Astrocytic Cebpd Regulates Pentraxin 3 Expression to Promote Fibrotic Scar Formation After Spinal Cord Injury.

Astroglial-fibrotic scars resulted from spinal cord injury affect motor and sensory function, leading to paralysis. In particular, the fibrotic scar is a main barrier that disrupts neuronal regeneration after spinal cord injury. However, the association between astrocytes and fibrotic scar formation is not yet understood. We have previously demonstrated that the transcriptional factor Cebpd contributes to astrogliosis, which promotes glial scar formation after spinal cord injury. Herein, we show that fibrotic scar formation was decreased in the epicenter region in Cebpd-/- mice after contusive spinal cord injury and astrocytic Cebpd promoted fibroblast migration through secretion of Ptx3. Furthermore, the expression of Mmp3 was increased under recombinant protein Ptx3 treatment in fibroblasts by observing microarray data, resulting in fibroblast migration. In addition, regulation of Mmp3 occurs through the NFκB signaling pathway by using an irreversible inhibitor of IκBα phosphorylation in pretreated fibroblasts. Of note, we used the synthetic peptide RI37, which blocks fibroblast migration and decreases fibroblast Mmp3 expression in IL-1ß-treated astrocyte conditioned media. Collectively, our data suggest that fibroblast migration can be affected by astrocytic Cebpd through the Ptx3/NFκB/Mmp3 axis pathway and that the RI37 peptide may act as a therapeutic medicine to inhibit fibrotic scar formation after spinal cord injury.

Mitochondrial Transplantation Attenuates Neural Damage and Improves Locomotor Function After Traumatic Spinal Cord Injury in Rats.

Mitochondrial dysfunction is a hallmark of secondary neuroinflammatory responses and neuronal death in spinal cord injury (SCI). Even though mitochondria-based therapy is an attractive therapeutic option for SCI, the efficacy of transplantation of allogeneic mitochondria in the treatment of SCI remains unclear. Herein, we determined the therapeutic effects of mitochondrial transplantation in the traumatic SCI rats. Compressive SCI was induced by applying an aneurysm clip on the T10 spinal cord of rats. A 100-µg bolus of soleus-derived allogeneic mitochondria labeled with fluorescent tracker was transplanted into the injured spinal cords. The results showed that the transplanted mitochondria were detectable in the injured spinal cord up to 28 days after treatment. The rats which received mitochondrial transplantation exhibited better recovery of locomotor and sensory functions than those who did not. Both the expression of dynamin-related protein 1 and severity of demyelination in the injured cord were reduced in the mitochondrial transplanted groups. Mitochondrial transplantation also alleviated SCI-induced cellular apoptosis and inflammation responses. These findings suggest that transplantation of allogeneic mitochondria at the early stage of SCI reduces mitochondrial fragmentation, neuroapoptosis, neuroinflammation, and generation of oxidative stress, thus leading to improved functional recovery following traumatic SCI.

Protease-Activated Receptor-1 Supports Locomotor Recovery by Biased Agonist Activated Protein C after Contusive Spinal Cord Injury.

Thrombin-induced secondary injury is mediated through its receptor, protease activated receptor-1 (PAR-1), by "biased agonism." Activated protein C (APC) acts through the same PAR-1 receptor but functions as an anti-coagulant and anti-inflammatory protein, which counteracts many of the effects of thrombin. Although the working mechanism of PAR-1 is becoming clear, the functional role of PAR-1 and its correlation with APC in the injured spinal cord remains to be elucidated. Here we investigated if PAR-1 and APC are determinants of long-term functional recovery after a spinal cord contusive injury using PAR-1 null and wild-type mice. We found that neutrophil infiltration and disruption of the blood-spinal cord barrier were significantly reduced in spinal cord injured PAR-1 null mice relative to the wild-type group. Both locomotor recovery and ability to descend an inclined grid were significantly improved in the PAR-1 null group 42 days after injury and this improvement was associated with greater long-term sparing of white matter and a reduction in glial scarring. Wild-type mice treated with APC acutely after injury showed a similar level of improved locomotor recovery to that of PAR-1 null mice. However, improvement of APC-treated PAR-1 null mice was indistinguishable from that of vehicle-treated PAR-1 null mice, suggesting that APC acts through PAR-1. Collectively, our findings define a detrimental role of thrombin-activated PAR-1 in wound healing and further validate APC, also acting through the PAR-1 by biased agonism, as a promising therapeutic target for spinal cord injury.

Astrocytic CCAAT/Enhancer-Binding Protein Delta Contributes to Glial Scar Formation and Impairs Functional Recovery After Spinal Cord Injury.

After spinal cord injury, inflammatory reaction induces the aggregation of astrocytes to form a glial scar that eventually blocks axonal regeneration. Transcription factor CCAAT/enhancer-binding protein delta (C/EBPδ) is a regulatory protein of genes responsive to inflammatory factors, but its role in glial scar formation after spinal cord injury remains unknown. By using a model of moderate spinal cord contusion injury at the mid-thoracic level, we found that C/EBPδ was expressed mostly in the reactive astrocytes bordering the lesion in wild-type mice from 7 days after the injury. C/EBPδ-deficient mice showed reduced glial scar formation, more residual white matter, and better motor function recovery compared with wild-type mice 28 days after the injury. Upon interleukin (IL)-1ß stimulation in vitro, the increased expression of C/EBPδ in reactive astrocytes inhibited RhoA expression and, subsequently, the ability of astrocyte migration. However, these reactive astrocytes also produced an increased amount of matrix metalloproteinase-3, which promoted the migration of non-IL-1ß-treated, inactive astrocytes. Although the involvement of other non-astroglial C/EBPδ cannot be entirely excluded, our studies suggest that astrocytic C/EBPδ is integral to the inflammatory cascades leading to glial scar formation after spinal cord injury.

The Possible Neuronal Mechanism of Acupuncture: Morphological Evidence of the Neuronal Connection between Groin A-Shi Point and Uterus.

Somatovisceral reflex suggested that the somatic stimulation could affect visceral function like acupuncture which treats diseases by stimulating acupoints. The neuronal connection between somatic point and visceral organ was not clear. Uterine pain referred to the groin region has long been recognized clinically. Wesselmann, using neurogenic plasma extravasation method, showed that uterine pain was referred to the groin region through a neuronal mechanism (Wesselmann and Lai 1997). This connection could be considered through the somatovisceral reflex pathway. However, the relay center of this pathway is still not clearly identified. In the present study, bee venom was injected in the groin region to induce central Fos expression to map the sensory innervation of groin region. Pseudorabies virus (PrV), a transneuronal tracer, was injected in the uterus to identify the higher motor control of the uterus. Immunohistochemistry staining revealed the Fos expression and PrV-infected double-labeled neurons in the nucleus of solitary tract (NTS), the dorsal motor nucleus of vagus (DMX), and the paraventricular hypothalamic nucleus (PVN). These results suggest a somatoparasympathetic neuronal connection (groin-spinal dorsal horn-NTS/DMX-uterus) and a somatosympathetic neuronal connection (groin-spinal dorsal horn-NTS-PVN-uterus). These two neuronal connections could be the prerequisites to the neuronal basis of the somatovisceral reflex and also the neuronal mechanism of acupuncture.

Delayed granulocyte colony-stimulating factor treatment promotes functional recovery in rats with severe contusive spinal cord injury.

STUDY DESIGN: We used a severe contusive spinal cord injury (SCI) model and electrophysiologic, motor functional, immunohistochemical, and electron microscopic examinations to analyze the neuroprotective effects of delayed granulocyte colony-stimulating factor (G-CSF) treatment. OBJECTIVE: To determine the neuroprotective effects of delayed G-CSF treatment using multimodality evaluations after severe contusive SCI in rats. SUMMARY OF BACKGROUND DATA: Despite some reports that G-CSF treatment in the acute stage of different central nervous system injury models was neuroprotective, it has not been determined whether delayed G-CSF treatment can promote neural recovery in severe contusive SCI. METHODS: Rats with severe contusive SCI were divided into 2 groups: G-CSF group rats were given serial subcutaneous injections of G-CSF, and control group rats (controls) were given only saline injections on postcontusion days 9 to 13. Using the Basso-Beattie-Bresnahan scale and cortical somatosensory evoked potentials, we recorded functional evaluations weekly. The spinal cords were harvested for protein and immunohistochemical analysis, and for electron microscopy examination. RESULTS: The preserved spinal cord area was larger in G-CSF group rats than in control group rats. Both sensory and motor functions improved after G-CSF treatment. Detachment and disruption of the myelin sheets in the myelinated axons were significantly decreased, and axons sprouted and regenerated. There were fewer microglia and macrophages in the G-CSF group than in the control group. The levels of brain-derived neurotrophic factor were comparable between the 2 groups. CONCLUSION: Delayed G-CSF treatment at the subacute stage of severe contusive SCI promoted spinal cord preservation and improved functional outcomes. The mechanism of G-CSF's protection may be related in part to attenuating the infiltration of microglia and macrophages.

Human umbilical mesenchymal stem cells promote recovery after ischemic stroke.

BACKGROUND AND PURPOSE: Stroke is a cerebrovascular defect that leads to many adverse neurological complications. Current pharmacological treatments for stroke remain unclear in their effectiveness, whereas stem cell transplantation shows considerable promise. Previously, we have shown that human umbilical mesenchymal stem cells (HUMSCs) can differentiate into neurons in neuronal-conditioned medium. Here we evaluate the therapeutic potential of HUMSC transplantation for ischemic stroke in rats. METHODS: Focal cerebral ischemia was produced by middle cerebral artery occlusion and reperfusion. The HUMSCs treated with neuronal-conditioned medium or not treated were transplanted into the ischemic cortex 24 hours after surgery. RESULTS: Histology and MRI revealed that rats implanted with HUMSCs treated with neuronal-conditioned medium or not treated exhibited a trend toward less infarct volume and significantly less atrophy compared with the control group, which received no HUMSCs. Moreover, rats receiving HUMSCs showed significant improvements in motor function, greater metabolic activity of cortical neurons, and better revascularization in the infarct cortex. Implanted HUMSCs, treated or not treated, survived in the infarct cortex for at least 36 days and released neuroprotective and growth-associated cytokines, including brain-derived neurotrophic factor, platelet-derived growth factor-AA, basic fibroblast growth factor, angiopoietin-2, CXCL-16, neutrophil-activating protein-2, and vascular endothelial growth factor receptor-3. CONCLUSIONS: Our results demonstrate the therapeutic benefits of HUMSC transplantation for ischemic stroke, likely due to the ability of the cells to produce growth-promoting factors. Thus, HUMSC transplantation may be an effective therapy in the future.

Matrix metalloproteinase-9 facilitates glial scar formation in the injured spinal cord.

In the injured spinal cord, a glial scar forms and becomes a major obstacle to axonal regeneration. Formation of the glial scar involves migration of astrocytes toward the lesion. Matrix metalloproteinases (MMPs), including MMP-9 and MMP-2, govern cell migration through their ability to degrade constituents of the extracellular matrix. Although MMP-9 is expressed in reactive astrocytes, its involvement in astrocyte migration and formation of a glial scar is unknown. Here we found that spinal cord injured, wild-type mice expressing MMPs developed a more severe glial scar and enhanced expression of chondroitin sulfate proteoglycans, indicative of a more inhibitory environment for axonal regeneration/plasticity, than MMP-9 null mice. To determine whether MMP-9 mediates astrocyte migration, we conducted a scratch wound assay using astrocytes cultured from MMP-9 null, MMP-2 null, and wild-type mice. Gelatin zymography confirmed the expression of MMP-9 and MMP-2 in wild-type cultures. MMP-9 null astrocytes and wild-type astrocytes, treated with an MMP-9 inhibitor, exhibited impaired migration relative to untreated wild-type controls. MMP-9 null astrocytes showed abnormalities in the actin cytoskeletal organization and function but no detectable untoward effects on proliferation, cellular viability, or adhesion. Interestingly, MMP-2 null astrocytes showed increased migration, which could be attenuated in the presence of an MMP-9 inhibitor. Collectively, our studies provide explicit evidence that MMP-9 is integral to the formation of an inhibitory glial scar and cytoskeleton-mediated astrocyte migration. MMP-9 may thus be a promising therapeutic target to reduce glial scarring during wound healing after spinal cord injury.

Abnormal growth of the corticospinal axons into the lumbar spinal cord of the hyt/hyt mouse with congenital hypothyroidism.

Thyroid hormone deficiency may cause severe neurological disorders resulting from developmental deficits of the central nervous system. The mutant hyt/hyt mouse, characterized by fetal-onset, life-long hypothyroidism resulting from a point mutation of the thyroid-stimulating hormone receptor of the thyroid gland, displays a variety of abnormalities in motor behavior that are likely associated with dysfunctions of specific brain regions and a defective corticospinal tract (CST). To test the hypothesis that fetal and neonatal hypothyroidism cause abnormal CST development, the growth of the CST was investigated in hypothyroid hyt/hyt mice and their euthyroid progenitors, the BALB/cByJ mice. Anterograde labeling with biotinylated dextran amine demonstrated a decrease in the number of CST axons in the hyt/hyt mouse at the first lumbar level at postnatal day (P) 10. After retrograde tracing with fast blue (FB), fewer FB-labeled neurons were found in the motor cortex, the red nucleus, and the lateral vestibular nucleus of the hyt/hyt mouse. At the fourth lumbar level, the hyt/hyt mouse also showed smaller CST cross-sectional areas and significantly lower numbers of unmyelinated axons, myelinated axons, and growth cones within the CST during postnatal development. At P10, the hyt/hyt mouse demonstrated significantly lower immunoreactivity of embryonic neural cell adhesion molecule in the CST at the seventh cervical level, whereas the expression of growth-associated protein 43 remained unchanged. Our study demonstrated an abnormal development of the CST in the hyt/hyt mouse, manifested by reduced axon quantity and retarded growth pattern at the lumbar spinal cord.

Matrix metalloproteinase-2 facilitates wound healing events that promote functional recovery after spinal cord injury.

Matrix metalloproteinases (MMPs) are proteolytic enzymes that are involved in both injury and repair mechanisms in the CNS. Pharmacological blockade of MMPs, limited to the first several days after spinal cord injury, improves locomotor recovery. This beneficial response is, however, lost when treatment is extended beyond the acutely injured cord to include wound healing and tissue remodeling. This suggests that some MMPs play a beneficial role in wound healing. To test this hypothesis, we investigated the role of MMP-2, which is actively expressed during wound healing, in white matter sparing and axonal plasticity, the formation of a glial scar, and locomotor recovery after spinal cord injury. MMP-2 increased between 7 and 14 d after injury, where it was immunolocalized in reactive astrocytes bordering the lesion epicenter. There was reduced white matter sparing and fewer serotonergic fibers, caudal to the lesion in injured MMP-2 null animals. MMP-2 deficiency also resulted in increased immunoreactivity to chondroitin sulfate proteoglycans and a more extensive astrocytic scar. Most importantly, locomotion in an open field, performance on a rotarod, and grid walking were significantly impaired in injured MMP-2 null mice. Our findings suggest that MMP-2 promotes functional recovery after injury by regulating the formation of a glial scar and white matter sparing and/or axonal plasticity. Thus, strategies exploiting MMPs as therapeutic targets must balance these beneficial effects during wound healing with their adverse interactions in the acutely injured spinal cord.

Development of the corticospinal tract in the mouse spinal cord: a quantitative ultrastructural analysis.

The growth of corticospinal tract (CST) axons was studied quantitatively at the 7th cervical (C7) and the 4th lumbar (L4) spinal segments in the balb/cByJ mice at the ages of postnatal day (P) 0, 2, 4, 6, 8, 10, 14, and 28. The cross-sectional area of the CST increased progressively with time. Unmyelinated axons, the most prominent CST element during early development, reached maximum at C7 and L4 on P14. Two phases of increase in the number of unmyelinated axons were observed at C7, while only one surge of axonal outgrowth was found at the L4 level. Pro-myelinated axons, defined as axons surrounded by only one layer of oligodendrocytic process, were first seen at P2 and P4 in the C7 and the L4 level, respectively, followed by a dramatic increase in the number of myelinated axons from P14 onwards at both spinal levels. Myelination of the CST axons occurred topographically in a dorsal-to-ventral pattern. The number of growth cones increased rapidly at the C7 level to reach its maximum at P4, while those at L4 increased steadily to the peak at P10. Growth cones with synapse-like junctions were occasionally observed in the growing CST. Degenerating axons and growth cones partly accounted for the massive axon loss at both spinal segments during CST development. Overall, the mouse CST elements changed dynamically in numbers during postnatal development, suggesting a vigorous growing and pruning activity in the tract. The mouse CST also showed a similar growth pattern to that of the rat CST.

Suitability of allogeneic sertoli cells for ex vivo gene delivery in the injured spinal cord.

Cell-based gene delivery for gene therapy offers the advantages of long-term stable expression of proteins without the safety concerns associated with viral vectors. However, issues of immune rejection prevent the widespread use of allogeneic cell implants. In this study, we determine if Sertoli cells, known for their immune privileged status, are suitable vehicles for allogeneic cell-based gene delivery into the injured spinal cord. As proof of concept, Sertoli cells were modified with recombinant adenovirus expressing enhanced green fluorescent protein (eGFP) or a human trophic factor, neurotrophin-3 (hNT-3), and eGFP. Genetically modified Sertoli cells retained their immunosuppressive ability in vitro, based upon lymphocyte proliferation assays, and were capable of generating biologically relevant levels of NT-3. Similarly, modified, allogeneic cells, implanted into the acutely injured spinal cord, reduced the early inflammatory response while producing significant levels of hNT-3 for at least 3 days after grafting. Moreover, these cells survived for at least 42 days after implantation in the injured cord. Together, these results demonstrate that Sertoli cells function in immunomodulation, can be engineered to produce bioactive molecules, and show long-term survival after implantation into the hostile environment of the acutely injured spinal cord. Such long-term survival represents an important first step toward developing an optimal cell-based delivery system that generates sustained expression of a therapeutic molecule.

Early profiles of axonal growth and astroglial response after spinal cord hemisection and implantation of Schwann cell-seeded guidance channels in adult rats.

We previously demonstrated that transplantation of Schwann cell-seeded channels promoted the regrowth of injured axons in the adult spinal cord. It is not clear, however, whether injured axons recapitulate the developmental scenarios to accomplish regeneration. In the present study, we investigated the early events associated with axonal regrowth after spinal cord hemisection at the eighth thoracic level and implantation of a Schwann cell-seeded minichannel in adult rats. Animals were sacrificed at postoperative days (PO) 2, 4, 7, and 14. Anterograde tracing with fluoro-ruby showed that regenerating axons grew into the graft prior to PO2 and reached the distal end of the channel at PO7. These axons expressed both embryonic neural cell adhesion molecule (E-NCAM) and growth associated protein-43 (GAP-43). Although the expression of E-NCAM decreased by PO7, that of GAP-43 remained high throughout the first 2 weeks after implantation. A close relation of vimentin-positive astroglia to the growing axons in the host tissue suggested a contact-mediated role of these cells in axon guidance. Aggregation of glial fibrillary acidic protein (GFAP)-positive astrocytes together with the increased expression of chondroitin sulfate proteoglycans (CSPGs) starting at PO7 appeared to inhibit axonal growth at the host-graft interface. Thus, adult regenerating axons and astroglia do express developmentally related molecules that may facilitate axonal growth into a permissive graft at the early phase of injury and regeneration. These results suggest that molecules and astroglia essential to development are both important in influencing axonal regrowth in the adult spinal cord.

Temporal and spatial distribution of growth-associated molecules and astroglial cells in the rat corticospinal tract during development.

To understand better the role of growth-promoting and -inhibiting molecules in the development of the corticospinal tract (CST), temporospatial expression of embryonic neural cell adhesion molecule (E-NCAM), growth-associated protein-43 (GAP-43), and chondroitin sulfate proteoglycan (CSPG) was studied in developing rats. Transverse sections of the seventh cervical (C7), seventh thoracic (T7), and fourth lumbar (L4) segments were examined at postnatal days (P) 2, 6, 10, 14, and 28. The highest E-NCAM immunoreactivity appeared at the C7 level on P2 and shifted caudally to the T7 on P6 and L4 on P10, which correlated closely with the time course of CST development. The peak expression of GAP-43 emerged at C7 on P2 and shifted to the T7 and L4 levels at a relatively lagging pace compared with that of E-NCAM. Conversely, a transient reduction in CSPG immunoreactivity was found within the CST at the C7 level on P2, T7 level on P6, and L4 level on P10, corresponding well with the arrival of CST-leading axons at these levels. Interestingly, higher levels of CSPG were found to surround the growing CST, suggesting a repulsive environment that channels the growth of CST. Moreover, a transition from immature to mature astrocytes in a rostrocaudal direction during CST development was evidenced by anti-vimentin and anti-glial fibrillary acidic protein (GFAP) immunostaining, suggesting a guidance role of immature astroglia in axonal outgrowth. Our study thus demonstrated dynamic changes of multiple growth-related molecules and astroglial environment that contribute to postnatal development of the CST.

Blood-spinal cord barrier after spinal cord injury: relation to revascularization and wound healing.

Spinal cord injury produces prominent disruption of the blood-spinal cord barrier. We have defined the blood-spinal cord barrier breakdown to the protein luciferase (61 kDa) in the acutely injured murine spinal cord and during revascularization. We show that newly formed and regenerating blood vessels that have abnormal permeability exhibit differential expression of the glucose-1 transporter (Glut-1), and that its expression is dependent on astrocytes. There was overt extravasation of luciferase within the first hour after injury, a period that coincided with marked tissue disruption within the epicenter of the lesion. Although there was a significant reduction in the number of blood vessels relative to controls by 24 hr after injury, abnormal barrier permeability remained significantly elevated. A second peak of abnormal barrier permeability at 3-7 days postinjury coincided with prominent revascularization of the epicenter. The barrier to luciferase was restored by 21 days postinjury and vascularity was similar to that of controls. During wound-healing process, the cord was reorganized into distinct domains. Between 14 and 21 days postinjury, each domain consisted primarily of nonneuronal cells, including macrophages. Astrocytes were limited characteristically to the perimeter of each domain. Only blood vessels affiliated closely with astrocytes in the perimeter expressed Glut-1, whereas blood vessels within each domain of the repairing cord did not express it. Together, these data demonstrate that both injured and regenerating vessels exhibit abnormal permeability and suggest that Glut-1 expression during revascularization is dependent on the presence of astrocytes.

Differential temporal expression of matrix metalloproteinases after spinal cord injury: relationship to revascularization and wound healing.

OBJECT: Matrix metalloproteinases (MMPs), particularly MMP-9/gelatinase B, promote early inflammation and barrier disruption after spinal cord injury (SCI). Early blockade of MMPs after injury provides neuroprotection and improves motor outcome. There is recent evidence, however, that MMP-9 and MMP-2/gelatinase A participate in later wound healing in the injured cord. The authors therefore examined the activity of these gelatinases during revascularization and glial scar formation in the contused murine spinal cord. METHODS: Gelatinase activity was evaluated using gelatin zymography 24 hours after a mild, moderate, or severe contusion injury. The active form of MMP-2 was not detected, whereas MMP-9 activity was evident in all SCI groups and rose with increasing injury severity. The temporal expression of gelatinases was then examined using gelatin zymography after a moderate SCI. The active form of MMP-9 was most prominent at 1 day, extended through the early period of revascularization, and returned to control by 14 days. The active form of MMP-2 appeared at 7 days postinjury and remained elevated compared with that documented in sham-treated mice for at least 21 days. Increased MMP-2 activity coincided with both revascularization and glial scar formation. Using in situ zymography, gelatinolytic activity was detected in the meninges, vascular elements, glia, and macrophage-like cells in the injured cord. Results of immunolabeling confirmed the presence of gelatinase in vessels during revascularization and in reactive astrocytes associated with glial scar formation. CONCLUSIONS: These findings suggest that although MMP-9 and -2 exhibit overlapping expression during revascularization, the former is associated with acute injury responses and the latter with formation of a glial scar.