Histology and histopathology Vol.28, nº 8 (2013)
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- PublicationOpen AccessEffects of the relationship between 65Zn and blood cells. A dynamic and morphological study(F. Hernández y Juan F. Madrid. Universidad de Murcia. Departamento de Biología Celular e Histología, 2013) Vera-Gil, A.; Pérez Castejón, M.J.; Whyte, J.; Cisneros, A.; Recreo, P.; Gascón, M.A.; Whyte, A.; Lahoz, M.; Pérez Castejón, M.C.We have studied the dynamic pathway of 65Zn and its autoradiographic location in blood cells, even at the ultra-structural level. We have found evidence that tends to confirm the old biochemical postulates about the capacity of this isotope to displace iron in the haemoglobin molecule. Recently, the bibliography has demonstrated that 57Co is also able to perform this displacement, but unlike 65Zn it does not invalidate the Redox function of the molecule. In the case of 65Zn, the mentioned displacement invalidates this function because the radionuclide can only use valence 2. We have also contributed evidence of erythrocytes destruction by the spleen after the incorporation of 65Zn, as well as the clearly marked degradation of haematic pigments inside the spleen.
- PublicationOpen AccessSpatial-temporal protein expression of inhibitor of differentiation-1 (Id1) during fetal embryogenesis and in different mouse and human cancer types(F. Hernández y Juan F. Madrid. Universidad de Murcia. Departamento de Biología Celular e Histología, 2013) Redrado, Miriam; Bodegas, Elena; Villaro, Ana Cristina; Nguewa, Paul A.; Lopez, Ines; Gil-Bazo, Ignacio; Calvo, AlfonsoInhibitor of differentiation-1 (Id1) plays a role in cell proliferation, acquisition of epithelial to mesenchymal transition (EMT) features and angiogenesis. Id1 was shown to be expressed in some tumor types, mainly in advanced dedifferentiated stages. However, recent studies using a validated and highly specific monoclonal antibody against Id1 have challenged many of the results obtained by immunohistochemistry. The goal of our work was to perform a thorough analysis of Id1 expression in mouse embryos and adult tissues, as well as healthy and malignant mouse and human samples using this validated antibody (Perk et al., 2006). Our results show that Id1 was highly expressed in the oropharyngeal cavity, lung, cartilage and skin of E14 and E15 mouse embryos, but expression was progressively reduced in more developed embryos. Immunostaining only remained in epithelial cells of the gut and uterus of adult mice. Mammary MMTV-Myc and MMTV-Myc/VEGF transgenic mouse tumors, and squamous cell carcinomas of the lung induced by N-nitroso-tris-chloroethylurea (NTCU) were highly positive for Id1, unlike their respective healthy counterparts. Id1 immunostaining in a human tissue microarray (TMA) revealed strong expression in cancers of the oral cavity, bladder and cervix. Some tumor specimens of esophagus, thyroid and breast were also strongly positive. Our results suggest that Id1 is an oncofetal protein highly expressed in particular tumor types that should be reanalyzed in future studies using large cohorts of patients to reassess its diagnostic/prognostic value. Moreover, MMTV-Mycand NTCU-induced tumors could serve as appropriate mouse models to study Id1 functions in breast and lung cancer, respectively.
- PublicationOpen AccessEffect of regenerative factor rich plasma, P substance and fetal calf serum on the growth of epithelial cells in the cornea. Comparative experimental study(F. Hernández y Juan F. Madrid. Universidad de Murcia. Departamento de Biología Celular e Histología, 2013) Márquez de Aracena del Cid, R.; Pérez Ordoñez, A.Purpose. The goal of this study was to evaluate the experimental effectiveness of Regenerative Factor Rich Plasma (RFRP) of human blood versus Fetal Bovim Serum (FBS) and neuropeptide Substance P (SP) on corneal epithelium cell proliferation. Method. Rabbit corneal epithelium cell (CCL-60) growth was compared between different RFRP fractions, FBS and with the neuropeptide Substance P. The ability of the RFRP fractions and SP to revert the inhibitory effect of the CsA was also evaluated. Results. All groups showed an increase (p<0.001) in corneal epithelial cell growth compared with the control group. The maximum capacity of cell growth was obtained with dilutions of 50% in the FBS, RFRP-I, RFRP -II, RFRP-III groups and with 100nM of SP. The highest growth was observed with 50% FBS, RFRP-I and RFRP-II. The group with SP and RFRP-III had significantly lower growth (p<0.001). When the NK1 receptor antagonist CsA was added at a dose of IC50, we found a significant decrease in cell growth (p<0.001) in all culture conditions, including the control group. The decrease was similar in all groups, but was especially pronounced in RFRP-II. RFRP I, II and III promoted growth more than SFB 10%. Conclusion. The RFRP of human blood promotes the growth of corneal epithelial cells in a significantly more efficient manner than FSB and SP. RFRP can be effective both in cell cultures and stem cell cultures.
- PublicationOpen AccessThe peculiar apoptotic behavior of skeletal muscle cells(F. Hernández y Juan F. Madrid. Universidad de Murcia. Departamento de Biología Celular e Histología, 2013) Salucci, Sara; Burattini, Sabrina; Baldassarri, ValentinaApoptosis plays an active role in maintaining skeletal muscle homeostasis. Its deregulation is involved in several skeletal muscle disorders such as dystrophies, myopathies, disuse and sarcopenia. The aim of this work was to study in vitro the apoptotic behavior induced by etoposide, staurosporine and hydrogen peroxide in the C2C12 skeletal muscle cell line, comparing myoblast vs myotube sensitivity, investigated by means of morphological and cytofluorimetric analyses. Myotubes appeared more resistant than myoblasts to apoptotic induction. In myoblasts treated with etoposide, nuclei with chromatin condensation were observed, in the presence of a diffuse DNA fragmentation, as shown by confocal microscopy. The latter also appeared in myotubes, where apoptotic and normal nuclei coexisted inside the same syncytium. After staurosporine treatment, myobalsts evidenced late apoptotic features and a high number of TUNEL-positive nuclei. Secondary necrosis appeared in myotubes, where myonuclei with cleaved DNA again coexisted with normal myonuclei. After H2O2 exposure, myotubes, differently from myoblasts, showed a poor sensitivity to cell death. Intriguingly, autophagic granules appeared abundantly in myotubes after each treatment. In myotubes, mitochondria were better preserved than in myoblasts since those which were damaged were probably degraded through autophagic processes. These findings demonstrate a scarce sensitivity of myotubes to apoptotic stimuli due to acquisition of an apoptosis-resistant phenotype during differentiation. The presence of nuclear-dependent “territorial” death domains in the syncytium could explain a slower death of myotubes compared to mononucleated cells. In addition, autophagy could preserve and protect muscle cell integrity against chemical stimuli, making C2C12 cells, in particular myotubes, more resistant to apoptosis induction.
- PublicationOpen AccessMouse models of human INAD by Pla2g6 deficiency(F. Hernández y Juan F. Madrid. Universidad de Murcia. Departamento de Biología Celular e Histología, 2013) Wada, Haruka; Kojo, Satoshi; Seino, Ken-ichiroInfantile neuroaxonal dystrophy (INAD) is a severe neurodegenerative disease characterized by its early onset. PLA2G6, which encodes a phospholipase A2, iPLA2ß, has been identified as a causative gene of INAD. iPLA2ß has been shown to be involved in various physiological and pathological processes, including immunity, cell death, and cell membrane homeostasis. Gene targeted mice with a null mutation of Pla2g6 develop the INAD phenotype as late as approximately 1 to 2 years after birth. Recently, another INAD mouse model, Pla2g6-INAD mice line, has been established. The Pla2g6-INAD mice bear a point mutation in the ankyrin repeat domain of Pla2g6 generated by N-ethylN-nitrosourea mutagenesis. These mutant mice develop severe motor dysfunction and hematopoietic abnormality in a manner following Mendelian law. The mice showed the abnormal gait and poor performance as early as 7 to 8 weeks of age, detected by hanging grip test. Neuropathological examination revealed widespread formation of spheroids containing tubulovesicular membranes similar to human INAD. Molecular and biochemical analysis revealed that the mutant mice expressed Pla2g6 mRNA and protein, but the mutated Pla2g6 protein had no glycerophospholipid-catalyzing enzyme activity. When analyzed the offspring which bear Pla2g6 knockout allele and Pla2g6-INAD allele, abnormal gait appeared slightly later than Pla2g6-INAD homozygotes but with earlier onset than the Pla2g6 knockout homozygotes. This result suggests that mutant Pla2g6 protein contributes to early onset of INAD symptoms in the absence of intact Pla2g6 protein. The analysis of various INAD mouse models may help to understand the pathogenesis of neurodegenerative diseases, including INAD.
- PublicationOpen AccessMorphological spectrum and clinical features of myopathies with tubular aggregates(F. Hernández y Juan F. Madrid. Universidad de Murcia. Departamento de Biología Celular e Histología, 2013) Funk, Fabian; Ceuterick-de Groote, Chantal; Martin, Jean-Jacques; Meinhardt, Axel; Taratuto, Ana L.; De Bleecker, Jan; Coster, Rudy Van; De Paepe, Boel; Schara, Ulrike; Vorgerd, Matthias; Häusler, Martin; Koppi, Stefan; Maschke, Matthias; De Jonghe, Peter; Maldergem, Lionel Van; Noel, Stéphane; Zimmermann, Christoph W.; Wirth, Stefan; Isenmann, Stefan; Stadler, Rudolf; Schröder, J. Michael; Schulz, Jörg B.; Weis, Joachim; Claeys, Kristl G.Tubular aggregates (TAs) are aggregates of densely packed tubules in human skeletal muscle fibers with particular histochemical and ultrastructural features that most probably arise from the sarcoplasmic reticulum. Some studies have shown an additional mitochondrial origin of TAs. We studied the histopathological spectrum and clinical features in a large cohort of patients with TAs in their muscle biopsy (106 biopsies), derived from our muscle biopsy archive (15,412 biopsies in total). In particular, we examined light microscopic, enzyme histochemical, immunohistochemical and ultrastructural features in the muscle biopsies, as well as the patients’ clinical data. We found TAs in 0.5% of all muscle biopsies. Based on the size of TAs, we identified two sub-groups: (1) myopathies with large TAs (29 biopsies) in type 2 fibers and sometimes also in type 1 fibers, absence of any other associated disorder, and a familial history in half of the cases, and (2) myopathies with small TAs (77 biopsies), exclusively in type 2 fibers, presence of another associated disease in the majority of patients and mostly no familial history. In the sub-group with large TAs, we observed a high variability of ultrastructural changes. The most frequent clinical symptom in both groups was limb muscle weakness. No significant differences in clinical presentation, age at onset or disease duration at the time of biopsy were found between the two groups. In conclusion, myopathies with TAs can be sub-divided into a group with large TAs, probably corresponding to the so-called primary TA myopathies, and into a group with small TAs as a feature of another underlying condition.
- PublicationOpen AccessMelatonin prevents dopaminergic cell loss induced by lentiviral vectors expressing A30P mutant alpha-synuclein(F. Hernández y Juan F. Madrid. Universidad de Murcia. Departamento de Biología Celular e Histología, 2013) Brito-Armas, J.M.; Baekelandt, V.; Castro-Hernández, J.R.; González-Hernández, T.; Rodríguez, M.; Castro Fuentes, RafaelTwo hallmarks of Parkinson’s disease (PD) are dopaminergic cell loss and the presence of cytoplasmic inclusions (Lewy bodies). Different point mutations in alpha-synuclein, the main constituent of Lewy bodies, have been identified in familial PD. Alpha-synuclein also constitutes one of the main components of Lewy bodies in sporadic cases of PD. Moreover, oxidant stress and generation of free radicals from both mitochondrial impairment and dopamine metabolism are considered to play critical roles in PD etiopathogenesis. Melatonin, a known potent antioxidant secreted by the pineal gland, may protect against the effect of several Parkinsonogenic compounds that are associated with progressive impairment of mitochondrial function and increased oxidative damage. However, the neuroprotective effect of melatonin has never been tested in the newly available genetic models of PD based on the viral expression of mutated alpha-synuclein. Lentiviral vectors encoding A30P mutant human alphasynuclein (lenti-A30P) were stereotactically injected into the right substantia nigra of adult male Sprague-Dawley rats and neuroprotection was examined by administration of melatonin or vehicle from two days before nigral administration of lenti-A30P until eight weeks after injection. It was found that lenti-A30P induced a significant TH+ cell-loss both in the medial and lateral substantia nigra versus the contrallateral side injected with lenti-eGFP. However, melatonin administration showed a total neuroprotective effect in both regions of the substantia nigra. In conclusion, the data here show that melatonin is neuroprotective against mutant alpha-synuclein-induced injury in the substantia nigra.
- PublicationOpen AccessIs tendinitis an inflammatory disease initiated and driven by pro-inflammatory cytokines such as interleukin 1ß?(F. Hernández y Juan F. Madrid. Universidad de Murcia. Departamento de Biología Celular e Histología, 2013) Mobasheri, Ali; Shakibaei, M.Tendonitis and tendinitis are terms used to describe an inflamed and painful tendon. Tendinopathy, is a descriptive term for describing clinical conditions arising from tendon injury and overuse both within and around tendons. The aim of this mini-review is to explore the role of pro-inflammatory cytokines, particularly interleukin-1ß (IL-1ß) in tendon disorders. A number of investigators including our group have proposed that pro-inflammatory cytokines such as IL-1ß are initiators of tendinopathies, stimulating inflammation, apoptosis and extracellular matrix (ECM) degradation. This is one of the reasons why IL-1ß is frequently used in culture models of tendon inflammation to study the inflammatory and catabolic responses of tenocytes. However, some researchers oppose this view and suggest that although IL-1ß may play a role in rheumatoid arthritis (RA) and osteoarthritis (OA), the involvement of IL-1ß in the development of tendinopathy is questionable. This mini-review discusses the relevant papers published in this area and summarises the evidence for and against the involvement of pro-inflammatory cytokines such as IL1ß in tendonitis. Reaching a consensus will be important for the development and refinement of biomimetic models of tendon inflammation and the formulation of new therapeutic strategies for the treatment of tendon injuries.
- PublicationOpen AccessDNA repair systems in rhabdomyosarcoma(F. Hernández y Juan F. Madrid. Universidad de Murcia. Departamento de Biología Celular e Histología, 2013) Tsioli, Panagiota G.; Patsouris, Efstratios S.; Giaginis, Constantinos; Theocharis, Stamatios E.Rhabdomyosarcoma (RMS) represents the most common soft tissue sarcoma in children and adolescent population. There are two major histological subtypes, embryonal (ERMS) and alveolar (ARMS), differing in cytogenetic and morphological features. RMS pathogenesis remains controversial and several cellular mechanisms and pathways have been implicated. Application of intense chemo- and radiotherapy improves survival rates for RMS patients, but significant efficacy has not been proved as DNA damage induced-resistance frequently occurs. The present review is aimed at summarizing the current evidence on DNA repair systems, implications in RMS development, focusing on gene expression alterations and point mutations of genes encoding for DNA repair enzymes. Understanding of DNA repair systems involvement in RMS pathogenesis could diversify RMS patients and provide novel individualized therapeutic targets.
- PublicationOpen AccessIschemic culture of dental pulp-derived cells is a useful model in which to investigate mechanisms of post-ischemic tissue recovery(F. Hernández y Juan F. Madrid. Universidad de Murcia. Departamento de Biología Celular e Histología, 2013) Agata, Hideki; Sumita, Yoshinori; Asahina, Izumi; Tojo, Arinobu; Kagami, HideakiDental pulp is a soft tissue characterized by unique regenerative properties. It is located in the center of each tooth, and is surrounded by hard tissue (dentin). Vascular access is limited to a small foramen at the root apex. Because of this anatomical limitation, dental pulp can easily lose its blood supply, causing the tissue to become ischemic. This occurs, for example, when a tooth is dislocated by traumatic injury or is subjected to inflammation. Since ischemia is caused by a critical shortage of oxygen and nutrients, ischemic damage is usually irreversible, even when the ischemic event is transient. However, unlike ischemia-sensitive organs such as the brain and heart, dental pulp is relatively ischemia-resistant, and recovers from ischemic injury by regenerating damaged tissue. The mechanisms by which this regeneration occurs are poorly understood, but are being investigated in cell culture models that mimic in vivo ischemic conditions using a combination of hypoxia and nutrient deprivation. Here, we review the use of ischemic cell culture to investigate the mechanisms of post-ischemic dental pulp tissue recovery.
- PublicationOpen AccessMonoclonal antibodies for immunodetection of fibrin deposits on cancer cells(F. Hernández y Juan F. Madrid. Universidad de Murcia. Departamento de Biología Celular e Histología, 2013) Schardt, Friedrich W.; Schmausser, Bernd; Bachmann, EvaThe progression of a tumor from benign to malign and localized to invasive and metastatic growth is the major cause of poor outcome of therapy in cancer patients. The deposition of fibrin along with other procoagulant molecules into the extracellular matrix obviously serves as a scaffold to support proliferation, migration and tumor cell growth as well as protection against the immune system. The use of antibodies as agents for the immunodetection of fibrin deposits in vivo has been hampered by anti-fibrin cross-reactivities with fibrinogen. For the immunohistochemical detection of fibrin we used highly specific monoclonal antibodies to a synthetic fibrinunique peptide, because the fibrin molecule shares many epitopes with fibrinogen. The monoclonal antibody was applied to adenocarcinoma of colon, mamma, pancreas, sarcoma and acute myeloic leukemia. In all tissue sections and cytospin preparations fibrin was identified in a direct apposition to the surface membranes of carcinoma and sarcoma cells, predominantly at the host-tumor interface and also in regions directly adjacent to zones of angiogenesis, whereas normal cells and tissue showed no deposits of fibrin. The findings will be supported by investigations that factors and components of the coagulation system could be detected in the tumor stroma and tumor cells. These factors are obviously produced and secreted by the malignant cells and deposited together with fibrinogen into the extracellular matrix. Our results show that basically all malignant cells examined, independently of ectodermal or mesenchymal derivation, themselves are the origin of hypercoagulability and fibrinolytic system inhibition
- PublicationOpen AccessVital staining of blood vessels and bile ducts with carboxyfluorescein diacetate succinimidyl ester: a novel tool for isolation of cholangiocytes(F. Hernández y Juan F. Madrid. Universidad de Murcia. Departamento de Biología Celular e Histología, 2013) Pryymachuk, Galyna; Polykandriotis, Elias; Schievenbusch, Stephanie; Arkudas, Andreas; Nierhoff, Dirk; Curth, Harald-Morten; Odenthal, Margarete; Horch, Raymund E.; Neiss, Wolfram F.; Goeser, Tobias; Steffen, Hans Michael; Toex, UlrichBackground and aim: Current methods for visualization of the blood vasculature, biliary tree and for isolation of vital cholangiocytes are afflicted with a plethora of technical difficulties, especially in mice. In this project, we propose a novel, reliable and straightforward alternative technique for histological demonstration of blood- and biliary systems and derivation of vital cholangiocytes. Methods: Intravital retrograde perfusion of bile ducts was performed in twenty wild type mice. Liver and gallbladder were exposed by median laparotomy. Using a venous catheter, the gallbladder was cannulated, a few millimeters of the liver edge were cropped to allow free outflow of the perfusate, and carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) solution was retrogradely infused. Thereafter, formaldehyde solution was either injected through the same catheter, or the liver was immediately dissociated into a single-cell suspension for FACS-analysis. Intravital perfusion of the vascular system was performed in ten Lewis rats by direct intra-arterial injection of CFDA-SE into the abdominal aorta. The specificity and sensitivity of CFDA-SE labeling was controlled using Indian ink or cytokeratin 19 immunohistochemistry respectively. Results: Upon histomorphological analysis of cryoand paraffin sections, strong fluorescence was noted in large and small bile ducts throughout the entire liver and in the vascular system after infusion of the CFDA-SE solution. In preliminary FACS-experiments, we succeeded in separating cholangiocytes based on combined CFDA-SE-staining and cell size. Conclusions: Visualization of liver architecture and the isolation of cholangiocytes is feasible using a fast and cost-effective method of retrograde perfusion and vital fluorescent labeling of mouse bile duct epithelium and vascular endothelium with CFDA-SE.
- PublicationOpen AccessFiber type composition of the architecturally distinct regions of human supraspinatus muscle: A cadaveric study(F. Hernández y Juan F. Madrid. Universidad de Murcia. Departamento de Biología Celular e Histología, 2013) Kim, S.Y.; Lunn, D.D.; Dyck, R.J.; Kirkpatrick, L.J.; Rosser, B.W.C.The human supraspinatus muscle is clinically important as it is frequently injured in older adults and the elderly. We have previously shown that the supraspinatus has a complex architecture with two distinct regions each consisting of three parts. Further we have found dynamic changes in architectural parameters such as fiber bundle length markedly vary between these regions. Fiber types of the supraspinatus have not been thoroughly investigated throughout its volume and are of interest to clinicians treating supraspinatus pathologies. In this study we investigated the distribution of fiber types within the distinct regions and parts of supraspinatus. Samples of supraspinatus were excised from six distinct parts of each muscle from five formalin embalmed specimens (one male, four female; mean age 77±11.1 years) free of tendon pathology. Samples were frozen in liquid nitrogen and then cryosectioned. Serial sections were labeled using immunohistochemical techniques and antibodies against fast or slow myosin heavy chain isoforms. The mean percentage of Type I (slow) fibers ranged from 56.73% to 63.97%. Results demonstrated significant variations in fiber type distribution. The middle part of the anterior region has a significantly greater percentage of Type I fibers compared to that of the posterior. The superficial part of the anterior region has a greater percentage of Type II (fast) fibers compared to the middle and deep parts. Findings aid in highlighting the distinct functions of the anterior and posterior regions, and prompt the need to re-evaluate assessment and treatment techniques established on a limited understanding of the fiber type distribution.
- PublicationOpen AccessNotch receptors in human choroid plexus tumors(F. Hernández y Juan F. Madrid. Universidad de Murcia. Departamento de Biología Celular e Histología, 2013) Beschorner, R.; Waidelich, J.; Trautmann, K.; Psaras, T.; Schittenhelm, J.Notch signaling plays a role in development and formation of the normal choroid plexus (nCP), and in formation of various tumors in humans. Activation of Notch3 has been reported to promote tumor growth in invasive gliomas and to initiate formation of choroid plexus tumors (CPT) in mice. We investigated the expression of all currently known Notch receptors (Notch 1-4) in 55 samples of nCP and 88 CPT, including 61 choroid plexus papillomas (CPP), 22 atypical CPP and 5 choroid plexus carcinomas by immunohistochemistry. Notch expression was semiquantitatively evaluated separately for membranous/cytoplasmic and for nuclear staining. In addition, we examined Her2 expression (EGFR2, Her2/neu, ErbB2, CD340) because of its functional link to Notch signaling. All samples were negative for Notch3. Membranous/cytoplasmic expression of Notch1 (p<0.0001) and Notch4 (p=0.046) was significantly higher, whereas Notch2 expression was significantly lower (p<0.0001) in nCP compared to CPT. Nuclear expression of Notch1, -2 and -4 was significantly higher in CPT compared to nCP (p<0.0001 each). Expression of Notch2 and Notch4 showed a shift from a prevailing membranous/cytoplasmic expression in nCP to a predominant nuclear expression in CPT. Her2 was weakly expressed in 42/84 CPT but only in 2/53 nCP (p=0.0001) and positively correlated with nuclear expression of Notch1, -2 and 4 in CPT. In summary, a shift between membranous/cytoplasmic (non-canonical signaling pathway) and nuclear expression (canonical signaling pathway) of Notch1, -2 and -4 and upregulation of Her2 indicate neoplastic transformation in human CP and may reveal new therapeutic approaches.