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The inhibitory environment of the injured spinal cord is an obstacle to functional recovery and axonal regeneration in adult mammals. We had previously shown that injection of adeno-associated virus (AAV) encoding the L1 cell adhesion molecule (AAV-L1) at the time of acute thoracic compression injury of adult mice promotes locomotor recovery, which is associated with ameliorated astrogliosis and improved axonal regeneration in the lumbar spinal cord. In the present study, we investigated whether delayed injection of AAV-L1, chondroitinase ABC (Chase), or the combination of the two agents into the mouse spinal cord 3 weeks after injury would also lead to improved recovery. The Basso Mouse Scale showed enhanced locomotor recovery 12 weeks after application of the agents in all treatment groups compared to the control group that was injected with AAV encoding green fluorescent protein (AAV-GFP). Investigation of hindlimb function using single-frame motion analysis revealed, however, that L1 overexpression, but not injection of Chase, improved voluntary movements without body weight support, whereas injection of Chase, but not L1 overexpression, enhanced body weight support during stepping. Mice with the combined application of AAV-L1 and Chase showed improvement in both parameters. Enhanced motor recovery after combined application correlated with increased densities of cholinergic and GABAergic terminals at motoneuronal cell bodies, and of lamina-specific glutamatergic sensory afferents 15 weeks after injury, indicating enhanced synaptic rearrangements in the lumbar spinal cord below the lesion site. These findings suggest that L1 overexpression combined with Chase application may contribute to the treatment of sub-chronic spinal cord injury.

Increased intracranial pressure (ICP) caused by edema following severe traumatic brain injury (TBI) or stroke contributes to high rates of mortality and morbidity. The search continues for more effective treatments that target the edema that contributes to increased ICP. We previously described the effect of the fixed charge density (FCD) of brain on its swelling behavior according to the Donnan effect. Here we show that reduction of brain tissue FCD is an effective means of reducing brain tissue swelling and edema in rat and porcine cortical brain tissue in vitro. The effect of enzymes directed at digesting candidate contributors to cellular FCD such as chondroitin sulfate proteoglycans (CSPGs), heparin sulfate proteoglycans (HSPGs), and DNA was examined in slices of the adult rat cortex. All enzymes were capable of decreasing FCD in the tissue by ?20%, and reducing tissue swelling over a 24?h period following dissection from ?60% to ?30%. Chondroitinase ABC (ChABC) was most effective at reducing dead brain tissue swelling in response to changes in ionic osmotic environments. ChABC reduced swelling in live slices of tissue even within the first 2?h following dissection. It also significantly reduced the FCD, initial tissue swelling, and volume change in response to hypotonic bathing solution in porcine cortical brain tissue. The use of ChABC to reduce tissue FCD may be an effective method for reducing brain edema and controlling ICP following injury.

Growth-inhibitory chondroitin sulfate proteoglycans (CSPG) are a primary target for therapeutic strategies after spinal cord injury because of their contribution to the inhibitory nature of glial scar tissue, a major barrier to successful axonal regeneration. Chondroitinase ABC (ChABC) digestion of CSPGs promotes axonal regeneration beyond a lesion site with subsequent functional improvement. ChABC also has been shown to promote sprouting of spared fibers but it is not clear if functional recovery results from such plasticity. Here we sought to better understand the roles rostral or caudal sprouting may play in ChABC-mediated functional improvement. To achieve this, ChABC or vehicle was injected rostral or caudal to a unilateral C5 injury. When injected rostral to a hemisection, ChABC promoted significant sprouting of 5HT+ fibers into dorsal and ventral horns. When ChABC was injected into tissue caudal to a hemisection, no additional sprouting was observed. When injected caudal to a hemicontusion injury, ChABC promoted sprouting of 5HT+ fibers into the ventral horn but not the dorsal horn. None of this sprouting resulted in a change in the synaptic component synapsin, nor did it impact performance in behavioral tests assessing motor function. These data suggest that ChABC-mediated sprouting of spared fibers does not necessarily translate into functional recovery.

Chondroitinase treatment of experimental spinal cord injury improves recovery of sensory, motor, and autonomic functions. Chondroitinase catalyzes the cleavage of glycosaminoglycans (GAGs) from the core proteins of chondroitin sulfate proteoglycans (CSPGs). Little is known about changes in production of these proteoglycans in the clinically relevant contusion model of spinal cord injury or if CSPG content is altered by chondroitinase treatment. Female Long-Evans rats were injured with a forceps contusion injury and treated on alternate days with chondroitinase ABCI or control enzyme via an intrathecal catheter. Spinal cords were analyzed at specific times after injury. The cord was divided in 4 mm long segments, one containing the lesion, two rostral and two caudal to the lesion. These segments were assessed for CSPG protein and message content (NG2, neurocan and phosphacan) by Western blotting and real-time PCR. CSPG protein content was increased by one day post injury for all CSPGs investigated, and was increased in all segments examined rostral and caudal to the lesion site. Significant increases in CSPG were observed with peak content detected at 7, 7 and 14 days post injury for NG2, neurocan and phosphacan, respectively. Chondroitinase treatment had little impact upon the CPSG protein content. Changes in message levels of these CSPGs are also reported. This demonstrates that expression patterns of CSPGs in contusion injury are similar to those surrounding surgical hemisection lesions and demonstrates that the sensory and motor function enhancing effects of chondroitinase are likely due to removal of GAG chains rather than reduction in CSPG content.

Chondroitin sulfate proteoglycans are synthesized and deposited in the spinal cord following injury. These proteoglycans may restrict regeneration and plasticity and contribute to the limited recovery seen after an injury. Chondroitinase, a bacterial enzyme that catalyzes the hydrolysis of the chondroitin chains on proteoglycans, has been shown to improve motor and sensory function following partial transection lesions of the spinal cord. To assess the effects of chondroitinase in a clinically relevant model of spinal cord injury, 128 female Long-Evans rats received either a severe, moderate, or mild contusion injury at the vertebral level T9/T10 with a forceps model and were treated for 2 weeks with chondroitinase ABCI at 0.06 Units per dose, penicillinase, or vehicle control via an intrathecal catheter placed near the injury. Motor behavior was measured by open-field testing of locomotion and bladder function monitored by measuring daily residual urine volumes. Animals treated with chondroitinase showed significant improvements in open-field locomotor activity as measured by the Basso, Beattie and Bresnahan scoring system after both severe and moderate SCI (p<0.05 and 0.01, respectively). No significant locomotor differences were observed in the mild injury group. In the moderate injury group, residual urine volumes were reduced with chondroitinase treatment by 2 weeks after injury (p<0.05) and in the severe injury group, by 6 weeks after injury (NS). These results demonstrate that chondroitinase is effective at promoting both somatic and autonomic motor recovery following a clinically relevant contusion spinal cord injury and is a candidate as a therapeutic for human spinal cord injury.