Spatial pattern modeling and discovery in biological images

Studying spatial arrangement and relationships in full tissue samples can improve our understanding of the various developmental/pathological processes that underlie proper organ or organism function. In particular, it has been found that neuronal or vascular structures are pervasive in many tissues, and oftentimes are spatially correlated with other cells. This work aims to discover those relationships, by extracting biological knowledge from cellular and sub-cellular imaging using spatial point process methods.In this dissertation, we present discoveries on spatial distributions and attributes of dendritic spines and retinal astrocytes, two crucial elements in the mammalian nervous system. Although little is known about the spatial distributions of either respective to their surroundings and attributes, this thesis attempts to pose some possible biological hypotheses based on strong statistical evidence, as well as further extend the tools used for spatial analysis. In particular, we develop a multitype version of the linear network K-function, a summary function used for measuring clustering or repulsion of point features existing on a linear network

Physiology and Pharmacology Glial Cell Contribution to Basal Vessel Diameter and Pressure-Initiated Vascular Responses in Rat Retina

PURPOSE. The purpose of this study was to test the hypothesis that retinal glial cells modify basal vessel diameter and pressure-initiated vascular regulation in rat retina. METHODS. In rats, L-2-aminoadipic acid (LAA, 10 nM) was intravitreally injected to inhibit glial cell activity. Twenty-four hours following injection, retinal glial intracellular calcium (Ca 2þ ) was labeled with the fluorescent calcium indicator Fluo-4/AM (F4, 1 mM). At 110 minutes after injection, intraocular pressure (IOP) was elevated from 20 to 50 mm Hg. Prior to and during IOP elevation, Ca 2þ and retinal vessel diameter were assessed using a spectral-domain optical coherence tomography/confocal scanning laser ophthalmoscope. Dynamic changes in Ca 2þ and diameter from IOP elevation were quantified. The response in LAA-treated eyes was compared with vehicle treated control eyes. RESULTS. L-2-Aminoadipic acid treatment significantly reduced F4-positive cells in the retina (LAA, 16 6 20 vs. control, 55 6 37 cells/mm 2 ; P ¼ 0.02). Twenty-four hours following LAA treatment, basal venous diameter was increased from 38.9 6 3.9 to 51.8 6 6.4 lm (P < 0.0001, n ¼ 20), whereas arterial diameter was unchanged (from 30.3 6 3.5 to 30.7 6 2.8 lm; P ¼ 0.64). In response to IOP elevation, LAA-treated eyes showed a smaller increase in glial cell Ca 2þ around both arteries and veins in comparison with control (P < 0.001 for both). There was also significantly greater IOP-induced vasoconstriction in both vessel types (P ¼ 0.05 and P ¼ 0.02, respectively; n ¼ 6 each). CONCLUSIONS. The results suggest that glial cells can modulate basal retinal venous diameter and contribute to pressure-initiated vascular responses

Autocrine and Paracrine Secretion of Vascular Endothelial Growth Factor in the Pre-Hypoxic Diabetic Retina

Vascular endothelial growth factor (VEGF) is well established as the main agent responsible for vascular leakage and angiogenesis in the diabetic retina. While VEGF can have positive effects on hyperglycemia stressed retinal tissues, it also plays a role in events progressing to the oxygen- stressed, i.e. hypoxic, diabetic retina. Some VEGF makes its way to the retina from systemic sources and some is produced locally within the eye. Hyperglycemia, oxidants, inflammation, and advanced glycation end-products are all stimulants to VEGF production, both in the hypoxic and the pre-hypoxic retina. Endothelial cells, pericytes, Müller cells, microglia, astrocytes, retinal pigment epithelium and neurons have all been known to produce VEGF at some point in retinal development or in disease. Excessive VEGF production in the early diabetic retina can lead to retinal exposure or mechanisms which exacerbate further damage. While Müller cells are likely the most significant producer of VEGF in the pre-hypoxic retina, other VEGF producing cells may also play a role due to their proximity to vessels or neurons. Study of the release of VEGF by retinal cells in hyperglycemia conditions, may help identify targets for early treatment and prevent the serious consequences of diabetic retinopathy

Probabilistic spatial analysis in quantitative microscopy with uncertainty-aware cell detection using deep Bayesian regression

The investigation of biological systems with three-dimensional microscopy demands automatic cell identification methods that not only are accurate but also can imply the uncertainty in their predictions. The use of deep learning to regress density maps is a popular successful approach for extracting cell coordinates from local peaks in a postprocessing step, which then, however, hinders any meaningful probabilistic output. We propose a framework that can operate on large microscopy images and output probabilistic predictions (i) by integrating deep Bayesian learning for the regression of uncertainty-aware density maps, where peak detection algorithms generate cell proposals, and (ii) by learning a mapping from prediction proposals to a probabilistic space that accurately represents the chances of a successful prediction. Using these calibrated predictions, we propose a probabilistic spatial analysis with Monte Carlo sampling. We demonstrate this in a bone marrow dataset, where our proposed methods reveal spatial patterns that are otherwise undetectable

Evaluation von Gliazellmarkern an einem organotypischen Hypoxiemodell der Netzhaut

Beim ZAV handelt es sich um eine ophthalmologische Notfallsituation. Der Patient erleidet innerhalb weniger Stunden einen irreversiblen Sehverlust. Eine Therapie gibt es derzeit nicht. Die neue Methode zur Erzeugung einer Ischämie in vitro mit Hilfe der Ischämiekammer ermöglicht es, einen ZAV nachzustellen und neue Therapieformen an Retinakulturen zu untersuchen. Sie wurde bereits von Matthias Blak erfolgreich in Bezug auf die retinalen Ganglienzellen getestet. Ziel dieser Arbeit war, die Auswirkungen einer Ischämie auf die Gliazellen der Retina zu untersuchen und zwei Therapieansätze zu testen. Dazu wurden immunhistochemische Färbungen durchgeführt. Eine Schwäche des in vitro Ischämiemodells ist die Tatsache, dass bereits die Axotomie einen Reiz darstellt, der Makro- und Mikroglia aktiviert. Die Glutamatversuche ergaben Ergebniswerte, die nicht für eine Aktivierung der Gliazellen oder eine Toxizität von Glutamat sprechen. Bei den Ischämieversuchen war kein eindeutiger Zusammenhang zwischen der Ischämiedauer oder dem Zeitpunkt der Auswertung und der Gliareaktion festzustellen. Signifikant mehr aktivierte Makroglia wurde nur bei einer Ischämiedauer von 120 Minuten beobachtet (Auswertung nach 48 Stunden). Der Mikrogliaanteil fiel signifikant bei 60 Minuten Ischämie. Anhand des Ischämiemodells wurden zwei Therapieansätze getestet. Zum einen die Hypothermie (10 °C, 20 °C, 30 °C) und zum anderen das Medikament CSA. Die Hypothermie-Behandlung (20 °C) während und nach einer 75minütigen Ischämie führte zur signifikant geringer ausgeprägten Müllerzellgliose. Die Mikrogliareaktion blieb unbeeinflusst. Eine CSA-Behandlung zeigte keine Verminderung der Müllerzellaktivierung nach Ischämiestress. Die Werte der Mikrogliauntersuchungen sprechen für einen aktivierenden Effekt des CSA auf die Mikroglia. Die in vitro Simulation einer Ischämie mit der neu entwickelten Ischämiekammer mag sich für die Untersuchung der retinalen Ganglienzellen eignen. In Bezug auf die Gliazellen stellt sie aber keine zuverlässige Methode dar. Es ist keine Korrelation zwischen dem Überleben der retinalen Ganglienzellen und der Ausprägung der Gliose erkennbar

Electrophysiological measures of optic nerve function

The contribution of retinal ganglion cells (RGCs) to human electroretinograms (ERGs) is known, but that of chicken (Gallus gallus domesticus) is not clear. This project seeks to determine the effect of RGC dysfunction on full-field flash ERGs in chickens using established protocols known to test RGC function in humans and other mammals. Chicks were treated to produce unilateral retinal dysfunction by surgical optic nerve section (ONS group) or by intravitreal injection of tetrodotoxin (TTX group) to block ganglion cell function. Contralateral eyes received sham treatments, consisting of sham surgery or injections of vehicle, phosphate buffered saline (PBS), respectively. For both groups, bilateral, full-field ERGs were recorded in dark-adapted (DA) birds (ONS: n=6; TTX: n=5) or in light-adapted (LA) birds (ONS: n=10; TTX: n=5) prior to the imposed treatment (at one-day post-hatch) and on days 3-, 5-, 7-, 14-, and 21- post-treatment on the same birds. In addition, bilateral, full-field, long-flash (150 ms) ERGs were recorded from light-adapted birds (ONS: n=8; TTX: n=5) prior to treatment (at one day post-hatch) and again at 3-, 14- and 21-days post-treatment. Interpolation and curve fitting, including Naka Rushton fitting, were used to report parameters of the ERG stimulus-response series such as maximum amplitudes (Vmax) and sensitivity (k, stimulus producing half Vmax). Cell counts (retinal histology) were conducted of the RGC and inner nuclear layers from histological sections of a separate group of 6 birds sacrificed at 21 days post-ONS. For both groups, the measures of the DA ERG stimulus-response series (dark-adapted Vmax and k, the oscillatory potential amplitudes, and the interpolated a-wave parameters) did not differ between the treated and sham-treated eyes in either treatment group. In addition, for both treatment groups, the negative waveform of the scotopic threshold response (STR), which reflects RGC function in most mammals, was not apparent in the chick ERGs to dim flashes. No differences between the eyes were v detected for the positive STR/DA b-wave to 0.01 cd.s/m2 flashes (ONS: p=0.59; TTX: p=0.21). Similarly, the photopic negative response (PhNR) following the light-adapted b-wave was small and showed no effect of either treatment (ONS: p=0.92; TTX: p=0.11). However, the offset positivity, the d-wave amplitude, was smaller in the treated eyes in both the ONS and TTX groups (ONS: p=0.008; TTX: p=0.03), but d-wave implicit times did not differ. Cell counts confirmed that RGCs were selectively lost following ONS (p<0.0001). This study suggests that the STR and PhNR do not reflect RGC functions in chickens, as they do in most mammals. Anatomical differences between the chicken and human retinae might underlie differences in the generation of ERG waveforms associated with ganglion cells. In particular, chicken eyes, and avian eyes in general, lack an inner retinal blood supply and associated intra-retinal astroglia which may be necessary for the generation of STR and PhNR waveforms. Finally, this thesis showed that, unlike in humans, the chicken d-wave may reflect the function of cells in the optic nerve including RGCs and cells of the centrifugal vision system in the chicken

Characterizing spatial distributions of astrocytes in the mammalian retina.

MotivationIn addition to being involved in retinal vascular growth, astrocytes play an important role in diseases and injuries, such as glaucomatous neuro-degeneration and retinal detachment. Studying astrocytes, their morphological cell characteristics and their spatial relationships to the surrounding vasculature in the retina may elucidate their role in these conditions.ResultsOur results show that in normal healthy retinas, the distribution of observed astrocyte cells does not follow a uniform distribution. The cells are significantly more densely packed around the blood vessels than a uniform distribution would predict. We also show that compared with the distribution of all cells, large cells are more dense in the vicinity of veins and toward the optic nerve head whereas smaller cells are often more dense in the vicinity of arteries. We hypothesize that since veinal astrocytes are known to transport toxic metabolic waste away from neurons they may be more critical than arterial astrocytes and therefore require larger cell bodies to process waste more efficiently.Availability and implementationA 1/8th size down-sampled version of the seven retinal image mosaics described in this article can be found on BISQUE (Kvilekval et al., 2010) at http://bisque.ece.ucsb.edu/client_service/view?resource=http://bisque.ece.ucsb.edu/data_service/dataset/6566968

Characterizing spatial distributions of astrocytes in the mammalian retina

MotivationIn addition to being involved in retinal vascular growth, astrocytes play an important role in diseases and injuries, such as glaucomatous neuro-degeneration and retinal detachment. Studying astrocytes, their morphological cell characteristics and their spatial relationships to the surrounding vasculature in the retina may elucidate their role in these conditions.ResultsOur results show that in normal healthy retinas, the distribution of observed astrocyte cells does not follow a uniform distribution. The cells are significantly more densely packed around the blood vessels than a uniform distribution would predict. We also show that compared with the distribution of all cells, large cells are more dense in the vicinity of veins and toward the optic nerve head whereas smaller cells are often more dense in the vicinity of arteries. We hypothesize that since veinal astrocytes are known to transport toxic metabolic waste away from neurons they may be more critical than arterial astrocytes and therefore require larger cell bodies to process waste more efficiently.Availability and implementationA 1/8th size down-sampled version of the seven retinal image mosaics described in this article can be found on BISQUE (Kvilekval et al., 2010) at http://bisque.ece.ucsb.edu/client_service/view?resource=http://bisque.ece.ucsb.edu/data_service/dataset/6566968