Analysis of Gene Expression Profiles Associated with Functional Recovery After Spinal Cord Injury Caused by Sema4D Knockdown in Oligodendrocytes

We previously showed that sema4D Knockdown in oligodendrocytes promotes functional recovery after spinal cord injury. In this paper, we examined gene expression profiles associated with functional recovery by PCR array. For general observation during first 4 weeks, we found that sema4D knockdown could reduce edema and stimulate SCEP. Further, PCR array analysis indicated sema4D knockdown in OPCs inhibited wound tissue angiogenesis and inflammation genes expression and upregulated axon regeneration genes expression at early phase. Our findings provided the molecular mechanism for its potential application.     

Oncostatin M Reduces Lesion Size and Promotes Functional Recovery and Neurite Outgrowth After Spinal Cord Injury

The family of interleukin (IL)-6 like cytokines plays an important role in the neuroinflammatory response to injury by regulating both neural as well as immune responses. Here, we show that expression of the IL-6 family member oncostatin M (OSM) and its receptor is upregulated after spinal cord injury (SCI). To reveal the relevance of increased OSM signaling in the pathophysiology of SCI, OSM was applied locally after spinal cord hemisection in mice. OSM treatment significantly improved locomotor recovery after mild and severe SCI. Improved recovery in OSM-treated mice was associated with a reduced lesion size. OSM significantly diminished astrogliosis and immune cell infiltration. Thus, OSM limits secondary damage after CNS trauma. In vitro viability assays demonstrated that OSM protects primary neurons in culture from cell death, suggesting that the underlying mechanism involves direct neuroprotective effects of OSM. Furthermore, OSM dose-dependently promoted neurite outgrowth in cultured neurons, indicating that the cytokine plays an additional role in CNS repair. Indeed, our in vivo experiments demonstrate that OSM treatment increases plasticity of serotonergic fibers after SCI. Together, our data show that OSM is produced at the lesion site, where it protects the CNS from further damage and promotes recovery.     

Plasminogen Activator Promotes Recovery Following Spinal Cord Injury

Plasminogen activators play an important role in synaptic plasticity associated with the crossed phrenic phenomenon (CPP) and recovery of respiratory function after spinal cord injury. A genetic approach using knockout mice lacking various genes in the plasminogen activator/plasmin system has shown that induction of urokinase plasminogen activator (uPA) is required during the first hour after a C2-hemisection for the acquisition of the CPP response. The uPA knockout mice do not show the structural remodeling of phrenic motor neuron synapses characteristic of the CPP response. As shown here uPA acts in a cell signaling manner via binding to its receptor uPAR rather than as a protease, since uPAR knockout mice or knock-in mice possessing a modified uPA that is unable to bind to uPAR both fail to generate a CPP and recover respiratory function. Microarray data and real-time PCR analysis of mRNAs induced in the phrenic motor nucleus after C2-hemisection in C57Bl/6 mice as compared to uPA knockout mice indicate a potential cell signaling cascade downstream possibly involving β-integrin and Src, and other pathways. Identification of these uPA-mediated signaling pathways may provide the opportunity to pharmacologically upregulate the synaptic plasticity necessary for recovery of phrenic motoneuron activity following cervical spinal cord injury.     

Restoring Cellular Energetics Promotes Axonal Regeneration and Functional Recovery after Spinal Cord Injury

1. Download : Download high-res image (201KB)
2. Download : Download full-size image

     

Clemastine Enhances Myelination,Delays Axonal Loss and Promotes Functional Recovery in Spinal Cord Injury

Recent evidence has shown that demyelination occurs along with axonal degeneration in spinal cord injury (SCI) during the secondary injury phase. Oligodendrocyte precursor cells (OPC) are present in the lesions but fail to differentiate into mature oligodendrocytes and form new myelin. Given the limited recovery of neuronal functions after SCI in adults without effective treatment available so far, it remains unknown whether enhancing OPC differentiation and myelination could benefit the recovery of SCI. To show the significance of myelin regeneration after SCI, the injury was treated with clemastine in the rat model. Clemastine is an FDA-approved drug that is potent in promoting oligodendrocyte differentiation and myelination in vivo, for four weeks following SCI. Motor function was assessed using sloping boards and grid walking tests and scored according to the Basso, Beattie, and Bresnahan protocol. The myelin integrity and protein expression were evaluated using transmission electron microscopy and immunofluorescence, respectively. The results indicated that clemastine treatment preserves myelin integrity, decreases loss of axons and improves functional recovery in the rat SCI model. The presented data suggest that myelination-enhancing strategies may serve as a potential therapeutic approach for the functional recovery in SCI.

     

Transplants of Human Mesenchymal Stem Cells Improve Functional Recovery After Spinal Cord Injury in the Rat

Human mesenchymal stem cells (hMSCs) derived from adult bone marrow represent a potentially useful source of cells for cell replacement therapy after nervous tissue damage. They can be expanded in culture and reintroduced into patients as autografts or allografts with unique immunologic properties. The aim of the present study was to investigate (i) survival, migration, differentiation properties of hMSCs transplanted into non-immunosuppressed rats after spinal cord injury (SCI) and (ii) impact of hMSC transplantation on functional recovery. Seven days after SCI, rats received i.v. injection of hMSCs (2×10⁶ in 0.5 mL DMEM) isolated from adult healthy donors. Functional recovery was assessed by Basso–Beattie–Bresnahan (BBB) score weekly for 28 days. Our results showed gradual improvement of locomotor function in transplanted rats with statistically significant differences at 21 and 28 days. Immunocytochemical analysis using human nuclei (NUMA) and BrdU antibodies confirmed survival and migration of hMSCs into the injury site. Transplanted cells were found to infiltrate mainly into the ventrolateral white matter tracts, spreading also to adjacent segments located rostro-caudaly to the injury epicenter. In double-stained preparations, hMSCs were found to differentiate into oligodendrocytes (APC), but not into cells expressing neuronal markers (NeuN). Accumulation of GAP-43 regrowing axons within damaged white matter tracts after transplantation was observed. Our findings indicate that hMSCs may facilitate recovery from spinal cord injury by remyelinating spared white matter tracts and/or by enhancing axonal growth. In addition, low immunogenicity of hMSCs was confirmed by survival of donor cells without immunosuppressive treatment.     

Adipose-Derived Stem Cells Expressing the Neurogenin-2 Promote Functional Recovery After Spinal Cord Injury in Rat

Neurogenin2 (Ngn2) is a proneural gene that directs neuronal differentiation of progenitor cells during development. This study aimed to investigate whether the use of adipose-derived stem cells (ADSCs) over-expressing the Ngn2 transgene (Ngn2–ADSCs) could display the characteristics of neurogenic cells and improve functional recovery in an experimental rat model of SCI. ADSCs from rats were cultured and purified in vitro, followed by genetically modified with the Ngn2 gene. Forty-eight adult female Sprague–Dawley rats were randomly assigned to three groups: the control, ADSCs, and Ngn2–ADSCs groups. The hind-limb motor function of all rats was recorded using the Basso, Beattie, and Bresnahan locomotor rating scale for 8 weeks. Moreover, hematoxylineosin staining and immunohistochemistry were also performed. After neural induction, positive expression rate of NeuN in Ngn2–ADSCs group was upon 90 %. Following transplantation, a great number of ADSCs was found around the center of the injury spinal cord at 1 and 4 weeks, which improved retention of tissue at the lesion site. Ngn2–ADSCs differentiated into neurons, indicated by the expression of neuronal markers, NeuN and Tuj1. Additionally, transplantation of Ngn2–ADSCs upregulated the trophic factors (brain-derived neurotrophic factor and vascular endothelial growth factor), and inhibited the glial scar formation, which was indicated by immunohistochemistry with glial fibrillary acidic protein. Finally, Ngn2–ADSCs-treated animals showed the highest functional recovery among the three groups. These findings suggest that transplantation of Ngn2-overexpressed ADSCs promote the functional recovery from SCI, and improve the local microenvironment of injured cord in a more efficient way than that with ADSCs alone.     

Autophagy Reduces Neuronal Damage and Promotes Locomotor Recovery via Inhibition of Apoptosis After Spinal Cord Injury in Rats

Autophagy is an intracellular catabolic mechanism that maintains the balance of proteins, lipids and aging organelles. 3-Methyladenine (3-MA) is a selective inhibitor of autophagy, whereas rapamycin, an antifungal agent, is a specific inducer of autophagy, inhibiting the protein mammalian target of rapamycin. In the present study, we examined the role of autophagy, inhibited by 3-MA and enhanced by rapamycin, in a model of acute spinal cord injury in rats. We found that rapamycin could significantly increase the expression of microtubule-associated protein 1 light chain 3 (LC3) and Beclin1 at the injury site. At the same time, the number of neurons and astrocytes with LC3 positive in the spinal cord was upregulated with time. In addition, administration of rapamycin produced an increase in the Basso, Beattie and Bresnahan scores of injured rats, indicating high recovery of locomotor function. Furthermore, expression of the proteins Bcl-2 and Bax was upregulated and downregulated, respectively. By contrast, the results for rats treated with 3-MA, which inhibits autophagy, were the opposite of those seen with the rapamycin-treated rats. These results show that induction of autophagy can produce neuroprotective effects in acute spinal cord injury in rats via inhibition of apoptosis.     

Photomechanical Wave-Driven Delivery of siRNAs Targeting Intermediate Filament Proteins Promotes Functional Recovery after Spinal Cord Injury in Rats

The formation of glial scars after spinal cord injury (SCI) is one of the factors inhibiting axonal regeneration. Glial scars are mainly composed of reactive astrocytes overexpressing intermediate filament (IF) proteins such as glial fibrillary acidic protein (GFAP) and vimentin. In the current study, we delivered small interfering RNAs (siRNAs) targeting these IF proteins to SCI model rats using photomechanical waves (PMWs), and examined the restoration of motor function in the rats. PMWs are generated by irradiating a light-absorbing material with 532-nm nanosecond laser pulses from a Q-switched Nd:YAG laser. PMWs can site-selectively increase the permeability of the cell membrane for molecular delivery. Rat spinal cord was injured using a weight-drop device and the siRNA(s) solutions were intrathecally injected into the vicinity of the exposed SCI, to which PMWs were applied. We first confirmed the substantial uptake of fluorescence-labeled siRNA by deep glial cells; then we delivered siRNAs targeting GFAP and vimentin into the lesion. The treatment led to a significant improvement in locomotive function from five days post-injury in rats that underwent PMW-mediated siRNA delivery. This was attributable to the moderate silencing of the IF proteins and the subsequent decrease in the cavity area in the injured spinal tissue.     

The Adhesion Molecule-Characteristic HNK-1 Carbohydrate Contributes to Functional Recovery After Spinal Cord Injury in Adult Zebrafish

The human natural killer cell antigen-1 (HNK-1) is functionally important in development, synaptic activity, and regeneration after injury in the nervous system of several mammalian species. It contains a sulfated glucuronic acid which is carried by neural adhesion molecules and expressed in nonmammalian species, including zebrafish, which, as opposed to mammals, spontaneously regenerate after injury in the adult. To evaluate HNK-1’s role in recovery of function after spinal cord injury (SCI) of adult zebrafish, we assessed the effects of the two HNK-1 synthesizing enzymes, glucuronyl transferase and HNK-1 sulfotransferase. Expression of these two enzymes was increased at the messenger RNA (mRNA) level 11 days after injury in the brainstem nuclei that are capable of regrowth of severed axons, namely, the nucleus of medial longitudinal fascicle and intermediate reticular formation, but not at earlier time points after SCI. mRNA levels of glucuronyl transferase and sulfotransferase were increased in neurons, not only of these nuclei but also in the spinal cord caudal to the injury site at 11 days. Mauthner neurons which are not capable of regeneration did not show increased levels of enzyme mRNAs after injury. Reducing protein levels of the enzymes by application of anti-sense morpholinos resulted in reduction of locomotor recovery for glucuronyl transferase, but not for HNK-1 sulfotransferase. The combined results indicate that HNK-1 is upregulated in expression only in those neurons that are intrinsically capable of regeneration and contributes to regeneration after spinal cord injury in adult zebrafish in the absence of its sulfate moiety.     

Granulocyte Colony-Stimulating Factor (G-CSF) Protects Oligpdendrocyte and Promotes Hindlimb Functional Recovery after Spinal Cord Injury in Rats

Background

Granulocyte colony-stimulating factor (G-CSF) is a protein that stimulates differentiation, proliferation, and survival of cells in the granulocytic lineage. Recently, a neuroprotective effect of G-CSF was reported in a model of cerebral infarction and we previously reported the same effect in studies of murine spinal cord injury (SCI). The aim of the present study was to elucidate the potential therapeutic effect of G-CSF for SCI in rats.

Methods

Adult female Sprague-Dawley rats were used in the present study. Contusive SCI was introduced using the Infinite Horizon Impactor (magnitude: 200 kilodyne). Recombinant human G-CSF (15.0 µg/kg) was administered by tail vein injection at 1 h after surgery and daily the next four days. The vehicle control rats received equal volumes of normal saline at the same time points.

Results

Using a contusive SCI model to examine the neuroprotective potential of G-CSF, we found that G-CSF suppressed the expression of pro-inflammatory cytokine (IL-1 beta and TNF- alpha) in mRNA and protein levels. Histological assessment with luxol fast blue staining revealed that the area of white matter spared in the injured spinal cord was significantly larger in G-CSF-treated rats. Immunohistochemical analysis showed that G-CSF promoted up-regulation of anti-apoptotic protein Bcl-Xl on oligpodendrocytes and suppressed apoptosis of oligodendrocytes after SCI. Moreover, administration of G-CSF promoted better functional recovery of hind limbs.

Conclusions

G-CSF protects oligodendrocyte from SCI-induced cell death via the suppression of inflammatory cytokines and up-regulation of anti-apoptotic protein. As a result, G-CSF attenuates white matter loss and promotes hindlimb functional recovery.     

Downregulation of USP4 Promotes Activation of Microglia and Subsequent Neuronal Inflammation in Rat Spinal Cord After Injury

Neurochemical Research - NF-κB is involved in the activation of microglia, which induces secondary spinal cord injury (SCI). This process involves the activation of NF-κB signaling...     

Naringin Treatment Improves Functional Recovery by Increasing BDNF and VEGF Expression, Inhibiting Neuronal Apoptosis After Spinal Cord Injury

The aim of this study was to determine the therapeutic efficacy of starting naringin treatment 1 day after spinal cord injury (SCI) in rat and to investigate the underlying mechanism. SCI was induced using the modified weight-drop method in Sprague-Dawley rats. The SCI animals were randomly divided into three groups: vehicle-treated group; 20 mg/kg naringin-treated group; 40 mg/kg naringin-treated group, and additionally with sham group (laminectomy only). Locomotors functional recovery was assessed during the 6 weeks post operation period by performing open-field locomotors tests and inclined-plane tests. At the end of the study, the segments of spinal cord encompassing the injury site were removed for histopathological analysis. Immunohistochemistry was performed to observe the expression of the brain-derived neurotrophic factor (BDNF). The expression of vascular endothelial growth factor (VEGF), B-cell CLL/lymphoma-2 (Bcl-2), BCL-2-associated X protein (Bax) and caspase-3 were detected by Western blot analysis. The apoptotic neural cells were assessed using the TUNEL method. The results showed that the naringin-treated animals had significantly better locomotor function recovery, less myelin loss, and higher expression of BDNF and VEGF. In addition, naringin treatment significantly increased in Bcl-2:Bax ratio, reduced the enzyme activity of caspase-3 and decreased the number of apoptotic cells after SCI. These findings suggest that naringin treatment starting 1 day after SCI can significantly improve locomotor recovery, and this neuroprotective effect may be related to the upregulation of BDNF and VEGF and the inhibition of neural apoptosis. Therefore, naringin may be useful as a promising therapeutic agent for SCI.     

Intrathecal Epigallocatechin Gallate Treatment Improves Functional Recovery After Spinal Cord Injury by Upregulating the Expression of BDNF and GDNF

This study aimed to investigate the therapeutic effects of epigallocatechin-3-gallate (EGCG) administered by subarachnoid injection following spinal cord injury (SCI) in rats and to explore the underlying mechanism. Sprague–Dawley rats were randomly divided into four groups of 12 as follows: a sham group (laminectomy only); a control group; a 10 mg/kg EGCG-treated group; and a 20 mg/kg EGCG-treated group. SCI was induced in the rats using the modified weight-drop method (10 g × 4 cm) at the T10 (10th thoracic vertebral) level. EGCG (10 or 20 mg/kg) or vehicle as control was administered by subarachnoid injection at lumbar level 4 immediately after SCI. Locomotor functional recovery was assessed during the four weeks post-operation using open-field locomotor tests and inclined-plane tests. At the end of the study, the segments of spinal cord encompassing the injury site were removed for histopathological analysis. Immunohistochemical and Western blot analyses were performed to observe the expression of: the B cell CLL/lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF). The results showed that the EGCG-treated animals had significantly better recovery of locomotor function, less myelin loss, greater Bcl-2 expression and attenuated Bax expression. In addition, the EGCG treatment significantly increased the expression of BDNF and GDNF after SCI. These findings suggest that EGCG treatment can significantly improve locomotor recovery, and this neuroprotective effect may be related to the up-regulation of BDNF and GDNF, and the inhibition of apoptosis-related proteins. Therefore, EGCG may be a promising therapeutic agent for SCI.     

Long-Term Changes in Spinal Cord Evoked Potentials After Compression Spinal Cord Injury in the Rat

SUMMARY 1. After traumatic spinal cord injury (SCI), histological and neurological consequences are developing for several days and even weeks. However, little is known about the dynamics of changes in spinal axonal conductivity. The aim of this study was to record and compare repeated spinal cord evoked potentials (SCEP) after SCI in the rat during a 4 weeks’ interval. These recordings were used: (i) for studying the dynamics of functional changes in spinal axons after SCI, and (ii) to define the value of SCEP as an independent outcome parameter in SCI studies.2. We have used two pairs of chronically implanted epidural electrodes for stimulation/recording. The electrodes were placed below and above the site of injury, respectively. Animals with implanted electrodes underwent spinal cord compression injury induced by epidural balloon inflation at Th8–Th9 level. There were five experimental groups of animals, including one control group (sham-operated, no injury), and four injury groups (different degrees of SCI).3. After SCI, SCEP waveform was either significantly reduced or completely lost. Partial recovery of SCEPs was observed in all groups. The onset and extent of recovery clearly correlated with the severity of injury.There was good correlation between quantitated SCEP variables and the volumes of the compressing balloon. However, sensitivity of electropohysiological parameters was inferior compared to neurological and morphometric outcomes.4. Our study shows for the first time, that the dynamics of axonal recovery depends on the degree of injury. After mild injury, recovery of signal is rapid. However, after severe injury, axonal conductivity can re-appear after as long as 2 weeks postinjury.In conclusion, SCEPs can be used as an independent parameter of outcome after SCI, but in general, the sensitivity of electrophysiological data were worse than standard morphological and neurological evaluations.     

LIN28 Expression in Rat Spinal Cord After Injury

LIN28, an RNA-binding protein, is known to be involved in the regulation of many cellular processes, such as embryonic stem cell proliferation, cell fate succession, developmental timing, and oncogenesis. However, its expression and function in central nervous system still unclear. In this study, we performed an acute spinal cord contusion injury (SCI) model in adult rats and investigated the dynamic changes of LIN28 expression in spinal cord. Western blot and immunohistochemistry analysis revealed that LIN28 was present in normal spinal cord. It gradually increased, reached a peak at 3 day, and then nearly declined to the basal level at 14 days after SCI. Double immunofluorescence staining showed that LIN28 immunoreactivity was found in neurons, astrocytes and a handful of microglia. Interestingly, LIN28 expression was increased predominantly in astrocytes but not in neurons. Moreover, the colocalization of LIN28 and proliferating cell nuclear antigen was detected after injury. Western blot showed that LIN28 participated in lipopolysaccharide (LPS) induced astrocytes inflammatory responses by NF-κB signaling pathway. These results suggested that LIN28 may be involved in the pathologic process of SCI, and further research is needed to have a good understanding of its function and mechanism.