Multifaceted Benefits of GDF11 Treatment in Spinal Cord Injury: In Vitro and In Vivo Studies.

Traumatic spinal cord injury (SCI) initiates a series of cellular and molecular events that include both primary and secondary injury cascades. This secondary cascade provides opportunities for the delivery of therapeutic intervention. Growth differentiation factor 11 (GDF11), a member of the transforming growth factor-ß (TGF-ß) superfamily, regulates various biological processes in mammals. The effects of GDF11 in the nervous system were not fully elucidated. Here, we perform extensive in vitro and in vivo studies to unravel the effects of GDF11 on spinal cord after injury. In vitro culture studies showed that GDF11 increased the survival of both neuronal and oligodendroglial cells but decreased microglial cells. In stressed cultures, GDF11 effectively inhibited LPS stimulation and also protected neurons from ischemic damage. Intravenous GDF11 administration to rat after eliciting SCI significantly improved hindlimb functional restoration of SCI rats. Reduced neuronal connectivity was evident at 6 weeks post-injury and these deficits were markedly attenuated by GDF11 treatment. Furthermore, SCI-associated oligodendroglial alteration were more preserved by GDF11 treatment. Taken together, GDF11 infusion via intravenous route to SCI rats is beneficial, facilitating its therapeutic application in the future.

The Application of an Omentum Graft or Flap in Spinal Cord Injury.

BACKGROUND: Spinal cord injury (SCI) causes a primary injury at the lesion site and triggers a secondary injury and prolonged inflammation. There has been no definitive treatment till now. Promoting angiogenesis is one of the most important strategies for functional recovery after SCI. The omentum, abundant in blood and lymph vessels, possesses the potent ability of tissue regeneration. METHODS: The present work examines the efficacy of autologous omentum, either as a flap (with vascular connection intact) or graft (severed vascular connection), on spinal nerve regeneration. After contusive SCI in rats, a thin sheath of omentum was grafted to the injured spinal cord. RESULTS: Omental graft improved behavior scores significantly from the 3rd to 6th week after injury (6th week, 5.5 ± 0.5 vs. 8.6 ± 1.3, p < 0.05). Furthermore, the reduction in cavity and the preservation of class III ß-tubulin-positive nerve fibers in the injury area was noted. Next, the free omental flap was transposed to a completely transected SCI in rats through a pre-implanted tunnel. The flap remained vascularized and survived well several weeks after the operation. At 16 weeks post-treatment, SCI rats with omentum flap treatment displayed the preservation of significantly more nerve fibers (p < 0.05) and a reduced injured cavity, though locomotor scores were similar. CONCLUSIONS: Taken together, the findings of this study indicate that treatment with an omental graft or transposition of an omental flap on an injured spinal cord has a positive effect on nerve protection and tissue preservation in SCI rats. The current data highlight the importance of omentum in clinical applications.

Characterizing the Neuroprotective Effects of S/B Remedy (Scutellaria baicalensis Georgi and Bupleurum scorzonerifolfium Willd) in Spinal Cord Injury.

The main causes of dysfunction after a spinal cord injury (SCI) include primary and secondary injuries that occur during the first minutes, hours, to days after injury. This treatable secondary cascade provides a window of opportunity for delivering therapeutic interventions. An S/B remedy (Scutellaria baicalensis Georgi and Bupleurum scorzonerifolfium Willd) has anti-inflammatory, cytoprotective, and anticarcinogenic effects in liver or neurodegenerative diseases. The present work examined the effect of S/B on injured spinal cord neurons in cultures and in vivo. S/B effectively reduced peroxide toxicity and lipopolysaccharide stimulation in both spinal cord neuron/glial and microglial cultures with the involvement of PKC and HSP70. The effect of S/B was further conducted in contusive SCI rats. Intraperitoneal injections of S/B to SCI rats preserved spinal cord tissues and effectively attenuated microglial activation. Consistently, S/B treatment significantly improved hindlimb functions of SCI rats. In the acute stage of injury, S/B treatment markedly reduced the levels of ED1 expression and lactate and had a tendency to decrease lipid peroxidation. Taken together, we demonstrated long-term hindlimb restoration alongside histological improvements with systemic S/B remedy treatment in a clinically relevant model of contusive SCI. Our findings highlight the potential of an S/B remedy for acute therapeutic intervention after SCI.

Improving the regenerative potential of olfactory ensheathing cells by overexpressing prostacyclin synthetase and its application in spinal cord repair.

BACKGROUND: Olfactory ensheathing cells (OEC), specialized glia that ensheathe bundles of olfactory nerves, have been reported as a favorable substrate for axonal regeneration. Grafting OEC to injured spinal cord appears to facilitate axonal regeneration although the functional recovery is limited. In an attempt to improve the growth-promoting properties of OEC, we transduced prostacyclin synthase (PGIS) to OEC via adenoviral (Ad) gene transfer and examined the effect of OEC with enhanced prostacyclin synthesis in co-culture and in vivo. Prostacyclin is a vasodilator, platelet anti-aggregatory and cytoprotective agent. RESULTS: Cultured OEC expressed high level of cyclooxygneases, but not PGIS. Infection of AdPGIS to OEC could selectively augument prostacyclin synthesis. When cocultured with either OEC or AdPGIS-OEC, neuronal cells were resistant to OGD-induced damage. The resulted OEC were further transplanted to the transected cavity of thoracic spinal cord injured (SCI) rats. By 6 weeks post-surgery, significant functional recovery in hind limbs occurred in OEC or AdPGIS-OEC transplanted SCI rats compared with nontreated SCI rats. At 10-12 weeks postgraft, AdPGIS-OEC transplanted SCI rats showed significantly better motor restoration than OEC transplanted SCI rats. Futhermore, regenerating fiber tracts in the distal spinal cord stump were found in 40-60% of AdPGIS-OEC transplanted SCI rats. CONCLUSIONS: Enhanced synthesis of prostacyclin in grafted OEC improved fiber tract regeneration and functional restoration in spinal cord injured rats. These results suggest an important potential of prostacyclin in stimulating OEC therapeutic properties that are relevant for neural transplant therapies.

Cytoprotective and anti-inflammatory effects of PAL31 overexpression in glial cells.

BACKGROUND: Acute spinal cord injury (SCI) leads to a series of reactive changes and causes severe neurological deficits. A pronounced inflammation contributes to secondary pathology after SCI. Astroglia respond to SCI by proliferating, migrating, and altering phenotype. The impact of reactive gliosis on the pathogenesis of SCI is not fully understood. Our previous study has identified an inflammatory modulating protein, proliferation related acidic leucine-rich protein (PAL31) which is upregulated in the microglia/macrophage of injured cords. Because PAL31 participates in cell cycle progression and reactive astroglia often appears in the injured cord, we aim to examine whether PAL31 is involved in glial modulation after injury. RESULTS: Enhanced PAL31 expression was shown not only in microglia/macrophages but also in spinal astroglia after SCI. Cell culture study reveal that overexpression of PAL31 in mixed glial cells or in C6 astroglia significantly reduced LPS/IFNγ stimulation. Further, enhanced PAL31 expression in C6 astroglia protected cells from H2O2 toxicity; however, this did not affect its proliferative activity. The inhibiting effect of PAL31 on LPS/IFNγ stimulation was observed in glia or C6 after co-culture with neuronal cells. The results demonstrated that the overexpressed PAL31 in glial cells protected neuronal damages through inhibiting NF-kB signaling and iNOS. CONCLUSIONS: Our data suggest that PAL31upregulation might be beneficial after spinal cord injury. Reactive gliosis might become a good target for future therapeutic interventions.

Recovery of neurological function of ischemic stroke by application of conditioned medium of bone marrow mesenchymal stem cells derived from normal and cerebral ischemia rats.

BACKGROUND: Several lines of evidence have demonstrated that bone marrow-derived mesenchymal stem cells (BM-MSC) release bioactive factors and provide neuroprotection for CNS injury. However, it remains elusive whether BM-MSC derived from healthy donors or stroke patients provides equal therapeutic potential. The present work aims to characterize BM-MSC prepared from normal healthy rats (NormBM-MSC) and cerebral ischemia rats (IschBM-MSC), and examine the effects of their conditioned medium (Cm) on ischemic stroke animal model. RESULTS: Isolated NormBM-MSC or IschBM-MSC formed fibroblastic like morphology and expressed CD29, CD90 and CD44 but failed to express the hematopoietic marker CD34. The number of colony formation of BM-MSC was more abundant in IschBM-MSC than in NormBM-MSC. This is in contrast to the amount of Ficoll-fractionated mononuclear cells from normal donor and ischemic rats. The effect of cm of BM-MSC was further examined in cultures and in middle cerebral artery occlusion (MCAo) animal model. Both NormBM-MSC Cm and IschBM-MSC Cm effectively increased neuronal connection and survival in mixed neuron-glial cultures. In vivo, intravenous infusion of NormBM-MSC Cm and IschBM-MSC Cm after stroke onset remarkably improved functional recovery. Furthermore, NormBM-MSC Cm and IschBM-MSC Cm increased neurogenesis and attenuated microglia/ macrophage infiltration in MCAo rat brains. CONCLUSIONS: Our data suggest equal effectiveness of BM-MSC Cm derived from ischemic animals or from a normal population. Our results thus revealed the potential of BM-MSC Cm on treatment of ischemic stroke.

Evaluating the toxic mechanism of 1,2-diacetylbenzene in neural cells/tissues: The favorable impact of silibinin.

1,2-diacetylbenzene (1,2-DAB) is a neurotoxic component of aromatic solvents commonly used in industrial applications that induces neuropathological changes in animals. This study unraveled the toxic impact of 1,2-DAB in nerve tissues, explant cultures, and neuron-glial cultures, and explored whether herbal products can mitigate its toxicity. The effects of DAB on axonal transport were studied in retinal explant cultures grown in a micro-patterned dish. The mitochondrial movement in the axons was captured using time-lapse video recordings. The results showed that 1,2-DAB, but not 1,3-DAB inhibited axonal outgrowth and mitochondrial movement in a dose-dependent manner. The toxicity of 1,2-DAB was further studied in spinal cord tissues and cultures. 1,2-DAB selectively induced modifications of microtubules and neurofilaments in spinal cord tissues. 1,2-DAB also potently induced cell damage in both neuronal and glial cultures. Further, 1,2-DAB-induced cellular ATP depletion precedes cell damage in glial cells. Interestingly, treatment with the herbal products silibinin or silymarin effectively mitigated 1,2-DAB-induced toxicity in spinal cord tissues and neuronal/glial cultures. Collectively, the molecular toxicity of 1,2-DAB in neural tissues involves protein modification, ATP depletion, and axonal transport defects, leading to cell death. Silibinin and silymarin show promising neuroprotective effects against 2-DAB-induced toxicity.

Minocycline exhibits synergism with conditioned medium of bone marrow mesenchymal stem cells against ischemic stroke.

Several lines of evidence show that a conditioned medium of bone marrow mesenchymal stem cells (BM-MSCcm) improve functional recovery after ischemic stroke but do not reduce ischemic lesions. It is important to develop a treatment strategy that can exhibit a synergistic effect with BM-MSCcm against ischemic stroke. In this study, the effect of BM-MSCcm and/or minocycline was examined in culture and in a middle cerebral artery occlusion (MCAo) animal model. In neuron-glial cultures, BM-MSCcm and combined treatment, but not minocycline, effectively increased neuronal connection and oligodendroglial survival. In contrast, minocycline and combined treatment, but not BM-MSCcm, reduced toxin-induced free radical production in cultures. Either minocycline or BM-MSCcm, or in combination, conferred protective effects against oxygen glucose deprivation-induced cell damage. In an in vivo study, BM-MSCcm and minocycline were administered to rats 2 h after MCAo. Monotherapy with BM-MSCcm or minocycline after ischemic stroke resulted in 9.4% or 17.5% reduction in infarction volume, respectively, but there was no significant difference. Interestingly, there was a 33.9% significant reduction in infarction volume by combined treatment with BM-MSCcm and minocycline in an in vivo study. The combined therapy also significantly improved grasping power, which was not altered by monotherapy. Furthermore, combined therapy increased the expression of neuronal nuclei in the peri-infarct area and hippocampus, and concurrently decreased the expression of ED1 in rat brain and the peri-infarct zone. Our data suggest that minocycline exhibits a synergistic effect with BM-MSCcm against ischemic stroke not only to improve neurological functional outcome but also to reduce cerebral infarction.

Stem cell transplantation and/or adenoviral glial cell line-derived neurotrophic factor promote functional recovery in hemiparkinsonian rats.

BACKGROUND: Parkinson's disease (PD) is a neurological disorder characterized by the progressive loss of midbrain dopamine (DA) neurons. Bone marrow mesenchymal stem cells (BMSCs) can differentiate into multiple cell types including neurons and glia. Transplantation of BMSCs is regarded as a potential approach for promoting neural regeneration. Glial cell line-derived neurotrophic factor (GDNF) can induce BMSC differentiation into neuron-like cells. This work evaluated the efficacy of nigral grafts of human BMSCs (hMSCs) and/or adenoviral (Ad) GDNF gene transfer in 6-hydroxydopamine (6-OHDA)-lesioned hemiparkinsonian rats. AIM: To evaluate the efficacy of nigral grafts of hMSCs and/or Ad-GDNF gene transfer in 6-OHDA-lesioned hemiparkinsonian rats. METHODS: We used immortalized hMSCs, which retain their potential for neuronal differentiation. hMSCs, preinduced hMSCs, or Ad-GDNF effectively enhanced neuronal connections in cultured neurons. In vivo, preinduced hMSCs and/or Ad-GDNF were injected into the substantia nigra (SN) after induction of a unilateral 6-OHDA lesion in the nigrostriatal pathway. RESULTS: Hemiparkinsonian rats that received preinduced hMSC graft and/or Ad-GDNF showed significant recovery of apomorphine-induced rotational behavior and the number of nigral DA neurons. However, DA levels in the striatum were not restored by these therapeutic treatments. Grafted hMSCs might reconstitute a niche to support tissue repair rather than contribute to the generation of new neurons in the injured SN. CONCLUSION: The results suggest that preinduced hMSC grafts exert a regenerative effect and may have the potential to improve clinical outcome.

Hydrogel-based zinc ion sensor on optical fiber with high resolution and application to neural cells.

Solid-state zinc ion sensor is developed with high enough resolution and reproducibility for the potential application in brain injury monitoring. An optical diffuser is incorporated into the zinc ion sensor based on optical fiber and hydrogel doped with the fluorescent zinc ion probe molecule meso-2,6-Dichlorophenyltripyrrinone (TPN-Cl2). The diffuser transforms the high-peak-intensity excitation light near the fiber end into a broad light with moderate local intensity to reduce the degradation of the probe molecule. Reversible detection can be reached for 1, 2, and 5 µM (10-6 Molar), with slopes 0.3, 0.6, and 0.8 respectively. This is the pathophysiological concentration range after brain injury. The sensor is applied to neuron-glial cultures and macrophage under the stimulation of lipopolysaccharide (LPS), KCl and oxygen/glucose deprivation (OGD) that reflect inflammation, depolarization and ischemia respectively, mimicking events after brain injury. The zinc ion level is raised to 4-5 µM after LPS treatment, and then reduced to <3 µM after the co-treatment with the herbal drug silymarin. The results suggest the conditions of the neural cells under stress can be monitored.

Attenuating Spinal Cord Injury by Conditioned Medium from Bone Marrow Mesenchymal Stem Cells.

Spinal cord injury (SCI) is a devastating neurological condition and might even result in death. However, current treatments are not sufficient to repair such damage. Bone marrow mesenchymal stem cells (BM-MSC) are ideal transplantable cells which have been shown to modulate the injury cascade of SCI mostly through paracrine effects. The present study investigates whether systemic administration of conditioned medium from MSCs (MSCcm) has the potential to be efficacious as an alternative to cell-based therapy for SCI. In neuron-glial cultures, MSC coculture effectively promoted neuronal connection and reduced oxygen glucose deprivation-induced cell damage. The protection was elicited even if neuron-glial culture was used to expose MSCcm, suggesting the effects possibly from released fractions of MSC. In vivo, intravenous administration of MSCcm to SCI rats significantly improved behavioral recovery from spinal cord injury, and there were increased densities of axons in the lesion site of MSCcm-treated rats compared to SCI rats. At early days postinjury, MSCcm treatment upregulated the protein levels of Olig 2 and HSP70 and also increased autophage-related proteins in the injured spinal cords. Together, these findings suggest that MSCcm treatment promotes spinal cord repair and functional recovery, possibly via activation of autophagy and enhancement of survival-related proteins.

Effect of enhanced prostacyclin synthesis by adenovirus-mediated transfer on lipopolysaccharide stimulation in neuron-glia cultures.

Prostacyclin (PGI2) is known as a short-lived, potent vasodilator and platelet anti-aggregatory eicosanoid. This work attempts to selectively augment PGI2 synthesis in neuron-glia cultures by adenoviral (Ad) gene transfer of PGI synthase (PGIS) or bicistronic cyclooxygenase 1 (COX-1)/PGIS and examines whether PGI2 confers protection against lipopolysaccharide (LPS) stimulation. Cultures released low levels of eicosanoids. Upon Ad-PGIS or Ad-COX-1/PGIS infection, cultures selectively increased prostacyclin release. Both PGIS- and COX-1/PGIS-overexpressed cultures contained fewer microglial numbers. Further, they significantly attenuated LPS-induced iNOS expression and lactate, nitric oxide, and TNF-alpha production. Taken together, enhanced prostacyclin synthesis in neuron-glial cultures reduced microglia number and suppressed LPS stimulation.

Enhanced prostacyclin synthesis by adenoviral gene transfer reduced glial activation and ameliorated dopaminergic dysfunction in hemiparkinsonian rats.

Prostacyclin (PGI2), a potent vasodilator and platelet antiaggregatory eicosanoid, is cytoprotective in cerebral circulation. It is synthesized from arachidonic acid (AA) by the sequential action of cyclooxygenase- (COX-) 1 or 2 and prostacyclin synthase (PGIS). Because prostacyclin is unstable in vivo, PGI2 analogs have been developed and demonstrated to protect against brain ischemia. This work attempts to selectively augment PGI2 synthesis in mixed glial culture or in a model of Parkinson's disease (PD) by direct adenoviral gene transfer of prostacyclin biosynthetic enzymes and examines whether it confers protection in cultures or in vivo. Confluent mixed glial cultures actively metabolized exogenous AA into PGE2 and PGD2. These PGs were largely NS398 sensitive and considered as COX-2 products. Gene transfer of AdPGIS to the cultures effectively shunted the AA catabolism to prostacyclin synthesis and concurrently reduced cell proliferation. Furthermore, PGIS overexpression significantly reduced LPS stimulation in cultures. In vivo, adenoviral gene transfer of bicistronic COX-1/PGIS to substantia nigra protected 6-OHDA- induced dopamine depletion and ameliorated behavioral deficits. Taken together, this study shows that enhanced prostacyclin synthesis reduced glial activation and ameliorated motor dysfunction in hemiparkinsonian rats. Prostacyclin may have a neuroprotective role in modulating the inflammatory response in degenerating nigra-striatal pathway.

Silymarin protects spinal cord and cortical cells against oxidative stress and lipopolysaccharide stimulation.

Contusive spinal cord injury (SCI) is a devastating event which leads to a loss of neurological function below the level of injury. A secondary degenerative process is initiated following acute SCI. This secondary cascade provides opportunities for the delivery of therapeutic interventions. Silymarin, a widely used "liver herb", is frequently used for the protection against various hepatobiliary problems. However, the effectiveness of silymarin in central nervous system (CNS), especially in spinal cord, is not firmly established. The present work evaluates the effects of silymarin and its major constituent, silybin, on oxidative stress and lipopolysaccharide (LPS) stimulation in primary neuronal/glial cell cultures and in vivo. Silymarin or silybin inhibited glial cell proliferation in a concentration-dependent manner. Furthermore, it protected glial cells against peroxide-induced reactive oxygen species (ROS) formation, ATP depletion, and cell damage. Interestingly, the inhibition of peroxide-induced ROS by silybin could be partially attenuated by inhibitors of NFκB or protein kinase C (PKC), suggesting an involvement of NFκB and PKC signaling pathways. In mixed neuronal/glial cell cultures from cerebral cortex or spinal cord, silymarin or silybin effectively attenuated peroxide-induced ROS formation, with silymarin being more effective than silybin, implicating other constituents of silymarin that may be involved. Consistently, silymarin reduced LPS-induced injures in spinal neuronal/glial cell cultures. In vivo, intrathecal administration of silymarin immediately after eliciting contusive SCI effectively improved hindlimb locomotor behavior in the rats. Taken together, silymarin or silybin shows promise in protecting the CNS cells from toxin- or injury-induced damages and might be used to treat head- or spinal cord-injuries related to free radical assault.

Enhanced expression of glycine N-methyltransferase by adenovirus-mediated gene transfer in CNS culture is neuroprotective.

Glycine N-methyltransferase (GNMT) is the most abundant hepatic methyltransferase and plays important roles in regulating methyl group metabolism. In the central nervous system, GNMT expression is low and its function has not been revealed. The present study examines the effect of GNMT overexpression by adenovirus-mediated transfer in cortical mixed neuron-glial cultures. Infection of adenovirus encoding green fluorescence protein to cultures demonstrates high preference for non-neuronal cells. Optimal GNMT overexpression in cultures by adenoviral GNMT (Ad-GNMT) infection not only induces protein kinase C phosphorylation, but also increases neuronal/oligodendroglial survival. Furthermore, these Ad-GNMT-infected cultures are significantly resistant to H(2)O(2) toxicity and lipopolysaccharide stimulation. Conditioned media from Ad-GNMT-infected microglia also significantly enhance neuronal survival. Taken together, enhanced GNMT expression in mixed neuronal-glial cultures is neuroprotective, most likely mediated through non-neuronal cells.

Dual effect of adenovirus-mediated transfer of BMP7 in mixed neuron-glial cultures: neuroprotection and cellular differentiation.

Bone morphogenetic proteins (BMPs), members of the TGF-beta superfamily, have been implicated in nervous system development and in response to injury. Previous studies have shown that recombinant BMP7 can enhance dendritic growth and protect cultured neurons from oxidative stress. Because of the presence of extracellular BMP antagonists, BMP7 seems to act locally. Therefore, the present study uses BMP7 overexpression using adenovirus (Ad)-mediated gene transfer to examine its effect in mixed neuronal cultures. Enhanced BMP7 expression selectively induces neuronal CGRP expression in a time-dependent manner. BMP7 overexpression not only significantly protects cultures from H2O2 toxicity but reduces lipopolysaccharide (LPS) stimulation. Concurrently, it profoundly reduces microglial numbers, but increases oligodendroglial and endothelial cells. Together, low-dose and continuously expressed BMP7 is both neuroprotective and differentiation-inductive.