chABC Treatment Research Articles

Chemonucleolysis combined with dynamic loading for inducing degeneration in bovine caudal intervertebral discs.

Chemonucleolysis has become an established method of producing whole organ culture models of intervertebral disc (IVD) degeneration. However, the field needs more side-by-side comparisons of the degenerative effects of the major enzymes used in chemonucleolysis towards gaining a greater understanding of how these organ culture models mimic the wide spectrum of characteristics observed in human degeneration. In the current work we induced chemonucleolysis in bovine coccygeal IVDs with 100µL of papain (65U/mL), chondroitinase ABC (chABC, 5U/mL), or collagenase II (col'ase, 0.5U/mL). Each enzyme was applied in a concentration projected to produce moderate levels of degeneration. After 7days of culture with daily dynamic physiological loading (0.02-0.2MPa, 0.2Hz, 2h), the cellular, biochemical and histological properties of the IVDs were evaluated in comparison to a PBS-injected control. Papain and collagenase, but not chABC, produced macroscopic voids in the tissues. Compared to day 0 intact IVDs, papain induced the greatest magnitude glycosaminoglycan (GAG) loss compared to chABC and col'ase. Papain also induced the greatest height loss (3%), compared to 0.7%, 1.2% and 0.4% for chABC, col'ase, and PBS, respectively. Cell viability in the region adjacent to papain and PBS-injection remained at nearly 100% over the 7-day culture period, whereas it was reduced to 60%-70% by chABC and col'ase. Generally, enzyme treatment tended to downregulate gene expression for major ECM markers, type I collagen (COL1), type II collagen (COL2), and aggrecan (ACAN) in the tissue adjacent to injection. However, chABC treatment induced an increase in COL2 gene expression, which was significant compared to the papain treated group. In general, papain and col'ase treatment tended to recapitulate aspects of advanced IVD degeneration, whereas chABC treatment captured aspects of early-stage degeneration. Chemonucleolysis of whole bovine IVDs is a useful tool providing researchers with a robust spectrum of degenerative changes and can be utilized for examination of therapeutic interventions.

CHARACTERIZATION OF NATIVE AND DECELLULARIZED PORCINE TENDON UNDER TENSION AND COMPRESSION: A CLOSER LOOK AT GLYCOSAMINOGLYCAN CONTRIBUTION TO TENDON MECHANICS

Decellularised porcine superflexor tendon (pSFT) has been demonstrated to be a suitable scaffold for anterior cruciate ligament reconstruction[1]. While the role of collagen in tendons is well known, the mechanical role of glycosaminoglycans (GAGs) is less clear and may be altered by the decellularisation process.To determine the effects of decellularisation on pSFT GAG content and mechanical function and to investigate the consequences of GAG loss in tensile and compressive loading.pSFTs were decellularised following previous techniques [2]. For GAG removal, native pSFTs were treated with chondroitinase ABC (ChABC; 0.1U/mL, 72h). Cell and GAG removal was validated using histology and quantitative assays. Native, decellularised and ChABC treated groups (n=6) were biomechanically characterised. In tension, specimens underwent stress relaxation and strength testing using previous protocols [1]. Stress relaxation data was fitted to a modified Maxwell-Weichert model to determine time-dependent (E1 & E2) and time-independent moduli (E0). The toe and linear region moduli (Etoe, Elinear), in addition to tensile strength (UTS) and failure strain were determined from strength testing. In compression, specimens underwent confined loading conditions (ramp at 10 s-1 to 10% strain and hold). The aggregate modulus (HA) and zero-strain permeability (k0) were determined using previous techniques [3]. Data was analysed by one-way ANOVA with Tukey post-hoc test to determine significant differences between test groups (p<0.05).Quantitative assays showed no GAG reduction post-decellularisation, but a significant reduction after ChABC treatment. HA was only significantly reduced in the ChABC group. k0 was significantly higher for the ChABC group compared to decellularised. E0 was significantly reduced in the decellularised group compared to native and ChABC groups, while E1 and E2 were not different between groups. Etoe, Elinear, UTS and failure strain were not different between groups.Decellularisation does not affect GAG content or impair mechanical function in pSFT. GAG loss adversely affects pSFT compressive properties, revealing major mechanical contribution under compression, but no significant role under tension.

Time and dose-dependent effects of chondroitinase ABC on growth of engineered cartilage

Tissue engineering techniques have been effective in developing cartilage-like tissues in vitro. However, many scaffold-based approaches to cultivating engineered cartilage have been limited by low collagen production, an impediment for attaining native functional load-bearing tensile mechanical properties. Enzymatic digestion of glycosaminoglycans (GAG) with chondroitinase ABC (chABC) temporarily suppresses the construct's GAG content and compressive modulus and increases collagen content. Based on the promising results of these early studies, the aim of this study was to further promote collagen deposition through more frequent chABC treatments. Weekly dosing of chABC at a concentration of 0.15U/mL resulted in a significant cell death, which impacted the ability of the engineered cartilage to fully recover GAG and compressive mechanical properties. In light of these findings, the influence of lower chABC dosage on engineered tissue (0.004 and 0.015U/mL) over a longer duration (one week) was investigated. Treatment with 0.004U/mL reduced cell death, decreased the recovery time needed to achieve native compressive mechanical properties and GAG content, and resulted in a collagen content that was 65% greater than the control. In conclusion, the results of this study demonstrate that longer chABC treatment (one week) at low concentrations can be used to improve collagen content in developing engineered cartilage more expediently than standard chABC treatments of higher chABC doses administered over brief durations.

Effect of chondroitinase ABC on adhesion and behavior of synovial membrane-derived mesenchymal stem cells in rabbit partial-thickness chondral defects

Transplanted cells may have difficulty attaching to the surface of partial-thickness chondral lesions because of the anti-adhesive properties of the proteoglycan rich matrix. Therefore, the current study attempts to evaluate the effect of chondroitinase ABC (chABC) on the adhesion and behavior of transplanted synovial membrane-derived mesenchymal stem cells (SDSCs) in rabbit partial-thickness chondral defects. In ex vivo adhesion experiments, chABC treatment (0.1 U/ml) was increased in SDSC attachment to the cartilage explants, and significantly diminished by pretreatment with neutralizing antibody against fibronectin. In the in vivo experiments, 1 day and 4 weeks after the chABC treatment (0.1 and 1 U/ml), the immunoreactivity (IR) against CS-56 (intact chondroitin sulfate antibody) was markedly decreased; however, the IR of 2B6 (stub of the chondroitin 4-sulfate chain), 3B3 (stub of the chondroitin 6-sulfate chain), and fibronectin was increased. At 12 weeks, this IR returned to normal except in the high-dose chABC-treated group (1 U/ml). Furthermore, the attachment of SDSCs to the chondral defects after chABC treatment was increased at 7 days compared with that in the chondral defects pretreated with saline. However, the tissue repaired by SDSCs was negatively stained for type II collagen at 12 weeks. In conclusion, these results showed that the exposure to fibronectin by chABC treatment enhances the attachment of SDSCs to partial-thickness chondral defects. However, the tissue regenerated by SDSCs showed lack of hyaline cartilage regeneration. Thus, to understand the fate of transplanted MSCs in cartilage defect is very important for successful cell therapies.

Perineuronal nets increase inhibitory GABAergic currents during the critical period in rats.

To investigate inhibitory γ-aminobutyric acid (GABA) ergic postsynaptic currents (IPSCs) and postsynaptic currents (PSCs) in layer IV of the rat visual cortex during the critical period and when plasticity was extended through dissolution of the perineuronal nets (PNNs). We employed 24 normal Long-Evans rats to study GABAA-PSC characteristics of neurons within layer IV of the visual cortex during development. The animals were divided into six groups of four rats according to ages at recording: PW3 (P21-23d), PW4 (P28-30d), PW5 (P35-37d), PW6 (P42-44d), PW7 (P49-51d), and PW8 (56-58d). An additional 24 chondroitin sulfate proteoglycan (CSPG) degradation rats (also Long-Evans) were generated by making a pattern of injections of chondroitinase ABC (chABC) into the visual cortex 1 week prior to recording at PW3, PW4, PW5, PW6, PW7, and PW8. Immunohistochemistry was used to identify the effect of chABC injection on CSPGs. PSCs were detected with whole-cell patch recordings, and GABAA receptor-mediated IPSCs were pharmacologically isolated. IPSC peak current showed a strong rise in the age-matched control group, peaked at PW5 and were maintained at a roughly constant value thereafter. Although there was a small increase in peak current for the chABC group with age, the peak currents continued to decrease with the delayed highest value at PW6, resulting in significantly different week-by-week comparison with normal development. IPSC decay time continued to increase until PW7 in the control group, while those in the chABC group were maintained at a stable level after an initial increase at PW4. Compared with normal rats, the decay times recorded in the chABC rats were always shorter, which differed significantly at each age. We did not observe any differences in IPSC properties between the age-matched control and penicillinase (P-ase) group. However, the change in IPSCs after chABC treatment was not reflected in the total PSCs or in basic membrane properties in layer IV of the rat visual cortex. Our results demonstrate that rather than rapidly increasing during the critical period for neuronal plasticity, IPSCs in layer IV of rat visual cortex are maintained at an immature level when PNNs are removed by chABC. This suggests that GABA receptor maturation involves the conformation of the CSPGs in PNNs.

Sialidase, Chondroitinase ABC, and Combination Therapy after Spinal Cord Contusion Injury

Axon regeneration in the central nervous system is severely hampered, limiting functional recovery. This is in part because of endogenous axon regeneration inhibitors that accumulate at the injury site. Therapeutic targeting of these inhibitors and their receptors may facilitate axon outgrowth and enhance recovery. A rat model of spinal cord contusion injury was used to test the effects of two bacterial enzyme therapies that target independent axon regeneration inhibitors, sialidase (Vibrio cholerae) and chondroitinase ABC (ChABC, Proteus vulgaris). The two enzymes, individually and in combination, were infused for 2 weeks via implanted osmotic pumps to the site of a moderate thoracic spinal cord contusion injury. Sialidase was completely stable, whereas ChABC retained>30% of its activity in vivo over the 2 week infusion period. Immunohistochemistry revealed that infused sialidase acted robustly throughout the spinal cord gray and white matter, whereas ChABC activity was more intense superficially. Sialidase treatment alone resulted in improved behavioral and anatomical outcomes. Rats treated exclusively with sialidase showed significantly increased hindlimb motor function, evidenced by higher Basso Beattie and Bresnahan (BBB) and BBB subscores, and fewer stepping errors on a horizontal ladder. Sialidase-treated rats also had increased serotonergic axons caudal to the injury. ChABC treatment, in contrast, did not enhance functional recovery or alter axon numbers after moderate spinal cord contusion injury, and dampened the response of sialidase in the dual enzyme treatment group. We conclude that sialidase infusion enhanced recovery from spinal cord contusion injury, and that combining sialidase with ChABC failed to improve outcomes.

Exogenous BDNF enhances the integration of chronically injured axons that regenerate through a peripheral nerve grafted into a chondroitinase-treated spinal cord injury site

Although axons lose some of their intrinsic capacity for growth after their developmental period, some axons retain the potential for regrowth after injury. When provided with a growth-promoting substrate such as a peripheral nerve graft (PNG), severed axons regenerate into and through the graft; however, they stop when they reach the glial scar at the distal graft-host interface that is rich with inhibitory chondroitin sulfate proteoglycans. We previously showed that treatment of a spinal cord injury site with chondroitinase (ChABC) allows axons within the graft to traverse the scar and reinnervate spinal cord, where they form functional synapses. While this improvement in outgrowth was significant, it still represented only a small percentage (<20%) of axons compared to the total number of axons that regenerated into the PNG. Here we tested whether providing exogenous brain-derived neurotrophic factor (BDNF) via lentivirus in tissue distal to the PNG would augment regeneration beyond a ChABC-treated glial interface. We found that ChABC treatment alone promoted axonal regeneration but combining ChABC with BDNF-lentivirus did not increase the number of axons that regenerated back into spinal cord. Combining BDNF with ChABC did increase the number of spinal cord neurons that were trans-synaptically activated during electrical stimulation of the graft, as indicated by c-Fos expression, suggesting that BDNF overexpression improved the functional significance of axons that did reinnervate distal spinal cord tissue.

Chondroitinase ABC promotes plasticity of spinal reflexes following peripheral nerve injury

Peripheral nerve transection, even with optimal repair, can result in an extensive disruption of central connectivity, which can lead to long-lasting impairments in motor and sensory function. We hypothesised that removal of spinal cord chondroitin sulphate proteoglycans (CSPGs) would promote plasticity in the spinal cord, allowing compensation for inaccurate peripheral reinnervation. In adult rats, the median and radial nerves were cut and repaired, either correctly (median to median and radial to radial), or incorrectly (median to radial and vice versa). This produced two levels of inaccuracy of peripheral reinnervation. Whole nerve recordings from a third brachial plexus nerve, the ulnar, were made during median or radial nerve stimulation. Low and high threshold reflexes were characterised in uninjured animals and a clear difference in the pattern of ulnar response to flexor (median) or extensor (radial) stimulation was established. This included the phenomenon of wind-up, where repetitive median nerve stimulation at supramaximal C-fibre threshold leads to a progressive increase in the number of spikes recorded. To achieve digestion of CSPGs a lentiviral vector expressing ChABC was delivered to the spinal cord via intraspinal injection. Following ChABC treatment, we found several indicators of reorganisation of central connections. Firstly, we found that the amplitude of a low threshold, polysynaptic reflex could be increased after nerve injury, only following treatment with ChABC. Secondly, wind-up of motor responses in the ulnar nerve to supramaximal stimulation of afferents in the median nerve, which collapses after nerve injury (to ~25% of uninjured value), could be restored by ChABC after correct repair (to ~90% of uninjured value). Thirdly, wind-up in ulnar motor axons to stimulation of radial nerve afferents, which is minimal in the uninjured state, becomes significantly stronger after nerve injury and ChABC treatment (a 10 fold increase). We propose that application of a plasticity-promoting treatment to the spinal cord allows the amplification of adaptive changes in response to inaccurate wiring in the periphery.

Functional regeneration of respiratory pathways after spinal cord injury

Spinal cord injuries (SCI) often occur at the cervical level above the phrenic motor pools, which innervate the diaphragm. Unfortunately, the untoward effects of impaired breathing are a leading cause of SCI-related death, underscoring the importance of developing strategies to restore respiratory activity. Here we show that after cervical SCI, there is upregulation of the perineuronal net (PNN) associated chondroitin sulfate proteoglycans (CSPGs) around phrenic motor neurons. Digestion of these potently inhibitory extracellular matrix molecules with Chondroitinase ABC (ChABC) can, by itself, promote plasticity of spared tracts and restore limited activity to the paralyzed diaphragm. However, when combined with application of a peripheral nerve autograft, ChABC treatment results in lengthy regeneration of serotonergic axons and other bulbospinal fibers with remarkable recovery of diaphragm function. Following recovery and initial transection of the bridge, there occurs an unusual, overall increased tonic diaphragmatic EMG activity, suggesting considerable remodeling of spinal cord circuitry after regeneration. This is followed by complete elimination of the restored activity proving that regeneration is critical for the return of function. Overall, these experiments present a way to profoundly restore function of a single muscle following debilitating CNS trauma, through both plasticity of spared tracts and regeneration of essential pathways.

Effect of orientation and targeted extracellular matrix degradation on the shear mechanical properties of the annulus fibrosus

The intervertebral disc experiences combinations of compression, torsion, and bending that subject the disc substructures, particularly the annulus fibrosus (AF), to multidirectional loads and deformations. Combined tensile and shear loading is a particularly important loading paradigm, as compressive loads place the AF in circumferential hoop tension, and spine torsion or bending induces AF shear. Yet the anisotropy of AF mechanical properties in shear, as well as important structure-function mechanisms governing this response, are not well-understood. The objective of this study, therefore, was to investigate the effects of tissue orientation and enzymatic degradation of glycosaminoglycan (GAG) and elastin on AF shear mechanical properties. Significant anisotropy was found: the circumferential shear modulus, Gθz, was an order of magnitude greater than the radial shear modulus, Grθ. In the circumferential direction, prestrain significantly increased the shear modulus, suggesting an important role for collagen fiber stretch in shear properties for this orientation. While not significant and highly variable, ChABC treatment to remove GAG increased the circumferential shear modulus compared to PBS control (p=0.15). Together with the established literature for tensile loading of fiber-reinforced GAG-rich tissues, the trends for changes in shear modulus with ChABC treatment reflect complex, structure-function relationships between GAG and collagen that potentially occur over several hierarchical scales. Elastase digestion did not significantly affect shear modulus with respect to PBS control; further contributing to the notion that circumferential shear modulus is dominated by collagen fiber stretch. The results of this study highlight the complexity of the structure-function relationships that govern the mechanical response of the AF in radial and circumferential shear, and provide new and more accurate data for the validation of material models and tissue-engineered disc replacements.

Partial functional recovery after complete spinal cord transection by combined chondroitinase and clenbuterol treatment

Spinal cord injury not only disrupts axonal tracts but also causes gliotic, fibrotic, and Schwannotic scarring with resulting deposition of chondroitin sulfate proteoglycans (CSPGs) which prevent axonal reconnection and recovery of locomotor function. Here, we determined whether recovery of locomotor function could be promoted after complete transection, by degrading CSPGs enzymatically within the injury site with chondroitinase ABC (chABC) together with treatment with the beta(2)-adrenoceptor agonist, clenbuterol, a neuroprotective agent which can promote regrowth of lower motoneurons. Partial recovery of locomotor function was observed 8-12 weeks postinjury only after combined chABC and clenbuterol treatment. The recovery of locomotor function coincided with the presence of axons caudal to the injury site arising from neurons of the reticular, vestibular, and red nuclei also only with combined chABC and clenbuterol treatment. Axons myelinated by Schwann cells were most prominent in the transection site in the combined treatment group. Clenbuterol treatment activated cAMP response element binding protein within retrogradely traced neurons which has been associated with axonal regrowth. ChABC treatment decreased scarring due to both CSPG and collagen deposition as well as the gap between intact regions of the spinal cord. ChABC also increased numbers of phagocytic cells which remove myelin debris as well as populations of astrocytes thereby aiding blood-spinal cord barrier reformation. Together the results suggest that chABC and clenbuterol can act synergistically to promote recovery of locomotor function.

Chondroitinase ABC has a long‐lasting effect on chondroitin sulphate glycosaminoglycan content in the injured rat brain

Chondroitin sulphate proteoglycans (CSPGs) are axon growth inhibitory molecules present in the glial scar that play a part in regeneration failure after damage to the CNS and which restrict CNS plasticity. Removal of chondroitin sulphate glycosaminoglycan (GAG) chains with chondroitinase-ABC (chABC) in models of CNS injury promotes both axon regeneration and plasticity. We have analysed the immediate and long-term effects of a single injection of chABC on CSPGs, GAGs and axon regeneration. We made unilateral nigrostriatal lesions in adult rats accompanied by an adjacent infusion of either chABC or a bacterial-derived control enzyme (penicillinase). Within 24 h of chABC treatment there was digestion of GAGs, including hyaluronan, and a reduction in neurocan in an area extending 1.5 mm around the injection site. Around 50% of GAG is inaccessible to chABC digestion, even in tissue digested in vitro, which probably represents intracellular stores. In control penicillinase treated animals, total GAGs recovered from the lesioned brains were up-regulated by 4-fold 7 days after injury and gradually decreased to normal at 28 days post-lesion. In chondroitinase-treated animals, the total GAG remained at low level throughout the 28-day experimental period. This suggests the persistence of active chABC for at least 10 days after injection which is able to digest CSPGs released from cells during this time. This was confirmed by immunological detection of enzyme for 10 days and by retrieval of active enzyme from the brain at 10 days after injection. Our results suggest that a single injection of chABC can produce an environment conducive to CNS repair for over 10 days.

Chondroitinase ABC Digestion of the Perineuronal Net Promotes Functional Collateral Sprouting in the Cuneate Nucleus after Cervical Spinal Cord Injury

Upregulation of extracellular chondroitin sulfate proteoglycans (CSPGs) after CNS injuries contributes to the impediment of functional recovery by restricting both axonal regeneration and synaptic plasticity. In the present study, the effect of degrading CSPGs with the application of the bacterial enzyme chondroitinase ABC (chABC) into the cuneate nucleus of rats partially denervated of forepaw dorsal column axons was examined. A dorsal column transection between the C6-C7 dorsal root entry zones was followed immediately by an ipsilateral brainstem injection of either chABC or a bacterial-derived control enzyme [penicillinase (P-ase)] and then subsequently (1 week later) followed with a second brainstem enzyme injection and cholera toxin B subunit (CTB) tracer injection into the ipsilateral forepaw digits and pads. After 1 additional week, the rats underwent electrophysiological receptive field mapping of the cuneate nucleus and/or anatomical evaluation. Examination of the brainstems of rats from each group revealed that CSPGs had been reduced after chABC treatment. Importantly, in the chABC-treated rats (but not in the P-ase controls), a significantly greater area of the cuneate nucleus was occupied by physiologically active CTB traced forepaw afferents that had been spared by the initial cord lesion. These results demonstrate, for the first time, a functional change directly linked to anatomical evidence of sprouting by spinal cord afferents after chABC treatment.