Characterization of proteoglycan 4 and hyaluronan composition and lubrication function of ovine synovial fluid following knee surgery

The objective of this study was to determine changes in (1) proteoglycan 4 (PRG4) and hyaluronan (HA) concentration, (2) HA molecular weight (MW) distribution, and (3) cartilage boundary lubricating ability of synovial fluid (SF) from surgical sham (SHAM), anterior cruciate ligament (ACL)/medial collateral ligament (MCL) transection, and lateral meniscectomy (MEN) in a post‐knee surgery ovine model. Ovine SF (oSF) was collected at euthanization 20 weeks after surgery, with the contralateral joint serving as the non‐operative control. PRG4 and HA concentration in oSF was measured by sandwich enzyme‐linked immunosorbent assay, and HA MW distribution by agarose gel electrophoresis. Cartilage boundary lubricating ability of oSF was measured by a cartilage–cartilage friction test. PRG4 and HA concentration in SHAM, ACL/MCL, and MEN oSF were similar in comparison to the contralateral control (CTRL) oSF. The HA MW distribution in the operated oSF for all ranges were similar to the respective CTRL oSF. The kinetic coefficients of friction in operated and CTRL oSF were similar in all groups, and were significantly lower than saline. These results indicate oSF lubricant composition and function at 20 weeks post‐knee surgery were similar to contralateral CTRL, and suggest earlier time points post surgery warrant further investigation. © 2013 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 31:1549–1554, 2013     

Impaired presynaptic function and elimination of synapses at premature stages during postnatal development of the cerebellum in the absence of CALEB (CSPG5/neuroglycan C)

Chicken acidic leucine‐rich EGF‐like domain‐containing brain protein (CALEB), also known as chondroitin sulfate proteoglycan (CSPG)5 or neuroglycan C, is a neural chondroitin sulfate‐containing and epidermal growth factor (EGF)‐domain‐containing transmembrane protein that is implicated in synaptic maturation. Here, we studied the role of CALEB within the developing cerebellum. Adult CALEB‐deficient mice displayed impaired motor coordination in Rota‐Rod experiments. Analysis of the neuronal connectivity of Purkinje cells by patch‐clamp recordings demonstrated impairments of presynaptic maturation of inhibitory synapses. GABAergic synapses on Purkinje cells revealed decreased evoked amplitudes, altered paired‐pulse facilitation and reduced depression after repetitive stimulation at early postnatal but not at mature stages. Furthermore, the elimination of supernumerary climbing fiber synapses on Purkinje cells was found to occur at earlier developmental stages in the absence of CALEB. For example, at postnatal day 8 in wild‐type mice, 54% of Purkinje cells had three or more climbing fiber synapses in contrast to mutants where this number was decreased to less than 25%. The basic properties of the climbing fiber Purkinje cell synapse remained unaffected. Using Sholl analysis of dye‐injected Purkinje cells we revealed that the branching pattern of the dendritic tree of Purkinje cells was not impaired in CALEB‐deficient mice. The alterations observed by patch‐clamp recordings correlated with a specific pattern and timing of expression of CALEB in Purkinje cells, i.e. it is dynamically regulated during development from a high chondroitin sulfate‐containing form to a non‐chondroitin sulfate‐containing form. Thus, our results demonstrated an involvement of CALEB in the presynaptic differentiation of cerebellar GABAergic synapses and revealed a new role for CALEB in synapse elimination in Purkinje cells.     

Differences in mRNA and protein expression of small proteoglycans in vaginal wall tissue from women with and without stress urinary incontinence

BACKGROUND: To investigate changes in mRNA and protein levels of biglycan (BGN), decorin (DCN) and fibromodulin (FMOD) in vaginal wall tissue from women with stress urinary incontinence (SUI) compared to menstrual-cycle matched continent women. METHODS: We determined mRNA expressions of BGN, DCN and FMOD by quantitative real-time PCR. They were localized in vaginal wall tissue by immunohistochemistry. We performed western blot analysis to examine protein expression. RESULTS: BGN, DCN and FMOD co-localized with collagen and elastin in the extracellular matrix (ECM) of vaginal wall tissue from both groups. The mRNA expression of FMOD was significantly lower in cases versus controls in the proliferative phase (P = 0.03). DCN mRNA expression in cases was higher in the proliferative (P = 0.05) and secretory phases (P = 0.02) versus controls. BGN mRNA expression showed no significant differences in either phase. Protein expression of FMOD in cases was lower in the proliferative phase versus controls (six out of nine pairs), whereas DCN and BGN protein expression in the secretory phase in cases was higher (seven out of nine pairs). CONCLUSION: BGN, DCN and FMOD expressions in vaginal wall tissue differ in women with SUI and are hormonally modulated. Differences in small proteoglycans may contribute to the altered pelvic floor connective tissues found in these women.     

Critical role of microvasculature basal lamina in ischemic brain injury

Cerebral vascular system can be divided into two categories: the macrovessels and microvessels. The microvessels consist of arterioles, capillaries and venules. There are three basic components in the microvasculature: endothelial cells, basal lamina and end-feet of astrocytes. The basal lamina is situated between the endothelial cells and the end-feet of astrocytes, and connects these two layers together. Damage to the basal lamina causes the dismantlement of microvascular wall structures, which in turn results in increase of microvascular permeability, hemorrhagic transformation, brain edema and compromise of the microcirculation. The present article reviews microvascular changes during ischemic brain injury, with emphasis on basal lamina damage.     

Quantitative T2* (T2 star) relaxation times predict site specific proteoglycan content and residual mechanics of the intervertebral disc throughout degeneration

Degeneration alters the biochemical composition of the disc, affecting the mechanical integrity leading to spinal instability. Quantitative T2* MRI probes water mobility within the macromolecular network, a potentially more sensitive assessment of disc health. We determined the relationship between T2* relaxation time and proteoglycan content, collagen content, and compressive mechanics throughout the degenerative spectrum. Eighteen human cadaveric lumbar (L4–L5) discs were imaged using T2* MRI. The T2* relaxation time at five locations (nucleous pulposus or NP, anterior annulus fibrosis or AF, posterior AF, inner AF, and outer AF) was correlated with sulfated‐glycosaminoglycan (s‐GAG) content, hydroxyproline content, and residual stress and strain at each location. T2* relaxation times were significantly correlated with s‐GAG contents in all test locations and were particularly strong in the NP (r = 0.944; p < 0.001) and inner AF (r = 0.782; p < 0.001). T2* relaxation times were also significantly correlated with both residual stresses and excised strains in the NP (r = 0.857; p < 0.001: r = 0.816; p < 0.001), inner AF (r = 0.535; p = 0.022: r = 0.516; p = 0.028), and outer AF (r = 0.668; p = 0.002: r = 0.458; p = 0.041). These strong correlations highlight T2* MRI's ability to predict the biochemical and mechanical health of the disc. T2* MRI assessment of disc health is a clinically viable tool showing promise as a biomarker for distinguishing degenerative changes. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:1083–1089, 2014.     

In the presence of danger：the extracellular matrix defensive response to central nervous system injury

Glial cells in the central nervous system （CNS） contribute to formation of the extracellular matrix, which provides adhesive sites, signaling molecules, and a diffusion barrier to enhance efficient on and axon potential propagation. In the normal adult CNS, the extracellular matrix （ECM） is relatively stable except in selected regions characterized by dynamic remodeling. However, after trauma such as a spinal cord injury or cortical contusion, the lesion epicenter becomes a focus of acute neuroinflammation. The activation of the surrounding glial cells leads to a dramatic change in the composition of the ECM at the edges of the lesion, creating a perilesion environment dominated by growth inhibitory molecules and restoration of the peripheral/ central nervous system border. An advantage of this response is to limit the invasion of damaging cells and diffusion of toxic molecules into the spared tissue regions, but this occurs at the cost of inhibiting migration of endogenous repair cells and preventing axonal regrowth. The following review was prepared by reading and discussing over 200 research articles in the field published in PubMed and selecting those with significant impact and/or controversial points. This article highlights structural and functional features of the normal adult CNS ECM and then focuses on the reactions of glial cells and changes in the perilesion border that occur following spinal cord or contusive brain injury. Current research strategies directed at modifying the inhibitory perilesion microenvironment without eliminating the protective functions of glial cell activation are discussed.     

Enhanced cognitive flexibility in reversal learning induced by removal of the extracellular matrix in auditory cortex

During brain maturation, the occurrence of the extracellular matrix (ECM) terminates juvenile plasticity by mediating structural stability. Interestingly, enzymatic removal of the ECM restores juvenile forms of plasticity, as for instance demonstrated by topographical reconnectivity in sensory pathways. However, to which degree the mature ECM is a compromise between stability and flexibility in the adult brain impacting synaptic plasticity as a fundamental basis for learning, lifelong memory formation, and higher cognitive functions is largely unknown. In this study, we removed the ECM in the auditory cortex of adult Mongolian gerbils during specific phases of cortex-dependent auditory relearning, which was induced by the contingency reversal of a frequency-modulated tone discrimination, a task requiring high behavioral flexibility. We found that ECM removal promoted a significant increase in relearning performance, without erasing already established—that is, learned—capacities when continuing discrimination training. The cognitive flexibility required for reversal learning of previously acquired behavioral habits, commonly understood to mainly rely on frontostriatal circuits, was enhanced by promoting synaptic plasticity via ECM removal within the sensory cortex. Our findings further suggest experimental modulation of the cortical ECM as a tool to open short-term windows of enhanced activity-dependent reorganization allowing for guided neuroplasticity.Structural remodeling and stabilization of synaptic networks are key mechanisms underlying learning in the adult brain. During early life, high structural and functional plasticity is required for the experience-shaped development of basic neuronal circuits (1). With brain maturation, juvenile plasticity of so-called critical or sensitive periods is decreased and is accompanied by the appearance of the brain’s extracellular matrix (ECM) and its specialized compact form named “perineuronal net” (PNN) enwrappping cell bodies and synaptic contacts (2, 3). Enzymatic degradation of the ECM in adult animals has been demonstrated to restore such forms of developmental (juvenile) plasticity with respect to topographical map plasticity in the visual cortex (4), fear-response–mediating circuits in the amygdala (5), spinal cord injuries (6, 7), and song learning circuits of zebra finches (8). In addition, enzymatic ECM removal altered several forms of synaptic plasticity in vitro and in vivo (9–12). However, even though structural stability of networks acquired during developmental phases is essential for neuronal efficiency, mechanisms allowing synaptic remodeling are key events during learning and memory formation throughout life (13). We recently demonstrated that endogenous proteases moderately digesting specific components of the ECM are regulated in an activity-dependent manner (2, 14) and ECM removal modulates synaptic short-term plasticity by synaptic exchange of postsynaptic glutamate receptors (10, 15). Further, ECM modulation enhances synaptic short-term plasticity by affecting voltage-dependent calcium channels (9). These findings challenge the view of the purely stabilizing role of ECM in the brain and indicate a potential regulatory switch for plastic network adaptations within the adult brain at the level of individual synapses by modulating the extracellular space (16). However, it remains open to what extent ECM modulations influence learning-related plasticity in the adult brain and its specific effects on behavior during a cognitive task.In the present study, we aimed at evaluating the potential role of experimental ECM removal within the auditory cortex (ACx) of Mongolian gerbils, which has been found to be particularly rich in ECM (17), during a cognitively demanding auditory go/no go shuttle-box task. We selected discrimination and reversal learning of frequency-modulated (FM) tones as a cognitive task, which necessarily requires ACx plasticity (18, 19). Injections of the ECM-degrading enzyme hyaluronidase (HYase) into the ACx after the first acquisition phase significantly enhanced subsequent reversal learning compared with sham-treated animals (injection of 0.9% saline). Particularly, after ECM degradation, animals abandoned the inappropriate discrimination strategy from the initial acquisition phase faster and thus promoted successful discrimination performance of the new contingency during reversal learning. ECM removal did not further influence the initial acquisition learning or interfere with already established—that is, learned—capacities in later learning stages, suggesting an enhanced activity-dependent neuroplastic reorganization of established synaptic networks in ACx during reversal learning.Our findings suggest that experimental degradation of the ECM in sensory cortex, although not affecting general sensory learning, does, however, enhance the cognitive flexibility that can build on the learned behaviors. Thereby, ECM degradation could be used as a tool for guided neuroplasticity, which might also bear therapeutic potential.     

Expression of polypeptide variants of receptor-type protein tyrosine phosphatase β: The secreted form,phosphacan, increases dramatically during embryonic development and modulates glial cell behavior in vitro

Glial cells express three splicing variants of a receptor-type protein tyrosine phosphatase called RPTPβ. Two are receptor forms that differ in a large extracellular domain. The third is a secreted proteoglycan called phosphacan that lacks the cytoplasmic phosphatase domains. We have now identified, by immunoblotting, proteins corresponding to these three forms of RPTPβ in rat C6 glioma cells and brain. The short receptor form is much more prevalent than the full-length receptor in C6 glioma cells. Phosphacan is much more abundant than either of the receptor forms in rat brain, and its expression increases progressively during embryonic development, while the receptor forms show only moderate changes. In contrast to the long form and phosphacan that were detected as proteoglycans, the short receptor form, lacking the large alternatively spliced domain, was not detected as a chondroitin sulfate proteoglycan. We recently showed that phosphacan binds to the neuron-glia cell adhesion molecule, Ng-CAM, and we now report that glia expressing RPTPβ adhere and extend processes on substrates coated with Ng-CAM. After one day in culture, however, the glia retract their processes and often lift off the substrate. Conditioned medium from glial cells, which contains large amounts of phosphacan, inhibits glial adhesion to Ng-CAM, and depletion of phosphacan from the conditioned medium by immunoadsorption reduces the inhibitory activity. The results show that phosphacan increases dramatically during development, and indicate that secreted forms of RPTPβ can modulate glial cell adhesion and behavior. © 1996 Wiley-Liss, Inc.