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1.  Two Faces of Chondroitin Sulfate Proteoglycan in Spinal Cord Repair: A Role in Microglia/Macrophage Activation 
PLoS Medicine  2008;5(8):e171.
Background
Chondroitin sulfate proteoglycan (CSPG) is a major component of the glial scar. It is considered to be a major obstacle for central nervous system (CNS) recovery after injury, especially in light of its well-known activity in limiting axonal growth. Therefore, its degradation has become a key therapeutic goal in the field of CNS regeneration. Yet, the abundant de novo synthesis of CSPG in response to CNS injury is puzzling. This apparent dichotomy led us to hypothesize that CSPG plays a beneficial role in the repair process, which might have been previously overlooked because of nonoptimal regulation of its levels. This hypothesis is tested in the present study.
Methods and Findings
We inflicted spinal cord injury in adult mice and examined the effects of CSPG on the recovery process. We used xyloside to inhibit CSPG formation at different time points after the injury and analyzed the phenotype acquired by the microglia/macrophages in the lesion site. To distinguish between the resident microglia and infiltrating monocytes, we used chimeric mice whose bone marrow-derived myeloid cells expressed GFP. We found that CSPG plays a key role during the acute recovery stage after spinal cord injury in mice. Inhibition of CSPG synthesis immediately after injury impaired functional motor recovery and increased tissue loss. Using the chimeric mice we found that the immediate inhibition of CSPG production caused a dramatic effect on the spatial organization of the infiltrating myeloid cells around the lesion site, decreased insulin-like growth factor 1 (IGF-1) production by microglia/macrophages, and increased tumor necrosis factor alpha (TNF-α) levels. In contrast, delayed inhibition, allowing CSPG synthesis during the first 2 d following injury, with subsequent inhibition, improved recovery. Using in vitro studies, we showed that CSPG directly activated microglia/macrophages via the CD44 receptor and modulated neurotrophic factor secretion by these cells.
Conclusions
Our results show that CSPG plays a pivotal role in the repair of injured spinal cord and in the recovery of motor function during the acute phase after the injury; CSPG spatially and temporally controls activity of infiltrating blood-borne monocytes and resident microglia. The distinction made in this study between the beneficial role of CSPG during the acute stage and its deleterious effect at later stages emphasizes the need to retain the endogenous potential of this molecule in repair by controlling its levels at different stages of post-injury repair.
Michal Schwartz and colleagues describe the role of chondroitin sulfate proteoglycan in the repair of injured tissue and in the recovery of motor function during the acute phase after spinal cord injury.
Editors' Summary
Background.
Every year, spinal cord injuries paralyze about 10,000 people in the United States. The spinal cord, which contains bundles of nervous system cells called neurons, is the communication superhighway between the brain and the body. Messages from the brain travel down the spinal cord to control movement, breathing, and other bodily functions; messages from the skin and other sensory organs travel up the spinal cord to keep the brain informed about the body. All these messages are transmitted along axons, long extensions on the neurons. The spinal cord is protected by the bones of the spine but if these are displaced or broken, the axons can be compressed or cut, which interrupts the information flow. Damage near the top of the spinal cord paralyzes the arms and legs (tetraplegia); damage lower down paralyzes the legs only (paraplegia). Spinal cord injuries also cause other medical problems, including the loss of bowel and bladder control. Currently there is no effective treatment for spinal cord injuries. Treatment with drugs to reduce inflammation has, at best, only modest effects. Moreover, because damaged axons rarely regrow, most spinal cord injuries are permanent.
Why Was This Study Done?
One barrier to recovery after a spinal cord injury seems to be an inappropriate immune response to the injury. After an injury, microglia (immune system cells that live in the nervous system), and macrophages (blood-borne immune system cells that infiltrate the injury) become activated. Microglia/macrophage activation can be either beneficial (the cells make IGF-1, a protein that stimulates axon growth) or destructive (the cells make TNF-α, a protein that kills neurons), so studies of microglia/macrophage activation might suggest ways to treat spinal cord injuries. Another possible barrier to recovery is “chondroitin sulfate proteoglycan” (CSPG). This is a major component of the scar tissue (the “glial scar”) that forms around spinal cord injuries. CSPG limits axon regrowth, so attempts have been made to improve spinal cord repair by removing CSPG. But if CSPG prevents spinal cord repair, why is so much of it made immediately after an injury? In this study, the researchers investigate this paradox by asking whether CSPG made in the right place and in the right amount might have a beneficial role in spinal cord repair that has been overlooked.
What Did the Researchers Do and Find?
The researchers bruised a small section of the spinal cord of mice to cause hind limb paralysis, and then monitored the recovery of movement in these animals. They also examined the injured tissue microscopically, looked for microglia and infiltrating macrophages at the injury site, and measured the production of IGF-1 and TNF-α by these cells. Inhibition of CSPG synthesis immediately after injury impaired the functional recovery of the mice and increased tissue loss at the injury site. It also altered the spatial organization of infiltrating macrophages at the injury site, reduced IGF-1 production by these microglia/macrophages, and increased TNF-α levels. In contrast, when CSPG synthesis was not inhibited until two days after the injury, the mice recovered well from spinal cord injury. Furthermore, the interaction of CSPG with a cell-surface protein called CD44 activated microglia/macrophages growing in dishes and increased their production of IGF-1 but not of molecules that kill neurons.
What Do These Findings Mean?
These findings suggest that, immediately after a spinal cord injury, CSPG is needed for the repair of injured neurons and the recovery of movement, but that later on the presence of CSPG hinders repair. The findings also indicate that CSPG has these effects, at least in part, because it regulates the activity and localization of microglia and macrophages at the injury site and thus modulates local immune responses to the damage. Results obtained from experiments done in animals do not always accurately reflect the situation in people, so these findings need to be confirmed in patients with spinal cord injuries. However, they suggest that the effect of CSPG on spinal cord repair is not an inappropriate response to the injury, as is widely believed. Consequently, careful manipulation of CSPG levels might improve outcomes for people with spinal cord injuries.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0050171.
The MedlinePlus encyclopedia provides information about spinal cord injuries; MedlinePlus provides an interactive tutorial and a list of links to additional information about spinal cord injuries (in English and Spanish)
The US National Institute of Neurological Disorders and Stroke also provides information about spinal cord injury (in English and Spanish)
Wikipedia has a page on glial scars (note: Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
doi:10.1371/journal.pmed.0050171
PMCID: PMC2517615  PMID: 18715114
2.  Extensive Neuronal Differentiation of Human Neural Stem Cell Grafts in Adult Rat Spinal Cord 
PLoS Medicine  2007;4(2):e39.
Background
Effective treatments for degenerative and traumatic diseases of the nervous system are not currently available. The support or replacement of injured neurons with neural grafts, already an established approach in experimental therapeutics, has been recently invigorated with the addition of neural and embryonic stem-derived precursors as inexhaustible, self-propagating alternatives to fetal tissues. The adult spinal cord, i.e., the site of common devastating injuries and motor neuron disease, has been an especially challenging target for stem cell therapies. In most cases, neural stem cell (NSC) transplants have shown either poor differentiation or a preferential choice of glial lineages.
Methods and Findings
In the present investigation, we grafted NSCs from human fetal spinal cord grown in monolayer into the lumbar cord of normal or injured adult nude rats and observed large-scale differentiation of these cells into neurons that formed axons and synapses and established extensive contacts with host motor neurons. Spinal cord microenvironment appeared to influence fate choice, with centrally located cells taking on a predominant neuronal path, and cells located under the pia membrane persisting as NSCs or presenting with astrocytic phenotypes. Slightly fewer than one-tenth of grafted neurons differentiated into oligodendrocytes. The presence of lesions increased the frequency of astrocytic phenotypes in the white matter.
Conclusions
NSC grafts can show substantial neuronal differentiation in the normal and injured adult spinal cord with good potential of integration into host neural circuits. In view of recent similar findings from other laboratories, the extent of neuronal differentiation observed here disputes the notion of a spinal cord that is constitutively unfavorable to neuronal repair. Restoration of spinal cord circuitry in traumatic and degenerative diseases may be more realistic than previously thought, although major challenges remain, especially with respect to the establishment of neuromuscular connections.
When neural stem cells from human fetal spinal cord were grafted into the lumbar cord of normal or injured adult nude rats, substantial neuronal differentiation was found.
Editors' Summary
Background.
Every year, spinal cord injuries, many caused by road traffic accidents, paralyze about 11,000 people in the US. This paralysis occurs because the spinal cord is the main communication highway between the body and the brain. Information from the skin and other sensory organs is transmitted to the brain along the spinal cord by bundles of neurons, nervous system cells that transmit and receive messages. The brain then sends information back down the spinal cord to control movement, breathing, and other bodily functions. The bones of the spine normally protect the spinal cord but, if these are broken or dislocated, the spinal cord can be cut or compressed, which interrupts the information flow. Damage near the top of the spinal cord can paralyze the arms and legs (tetraplegia); damage lower down paralyzes the legs only (paraplegia). Spinal cord injuries also cause many other medical problems, including the loss of bowel and bladder control. Although the deleterious effects of spinal cord injuries can be minimized by quickly immobilizing the patient and using drugs to reduce inflammation, the damaged nerve fibers never regrow. Consequently, spinal cord injury is permanent.
Why Was This Study Done?
Scientists are currently searching for ways to reverse spinal cord damage. One potential approach is to replace the damaged neurons using neural stem cells (NSCs). These cells, which can be isolated from embryos and from some areas of the adult nervous system, are able to develop into all the specialized cells types of the nervous system. However, because most attempts to repair spinal cord damage with NSC transplants have been unsuccessful, many scientists believe that the environment of the spinal cord is unsuitable for nerve regeneration. In this study, the researchers have investigated what happens to NSCs derived from the spinal cord of a human fetus after transplantation into the spinal cord of adult rats.
What Did the Researchers Do and Find?
The researchers injected human NSCs that they had grown in dishes into the spinal cord of intact nude rats (animals that lack a functioning immune system and so do not destroy human cells) and into nude rats whose spinal cord had been damaged at the transplantation site. The survival and fate of the transplanted cells was assessed by staining thin slices of spinal cord with an antibody that binds to a human-specific protein and with antibodies that recognize proteins specific to NSCs, neurons, or other nervous system cells. The researchers report that the human cells survived well in the adult spinal cord of the injured and normal rats and migrated into the gray matter of the spinal cord (which contains neuronal cell bodies) and into the white matter (which contains the long extensions of nerve cells that carry nerve impulses). 75% and 60% of the human cells in the gray and white matter, respectively, contained a neuron-specific protein six months after transplantation but only 10% of those in the membrane surrounding the spinal cord became neurons; the rest developed into astrocytes (another nervous system cell type) or remained as stem cells. Finally, many of the human-derived neurons made the neurotransmitter GABA (one of the chemicals that transfers messages between neurons) and made contacts with host spinal cord neurons.
What Do These Findings Mean?
These findings suggest that human NSC grafts can, after all, develop into neurons (predominantly GABA-producing neurons) in normal and injured adult spinal cord and integrate into the existing spinal cord if the conditions are right. Although these animal experiments suggest that NSC transplants might help people with spinal injuries, they have some important limitations. For example, the spinal cord lesions used here are mild and unlike those seen in human patients. This and the use of nude rats might have reduced the scarring in the damaged spinal cord that is often a major barrier to nerve regeneration. Furthermore, the researchers did not test whether NSC transplants provide functional improvements after spinal cord injury. However, since other researchers have also recently reported that NSCs can grow and develop into neurons in injured adult spinal cord, these new results further strengthen hopes it might eventually be possible to use human NSCs to repair damaged spinal cords.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/doi:10.1371/journal.pmed.0040039.
The US National Institute of Neurological Disorders and Stroke provides information on spinal cord injury and current spinal cord research
Spinal Research (a UK charity) offers information on spinal cord injury and repair
The US National Spinal Cord Injury Association Web site contains factsheets on spinal cord injuries
MedlinePlus encyclopedia has pages on spinal cord trauma and interactive tutorials on spinal cord injury
The International Society for Stem Cell Research offers information on all sorts of stem cells including NSCs
The US National Human Neural Stem Cell Resource provides information on human NSCs, including the current US government's stance on stem cell research
doi:10.1371/journal.pmed.0040039
PMCID: PMC1796906  PMID: 17298165
3.  Templated Agarose Scaffolds for the Support of Motor Axon Regeneration Into Sites of Complete Spinal Cord Transection 
Biomaterials  2012;34(5):1529-1536.
Bioengineered scaffolds have the potential to support and guide injured axons after spinal cord injury, contributing to neural repair. In previous studies we have reported that templated agarose scaffolds can be fabricated into precise linear arrays and implanted into the partially injured spinal cord, organizing growth and enhancing the distance over which local spinal cord axons and ascending sensory axons extend into a lesion site. However, most human injuries are severe, sparing only thin rims of spinal cord tissue in the margins of a lesion site. Accordingly, in the present study we examined whether template agarose scaffolds seeded with bone marrow stromal cells secreting Brain-Derived Neurotrophic Factor (BDNF) would support regeneration into severe, complete spinal cord transection sites. Moreover, we tested responses of motor axon populations originating from the brainstem. We find that templated agarose scaffolds support motor axon regeneration into a severe spinal cord injury model and organize axons into fascicles of highly linear configuration. BDNF significantly enhances axonal growth. Collectively, these findings support the feasibility of scaffold implantation for enhancing central regeneration after even severe central nervous system injury.
doi:10.1016/j.biomaterials.2012.10.070
PMCID: PMC3518618  PMID: 23182350
4.  Comparison of polymer scaffolds in rat spinal cord: A step toward quantitative assessment of combinatorial approaches to spinal cord repair 
Biomaterials  2011;32(32):8077-8086.
The transected rat thoracic (T9/10) spinal cord model is a platform for quantitatively compa0ring biodegradable polymer scaffolds. Schwann cell-loaded scaffolds constructed from poly (lactic co-glycolic acid) (PLGA), poly(ε-caprolactone fumarate) (PCLF), oligo(polyethylene glycol) fumarate (OPF) hydrogel or positively charged OPF (OPF+) hydrogel were implanted into the model. We demonstrated that the mechanical properties (3-point bending and stiffness) of OPF and OPF+ hydrogels closely resembled rat spinal cord. After one month, tissues were harvested and analyzed by morphometry of neurofilament-stained sections at rostral, midlevel, and caudal scaffold. All polymers supported axonal growth. Significantly higher numbers of axons were found in PCLF (P < 0.01) and OPF+ (P < 0.05) groups, compared to that of the PLGA group. OPF+ polymers showed more centrally distributed axonal regeneration within the channels while other polymers (PLGA, PCLF and OPF) tended to show more evenly dispersed axons within the channels. The centralized distribution was associated with significantly more axons regenerating (P < 0.05). Volume of scar and cyst rostral and caudal to the implanted scaffold was measured and compared. There were significantly smaller cyst volumes in PLGA compared to PCLF groups. The model provides a quantitative basis for assessing individual and combined tissue engineering strategies.
doi:10.1016/j.biomaterials.2011.07.029
PMCID: PMC3163757  PMID: 21803415
OPF; PLGA; PCLF; axon regeneration; spinal cord injury; Schwann cell
5.  Neural Stem Cell– and Schwann Cell–Loaded Biodegradable Polymer Scaffolds Support Axonal Regeneration in the Transected Spinal Cord 
Tissue Engineering. Part A  2009;15(7):1797-1805.
Biodegradable polymer scaffolds provide an excellent approach to quantifying critical factors necessary for restoration of function after a transection spinal cord injury. Neural stem cells (NSCs) and Schwann cells (SCs) support axonal regeneration. This study examines the compatibility of NSCs and SCs with the poly-lactic-co-glycolic acid polymer scaffold and quantitatively assesses their potential to promote regeneration after a spinal cord transection injury in rats. NSCs were cultured as neurospheres and characterized by immunostaining for nestin (NSCs), glial fibrillary acidic protein (GFAP) (astrocytes), βIII-tubulin (immature neurons), oligodendrocyte-4 (immature oligodendrocytes), and myelin oligodendrocyte (mature oligodendrocytes), while SCs were characterized by immunostaining for S-100. Rats with transection injuries received scaffold implants containing NSCs (n = 17), SCs (n = 17), and no cells (control) (n = 8). The degree of axonal regeneration was determined by counting neurofilament-stained axons through the scaffold channels 1 month after transplantation. Serial sectioning through the scaffold channels in NSC- and SC-treated groups revealed the presence of nestin, neurofilament, S-100, and βIII tubulin–positive cells. GFAP-positive cells were only seen at the spinal cord–scaffold border. There were significantly more axons in the NSC- and SC- treated groups compared to the control group. In conclusion, biodegradable scaffolds with aligned columns seeded with NSCs or SCs facilitate regeneration across the transected spinal cord. Further, these multichannel biodegradable polymer scaffolds effectively serve as platforms for quantitative analysis of axonal regeneration.
doi:10.1089/ten.tea.2008.0364
PMCID: PMC2792101  PMID: 19191513
6.  Astrocytes derived from glial-restricted precursors promote spinal cord repair 
Journal of Biology  2006;5(3):7.
Background
Transplantation of embryonic stem or neural progenitor cells is an attractive strategy for repair of the injured central nervous system. Transplantation of these cells alone to acute spinal cord injuries has not, however, resulted in robust axon regeneration beyond the sites of injury. This may be due to progenitors differentiating to cell types that support axon growth poorly and/or their inability to modify the inhibitory environment of adult central nervous system (CNS) injuries. We reasoned therefore that pre-differentiation of embryonic neural precursors to astrocytes, which are thought to support axon growth in the injured immature CNS, would be more beneficial for CNS repair.
Results
Transplantation of astrocytes derived from embryonic glial-restricted precursors (GRPs) promoted robust axon growth and restoration of locomotor function after acute transection injuries of the adult rat spinal cord. Transplantation of GRP-derived astrocytes (GDAs) into dorsal column injuries promoted growth of over 60% of ascending dorsal column axons into the centers of the lesions, with 66% of these axons extending beyond the injury sites. Grid-walk analysis of GDA-transplanted rats with rubrospinal tract injuries revealed significant improvements in locomotor function. GDA transplantation also induced a striking realignment of injured tissue, suppressed initial scarring and rescued axotomized CNS neurons with cut axons from atrophy. In sharp contrast, undifferentiated GRPs failed to suppress scar formation or support axon growth and locomotor recovery.
Conclusion
Pre-differentiation of glial precursors into GDAs before transplantation into spinal cord injuries leads to significantly improved outcomes over precursor cell transplantation, providing both a novel strategy and a highly effective new cell type for repairing CNS injuries.
doi:10.1186/jbiol35
PMCID: PMC1561531  PMID: 16643674
7.  Transplantation of Specific Human Astrocytes Promotes Functional Recovery after Spinal Cord Injury 
PLoS ONE  2011;6(3):e17328.
Repairing trauma to the central nervous system by replacement of glial support cells is an increasingly attractive therapeutic strategy. We have focused on the less-studied replacement of astrocytes, the major support cell in the central nervous system, by generating astrocytes from embryonic human glial precursor cells using two different astrocyte differentiation inducing factors. The resulting astrocytes differed in expression of multiple proteins thought to either promote or inhibit central nervous system homeostasis and regeneration. When transplanted into acute transection injuries of the adult rat spinal cord, astrocytes generated by exposing human glial precursor cells to bone morphogenetic protein promoted significant recovery of volitional foot placement, axonal growth and notably robust increases in neuronal survival in multiple spinal cord laminae. In marked contrast, human glial precursor cells and astrocytes generated from these cells by exposure to ciliary neurotrophic factor both failed to promote significant behavioral recovery or similarly robust neuronal survival and support of axon growth at sites of injury. Our studies thus demonstrate functional differences between human astrocyte populations and suggest that pre-differentiation of precursor cells into a specific astrocyte subtype is required to optimize astrocyte replacement therapies. To our knowledge, this study is the first to show functional differences in ability to promote repair of the injured adult central nervous system between two distinct subtypes of human astrocytes derived from a common fetal glial precursor population. These findings are consistent with our previous studies of transplanting specific subtypes of rodent glial precursor derived astrocytes into sites of spinal cord injury, and indicate a remarkable conservation from rat to human of functional differences between astrocyte subtypes. In addition, our studies provide a specific population of human astrocytes that appears to be particularly suitable for further development towards clinical application in treating the traumatically injured or diseased human central nervous system.
doi:10.1371/journal.pone.0017328
PMCID: PMC3047562  PMID: 21407803
8.  Development of a tissue-engineered composite implant for treating traumatic paraplegia in rats 
European Spine Journal  2005;15(2):234-245.
This study was designed to assess a new composite implant to induce regeneration of injured spinal cord in paraplegic rats following complete cord transection. Neuronal xenogeneic cells from biopsies of adult nasal olfactory mucosa (NOM) of human origin, or spinal cords of human embryos, were cultured in two consecutive stages: stationary cultures in a viscous semi-solid gel (NVR-N-Gel) and in suspension on positively charged microcarriers (MCs). A tissue-engineered tubular scaffold, containing bundles of parallel nanofibers, was developed. Both the tube and the nanofibers were made of a biodegradable dextran sulphate–gelatin co-precipitate. The suturable scaffold anchored the implant at the site of injury and provided guidance for the regenerating axons. Implants of adult human NOM cells were implanted into eight rats, from which a 4 mm segment of the spinal cord had been completely removed. Another four rats whose spinal cords had also been transected were implanted with a composite implant of cultured human embryonic spinal cord cells. Eight other cord-transected rats served as a control group. Physiological and behavioral analysis, performed 3 months after implantation, revealed partial recovery of function in one or two limbs in three out of eight animals of the NOM implanted group and in all the four rats that were implanted with cultured human embryonic spinal cord cells. Animals of the control group remained completely paralyzed and did not show transmission of stimuli to the brain. The utilization of an innovative composite implant to bridge a gap resulting from the transection and removal of a 4 mm spinal cord segment shows promise, suggesting the feasibility of this approach for partial reconstruction of spinal cord lesions. Such an implant may serve as a vital bridging station in acute and chronic cases of paraplegia.
doi:10.1007/s00586-005-0981-8
PMCID: PMC3489403  PMID: 16292587
Olfactory mucosa; Spinal cord; Transection; Transplantation
9.  Evaluation of Biomaterials for Bladder Augmentation using Cystometric Analyses in Various Rodent Models 
Renal function and continence of urine are critically dependent on the proper function of the urinary bladder, which stores urine at low pressure and expels it with a precisely orchestrated contraction. A number of congenital and acquired urological anomalies including posterior urethral valves, benign prostatic hyperplasia, and neurogenic bladder secondary to spina bifida/spinal cord injury can result in pathologic tissue remodeling leading to impaired compliance and reduced capacity1. Functional or anatomical obstruction of the urinary tract is frequently associated with these conditions, and can lead to urinary incontinence and kidney damage from increased storage and voiding pressures2. Surgical implantation of gastrointestinal segments to expand organ capacity and reduce intravesical pressures represents the primary surgical treatment option for these disorders when medical management fails3. However, this approach is hampered by the limitation of available donor tissue, and is associated with significant complications including chronic urinary tract infection, metabolic perturbation, urinary stone formation, and secondary malignancy4,5.
Current research in bladder tissue engineering is heavily focused on identifying biomaterial configurations which can support regeneration of tissues at defect sites. Conventional 3-D scaffolds derived from natural and synthetic polymers such as small intestinal submucosa and poly-glycolic acid have shown some short-term success in supporting urothelial and smooth muscle regeneration as well as facilitating increased organ storage capacity in both animal models and in the clinic6,7. However, deficiencies in scaffold mechanical integrity and biocompatibility often result in deleterious fibrosis8, graft contracture9, and calcification10, thus increasing the risk of implant failure and need for secondary surgical procedures. In addition, restoration of normal voiding characteristics utilizing standard biomaterial constructs for augmentation cystoplasty has yet to be achieved, and therefore research and development of novel matrices which can fulfill this role is needed.
In order to successfully develop and evaluate optimal biomaterials for clinical bladder augmentation, efficacy research must first be performed in standardized animal models using detailed surgical methods and functional outcome assessments. We have previously reported the use of a bladder augmentation model in mice to determine the potential of silk fibroin-based scaffolds to mediate tissue regeneration and functional voiding characteristics.11,12 Cystometric analyses of this model have shown that variations in structural and mechanical implant properties can influence the resulting urodynamic features of the tissue engineered bladders11,12. Positive correlations between the degree of matrix-mediated tissue regeneration determined histologically and functional compliance and capacity evaluated by cystometry were demonstrated in this model11,12. These results therefore suggest that functional evaluations of biomaterial configurations in rodent bladder augmentation systems may be a useful format for assessing scaffold properties and establishing in vivo feasibility prior to large animal studies and clinical deployment. In the current study, we will present various surgical stages of bladder augmentation in both mice and rats using silk scaffolds and demonstrate techniques for awake and anesthetized cystometry.
doi:10.3791/3981
PMCID: PMC3486757  PMID: 22907252
Bioengineering; Issue 66; Medicine; Biomedical Engineering; Physiology; Silk; bladder tissue engineering; biomaterial; scaffold; matrix; augmentation; cystometry
10.  Combining Peripheral Nerve Grafts and Chondroitinase Promotes Functional Axonal Regeneration in the Chronically Injured Spinal Cord 
Because there currently is no treatment for spinal cord injury, most patients are living with long-standing injuries. Therefore, strategies aimed at promoting restoration of function to the chronically injured spinal cord have high therapeutic value. For successful regeneration, long-injured axons must overcome their poor intrinsic growth potential as well as the inhibitory environment of the glial scar established around the lesion site. Acutely injured axons that regenerate into growth-permissive peripheral nerve grafts (PNGs) reenter host tissue to mediate functional recovery if the distal graft– host interface is treated with chondroitinase ABC (ChABC) to cleave inhibitory chondroitin sulfate proteoglycans in the scar matrix. To determine whether a similar strategy is effective for a chronic injury, we combined grafting of a peripheral nerve into a highly relevant, chronic, cervical contusion site with ChABC treatment of the glial scar and glial cell line-derived neurotrophic factor (GDNF) stimulation of long-injured axons. We tested this combination in two grafting paradigms: (1) a peripheral nerve that was grafted to span a chronic injury site or (2) a PNG that bridged a chronic contusion site with a second, more distal injury site. Unlike GDNF–PBS treatment, GDNF–ChABC treatment facilitated axons to exit the PNG into host tissue and promoted some functional recovery. Electrical stimulation of axons in the peripheral nerve bridge induced c-Fos expression in host neurons, indicative of synaptic contact by regenerating fibers. Thus, our data demonstrate, for the first time, that administering ChABC to a distal graft interface allows for functional axonal regeneration by chronically injured neurons.
doi:10.1523/JNEUROSCI.3641-09.2009
PMCID: PMC2824589  PMID: 19940184
11.  Repair of injured spinal cord using biomaterial scaffolds and stem cells 
The loss of neurons and degeneration of axons after spinal cord injury result in the loss of sensory and motor functions. A bridging biomaterial construct that allows the axons to grow through has been investigated for the repair of injured spinal cord. Due to the hostility of the microenvironment in the lesion, multiple conditions need to be fulfilled to achieve improved functional recovery. A scaffold has been applied to bridge the gap of the lesion as contact guidance for axonal growth and to act as a vehicle to deliver stem cells in order to modify the microenvironment. Stem cells may improve functional recovery of the injured spinal cord by providing trophic support or directly replacing neurons and their support cells. Neural stem cells and mesenchymal stem cells have been seeded into biomaterial scaffolds and investigated for spinal cord regeneration. Both natural and synthetic biomaterials have increased stem cell survival in vivo by providing the cells with a controlled microenvironment in which cell growth and differentiation are facilitated. This optimal multi‒disciplinary approach of combining biomaterials, stem cells, and biomolecules offers a promising treatment for the injured spinal cord.
doi:10.1186/scrt480
PMCID: PMC4282172  PMID: 25157690
12.  Mesenchymal stromal cells integrate and form longitudinally-aligned layers when delivered to injured spinal cord via a novel fibrin scaffold 
Neuroscience Letters  2014;569(100):12-17.
Highlights
•MSCs can be delivered to injured spinal cord through use of a fibrin scaffold.•Scaffold-delivered MSCs form longitudinally-aligned layers over the lesion site.•Regenerating axons enter scaffold-delivered grafts and grow longitudinally.•MSCs delivered via injection orient perpendicular to the plane of the spinal cord.•Regenerating axons in injected grafts grow perpendicular to the plane of the cord.
Mesenchymal stromal cells (MSCs) have been shown to promote healing and regeneration in a number of CNS injury models and therefore there is much interest in the clinical use of these cells. For spinal cord injuries, a standard delivery method for MSCs is intraspinal injection, but this can result in additional injury and provides little control over how the cells integrate into the tissue. The present study examines the use of a novel fibrin scaffold as a new method of delivering MSCs to injured spinal cord. Use of the fibrin scaffold resulted in the formation of longitudinally-aligned layers of MSCs growing over the spinal cord lesion site. Host neurites were able to migrate into this MSC architecture and grow longitudinally. The length of the MSC bridge corresponded to the length of the fibrin scaffold. MSCs that were delivered via intraspinal injection were mainly oriented perpendicular to the plane of the spinal cord and remained largely restricted to the lesion site. Host neurites within the injected MSC graft were also oriented perpendicular to the plane of the spinal cord.
doi:10.1016/j.neulet.2014.03.023
PMCID: PMC4015360  PMID: 24680849
Spinal cord injury; Mesenchymal stromal cells; Cell scaffold; Fibrin; MSCs, mesenchymal stromal cells; EDC, N-(3-dimethylaminopropyl)-N-ethyl-carbodiimide hydrochloride; NHS, N-hydroxysuccinimide
13.  Allografts of the Acellular Sciatic Nerve and Brain-Derived Neurotrophic Factor Repair Spinal Cord Injury in Adult Rats 
PLoS ONE  2012;7(8):e42813.
Objective
We aimed to investigate whether an innovative growth factor-laden scaffold composed of acellular sciatic nerve (ASN) and brain-derived neurotrophic factor (BDNF) could promote axonal regeneration and functional recovery after spinal cord injury (SCI).
Methods
Following complete transection at the thoracic level (T9), we immediately transplanted the grafts between the stumps of the severed spinal cords. We evaluated the functional recovery of the hindlimbs of the operated rats using the BBB locomotor rating scale system every week. Eight weeks after surgery, axonal regeneration was examined using the fluorogold (FG) retrograde tracing method. Electrophysiological analysis was carried out to evaluate the improvement in the neuronal circuits. Immunohistochemistry was employed to identify local injuries and recovery.
Results
The results of the Basso-Beattie-Bresnahan (BBB) scale indicated that there was no significant difference between the individual groups. The FG retrograde tracing and electrophysiological analyses indicated that the transplantation of ASN-BDNF provided a permissive environment to support neuron regeneration.
Conclusion
The ASN-BDNF transplantation provided a promising therapeutic approach to promote axonal regeneration and recovery after SCI, and can be used as part of a combinatory treatment strategy for SCI management.
doi:10.1371/journal.pone.0042813
PMCID: PMC3429476  PMID: 22952613
14.  Neurosurgery concepts: Key perspectives on dendritic cell vaccines, metastatic tumor treatment, and radiosurgery 
Background:
This is a laboratory study to investigate the effect of adding brain-derived-neurotrophic factor (BDNF) in a poly (N-isopropylacrylamide-g-poly (ethylene glycol) scaffold and its effect on spinal cord injury in a rat model.
Methods:
This is a laboratory investigation of a spinal cord injury in a rat model. A dorsolateral funiculotomy was used to disrupt the dorsolateral funiculus and rubrospinal tract. Animals were then injected with either the scaffold polymer or scaffold polymer with BDNF. Postoperatively, motor functions were assessed with single pellet reach to grasp task, stair case reaching task and cylinder task. Histological study was also performed to look at extent of glial scar and axonal growth.
Results:
Animals received BDNF containing polymer had an increased recovery rate of fine motor function of forelimb, as assessed by stair case reaching task and single pellet reach to grasp task compared with control animals that received the polymer only. There is no significant difference in the glial scar formation. BDNF treated animals also had increased axon growth including increase in the number and length of the rubrospinal tract axons.
Conclusion:
BDNF delivered via a scaffold polymer results in increased recovery rate in forelimb motor function in an experimental model of spinal cord injury, possibly through a promotion of growth of axons of the rubrospinal tract.
doi:10.4103/2152-7806.149389
PMCID: PMC4310042  PMID: 25657859
Radiosurgery; Gamma Knife; metastasis; immunotherapy; glioblastoma; dens fracture; meningiomas
15.  Axon Regeneration through Scaffold into Distal Spinal Cord after Transection 
Journal of neurotrauma  2009;26(10):1759-1771.
We employed Fast Blue (FB) axonal tracing to determine the origin of regenerating axons after thoracic spinal cord transection injury in rats. Schwann cell (SC)-loaded, biodegradable, poly(lactic-co-glycolic acid) (PLGA) scaffolds were implanted after transection. Scaffolds loaded with solubilized basement membrane preparation (without SCs) were used for negative controls, and nontransected cords were positive controls. One or 2 months after injury and scaffold implantation, FB was injected 0–15 mm caudal or about 5 mm rostral to the scaffold. One week later, tissue was harvested and the scaffold and cord sectioned longitudinally (30 μm) on a cryostat. Trans-scaffold labeling of neuron cell bodies was identified with confocal microscopy in all cell-transplanted groups. Large (30–50 μm diameter) neuron cell bodies were predominantly labeled in the ventral horn region. Most labeled neurons were seen 1–10 mm rostral to the scaffold, although some neurons were also labeled in the cervical cord. Axonal growth occurred bidirectionally after cord transection, and axons regenerated up to 14 mm beyond the PLGA scaffolds and into distal cord. The extent of FB labeling was negatively correlated with distance from the injection site to the scaffold. Electron microscopy showed myelinated axons in the transverse sections of the implanted scaffold 2 months after implantation. The pattern of myelination, with extracellular collagen and basal lamina, was characteristic of SC myelination. Our results show that FB labeling is an effective way to measure the origin of regenerating axons.
doi:10.1089/neu.2008-0610
PMCID: PMC2763055  PMID: 19413501
axonal tracing; biodegradable polymers; Fast Blue; Schwann cells; spinal cord injury
16.  Axon Regeneration through Scaffold into Distal Spinal Cord after Transection 
Journal of Neurotrauma  2009;26(10):1759-1771.
Abstract
We employed Fast Blue (FB) axonal tracing to determine the origin of regenerating axons after thoracic spinal cord transection injury in rats. Schwann cell (SC)-loaded, biodegradable, poly(lactic-co-glycolic acid) (PLGA) scaffolds were implanted after transection. Scaffolds loaded with solubilized basement membrane preparation (without SCs) were used for negative controls, and nontransected cords were positive controls. One or 2 months after injury and scaffold implantation, FB was injected 0–15 mm caudal or about 5 mm rostral to the scaffold. One week later, tissue was harvested and the scaffold and cord sectioned longitudinally (30 μm) on a cryostat. Trans-scaffold labeling of neuron cell bodies was identified with confocal microscopy in all cell-transplanted groups. Large (30–50 μm diameter) neuron cell bodies were predominantly labeled in the ventral horn region. Most labeled neurons were seen 1–10 mm rostral to the scaffold, although some neurons were also labeled in the cervical cord. Axonal growth occurred bidirectionally after cord transection, and axons regenerated up to 14 mm beyond the PLGA scaffolds and into distal cord. The extent of FB labeling was negatively correlated with distance from the injection site to the scaffold. Electron microscopy showed myelinated axons in the transverse sections of the implanted scaffold 2 months after implantation. The pattern of myelination, with extracellular collagen and basal lamina, was characteristic of SC myelination. Our results show that FB labeling is an effective way to measure the origin of regenerating axons.
doi:10.1089/neu.2008.0610
PMCID: PMC2763055  PMID: 19413501
axonal tracing; biodegradable polymers; Fast Blue; Schwann cells; spinal cord injury
17.  Precursor Cell Biology and the Development of Astrocyte Transplantation Therapies: Lessons from Spinal Cord Injury 
Neurotherapeutics  2011;8(4):677-693.
This review summarizes current progress on development of astrocyte transplantation therapies for repair of the damaged central nervous system. Replacement of neurons in the injured or diseased central nervous system is currently one of the most popular therapeutic goals, but if neuronal replacement is attempted in the absence of appropriate supporting cells (astrocytes and oligodendrocytes), then the chances of restoring neurological functional are greatly reduced. Although the past 20 years have offered great progress on oligodendrocyte replacement therapies, astrocyte transplantation therapies have been both less explored and comparatively less successful. We have now developed successful astrocyte transplantation therapies by pre-differentiating glial restricted precursor (GRP) cells into a specific population of GRP cell-derived astrocytes (GDAs) by exposing the GRP cells to bone morphogenetic protein-4 (BMP) prior to transplantation. When transplanted into transected rat spinal cord, rat and human GDAsBMP promote extensive axonal regeneration, rescue neuronal cell survival, realign tissue structure, and restore behavior to pre-injury levels on a grid-walk analysis of volitional foot placement. Such benefits are not provided by GRP cells themselves, demonstrating that the lesion environment does not direct differentiation in a manner optimally beneficial for the restoration of function. Such benefits also are not provided by transplantation of a different population of astrocytes generated from GRP cells exposed to ciliary neurotrophic factor (GDAsCNTF), thus providing the first transplantation-based evidence of functional heterogeneity in astrocyte populations. Moreover, lessons learned from the study of rat cells are strongly predictive of outcomes using human cells. Thus, these studies provide successful strategies for the use of astrocyte transplantation therapies for restoration of function following spinal cord injury.
Electronic supplementary material
The online version of this article (doi:10.1007/s13311-011-0071-z) contains supplementary material, which is available to authorized users.
doi:10.1007/s13311-011-0071-z
PMCID: PMC3210359  PMID: 21918888
Glial-restricted precursor cells; Glial precursor cell-derived astrocyte; Spinal cord injury; Regeneration; Astrocyte transplantation therapy; Astrocyte heterogeneity
18.  Precursor Cell Biology and the Development of Astrocyte Transplantation Therapies: Lessons from Spinal Cord Injury 
Neurotherapeutics  2011;8(4):677-693.
This review summarizes current progress on development of astrocyte transplantation therapies for repair of the damaged central nervous system. Replacement of neurons in the injured or diseased central nervous system is currently one of the most popular therapeutic goals, but if neuronal replacement is attempted in the absence of appropriate supporting cells (astrocytes and oligodendrocytes), then the chances of restoring neurological functional are greatly reduced. Although the past 20 years have offered great progress on oligodendrocyte replacement therapies, astrocyte transplantation therapies have been both less explored and comparatively less successful. We have now developed successful astrocyte transplantation therapies by pre-differentiating glial restricted precursor (GRP) cells into a specific population of GRP cell-derived astrocytes (GDAs) by exposing the GRP cells to bone morphogenetic protein-4 (BMP) prior to transplantation. When transplanted into transected rat spinal cord, rat and human GDAsBMP promote extensive axonal regeneration, rescue neuronal cell survival, realign tissue structure, and restore behavior to pre-injury levels on a grid-walk analysis of volitional foot placement. Such benefits are not provided by GRP cells themselves, demonstrating that the lesion environment does not direct differentiation in a manner optimally beneficial for the restoration of function. Such benefits also are not provided by transplantation of a different population of astrocytes generated from GRP cells exposed to ciliary neurotrophic factor (GDAsCNTF), thus providing the first transplantation-based evidence of functional heterogeneity in astrocyte populations. Moreover, lessons learned from the study of rat cells are strongly predictive of outcomes using human cells. Thus, these studies provide successful strategies for the use of astrocyte transplantation therapies for restoration of function following spinal cord injury.
Electronic supplementary material
The online version of this article (doi:10.1007/s13311-011-0071-z) contains supplementary material, which is available to authorized users.
doi:10.1007/s13311-011-0071-z
PMCID: PMC3210359  PMID: 21918888
Glial-restricted precursor cells; Glial precursor cell-derived astrocyte; Spinal cord injury; Regeneration; Astrocyte transplantation therapy; Astrocyte heterogeneity
19.  Small molecule protein tyrosine phosphatase inhibition as a neuroprotective treatment following spinal cord injury in adult rats 
Spinal cord injury causes progressive secondary tissue degeneration leaving many injured people with neurological disabilities. There are no satisfactory neuroprotective treatments. Protein tyrosine phosphatases inactivate neurotrophic factor receptors and downstream intracellular signaling molecules. Thus, we tested whether the peroxovanadium compound bpV(phen), a stable, potent and selective protein tyrosine phosphatase inhibitor, would be neuroprotective following a thoracic spinal cord contusion in adult rats. Intrathecal bpV(phen) infusions through a lumbar puncture rescued dorsal column sensory axons innervating the nucleus gracilis and white matter at the injury epicenter. At the most effective dose, essentially all of these axons and most of the white matter at the epicenter were spared (vs. ~60% with control infusions). BpV(phen) treatments started 4 hours after contusion were fully effective. This treatment greatly improved and normalized sensory-motor function in a grid walking test and provided complete axonal protection over 6 weeks. The treatment rescued sensory evoked potentials which disappeared after dorsal column transection. BpV(phen) affected early degenerative mechanisms, as the main effects were seen at 7 days and lasted beyond the treatment period. The neuroprotection appeared to be mediated by rescue of blood vessels. BpV(phen) reduced apoptosis of cultured endothelial cells. These results show that a small molecule, used in a clinically relevant manner, reduces loss of long-projecting axons, myelin, blood vessels and function in a model relevant to the most common type of spinal cord injury in humans. They reveal a novel mechanism of spinal cord degeneration involving protein tyrosine phosphatases that can be targeted with therapeutic drugs.
doi:10.1523/JNEUROSCI.1826-08.2008
PMCID: PMC2678912  PMID: 18632933
axon; blood vessel; degeneration; myelin; neuroprotection; sensory
20.  Infiltrating Blood-Derived Macrophages Are Vital Cells Playing an Anti-inflammatory Role in Recovery from Spinal Cord Injury in Mice 
PLoS Medicine  2009;6(7):e1000113.
Using a mouse model of spinal injury, Michal Schwartz and colleagues tested the effect of macrophages on the recovery process and demonstrate an important anti-inflammatory role for a subset of infiltrating monocyte-derived macrophages that is dependent upon their expression of interleukin 10.
Background
Although macrophages (MΦ) are known as essential players in wound healing, their contribution to recovery from spinal cord injury (SCI) is a subject of debate. The difficulties in distinguishing between different MΦ subpopulations at the lesion site have further contributed to the controversy and led to the common view of MΦ as functionally homogenous. Given the massive accumulation in the injured spinal cord of activated resident microglia, which are the native immune occupants of the central nervous system (CNS), the recruitment of additional infiltrating monocytes from the peripheral blood seems puzzling. A key question that remains is whether the infiltrating monocyte-derived MΦ contribute to repair, or represent an unavoidable detrimental response. The hypothesis of the current study is that a specific population of infiltrating monocyte-derived MΦ is functionally distinct from the inflammatory resident microglia and is essential for recovery from SCI.
Methods and Findings
We inflicted SCI in adult mice, and tested the effect of infiltrating monocyte-derived MΦ on the recovery process. Adoptive transfer experiments and bone marrow chimeras were used to functionally distinguish between the resident microglia and the infiltrating monocyte-derived MΦ. We followed the infiltration of the monocyte-derived MΦ to the injured site and characterized their spatial distribution and phenotype. Increasing the naïve monocyte pool by either adoptive transfer or CNS-specific vaccination resulted in a higher number of spontaneously recruited cells and improved recovery. Selective ablation of infiltrating monocyte-derived MΦ following SCI while sparing the resident microglia, using either antibody-mediated depletion or conditional ablation by diphtheria toxin, impaired recovery. Reconstitution of the peripheral blood with monocytes resistant to ablation restored the lost motor functions. Importantly, the infiltrating monocyte-derived MΦ displayed a local anti-inflammatory beneficial role, which was critically dependent upon their expression of interleukin 10.
Conclusions
The results of this study attribute a novel anti-inflammatory role to a unique subset of infiltrating monocyte-derived MΦ in SCI recovery, which cannot be provided by the activated resident microglia. According to our results, limited recovery following SCI can be attributed in part to the inadequate, untimely, spontaneous recruitment of monocytes. This process is amenable to boosting either by active vaccination with a myelin-derived altered peptide ligand, which indicates involvement of adaptive immunity in monocyte recruitment, or by augmenting the naïve monocyte pool in the peripheral blood. Thus, our study sheds new light on the long-held debate regarding the contribution of MΦ to recovery from CNS injuries, and has potentially far-reaching therapeutic implications.
Please see later in the article for Editors' Summary
Editors' Summary
Background
Every year, spinal cord injuries paralyze about 11,000 people in the US. The spinal cord, which contains bundles of nervous system cells called neurons, is the communication highway between the brain and the body. Messages from the brain travel down the spinal cord to control movement, breathing and other bodily functions; messages from the skin and other sensory organs travel up the spinal cord to keep the brain informed about the body. The bones of the spine normally protect the spinal cord but, if these are broken or displaced, the spinal cord can be cut or compressed, which interrupts the information flow. Damage near the top of the spinal cord paralyzes the arms and legs (tetraplegia); damage lower down paralyzes the legs only (paraplegia). Spinal cord injuries also cause other medical problems, including the loss of bladder and bowel control. Currently, there is no effective treatment for spinal cord injuries, which usually cause permanent disability because the damaged nerve fibers rarely regrow.
Why Was This Study Done?
After a spinal cord injury, immune system cells called macrophages accumulate at the injury site. Some of these macrophages—so-called monocyte-derived macrophages—come into (infiltrate) the spinal cord from the blood in response to the injury, whereas others—microglia—are always in the nervous system. Although macrophages are essential for wound healing in other parts of the body, it is unclear whether they have good or bad effects in the spinal cord. Many experts believe that immune system cells hinder healing in the spinal cord and should be suppressed or eliminated, but other scientists claim that macrophages secrete factors that stimulate nerve regrowth. Furthermore, although some macrophages elsewhere in the body have proinflammatory (potentially deleterious) effects, others have anti-inflammatory (beneficial) effects. So do the infiltrating monocyte-derived macrophages and the resident microglia (which are proinflammatory) have different functions at spinal cord injury sites? In this study, the researchers try to answer this important question.
What Did the Researchers Do and Find?
The researchers bruised a small section of the spinal cord of adult mice and then investigated the effect of infiltrating monocyte-derived macrophages on the recovery process. Monocyte-derived macrophages and microglia cannot be distinguished using standard staining techniques so to study their behavior after spinal cord injury the researchers introduced labeled monocyte-derived macrophages into their experimental animals by using adoptive transfer (injection of genetically labeled monocytes into the animals) or by making bone marrow chimeras. In this second technique, the animals' monocyte-derived macrophages (but not their microglia) were killed by irradiating the animals before injection of genetically labeled bone marrow, the source of monocytes. Using these approaches, the researchers found that monocyte-derived macrophages collected at the margins of spinal cord injury sites whereas microglia accumulated throughout the sites. When the pool of monocyte-derived macrophages in the mice was increased by adoptive transfer or by using a technique called “CNS-specific vaccination,” more monocyte-derived macrophages infiltrated the injury site and the animals' physical recovery from injury improved. Conversely, removal of the infiltrating monocyte-derived macrophages from the injury site reduced the animals' physical recovery. Other experiments indicated that the infiltrating monocyte-derived macrophages have a beneficial, local anti-inflammatory effect that is dependent on their expression of interleukin-10 (an anti-inflammatory signaling molecule).
What Do These Findings Mean?
These findings provide new information about the contribution of monocyte-derived macrophages to spontaneous recovery from spinal cord injury, a contribution that has long been debated. In particular, the findings suggest that this subset of macrophages (but not the resident microglia) has a beneficial effect on spinal cord injuries that is mediated by their production of the anti-inflammatory molecule interleukin-10. The findings also show that the effect of these monocyte-derived macrophages can be boosted, at least in mice. Although results obtained in experiments done in animals do not always accurately reflect what happens in people, this new understanding of the different functions of microglia and infiltrating monocyte-derived macrophages after injury to the spinal cord may eventually lead to the development of better treatments for spinal cord injuries.
Additional Information
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1000113.
The MedlinePlus encyclopedia provides information about spinal cord injuries (in English and Spanish)
The US National Institute of Neurological Disorders and Stroke provides detailed information about spinal cord injury, including information on current research into the problem (in English and Spanish)
MedlinePlus provides an interactive tutorial on spinal cord injury and a list of links to additional information (in English and Spanish)
doi:10.1371/journal.pmed.1000113
PMCID: PMC2707628  PMID: 19636355
21.  Peripheral Nerve Grafts after Cervical Spinal Cord Injury in Adult Cats 
Experimental neurology  2010;225(1):173-182.
Peripheral nerve grafts (PNG) into the rat spinal cord support axon regeneration after acute or chronic injury, with synaptic reconnection across the lesion site and some level of behavioral recovery. Here, we grafted a peripheral nerve into the injured spinal cord of cats as a preclinical treatment approach to promote regeneration for eventual translational use. Adult female cats received a partial hemisection lesion at the cervical level (C7) and immediate apposition of an autologous tibial nerve segment to the lesion site. Five weeks later, a dorsal quadrant lesion was performed caudally (T1), the lesion site treated with Chondroitinase ABC two days later to digest growth inhibiting extracellular matrix molecules, and the distal end of the PNG apposed to the injury site. After 4–20 weeks, the grafts survived in 10/12 animals with several thousand myelinated axons present in each graft. The distal end of 9/10 grafts was well apposed to the spinal cord and numerous axons extended beyond the lesion site. Intraspinal stimulation evoked compound action potentials in the graft with an appropriate latency illustrating normal axonal conduction of the regenerated axons. Although stimulation of the PNG failed to elicit responses in the spinal cord distal to the lesion site, the presence of c-Fos immunoreactive neurons close to the distal apposition site indicates that regenerated axons formed functional synapses with host neurons. This study demonstrates the successful application of a nerve grafting approach to promote regeneration after spinal cord injury in a non-rodent, large animal model.
doi:10.1016/j.expneurol.2010.06.011
PMCID: PMC2922456  PMID: 20599980
spinal cord injury; regeneration; peripheral nerve graft; cat; c-Fos; chondroitinase
22.  Rigid Fixation of the Spinal Column Improves Scaffold Alignment and Prevents Scoliosis in the Transected Rat Spinal Cord 
Spine  2008;33(24):E914-E919.
Study Design
A controlled study to evaluate a new technique for spinal rod fixation after spinal cord injury in rats. Alignment of implanted tissue-engineered scaffolds was assessed radiographically and by magnetic resonance imaging.
Objective
To evaluate the stability of implanted scaffolds and the extent of kyphoscoliotic deformities after spinal fixation.
Summary of Background Data
Biodegradable scaffolds provide an excellent platform for the quantitative assessment of cellular and molecular factors that promote regeneration within the transected cord. Successful delivery of scaffolds to the damaged cord can be hampered by malalignment following transplantation, which in turn, hinders the assessment of neural regeneration.
Methods
Radio-opaque barium sulfate-impregnated poly-lactic-co-glycolic acid scaffolds were implanted into spinal transection injuries in adult rats. Spinal fixation was performed in one group of animals using a metal rod fixed to the spinous processes above and below the site of injury, while the control group received no fixation. Radiographic morphometry was performed after 2 and 4 weeks, and 3-dimensional magnetic resonance microscopy analysis 4 weeks after surgery.
Results
Over the course of 4 weeks, progressive scoliosis was evident in the unfixed group, where a Cobb angle of 8.13 ± 2.03° was measured. The fixed group demonstrated significantly less scoliosis, with a Cobb angle measurement of 1.89 ± 0.75° (P = 0.0004). Similarly, a trend for less kyphosis was evident in the fixed group (7.33 ± 1.68°) compared with the unfixed group (10.13 ± 1.46°). Quantitative measurements of the degree of malalignment of the scaffolds were also significantly less in the fixed group (5 ± 1.23°) compared with the unfixed group (11 ± 2.82°) (P = 0.0143).
Conclusion
Radio-opaque barium sulfate allows for visualization of scaffolds in vivo using radiographic analysis. Spinal fixation was shown to prevent scoliosis, reduce kyphosis, and reduce scaffold malalignment within the transected rat spinal cord. Using a highly optimized model will increase the potential for finding a therapy for restoring function to the injured cord.
doi:10.1097/BRS.0b013e318186b2b1
PMCID: PMC2773001  PMID: 19011531
spine fixation; transection spinal cord injury; scaffold; scoliosis
23.  Transection method for shortening the rat spine and spinal cord 
Previous studies have presented evidence which indicates that the regeneration of axons in the spinal cord occurs following spinal cord transection in young rats. However, in a transection-regeneration model, the completeness of the transection is often a matter of dispute. We established a method for shortening the rat spine and spinal cord to provide a spinal cord injury (SCI) model in which there was no doubt about whether the axonal transection was complete. In the future, this model may be applied to the chronic period of complete paralysis following SCI. Adult, female Wistar rats (220–250g) were used in the study. The spinal cord was exposed and a 4-mm-long segment of the spinal cord was removed at Th8. Subsequently, the Th7/8 and Th8/9 discs were cut between the stumps of the spinal cord to remove the Th8 vertebra. The stitches which had been passed through the 7th and 9th ribs bilaterally were tied gradually to bring together the stumps of the spinal cord. Almost all the rats survived until the end of the experiment. Uncoordinated movements of the hind limbs in locomotion were observed at 4 weeks after surgery. However coordinated movements of the hind limbs in locomotion were not observed until the end of the experiment. After 12 weeks, an intracardiac perfusion was performed to remove the thoracic spine and the spinal cord. There were no signs of infection. The bone fusion of the Th7 and Th9 vertebrae was observed to be complete in all specimens and the alignment of the thoracic spine was maintained. The spinal canal was also correctly reconstituted. The stumps of the spinal cord were connected. Light microscopy of the cord showed that scar tissue intervened at the connection site. Cavitation inhibiting the axonal regeneration was also observed. This model was also made on the assumption that glial scar tissue inhibits axonal regeneration in chronic SCI. Axonal regeneration was not observed across the transected spinal cord in this model. Attempts should be made to minimize the damage to the spinal cord and the surgery time for successful axonal regeneration to occur. The model developed in this study may be useful in the study of axonal regeneration in SCI.
doi:10.3892/etm.2012.841
PMCID: PMC3570119  PMID: 23403404
spinal cord injury; animal model; rat
24.  LAR is a functional receptor for CSPG axon growth inhibitors 
CSPGs (chondroitin sulfate proteoglycans) are a family of extracellular matrix molecules with various functions in regulating tissue morphogenesis, cell division and axon guidance. A number of CSPGs are highly upregulated by reactive glial scar tissues after injuries and form a strong barrier for axonal regeneration in the adult vertebrate CNS. Although CSPGs may negatively regulate axonal growth via binding and altering activity of other growth-regulating factors, the molecular mechanisms by which CSPGs restrict axonal elongation are not well understood. Here, we identified a novel receptor mechanism whereby CSPGs inhibit axonal growth via interactions with neuronal transmembrane LAR (the leukocyte common antigen-related phosphatase). CSPGs bind LAR with high affinity in transfected COS-7 cells and co-immunoprecipitate with LAR expressed in various tissues including the brain and spinal cord. CSPG stimulation enhances activity of LAR phosphatase in vitro. Deletion of LAR in knockout mice or blockade of LAR with sequence-selective peptides significantly overcomes neurite growth restrictions of CSPGs in neuronal cultures. Intracellularly, CSPG-LAR interaction mediates axonal growth inhibition of neurons partially via inactivating Akt and activating RhoA signals. Systemic treatments with LAR-targeting peptides in mice with thoracic spinal cord transection injuries induce significant axon growth of descending serotonergic fibers in the vicinity of the lesion and beyond in the caudal spinal cord and promote locomotor functional recovery. Identification of LAR as a novel CSPG functional receptor provides a therapeutic basis for enhancing axonal regeneration and functional recovery after CNS injuries in adult mammals.
doi:10.1523/JNEUROSCI.1737-11.2011
PMCID: PMC3220601  PMID: 21976490
Chondroitin sulfate proteoglycans; LAR; receptor; axon regeneration; spinal cord injury
25.  Meningeal cells and glia establish a permissive environment for axon regeneration after spinal cord injury in newts 
Neural Development  2011;6:1.
Background
Newts have the remarkable ability to regenerate their spinal cords as adults. Their spinal cords regenerate with the regenerating tail after tail amputation, as well as after a gap-inducing spinal cord injury (SCI), such as a complete transection. While most studies on newt spinal cord regeneration have focused on events occurring after tail amputation, less attention has been given to events occurring after an SCI, a context that is more relevant to human SCI. Our goal was to use modern labeling and imaging techniques to observe axons regenerating across a complete transection injury and determine how cells and the extracellular matrix in the injury site might contribute to the regenerative process.
Results
We identify stages of axon regeneration following a spinal cord transection and find that axon regrowth across the lesion appears to be enabled, in part, because meningeal cells and glia form a permissive environment for axon regeneration. Meningeal and endothelial cells regenerate into the lesion first and are associated with a loose extracellular matrix that allows axon growth cone migration. This matrix, paradoxically, consists of both permissive and inhibitory proteins. Axons grow into the injury site next and are closely associated with meningeal cells and glial processes extending from cell bodies surrounding the central canal. Later, ependymal tubes lined with glia extend into the lesion as well. Finally, the meningeal cells, axons, and glia move as a unit to close the gap in the spinal cord. After crossing the injury site, axons travel through white matter to reach synaptic targets, and though ascending axons regenerate, sensory axons do not appear to be among them. This entire regenerative process occurs even in the presence of an inflammatory response.
Conclusions
These data reveal, in detail, the cellular and extracellular events that occur during newt spinal cord regeneration after a transection injury and uncover an important role for meningeal and glial cells in facilitating axon regeneration. Given that these cell types interact to form inhibitory barriers in mammals, identifying the mechanisms underlying their permissive behaviors in the newt will provide new insights for improving spinal cord regeneration in mammals.
doi:10.1186/1749-8104-6-1
PMCID: PMC3025934  PMID: 21205291

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