Kåre E. Tvedt

Closely related colon cancer cell lines display different sensitivity to polyunsaturated fatty acids, accumulate different lipid classes and downregulate sterol regulatory element‐binding protein 1

N‐6 polyunsaturated fatty acids (PUFAs) may be associated with increased risk of colon cancer, whereas n‐3 PUFAs may have a protective effect. We examined the effects of docosahexaenoic acid (DHA), eicosapentaenoic acid and arachidonic acid on the colon carcinoma cell lines SW480 derived from a primary tumour, and SW620 derived from a metastasis of the same tumour. DHA had the strongest growth‐inhibitory effect on both cell lines. SW620 was relatively more growth‐inhibited than SW480, but SW620 also had the highest growth rate in the absence of PUFAs. Flow cytometry revealed an increase in the fraction of cells in the G2/M phase of the cell cycle, particularly for SW620 cells. Growth inhibition was apparently not caused by increased lipid peroxidation, reduced glutathione or low activity of glutathione peroxidase. Transmission electron microscopy revealed formation of cytoplasmic lipid droplets after DHA treatment. In SW620 cells an eightfold increase in total cholesteryl esters and a 190‐fold increase in DHA‐containing cholesteryl esters were observed after DHA treatment. In contrast, SW480 cells accumulated DHA‐enriched triglycerides. Arachidonic acid accumulated in a similar manner, whereas the nontoxic oleic acid was mainly incorporated in triglycerides in both cell lines. Interestingly, nuclear sterol regulatory element‐binding protein 1 (nSREBP1), recently associated with cell growth regulation, was downregulated after DHA treatment in both cell lines. Our results demonstrate cell‐specific mechanisms for the processing and storage of cytotoxic PUFAs in closely related cell lines, and suggest downregulation of nSREBP1 as a possible contributor to the growth inhibitory effect of DHA.

Gut luminal microdialysis of glycerol as a marker of intestinal ischemic injury and recovery

Objective:To evaluate microdialysis as a method to assess different degrees of intestinal damage and recovery during ischemia and reperfusion; to evaluate information obtained from microdialysis catheters in the peritoneum, the gut wall, and the gut lumen. Design:Randomized, controlled animal experiment. Setting:University laboratory animal center. Subjects:Twenty-seven domestic pigs. Interventions:The superior mesenteric artery was cross-clamped for 60 mins (n = 14) or 120 mins (n = 10) followed by 2 or 4 hrs of reperfusion. Three pigs served as controls. Measurements and Main Results:Intestinal mucosal integrity was assessed by morphometry, adenosine triphosphate in the gut wall, and permeability of 14C-polyethylene glycol. Lactate, glycerol, pyruvate, and glucose were measured by microdialysis. Changes in adenosine triphosphate, permeability, or lactate did not correlate to different extents of intestinal damage caused by 60 or 120 mins of ischemia. During the reperfusion period, pigs with 60 mins of intestinal ischemia showed a faster recovery of these variables than pigs with 120 mins of intestinal ischemia. Glycerol increased with increasing duration of the ischemic insult. After 60 mins of intestinal ischemia, glycerol in the gut lumen decreased toward baseline but remained high after 120 mins of intestinal ischemia. There was a good correlation between gut luminal glycerol and recovery of mucosal damage throughout the reperfusion period. In the peritoneal cavity, both glycerol and lactate decreased to baseline relatively shortly after onset of reperfusion independent of the duration of intestinal ischemia. Conclusions:Microdialysis of glycerol provides information about the extent and severity of intestinal damage after ischemia and about the ensuing recovery. The gut lumen is to be preferred as a site for placement of microdialysis catheters.

In vivo MRI of olfactory ensheathing cell grafts and regenerating axons in transplant mediated repair of the adult rat optic nerve

The purpose of the present study was to use magnetic resonance imaging (MRI) as a tool for monitoring transplant‐mediated repair of the adult rat visual pathway. We labelled rat olfactory ensheathing cells (OECs) using micron‐sized particles of iron oxide (MPIO) and transplanted them by: i) intravitreal injection (ivit) and ii) intra‐optic nerve (ON) injection (iON) in adult rats with ON crush (ONC) injury. We applied T2‐weighted MRI and manganese‐enhanced MRI (MEMRI) to visualise transplanted cells and ON axons at specific times after injury and cell engraftment. Our findings demonstrate that ivit MPIO‐labelled OECs are unequivocally detected by T2‐weighted MRI in vivo and that the T1‐weighted 3D FLASH sequence applied for MEMRI facilitates simultaneous visualisation of Mn2+−enhanced regenerating retinal ganglion cell (RGC) axons and MPIO‐labelled OEC grafts. Furthermore, analysis of MRI data and ultrastructural findings supports the hypothesis that iON OEC transplants mediate regeneration and remyelination of RGC axons post injury. Copyright

Quick sampling and perpendicular cryosectioning of cell monolayers for the X-ray microanalysis of diffusible elements.

A quick sampling and preparation method for freezing of cell monolayers is described. The cells are grown on a large Formvar film supported by a frame of polystyrene. A polyvinylpyrrolidone (PVP) solution is applied to one side of the film forming a flat disc when frozen with a pair of pliers precooled in liquid nitrogen. The PVP solution provides the specimen with sufficient strength and may be used as an elemental standard for absolute quantification if salts of known concentrations are added. Manipulation of the cells prior to freezing is thus restricted to a minimum, which eliminates possible harmful treatments like scraping and centrifugation. The procedure is quickly performed, the freezing being completed within 30 s of the cells having been removed from the culture well. The analytical results reveal low and stable Na: K ratios. Our results confirm that cells in vitro are comparable to cells in vivo with respect to elemental composition.

Transepithelial transport of morphine and mannitol in Caco‐2 cells: the influence of chitosans of different molecular weights and degrees of acetylation

The object of this study was to compare the effect of chitosans of different number‐average molecular weights (MWs) and degrees of acetylation (FA) on transepithelial transport of morphine in Caco‐2 cells. Caco‐2 monolayers on polycarbonate (PC) membranes (0.5cm2) were incubated with morphine (10μ) or mannitol (55μ) for 180 min. Samples for analysis of morphine (LCMSMS) and mannitol (liquid scintillation) were drawn at 45, 90, 120 and 180 min. Transepithelial electrical resistance (TEER) and transmission electron microscopy were used to monitor cell integrity. In controls, morphine transport was half that of mannitol. Chitosans affected the transport of morphine and mannitol similarly. For chitosans with similar FA (0.32‐0.43) and varying MWs (7–200 kD), transport was increased at MWs of 29 kD or more. Among chitosans of similar MWs (180–300 kD) and varying FA (0.01‐0.61), those with the highest Fa (0.61) had the least effect, while chitosans with Fa/MW 0.01/250 and 0.17/300 promoted the greatest transport. An FA/MW of 0.32/200 and 0.43/170 induced a high and stable transport rate. Chitosans may enhance transepithelial transport of morphine by the same mechanism as for mannitol. Chitosans with FA of 0.3‐0.4 and MW of approx. 200 kD seem favourable in this respect.

A section press and low elemental support for enhanced preparation of freeze‐dried cryosections

A press‐ and specimen holder system is described, whereby thin, freeze‐dried cryosections can be obtained more conveniently and with greater protection against contamination and harmful temperature fluctuations. The use of a grid and retainer assembly as the basic working unit, greatly facilitates low temperature work particularly when delicate grids are preferable from an analytical point of view. A new press has been constructed in order to keep the sections permanently pressed during freeze‐drying and rewarming. The press also protects the sections during transfer to an external freeze‐dryer.

Labelling of olfactory ensheathing cells with micron-sized particles of iron oxide and detection by MRI

A crucial issue in transplant-mediated repair of the damaged central nervous system (CNS) is serial non-invasive imaging of the transplanted cells, which has led to interest in the application of magnetic resonance imaging (MRI) combined with designated intracellular magnetic labels for cell tracking. Micron-sized particles of iron oxide (MPIO) have been successfully used to track cells by MRI, yet there is relatively little known about either their suitability for efficient labelling of specific cell types, or their effects on cell viability. The purpose of this study was to develop a suitable MPIO labelling protocol for olfactory ensheathing cells (OECs), a type of glia used to promote the regeneration of CNS axons after transplantation into the injured CNS. Here, we demonstrate an OEC labelling efficiency of >90% with an MPIO incubation time as short as 6 h, enabling intracellular particle uptake for single-cell detection by MRI without affecting cell proliferation, migration and viability. Moreover, MPIO are resolvable in OECs transplanted into the vitreous body of adult rat eyes, providing the first detailed protocol for efficient and safe MPIO labelling of OECs for non-invasive MRI tracking of transplanted OECs in real time for use in studies of CNS repair and axon regeneration.

Carcinoma Metastasis - An Approach to Models

Background: Epithelium is separated from other tissues in the body by the basal membrane. When respecting this boundary, atypical epithelial growth does not cause serious illness in most cases. Therefore, carcinoma in situ is considered to be a non-malignant condition. However, the situation is quite different if the epithelial cells do not respect the natural boundaries in the tissue, a condition that is often referred to as cancer. Uncontrolled invasive growth is indeed the main characteristic of malignancy, and metastasis is in most cases the reason why cancer patients die. Materials and methods: The purpose of this article is to highlight how carcinoma cells, nature, can be classified in three steps, in respect to the first local infiltration of malignant epithelium. The literature referred to is selected on the basis that the views advocated are not controversial, and not represent individual findings. Moreover, some considerations are based on the authors own experience in clinical and molecular basic research. Results: The main characteristics of invasive cellular behavior are modified adhesion and a transition from fixed cells to a migratory phenotype. Invasion is made possible by the degradation of extracellular components. We only know fragments of the gene and phenotypic changes that enable cancers origin of behavior, but there is evidence that chemokines play a central role in the directional spread of motile cells. However, the most common characteristic of carcinoma cells is their loss of cell polarity. Interpretation: The complexity of multicellular organisms is staggering. Artificial and highly simplified model systems are therefore cancer researcher’s most important tools. To be significant, such results must be translated and verified to the in vivo situation. In addition, generality in cancer research finding must be given greater importance, since individual results, not can form the basis for new treatment regimes. Today’s biggest challenge for researchers is therefore to be able to collate the enormous diversity of molecular biological knowledge that daily runs.

Can Drying Be an Alternative Tissue Preservation Method in Cancer Research Biobanking

The currently available methods for conservation of biobank material are mainly based on formalin fixation or the use of different freezing techniques. For molecular biological analysis, it is common to use quick freezing and low-temperature storage of the tissue materiel. This is a very energy-intensive and expensive method that requires advanced infrastructure, including monitoring and control procedures. The purpose of this work has been to study drying as an alternative process to cryogenic storage of undried biobank material, especially for use in cancer research groups. Fast freezing has been shown to be suitable to preserve the integrity of RNAs, while traditional formalin fixation preserves proteins and thus morphology in a good way. Various fresh-harvested murine tissues, such as lung, heart, skeletal muscle, liver, and kidney, were quickly frozen in liquid nitrogen and then subsequently dried at +5°C and −10°C, respectively, in a heat pump dryer. After drying, the RNA integrity was measured. The dried material was then stored for five months at +4°C and −20°C in commercial refrigerators, with subsequent measurement of RNA integrity. Dried materials were also evaluated with light microscopy and by electron microscopy with respect to tissue and cell structure. The same pattern was found for all five murine tissues. We conclude that drying at temperatures below 0°C is most careful to preserve the RNA integrity, with approximately the same RIN score of dried and non-dried samples for all five tissues. What characterized the general pattern of stored samples is that drying leads to a preservation of RNA integrity. Moreover, architecture in tissue resembled normal sections prepared from fresh tissue. In some places in the rim of the tissue sample, the lung tissue revealed alveolar-like morphology. In the electron microscope, few organelles other than the nuclei could be identified. Drying of biological material is a promising and cost-effective method for biobanks that store tissue, compared to cryogenic storage of undried material. Degradation of RNA, measured by the RIN number, is a critical factor in storing biobank tissue. In low-temperature dried material, the RIN factor is at the same level as storage of undried material at cryogenic temperatures, which is the common way of storing biobank material today. In this study, a heat pump dryer was used successfully to establish drying temperatures below and above the freezing point of the material. Further work has to be done in order to study different drying methods, drying conditions, and drying costs.

Post-ischaemic restituted intestinal mucosa is more resistant to further ischaemia than normal mucosa in the pig.

Objective. Ischaemic preconditioning may protect the intestine from subsequent prolonged ischaemia. This study evaluates whether a much longer initial ischaemia, encountered clinically, may modify intestinal resistance to further ischaemia in a pig model. Material and methods. After cross‐clamping of the superior mesenteric artery for 1 h, the intestine was either reperfused for 8 h or a second cross‐clamping for 1 h was performed at 4 h of reperfusion. Based on microarray analysis of intestinal samples at 1, 4 and 8 h of reperfusion, mRNA of selected genes was measured with QRT‐PCR. Results. The first ischaemic period caused exfoliation of surface epithelial cells from the basement membrane comprising about 90 % of the villi tips, a marked increase in permeability and depletion of ATP. The second ischaemic challenge caused about 30 % less denudation of the basement membrane (p = 0.008), no increase in permeability (p = 0.008) and less depletion of ATP (p = 0.039). mRNAs for superoxide dismutase 2, heat shock proteins and signal transducer and activator of transcription 3, which may protect against ischaemia/reperfusion injury, were up‐regulated throughout the reperfusion period. mRNAs for matrix metalloproteinase 1, connexin 43 and peripheral myelin 22, which may be associated with cell migration or tight junctions, showed a particular up‐regulation at 4 h of reperfusion. Conclusion. One hour of initial ischaemia followed by 4 h of reperfusion is associated with increased intestinal resistance to further ischaemia. The differential regulation of genes identified in this study provides working hypotheses for mechanisms behind this observation.