Furthermore, in vitro extracellular environment, alters the cell's natural characteristics and function 9 , which may impair the quality of biological material, but stem cells' nutritional requirements have not been identified yet Signaling pathways of apoptosis, cell's survival and growth has a close correlation with glucose and glutamine metabolic use 13 , In order to preserve cells' natural characteristics following transplantation, and based on nutrients metabolic importance, the present study aims to evaluate microscopic behavior and viability of DPSc under glucose and glutamine different concentrations and deprivation.
Human tooth tissues were minced in isolated pieces from epithelial or mesenchymal tissues from periodontal ligament or pulp, respectively. The medium was replaced twice a week until cells confluence was obtained. Following proliferation of cell culture, cells were cultured in a chemically defined culture medium DMEM, Invitrogen r Carlsbad, California. Commercially available formulas were utilized with different concentrations of Glucose and Glutamine: Low Glc Glucose: 1.
All of the experiments were performed within 14 days of a previous culture. The proliferation and number of cells were both analyzed by an automated cell counter Countess - Invitrogen r Carlsbad, California. Trypan blue solution diluted, according to the manufacturer's instructions, was used to identify live and dead cells, and the total cells' number.
The evolution of cell culture in this study was conducted using an inverted optical microscope Axiovert 40C Carl Zeiss r , Gottingen, Germany in addition to AxioVision software AxioVision Software r 4. Cells were cultured on chambered coverglass slides throughout the 14 days of the study.
Structural analyses were performed using Alexa Fluor for cytoskeleton, Dapi for nucleus, and Mitotracker for mitocondria Invitrogen, Carlsbad, California following the manufacturer's instructions. The Methylthiazoltetrazolium test MTT was conducted to determine cellular viability. Mitochondrial activity, determined by the amount of blue crystal's formation, was read on Elisa spectrophotometer wavelength nm in 96 well-plates Afterwards, multiple unilateral comparisons were accomplished using a non-parametric version of the Dunnett test.
Descriptive statistics results showed that the Low Glc culture medium promoted better patterns of viability, and Gln culture medium exhibited poorer results.
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In the serum-supplemented culture media, the patterns were similar, except for Low Glc medium that showed an important decrease Figure 1. A non-parametric version of Dunnett test demonstrated a significant superiority of cell viability in serum absence of Low Glc culture medium.
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Non-parametric version of Dunnett's test. A chi-square test was applied to analyze the effect of culture media and FBS on the dead cells' number. Although the Low Glc medium in serum absence illustrated a superior pattern of cell viability, we observed a considerable number of dead cells. These results suggest a small group selection from a viable cell population. The serum supplemented culture medium decreased cell death percentage in all culture medium groups Figure 5. These data show the presence of non-characterized elements in FBS, affecting cell culture when associated with other nutrients.
From a different perspective, nutrient flow alterations of glucose and glutamine modify the metabolic activity so as to induce direct effects on cell's death. Microscopic evaluation of cell architecture denotes a misrepresented behavior in response to culture media composition, showing that FBS presence may induce transformations in the cellular morphology Figure 6. Gln culture medium promotes a disappearance process of the extracellular matrix in several phases, modifying the disposition of cells and forming amorphous and complexes structures in serum supplemented B1,2 and serum absence B3,B4 groups.
Low doses of glucose in a serum-free culture medium favored the evolution of cells in this study, preserving their natural characteristics in the serum-free Low Glc culture medium D3,D4. In contrast, FBS addition to this medium has induced the loss of extracellular matrix and the change in cells' form D1,D2. In both groups, serum supplemented 1,2 and serum absence 3,4 alterations were observed in conventional patterns of cell's behavior.
The Glc culture medium displayed cell adherence discontinuity A1-A3 , showing a rounded shape, and cells partially detached, with small clusters were formed A4. Gln culture medium promoted extracellular matrix degradation in serum supplemented B1,B2 and serum absence B3,B4 , adhesion damages, and unstable overlap of cells B1,B3,B4.
Hence, these complex and amorphous structures suggest a helpful role of glucose in extracellular matrix synthesis.
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Low Glc culture medium demonstrated supreme microscopic outline in serum absence, preserving the culture architecture, without signals of apoptosis or damage D3, D4. In the serum supplemented group D1, D2 , important changes occurred, with total detachment of cells in culture, clusters formation, and cell rounding due to extracellular matrix absence.
Confocal microscopy did not demonstrated specific alterations in relation to nutrient concentrations in the circumstances of this study. Nevertheless, FBS promoted transformations in the cell structures studied here Figure 7.
Cytoskeleton presented significant structural damages and rounding, outside mitochondrial fragmentation and a discrete alteration in nucleus organization A1, A2, A3. The serum absence groups Figure 8 also exhibited some structural impairment. However, this occurred in a smaller scale, preserving part of the classic configuration of the cellular structures. Extensions of cytoskeleton were present, supporting cell adherence, cell to cell communication, and proliferation B1, B2, B3, B4 , despite eventual variations in the cytoskeleton contour B1, B3.
According to the established conditions and statistical tests performed in this study, it is possible to state that glucose and glutamine deprivation or their different concentrations promoted important alterations on cell behavior and viability. Normal components of adult female filarial nematodes. Ovaries of an onchocercal worm, B. Earliest stage of filarial ova, C. Morulae stages filling two uterine horns of a healthy female filarial worm, D. Mature morulae inside individual egg shells, E.
Coiled microfilariae contained within egg shells, F.
Fully stretched microfilariae ready for release from the uterus. Different ways of assessing the state of worms in histological sections have been used. Examples of typical degenerative components of adult female filarial nematodes. Filarial nematode with damage to the body wall. Filarial nematode with damaged horns of the uterus with early calcification blue staining of the uterine content , C.
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A degenerating worm with the morula stages unable to form. Disrupting early morulae forms E. Uterine horn of a damaged filarial worm containing many degenerating forms, F. Calcified casts of a dead filarial worm embedding in a chronic tissue response nodule. Much of what has been described for assessing parasites in in vivo situations applies to those parasites that have been obtained from culture systems.
Here it is important to acknowledge that worms maintained in vitro are already in an unnatural environment and this can affect certain anatomical components more than others. The wall and cuticle of cultured worms often, even in the control samples, can have degenerative changes that are induced by this unusual environment.
If worms have been isolated using tissue digestive methods before culturing then the changes due to the processing are consequently even more common. This collection of worms, optimally a minimum of 5 worms, is then regarded statistically as one entity as are in vivo nodules. The use of in situ markers is an important new approach being developed for assessing degenerative changes in nematodes but to date, there are still relatively few studies that address this issue in any great depth. One that has nevertheless been extensively described is the continuing presence of the required endosymbiont Wolbachia —usually identified by using labeled antibody markers against a primary antigen WSP of this bacterium.
The presence of these organisms has been used as an indicator of the viability of the adult filarial worm. It must be recognized that this endosymbiont is not uniformly distributed along the worm and it is therefore relatively easy to be misled by only observing relatively few histological sections of the worm, many of which may naturally not actually contain this organism. There are several studies that have described several enzymes that appear to be present in filarial worms [ 14 ], and it is likely that specific enzymes will be identified soon whose presence could act as reliable indicators of worm viability.
Biochemical approaches have been used to assess the viability of in vitro worms for many years—the formazan assay being commonly used [ 15 — 17 ]. Although it is not a major purview of this discussion here to go into the wider area of laboratory and rapid test systems, it is nevertheless important to note that there is a considerable amount of experience over many years with the use of immunological markers, such as circulating antigens and host antibody responses for the diagnosis and epidemiological assessment of filarial infections, both in humans, dogs, and other animals [ 19 ].
The use of samples of urine and saliva for these assays has been attempted but with varying and unfortunately rather unuseful results to date. This is an area of research that is vital to the efforts to eliminate the major parasitic diseases across the world. It is likely that in the next few years specific circulating markers will be identified in blood, or hopefully for ease of collection in urine or in saliva. This would provide a more practical way to assesses populations in epidemiological studies and lead us more quickly to the global goal of eliminating nematodes for affected populations.
There are still many aspects of measuring the viability of nematodes that need improving. A major challenge is to determine when a population of worms that have been subjected to an intervention, e. As described above it is relatively easy to detect alterations if they are physically obvious e. Optimally it would be extremely useful to define a single change or a simple collection of changes that reflect permanent irreversible damage.
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In the case of filariae it is likely that the uterus the biggest organ in the female is a useful indicator site for detecting damage and defining permanent damage. The body wall is also an important target organ for this role but it is an organ that is easily artificially altered by many of the isolation techniques used in preparing the worm samples for studies, especially those for in vitro studies.
Another reason for focusing on the uterus is that interruption of the reproductive capability is a major intervention goal for the three major filarial infections of humans and would be extremely valuable to the success of the current global elimination programs for these two infections. The assessment approach used needs to be driven by the question being asked when deciding on the method and focus of any assessment.
The complexity of the life cycle of nematodes necessitates carefully focusing investigations on stages or anatomical components that are likely to prove useful and provide practical information. It is also important to distinguish between estimating parasite loads in an infected host and the measuring of direct effects on a parasite stage; although these two questions may be intimately linked they are not necessarily the same nor necessarily use the same method for assessment.
An obvious area of basic research that would greatly enhance the needs for developing better techniques for assessing parasite viability is advancing the knowledge of the basic physiological, biochemical and functional characteristics of nematodes and any species differences. Using C. This kind of research is difficult to maintain in the present world where research funding is difficult to acquire, but nevertheless, the acquisition of more detailed information in this subject would undoubtedly be highly valuable.
There is a tendency to interpret cell death, tissue damage and other pathological processes from the perspective of what we know about these processes in mammalian organisms and it would be extremely useful to know if there are similar or different processes occurring in metazoans.
Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution 3. Help us write another book on this subject and reach those readers.
Login to your personal dashboard for more detailed statistics on your publications. Edited by Mohammad Manjur Shah. We are IntechOpen, the world's leading publisher of Open Access books. Built by scientists, for scientists. Our readership spans scientists, professors, researchers, librarians, and students, as well as business professionals. Downloaded: Abstract The assessment of nematodes as they generate and die is not a simple thing to do due in part to the complexity of the organism, and the fact that still relatively little is known about their physiology and internal biology.
Keywords filarial nematodes assessment viability death histopathology. Introduction Nematodes commonly infect humans, animals, and plants in all the ecosystems from the tropics to the polar regions; they can cause significant damage and consequently are responsible for some of the major chronic infections of these hosts.
Filarial nematodes Filariae are a very diverse group of nematodes that infect a very wide range of specific hosts. Indirect clinical assessment of population endemicity Adult worms are central to the assessment the longevity of nematode infections, and this is particularly so in filarial infections, e.
Imaging of parasites in vivo There are certain specific occasions in filarial infections where the adult worms can be seen by careful direct observation or using a diagnostic instrument. Isolation of worms from host tissues In the case of onchocerciasis, the surgical removal of the fibrous subcutaneous nodules, and is in fact regarded as a therapeutic step, as it removes at least some of the fecund females; removal of nodules is also in some cases also a definitive diagnostic step.
Assessing the changes in parasites Alterations in worms that are extensively changed can often be seen by directly looking at the whole worm either in situ or in vivo. Sign in Register Subscribe. Decision Support. CPD articles. Nurse's invention helps with grading of pressure ulcers New grading wheel ensures patients receive appropriate and swift treatment. Documentation and record-keeping in pressure ulcer management National and international guidelines recommend the use of clinical assessments and Preventing pressure ulcers in patients in intensive care This article discusses the prevention and management of pressure ulcers in intensive care Strong views on mandatory staff vaccination show opinion in the profession is polarised.
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