Dana M. McTigue

The life, death, and replacement of oligodendrocytes in the adult CNS

Oligodendrocytes (OLs) are mature glial cells that myelinate axons in the brain and spinal cord. As such, they are integral to functional and efficient neuronal signaling. The embryonic lineage and postnatal development of OLs have been well‐studied and many features of the process have been described, including the origin, migration, proliferation, and differentiation of precursor cells. Less clear is the extent to which OLs and damaged/dysfunctional myelin are replaced following injury to the adult CNS. OLs and their precursors are very vulnerable to conditions common to CNS injury and disease sites, such as inflammation, oxidative stress, and elevated glutamate levels leading to excitotoxicity. Thus, these cells become dysfunctional or die in multiple pathologies, including Alzheimer’s disease, spinal cord injury, Parkinson’s disease, ischemia, and hypoxia. However, studies of certain conditions to date have detected spontaneous OL replacement. This review will summarize current information on adult OL progenitors, mechanisms that contribute to OL death, the consequences of their loss and the pathological conditions in which spontaneous oligodendrogenesis from endogenous precursors has been observed in the adult CNS.

Bone marrow transplants provide tissue protection and directional guidance for axons after contusive spinal cord injury in rats

Contusive spinal cord injury (SCI) produces large fluid-, debris- and inflammatory cell-filled cystic cavities that lack structure to support significant axonal regeneration. The recent discovery of stem cells capable of generating central nervous system (CNS) tissues, coupled with success in neurotransplantation strategies, has renewed hope that repair and recovery from CNS trauma is possible. Based on results from several studies using bone marrow stromal cells (MSCs) to promote CNS repair, we transplanted MSCs into the rat SCI lesion cavity to further investigate their effects on functional recovery, lesion morphology, and axonal growth. We found that transplanted MSCs induced hindlimb airstepping--a spontaneous locomotor movement associated with activation of the stepping control circuitry--but did not alter the time course or extent of overground locomotor recovery. Using stereological techniques to describe spinal cord anatomy, we show that MSC transplants occupied the lesion cavity and were associated with preservation of host tissue and white matter (myelin), demonstrating that these cells exert neuroprotective effects. The tissue matrix formed by MSC grafts supported greater axonal growth than that found in specimens without grafts. Moreover, uniform random sampling of axon profiles revealed that the majority of neurites in MSC grafts were oriented with their long axis parallel to that of the spinal cord, suggesting longitudinally directed growth. Together, these studies support further investigation of marrow stromal cells as a potential SCI repair strategy.

Selective chemokine mRNA accumulation in the rat spinal cord after contusion injury

Following traumatic injury to the spinal cord, hematogenous inflammatory cells including neutrophils, monocytes, and lymphocytes infiltrate the lesion in a distinct temporal sequence. To examine potential mechanisms for their recruitment, we measured chemokine mRNAs in the contused rat spinal cord, using specific and sensitive reverse transcriptase polymerase chain reaction (RT‐PCR) dot‐blot hybridization assays. The neutrophil chemoattractant GRO‐α was 30‐fold higher than control values at 6 hr postinjury and decayed rapidly thereafter. LIX, a highly related α‐chemokine, also was elevated early postinjury. Monocyte chemoattractant peptide (MCP)‐1 and MCP‐5 mRNAs, potent chemoattractants for monocytes, were significantly elevated at the lesion epicenter at 12 and 24 hr postinjury and declined thereafter. Interferon‐γ‐inducible protein, 10 kDa (IP‐10), chemoattractant towards activated T‐lymphocytes, was significantly elevated at 6 and 12 hr postinjury. The dendritic cell chemoattractant MIP‐3α also was increased, perhaps contributing to the development of T‐cell autoreactivity to neural components after spinal cord injury (SCI) in rats. Other β‐chemokines, including MIP‐1α and RANTES (regulated on expression normal T‐cell expressed and secreted), were minimally affected by SCI. Expression of chemokines, therefore, directly precedes the influx of target neutrophils, monocytes, and T‐cells into the spinal cord postinjury, as noted previously. Thus, selective chemokine expression may be integral to inflammatory processes within the injured spinal cord as a mechanism of recruitment for circulating leukocytes. J. Neurosci. Res. 53:368–376, 1998.

Prominent oligodendrocyte genesis along the border of spinal contusion lesions

Oligodendrocyte (OL) loss and axon demyelination occur after spinal cord injury (SCI). OLs may be replaced, however, by proliferating NG2+ progenitor cells. Indeed, new OLs have been noted in ventral white matter after SCI. Since tissue adjacent to lesion cavities is exposed to different mediators compared with outlying spared tissue, the authors used a rat SCI model to compare NG2 cell proliferation and OL genesis adjacent to lesion cavities with that in spared tissue closer to meninges. NG2 cells proliferated throughout the first week postinjury and accumulated along lesion borders, especially within gray matter. By 3 days postinjury (dpi), new OLs were detected throughout the cross‐sections; between 4 and 7 dpi, however, oligogenesis was restricted to lesion borders. New OLs derived from cells proliferating during 1–7 dpi increased dramatically by 14 dpi; most were located along lesion borders and in spared gray matter. Oligogenesis continued along lesion borders during the second week postinjury. Overall OL numbers were reduced at 3 dpi in spared tissue, but rebounded to normal levels by 14 dpi. Surprisingly, lesion borders maintained normal OL numbers at 3 dpi, which then rose to exceed preinjury levels at 7 and 14 dpi. These results indicate that oligogenesis is protracted after SCI and leads to increased OL numbers. Most new OLs are formed in regions of greatest NG2 cell proliferation. Thus, the adult spinal cord spontaneously develops a dynamic gliogenic zone along lesion borders.

Localization of transforming growth factor-β1 and receptor mRNA after experimental spinal cord injury

Transforming growth factor-beta1 (TGFbeta1) is a cytokine/growth factor found within the pathological central nervous system. TGFbeta1 has been shown to inhibit the release of cytotoxic molecules from microglia and macrophages, decrease astrocyte proliferation, and promote neuron survival. Because of the relevance of these actions to spinal cord injury, we examined TGFbeta1 and its receptors betaRI and betaRII mRNA levels and localization within the contused rat spinal cord using in situ hybridization. At the lesion site, TGFbeta1 mRNA peaked at 7 days postinjury and declined thereafter. Temporal and spatial localization of the betaRI and betaRII receptor mRNA closely mimicked that for TGFbeta1 in the epicenter. TGFbeta1, betaRI, and betaRII mRNAs also were elevated rostral and caudal to the injury, especially in regions known to contain activated microglia and degenerating axon profiles. Immunohistochemical staining of nearby sections confirmed that the highest levels of TGFbeta1 and receptor mRNA corresponded to regions filled with activated microglia and macrophages. The similar expression pattern of TGFbeta1, betaRI, and betaRII mRNA within the injured spinal cord suggests a local site of action. Since TGFbeta1 can act as an immunosuppressant as well as a stimulant for growth factors and neurite sprouting, it likely plays an important role, both temporally and spatially, in orchestrating postinjury events within the spinal cord.

The PPAR gamma agonist Pioglitazone improves anatomical and locomotor recovery after rodent spinal cord injury

Traumatic spinal cord injury (SCI) is accompanied by a dramatic inflammatory response, which escalates over the first week post-injury and is thought to contribute to secondary pathology after SCI. Peroxisome proliferator-activated receptors (PPAR) are widely expressed nuclear receptors whose activation has led to diminished pro-inflammatory cascades in several CNS disorders. Therefore, we examined the efficacy of the PPARgamma agonist Pioglitazone in a rodent SCI model. Rats received a moderate mid-thoracic contusion and were randomly placed into groups receiving vehicle, low dose or high dose Pioglitazone. Drug or vehicle was injected i.p. at 15 min post-injury and then every 12 h for the first 7 days post-injury. Locomotor function was followed for 5 weeks using the BBB scale. BBB scores were greater in treated animals at 7 days post-injury and significant improvements in BBB subscores were noted, including better toe clearance, earlier stepping and more parallel paw position. Stereological measurements throughout the lesion revealed a significant increase in rostral spared white matter in both Pioglitazone treatment groups. Spinal cords from the high dose group also had significantly more gray matter sparing and motor neurons rostral and caudal to epicenter. Thus, our results reveal that clinical treatment with Pioglitazone, an FDA-approved drug used currently for diabetes, may be a feasible and promising strategy for promoting anatomical and functional repair after SCI.

NG2 colocalizes with axons and is expressed by a mixed cell population in spinal cord lesions.

The NG2 proteoglycan is of general interest after spinal cord injury because it is expressed by oligodendrocyte progenitors (OPCs), which contribute to central nervous system remyelination; however, NG2 may inhibit axon regeneration. We and others have examined the spatiotemporal expression of NG2 after spinal cord injury (SCI). Here, we extend those observations and provide a comprehensive analysis of the distribution, phenotype, and colocalization of NG2 cells with axons in a clinically relevant model of spinal contusion. Because contusion models mimic the majority of human SCI, this information is important for understanding endogenous processes that promote and/or prevent repair. The data demonstrate that NG2 levels rise significantly between 3 and 7 days postinjury (dpi) and remain elevated chronically throughout the lesions. NG2 within the lesions could be derived from an array of infiltrating cells; thus, a panel of antibodies was used to investigate NG2 cell phenotypes. First, platelet-derived growth factor-&agr; receptor (PDGF&agr;R) colocalization was examined because OPCs normally express both markers. PDGF&agr;R cells were present in lesions at all times examined. However, only 37% of NG2 cells coexpressed PDGF&agr;R at 14 dpi, which dropped to <1% by 70 dpi. This contrasts with the nearly complete overlap in spared tissue surrounding the lesion. In contrast, 40% to 60% of NG2 cells expressed p75 and approximately 84% expressed Sox10, suggesting that many NG2 cells were nonmyelinating Schwann cells. Despite rising levels of NG2, we noted robust and sustained axon growth into the lesions, many of which were located along NG2 profiles. Thus, spinal contusion produces an NG2-rich environment into which axons grow and in which the source of NG2 appears considerably different from that in surrounding spared tissue.

Pegylated Brain-Derived Neurotrophic Factor Shows Improved Distribution into the Spinal Cord and Stimulates Locomotor Activity and Morphological Changes after Injury

The neurotrophin brain-derived neurotrophic factor (BDNF) shows promise for the treatment of central nervous system (CNS) trauma and disease. Effective delivery methods are required, however, for BDNF to be useful as a therapeutic agent. To this end, we examined the penetration of intrathecally infused N-terminal pegylated BDNF (peg-BDNF) compared to similar infusion of native BDNF after spinal cord injury (SCI). Pegylation dramatically improved delivery of BDNF to the spinal cord and induced the expression of Fos in spinal cord neurons. To test whether enhanced delivery would improve the modest effects on behavioral recovery and axonal outgrowth observed with native BDNF infusion, we assessed the efficacy of 2-week 25 microg/day peg-BDNF treatment, beginning 12-24 h (early) or 15 days (delayed) after midthoracic spinal contusion. Similar to native BDNF, early treatment with peg-BDNF accelerated the recovery of stepping in the open-field and acutely stimulated locomotor central pattern generator activity, as seen by the activation of hindlimb airstepping during either period of administration. The infusion of peg-BDNF, regardless of the timing of delivery, was related to enhanced sprouting of putative cholinergic fibers, like that observed after high dose native BDNF treatment. Despite improved delivery, however, neither axonal responses nor the extent of locomotor recovery were enhanced compared to native BDNF treatment. This suggests that alternative strategies, such as neurotrophin treatment in conjunction with cell transplantation techniques, or treatment nearer the cell bodies of target neurons might be employed in an attempt to effect significant repair after SCI.

Oligodendrocyte generation is differentially influenced by toll-like receptor (TLR) 2 and TLR4-mediated intraspinal macrophage activation.

Oligodendrocytes are vulnerable to CNS injury and disease. Because oligodendrocytes myelinate CNS axons, their death leads to demyelination and impaired axon conductance, which in turn contribute to neurologic deficits. Replacing oligodendrocytes requires proliferation and differentiation of endogenous NG2+ progenitor cells, a process that can be potently influenced by activated macrophages, which are present in most CNS pathologies. To examine the relationship between oligodendrocyte generation and macrophage activation in vivo, we compared the extent of oligodendrocyte loss and NG2 cell proliferation and differentiation after intraspinal microinjection of lipopolysaccharide (a Toll-like receptor-4 agonist) or zymosan (Toll-like receptor-2 agonist) in rats. Controls included injecting vehicle (sterile PBS; negative control) or lysolecithin (positive control for NG2 cell proliferation and oligodendrocyte differentiation). By 14 days postinjection, lipopolysaccharide injection sites displayed a sigficant rise in NG2 cell proliferation and oligodendrocyte differentiation, which exceeded that in vehicle and lysolecithin injections. Additionally, upregulated ciliary neurotrophic factor expression was present in lipopolysaccharide lesions. In contrast, zymosan-activated macrophages produced complete oligodendrocyte loss without stimulating NG2 cell proliferation, oligodendrocyte replacement, or ciliary neurotrophic factor expression. Zymosan also evoked a delayed lesion expansion and primary demyelination of intact myelinated axons around the lesions. These results clearly delineate the dichotomous potential of macrophage activation for influencing NG2 cell proliferation and oligodendrocyte differentiation. Because endogenous Toll-like receptor ligands are often present in injured CNS tissue, these results shed light on possible mechanisms that restrict oligodendrocyte replacement to specific domains of CNS trauma or disease sites.

Regional heterogeneity in astrocyte responses following contusive spinal cord injury in mice.

Astrocytes and their precursors respond to spinal cord injury (SCI) by proliferating, migrating, and altering phenotype. This contributes to glial scar formation at the lesion border and gliosis in spared gray and white matter. The present study was undertaken to evaluate astrocyte changes over time and determine when and where interventions might be targeted to alter the astrocyte response. Bromodeoxyuridine (BrdU) was administered to mice 3 days after SCI, and cells expressing BrdU and the astrocyte marker, glial fibrillary acidic protein (GFAP), were counted at 3, 7, and 49 days post‐injury (DPI). BrdU‐labeled cells accumulated at the lesion border by 7 DPI and approximately half of these expressed GFAP. In spared white matter, the total number of BrdU+ cells decreased, while the percentage of BrdU+ cells expressing GFAP increased at 49 DPI. Phenotypic changes were examined using the progenitor marker nestin, the radial glial marker, brain lipid binding protein (BLBP), and GFAP. Nestin was upregulated by 3 DPI and declined between 7 and 49 DPI in all regions, and GFAP increased and remained above naïve levels at all timepoints. BLBP increased early and remained high along the lesion border and spared white matter, but was expressed transiently by cells lining the central canal and in a unique population of small cells found within the lesion and in gray matter rostral and caudal to the border. The results demonstrate that the astrocyte response to SCI is regionally heterogeneous, and suggests astrocyte populations that could be targeted by interventions. J. Comp. Neurol. 518:1370–1390, 2010.