Journal of Rehabilitation Research and Development Vol. 40, No. 4, July/August 2003, Supplement Pages ?–? Transplantation strategies to promote repair of the injured spinal cord Mary Bartlett Bunge, PhD; Damien D. Pearse, PhD The Miami Project to Cure Paralysis, Departments of Cell Biology and Anatomy and Neurological Surgery, University of Miami School of Medicine, Miami, FL Abstract—This review describes the results of the transplanta- immunomodulatory interventions. After that, strategies tion of Schwann cells and olfactory ensheathing glia in combi- to promote regrowth of axons and restore function will nation with other interventions. The complete transection injury involve multiple approaches: (1) reducing scar forma- model was used to test the combination of Schwann cell tion, thereby diminishing the accumulation of proteogly- bridges with methylprednisolone, neurotrophins, or olfactory can molecules known to be inhibitory to axonal growth; ensheathing glia. The contusion injury model was used to com- (2) overcoming additional inhibitory molecules, myelin- pare Schwann cell and olfactory ensheathing glia transplanta- related constituents, that also stymie axonal extension; tion and to examine the results of combining Schwann cell transplants with elevated levels of cyclic adenosine monophos- (3) awakening damaged nerve cells to regenerate axons; phate. The combination strategies were more effective than cell transplantation alone. The improved regeneration response usu- ally involved a reduction in secondary tissue loss, axonal regen- eration from brainstem neurons, an increase in myelinated Abbreviations: ATP = adenosine triphosphate, BBB = Basso, fibers in the transplant, the exit of regenerated fibers from the Beattie, Bresnahan rating scale, BDNF = brain-derived neu- transplant into the contiguous cord, and an improvement in rotrophic factor, cAMP = cyclic adenosine monophosphate, locomotor function. cDNA = complementary deoxyribonucleic acid, CNS = central nervous system, CREB = cAMP-response element binding protein, db-cAMP = di-butyryl cAMP, GTP = guanosine triph- osphate, MAG = myelin-associated glycoprotein, NF-κB = Key words: central nervous system (CNS) regeneration, contu- nuclear factor kappa B, NT-3 = neurotrophin-3, OEG = olfac- sion injury, cyclic adenosine monophosphate, methylpredniso- tory ensheathing glia, PKA = protein kinase A, SC = Schwann lone, neurotrophins, olfactory ensheathing glia, raphespinal cell, TNF-α = tumor necrosis factor-α, WGA-HRP = wheat tract, Schwann cells, spinal cord transection. germ agglutinin horseradish peroxydase. This material was based on work supported by the Christopher Reeve Paralysis Foundation, the National INTRODUCTION Institutes of Health/National Institute of Neurological Diseases and Stroke grants N09923 and 38665, The Miami Changes in injured spinal cord tissue start rapidly Project to Cure Paralysis, and the Buoniconti Fund. and are varied and many. It is likely, therefore, that effec- Address all correspondence and requests for reprints to Mary Bartlett Bunge, PhD, The Miami Project to Cure Paralysis, tive therapeutic strategies will consist of a series of inter- University of Miami School of Medicine, P.O. Box 016960, ventions. First, secondary tissue loss should be prevented Miami, FL 33101; 305-243-4596; fax: 305-243-3923; email: early through neuroprotective, anti-inflammatory, or [email protected]. 1 2 Journal of Rehabilitation Research and Development Vol. 40, No. 4, 2003, Supplement (4) providing sustenance to the nerve cells that are regenerated axons from transplants into the spinal cord separated from their targets and, thus, are bereft of [17,18]. There are also important new approaches being trophicfactors; (5) facilitating axonal growth across the tested in training and rehabilitation studies. The limitation site of injury; (6) guiding axonal growth to appropriate of space in this article precludes additional discussion of spinal cord regions; (7) enabling formation of new con- new interventions being examined in other laboratories. nections; and, finally, (8) retraining the nervous system Accordingly, the reference list is abbreviated. What fol- to use the therapeutic interventions. Interestingly, in the lows is an overview of studies conducted in our labora- early 1900s, Ramón y Cajal [1] speculated that to correct tory at The Miami Project to Cure Paralysis. the central nervous system (CNS) deficiency in repair, we must “give to the sprouts, by means of adequate ali- mentation, a vigorous capacity for growth; and, place in COMPLETE TRANSECTION/SCHWANN CELL front of the disoriented nerve cones and in the thickness BRIDGE MODEL of the tracks of the white matter and neuronic foci, spe- cific orienting substances.” This was undoubtedly the We have, over the last decade, explored the efficacy first suggestion of a combination strategy to effect repair of grafted SCs to repair the injured spinal cord in the in the adult CNS. adult rat. SCs have long been known to be key for the There are now exciting investigations under way in regeneration that occurs in the peripheral nervous system. many laboratories to devise reparative strategies to SCs in peripheral nerves everywhere either ensheathe address the aforementioned challenges for spinal cord axons with their cytoplasm or myelinate axons. When an repair. Surface receptors on growing nerve fibers that axon is damaged and it then degenerates, SCs neverthe- respond to inhibitory factors are being blocked by pre- less remain in their tunnels of extracellular matrix, and it senting antibodies to the inhibitory factors [2] or frag- is into these tunnels that the nerve fibers regenerate. ments thereof [3], or by introducing competitive Also, SCs secrete growth factors and extracellular matrix antagonist peptides [4], thereby enabling the axons to components that are known to promote nerve fiber grow through an inhibitory milieu. Another approach is to growth [19]. SCs function in the CNS and have been modulate intracellular signaling to (a) interfere with cas- shown to be more effective when genetically engineered cades that are initiated after a growing nerve fiber to produce more growth factors than they normally do. encounters an inhibitory molecule (such as targeting the Another advantage of SCs is their accessability. They Rho family of GTPases that regulate actin-mediated could be obtained from a piece of peripheral nerve from a motility [5,6]), or (b) turn on pathways responsible for spinal cord injured person, expanded to very large num- initiating axon growth and to enable fibers to grow bers in tissue culture (which is now possible), and then be through inhibitory environments (such as elevating levels transplanted into the area of injury in the same person of cyclic adenosine monophosphate (cAMP)). Another without the potential of immune rejection. pursuit to overcome inhibitory factors is to deliver We have studied two models, the complete transec- enzymes that prevent the formulation of or degrade chon- tion and the contusion models. Both have advantages and droitin sulfate proteoglycans as they accumulate near the disadvantages. One of the strong advantages of the com- site of injury [7,8]. Other studies are investigating ways plete transection model is the availability of unambigu- to promote axonal growth across the area of injury; trans- ous results in detecting regenerated fibers below the area plantation plays a major role in these studies. Implanta- of injury. In a contusion injury, spared fibers exist around tion of pieces of peripheral nerve [9], fetal tissue [10], the spinal cord perimeter, which complicates the evalua- olfactory ensheathing glia (OEG) [11,12], and Schwann tion of regenerated fibers. The contusion injury, highly cell (SC) bridges [13], for example, are being assessed. clinically relevant, leads to the development of a large Transplants are often tested in conjunction with the neu- cavity over weeks in both rats and humans. rotrophins, brain-derived neurotrophic factor (BDNF) An early complete transection/SC bridge paradigm and neurotrophin 3 (NT-3). These neurotrophins have used a cable of six million SCs encased within a polymer been shown to awaken neurons to regrow axons [14], to channel, into which both stumps of the severed spinal increase numbers and different types of axons that grow cord were inserted [20]. SCs and the channel were into transplants [10,15,16], and to promote the growth of implanted into a gap created at the thoracic 8–11 levels in 3 BUNGE and PEARSE. Transplant strategies for SCI repair an adult Fischer rat spinal cord. One month later, it was was labeling of a mean of 92 neurons in the brainstem. In observed that axons grew onto the SC bridge from both other experiments [25], SCs were transduced with a stumps, there was mean of over 1000 spinal cord neurons human prepro BDNF cDNA that was introduced by that responded by regenerating axons onto the bridge, means of a retrovirus. This paradigm differed somewhat there was a mean of nearly 2000 myelinated axons on the from the one described above, in that the spinal cord was bridge, and there were over eight times more unmyeli- transected and the SCs (transduced or untreated) were nated axons on the bridge. However, there was a minimal deposited in the distal stump to create a 5 mm-long trail, response from brainstem neurons, and axons were not as well as in the transection site. We found, one month observed to leave the transplant. We next considered later, that the trails had remained