Michal Miko

Telocytes (interstitial Cajal-like cells) in human Fallopian tubes.

BACKGROUND Telocytes represent a relatively newly discovered population of cells found within the various tissues and organs, including Fallopian tubes. It is presumed that telocytes could serve as a sensor of hormone levels or regulate activity of muscle peristaltic movement. METHODS Tissue sections from anatomically different parts of Fallopian tubes of 48 women (age 48.8±9.1) were stained for the expression of five different antigens: c-kit (CD117), CD34, vimentin, podoplanin (D2-40) and Dog-1. RESULTS Telocytes form a network associated with the smooth muscle cells. From the mentioned antibodies, only anti-c-kit (CD117) seems to be relatively selective specific to the telocytes, others react also with numerous other cells and tissue structures. Our results when using antibodies against podoplanin and Dog-1 are in dissonance with recent literature - with regards to our results, they are not suitable for detection of telocytes. CONCLUSION Methods of immunohistochemistry are suitable for identification of telocytes in Fallopian tubes. C-kit (CD117) antigens are useful for routine identification of telocytes in histological sections. This antigen can be combined with CD34 or vimentin in cases of double staining immunohistochemistry.

Ultrastructural analysis of different human mesenchymal stem cells after in vitro expansion: a technical review

Transmission electron microscopy reveals ultrastructural details of cells, and it is a valuable method for studying cell organelles. That is why we used this method for detailed morphological description of different adult tissue-derived stem cells, focusing on the morphological signs of their functions (proteosynthetic activity, exchange with external environment, etc.) and their comparison. Preparing a specimen from the cell culture suitable for transmission electron microscopy is, however, much more challenging than routine tissue processing for normal histological examination. There are several issues that need to be solved while working with cell pellets instead of solid tissue. Here we describe a simple protocol for the isolation and culture of mesenchymal stem cells from different adult tissues, with applications to stem cell biology and regenerative medicine. Since we are working with population of cells that was obtained after many days of passaging, very efficient and gentle procedures are highly necessary. We demonstrated that our semi-conservative approach regarding to histological techniques and processing of cells for transmission electron microscopy is a well reproducible procedure which results in quality pictures and images of cell populations with minimum distortions and artifacts. We also commented about riskiest steps and histochemical issues (e.g., precise pH, temperature) while preparing the specimen. We bring full and detailed procedures of fixation, post-fixation, infiltration, embedding, polymerization and contrasting of cell obtained from in vitro cell and tissue cultures, with modifications according to our experience. All this steps are essential for us to know more about adult stem cells derived from different sources or about other random cell populations. The knowledge about detailed ultra-structure of adult stem cells cultured in vitro are also essential for their using in regenerative medicine and tissue engineering.

Ultrastructural changes of kidney in diabetic rats.

OBJECTIVES To demonstrate histopathological changes in diabetic kidneys of the rat on ultrastructural level with focusing on glomerular cellular environment. METHODS Male Wistar rats were administered streptozotocin and after 8 weeks, their kidneys were studied using qualitative transmission electron microscope analysis. RESULTS In diabetic kidneys, the presence of irregularly thickened glomerular membrana basalis (GBM), often up to six times as compared to healthy kidney GBM, mainly around peripheral capillary loops, was striking. We also demonstrated lamination and split appearance of GBM, with presence of delicate fibrillar structure, which was absent within normal GBM. Accumulation of mesangial matrix was found only occasionally. CONCLUSION Model may be useful to better understand the mechanisms that play role in the progression of DM-induced renal disease and to comprehend the morphological changes, which this process leaves within the tissue. The clear association between ultra-structure and functional markers may not be necessary (Fig. 4, Ref. 27).

Cholinesterase activity in quail primary lymphoid organs

The present histochemical study was carried out to analyze the distribution and topography of acetylcholinepositive nerve fibers in the thymus and bursa of Fabricius of quails. The AChE-positive nerve fibers were demonstrated by direct thiocholine histochemical method. Nerve fibers present in the thymuses form periarterial nerve plexuses located mostly in the interlobular septa and on the cortico-medullary junction. Vessels-independent nerve fibers occur also in the parenchyma of thymic medulla, but rarely in parenchyma of the cortex. Within the connective tissue between the bursa of Fabricius and the wall of proctodeum we observed conspicuous AChE-positive ganglia, often in close relationship to greater arteries. Within the wall of bursa of Fabricius, AChE-positive nerve fibers create nerve plexuses around arteries. We observed a close relationship between lymphoid follicules in bursal submucosa and mucosa and AChE-positive nerve fibers. Nerve fibers create a ring around lymphoid follicles, but do not penetrate into the germinal center of the follicle. Arteries inside quail thymuses and bursae of Fabricius contain rich AChE-positive nerve plexuses, when compared to the veins, which have a very poor presence of AChE-positive nerves. According to lesser presence and decreased density of AChE-positive nerve fibers in older animals, we described age-dependent changes in both quail primary lymphoid organs.

Two nuclei inside a single cardiac muscle cell. More questions than answers about the binucleation of cardiomyocytes

Abstract Human cardiac muscle cells are the most physically energetic cells in the body, and according to various researchers they contain two nuclei in 25–40%. In humans, the heart during prenatal development consists mainly of cardiomyocytes with one nucleus. Just before birth, binucleation begins and can extend into early neonatal life. The physiological importance of binucleation is still poorly understood. In this critical review, we provide a summary of the latest research on binucleation of cardiac muscle cells, with special emphasis on the potential application of such knowledge to the fields of regenerative medicine. We summed up and discussed about ten possible biological arguments why binucleation may be beneficial for cardiac muscle cells as well as for the whole myocardium. These arguments include increase of gene expression, purposeful cell shaping, increase of metabolic activity, energy-saving growth and function, need for organ growth despite of telomere depletion, adaptation to stress (tissue regeneration), prevention of overgrowth – organ shaping, prevention of aneuploidy, terminally differentiated state (cardiomyocytes exit the cell cycle, end of proliferation activity); or, we hypothesize, binucleation is just an unwanted side product.

Effect of chronic intake of liquid nutrition on stomach and duodenum morphology

Changes in the quantity and/or quality of food intake have been shown to be associated with morphological and functional alterations of the gastrointestinal system. To examine this, we investigated the effect of chronic liquid nutrition intake (Fresubin) on stomach and duodenum morphology in Wistar rats fed liquid nutrition during different developmental periods. We used four groups of rats: a) control group (CON) fed pelleted chow for 130days; b) liquid nutrition group (LN) fed liquid nutrition for 130days; c) liquid nutrition juvenile group (LNJ) fed liquid nutrition for 70days and then pelleted food for 60days; d) liquid nutrition adult group (LNA) fed pelleted chow for 70days and then liquid nutrition for 60days. We found that LN and LNA rats showed a significant reduction of empty stomach mass compared to CON animals, while stomach and duodenal longitudinal muscle layer thickness did not differ between groups. Villus height was increased only in LNA animals, while villus width was increased in both LN and LNA rats. Crypt depth was reduced in LNJ. However, liquid nutrition intake did not affect villus height/crypt depth ratio, nor number of goblet cells. We found that chronic intake of liquid nutrition affects some morphological parameters of the stomach and duodenum but these changes were not homogenous between experimental groups. Interestingly, transition from liquid nutrition to solid food reversed the alterations of stomach weight as well as villus width induced by intake of liquid nutrition in LNA rats. Our data indicate that morphological and functional changes in the gastrointestinal system induced by qualitative and quantitative changes in food intake are at least partially reversible. Therefore, specific diets may be used potentially as adjuvant treatment for modulating the progression of gastrointestinal diseases by affecting stomach and small intestine morphology.

Histologic Examination of Peripheral Nerves

Tissue from peripheral nerves is generally studied as a processed and stained section and examined with the light or electron microscope. For the peripheral nerve as a specimen to reach its final destination on the stage of a light microscope as a slide, it needs to undergo a long process, which is called tissue preparation. This process can be subdivided into 10 actions: sampling or dissection, fixation, dehydration, clearing, infiltration and embedding, cutting, deparaffinization, staining, mounting, and finalization of the slide. From the point of view of histologic examination of peripheral nerves, it is important that the nerve is composed of nerve fibers, which are threadlike extensions of a nerve cell (the neuron) and consist of axons and myelin sheaths (if present) and of connective tissue sheaths (endoneurium, perineurium, and epineurium), which are organized in distinct patterns. Several staining methods are used (and discussed in this chapter) for the visualization of tissues present within peripheral nerves.