p16INK4a and its regulator miR-24 link senescence and chondrocyte terminal differentiation-associated matrix remodeling in osteoarthritis.

INTRODUCTION: Recent evidence suggests that tissue accumulation of senescent p16INK4a-positive cells during the life span would be deleterious for tissue functions and could be the consequence of inherent age-associated disorders. Osteoarthritis (OA) is characterized by the accumulation of chondrocytes expressing p16INK4a and markers of the senescence-associated secretory phenotype (SASP), including the matrix remodeling metalloproteases MMP1/MMP13 and pro-inflammatory cytokines interleukin-8 (IL-8) and IL-6. Here, we evaluated the role of p16INK4a in the OA-induced SASP and its regulation by microRNAs (miRs). METHODS: We used IL-1-beta-treated primary OA chondrocytes cultured in three-dimensional setting or mesenchymal stem cells differentiated into chondrocyte to follow p16INK4a expression. By transient transfection experiments and the use of knockout mice, we validate p16INK4a function in chondrocytes and its regulation by one miR identified by means of a genome-wide miR-array analysis. RESULTS: p16INK4a is induced upon IL-1-beta treatment and also during in vitro chondrogenesis. In the mouse model, Ink4a locus favors in vivo the proportion of terminally differentiated chondrocytes. When overexpressed in chondrocytes, p16INK4a is sufficient to induce the production of the two matrix remodeling enzymes, MMP1 and MMP13, thus linking senescence with OA pathogenesis and bone development. We identified miR-24 as a negative regulator of p16INK4a. Accordingly, p16INK4a expression increased while miR-24 level was repressed upon IL-1-beta addition, in OA cartilage and during in vitro terminal chondrogenesis. CONCLUSIONS: We disclosed herein a new role of the senescence marker p16INK4a and its regulation by miR-24 during OA and terminal chondrogenesis.

[A potential contribution of virtual slides and automatic image processing to the estimation of the vascularization of cranial sutures]. / Apport potentiel des lames virtuelles et de l'analyse d'images automatique dans l'estimation de la vascularisation des sutures crâniennes.

AIM: Determining age at the time of death is a difficult problem in forensic practice. The study of the vascularization of cranial sutures is an original approach, which may mark the process of synostosis associated with aging. Counting sections of blood vessels on a histological section of cranial suture, raises however a number of practical problems related to the quality of the preparation and to the method of quantification. The purpose of this study is to illustrate the potential contribution of an automatic analysis of virtual slides to overcome these problems. MATERIALS AND METHODS: The proposed method of analysis is illustrated from three samples of frontosphenoidal suture whose vessels were immunostained after decalcification of bone tissue. A high resolution image (x 20 objective and microscopic resolution of 0.5 microm) of each microscopic preparation was acquired through a microscopic scanning device. The automatic image analysis protocol takes advantage of a processing of virtual slides at two resolutions. RESULTS: The chosen strategy ensures the identification of the specific area of interest, the enumeration of blood vessel sections on the whole preparation and the visual control of the detected structures. CONCLUSION: The quantitative estimate of the vascularization of a large structure, such as a cranial suture, can benefit from scanning and fully automatic processing of virtual slides. The automatic analysis requires however an optimal preparation of tissues; decalcification and immunohistochemical staining must be done with great care.

Apoptosis of neurons in cardiovascular autonomic centres triggered by inducible nitric oxide synthase after death from septic shock.

BACKGROUND: Results of experimental and clinical studies have shown that septic shock is associated with cardiovascular autonomic failure. Thus, we aimed to investigate the existence of ischaemia and apoptosis within the cerebral autonomic centres that control the cardiovascular system in patients with septic shock. METHODS: In a prospective cohort study, we did post-mortem examinations of supraoptic and paraventricular nuclei, cerebral amygdala, locus coeruleus, and medullary autonomic nuclei in 19 patients with septic shock, seven with non-septic shock and five who died suddenly from extracranial injury. Ischaemic and apoptotic neurons and microglial cells, and expression of tumour necrosis factor alpha (TNFalpha) and inducible nitric oxide synthase (iNOS) were scored. FINDINGS: Ischaemic, neuronal, and microglial apoptosis scores differed between groups (p=0.0007, p<0.0001, and p=0.0037, respectively) and were higher in patients with septic shock than in those with non-septic shock (p=0.0033, p=0.0005, and p=0.0235, respectively), and extra-cranial injury related deaths (p=0.0027, p=0.0007, and p=0.0045, respectively). There was little microglial activation and glial expression of TNFalpha. The scores for endothelial iNOS expression were different between the three groups (p<0.0001), and were higher in septic shock than in non-septic shock (p=0.0009) and than in extracranial injury related deaths (p=0.0007). Vascular expression of iNOS also correlated (Spearman tau=0.57) with autonomic-centre neuronal apoptosis in the combined septic and non-septic shock group. INTERPRETATION: Septic shock is associated with neuronal and glial apoptosis within the autonomic centres, which is strongly associated with endothelial iNOS expression.