Transcriptional profiling of fetal hypothalamic TRH neurons

RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are.AbstractBackgroundDuring murine hypothalamic development, different neuroendocrine cell phenotypes are generated in overlapping periods; this suggests that cell-type specific developmental programs operate to achieve complete maturation. A balance between programs that include cell proliferation, cell cycle withdrawal as well as epigenetic regulation of gene expression characterizes neurogenesis. Thyrotropin releasing hormone (TRH) is a peptide that regulates energy homeostasis and autonomic responses. To better understand the molecular mechanisms underlying TRH neuron development, we performed a genome wide study of its transcriptome during fetal hypothalamic development. ResultsIn primary cultures, TRH cells constitute 2% of the total fetal hypothalamic cell population. To purify these cells, we took advantage of the fact that the segment spanning -774 to +84 bp of the Trh gene regulatory region confers specific expression of the green fluorescent protein (GFP) in the TRH cells. Transfected TRH cells were purified by fluorescence activated cell sorting, various cell preparations pooled, and their transcriptome compared to that of GFP- hypothalamic cells. TRH cells undergoing the terminal phase of differentiation, expressed genes implicated in protein biosynthesis, intracellular signaling and transcriptional control. Among the transcription-associated transcripts, we identified the transcription factors Klf4, Klf10 and Atf3, which were previously uncharacterized within the hypothalamus. ConclusionTo our knowledge, this is one of the first reports identifying transcripts with a potentially important role during the development of a specific hypothalamic neuronal phenotype. This genome-scale study forms a rational foundation for identifying genes that might participate in the development and function of hypothalamic TRH neurons.Published versio

The impact of surgical delay on resectability of colorectal cancer: An international prospective cohort study

AIM: The SARS-CoV-2 pandemic has provided a unique opportunity to explore the impact of surgical delays on cancer resectability. This study aimed to compare resectability for colorectal cancer patients undergoing delayed versus non-delayed surgery. METHODS: This was an international prospective cohort study of consecutive colorectal cancer patients with a decision for curative surgery (January-April 2020). Surgical delay was defined as an operation taking place more than 4 weeks after treatment decision, in a patient who did not receive neoadjuvant therapy. A subgroup analysis explored the effects of delay in elective patients only. The impact of longer delays was explored in a sensitivity analysis. The primary outcome was complete resection, defined as curative resection with an R0 margin. RESULTS: Overall, 5453 patients from 304 hospitals in 47 countries were included, of whom 6.6% (358/5453) did not receive their planned operation. Of the 4304 operated patients without neoadjuvant therapy, 40.5% (1744/4304) were delayed beyond 4 weeks. Delayed patients were more likely to be older, men, more comorbid, have higher body mass index and have rectal cancer and early stage disease. Delayed patients had higher unadjusted rates of complete resection (93.7% vs. 91.9%, P = 0.032) and lower rates of emergency surgery (4.5% vs. 22.5%, P < 0.001). After adjustment, delay was not associated with a lower rate of complete resection (OR 1.18, 95% CI 0.90-1.55, P = 0.224), which was consistent in elective patients only (OR 0.94, 95% CI 0.69-1.27, P = 0.672). Longer delays were not associated with poorer outcomes. CONCLUSION: One in 15 colorectal cancer patients did not receive their planned operation during the first wave of COVID-19. Surgical delay did not appear to compromise resectability, raising the hypothesis that any reduction in long-term survival attributable to delays is likely to be due to micro-metastatic disease

Nanomedicine in the Face of Parkinson&rsquo;s Disease: From Drug Delivery Systems to Nanozymes

The complexity and overall burden of Parkinson&rsquo;s disease (PD) require new pharmacological approaches to counteract the symptomatology while reducing the progressive neurodegeneration of affected dopaminergic neurons. Since the pathophysiological signature of PD is characterized by the loss of physiological levels of dopamine (DA) and the misfolding and aggregation of the alpha-synuclein (&alpha;-syn) protein, new proposals seek to restore the lost DA and inhibit the progressive damage derived from pathological &alpha;-syn and its impact in terms of oxidative stress. In this line, nanomedicine (the medical application of nanotechnology) has achieved significant advances in the development of nanocarriers capable of transporting and delivering basal state DA in a controlled manner in the tissues of interest, as well as highly selective catalytic nanostructures with enzyme-like properties for the elimination of reactive oxygen species (responsible for oxidative stress) and the proteolysis of misfolded proteins. Although some of these proposals remain in their early stages, the deepening of our knowledge concerning the pathological processes of PD and the advances in nanomedicine could endow for the development of potential treatments for this still incurable condition. Therefore, in this paper, we offer: (i) a brief summary of the most recent findings concerning the physiology of motor regulation and (ii) the molecular neuropathological processes associated with PD, together with (iii) a recapitulation of the current progress in controlled DA release by nanocarriers and (iv) the design of nanozymes, catalytic nanostructures with oxidoreductase-, chaperon, and protease-like properties. Finally, we conclude by describing the prospects and knowledge gaps to overcome and consider as research into nanotherapies for PD continues, especially when clinical translations take place

Intrastriatal Grafting of Chromospheres: Survival and Functional Effects in the 6-OHDA Rat Model of Parkinson's Disease

<div><p>Cell replacement therapy in Parkinson’s disease (PD) aims at re-establishing dopamine neurotransmission in the striatum by grafting dopamine-releasing cells. Chromaffin cell (CC) grafts produce some transitory improvements of functional motor deficits in PD animal models, and have the advantage of allowing autologous transplantation. However, CC grafts have exhibited low survival, poor functional effects and dopamine release compared to other cell types. Recently, chromaffin progenitor-like cells were isolated from bovine and human adult adrenal medulla. Under low-attachment conditions, these cells aggregate and grow as spheres, named chromospheres. Here, we found that bovine-derived chromosphere-cell cultures exhibit a greater fraction of cells with a dopaminergic phenotype and higher dopamine release than CC. Chromospheres grafted in a rat model of PD survived in 57% of the total grafted animals. Behavioral tests showed that surviving chromosphere cells induce a reduction in motor alterations for at least 3 months after grafting. Finally, we found that compared with CC, chromosphere grafts survive more and produce more robust and consistent motor improvements. However, further experiments would be necessary to determine whether the functional benefits induced by chromosphere grafts can be improved, and also to elucidate the mechanisms underlying the functional effects of the grafts.</p></div

Comparison between chromosphere grafts and CC grafts.

<p>(<b>a</b>) Percentage of change in circling behavior induced by amphetamine in 6-OHDA lesioned animals with either chromosphere (n = 7, orange) or CC (n = 8, purple) grafts and in a 6-OHDA lesioned group (n = 6). Each bar represents the mean ± SEM. The dotted horizontal line denotes a reduction in turn number of 50%. Significant differences in chromaffin grafted animals were observed at 2 and 8 wpg when compared to the lesioned group without graft (repeated measures multivariate ANOVA, P < 0.05, F = 6.991, DF = 23, p < 0.0001; followed by Tukey´s multiple comparisons <i>post hoc</i> test, p < 0.05*, p < 0.01** and p < 0.001***) (black asterisks), but not when compared with their circling behavior before grafting (repeated measures ANOVA, F = 1.142, r² = 0.1402, p = 0.3485). (<b>b</b>) The survival of chromaffin grafted cells was determined by directly counting TH⁺ cells in immunostained coronal brain slices. The dots represent the number of surviving grafted cells from single animals, and the bars denote the mean ± SEM. The dotted horizontal line represents the number of cells equivalent to 1 x 10³. A significant difference was observed between chromaffin and chromosphere cell survival (Unpaired t-test, P > 0.05, t = 3.572, df = 10, p = 0.0051**). (<b>c</b>) Percentage of dopaminergic cells in the lesioned SNpc in relation to the non-lesioned hemisphere in animals unilaterally lesioned with 6OHDA and grafted with either CC (grey) or chromospheres (black). The number of TH⁺ cells in the SNpc of both the lesioned and non-lesioned sides was determined by direct cell counting of TH immunostained brain slices. Data was normalized to the number of counted TH⁺ cells in the non-lesioned SNpc. Each data point represents an individual animal, and the mean ± SEM is indicated by solid horizontal lines. The dotted horizontal line denotes a loss of 20% of TH⁺ cells in the lesioned hemisphere compared to the non-lesioned SNpc. No significant differences were observed (two-tailed unpaired t-test) between animals grafted with either chromospheres or CC, suggesting that the degree of lesion induced by the 6-OHDA toxin was similar in all the analyzed animals.</p

Survival and motor efficacy of chromosphere cell grafts.

<p>Survival and motor efficacy of chromosphere cell grafts.</p

Estimation of chromosphere graft survival.

<p>(<b>a</b>) One out of four of the total coronal sections of the striatum (represented by dotted squares) were selected for immunofluorescence staining for TH. The antero-posterior extension of the graft along the striatum was estimated by determining the number of coronal sections of the striatum in which grafted cells could still be visualized, as schematized in the figure. As time after grafting progressed, the number of slices with grafted cells became smaller, indicating an antero-posterior shrinking of the grafted area due to cell death. (<b>b</b>) Quantification of the TH⁺ area of a graft. TH⁺ immunofluorescence reconstructions of the striatum (left panel) at 10x magnification were transformed into 8-bit images (middle panel), followed by binary transformation (right panel) to easily quantify the total area of the graft in each striatal section using the area quantification option in ImageJ. The insert framed by a dotted white square in the middle panel highlights the region containing the graft over which we quantified the TH⁺ area. (<b>c</b>) Graph of the estimated TH⁺ area <i>vs</i> the number of TH⁺ cells counted directly. Each data point represents the number of cells or the TH⁺ area determined for a single coronal section. Data points from grafts evaluated at 1, 2, 4 or 12 wpg are shown in different colors. The solid line is the linear regression to the data, with parameters shown at the bottom right. The dotted lines denote the uncertainty of the linear fit. (<b>d</b>) Comparison of the estimation (denoted by letter E in the graph) of graft total cell number using the extrapolation of measured TH⁺ area to the linear regression in (<b>c</b>) with the total number of TH⁺ cells directly counted (denoted by C in the graph) shows that the approximate method yields very similar results to the direct method with no statistical differences (multivariate ANOVA, P < 0.05, F = 0.0043, p = 0.9480).</p

Motor performance during the beam test at 1, 4 and 12 weeks post-grafting.

<p>(<b>a-c, left</b>) The total time (seconds) that the animals took to complete the test and (<b>a-c, right</b>) the time during which the animals remained immobile (no-movement time) while the test was on-going were measured in four different groups. The performance of each animal was evaluated in all beam widths (3, 6, 12, 18 and 24 mm) in each experimental trial. The following groups were included in the experiment: control (n = 8, gray), Sham (n = 8 blue), 6-OHDA (n = 7, black), 6-OHDA + chromosphere grafts (n = 8, orange). Evaluations in all groups were carried out periodically for 3 months after the grafting surgery in the test group. Only the evaluations obtained after 1-week, 4-weeks and 12-weeks post-grafting are shown, the rest of the evaluations (2, 6 and 8 wpg) are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160854#pone.0160854.s002" target="_blank">S2 Fig</a>. Empty orange bars are the measurements from the grafted animal group obtained after the 6-OHDA-lession procedure but before grafting. Significant differences were observed between the total time and no movement time measured before grafting compared with the total time and no movement time of the same group after grafting (orange asterisks) (repeated measures multivariate ANOVA, p < 0.05, F = 5.349, DF = 4, p = 0.0018; followed by Bonferroni´s multiple comparisons <i>post hoc</i> test, p < 0.01** and p < 0.001***). Also, significant was the difference in some evaluations in both the total and no movement time between 6-OHDA lesioned animals without graft and 6-OHDA lesioned animals with chromospheres, control and sham groups (black asterisks) (repeated measures multivariate ANOVA, P < 0.05, F = 36.17, DF = 7, < 0.0001; followed by Bonferroni´s multiple comparisons <i>post hoc</i> test, p < 0.05*, p < 0.01** and p < 0.001***). Error bars are the SEM.</p

Survival efficacy of chromosphere cell grafts.

<p>Survival efficacy of chromosphere cell grafts.</p