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1.  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
2.  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
3.  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
4.  Early Response of Endogenous Adult Neural Progenitor Cells to Acute Spinal Cord Injury in Mice 
Stem cells (Dayton, Ohio)  2005;24(4):1011-1019.
Adult neural progenitor cells (NPCs) are an attractive source for functional replacement in neurodegenerative diseases and traumatic central nervous systems (CNS) injury. It has been shown that transplantation of neural stem cells or NPCs into the lesioned region partially restores CNS function. However, the capacity of endogenous NPCs in neuronal replacement and functional recovery of spinal cord injury (SCI) is apparently poor. More significantly, the early response of endogenous adult NPCs to SCI remains largely undefined. A comprehensive understanding of the temporal and spatial responses of NPCs to SCI is essential for the development of effective NPC therapy to restore neurological function. To this end, we have analyzed the early organization, distribution, and potential function of NPCs in response to SCI using nestin promoter controlled LacZ reporter transgenic mice. We showed that there was an increase of NPC proliferation, migration, and neurogenesis in adult spinal cord after traumatic compression SCI. The proliferation of NPCs detected by BrdU incorporation and LacZ staining was restricted to the ependymal zone (EZ) of the central canal. During acute SCI, NPCs in the EZ of the central canal migrated vigorously toward the dorsal direction, where the compression lesion is generated. The optimal NPC migration occurred in the adjacent region close to the epicenter. More significantly, there was an increased de novo neurogenesis from NPCs 24 hours after SCI. The enhanced proliferation, migration, and neurogenesis of (from) endogenous NPCs in the adult spinal cord in response to SCI suggest a potential role for NPCs in attempting to restore SCI-mediated neuronal dysfunction.
doi:10.1634/stemcells.2005-0249
PMCID: PMC1857304  PMID: 16339643
Neural progenitor cells; Neurogenesis; Astrogenesis; Spinal cord Injury; Nestin; Radial glia
5.  ADAM8 is selectively upregulated in endothelial cells and is associated with angiogenesis after spinal cord injury in adult mice 
Endothelial cell (EC) loss and subsequent angiogenesis occurs over the first week after spinal cord injury (SCI). To identify molecular mechanisms that could be targeted with intravenous (i.v.) treatments we determined whether transmembrane A Disintegrin And Metalloprotease (ADAM) proteins are expressed in ECs of the injured spinal cord. ADAMs bind to integrins which are important for EC survival and angiogenesis. Female adult C57Bl/6 mice with a spinal cord contusion had progressively more ADAM8 (CD156) immunostaining in blood vessels and individual ECs between 1 and 28 days following injury. Uninjured spinal cords had little ADAM8 staining. The increase in ADAM8 mRNA and protein was confirmed in spinal cord lysates, and ADAM8 mRNA was present in FACS-enriched ECs. ADAM8 co-localized extensively and exclusively with the EC marker PECAM and also with i.v. injected lectins. I.v. injected isolectin B4 (IB4) labels a subpopulation of blood vessels at and within the injury epicenter 3-7 days after injury, coincident with angiogenesis. Both ADAM8 and the proliferation marker Ki-67 were present in IB4-positive microvessels. ADAM8-positive proliferating cells were seen at the leading end of IB4-positive blood vessels. Angiogenesis was confirmed by BrdU incorporation, binding of i.v. injected nucleolin antibodies, and MT1-MMP immunostaining in a subset of blood vessels. These data suggest that ADAM8 is vascular-selective and plays a role in proliferation and/or migration of ECs during angiogenesis following SCI.
doi:10.1002/cne.21902
PMCID: PMC2746684  PMID: 19003792
angiogenesis; blood vessel; contusion; disintegrin; metalloprotease; neovascularization; nucleolin; proliferation
6.  Ex Vivo VEGF Delivery by Neural Stem Cells Enhances Proliferation of Glial Progenitors, Angiogenesis, and Tissue Sparing after Spinal Cord Injury 
PLoS ONE  2009;4(3):e4987.
The present study was undertaken to examine multifaceted therapeutic effects of vascular endothelial growth factor (VEGF) in a rat spinal cord injury (SCI) model, focusing on its capability to stimulate proliferation of endogenous glial progenitor cells. Neural stem cells (NSCs) can be genetically modified to efficiently transfer therapeutic genes to diseased CNS. We adopted an ex vivo approach using immortalized human NSC line (F3 cells) to achieve stable and robust expression of VEGF in the injured spinal cord. Transplantation of NSCs retrovirally transduced to overexpress VEGF (F3.VEGF cells) at 7 days after contusive SCI markedly elevated the amount of VEGF in the injured spinal cord tissue compared to injection of PBS or F3 cells without VEGF. Concomitantly, phosphorylation of VEGF receptor flk-1 increased in F3.VEGF group. Stereological counting of BrdU+ cells revealed that transplantation of F3.VEGF significantly enhanced cellular proliferation at 2 weeks after SCI. The number of proliferating NG2+ glial progenitor cells (NG2+/BrdU+) was also increased by F3.VEGF. Furthermore, transplantation of F3.VEGF increased the number of early proliferating cells that differentiated into mature oligodendrocytes, but not astrocytes, at 6 weeks after SCI. F3.VEGF treatment also increased the density of blood vessels in the injured spinal cord and enhanced tissue sparing. These anatomical results were accompanied by improved BBB locomotor scores. The multifaceted effects of VEGF on endogenous gliogenesis, angiogenesis, and tissue sparing could be utilized to improve functional outcomes following SCI.
doi:10.1371/journal.pone.0004987
PMCID: PMC2656622  PMID: 19319198
7.  Chondroitinase and Growth Factors Enhance Activation and Oligodendrocyte Differentiation of Endogenous Neural Precursor Cells after Spinal Cord Injury 
PLoS ONE  2012;7(5):e37589.
The adult spinal cord harbours a population of multipotent neural precursor cells (NPCs) with the ability to replace oligodendrocytes. However, despite this capacity, proliferation and endogenous remyelination is severely limited after spinal cord injury (SCI). In the post-traumatic microenvironment following SCI, endogenous spinal NPCs mainly differentiate into astrocytes which could contribute to astrogliosis that exacerbate the outcomes of SCI. These findings emphasize a key role for the post-SCI niche in modulating the behaviour of spinal NPCs after SCI. We recently reported that chondroitin sulphate proteoglycans (CSPGs) in the glial scar restrict the outcomes of NPC transplantation in SCI by reducing the survival, migration and integration of engrafted NPCs within the injured spinal cord. These inhibitory effects were attenuated by administration of chondroitinase (ChABC) prior to NPC transplantation. Here, in a rat model of compressive SCI, we show that perturbing CSPGs by ChABC in combination with sustained infusion of growth factors (EGF, bFGF and PDGF-AA) optimize the activation and oligodendroglial differentiation of spinal NPCs after injury. Four days following SCI, we intrathecally delivered ChABC and/or GFs for seven days. We performed BrdU incorporation to label proliferating cells during the treatment period after SCI. This strategy increased the proliferation of spinal NPCs, reduced the generation of new astrocytes and promoted their differentiation along an oligodendroglial lineage, a prerequisite for remyelination. Furthermore, ChABC and GF treatments enhanced the response of non-neural cells by increasing the generation of new vascular endothelial cells and decreasing the number of proliferating macrophages/microglia after SCI. In conclusions, our data strongly suggest that optimization of the behaviour of endogenous spinal NPCs after SCI is critical not only to promote endogenous oligodendrocyte replacement, but also to reverse the otherwise detrimental effects of their activation into astrocytes which could negatively influence the repair process after SCI.
doi:10.1371/journal.pone.0037589
PMCID: PMC3358255  PMID: 22629425
8.  Delayed Administration of a Bio-Engineered Zinc-Finger VEGF-A Gene Therapy Is Neuroprotective and Attenuates Allodynia Following Traumatic Spinal Cord Injury 
PLoS ONE  2014;9(5):e96137.
Following spinal cord injury (SCI) there are drastic changes that occur in the spinal microvasculature, including ischemia, hemorrhage, endothelial cell death and blood-spinal cord barrier disruption. Vascular endothelial growth factor-A (VEGF-A) is a pleiotropic factor recognized for its pro-angiogenic properties; however, VEGF has recently been shown to provide neuroprotection. We hypothesized that delivery of AdV-ZFP-VEGF – an adenovirally delivered bio-engineered zinc-finger transcription factor that promotes endogenous VEGF-A expression – would result in angiogenesis, neuroprotection and functional recovery following SCI. This novel VEGF gene therapy induces the endogenous production of multiple VEGF-A isoforms; a critical factor for proper vascular development and repair. Briefly, female Wistar rats – under cyclosporin immunosuppression – received a 35 g clip-compression injury and were administered AdV-ZFP-VEGF or AdV-eGFP at 24 hours post-SCI. qRT-PCR and Western Blot analysis of VEGF-A mRNA and protein, showed significant increases in VEGF-A expression in AdV-ZFP-VEGF treated animals (p<0.001 and p<0.05, respectively). Analysis of NF200, TUNEL, and RECA-1 indicated that AdV-ZFP-VEGF increased axonal preservation (p<0.05), reduced cell death (p<0.01), and increased blood vessels (p<0.01), respectively. Moreover, AdV-ZFP-VEGF resulted in a 10% increase in blood vessel proliferation (p<0.001). Catwalk™ analysis showed AdV-ZFP-VEGF treatment dramatically improves hindlimb weight support (p<0.05) and increases hindlimb swing speed (p<0.02) when compared to control animals. Finally, AdV-ZFP-VEGF administration provided a significant reduction in allodynia (p<0.01). Overall, the results of this study indicate that AdV-ZFP-VEGF administration can be delivered in a clinically relevant time-window following SCI (24 hours) and provide significant molecular and functional benefits.
doi:10.1371/journal.pone.0096137
PMCID: PMC4028194  PMID: 24846143
9.  Sildenafil Improves Epicenter Vascular Perfusion but not Hindlimb Functional Recovery after Contusive Spinal Cord Injury in Mice 
Journal of Neurotrauma  2012;29(3):528-538.
Abstract
Nitric oxide (NO) is an important regulator of vasodilation and angiogenesis in the central nervous system (CNS). Signaling initiated by the membrane receptor CD47 antagonizes vasodilation and angiogenesis by inhibiting synthesis of cyclic guanosine monophosphate (cGMP). We recently found that deletion of CD47 led to significant functional locomotor improvements, enhanced angiogenesis, and increased epicenter microvascular perfusion in mice after moderate contusive spinal cord injury (SCI). We tested the hypothesis that improving NO/cGMP signaling within the spinal cord immediately after injury would increase microvascular perfusion, angiogenesis, and functional recovery, with an acute, 7-day administration of the cGMP phosphodiesterase 5 (PDE5) inhibitor sildenafil. PDE5 expression is localized within spinal cord microvascular endothelial cells and smooth muscle cells. While PDE5 antagonism has been shown to increase angiogenesis in a rat embolic stroke model, sildenafil had no significant effect on angiogenesis at 7 days post-injury after murine contusive SCI. Sildenafil treatment increased cGMP concentrations within the spinal cord and improved epicenter microvascular perfusion. Basso Mouse Scale (BMS) and Treadscan analyses revealed that sildenafil treatment had no functional consequence on hindlimb locomotor recovery. These data support the hypothesis that acutely improving microvascular perfusion within the injury epicenter by itself is an insufficient strategy for improving functional deficits following contusive SCI.
doi:10.1089/neu.2011.2036
PMCID: PMC3278821  PMID: 21970599
microvasculature; nitric oxide; phosphodiesterase 5; sildenafil; spinal cord injury
10.  Neural Progenitor Cell Implants Modulate Vascular Endothelial Growth Factor and Brain-Derived Neurotrophic Factor Expression in Rat Axotomized Neurons 
PLoS ONE  2013;8(1):e54519.
Axotomy of central neurons leads to functional and structural alterations which largely revert when neural progenitor cells (NPCs) are implanted in the lesion site. The new microenvironment created by NPCs in the host tissue might modulate in the damaged neurons the expression of a high variety of molecules with relevant roles in the repair mechanisms, including neurotrophic factors. In the present work, we aimed to analyze changes in neurotrophic factor expression in axotomized neurons induced by NPC implants. For this purpose, we performed immunofluorescence followed by confocal microscopy analysis for the detection of vascular endothelial growth factor (VEGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and nerve growth factor (NGF) on brainstem sections from rats with axotomy of abducens internuclear neurons that received NPC implants (implanted group) or vehicle injections (axotomized group) in the lesion site. Control abducens internuclear neurons were strongly immunoreactive to VEGF and BDNF but showed a weak staining for NT-3 and NGF. Comparisons between groups revealed that lesioned neurons from animals that received NPC implants showed a significant increase in VEGF content with respect to animals receiving vehicle injections. However, the immunoreactivity for BDNF, which was increased in the axotomized group as compared to control, was not modified in the implanted group. The modifications induced by NPC implants on VEGF and BDNF content were specific for the population of axotomized abducens internuclear neurons since the neighboring abducens motoneurons were not affected. Similar levels of NT-3 and NGF immunolabeling were obtained in injured neurons from axotomized and implanted animals. Among all the analyzed neurotrophic factors, only VEGF was expressed by the implanted cells in the lesion site. Our results point to a role of NPC implants in the modulation of neurotrophic factor expression by lesioned central neurons, which might contribute to the restorative effects of these implants.
doi:10.1371/journal.pone.0054519
PMCID: PMC3548797  PMID: 23349916
11.  Pre-Evaluated Safe Human iPSC-Derived Neural Stem Cells Promote Functional Recovery after Spinal Cord Injury in Common Marmoset without Tumorigenicity 
PLoS ONE  2012;7(12):e52787.
Murine and human iPSC-NS/PCs (induced pluripotent stem cell-derived neural stem/progenitor cells) promote functional recovery following transplantation into the injured spinal cord in rodents. However, for clinical applicability, it is critical to obtain proof of the concept regarding the efficacy of grafted human iPSC-NS/PCs (hiPSC-NS/PCs) for the repair of spinal cord injury (SCI) in a non-human primate model. This study used a pre-evaluated “safe” hiPSC-NS/PC clone and an adult common marmoset (Callithrix jacchus) model of contusive SCI. SCI was induced at the fifth cervical level (C5), followed by transplantation of hiPSC-NS/PCs at 9 days after injury. Behavioral analyses were performed from the time of the initial injury until 12 weeks after SCI. Grafted hiPSC-NS/PCs survived and differentiated into all three neural lineages. Furthermore, transplantation of hiPSC-NS/PCs enhanced axonal sparing/regrowth and angiogenesis, and prevented the demyelination after SCI compared with that in vehicle control animals. Notably, no tumor formation occurred for at least 12 weeks after transplantation. Quantitative RT-PCR showed that mRNA expression levels of human neurotrophic factors were significantly higher in cultured hiPSC-NS/PCs than in human dermal fibroblasts (hDFs). Finally, behavioral tests showed that hiPSC-NS/PCs promoted functional recovery after SCI in the common marmoset. Taken together, these results indicate that pre-evaluated safe hiPSC-NS/PCs are a potential source of cells for the treatment of SCI in the clinic.
doi:10.1371/journal.pone.0052787
PMCID: PMC3531369  PMID: 23300777
12.  The temporal and spatial profiles of cell loss following experimental spinal cord injury: effect of antioxidant therapy on cell death and functional recovery 
BMC Neuroscience  2013;14:146.
Background
Traumatic spinal cord injury (SCI)-induced overproduction of endogenous deleterious substances triggers secondary cell death to spread damage beyond the initial injury site. Substantial experimental evidence supports reactive species (RS) as important mediators of secondary cell death after SCI. This study established quantitative temporal and spatial profiles of cell loss, characterized apoptosis, and evaluated the effectiveness of a broad spectrum RS scavenger - Mn (III) tetrakis (4-benzoic acid) porphyrin (MnTBAP) and a combination of MnTBAP plus nitro-L-arginine to prevent cell loss and neurological dysfunction following contusion SCI to the rat spinal cord.
Results
By counting the number of surviving cells in spinal cord sections removed at 1, 6, 12, 24, 48, 72 h and 1 week post-SCI and at 0 – 4 mm from the epicenter, the temporal and spatial profiles of motoneuron and glia loss were established. Motoneurons continued to disappear over a week and the losses decreased with increasing distance from the epicenter. Significant glia loss peaked at 24 to 48 h post-SCI, but only at sections 0–1.5 mm from the epicenter. Apoptosis of neurons, motoneurons and astrocytes was characterized morphologically by double immuno-staining with cell-specific markers and apoptosis indicators and confirmed by transmission electron microscopy. DNA laddering, ELISA quantitation and caspase-3 activation in the spinal cord tissue indicated more intense DNA fragments and greater caspase-3 activation in the epicenter than at 1 and 2 cm away from the epicenter or the sham-operated sections. Intraperitoneal treatment with MnTBAP + nitro-L-arginine significantly reduced motoneuron and cell loss and apoptosis in the gray and white matter compared with the vehicle-treated group. MnTBAP alone significantly reduced the number of apoptotic cells and improved functional recovery as evaluated by three behavioral tests.
Conclusions
Our temporal and spatial profiles of cell loss provide data bases for determining the time and location for pharmacological intervention. Our demonstration that apoptosis follows SCI and that MnTBAP alone or MnTBAP + nitro-L-arginine significantly reduces apoptosis correlates SCI-induced apoptosis with RS overproduction. MnTBAP significantly improved functional recovery, which strongly supports the important role of antioxidant therapy in treating SCI and the candidacy of MnTBAP for such treatment.
doi:10.1186/1471-2202-14-146
PMCID: PMC3924333  PMID: 24238557
Antioxidant therapy; Apoptotic cell death; Behavioral test; Mn (III) tetrakis (4-benzoic acid) porphyrin; Nitro-L-arginine; Secondary spinal cord injury
13.  Fibrin-based tissue engineering scaffolds enhance neural fiber sprouting and delays the accumulation of reactive astrocytes at the lesion in a subacute model of spinal cord injury† 
The purpose of this study was to evaluate the effects of fibrin scaffolds on subacute rat spinal cord injury (SCI). Long Evans rats were anesthetized and underwent a dorsal hemisection injury, two weeks later the injury site was re-exposed, scar tissue was removed, and a fibrin scaffold was implanted into the wound site. An effective method for fibrin scaffold implantation following subacute SCI was investigated based on the presence of fibrin within the lesion site and morphological analysis 1 week after implantation. Pre-polymerized fibrin scaffolds were found to be present within the lesion site 1 week after treatment and were used for the remainder of the study. Fibrin scaffolds were then implanted for 2 and 4 weeks, after which spinal cords were harvested and evaluated using markers for neurons, astrocytes, and chondroitin sulfate proteoglycans. Compared to untreated control, the fibrin-treated group had significantly higher levels of neural fiber staining in the lesion site at 2 and 4 weeks after treatment, and the accumulation of glial fibrillary acidic protein (GFAP) positive reactive astrocytes surrounding the lesion was delayed. These results show that fibrin is conducive to regeneration and cellular migration, and illustrates the advantage of using fibrin as a scaffold for drug delivery and cell-based therapies for SCI.
doi:10.1002/jbm.a.32343
PMCID: PMC2787862  PMID: 19165795
nerve regeneration; glial scar; CNS scaffold; spinal cord regeneration
14.  A Method to Assess Target Gene Involvement in Angiogenesis In Vitro and In Vivo Using Lentiviral Vectors Expressing shRNA 
PLoS ONE  2014;9(4):e96036.
Current methods to study angiogenesis in cancer growth and development can be difficult and costly, requiring extensive use of in vivo methodologies. Here, we utilized an in vitro adipocyte derived stem cell and endothelial colony forming cell (ADSC/ECFC) co-culture system to investigate the effect of lentiviral-driven shRNA knockdown of target genes compared to a non-targeting shRNA control on cord formation using High Content Imaging. Cord formation was significantly reduced following knockdown of the VEGF receptor VEGFR2 in VEGF-driven cord formation and the FGF receptor FGFR1 in basic FGF (bFGF)-driven cord formation. In addition, cord formation was signifcantly reduced following knockdown of the transcription factor forkhead box protein O1 (FOXO1), a protein with known positive effects on angiogenesis and blood vessel stabilization in VEGF- and bFGF-driven cord formation. Lentiviral shRNA also demonstrated utility for stable knockdown of VEGFR2 and FOXO1 in ECFCs, allowing for interrogation of protein knockdown effects on in vivo neoangiogenesis in a Matrigel plug assay. In addition to interrogating the effect of gene knockdown in endothelial cells, we utilized lentiviral shRNA to knockdown specificity protein 1 (SP1), a transcription factor involved in the expression of VEGF, in U-87 MG tumor cells to demonstrate the ability to analyze angiogenesis in vitro in a tumor-driven transwell cord formation system and in tumor angiogenesis in vivo. A significant reduction in tumor-driven cord formation, VEGF secretion, and in vivo tumor angiogenesis was observed upon SP1 knockdown. Therefore, evaluation of target gene knockdown effects in the in vitro co-culture cord formation assay in the ADSC/ECFC co-culture, ECFCs alone, and in tumor cells translated directly to in vivo results, indicating the in vitro method as a robust, cost-effective and efficient in vitro surrogate assay to investigate target gene involvement in endothelial or tumor cell function in angiogenesis.
doi:10.1371/journal.pone.0096036
PMCID: PMC3997504  PMID: 24759702
15.  Stability, Disposition, and Penetration of Catalytic Antioxidants Mn-Porphyrin and Mn-Salen and of Methylprednisolone in Spinal Cord Injury 
This study measured the time courses of concentration changes following administration of the catalytic antioxidants Mn (III) tetrakis (4-benzoic acid) porphyrin (MnTBAP) and Mn (III) 3-methoxy N, N' bis (salicyclidene) ethylenediamine chloride (EUK-134) in blood and cerebrospinal fluid (CSF) of rats with a spinal cord injury (SCI) and sham controls. Parallel measurements were made for methylprednisolone, the only drug presently used clinically for treating SCI. The time courses kinetically characterized the agents in their stability, disposition, and ability to penetrate the blood–spinal cord barrier (BSB). In both the SCI and control groups, MnTBAP was stable in CSF and in blood across the collection periods (10 h and 24 h, respectively) following administration. In the blood, [EUK-134] and [methylprednisolone] rapidly declined to near basal concentrations at 4 h and 2 h, respectively, post-administration. Therefore the order of stability in CSF and blood was MnTBAP >> EUK-134 > methylprednisolone. The maximum CSF/blood concentration ratios for EUK-134, methylprednisolone and MnTBAP post-administration were: 32 ± 3.1%, 19.2 ± 6.4%, and 4.42 ± 0.73% in the injured rats, and 22 ± 6.5%, 17.8 ± 2.9%, and 1.0 ± 0.5% in the sham control animals. This suggests an order of BSB penetration of EUK-134 > methylprednisolone >> MnTBAP. Despite much lower penetration by MnTBAP compared with EUK-134 and methylprednisolone, a lower dose of MnTBAP because of its stability provided a higher concentration in CSF than did the other agents given at higher doses. This finding supports further exploration of MnTBAP as a potential treatment for SCI.
PMCID: PMC4288768  PMID: 22640221
MnTBAP; EUK-134; Methylprednisolone; Blood–spinal cord barrier; Spinal cord injury; Stability and disposition; Antioxidant therapy; Mn-containing catalytic antioxidants
16.  Human Embryonic Stem Cell-Derived Oligodendrocyte Progenitors Aid in Functional Recovery of Sensory Pathways following Contusive Spinal Cord Injury 
PLoS ONE  2012;7(10):e47645.
Background
Transplantations of human stem cell derivatives have been widely investigated in rodent models for the potential restoration of function of neural pathways after spinal cord injury (SCI). Studies have already demonstrated cells survival following transplantation in SCI. We sought to evaluate survival and potential therapeutic effects of transplanted human embryonic stem (hES) cell-derived oligodendrocyte progenitor cells (OPCs) in a contusive injury in rats. Bioluminescence imaging was utilized to verify survivability of cells up to 4 weeks, and somatosensory evoked potential (SSEPs) were recorded at the cortex to monitor function of sensory pathways throughout the 6-week recovery period.
Principal Findings
hES cells were transduced with the firefly luciferase gene and differentiated into OPCs. OPCs were transplanted into the lesion epicenter of rat spinal cords 2 hours after inducing a moderate contusive SCI. The hES-treatment group showed improved SSEPs, including increased amplitude and decreased latencies, compared to the control group. The bioluminescence of transplanted OPCs decreased by 97% in the injured spinal cord compared to only 80% when injected into an uninjured spinal cord. Bioluminescence increased in both experimental groups such that by week 3, no statistical difference was detected, signifying that the cells survived and proliferated independent of injury. Post-mortem histology of the spinal cords showed integration of human cells expressing mature oligodendrocyte markers and myelin basic protein without the expression of markers for astrocytes (GFAP) or pluripotent cells (OCT4).
Conclusions
hES-derived OPCs transplanted 2 hours after contusive SCI survive and differentiate into OLs that produce MBP. Treated rats demonstrated functional improvements in SSEP amplitudes and latencies compared to controls as early as 1 week post-injury. Finally, the hostile injury microenvironment at 2 hours post-injury initially caused increased cell death but did not affect the long-term cell proliferation or survival, indicating that cells can be transplanted sooner than conventionally accepted.
doi:10.1371/journal.pone.0047645
PMCID: PMC3473046  PMID: 23091637
17.  Molecular basis of vascular events following spinal cord injury  
Journal of Medicine and Life  2010;3(3):254-261.
The aim of this article is to analyze the effects of the molecular basis of vascular events following spinal cord injury and their contribution in pathogenesis.
First of all, we reviewed the anatomy of spinal cord vessels.
The pathophysiology of spinal cord injuries revealed two types of pathogenic mechanisms. The primary event, the mechanic trauma, results in a disruption of neural and vascular structures into the spinal cord. It is followed by secondary pathogenesis that leads to the progression of the initial lesion. We reviewed vascular responses following spinal cord injury, focusing on both primary and secondary events. The intraparenchymal hemorrhage is a direct consequence of trauma; it has a typical pattern of distribution into the contused spinal cord, inside the gray matter and, it is radially extended into the white matter. The intraparenchymal hemorrhage is restricted to the dorsal columns, into adjacent rostral and caudal spinal segments. Distribution of chronic lesions overlaps the pattern of the early intraparenchymal hemorrhage. We described the mechanisms of action, role, induction and distribution of the heme oxygenase isoenzymes 1 and 2. Posttraumatic inflammatory response contributes to secondary pathogenesis. We analyzed the types of cells participating in the inflammatory response, the moment of appearance after the injury, the decrease in number, and the nature of their actions. The disruption of the blood–spinal cord barrier is biphasic. It exposes the spinal cord to inflammatory cells and to toxic effects of other molecules. Endothelin 1 mediates oxidative stress into the spinal cord through the modulation of spinal cord blood flow. The role of matrix metalloproteinases in blood–spinal cord barrier disruption, inflammation, and angiogenesis are reviewed.
PMCID: PMC3018992  PMID: 20945816
intraparenchymal hemorrhage; heme oxygenase 1; heme oxygenase 2; posttraumatic inflammatory response; blood–spinal cord barrier; endothelin 1; matrix metalloproteinasis
18.  Developmental and injury-induced expression of α1β1 and α6β1 integrins in the rat spinal cord 
Brain research  2006;1130(1):54-66.
Loss and damage to blood vessels are thought to contribute to secondary tissue loss after spinal cord injury. Integrins might be therapeutic targets to protect the vasculature and/or promote angiogenesis, as their activation can promote tubule formation and survival of endothelial cells in vitro. Here, we show that immunostaining with an antibody against the α1β1 integrin heterodimer is present only in blood vessels from postnatal day 1 (P1) through adulthood in Sprague-Dawley rats. After a spinal cord contusion at T9 in adults, the area of α1β1 integrin positive blood vessels increases within 11 mm from the injury site at 3 days post-injury and remains prominent within the injured core only at 7 days. Staining for the α6β1 integrin heterodimer increases in blood vessels between P10 and adulthood and is present in preganglionic neurons of the intermediolateral cell column (IML) at all ages. The α6β1 integrin is also expressed by motor neurons postnatally, and oligodendrocyte precursors (OPCs), as previously reported. After the contusion, the area of α6β1 stained blood vessels was increased at 3 days and most prominently, 1 mm from the injury site, followed by a significant reduction at 7 days, when α6β1 integrin staining is most prominent around the injured core. Staining is also present in a subset of microglia and/or macrophages. These results raise the possibility that α1β1 and α6β1 integrins in blood vessels might be targeted to reduce blood vessel loss and promote angiogenesis, which may promote tissue sparing after spinal cord injury.
doi:10.1016/j.brainres.2006.10.072
PMCID: PMC1794000  PMID: 17161391
angiogenesis, blood vessels; oligodendrocyte precursor; preganglionic neuron; spinal cord injury
19.  Establishing a model spinal cord injury in the African green monkey for the preclinical evaluation of biodegradable polymer scaffolds seeded with human neural stem cells 
Journal of neuroscience methods  2010;188(2):258-269.
Given the involvement of post-mitotic neurons, long axonal tracts and incompletely elucidated injury and repair pathways, spinal cord injury (SCI) presents a particular challenge for the creation of preclinical models to robustly evaluate longitudinal changes in neuromotor function in the setting in the presence and absence of intervention. While rodent models exhibit high degrees of spontaneous recovery from SCI injury, animal care concerns preclude complete cord transections in non-human primates and other larger vertebrate models. To overcome such limitations a segmental thoracic (T9–T10) spinal cord hemisection was created and characterized in the African green monkey. Physiological tolerance of the model permitted behavioral analyses for a prolonged period post-injury, extending to predefined study termination points at which histological and immunohistochemical analyses were performed. Four monkeys were evaluated (one receiving no implant at the lesion site, one receiving a poly(lactide-co-glycolide) (PLGA) scaffold, and two receiving PLGA scaffolds seeded with human neural stem cells (hNSC)). All subjects exhibited Brown-Séquard syndrome 2 days post-injury consisting of ipsilateral hindlimb paralysis and contralateral hindlimb hypesthesia with preservation of bowel and bladder function. A 20-point observational behavioral scoring system allowed quantitative characterization of the levels of functional recovery. Histological endpoints including silver degenerative staining and Iba1 immunohistochemistry, for microglial and macrophage activation, were determined to reliably define lesion extent and correlate with neurobehavioral data, and justify invasive telemetered electromyographic and kinematic studies to more definitively address efficacy and mechanism.
doi:10.1016/j.jneumeth.2010.02.019
PMCID: PMC4157751  PMID: 20219534
Spinal cord injury; African green monkey; Non-human primate; Stem cells; Biomaterials; Injury model; Behavioral scoring
20.  Novel Combination Strategies to Repair the Injured Mammalian Spinal Cord 
Summary:
Due to the varied and numerous changes in spinal cord tissue following injury, successful treatment for repair may involve strategies combining neuroprotection (pharmacological prevention of some of the damaging intracellular cascades that lead to secondary tissue loss), axonal regeneration promotion (cell transplantation, genetic engineering to increase growth factors, neutralization of inhibitory factors, reduction in scar formation), and rehabilitation. Our goal has been to find effective combination strategies to improve outcome after injury to the adult rat thoracic spinal cord. Combination interventions tested have been implantation of Schwann cells (SCs) plus neuroprotective agents and growth factors administered in various ways, olfactory ensheathing cell (OEC) implantation, chondroitinase addition, or elevation of cyclic AMP. The most efficacious strategy in our hands for the acute complete transection/SC bridge model, including improvement in locomotion [Basso, Beattie, Bresnahan Scale (BBB)], is the combination of SCs, OECs, and chondroitinase administration (BBB 2.1 vs 6.6, 3 times more myelinated axons in the SC bridge, increased serotonergic axons in the bridge and beyond, and significant correlation between the number of bridge myelinated axons and functional improvement). We found the most successful combination strategy for a subacute spinal cord contusion injury (12.5–mm, 10–g weight, MASCIS impactor) to be SCs and elevation of cyclic AMP (BBB 10.4 vs 15, significant increases in white matter sparing, in myelinated axons in the implant, and in responding reticular formation and red and raphe nuclei, and a significant correlation between the number of serotonergic fibers and improvement in locomotion). Thus, in two injury paradigms, these combination strategies as well as others studied in our laboratory have been found to be more effective than SCs alone and suggest ways in which clinical application may be developed.
PMCID: PMC2565567  PMID: 18795474
Spinal cord injuries; Schwann cells; Olfactory ensheathing cells; Axonal regeneration; Chondroitinase; Cyclic adenosine monophosphate
21.  Short Term Interactions with Long Term Consequences: Modulation of Chimeric Vessels by Neural Progenitors 
PLoS ONE  2012;7(12):e53208.
Vessels are a critical and necessary component of most tissues, and there has been substantial research investigating vessel formation and stabilization. Several groups have investigated coculturing endothelial cells with a second cell type to promote formation and stabilization of vessels. Some have noted that long-term vessels derived from implanted cocultures are often chimeric consisting of both host and donor cells. The questions arise as to whether the coculture cell might impact the chimeric nature of the microvessels and can modulate the density of donor cells over time. If long-term engineered microvessels are primarily of host origin, any impairment of the host's angiogenic ability has significant implications for the long-term success of the implant. If one can modulate the host versus donor response, one may be able to overcome a host's angiogenic impairment. Furthermore, if one can modulate the donor contribution, one may be able to engineer microvascular networks to deliver molecules a patient lacks systemically for long times. To investigate the impact of the cocultured cell on the host versus donor contributions of endothelial cells in engineered microvascular networks, we varied the ratio of the neural progenitors to endothelial cells in subcutaneously implanted poly(ethylene glycol)/poly-L-lysine hydrogels. We found that the coculture of neural progenitors with endothelial cells led to the formation of chimeric host-donor vessels, and the ratio of neural progenitors has a significant impact on the long term residence of donor endothelial cells in engineered microvascular networks in vivo even though the neural progenitors are only present transiently in the system. We attribute this to the short term paracrine signaling between the two cell types. This suggests that one can modulate the host versus donor contributions using short-term paracrine signaling which has broad implications for the application of engineered microvascular networks and cellular therapy more broadly.
doi:10.1371/journal.pone.0053208
PMCID: PMC3531360  PMID: 23300890
22.  Spinal Activation of Serotonin 1A Receptors Enhances Latent Respiratory Activity After Spinal Cord Injury 
Background/Objective:
Hemisection of the cervical spinal cord results in paralysis of the ipsilateral hemidiaphragm. Removal of sensory feedback through cervical dorsal rhizotomy activates latent respiratory motor pathways and restores hemidiaphragm function. Because systemic administration of serotonin 1A receptor (5HT1A) agonists reversed the altered breathing patterns after spinal cord injury (SCI), we predicted that 5HT1A receptor activation after SCI (C2) would activate latent crossed motor pathways. Furthermore, because 5HT1A receptors are heavily localized to dorsal horn neurons, we predicted that spinal administration of 5HT1A agonists should also activate latent motor pathways.
Methods:
Hemisection of the C2 spinal cord was performed 24 to 48 hours, 1 week, or 16 weeks before experimentation. Bilateral phrenic nerve activity was recorded in anesthetized, vagotomized, paralyzed Sprague-Dawley rats, and 8-OH-DPAT (5HT1A agonist) was applied to the dorsal aspect of the cervical spinal cord (C3–C7) or injected systemically.
Results:
Systemic administration of 8-OH-DPAT led to a significant increase in phrenic frequency and amplitude, whereas direct application to the spinal cord increased phrenic amplitude alone. Both systemic and spinal administration of 8-OH-DPAT consistently activated latent crossed phrenic activity. 8-OH-DPAT induced a greater respiratory response in spinal injured rats compared with controls.
Conclusion:
The increase in crossed phrenic output after application of 8-OH-DPAT to the spinal cord suggests that dorsal horn inputs, respiratory and/or nonrespiratory, may inhibit phrenic motor output, especially after SCI. These findings support the idea that the administration of 5HT1A agonists may be a beneficial therapy in enhancing respiratory neural output in patients with SCI.
PMCID: PMC1864797  PMID: 16739558
Spinal cord injuries; Serotonin 1A receptor; Rats; Respiration; Crossed phrenic pathway; Sensory afferents; 5HT1A agonist
23.  Effect of VEGF Treatment on the Blood-Spinal Cord Barrier Permeability in Experimental Spinal Cord Injury: Dynamic Contrast-Enhanced Magnetic Resonance Imaging 
Journal of Neurotrauma  2009;26(7):1005-1016.
Abstract
Compromised blood-spinal cord barrier (BSCB) is a factor in the outcome following traumatic spinal cord injury (SCI). Vascular endothelial growth factor (VEGF) is a potent stimulator of angiogenesis and vascular permeability. The role of VEGF in SCI is controversial. Relatively little is known about the spatial and temporal changes in the BSCB permeability following administration of VEGF in experimental SCI. Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) studies were performed to noninvasively follow spatial and temporal changes in the BSCB permeability following acute administration of VEGF in experimental SCI over a post-injury period of 56 days. The DCE-MRI data was analyzed using a two-compartment pharmacokinetic model. Animals were assessed for open field locomotion using the Basso-Beattie-Bresnahan score. These studies demonstrate that the BSCB permeability was greater at all time points in the VEGF-treated animals compared to saline controls, most significantly in the epicenter region of injury. Although a significant temporal reduction in the BSCB permeability was observed in the VEGF-treated animals, BSCB permeability remained elevated even during the chronic phase. VEGF treatment resulted in earlier improvement in locomotor ability during the chronic phase of SCI. This study suggests a beneficial role of acutely administered VEGF in hastening neurobehavioral recovery after SCI.
doi:10.1089/neu.2008.0860
PMCID: PMC2857512  PMID: 19226205
BBB score; blood-spinal cord barrier; DCE-MRI; spinal cord injury; VEGF
24.  VEGF and FGF-2 delivery from spinal cord bridges to enhance angiogenesis following injury 
The host response to spinal cord injury can lead to an ischemic environment that can induce cell death and limits cell transplantation approaches to promote spinal cord regeneration. Spinal cord bridges that provide a localized and sustained release of VEGF and FGF-2 were investigated for their ability to promote angiogenesis and nerve growth within the injury. Bridges were fabricated by fusion of poly(lactide-co-glycolide) microspheres using a gas foaming/particulate leaching technique, and proteins were incorporated by encapsulation into the microspheres and/or mixing with the microspheres before foaming. Compared to the mixing method, encapsulation reduced the losses during leaching and had a slower protein release, while VEGF was released more rapidly than FGF-2. In vivo implantation of bridges loaded with VEGF enhanced the levels of VEGF within the injury at one week, and bridges releasing VEGF and FGF-2 increased the infiltration of endothelial cells and the formation of blood vessel at 6 weeks post implantation. Additionally, substantial neurofilament staining was observed within the bridge; however, no significant difference was observed between bridges with or without protein. Bridges releasing angiogenic factors may provide an approach to overcome an ischemic environment that limits regeneration and cell transplantation based approaches.
doi:10.1002/jbm.a.33112
PMCID: PMC3190227  PMID: 21630429
Spinal cord injury; angiogenesis; spinal cord bridges; PLG; protein delivery
25.  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

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