A rapid and accurate method to quantify neurite outgrowth from cell and tissue cultures: Two image analytic approaches using adaptive thresholds or machine learning.

BACKGROUND: Assessments of axonal outgrowth and dendritic development are essential readouts in many in vitro models in the field of neuroscience. Available analysis software is based on the assessment of fixed immunolabelled tissue samples, making it impossible to follow the dynamic development of neurite outgrowth. Thus, automated algorithms that efficiently analyse brightfield images, such as those obtained during time-lapse microscopy, are needed. NEW METHOD: We developed and validated algorithms to quantitatively assess neurite outgrowth from living and unstained spinal cord slice cultures (SCSCs) and dorsal root ganglion cultures (DRGCs) based on an adaptive thresholding approach called NeuriteSegmantation. We used a machine learning approach to evaluate dendritic development from dissociate neuron cultures. RESULTS: NeuriteSegmentation successfully recognized axons in brightfield images of SCSCs and DRGCs. The temporal pattern of axonal growth was successfully assessed. In dissociate neuron cultures the total number of cells and their outgrowth of dendrites were successfully assessed using machine learning. COMPARISON WITH EXISTING METHODS: The methods were positively correlated and were more time-saving than manual counts, having performing times varying from 0.5-2 min. In addition, NeuriteSegmentation was compared to NeuriteJ®, that uses global thresholding, being more reliable in recognizing axons in areas of intense background. CONCLUSION: The developed image analysis methods were more time-saving and user-independent than established approaches. Moreover, by using adaptive thresholding, we could assess images with large variations in background intensity. These tools may prove valuable in the quantitative analysis of axonal and dendritic outgrowth from numerous in vitro models used in neuroscience.

Spectral sensitivity of the electroretinogram b-wave in dark-adapted Prussian carp (Carassius gibelio Bloch, 1782).

One of the purposes of this study was to examine whether b-wave measurements can be used in the evaluation of scotopic spectral sensitivity in Prussian carp measurements when the eyes were surgically deprived of cornea, lens, and most of the vitreous. Another goal was testing the new fitting procedure for A2-based photopigments. Using fitted amplitude-log intensity functions for threshold calculation, and two models for computer-assisted fitting of spectral sensitivity curves, no significant differences in λ(max) were found between rod photopigments and b-wave-based spectral sensitivity.

Sperm DNA integrity testing: big halo is a good predictor of embryo quality and pregnancy after conventional IVF.

Sperm DNA integrity is a sperm functional parameter of male fertility evaluation. Two parameters of sperm DNA integrity were observed: DNA damage expressed as DNA fragmentation index (DFI) and percentage of the DNA undamaged spermatozoa expressed as big halo. Halosperm test was used for sperm DNA integrity determination. The aim of this study was to evaluate which DNA integrity parameter is better as an embryo quality and pregnancy prognostic parameter after the conventional IVF. We evaluated two embryo groups (positive and negative group) according to the 3rd day cumulative embryo score. Big halo and DFI, as we expected, showed good correlation (r = -0.69; p < 0.001). Receiver operating characteristic (ROC) analyses show that DFI and big halo are significant (p < 0.001) as prognostic parameters of embryo quality. ROC curves comparison of DFI and big halo revealed the AUC value for big halo to be significantly higher (DFI AUC = 0.71 vs. big halo AUC = 0.83; p = 0.025) than for DFI. Big halo was found to be the only independent predictor of embryo quality. Sperm DNA integrity both parameters are good prognostic parameters of embryo quality after the conventional IVF where big halo seems to be better. ROC analyses show DFI and big halo as significant prognostic parameters for achieved pregnancy (AUC ± SE for DFI was 0.67 ± 0.06 and 0.75 ± 0.06 for big halo). To our knowledge, this is the first study demonstrating the correlation between sperm DNA undamaged rate expressed as big halo parameter and semen characteristics as well as the influence on fertilization rate, embryo quality and pregnancy in conventional IVF.

Relationship between 4-day air mass back trajectories and metallic components in PM(10) and PM (2.5) particle fractions in Zagreb Air, Croatia.

This paper presents a study on the relationship between trace metal concentrations and the state of the atmosphere at the Croatian EMEP station Puntijarka, Zagreb. PM(10) and PM(2.5) particle fractions are hazardous in terms of morbidity and hospitalization due to cardiovascular and respiratory diseases, and in terms of total mortality. In Zagreb, PM(10) and PM(2.5) monitoring started on a daily basis at a sampling site located in the northern, residential part of the city. Trace metal concentrations were determined from daily samples. Air mass back trajectories were used to determine particulate air pollution from local and remote sources. The investigation has shown a statistically significant association between air mass back trajectories and metallic air concentration levels.

The d-wave in fish and the state of light adaptation.

Comparative electroretinographic studies of the d-wave evoked with long duration photo stimuli in dark- and light-adapted fish species (three marine and three freshwater) were performed. At the end of prolonged photo-stimulation in scotopic conditions a negative d-wave appears in electroretinograms of dogfish shark, eel and goldfish diminishing and eventually changing with intensity of photo-stimulation, while in rudd it only increases. Dark-adapted electroretinograms of two percids (perch and painted comber) exhibit a positive d-wave that approaches the b-wave amplitude under bright photopic conditions. Judging from the d-wave, only the rod pathway is active in dark-adapted dogfish shark, eel, and goldfish. Under the light adaptation, cone pathways are active in eel and goldfish, whereas the positive response to the end of light stimuli in dogfish shark could be explained by independent ON and OFF pathways from outer to inner retina via bipolar cells. In the case of two percids, dark adaptation has no influence on cone pathways. The d-wave of rudd behaves like cone-driven d-waves but in opposite sign. The data thus show that the d-wave form, amplitude and sign depend on interconnection of ON and OFF pathways as determined by the state of adaptation and/or type of photoreceptor.