Chondroitin Sulfate Proteoglycan Levels Research Articles

Selective Disruption of Perineuronal Nets in Mice Lacking Crtl1 is Sufficient to Make Fear Memories Susceptible to Erasure

The ability to store, retrieve, and extinguish memories of adverse experiences is an essential skill for animals’ survival. The cellular and molecular factors that underlie such processes are only partially known. Using chondroitinase ABC treatment targeting chondroitin sulfate proteoglycans (CSPGs), previous studies showed that the maturation of the extracellular matrix makes fear memory resistant to deletion. Mice lacking the cartilage link protein Crtl1 (Crtl1-KO mice) display normal CSPG levels but impaired CSPG condensation in perineuronal nets (PNNs). Thus, we asked whether the presence of PNNs in the adult brain is responsible for the appearance of persistent fear memories by investigating fear extinction in Crtl1-KO mice. We found that mutant mice displayed fear memory erasure after an extinction protocol as revealed by analysis of freezing and pupil dynamics. Fear memory erasure did not depend on passive loss of retention; moreover, we demonstrated that, after extinction training, conditioned Crtl1-KO mice display no neural activation in the amygdala (Zif268 staining) in comparison to control animals. Taken together, our findings suggest that the aggregation of CSPGs into PNNs regulates the boundaries of the critical period for fear extinction.

Emerging Therapeutic Role of Chondroitinase (ChABC) in Neurological Disorders and Cancer

Abstract: Proteoglycans are essential biomacromolecules that participate in matrix structure and organization, cell proliferation and migration, and cell surface signal transduction. However, their roles in physiology, particularly in CNS, remain incompletely deciphered. Numerous studies highlight the elevated levels of chondroitin sulphate proteoglycans (CSPGs) in various diseases, like cancers, and neurological disorders, like spinal cord injury (SCI), traumatic brain damage, neurodegenerative diseases, and are mainly implicated to hinder tissue repair. In such a context, chondroitinase ABC (ChABC), a therapeutic enzyme, has shown immense hope to treat these diseases in several preclinical studies, primarily attributed to the digestion of the side chains of the proteoglycan chondroitin sulphate (CS) molecule. Despite extensive research, the progress in evolution of the concept of therapeutic targeting of proteoglycans is still in its infancy. This review thus provides fresh insights into the emerging therapeutic applications of ChABC in various diseases apart from SCI and the underlying mechanisms.

Emerging role of neuregulin-1beta1 in pathogenesis and progression of multiple sclerosis

Emerging role of neuregulin-1beta1 in pathogenesis and progression of multiple sclerosis

Inhibition of central axon regeneration: perspective from chondroitin sulfate proteoglycans in lamprey spinal cord injury.

Inhibition of central axon regeneration: perspective from chondroitin sulfate proteoglycans in lamprey spinal cord injury.

Inhibition of Autophagy Flux Promotes Secretion of Chondroitin Sulfate Proteoglycans in Primary Rat Astrocytes.

Following spinal cord injury (SCI), reactive astrocytes in the glial scar produce high levels of chondroitin sulfate proteoglycans (CSPGs), which are known to inhibit axonal regeneration. Transforming growth factor beta (TGFβ) is a well-known factor that induces the production of CSPGs, and in this study, we report a novel mechanism underlying TGFβ's effects on CSPG secretion in primary rat astrocytes. We observed increased TGFβ-induced secretion of the CSPGs neurocan and brevican, and this occurred simultaneously with inhibition of autophagy flux. In addition, we show that neurocan and brevican levels are further increased when TGFβ is administered in the presence of an autophagy inhibitor, Bafilomycin-A1, while they are reduced when cells are treated with a concentration of rapamycin that is not sufficient to induce autophagy. These findings suggest that TGFβ mediates its effects on CSPG secretion through autophagy pathways. They also represent a potential new approach to reduce CSPG secretion in vivo by targeting autophagy pathways, which could improve axonal regeneration after SCI.

A Shift in Tissue Stiffness During Hippocampal Maturation Correlates to the Pattern of Neurogenesis and Composition of the Extracellular Matrix.

Aging changes the mechanical properties of brain tissue, such as stiffness. It has been proposed that the maintenance and differentiation of neural stem cells (NSCs) are regulated in accordance with extracellular stiffness. Neurogenesis is observed in restricted niches, including the dentate gyrus (DG) of the hippocampus, throughout mammalian lifetimes. However, profiles of tissue stiffness in the DG in comparison with the activity of NSCs from the neonatal to the matured brain have rarely been addressed so far. Here, we first applied ultrasound-based shear-wave elasticity imaging (SWEI) in living animals to assess shear modulus as in vivo brain stiffness. To complement the assay, atomic force microscopy (AFM) was utilized to determine the Young’s modulus in the hippocampus as region-specific stiffness in the brain slice. The results revealed that stiffness in the granule cell layer (GCL) and the hilus, including the subgranular zone (SGZ), increased during hippocampal maturation. We then quantified NSCs and immature neural cells in the DG with differentiation markers, and verified an overall decrease of NSCs and proliferative/immature neural cells along stages, showing that a specific profile is dependent on the subregion. Subsequently, we evaluated the amount of chondroitin sulfate proteoglycans (CSPGs), the major extracellular matrix (ECM) components in the premature brain by CS-56 immunoreactivity. We observed differential signal levels of CSPGs by hippocampal subregions, which became weaker during maturation. To address the contribution of the ECM in determining tissue stiffness, we manipulated the function of CSPGs by enzymatic digestion or supplementation with chondroitin sulfate, which resulted in an increase or decrease of stiffness in the DG, respectively. Our results illustrate that stiffness in the hippocampus shifts due to the composition of ECM, which may affect postnatal neurogenesis by altering the mechanical environment of the NSC niche.

Hypomorphic zebrafish models mimic the musculoskeletal phenotype of β4GalT7-deficient Ehlers-Danlos syndrome

β4GalT7 is a transmembrane Golgi enzyme, encoded by B4GALT7, that plays a pivotal role in the proteoglycan linker region formation during proteoglycan biosynthesis. Defects in this enzyme give rise to a rare autosomal recessive form of Ehlers-Danlos syndrome (EDS), currently known as ‘spondylodysplastic EDS (spEDS-B4GALT7)’. This EDS subtype is mainly characterized by short stature, hypotonia and skeletal abnormalities, thereby illustrating its pleiotropic importance during human development. Insights into the pathogenic mechanisms underlying this disabling disease are very limited, in part due to the lack of a relevant in vivo model.As the majority of mutations identified in patients with spEDS-B4GALT7 are hypomorphic, we generated zebrafish models with partial loss of B4galt7 function, including different knockdown (morphant) and mosaic knockout (crispant) b4galt7 zebrafish models and studied the morphologic, functional and molecular aspects in embryonic and larval stages.Morphant and crispant zebrafish show highly similar morphological abnormalities in early development including a small, round head, bowed pectoral fins, short body-axis and mild developmental delay. Several craniofacial cartilage and bone structures are absent or strongly misshapen. In addition, the total amount of sulfated glycosaminoglycans is significantly diminished and particularly heparan and chondroitin sulfate proteoglycan levels are greatly reduced. We also show impaired cartilage patterning and loss of chondrocyte organization in a cartilage-specific Tg(Col2a1aBAC:mcherry) zebrafish reporter line. The occurrence of the same abnormalities in the different models confirms these are specifically caused by B4galt7 deficiency. A disturbed actin pattern, along with a lack of muscle tone, was only noted in morphants in which translation of b4galt7 was blocked.In conclusion, we generated the first viable animal models for spEDS-B4GALT7, and show that in early development the human spEDS-B4GALT7 phenotype is faithfully mimicked in these zebrafish models. Our findings underscore a key role for β4GalT7 in early development of cartilage, bone and muscle. These models will lead to a better understanding of spEDS-B4GALT7 and can be used in future efforts focusing on therapeutic applications.

Local delivery of stabilized chondroitinase ABC degrades chondroitin sulfate proteoglycans in stroke-injured rat brains

Central nervous system (CNS) injuries, such as stroke and spinal cord injuries, result in the formation of a proteoglycan-rich glial scar, which acts as a barrier to axonal regrowth and limits the regenerative capacity of the CNS. Chondroitinase ABC (ChABC) is a potent bacterial enzyme that degrades the chondroitin sulfate proteoglycan (CSPG) component of the glial scar and promotes tissue recovery; however, its use is significantly limited by its inherent instability at physiological temperatures. Here, we demonstrate that ChABC can be stabilized using site-directed mutagenesis and covalent modification with poly(ethylene glycol) chains (i.e. PEGylation). Rosetta protein structure modeling was used to screen >20,000 single point mutations, and four potentially stabilizing mutations were tested in vitro. One of the mutations, N1000G (asparagine ➔ glycine at residue 1000), significantly improved the long-term activity of the protein, doubling its functional half-life. PEGylation of this ChABC mutant inhibited unfolding and aggregation and resulted in prolonged bioactivity with a 10-fold increase in activity compared to the unmodified protein after two days. Local, affinity-controlled release of the modified protein (PEG-N1000G-ChABC) was achieved by expressing it as a fusion protein with Src homology 3 (SH3) and delivering the protein from a methylcellulose hydrogel modified with SH3 binding peptides. This affinity-based release strategy provided sustained PEG-N1000G-ChABC-SH3 release over several days in vitro. Direct implantation of the hydrogel delivery vehicle containing stabilized PEG-N1000G-ChABC-SH3 onto the rat brain cortex in a sub-acute model of stroke resulted in significantly reduced CSPG levels in the penumbra of 49% at 14 and 40% at 28 days post-injury compared to animals treated with the vehicle alone.

Sox9 knockout mice have improved recovery following stroke

The partial recovery that can occur after a stroke has been attributed to structural and functional plasticity that compensate for damage and lost functions. This plasticity is thought to be limited in part by the presence of growth inhibitors in the central nervous system. Blocking or reducing signals from inhibitors of axonal sprouting such as Nogo and chondroitin sulfate proteoglycans (CSPGs) increases post-stroke axonal sprouting and improves recovery. We previously identified the transcription factor SOX9 as a key up-regulator of CSPG production and demonstrated that conditional Sox9 ablation leads to increased axonal sprouting and improved recovery after spinal cord injury. In the present study we evaluate the effect of conditional Sox9 ablation in a transient middle cerebral artery occlusion (MCAO) model of stroke. We demonstrate that conditional Sox9 ablation leads to reduced CSPG levels, increased tissue sparing and improved post-stroke neurological recovery. Anterograde tract tracing studies demonstrate that in the Sox9 conditional knockout mice corticorubral and corticospinal projections from the contralateral, uninjured cortex increase projections to targets in the midbrain and spinal cord denervated by the injury. These results suggest that targeting SOX9 is a viable strategy to promote reparative axonal sprouting, neuroprotection and recovery after stroke.

Attenuation of Robust Glial Scar Formation Facilitates Functional Recovery in Animal Models of Chronic Nerve Compression Injury.

Late surgery for chronic nerve compression injuries usually improves sensation but rarely reverses motor atrophy. We hypothesized that a persistent glial scar after chronic nerve compression injury might account for poor motor recovery and that degradation of the glial scar as an adjunct to surgical decompression would improve functional recovery. A previously described model of chronic nerve compression injury was created in C57BL/6 mice and Sprague-Dawley rats, and the nerves were harvested early or late after electrophysiological confirmation of the injury. Western blot, polymerase chain reaction, and quantitative immunohistochemical analyses were performed to determine levels of chondroitin sulfate proteoglycans and extracellular matrix molecules. Subsets of mice were treated either with surgical decompression alone or with decompression coupled with intraepineurial injection of a low dose (0.1 μgμL) or a high dose (0.2 μg/μL) of chondroitinase ABC at 6 weeks after injury. Aggrecan showed the greatest change in mRNA and protein levels at the early and late time points following creation of the chronic nerve compression injury. Quantitative immunohistochemical analysis revealed early aggrecan upregulation localized primarily to the endoneurium and late upregulation localized to the perineurium and epineurium (p < 0.0105). Quantitative immunohistochemical analysis for collagen IV, laminin-α2, and fibronectin also showed early upregulation with perineurial scarring. Quantitative immunohistochemical analysis and Western blot analysis for aggrecan demonstrated a marked increase in the endoneurium at the early time points and upregulation of expression in the epineurium and perineurium at the late time points. Decompression along with intraepineurial injection of high-dose chondroitinase ABC at 6 weeks after creation of the compression injury resulted in marked attenuation of decorin and aggrecan expression with functional improvement in nerve conduction velocity. Significant upregulation of chondroitin sulfate proteoglycans and other extracellular matrix components contributes to the pathogenesis of compression neuropathies in murine models. The administration of chondroitinase ABC degrades these chondroitin sulfate proteoglycans and improves functional recovery after chronic nerve compression injury; thus, it can be considered as a possible therapeutic adjunct.

Anabolic role of lysyl oxidase like-2 in cartilage of knee and temporomandibular joints with osteoarthritis

BackgroundLysyl oxidase like-2 (LOXL2) is a copper-dependent amine oxidase. Our previous studies showed that LOXL2 is elevated during mouse fracture healing. The goal of this study was to evaluate the potential of LOXL2 to act as an anabolic agent in cartilage affected by osteoarthritis (OA).MethodsLOXL2 was visualized in tissues from human knee and hip joints and temporomandibular joints (TMJ) by immunofluorescence. The activity of LOXL2 in human articular and TMJ chondrocytes was assessed by cell-based assays, microarray analysis, and RT-qPCR, and LOXL2-mediated activation of NF-κB and extracellular signal-related kinase (ERK) signaling pathways was measured by western blotting. To examine LOXL2-induced effect in vivo, we implanted Matrigel-imbedded human chondrocytes into nude mice and exposed them to exogenous LOXL2 for 6 weeks. Finally, LOXL2-induced effects on collagen type 2 α1 (COL2A1) and phospho-SMAD2/3 were evaluated by immunofluorescence analysis.ResultsLOXL2 staining was detected in damaged regions of human TMJ, hip and knee joints affected by OA. Stimulation with transforming growth factor (TGF)-β1 upregulated LOXL2 expression, while pro-inflammatory cytokines IL-1β and TNF-α downregulated LOXL2, in human chondrocytes. Viral transduction of LOXL2 in OA chondrocytes increased the mRNA levels of chondroitin sulfate proteoglycan (CSPG4), aggrecan (ACAN), sex determining region Y-box containing gene 9 (SOX9), and COL2A1 but reduced the levels of extracellular matrix (ECM)-degrading enzymes matrix metalloproteinase (MMP)1, MMP3, and MMP13. Further, forced expression of LOXL2 promoted chondrogenic lineage-specific gene expression, increased the expression of COL2A1 in the presence of TNF-α, and inhibited chondrocyte apoptosis. LOXL2 expression also inhibited IL-1β-induced phospho-NF-κB/p65 and TGF-β1-induced ERK1/2 phosphorylation. Matrigel constructs of human chondrocytes from the knee joint and TMJ implanted in nude mice showed anabolic responses after LOXL2 transduction, including increased expression of SOX9, ACAN, and COL2A1. Finally, immunofluorescence staining revealed co-localization of LOXL2 with SOX9 in the nuclei of cells in the implants, decreased phospho-SMAD2/3, and increased COL2A1 staining.ConclusionOur results suggest that although LOXL2 is upregulated in cartilage affected by OA, this may be a protective response that promotes anabolism while inhibiting specific catabolic responses in the pathophysiology of OA.

Local delivery of chondroitinase ABC with or without stromal cell-derived factor 1α promotes functional repair in the injured rat spinal cord.

Traumatic spinal cord injury (SCI) is a devastating event for which functional recovery remains elusive. Due to the complex nature of SCI pathology, a combination treatment strategy will likely be required for success. We hypothesized that tissue and functional repair would be achieved in a rat model of impact-compression SCI by combining degradation of the glial scar, using chondroitinase ABC (ChABC), with recruitment of endogenous neural precursor cells (NPCs), using stromal cell-derived factor 1α (SDF). To test this hypothesis, we designed a crosslinked methylcellulose hydrogel (XMC) for minimally invasive, localized, and sustained intrathecal drug delivery. ChABC was released from XMC using protein-peptide affinity interactions while SDF was delivered by electrostatic affinity interactions from polymeric nanoparticles embedded in XMC. Rats with SCI were treated acutely with a combination of SDF and ChABC, SDF alone, ChABC alone, or vehicle alone, and compared to injury only. Treatment with ChABC, both alone and in combination with SDF, resulted in faster and more sustained behavioural improvement over time than other groups. The significantly reduced chondroitin sulfate proteoglycan levels and greater distribution of NPCs throughout the spinal cord tissue with ChABC delivery, both alone and in combination with SDF, may explain the improved locomotor function. Treatment with SDF alone had no apparent effect on NPC number or distribution nor synergistic effect with ChABC delivery. Thus, in this model of SCI, tissue and functional repair is attributed to ChABC.

Conditional Sox9 ablation improves locomotor recovery after spinal cord injury by increasing reactive sprouting

The absence of axonal regeneration after spinal cord injury (SCI) has been attributed to the up-regulation of axon-repelling molecules, such as chondroitin sulfate proteoglycans (CSPGs) present in the glial scar that forms post-SCI. We previously identified the transcription factor SOX9 as a key up-regulator of CSPG production and also demonstrated that conditional Sox9 ablation leads to decreased CSPG levels and improved recovery of hind limb function after SCI. We herein demonstrate increased neural input onto spinal neurons caudal to the lesion in spinal cord injured Sox9 conditional knock out mice as indicated by increased levels of the presynaptic markers synaptophysin and vesicular glutamate transporter 1 (VGLUT1) compared to controls. Axonal sparing, long-range axonal regeneration and reactive sprouting were investigated as possible explanations for the increase in neural inputs caudal to the lesion and for the improved locomotor outcomes in spinal cord-injured Sox9 conditional knock out mice. Whereas retrograde tract-tracing studies failed to reveal any evidence for increased axonal sparing or for long-range regeneration in the Sox9 conditional knock out mice, anterograde tract-tracing experiments demonstrated increased reactive sprouting caudal to the lesion after SCI. Finally we demonstrate that application of a broad spectrum MMP inhibitor to reduce CSPG degradation in Sox9 conditional knock out mice prevents the improvements in locomotor recovery observed in untreated Sox9 conditional knock out mice. These results suggest that improved recovery of locomotor function in Sox9 conditional knock out mice after SCI is due to increased reactive sprouting secondary to reduced CSPG levels distal to the lesion.

Local Delivery of High-Dose Chondroitinase ABC in the Sub-Acute Stage Promotes Axonal Outgrowth and Functional Recovery after Complete Spinal Cord Transection.

Chondroitin sulfate proteoglycans (CSPGs) are glial scar-associated molecules considered axonal regeneration inhibitors and can be digested by chondroitinase ABC (ChABC) to promote axonal regeneration after spinal cord injury (SCI). We previously demonstrated that intrathecal delivery of low-dose ChABC (1 U) in the acute stage of SCI promoted axonal regrowth and functional recovery. In this study, high-dose ChABC (50 U) introduced via intrathecal delivery induced subarachnoid hemorrhage and death within 48 h. However, most SCI patients are treated in the sub-acute or chronic stages, when the dense glial scar has formed and is minimally digested by intrathecal delivery of ChABC at the injury site. The present study investigated whether intraparenchymal delivery of ChABC in the sub-acute stage of complete spinal cord transection would promote axonal outgrowth and improve functional recovery. We observed no functional recovery following the low-dose ChABC (1 U or 5 U) treatments. Furthermore, animals treated with high-dose ChABC (50 U or 100 U) showed decreased CSPGs levels. The extent and area of the lesion were also dramatically decreased after ChABC treatment. The outgrowth of the regenerating axons was significantly increased, and some partially crossed the lesion site in the ChABC-treated groups. In addition, retrograde Fluoro-Gold (FG) labeling showed that the outgrowing axons could cross the lesion site and reach several brain stem nuclei involved in sensory and motor functions. The Basso, Beattie and Bresnahan (BBB) open field locomotor scores revealed that the ChABC treatment significantly improved functional recovery compared to the control group at eight weeks after treatment. Our study demonstrates that high-dose ChABC treatment in the sub-acute stage of SCI effectively improves glial scar digestion by reducing the lesion size and increasing axonal regrowth to the related functional nuclei, which promotes locomotor recovery. Thus, our results will aid in the treatment of spinal cord injury.

Combination therapy of stem cell derived neural progenitors and drug delivery of anti-inhibitory molecules for spinal cord injury

Regeneration of lost synaptic connections following spinal cord injury (SCI) is limited by local ischemia, cell death, and an excitotoxic environment, which leads to the development of an inhibitory glial scar surrounding a cystic cavity. While a variety of single therapy interventions provide incremental improvements to functional recovery after SCI, they are limited; a multifactorial approach that combines several single therapies may provide a better chance of overcoming the multitude of obstacles to recovery. To this end, fibrin scaffolds were modified to provide sustained delivery of neurotrophic factors and anti-inhibitory molecules, as well as encapsulation of embryonic stem cell-derived progenitor motor neurons (pMNs). In vitro characterization of this combination scaffold confirmed that pMN viability was unaffected by culture alongside sustained delivery systems. When transplanted into a rat sub-acute SCI model, fibrin scaffolds containing growth factors (GFs), anti-inhibitory molecules without pMNs, or pMNs with GFs had lower chondroitin sulfate proteoglycan levels compared to scaffolds containing anti-inhibitory molecules with pMNs. Scaffolds containing pMNs, but not anti-inhibitory molecules, showed survival, differentiation into neuronal cell types, axonal extension in the transplant area, and the ability to integrate into host tissue. However, the combination of pMNs with sustained-delivery of anti-inhibitory molecules led to reduced cell survival and increased macrophage infiltration. While combination therapies retain potential for effective treatment of SCI, further work is needed to improve cell survival and to limit inflammation. Statement of SignificanceSpinal cord injury (SCI) creates a highly complex inhibitory environment with a multitude of obstacles that limit recovery. Many therapeutic options have been developed to overcome single obstacles, but single therapies typically only lead to limited functional improvement. Therefore combination therapies may improve recovery by targeting several inhibitory obstacles simultaneously. The present study used biomaterial scaffolds to combine the sustained release of anti-inhibitory molecules and growth factors with cell transplantation of highly purified progenitor motor neurons. This expands upon previously established biomaterial scaffolds by supporting surviving cells, limiting inhibition from the extracellular environment, and replenishing lost cell populations. We show that while promising, certain combinations may exacerbate negative side-effects instead of augmenting positive features.

Sox9 knock‐down in mice improves locomotor recovery after spinal cord injury by reducing CSPG levels and facilitating reactive sprouting

We have identified the transcription factor SOX9 as a key regulator of chondroitin sulfate proteoglycan (CSPG) production in the injured spinal cord. CSPGs in the lesion epicenter are produced by reactive astrocytes responding to the injury. CSPGs are also key components of the perineuronal matrix that surround the cell bodies and dendrites of many neurons in the central nervous system. Thus CSPGs may limit plasticity after SCI by inhibiting regeneration of axons through the glial scar or by inhibiting reactive sprouting at deafferented targets distant to the lesion. We have demonstrated reduced CSPG levels and improved locomotor recovery in spinal cord‐injured Sox9 conditional knockout mice. Herein we investigated sparing, long‐range regeneration and reactive sprouting as possible explanations for the improved locomotor outcomes in Sox9 knockout mice after SCI. We herein demonstrate that the improved recovery of locomotor function in Sox9 conditional knockout mice after SCI is due to increased reactive sprouting secondary to reduced CSPG levels distal to the lesion.

Expression of hyaluronidase-4 in a rat spinal cord hemisection model.

Study DesignExamination of hyaluronidase-4 (Hyal-4) expression in a rat spinal cord hemisection model.PurposeTo determine the status of Hyal-4 expression after hemisection of the spinal cord, and the relationship between its expression and that of chondroitin sulfate proteoglycans (CSPGs).Overview of LiteratureCSPGs are expressed at the site of spinal cord injury and inhibit axon regeneration. Administration of exogenous chrondroitinase ABC (ChABC), derived from bacteria, digested CSPGs and promoted axonal regrowth. Using a rat hemisection model, we have demonstrated peak CSPGs levels at by 3 weeks after injury but then decreased spontaneously. Could there be an endogenous enzyme similar to ChABC in the spinal cord? It has been suggested that Hyal-4 is involved in CSPG degradation.MethodsA rat hemisection model was prepared and spinal cord frozen sections were prepared at 4 days and 1, 2, 3, 4, 5, and 6 weeks post-cordotomy and stained for CSPGs and Hyal-4 and subjected to Western blotting.ResultsCSPGs appeared at the injury site at 4 days after hemisection, reached a peak after 3 weeks, and then decreased. Hyal-4 was observed around the injury site from 4 days after cordotomy and increased until after 5-6 weeks. Double staining showed Hyal-4 around CSPGs. Western blotting identified a band corresponding to Hyal-4 from 4 days after hemisection.ConclusionsHyal-4 was expressed in a rat hemisection model in areas surrounding CSPGs, and as its peak was delayed compared with that of CSPGs. These results suggest the involvement of Hyal-4 in the digestion of CSPGs.

The thymoquinone-induced production of reactive oxygen species promotes dedifferentiation through the ERK pathway and inflammation through the p38 and PI3K pathways in rabbit articular chondrocytes.

Dedifferentiation and inflammation are major features of cartilage degeneration during the pathogenesis of osteoarthritis (OA). Thymoquinone (TQ) is the major compound of black seed oil isolated from Nigella sativa with various beneficial or harmful effects on several diseases; however, its effects on the dedifferentiation and inflammation of chondrocytes have not yet been characterized. In the present study, we investigated whether TQ regulates the dedifferentiation and inflammation of rabbit articular chondrocytes, focusing on the production of reactive oxygen species (ROS) in rabbit articular chondrocytes. TQ induced the generation of ROS in a dose-dependent manner, as shown by staining with the fluorescent probe, 2′–7′-dichlorofluorescein diacetate. We confirmed that TQ induced dedifferentiation by measuring the loss of type II collagen and the reduction in chondroitin sulfate proteoglycan levels. TQ also caused inflammation by inducing the expression of cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2). The antioxidant, N-acetyl cysteine (NAC), prevented the dedifferentiation and inflammation which was generated by the TQ-induced production of ROS. Furthermore, TQ caused a dose-dependent increase in p38, phosphorylated extracellular signal-regulated kinase (p-ERK) and phosphoinositide 3-kinase (PI3K) expression. NAC abrogated this effect and attenuated the dedifferentiation and inflammation which was generated by the TQ-induced production of ROS. To identify the ROS-regulated pathways, we treated the chondrocytes with the p38 inhibitor, SB203580, the MEK inhibitor, PD98059, and the PI3K inhibitor, LY294002. PD98059 inhibited the TQ-induced dedifferentiation and SB203580 and LY294002 prevented the TQ-induced inflammation. These findings suggest that the TQ-induced production of ROS causes dedifferentiation through the ERK pathway and inflammation through the PI3K and p38 pathways in rabbit articular chondrocytes.

Assessment of Nogo-66 Receptor 1 Function In Vivo After Spinal Cord Injury

Neuronal Nogo-66 receptor 1 (NgR1) has attracted attention as a converging point for mediating the effects of myelin-associate inhibitory ligands in the central nervous system, establishing the growth-restrictive environment, and limiting axon regeneration after traumatic injury. To investigate the factors that may be contributing to the discrepancy in the importance of NgR1, which has been undermined by several studies that have shown the lack of substantial axon regeneration after spinal cord injury (SCI) in NgR1-knockout or -knockdown animal models. We used mice carrying either a homozygous or heterozygous null mutation in the NgR1 gene and subjected them to either a moderate or severe SCI. Locomotor function assessments revealed that the level of functional recovery is affected by the degree of injury suffered. NgR1 ablation enhanced local collateral sprouting in the mutant mice. Reactive astrocytes and chondroitin sulfate proteoglycans (CSPGs) are upregulated surrounding the injury site. Matrix metalloproteinase-9, which has been shown to degrade CSPGs, was significantly upregulated in the homozygous mutant mice compared with the heterozygous or wild-type mice. However, CSPG levels remained higher in the homozygous compared with the heterozygous mice, suggesting that CSPG-degrading activity of matrix metalloproteinase-9 may require the presence of NgR1. Genetic ablation of NgR1 may lead to significant recovery in locomotor function after SCI. The difference in locomotor recovery we observed between the groups that suffered various degrees of injury suggests that injury severity may be a confounding factor in functional recovery after SCI.

Fgf2 improves functional recovery—decreasing gliosis and increasing radial glia and neural progenitor cells after spinal cord injury

ObjectivesA major impediment for recovery after mammalian spinal cord injury (SCI) is the glial scar formed by proliferating reactive astrocytes. Finding factors that may reduce glial scarring, increase neuronal survival, and promote neurite outgrowth are of major importance for improving the outcome after SCI. Exogenous fibroblast growth factor (Fgf) has been shown to decrease injury volume and improve functional outcome; however, the mechanisms by which this is mediated are still largely unknown.MethodsIn this study, Fgf2 was administered for 2 weeks in mice subcutaneously, starting 30 min after spinal cord hemisection.ResultsFgf2 treatment decreased the expression of TNF-a at the lesion site, decreased monocyte/macrophage infiltration, and decreased gliosis. Fgf2 induced astrocytes to adopt a polarized morphology and increased expression of radial markers such as Pax6 and nestin. In addition, the levels of chondroitin sulfate proteoglycans (CSPGs), expressed by glia, were markedly decreased. Furthermore, Fgf2 treatment promotes the formation of parallel glial processes, “bridges,” at the lesion site that enable regenerating axons through the injury site. Additionally, Fgf2 treatment increased Sox2-expressing cells in the gray matter and neurogenesis around and at the lesion site. Importantly, these effects were correlated with enhanced functional recovery of the left paretic hind limb.ConclusionsThus, early pharmacological intervention with Fgf2 following SCI is neuroprotective and creates a proregenerative environment by the modulation of the glia response.