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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...

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

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 T(2)-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 T(2)-weighted MRI in vivo and that the T(1)-weighted 3D FLASH sequence applied for MEMRI facilitates simultaneous visualisation of Mn(2+-) 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.

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.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.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.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.

Rationale and Objectives Studies have shown that water-soluble radiographic contrast media may be useful as intestinal permeability probes. The current study was undertaken to provide information about the route by which such a permeability probe crosses the mucosal epithelium. Materials and Methods The authors administered 3 mL of iodixanol (iodine, 320 mg/mL) by means of an enema into the colons of (a) rats with experimental colitis induced 14 days earlier and (b) normal rats. At various intervals after the enema, laparotomy was performed and the colon was identified and longitudinally split immediately before cryofixation, without prior interruption of circulation. Freeze-dried cryosections were studied by means of electron microscopy and x-ray microanalysis. Results Approximately equal concentrations of iodine were found intracellularly and between the epithelial cells of the colonic mucosa. Ninety percent of analyzed cells had a normal sodium-potassium ratio and low levels of intracellular chlorine, indicating intact cell membranes. Nevertheless, many of these cells, in specimens from both normal rats and rats with colitis, contained high intracellular iodine concentrations. Conclusion The transcellular route seems to be at least as important as the paracellular route for permeation of the highly water-soluble molecule iodixanol through the mucosal epithelium of both normal and inflamed rat colon. This finding may contradict previous findings in other water-soluble substances.

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.

Background: In recent years, many investigators have focused on the development of drugs with the potential to influence the malignant phenotype. Clearly, our understanding of molecular and cellular processes behind such mechanisms has increased substantially through the past decades. This knowledge has led to an extensive search for targeted carcinoma treatment.Methods: This review is based on a qualified selection of articles published in internationally recognized journals. Some considerations are also based on the authors’ own experience in clinical and molecular-targeted basic research.Results: Inhibitors of tyrosine kinases (TKIs) and selective gene expressional regulators, small interfering RNAs, give hope for advances in the treatment of various carcinomas. However, it seems clear that the main challenge also in such therapy is the delivery, toxicity, uptake, and stability of small targeted molecules. These must be dealt with in order to unleash the potential of individual therapy.Conclusion: Development of TKIs and siRNAs, with effects better than or at least similar to conventional therapy must be given high priority. Tolerable side effects are also critical to successful implementation. Only through such efforts may such treatment be fully developed and find its place in the treatment of malignant tumours, solely, as primary choice, or in combination with conventional cancer therapy.

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 T(2)-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 T(2)-weighted MRI in vivo and that the T(1)-weighted 3D FLASH sequence applied for MEMRI facilitates simultaneous visualisation of Mn(2+-) 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.

A method developed for X-ray microanalysis (XRMA) of cell monolayers cultured on Formvar film has been shown to be well suited for the study of explant cultures of human prostate. Adherence and epithelial cell outgrowth occur as readily on the formvar as on ordinary tissue culture plastic, giving rise to cultures with the same morphological characteristics. Conventional transmission electron microscopy revealed intracytoplasmic granules with the appearance of secretory vesicles. XRMA of electron-dense intracytoplasmic granules in freeze-dried cryosections showed significant local sequestration of calcium, but not of magnesium or zinc, elements that have previously been shown to colocalize with calcium in secretory granules in vivo. It is concluded that some aspects of the secretory phenotype are supported in this in vitro model. The factors regulating the expression of a differentiated phenotype in prostatic epithelium await further elucidation. XRMA may be useful in assessing the effects on secretory differentiation induced by variations in the culture conditions.

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.

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.

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.Randomized, controlled animal experiment.University laboratory animal center.Twenty-seven domestic pigs.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.Intestinal mucosal integrity was assessed by morphometry, adenosine triphosphate in the gut wall, and permeability of C-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.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.

Background The mucosal surface epithelium is an essential part of the functional intestinal barrier, but its structural response to ischemia/reperfusion is only partly characterized. The purpose of this study was to provide a detailed morphological evaluation of intestinal surface epithelium after aortic cross-clamping. Material and methods Pigs were subjected to thoracic aortic cross-clamping for 60 min and subsequent reperfusion for 120 min. Tissue blood flow and high-energy phosphates were measured with microspheres and HPLC, respectively. Urinary excretion of 14C polyethylene glycol (MW 4000 Da) (PEG-4000), loaded into an intestinal loop, provided an index of intestinal permeability. Results Jejunal blood flow was restored at 10 min after aortic declamping. Denudation of the basement membrane of the intestinal villi tips, as a consequence of epithelial shedding, increased markedly during the initial 60 min of reperfusion (P = 0.002). During the following 45 min, the denuded basement membrane was partly covered with low cuboidal and squamous-shaped cells extending lamellipodia over a wavy basement membrane. Restoration of ATP at 60 min after aortic declamping correlated inversely to the extent of denuded basement membrane (r = 0.75, P = 0.032). Permeability of PEG-4000 increased markedly after aortic declamping and was linearly correlated to the area of denuded basement membrane (r = 0.87, P = 0.01). Conclusions Reperfusion for 2 h after aortic cross-clamping is associated with initial aggravation of ischemia-induced injury in the porcine jejunum, but thereafter with restitution of the surface epithelium. Restoration of ATP may be important to avoid intestinal injury after ischemia. Increased permeability of a macromolecule in response to reperfusion is closely correlated to injury of the surface epithelium.

Studies have shown that water-soluble radiographic contrast media may be useful as intestinal permeability probes. The current study was undertaken to provide information about the route by which such a permeability probe crosses the mucosal epithelium.The authors administered 3 mL of iodixanol (iodine, 320 mg/mL) by means of an enema into the colons of (a) rats with experimental colitis induced 14 days earlier and (b) normal rats. At various intervals after the enema, laparotomy was performed and the colon was identified and longitudinally split immediately before cryofixation, without prior interruption of circulation. Freeze-dried cryosections were studied by means of electron microscopy and x-ray microanalysis.Approximately equal concentrations of iodine were found intracellularly and between the epithelial cells of the colonic mucosa. Ninety percent of analyzed cells had a normal sodium-potassium ratio and low levels of intracellular chlorine, indicating intact cell membranes. Nevertheless, many of these cells, in specimens from both normal rats and rats with colitis, contained high intracellular iodine concentrations.The transcellular route seems to be at least as important as the paracellular route for permeation of the highly water-soluble molecule iodixanol through the mucosal epithelium of both normal and inflamed rat colon. This finding may contradict previous findings in other water-soluble substances.

In order to make needle biopsies more readily applicable to cryo-ultramicrotomy and x-ray microanalysis, we have developed a method which permits biopsy specimens to be frozen immediately after removal from the patients, using metal mirror freezing.Then tissue sample is quickly transferred from the biopsy needle to a formvar film which in advance has been supplied with a drop of standard solution containing PVP and inorganic salts. Freezing is performed by clamping the sample between the cylindrical copper jaws of a pair of pliers, precooled in liquid nitrogen (fig.l). The flat specimen is cut in a Reichert-Jung Ultracut/FC4 cryosystem. The section is collected on a grid/retainer assembly, which is mounted inside a. transportable press. Secured between two grids inside the press, the sections are transferred to an external freeze-drier.This system makes it possible to freeze,even small pieces of tissue using the metal mirror freezing method. The pliers produce a flat specimen containing the tissue and the elemental standard, with two well-frozen surfaces which readily can be sectioned using a flat specimen holder.

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.

A method for clamp-freezing of small biopsies for ultrathin cryosectioning is described. A formvar film is used to support the specimen before freezing with a pair of pliers, precooled in liquid nitrogen. This method makes it possible to align the piece of tissue close to a drop of standard solution and freeze the two simultaneously. After clamping, the flat, frozen sample contains both the piece of tissue and the standard in one solid block, which may thus be sectioned as a whole. The method has been used in the surgical theater, yielding good results with needle biopsies of the prostate and cystoscopically obtained biopsies of bladder tumors. The time lapse between excision and freezing may be less than 30 seconds, which represents a great improvement compared with the far more time-consuming mincing of tissue and mounting of the pieces on metal pins. From the frozen specimen, a large number of sections with good morphologic characteristics may be produced. The method was evaluated with the use of rat liver and lactating mammary tissue. Directly frozen tissue with intact blood supply was compared with biopsies prepared as described. Redistribution of Ca could not be demonstrated in either specimen type. However, the results indicate that the monovalent ions are far more mobile, and a significant increase of the Na:K ratios was observed in the biopsies compared with directly frozen tissue.

A 61-year-old man with idiopathic haemochromatosis and eye symptoms is described. The examination showed microaneurysms scattered over the bulbar conjunctiva, and some discrete pigmentations in the superficial tissue in the perilimbal area. Each cornea revealed a light brown, ellipsoid opacity with the longest diameter horizontally, seemingly being located in the corneal epithelium and/or the anterior part of the stroma. A corneal biopsy was examined by light and electron microscopy. Two different types of granules were found mainly located to the Bowman's membrane. X-ray microanalysis showed no detectable amounts of iron, but high concentrations of phosphorus and calcium, and in addition magnesium in one type of the granules. Treatment with EDTA improved the vision considerably.

X-ray microanalysis has been performed on freeze-dried cryosections of normal, hyperplastic, and neoplastic human prostates. Needle biopsies from prostates suspected to be malignant were collected from ten patients. Seven of these patients had fully developed nodular hyperplasia, whereas the remaining three specimens were histologically classified as moderately differentiated adenocarcinomas. Prostates with normal light microscopic appearance were obtained from two brain-dead kidney donors. The concentration of calcium in secretory vesicles was found to be several orders of magnitude higher than the concentration of magnesium and zinc in all of the glands studied, which is consistent with calcium being the major cation secreted by the prostatic acinar cells. Some elderly prostates and neoplastic prostates revealed even lower mean zinc:calcium ratios in secretory vesicles. Even though both elements varied considerably, the wide variation of the zinc:calcium ratios was due mainly to variations in the concentration of zinc. Large, electron-dense bodies occasionally were found in the cytoplasm of prostatic acinar cells. These bodies contained high concentrations of sulphur which by far was the dominating element.