Model prediction and validation of an order mechanism controlling the spatio-temporal phenotype of early hepatocellular carcinoma

The aggressiveness of a tumor may be reflected by its micro-architecture. To gain a deeper understanding of the mechanisms controlling spatial organization of tumors at early stages after tumor initiation, we used an agent-based spatio-temporal model previously established to simulate features of liver regeneration. Here, this model was further developed to simulate scenarios in early tumor development, when individual initiated hepatocytes gain increased proliferation capacity. The model simulations were performed in realistic liver microarchitectures obtained from 3D reconstruction of confocal laser scanning micrographs. Interestingly, the here established model predicted that initially initiated hepatocytes arrange in elongated patterns. Only when the tumor progresses to cell numbers of approximately 4,000, it adopts spherical structures. This model prediction was validated by the analysis of initiated cells in a rat liver tumor initiation study using single doses of 250 mg/kg of the genotoxic carcinogen Nnitrosomorpholine (NNM). Indeed, small clusters of GST-P positive cells induced by NNM were elongated, almost columnar, while larger GDT-P positive foci of approximately the size of liver lobuli, adopted spherical shapes. Simulation of numerous possible mechanisms demonstrated that only hepatocyte-sinusoidal-alignment (HSA), a previously discovered order mechanism involved in coordination of liver tissue architecture, could explain the experimentally observed initial deviation from sphericalshape. The present study demonstrates that the architecture of small hepatocellular tumor cell clusters early after initiation is still controlled by physiological control mechanisms. However, this coordinating influence is lost when the tumor grows to approximately 4,000 cells, leading to further growth in spherical shape. Our findings stress the potential importance of organ micro-architecture in understanding tumor phenotypes

Chondrocyte Apoptosis after Simulated Intraarticular Fracture: A Comparison of Histologic Detection Methods

Accurate evaluation of programmed cell death, or apoptosis, in chondrocytes is essential to studying cartilage injury. We evaluated four methods of detecting chondrocyte-programmed cell death in formalin-fixed, paraffin-embedded cartilage after experimental osteochondral fracture. Human osteochondral explants were subjected to experimental fracture in a manner known to induce high levels of chondrocyte-programmed cell death. After 4 days in culture, specimens were fixed and analyzed for programmed cell death using: (1) terminal deoxynucleotidyl transferase end labeling; (2) DNA denaturation analysis using an antibody specific for single-stranded DNA; (3) immunohistochemistry using antisera specific for active caspase-3; and (4) in situ oligonucleotide ligation. Quantitative analysis of programmed cell death levels for each technique was performed comparing injured and uninjured areas of cartilage. We observed differences between injured and uninjured areas of cartilage using the four methods. Human cartilage fixed in zinc-formalin and embedded in paraffin is amenable to programmed cell death analysis using any of four independent methods, each of which ostensibly has some advantages in terms of assaying different steps along the apoptotic pathway. Using the protocols described in this article, investigators may have additional tools to identify and quantify chondrocytes undergoing programmed cell death after experimental cartilage injury

Colon tumor promotion, is it a selection process? Effects of cholate, phytate, and food restriction in rats on proliferation and apoptosis in normal and aberrant crypts

Promotion would suppose the selection of initiated cells. We tested the selection of aberrant crypt cells by cholic acid, a colon cancer promoter, and the effect of protectors, phytate and food restriction. After an azoxymethane injection, rats were allocated to a control diet, or to supplements of cholic acid, sodium phytate, or to a 50% food restriction. The proliferation and apoptosis of 1200 crypts were assessed, after immuno-staining for BrdU. Cholic acid increased the proliferation of aberrant crypts but not of normal crypts. Phytate and food restriction decreased the proliferation of normal crypts, but not of aberrant crypts. Apoptosis was not affected by diets. Results support the hypothesis that cholic acid can select initiated cells in the colon

Erythropoietin reduces neuronal cell death and hyperalgesia induced by peripheral inflammatory pain in neonatal rats

Painful stimuli during neonatal stage may affect brain development and contribute to abnormal behaviors in adulthood. Very few specific therapies are available for this developmental disorder. A better understanding of the mechanisms and consequences of painful stimuli during the neonatal period is essential for the development of effective therapies. In this study, we examined brain reactions in a neonatal rat model of peripheral inflammatory pain. We focused on the inflammatory insult-induced brain responses and delayed changes in behavior and pain sensation. Postnatal day 3 pups received formalin injections into the paws once a day for 3 days. The insult induced dysregulation of several inflammatory factors in the brain and caused selective neuronal cell death in the cortex, hippocampus and hypothalamus. On postnatal day 21, rats that received the inflammatory nociceptive insult exhibited increased local cerebral blood flow in the somatosensory cortex, hyperalgesia, and decreased exploratory behaviors. Based on these observations, we tested recombinant human erythropoietin (rhEPO) as a potential treatment to prevent the inflammatory pain-induced changes. rhEPO treatment (5,000 U/kg/day, i.p.), coupled to formalin injections, ameliorated neuronal cell death and normalized the inflammatory response. Rats that received formalin plus rhEPO exhibited normal levels of cerebral blood flow, pain sensitivity and exploratory behavior. Treatment with rhEPO also restored normal brain and body weights that were reduced in the formalin group. These data suggest that severe inflammatory pain has adverse effects on brain development and rhEPO may be a possible therapy for the prevention and treatment of this developmental disorder

Regulated mitochondrial DNA replication during oocyte maturation is essential for successful porcine embryonic development.

Cellular ATP is mainly generated through mitochondrial oxidative phosphorylation, which is dependent on mitochondrial DNA (mtDNA). We have previously demonstrated the importance of oocyte mtDNA for porcine and human fertilization. However, the role of nuclear-encoded mitochondrial replication factors during oocyte and embryo development is not yet understood. We have analyzed two key factors, mitochondrial transcription factor A (TFAM) and polymerase gamma (POLG), to determine their role in oocyte and early embryo development. Competent and incompetent oocytes, as determined by brilliant cresyl blue (BCB) dye, were assessed intermittently during the maturation process for TFAM and POLG mRNA using real-time RT-PCR, for TFAM and POLG protein using immunocytochemistry, and for mtDNA copy number using real-time PCR. Analysis was also carried out following treatment of maturing oocytes with the mtDNA replication inhibitor, 2',3'-dideoxycytidine (ddC). Following in vitro fertilization, preimplantation embryos were also analyzed. Despite increased levels of TFAM and POLG mRNA and protein at the four-cell stage, no increase in mtDNA copy number was observed in early preimplantation development. To compensate for this, mtDNA appeared to be replicated during oocyte maturation. However, significant differences in nuclear-encoded regulatory protein expression were observed between BCB(+) and BCB(-) oocytes and between untreated oocytes and those treated with ddC. These changes resulted in delayed mtDNA replication, which correlated to reduced fertilization and embryonic development. We therefore conclude that adherence to the regulation of the timing of mtDNA replication during oocyte maturation is essential for successful embryonic development

Investigation of cerebral autoregulation in the newborn piglet during anaesthesia and surgery

The relationship between cerebral autoregulation (CA) and the neurotoxic effects of anaesthesia with and without surgery is investigated. Newborn piglets were randomly assigned to receive either 6 h of anaesthesia (isoflurane) or the same with an additional hour of minor surgery. The effect of the spontaneous changes in mean arterial blood pressure (MABP) on the cerebral haemodynamics (oxy- and deoxy-haemoglobin, HbO2 and Hb) was measured using transverse broadband near-infrared spectroscopy (NIRS). A marker for impaired CA, concordance between MABP and intravascular oxygenation (HbD = HbO2 - Hb) in the ultra-low frequency domain (0.0018-0.0083 Hz), was assessed using coherence analysis. Presence of CA impairment was not significant but found to increase with surgical exacerbation. The impairment did not correlate with histological outcome (presence of cell death, apoptosis and microglial activation in the brain)