Degradation of Chondroitin Sulfate Proteoglycan Enhances the Neurite-Promoting Potential of Spinal Cord Tissue Jian Zuo, Debbie Neubauer, Kelly Dyess, TobyA. Ferguson, and David Muir 1 Department of Pediatrics, Neurology Division, and Department of Neuroscience, University of Florida Brain Institute and College of Medicine, Gainesville, Florida 32610-0296 Received May 20, 1998; accepted September 4, 1998 The contribution of chondroitin sulfate proteogly- can (CSPG) in the suppression of axonal growth in rat spinal cord has been examined by means of an in vitro bioassay in which regenerating neurons are grown on tissue section substrata. Dissociated embryonic chick dorsal root ganglionic neurons were grown on normal and injured adult spinal cord tissue sections treated with chondroitinases. Neuritic growth on normal spi- nal cord tissue was meager. However, both the percent- age of neurons with neurites and the average neurite length were substantially greater on sections treated with chondroitinase ABC. Enzymes that specifically degraded dermatan sulfate or hyaluronan were ineffec- tive. Neuritic growth was significantly greater on in- jured (compared to normal) spinal cord and a further dramatic increase resulted from chondroitinase ABC treatment. Neurites grew equally within white and gray matter regions after chondroitinase treatment. Observed increases in neurite outgrowth on chondroiti- nase-treated tissues were largely inhibited in the pres- ence of function-blocking laminin antibodies. These findings indicate that inhibitory CSPG is widely distrib- uted and predominant in both normal and injured spinal cord tissues. Additionally, inhibitory CSPG is implicated in negating the potential stimulatory ef- fects of laminin that might otherwise support spinal cord regeneration.  1998 Academic Press Key Words: chondroitin sulfate proteoglycan; lami- nin; neuronal regeneration; neurite inhibitor; rat spi- nal cord injury; cryoculture. INTRODUCTION The failure of adult mammalian spinal cord to sup- port axonal regeneration has been attributed to a paucity of neurite-promoting molecules and an abun- dance of inhibitory components associated with glial cell surfaces and the surrounding extracellular matrix (ECM). Laminin has a potent neurite-promoting func- tion and plays an important role in axonal growth and guidance in the developing nervous system (23, 33). The prevalence of laminin is also a major determinant of axonal growth in regenerative nervous systems and its abundance in the basal lamina of peripheral nerve contributes to the success of nerve regeneration (10, 46). In contrast, laminin is scarce in the parenchyma of the normal adult CNS, where it is only found associated with astrocytic foot processes, vascular basement mem- branes, and leptomeningial elements, and certain lami- nin chains are expresses by neurons as well (2, 20, 21, 43). Following CNS trauma, enhanced expression of ECM molecules by a subpopulation of astrocytes appears to be one of the earliest molecular changes at sites of injury. Numerous laminin-positive astrocytes appear which assemble basal lamina associated with newly formed glial limitans and within the matrix of the lesion and glial scar (18, 22, 43). It remains unclear if these laminin-rich structures can benefit axonal regen- eration since growth has only been associated with these ECM structures in isolated instances (32, 43). In the past decade convincing evidence indicates that axonal regeneration within the brain and spinal cord may be prevented by a preponderance of inhibitory molecules. Potential growth inhibitory properties have been reported for several cell surface and myelin- associated proteins as well as for certain ECM macro- molecules (8, 30, 38). Chondroitin sulfate proteoglycans (CSPGs) are particularly abundant in the nervous system and are highly expressed in CNS injury (re- viewed in 16). Emerging evidence indicates CSPGs can profoundly influence axonal development and regenera- tion. Certain CSPG are growth-inhibitory molecules which, at high concentration relative to growth- promoting signals, may suppress axonal growth. Fur- thermore, CSPGs have been found to inhibit the neurite- promoting activity of purified laminin as well as laminin within CNS scar tissue and nerve endoneurium (27, 29, 41, 49). CSPGs are candidate molecules for regulating axonal growth through inhibitory mechanisms in nor- 1 To whom correspondence and reprint requests should be ad- dressed. Fax: 352-392-9520. E-mail: muir@ufbi.ufl.edu. EXPERIMENTAL NEUROLOGY 154, 654–662 (1998) ARTICLE NO. EN986951 654 0014-4886/98 $25.00 Copyright  1998 by Academic Press All rights of reproduction in any form reserved.