Combining motion analysis and microfluidics--a novel approach for detecting whole-animal responses to test substances.

Small, early life stages, such as zebrafish embryos are increasingly used to assess the biological effects of chemical compounds in vivo. However, behavioural screens of such organisms are challenging in terms of both data collection (culture techniques, drug delivery and imaging) and data evaluation (very large data sets), restricting the use of high throughput systems compared to in vitro assays. Here, we combine the use of a microfluidic flow-through culture system, or BioWell plate, with a novel motion analysis technique, (sparse optic flow - SOF) followed by spectral analysis (discrete Fourier transformation - DFT), as a first step towards automating data extraction and analysis for such screenings. Replicate zebrafish embryos housed in a BioWell plate within a custom-built imaging system were subject to a chemical exposure (1.5% ethanol). Embryo movement was videoed before (30 min), during (60 min) and after (60 min) exposure and SOF was then used to extract data on movement (angles of rotation and angular changes to the centre of mass of embryos). DFT was subsequently used to quantify the movement patterns exhibited during these periods and Multidimensional Scaling and ANOSIM were used to test for differences. Motion analysis revealed that zebrafish had significantly altered movements during both the second half of the alcohol exposure period and also the second half of the recovery period compared to their pre-treatment movements. Manual quantification of tail flicking revealed the same differences between exposure-periods as detected using the automated approach. However, the automated approach also incorporates other movements visible in the organism such as blood flow and heart beat, and has greater power to discern environmentally-driven changes in the behaviour and physiology of organisms. We suggest that combining these technologies could provide a highly efficient, high throughput assay, for assessing whole embryo responses to various drugs and chemicals

Short-course radiation plus temozolomide in elderly patients with glioblastoma

Glioblastoma is associated with a poor prognosis in the elderly. Survival has been shown to increase among patients 70 years of age or younger when temozolomide chemotherapy is added to standard radiotherapy (60 Gy over a period of 6 weeks). In elderly patients, more convenient shorter courses of radiotherapy are commonly used, but the benefit of adding temozolomide to a shorter course of radiotherapy is unknown

Optic flow movement patterns of an individual zebrafish embryo (individual No. 4) at the 22-somite stage for the time period of the whole experiment (pre-exposure, ethanol and recovery periods).

<p>The stars indicate manually observed tail-flick movements.</p

Mean number of tail flicks performed by <i>Danio rerio</i> embryos during the five treatment periods as obtained through manual quantification (mean ± 1.S.E.).

<p>Values with different letters are significantly different (Repeated Measures ANOVA followed by Bonferroni pair wise comparisons, p<0.05). For significance levels please refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113235#pone-0113235-t001" target="_blank">Table 1</a>.</p

Movement patterns of two embryos during the time period of the experiment.

<p>Embryo 1 and Embryo 2 exhibited different movement responses to ethanol exposure, Embryo 1 increasing tail flick frequency markedly whereas the response of Embryo 2 was more subtle. A+B: Frame by frame optic flow parameters (red – positive angle movements, blue – negative angle movements, green – X coordinate of centre of mass, yellow – Y coordinate of centre of mass). C: tail flick frequency (tail flicks per 30 sec).</p

Schematic representation of the experimental design.

<p>One cycle represents the time period taken to record all 15 embryos once (7.5 min). The experiment is divided into five time periods: ”pre-exposure period” (recording cycle 1–4: aerated, deionized water), “ethanol period” (divided into two groups “ethanol 1” (recording cycle 5–8) and “ethanol 2” (recording cycle 9–12); 1.5% ethanol solution) and “recovery period” (divided into two groups “recovery 1” (recording cycle 13–16) and “recovery 2” (recording cycle 17–20); aerated, deionized water).</p