OBJECTIVE: To evaluate the range of activation changes of polymorphonuclear leukocytes (PMN) and the ratio of apoptosis and necrosis in synovial effusions of patients with various arthropathies, and to reveal possible correlations with clinical variants of joint inflammation.
METHODS: Synovial effusions were aspirated from the knee joints of patients with rheumatoid arthritis (RA, 28 cases), and seronegative spondyloarthritides (SSA): Reiter's disease (RD, 9 cases), peripheral form of the ankylosing spondyloarthritis (6 cases) and psoriatic arthritis (6 cases); and primary osteoarthritis (OA, 9 cases). Cytospin preparations were processed for transmission electron microscopy and assessed for the incidence of apoptosis, necrosis, and cytophagocytic cells (CPC) in the synovial fluid (SF). The range of activation changes of the neutrophil granulocytes, the dominating cell population in the arthritic SF, was evaluated.
RESULTS: In all arthropathies under investigation most of the synovial effusion cells had intact ultrastructure with a certain amount of apoptotic cells dominating over the cells with signs of necrosis, and a few CPC. The highest rate of apoptosis was discovered in the synovial effusions of patients with RA, the lowest in those with OA, while the rate of CPC among the inflammatory joint diseases was the lowest in RA. In RA the current disease activity correlated with the incidence of apoptotic cells and CPC, while the clinical stage was related only to the CPC rate. These data suggest that in RA, despite exposure to the anti-apoptotic signals, apoptosis of the synovial effusion PMN is maintained at a significantly higher level than in non-rheumatoid arthropathies, both inflammatory (SSA) and degenerative (OA), providing elimination of the neutrophils accumulating in the joint cavity and thus stimulating resolution of the joint inflammation.
OBJECTIVE: To determine in situ using TEM the balance of apoptosis and necrosis in the articular cartilage of patients with inflammatory (rheumatoid arthritis and seronegative spondyloarthritis) and degenerative (osteoarthritis) joint diseases and to establish possible correlation between the cell death rate and the matrix vesicles formation.
METHODS: Cartilage samples of the knee joint were obtained from patients with rheumatoid arthritis (RA, 18 cases), osteoarthritis (OA, 22 cases), Reiter's disease (RD, 9 cases), peripheral form of the ankylosing spondyloarthritis (AS, 6 cases) and psoriatic arthritis (PA, 6 cases) during arthroscopy or knee surgery. Normal samples taken from autopsy cases without a history of joint diseases were used as control. Samples were processed for TEM with subsequent semi-quantitative estimation of the cell death rate in the superficial, middle and deep zone of non-calcified articular cartilage, and computer-aided ultramorphometric evaluation of the matrix vesicles of different types.
RESULTS: Both apoptotic and necrotic cell death could be identified in the cartilage of patients with inflammatory joint diseases, including seronegative spondyloarthritides and degenerative arthropathies. Apoptosis dominated over necrosis in all examined arthritides, including RA patients in which necrosis of the chondrocyte was the most frequent among arthropathies, while the highest apoptotic cell death rate was discovered in OA in which it correlated with the volume and numeric density of the matrix vesicles. These data provide evidence that apoptosis may contribute to the cartilage breakdown not only in RA and OA but also in the seronegative spondyloarthritides, which had a significantly higher apoptotic rate than the normal cartilage.
An indigenous contraceptive herbal formulation consisting of a mixture of Lepidagathis longifolia, Palaquium sp and Phyllagathis rotundifolia is being used by the Temuan Aborigins of Malaysia. Although the previous studies demonstrated that this contraceptive herbal formulation causes anovulatory estrous cycle, altered circulating hormone levels and fetal resorption in rats, but the effects of this formulation on the gonadotrphs of the pituitary gland are yet to be evaluated. The present study was designed to observe the morphometric changes of the gonadotrophs and the plasma concentrations of follicle stimulating hormone and leutinizing hormone. Thirty five Sprague-Dawley adult female rats were randomly divided into 5 groups. Experimental animals were given a combined herbal extract or individual herbal extract at a dose of 540 mg/kg/day subcutaneously for 7 days. Immunostained gonadotrophs were studied by using image analyzer. FSH and LH serum concentrations were determined using RIA. The FSH and LH concentrations were low in animals that received combined herbal extract (p<0.01). FSH concentration was noted to be significantly low in animals that received P. rotundifolia (p<0.05). The mean cell area and cell density of gonadotrophs of animals that received combined herbal extract were significantly low compared to control group (p<0.05). It was concluded that the herbal extracts do suppress the production of gonaotrophins along with the demonstrable suppresive effect on the FSH cells.
Steroid-induced ocular hypertension (SIOH) is associated with topical and systemic use of steroids. However, SIOH-associated anterior and posterior segment morphological changes in rats have not been described widely. Here we describe the pattern of intraocular pressure (IOP) changes, quantitative assessment of trabecular meshwork (TM) and retinal morphological changes and changes in retinal redox status in response to chronic dexamethasone treatment in rats. We also evaluated the responsiveness of steroid-pretreated rat eyes to 5 different classes of antiglaucoma drugs that act by different mechanisms. Up to 80% of dexamethasone treated animals achieved significant and sustained IOP elevation. TM thickness was significantly increased and number of TM cells was significantly reduced in SIOH rats compared to the vehicle-treated rats. Quantitative assessment of retinal morphology showed significantly reduced thickness of ganglion cell layer (GCL) and inner retina (IR) in SIOH rats compared to vehicle-treated rats. Estimation of retinal antioxidants including catalase, superoxide dismutase and glutathione showed significantly increased retinal oxidative stress in SIOH animals. Furthermore, steroid-treated eyes showed significant IOP lowering in response to treatment with 5 different drug classes. This indicated the ability of SIOH eyes to respond to drugs acting by different mechanisms. In conclusion, SIOH was associated with significant morphological changes in TM and retina and retinal redox status. Additionally, SIOH eyes also showed IOP lowering in response to drugs that act by different mechanisms of action. Hence, SIOH rats appear to be an inexpensive and noninvasive model for studying the experimental antiglaucoma drugs for IOP lowering and neuroprotective effects.
Experiments involving short-term space flight have shown an adverse effect on the physiology, morphology and functions of cells investigated. The causes for this effect on cells are: microgravity, temperature fluctuations, mechanical stress, hypergravity, nutrient restriction and others. However, the extent to which these adverse effects can be repaired by short-term space flown cells when recultured in conditions of normal gravity remains unclear. Therefore this study aimed to investigate the effect of short-term spaceflight on cytoskeleton distribution and recovery of cell functions of normal human osteoblast cells. The ultrastructure was evaluated using ESEM. Fluorescent staining was done using Hoechst, Mito Tracker CMXRos and Tubulin Tracker Green for cytoskeleton. Gene expression of cell functions was quantified using qPCR. As a result, recovered cells did not show any apoptotic markers when compared with control. Tubulin volume density (p<0.001) was decreased significantly when compared to control, while mitochondria volume density was insignificantly elevated. Gene expression for IL-6 (p<0.05) and sVCAM-1 (p<0.001) was significantly decreased while alkaline phosphatase (p<0.001), osteocalcin and sICAM (p<0.05) were significantly increased in the recovered cells compared to the control ones. The changes in gene and protein expression of collagen 1A, osteonectin, osteoprotegerin and beta-actin, caused by short-term spaceflight, were statistically not significant. These data indicate that short term space flight causes morphological changes in osteoblast cells which are consistent with hypertrophy, reduced cell differentiation and increased release of monocyte attracting proteins. The long-term effect of these changes on bone density and remodeling requires more detailed studies.
Quantitative immunohistochemical methods were used to assess activation of the hypothalamo-hypophyseal-adrenocortical system at the level of its central component - the adenohypophysis - in the growing body during chronic exposure to psychoemotional stressors of different strengths. Sprague-Dawley rats aged 30 days were subjected to "mild" or "severe" immobilization stress for 5 h per day for seven days. Animals were decapitated at the end of the last stress session and the endocrine glands (hypophysis, adrenals) were harvested, weighed, and embedded in paraffin; sections were stained with hematoxylin and eosin, and also immunohistochemically using monoclonal antibodies to adrenocorticotropic hormone (ACTH) and proliferating cell nuclear antigen (PCNA) following by automated image analysis. These studies showed that stress-associated hyperplasia of corticotropocytes in rats of pubertal age was due more to the differentiation of existing immature precursor cells than to cell proliferation.
Microgravity, hypergravity, vibration, ionizing radiation and temperature fluctuations are major factors of outer space flight affecting human organs and tissues. There are several reports on the effect of space flight on different human cell types of mesenchymal origin while information regarding changes to vascular endothelial cells is scarce. Ultrastructural and cytophysiological features of macrovascular endothelial cells in outer space flight and their persistence during subsequent culturing were demonstrated in the present investigation. At the end of the space flight, endothelial cells displayed profound changes indicating cytoskeletal lesions and increased cell membrane permeability. Readapted cells of subsequent passages exhibited persisting cytoskeletal changes, decreased metabolism and cell growth indicating cellular senescence.
The spinal nucleus of the accessory nerve (SNA) comprises the group of somata (perikarya) of motor neurons that supply the sternocleidomastoid and trapezius muscles. There are many conflicting views regarding the longitudinal extent and topography of the SNA, even in the same species, and these disagreements prompted the present investigation. Thirty Sprague-Dawley rats (15 males, 15 females) were used. The SNA was localized by retrograde axonal transport of horseradish peroxidase. Longitudinally, the SNA was found to be located in the caudal part (caudal 0.9-1.2 mm) of the medulla oblongata, the whole lengths of cervical spinal cord segments C1, C2, C3, C4, C5 and rostral fourth of C6. In the caudal part of the medulla oblongata, the SNA was represented by a group of perikarya of motor neurons lying immediately ventrolateral to the pyramidal fibres that were passing dorsolaterally after their decussation. In the spinal cord, the motor neuronal somata of the SNA were located in the dorsomedial and central columns at C1, in the dorsomedial, central and ventrolateral columns at C2 and in the ventrolateral column only at C3, C4, C5 and rostral quarter of C6. The perikarya of motor neurons supplying the sternocleidomastoid were located in the caudal part (caudal 0.9-1.2 mm) of the medulla oblongata ventrolateral to the pyramidal fibres that were passing dorsolaterally after their decussation. They were also located in the dorsomedial and central columns at C1, in the dorsomedial, central and ventrolateral columns at C2 and only in the ventrolateral column at the rostral three-quarters of C3. The perikarya of motor neurons supplying the trapezius muscle were located in the ventrolateral column only in the caudal three-quarters of C2, the whole lengths of C3, C4 and C5, and in the rostral quarter of C6.
The aim of the study was to investigate the location of motor neuron somata of geniohyoid muscle in rat. Nine Sprague-Dawley rats were used in this study. Operations were performed under general anaesthesia. Nembutal sodium, 40 mg per kg intraperitoneally was used for anaesthesia. 0.02 to 0.05 ml of 30% horseradish peroxidase (Sigma Type VI) solution in normal saline was injected into the exposed right geniohyoid muscle. After 48 hr, the animals were fixed by perfusion through left ventricle of heart, first by 100 ml normal saline and then with 500 ml of 1.25% glutaraldehyde and 1% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, at room temperature, and finally with 500 ml of 10% sucrose in the same buffer at 4°C. The medulla oblongata and first cervical segment of spinal cord were removed, kept in 10% sucrose in above phosphate buffer at 4°C for 24 hr. Thereafter, their serial transverse sections were cut in a cryostat at a thickness of 60 μm. The sections were treated according to tetramethyl benzidine (TMB)-horseradish peroxidase (HRP) method. HRP-labelled neuron somata were observed at the following sites: (a) In ventral part of right main hypoglossal nucleus in upper two-thirds of the closed part of medulla oblongata. (b) In ventrolateral subnucleus of hypoglossal nucleus in lower third of closed part of medulla oblongata. (c) At spinomedullary junction, they were located in dorsomedial part of right ventral grey column; a few were also seen here scattered on right side of central canal and among corticospinal fibres.
Exposure of Normal Human Osteoblast cells (NHOst) to a period of hypothermia may interrupt their cellular functions, lead to changes in bone matrix and disrupt the balance between bone formation and resorption, resulting in bone loss or delayed fracture healing. To investigate this possibility, we exposed NHOst cells to moderate (35 °C) and severe (27 °C) hypothermia for 1, 12, 24 and 72 h. The effects of hypothermia with respect to cell cytoskeleton organization, metabolic activity and the expression of cold shock chaperone proteins, osteoblast transcription factors and functional markers, were examined. Our findings showed that prolonged moderate hypothermia retained the polymerization of the cytoskeletal components. NHOst cell metabolism was affected differently according to hypothermia severity. The osteoblast transcription factors Runx2 and osterix were necessary for the transcription and translation of bone matrix proteins, where alkaline phosphatase (Alp) activity and osteocalcin (OCN) bone protein were over expressed under hypothermic conditions. Consequently, bone mineralization was stimulated after exposure to moderate hypothermia for 1 week, indicating bone function was not impaired. The cold shock chaperone protein Rbm3 was significantly upregulated (p<0.001) during the cellular stress adaption under hypothermic conditions. We suggest that Rbm3 has a dual function: one as a chaperone protein that stabilizes mRNA transcripts and a second one in enhancing the transcription of Alp and Ocn genes. Our studies demonstrated that hypothermia permitted the in vitro maturation of NHOst cells probably through an osterix-dependent pathway. For that reason, we suggest that moderate hypothermia can be clinically applied to counteract heat production at the fracture site that delays fracture healing.
Steroid-induced hypertension and glaucoma is associated with increased extracellular meshwork (ECM) deposition in trabecular meshwork (TM). Previous studies have shown that single drop application of trans-resveratrol lowers IOP in steroid-induced ocular hypertensive (SIOH) rats. This IOP lowering is attributed to activation of adenosine A1 receptors, which may lead to increased matrix metalloproteinase (MMP)-2 activity. This study evaluated the effect of repeated topical application of trans-resveratrol for 21 days in SIOH animals on IOP, changes in MMP-2 level in aqueous humor, trabecular meshwork and retinal morphology and retinal redox status. We observed that treatment with trans-resveratrol results in significant and sustained IOP reduction in SIOH rats. This IOP reduction is associated with significantly higher aqueous humor total MMP-2 level; significantly reduced TM thickness and increased number of TM cells. Treatment with trans-resveratrol also significantly increased ganglion cell layer (GCL) thickness, the linear cell density in the GCL and inner retina thickness; and significantly reduced retinal oxidative stress compared to the SIOH vehicle-treated group. In conclusion, repeated dose topical application of trans-resveratrol produces sustained IOP lowering effect, which is associated with increased level of aqueous humor MMP-2, normalization of TM and retinal morphology and restoration of retinal redox status.
Morphological and phenotypical signs of cultured readaptation osteoblasts were studied after a short-term space mission. The ultrastructure and phenotype of human osteoblasts after Soyuz TMA-11 space flight (2007) were evaluated by scanning electron microscopy, laser confocal microscopy, and ELISA. The morphofunctional changes in cell cultures persisted after 12 passages. Osteoblasts retained the drastic changes in their shape and size, contour deformation, disorganization of the microtubular network, redistribution of organelles and specialized structures of the plasmalemma in comparison with the ground control cells. On the other hand, the expression of osteoprotegerin and osteocalcin (bone metabolism markers) increased; the expression of bone resorption markers ICAM-1 and IL-6 also increased, while the expression of VCAM-1 decreased. Hence, space flight led to the development of persistent shifts in cultured osteoblasts indicating injuries to the cytoskeleton and the phenotype changes, indicating modulation of bone metabolism biomarkers.
Conference abstracts: Malaysia in affiliation
(1). PO-211. AGE-SPECIFIC STRESS-MODULATED
CHANGES OF SPLENIC IMMUNOARCHITECTURE
IN THE GROWING BODY. Marina Yurievna Kapitonova, Syed Baharom Syed Ahmad Fuad, Flossie Jayakaran; Faculty of Medicine, Universiti Teknologi MARA, Shah Alam, Malaysia
syedbaharom@salam.uitm.edu.my
(2). PO-213. A DETAILED OSTEOLOGICAL STUDY OF THE ANOMALOUS GROOVES NEAR THE
MASTOID NOTCH OF THE SKULL. ISrijit Das, 2Normadiah Kassim, lAzian Latiff, IFarihah Suhaimi, INorzana Ghafar, lKhin Pa Pa Hlaing, lIsraa Maatoq, IFaizah Othman; I Department of Anatomy, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia; 2 Department of Anatomy, Universiti Malaya, Kuala Lumpur, Malaysia. das_sri jit23@rediffmail.com
(3). PO-21S. FIRST LUMBRICAL MUSCLE OF THE
PALM: A DETAILED ANATOMICAL STUDY WITH
CLINICAL IMPLICATIONS. Srijit Das, Azian Latiff, Parihah Suhaimi, Norzana Ghafar, Khin Pa Pa Hlaing, Israa Maatoq, Paizah Othman; Department of Anatomy, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia. das_srijit23@rediffmail.com
(4). PO-336. IMPROVEMENT IN EXPERIMENTALLY
INDUCED INFRACTED CARDIAC FUNCTION
FOLLOWING TRANSPLANTATION OF HUMAN
UMBILICAL CORD MATRIX-DERIVED
MESENCHYMAL CELLS. lSeyed Noureddin Nematollahi-Mahani, lMastafa Latifpour, 2Masood Deilami, 3Behzad Soroure-Azimzadeh, lSeyed
Hasan Eftekharvaghefi, 4Fatemeh Nabipour, 5Hamid
Najafipour, 6Nouzar Nakhaee, 7Mohammad Yaghoobi, 8Rana Eftekharvaghefi, 9Parvin Salehinejad, IOHasan Azizi; 1 Department of Anatomy, Kerman University of Medical Sciences, Kerman, Iran; 2 Department of Cardiosurgery, Hazrat-e Zahra Hospital, Kerman, Iran; 3 Department of Cardiology, Kerman University of Medical Sciences, Kerman, Iran; 4 Department of Pathology, Kerman University of Medical Sciences, Kerman, Iran; 5 Department of Physiology, Kerman University of Medical Sciences, Kerman, Iran; 6 Department of Neuroscience Research Center, Kerman University of Medical Sciences, Kerman, Iran; 7 Department
of Biotechnology, Research Institute of Environmental Science, International Center for Science, High Technology & Environmental Science, Kerman, Iran; 8 Students Research Center, Kerman University of Medical Sciences, Kerman, Iran; 9 Institute of Bioscience, University Putra Malaysia,
Kuala Lumpur, Malaysia; 10 Department of Stem Cell, Cell Science Research Center, Royan Institute, ACECR, Tehran, Iran. nnematollahi@kmu.ac.ir
(5).