5Y cells with maximal effect at 10(-7) M. This effect of raloxifene was imitated by an agonist (±)-1-[(3aR*,4S*,9bS*)-4-(6-bromo-1,3-benzodioxol-5-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinolin-8-yl]-ethanone of G protein-coupled estrogen receptor-1 (GPER) and blocked by an antagonist (3aS*,4R*,9bR*)-4-(6-bromo-1,3-benzodioxol-5-yl)-3a,4,5,9b-3H-cyclopenta[c]quinoline of GPER and by GPER silencing. Raloxifene induced a time-dependent increase in the level of phosphorylated ERK1 and ERK2, by a mechanism blocked by the GPER antagonist. The treatment of SH-SY5Y cells with either a MAPK/ERK kinase 1/2-specific inhibitor (1,4-diamino-2, 3-dicyano-1,4-bis(2-aminophenylthio)butadine) or a protein kinase C inhibitor (sotrastaurin) blocked the effects of raloxifene on the phosphorylation of ERK1/2 and the regulation of AVP mRNA levels. These results reveal a mechanism mediating the regulation of AVP expression by raloxifene, involving the activation of GPER, which in turn activates protein kinase C, MAPK/ERK kinase, and ERK. The regulation of AVP by raloxifene and GPER may have implications for the treatment of blood hypertension(.).

Aromatase inhibition exacerbates pain and reactive gliosis in the dorsal horn of the spinal cord of female rats caused by spinothalamic tract injury.

Central pain syndrome is characterized by severe and excruciating pain resulting from a lesion in the central nervous system. Previous studies have shown that estradiol decreases pain and that inhibitors of the enzyme aromatase, which synthesizes estradiol from aromatizable androgens, increases pain sensitivity. In this study we have assessed whether aromatase expression in the dorsal horns of the spinal cord is altered in a rat model of central pain syndrome, induced by the unilateral electrolytic lesion of the spinothalamic tract. Protein and mRNA levels of aromatase, as well as the protein and mRNA levels of estrogen receptors α and ß, were increased in the dorsal horn of female rats after spinothalamic tract injury, suggesting that the injury increased estradiol synthesis and signaling in the dorsal horn. To determine whether the increased aromatase expression in this pain model may participate in the control of pain, mechanical allodynia thresholds were determined in both hind paws after the intrathecal administration of letrozole, an aromatase inhibitor. Aromatase inhibition enhanced mechanical allodynia in both hind paws. Because estradiol is known to regulate gliosis we assessed whether the spinothalamic tract injury and aromatase inhibition regulated gliosis in the dorsal horn. The proportion of microglia with a reactive phenotype and the number of glial fibrillary acidic protein-immunoreactive astrocytes were increased by the injury in the dorsal horn. Aromatase inhibition enhanced the effect of the injury on gliosis. Furthermore, a significant a positive correlation of mechanical allodynia and gliosis in the dorsal horn was detected. These findings suggest that aromatase is up-regulated in the dorsal horn in a model of central pain syndrome and that aromatase activity in the spinal cord reduces mechanical allodynia by controlling reactive gliosis in the dorsal horn.

Estradiol attenuates spinal cord injury-induced pain by suppressing microglial activation in thalamic VPL nuclei of rats.

In our previous study we showed that central pain syndrome (CPS) induced by electrolytic injury caused in the unilateral spinothalamic tract (STT) is a concomitant of glial alteration at the site of injury. Here, we investigated the activity of glial cells in thalamic ventral posterolateral nuclei (VPL) and their contribution to CPS. We also examined whether post-injury administration of a pharmacological dose of estradiol can attenuate CPS and associated molecular changes. Based on the results,in the ipsilateral VPL the microglial phenotype switched o hyperactive mode and Iba1 expression was increased significantly on days 21 and 28 post-injury. The same feature was observed in contralateral VPL on day 28 (P<.05). These changes were strongly correlated with the onset of CPS (r(2)=0.670). STT injury did not induce significant astroglial response in both ipsilateral and contralateral VPL. Estradiol attenuated bilateral mechanical hypersensitivity 14 days after STT lesion (P<.05). Estradiol also suppressed microglial activation in the VPL. Taken together, these findings indicate that selective STT lesion induces bilateral microglia activation in VPL which might contribute to mechanical hypersensitivity. Furthermore, a pharmacological dose of estradiol reduces central pain possibly via suppression of glial activity in VPL region.