tretinoin and Spinal-Cord-Injuries

Experiments with sciatic nerve lesions and spinal cord contusion injury demonstrate that the retinoic acid (RA) signaling cascade is activated by these traumatic events. In both cases the RA-synthesizing enzyme is RALDH-2. In the PNS, lesions cause RA-induced gene transcription, intracellular translocation of retinoid receptors, and increased transcription of CRBP-I, CRABP-II, and retinoid receptors. The activation of RARbeta appears to be responsible for neurotrophic and neuritogenic effects of RA on dorsal root ganglia and embryonic spinal cord. While the physiological role of RA in the injured nervous system is still under investigation three domains of functions are suggested: (1) neuroprotection and support of axonal growth, (2) modulation of the inflammatory reaction by microglia/macrophages, and (3) regulation of glial differentiation. Few studies have been performed to support nerve regeneration with RA signals in vivo, but a large number of experiments with neuronal and glial cell cultures and spinal cord explants point to beneficial effects of RA, so that future therapeutic approaches will likely focus on the activation of RA signaling.

Topics: Animals; Humans; Nerve Regeneration; Neurons; Sciatic Nerve; Signal Transduction; Spinal Cord Injuries; Tretinoin

Spinal cord injury (SCI) is a significant public health issue that imposes numerous burdens on patients and society. Uncontrolled excessive inflammation in the second pathological phase of SCI can aggravate the injury. In this paper, we hypothesized that suppressing inflammatory pathways via autophagy could aid functional recovery, and prevent spinal cord tissue degeneration following SCI. To this end, we examined the effects of intrathecal injection of all-trans retinoic acid (ATRA)-preconditioned bone marrow mesenchymal stem cells (BM-MSCs) (ATRA-MSCs) on autophagy activity and the HMGB1/NF-κB/NLRP3 inflammatory pathway in an SCI rat model. This study demonstrated that SCI increased the expression of Beclin-1 (an autophagy-related gene) and NLRP3 inflammasome components such as NLRP3, ASC, Caspase-1, and pro-inflammatory cytokines IL-1β, IL-18, IL-6, and TNF-α. Additionally, following SCI, the protein levels of key autophagy factors (Beclin-1 and LC3-II) and HMGB1/NF-κB/NLRP3 pathway factors (HMGB1, p-NF-κB, NLRP3, IL-1β, and TNF-α) increased. Our findings indicated that ATRA-MSCs enhanced Beclin-1 and LC3-II levels, regulated the HMGB1/NF-κB/NLRP3 pathway, and inhibited pro-inflammatory cytokines. These factors improved hind limb motor activity and aided in the survival of neurons. Furthermore, ATRA-MSCs demonstrated greater beneficial effects than MSCs in treating spinal cord injury. Overall, ATRA-MSC treatment revealed beneficial effects on the damaged spinal cord by suppressing excessive inflammation and activating autophagy. Further research and investigation of the pathways involved in SCI and the use of amplified stem cells may be beneficial for future clinical use.

Topics: Animals; Autophagy; Beclin-1; HMGB1 Protein; Humans; Inflammation; Mesenchymal Stem Cells; NF-kappa B; NLR Family, Pyrin Domain-Containing 3 Protein; Rats; Spinal Cord Injuries; Tretinoin; Tumor Necrosis Factor-alpha

Motor neuron diseases such as spinal cord injuries and amyotrophic lateral sclerosis are known as the most common disorders worldwide. Using stem cells (e.g., human umbilical cord blood mesenchymal stem cells) is currently a potent medical approach for modulating the impact of neural damages and regeneration of spinal cord injuries. MicroRNAs (miRNA) are taken into account as principal regulators during differentiation. The miRNAs play a significant role in stem cell self-renewal and fate determination. There are few studies on how miRNAs regulate neural differentiation in stem cells. The purpose of this study is to explore miRNA profiles of CB-MSCs during differentiation into motor neuron-like cells. Human CB-MSCs were isolated and characterized using flow cytometry. Cell differentiation has been induced by combining retinoic acid (RA) and sonic hedgehog (Shh) in a two-step protocol for 14 days. Then, cell differentiation was confirmed by immunocytochemistry and flow cytometry. The miRNA was analyzed using Illumina/Solexa sequencing platform. In this regard, three libraries were prepared to investigate the effect of these two biological morphogens on the miRNA profile of the differentiating cells. These libraries were Control (non-treated CB-MSCs), Test 1 (RA + /Shh +), and Test 2 (RA-/Shh-). Quantitative RT-PCR was employed to verify miRNA expression. CB-MSCs were spindle-shaped in morphology, and they did not express hematopoietic markers. After differentiation, the cells expressed motor neuron markers (i.e., Islet-1, SMI-32, and ChAT) at the protein level after 14 days. The analysis of miRNA sequencing demonstrated a significant up-regulation of miR-9-5p and miR-324-5p in Test 1 (RA + /Shh +). Also, there is a considerable down-regulation of mir-137 and let-7b in Test 2 (RA-/Shh-). These results have been obtained by comparing them with the Control library. Indeed, they were responsible for neuron and motor neuron differentiation and suppression of proliferation in neural progenitor cells. Furthermore, significant up-regulation was detected in some novel microRNAs involved in cholinergic, JAK-STAT, and Hedgehog and MAPK signaling pathways. CB-MSCs are potent to express motor neuron markers. This procedure has been performed by developing a two-week protocol and employing Shh and RA. The miRNA profile analysis showed a significant up-regulation in the expression of some miRs involved in neuron differentiation and motor neuron maturation. MiR-9-5p and m

Topics: Cell Differentiation; Cholinergic Agents; Fetal Blood; Hedgehog Proteins; Humans; Mesenchymal Stem Cells; MicroRNAs; Motor Neurons; Spinal Cord Injuries; Tretinoin

Human adipose-derived stem cells (hADSCs) are increasingly presumed to be a prospective stem cell source for cell replacement therapy in various degenerative and/or traumatic diseases. The potential of trans-differentiating hADSCs into motor neuron cells indisputably provides an alternative way for spinal cord injury (SCI) treatment. In the present study, a stepwise and efficient hADSC trans-differentiation protocol with retinoic acid (RA), sonic hedgehog (SHH), and neurotrophic factors were developed. With this protocol hADSCs could be converted into electrophysiologically active motoneuron-like cells (hADSC-MNs), which expressed both a cohort of pan neuronal markers and motor neuron specific markers. Moreover, after being primed for neuronal differentiation with RA/SHH, hADSCs were transplanted into SCI mouse model and they survived, migrated, and integrated into injured site and led to partial functional recovery of SCI mice. When ablating the transplanted hADSC-MNs harboring HSV-TK-mCherry overexpression system with antivirial Ganciclovir (GCV), functional relapse was detected by motor-evoked potential (MEP) and BMS assays, implying that transplanted hADSC-MNs participated in rebuilding the neural circuits, which was further confirmed by retrograde neuronal tracing system (WGA). GFP-labeled hADSC-MNs were subjected to whole-cell patch-clamp recording in acute spinal cord slice preparation and both action potentials and synaptic activities were recorded, which further confirmed that those pre-conditioned hADSCs indeed became functionally active neurons in vivo. As well, transplanted hADSC-MNs largely prevented the formation of injury-induced cavities and exerted obvious immune-suppression effect as revealed by preventing astrocyte reactivation and favoring the secretion of a spectrum of anti-inflammatory cytokines and chemokines. Our work suggests that hADSCs can be readily transformed into MNs in vitro, and stay viable in spinal cord of the SCI mouse and exert multi-therapeutic effects by rebuilding the broken circuitry and optimizing the microenvironment through immunosuppression.

Topics: Animals; Cell Differentiation; Cell Transdifferentiation; Disease Models, Animal; Hedgehog Proteins; Humans; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Mice; Motor Neurons; Nerve Growth Factors; Spinal Cord Injuries; Tretinoin

In the CNS, oligodendrocytes are responsible for myelin formation and maintenance. Following spinal cord injury, oligodendrocyte loss and an inhibitory milieu compromise remyelination and recovery. Here, we explored the role of retinoic acid receptor-beta (RARβ) signaling in remyelination. Using a male Sprague Dawley rat model of PNS-CNS injury, we show that oral treatment with a novel drug like RARβ agonist, C286, induces neuronal expression of the proteoglycan decorin and promotes myelination and differentiation of oligodendrocyte precursor cells (NG2

Topics: Animals; Decorin; ErbB Receptors; Exosomes; Myelin Sheath; Nerve Regeneration; Oligodendroglia; Rats; Rats, Sprague-Dawley; Receptors, Retinoic Acid; Signal Transduction; Spinal Cord Injuries; Tretinoin

Spinal cord injury (SCI) induces the disruption of the blood-spinal cord barrier (BSCB), which leads to infiltration of blood cells, inflammatory responses and neuronal cell death, with subsequent development of spinal cord secondary damage. Recent reports pointed to an important role of retinoic acid (RA), the active metabolite of the vitamin A, in the induction of the blood-brain barrier (BBB) during human and mouse development, however, it is unknown whether RA plays a role in maintaining BSCB integrity under the pathological conditions such as SCI. In this study, we investigated the BSCB protective role of RA both in vivo and in vitro and demonstrated that autophagy are involved in the BSCB protective effect of RA. Our data show that RA attenuated BSCB permeability and also attenuated the loss of tight junction molecules such as P120, β-catenin, Occludin and Claudin5 after injury in vivo as well as in brain microvascular endothelial cells. In addition, RA administration improved functional recovery of the rat model of trauma. We also found that RA could significantly increase the expression of LC3-II and decrease the expression of p62 both in vivo and in vitro. Furthermore, combining RA with the autophagy inhibitor chloroquine (CQ) partially abolished its protective effect on the BSCB and exacerbated the loss of tight junctions. Together, our studies indicate that RA improved functional recovery in part by the prevention of BSCB disruption via the activation of autophagic flux after SCI.

Topics: Animals; Autophagy; beta Catenin; Brain; Catenins; Cells, Cultured; Claudins; Delta Catenin; Female; Humans; Microvessels; Motor Activity; Occludin; Permeability; Rats, Sprague-Dawley; Spinal Cord; Spinal Cord Injuries; Tretinoin

Spinal cord injury (SCI) induces the disruption of the blood-spinal cord barrier (BSCB) which leads to infiltration of blood cells, an inflammatory response, and neuronal cell death, resulting spinal cord secondary damage. Retinoic acid (RA) has a neuroprotective effect in both ischemic brain injury and SCI, however the relationship between BSCB disruption and RA in SCI is still unclear. In this study, we demonstrated that autophagy and ER stress are involved in the protective effect of RA on the BSCB. RA attenuated BSCB permeability and decreased the loss of tight junction (TJ) molecules such as P120, β-catenin, Occludin and Claudin5 after injury in vivo as well as in Brain Microvascular Endothelial Cells (BMECs). Moreover, RA administration improved functional recovery in the rat model of SCI. RA inhibited the expression of CHOP and caspase-12 by induction of autophagic flux. However, RA had no significant effect on protein expression of GRP78 and PDI. Furthermore, combining RA with the autophagy inhibitor chloroquine (CQ) partially abolished its protective effect on the BSCB via exacerbated ER stress and subsequent loss of tight junctions. Taken together, the neuroprotective role of RA in recovery from SCI is related to prevention of of BSCB disruption via the activation of autophagic flux and the inhibition of ER stress-induced cell apoptosis. These findings lay the groundwork for future translational studies of RA for CNS diseases, especially those related to BSCB disruption.

Topics: Animals; Apoptosis; Blood-Brain Barrier; Cell Survival; Cells, Cultured; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Female; Humans; Rats; Rats, Sprague-Dawley; Spinal Cord Injuries; Tretinoin

Transplantation of bone marrow mesenchymal stem cells (MSCs) promotes functional recovery in multiple sclerosis (MS) patients and in a murine model of MS. However, there is only a modicum of information on differentiation of grafted MSCs into oligodendrocyte-like cells in MS. The purpose of this study was to transplant neurotrophin-3 (NT-3) and retinoic acid (RA) preinduced MSCs (NR-MSCs) into a demyelinated spinal cord induced by ethidium bromide and to investigate whether EA treatment could promote NT-3 secretion in the demyelinated spinal cord. We also sought to determine whether increased NT-3 could further enhance NR-MSCs overexpressing the tyrosine receptor kinase C (TrkC) to differentiate into more oligodendrocyte-like cells, resulting in increased remyelination and nerve conduction in the spinal cord. Our results showed that NT-3 and RA increased transcription of TrkC mRNA in cultured MSCs. EA increased NT-3 levels and promoted differentiation of oligodendrocyte-like cells from grafted NR-MSCs in the demyelinated spinal cord. There was evidence of myelin formation by grafted NR-MSCs. In addition, NR-MSC transplantation combined with EA treatment (the NR-MSCs + EA group) reduced demyelination and promoted remyelination. Furthermore, the conduction of cortical motor-evoked potentials has improved compared to controls. Together, our data suggest that preinduced MSC transplantation combined with EA treatment not only increased MSC differentiation into oligodendrocyte-like cells forming myelin sheaths, but also promoted remyelination and functional improvement of nerve conduction in the demyelinated spinal cord.

Topics: Animals; Cell Differentiation; Electroacupuncture; Male; Mice; Multiple Sclerosis; Neurotrophin 3; Oligodendroglia; Rats; Rats, Sprague-Dawley; Spinal Cord Injuries; Tretinoin

To observe the effect of transplantation of neural stem cells (NSCs) induced by all-trans-retinoic acid (ATRA) combined with glial cell line derived neurotrophic factor (GDNF) and chondroitinase ABC (ChABC) on the neurological functional recovery of injured spinal cord in Sprague Dawley (SD) rats.. Sixty adult SD female rats, weighing 200-250 g, were randomly divided into 5 groups (n = 12): sham operation group (group A), SCI model group (group B), NSCs+GDNF treatment group (group C), NSCs+ChABC treatment group (group D), and NSCs+GDNF+ChABC treatment group (group E). T10 segmental transversal injury model of the spinal cord was established except group A. NSCs induced by ATRA and marked with BrdU were injected into the site of injury at 8 days after operation in groups C-E. Groups C-E were treated with GDNF, ChABC, and GDNF+ChABC respectively at 8-14 days after operation; and group A and B were treated with the same amount of saline solution. Basso Beattie Bresnahan (BBB) score and somatosensory evoked potentials (SEP) test were used to study the functional improvement at 1 day before remodeling, 7 days after remodeling, and at 1, 2, 5, and 8 weeks after transplantation. Immunofluorescence staining and HE staining were performed to observe the cells survival and differentiation in the spinal cord.. Five mouse died but another rats were added. At each time point after modeling, BBB score of groups B, C, D, and E was significantly lower than that of group A, and SEP latent period was significantly longer than that of group A (P < 0.05), but no difference was found among groups B, C, D, and E at 7 days after remodeling and 1 week after transplantation (P > 0.05). BBB score of groups C, D, and E was significantly higher than that of group B, and SEP latent period was significantly shorter than that of group B at 2, 5, and 8 weeks after transplantation (P < 0.05); group E had higher BBB score and shorter SEP latent period than groups C and D at 5 and 8 weeks, showing significant difference (P < 0.05). HE staining showed that there was a clear boundary between gray and white matter of spinal cord and regular arrangement of cells in group A; there were incomplete vascular morphology, irregular arrangement of cells, scar, and cysts in group B; there were obvious cell hyperplasia and smaller cysts in groups C, D, and E. BrdU positive cells were not observed in groups A and B, but could be found in groups C, D and E. Group E had more positive cells than groups C and D, and difference was significant (P < 0.05). The number of glial fibrillary acidic protein positive cells of groups C, D, and E was significantly less than that of groups A and B, and it was significantly less in group E than groups C and D (P < 0.05). The number of microtubule-associated protein 2 positive cells of groups C, D, and E was significantly more than that of groups A and B, and it was significantly more in group E than groups C and D (P < 0.05).. The NSCs transplantation combined with GDNF and ChABC could significantly promote the functional recovery of spinal cord injury, suggesting that GDNF and ChABC have a synergistic effect in the treatment of spinal cord injury.

Topics: Animals; Cell Differentiation; Chondroitin ABC Lyase; Cicatrix; Female; Glial Cell Line-Derived Neurotrophic Factor; Glial Fibrillary Acidic Protein; Mice; Neural Stem Cells; Random Allocation; Rats; Rats, Sprague-Dawley; Recovery of Function; Spinal Cord Injuries; Stem Cell Transplantation; Tretinoin

Neural stem cells (NSCs) provide promising therapeutic potential for cell replacement therapy in spinal cord injury (SCI). However, high increases of cell viability and poor control of cell differentiation remain major obstacles. In this study, we have developed a non-woven material made of co-electrospun fibers of poly L-lactic acid and gelatin with a degradation rate and mechanical properties similar to peripheral nerve tissue and investigated their effect on cell survival and differentiation into motor neuronal lineages through the controlled release of retinoic acid (RA) and purmorphamine. Engineered Neural Stem-Like Cells (NSLCs) seeded on these fibers, with and without the instructive cues, differentiated into β-III-tubulin, HB-9, Islet-1, and choactase-positive motor neurons by immunostaining, in response to the release of the biomolecules. In addition, the bioactive material not only enhanced the differentiation into motor neuronal lineages but also promoted neurite outgrowth. This study elucidated that a combination of electrospun fiber scaffolds, neural stem cells, and controlled delivery of instructive cues could lead to the development of a better strategy for peripheral nerve injury repair.

Topics: Cell Differentiation; Cell Survival; Cells, Cultured; Gelatin; Humans; Lactic Acid; Microscopy, Electron, Scanning; Microscopy, Electron, Transmission; Morpholines; Motor Neurons; Nerve Tissue; Neural Stem Cells; Polyesters; Polymers; Purines; Spinal Cord Injuries; Tissue Scaffolds; Tretinoin

In the past decades, mesenchymal stem cells (MSCs) as a promising cell candidate have received the most attention in the treatment of spinal cord injury (SCI). However, due to the low survival rate and low neural differentiation rate, the grafted MSCs do not perform well as one would have expected. In the present study, we tested a combinational therapy to improve on this situation. MSCs were loaded into three-dimensional gelatin sponge (GS) scaffold. After 7 days of induction with neurotrophin-3 (NT-3) and retinoic acid (RA) in vitro, we observed a significant increase in TrkC mRNA transcription by Real-time PCR and this was confirmed by in situ hybridization. The expression of TrkC was also confirmed by Western blot and immunohistochemistry. Differentiation potential of MSCs in vitro into neuron-like cells or oligodendrocyte-like cells was further demonstrated by using immunofluorescence staining. The pre-induced MSCs seeding in GS scaffolds were then grafted into the transected rat spinal cord. One day after grafting, Governor Vessel electro-acupuncture (GV-EA) treatment was applied to rats in the NR-MSCs + EA group. At 30 days after GV-EA treatment, it found that the grafted MSCs have better survival rate and neuron-like cell differentiation compared with those without GV-EA treatment. The sustained TrkC expression in the grafted MSCs as well as increased NT-3 content in the injury/graft site by GV-EA suggests that NT-3/TrkC signaling pathway may be involved in the promoting effect. This study demonstrates that GV-EA and pre-induction with NT-3 and RA together may promote the survival and differentiation of grafted MSCs in GS scaffold in rat SCI.

Topics: Acupuncture Therapy; Animals; Antineoplastic Agents; Apoptosis; Blotting, Western; Bone Marrow; Cell Differentiation; Cell Proliferation; Cells, Cultured; Gelatin; Immunoenzyme Techniques; Male; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Nerve Regeneration; Neurotrophin 3; Porifera; Rats; Rats, Sprague-Dawley; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Spinal Cord Injuries; Tissue Scaffolds; Tretinoin

Human mesenchymal stem cells (MSCs) are easy to expand, are relatively safe, and can be transplanted in allogeneic recipients as off-the-shelf cells. MSCs can be induced to form functional peptidergic neurons and express the neurotransmitter gene, TAC1. Expression of TAC1 requires that the repressor gene, RE-1 silencing transcription factor (REST), is decreased. This study investigated the molecular pathway in TAC1 induction as MSCs differentiated into neurons and then applied the findings in a model of spinal cord injury (SCI) in zebrafish. We studied the developmental roles of the 2 cAMP response element (CRE) sites: CRE1 and CRE2. Activator protein-1 (AP-1) binding site overlaps with CRE2 (CRE2/AP-1). Reporter gene studies with the 5' regulatory region of TAC1 containing wild-type or mutant CRE sites and, parallel studies with ectopically expressed inhibitor of cAMP proteins (inducible cAMP early repressor) indicated that CRE1 and CRE2/AP-1 are activated at days 6 and 12, respectively. Studies with protein kinase-A (PKA) and Jun N-terminal kinase (JNK) inhibitors in the reporter gene studies, chromatin immunoprecipation assay, and ectopic expression of REST indicated the following pathways: Decrease of REST activated upstream c-Jun N-terminal kinase (JNK). In turn, JNK activated ATF-2 and AP-1 for interaction with CRE1 and CRE2/AP-1, respectively. To apply the finding to SCI, we transplanted 6-day-induced MSCs in transgenic HB9-GFP zebrafish larvae with SCI, in the presence or absence of JNK inhibitors. Imaging and functional studies showed significant improvement in the fish. The repair mechanism involved the activation of JNK. The findings have long-term implications for SCI repair with MSCs.

Topics: Activating Transcription Factors; Animals; Binding Sites; Cells, Cultured; Co-Repressor Proteins; Cyclic AMP Response Element-Binding Protein; Fibroblast Growth Factor 2; Gene Expression Regulation; Genes, Reporter; Humans; Models, Biological; Nerve Tissue Proteins; Protein Binding; Proto-Oncogene Proteins c-jun; Repressor Proteins; Response Elements; Signal Transduction; Spinal Cord Injuries; Tachykinins; Transcription Factor AP-1; Transplantation, Heterologous; Tretinoin; Zebrafish

The objective of this study was to determine whether amniotic fluid levels of glial acidic fibrillary protein (GFAP) would reflect myelomeningocele-related neurodegeneration in the rat model of retinoic acid-induced myelomeningocele, which is a model that is very similar to human myelomeningocele and develops the entire spectrum of disease severity including features of the Chiari II malformation.. Time-dated (embryonic day 10) pregnant Sprague-Dawley rats were gavage fed 60 mg/kg/bodyweight retinoic acid that had been dissolved in olive oil or olive oil alone. Myelomeningocele, retinoic acid-exposed no myelomeningocele, and control fetuses were harvested at specific time points throughout gestation. A standard set of pinching tests was performed to interrogate the sensorimotor reflex arc of hindpaws and tails. Amniotic fluid-GFAP levels were analyzed by standard enzyme-linked immunosorbent assay techniques.. Amniotic fluid-GFAP levels were similar between groups at embryonic days 14, 16, and 18, respectively. Compared with control fetuses, amniotic fluid GFAP levels were significantly increased in myelomeningocele fetuses at embryonic days 20 and 22 (P < .001). Defect size (P < .001), presence of clubfoot deformity (P = .0004), and absence of sensorimotor function (P < .01) at embryonic day 22 correlated with amniotic fluid-GFAP levels.. Amniotic fluid-GFAP levels appear to correlate with spinal cord injury as gestation proceeds in fetal rats with myelomeningocele.

Topics: Amniotic Fluid; Animals; Biomarkers; Chi-Square Distribution; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Fetus; Glial Fibrillary Acidic Protein; Immunohistochemistry; Meningomyelocele; Rats; Rats, Sprague-Dawley; Spinal Cord Injuries; Tretinoin

The suppression of cell apoptosis using a biodegradable scaffold to replace the missing or altered extracellular matrix (ECM) could increase the survival of transplanted cells and thus increase the effectiveness of cell therapy.. We studied the best conditions for the proliferation and differentiation of human bone marrow stromal cells (hBMSC) when cultured on different biologic scaffolds derived from fibrin and blood plasma, and analyzed the best concentrations of fibrinogen, thrombin and calcium chloride for favoring cell survival. The induction of neural differentiation of hBMSC was done by adding to these scaffolds different growth factors, such as nerve growth factor (NGF), brain-derived-neurotrophic factor (BDNF) and retinoic acid (RA), at concentrations of 100 ng/mL (NGF and BDNF) and 1 micro/mL (RA), over 7 days.. Although both types of scaffold allowed survival and neural differentiation of hBMSC, the results showed a clear superiority of platelet-rich plasma (PRP) scaffolds, mainly after BDNF administration, allowing most of the hBMSC to survive and differentiate into a neural phenotype.. Given that clinical trials for spinal cord injury using hBMSC are starting, these findings may have important clinical applications.

Topics: Biocompatible Materials; Bone Marrow Cells; Brain-Derived Neurotrophic Factor; Cell Differentiation; Cell Proliferation; Cell Survival; Cells, Cultured; Fibrin; Humans; In Situ Nick-End Labeling; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Middle Aged; Nerve Growth Factor; Neurons; Platelet-Rich Plasma; Spinal Cord Injuries; Stromal Cells; Tissue Scaffolds; Tretinoin

Early rises of pro-inflammatory cytokines play a key role in tissue damage and has detrimental consequences for functional outcome after spinal cord injury (SCI). All-trans retinoic acid (RA) has been shown to be a therapeutic agent reducing cytokine expression in vitro, but its use may be limited due to adverse side effects associated with systemic delivery. Local delivery of RA may circumvent adverse side effects, but may simultaneously reduce the therapeutic benefits of the therapy. Here, we investigated whether local or systemic RA treatment differentially affected pro-inflammatory cytokine expression early after rat SCI. Pro-inflammatory cytokines IL-1β, IL-6 and TNFα were investigated at 6h after moderate contusion injury of the thoracic (T9) spinal cord, when mRNA levels are known to peak. Rats were either treated with intrathecal RA (0, 2.5, 10, or 100ng) or received an intraperitoneal injection of RA (15mg/kg bodyweight). Surprisingly intrathecal RA up to amounts of 100ng did not attenuate SCI-induced increases in gene-expression of pro-inflammatory cytokines. In contrast, intraperitoneal RA rendered a 60%, 35% and 58% reduction of IL-1β, IL-6 and TNFα mRNA levels, respectively. Although local doses higher than 100ng RA may reduce pro-inflammatory cytokine gene-expression, such doses precipitate and possibly increase risks of adverse side effects. We conclude that in contrast to systemic delivery, intrathecal administration of RA up to doses of 100ng is ineffective in reducing early pro-inflammatory cytokine gene-expression. Future studies are required to investigate the effects of single intraperitoneal RA treatment on long-term SCI outcome.

Topics: Animals; Female; Inflammation; Injections, Intraperitoneal; Injections, Spinal; Interleukin-1beta; Interleukin-6; Neuroimmunomodulation; Rats; Rats, Sprague-Dawley; RNA, Messenger; Spinal Cord Injuries; Tretinoin; Tumor Necrosis Factor-alpha

Implantation of marrow-derived mesenchymal stem cells (MSCs) is the most promising therapeutic strategy for the treatment of spinal cord injury (SCI), especially because of their potential for clinical application, such as the avoidance of immunologic rejection, their strong secretory properties, and their plasticity for developing into neural cells. However, the recovery from SCI after MSC implantation is minimal due to their limited capacity for the reduction of cystic cavitation, for the axonal regeneration and their uncertain neural plasticity in the spinal cord. We previously pretreated MSCs with all-trans retinoic acid (RA) in vitro. Then we genetically modified them to overexpress neurotrophin-3 (NT-3) via a recombinant adenoviral vector (Adv). This combined treatment not only permitted more neuronal differentiation of MSCs, but stimulated more NT-3 secretion prior to grafting, according to our previous and present results. When these cells were implanted into the transected spinal cord of rats, the animals had some improvement (both functionally and structurally), including the recovery of hindlimb locomotor function, shown by the highest Basso, Beattie, and Bresnahan (BBB) scores, as well as dramatically reduced cavity volume, clear axonal regeneration and more neuronal survival. In contrast, simple MSC implantation is not a very effective therapy for spinal transection. However, the neuronal differentiation of MSCs after treatment with a combination of Adv-mediated NT-3 gene transfer and RA was only mildly improved in vivo.

Topics: Adenoviridae; Adult Stem Cells; Animals; Bone Marrow Cells; Cell Differentiation; Female; Genetic Vectors; Immunohistochemistry; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Nerve Regeneration; Neurotrophin 3; Rats; Rats, Sprague-Dawley; Recovery of Function; Spinal Cord Injuries; Transfection; Tretinoin; Vitamins

The present study investigated the effect of 4[(5,6,7,8-tetrahydro-5,5,8,8,-tetramethyl-2-naphthalenyl)carbamoyl] benzoic acid (Am-80), a synthetic retinoid, on spinal cord injury (SCI) in rats. Treatment with Am-80 (orally and subcutaneously) significantly promoted recovery from SCI-induced motor dysfunction. On day 28 after injury, the lesion cavity was markedly reduced, while the expression of myelin basic protein (MBP; myelin), betaIIItubulin (neuron), and glial fibrillary acidic protein (GFAP; astrocyte) was increased, in comparison with SCI controls. Interestingly, expression of neurotrophin receptor, tyrosine kinase B (TrkB) was over 3-fold higher after Am-80 treatment than in SCI controls. A lot of TrkB-positive cells as well as brain-derived neurotrophic factor (BDNF)-positive ones were observed around the injured site. Am-80 (10 microM) combined with BDNF (100 ng/ml) promoted extensive neurite outgrowth and TrkB gene expression by cultured SH-SY5Y cells, as did all-trans retinoic acid (ATRA). Thymidine incorporation was dramatically suppressed, but there was little effect on cell viability. These findings suggest that Am-80 has the potential to be used for treating neurodegenerative disorders, including SCI. Its efficacy may be partly ascribed to promotion of cell viability and differentiation of neural stem cells through increased TrkB expression.

Topics: Animals; Benzoates; Biomarkers; Blotting, Western; Cell Differentiation; Cell Line, Tumor; Female; Hindlimb; Humans; Keratolytic Agents; Locomotion; Movement Disorders; Nerve Tissue Proteins; Neurons; Rats; Rats, Sprague-Dawley; Retinoids; Spinal Cord Injuries; Tetrahydronaphthalenes; Tretinoin

The physiological reactions after spinal cord injury are accompanied by local synthesis of the transcriptional activator retinoic acid (RA). RA exerts its effects by binding to retinoic acid receptors (RAR) which heterodimerize with retinoid X receptors (RXR) and then act as ligand-activated transcription factors. To identify possible cellular targets of RA we investigated protein levels and cellular distribution of retinoid receptors in the rat spinal cord at 4, 7, 14 and 21 days after a contusion injury. In the nonlesioned spinal cord, immunoreactivity for RARalpha, RXRalpha, RXRbeta and RXRgamma was localized in the cytosol of neurons, that of RXRalpha and RXRbeta in astrocytes and that of RARalpha, RXRalpha and RXRgamma in some oligodendrocytes. After contusion injury RARalpha and all RXRs appeared in the cell nuclei of reactive microglia and macrophages. This nuclear staining began at 4 days, was most prominent at 7 and 14 days and had decreased at 21 days after injury. A similar nuclear translocation was also observed for the RARalpha, RXRalpha and RXRbeta staining in neurons situated around the border of the contusion. These observations suggest that RA participates as a signal for the physiological responses of microglia and neurons after CNS injury.

Topics: 2',3'-Cyclic-Nucleotide Phosphodiesterases; Animals; Behavior, Animal; Blotting, Western; Ectodysplasins; Gene Expression Regulation; Glial Fibrillary Acidic Protein; Immunohistochemistry; Indoles; Macrophages; Male; Membrane Proteins; Microtubule-Associated Proteins; Models, Biological; Neurons; Rats; Rats, Inbred Lew; Receptors, Retinoic Acid; Signal Transduction; Spinal Cord Injuries; Time Factors; Tretinoin; Tumor Necrosis Factors

Retinoic acid (RA) promotes growth and differentiation in many developing tissues but less is known about its influence on CNS regeneration. We investigated the possible involvement of RA in rat spinal cord injury (SCI) using the New York University (NYU) impactor to induce mild or moderate spinal cord contusion injury. Changes in RA at the lesion site were determined by measuring the activity of the enzymes for its synthesis, the retinaldehyde dehydrogenases (RALDHs). A marked increase in enzyme activity occurred by day 4 and peaked at days 8-14 following the injuries. RALDH2 was the only detectable RALDH present in the control or injured spinal cord. The cellular localization of RALDH2 was identified by immunostaining. In the noninjured spinal cord, RALDH2 was detected in oligodendroglia positive for the markers RIP and CNPase. Expression was also intense in the arachnoid membrane surrounding the spinal cord. After SCI the increase in RALDH2 was independent of the RIP- and CNPase-positive cells, which were severely depleted. Instead, RALDH2 was present in a cell type not previously identified as capable of synthesizing RA, that expressed NG2 and that was negative for markers of astrocytes, oligodendroglia, microglia, neurons, Schwann cells and immature lymphocytes. We postulate that the RALDH2- and NG2-positive cells migrate into the injured sites from the adjacent arachnoid membrane, where the RALDH2-positive cells proliferate substantially following SCI. These findings indicate that close correlations exist between RA synthesis and SCI and that RA may play a role in the secondary events that follow acute SCI.

Topics: Aldehyde Oxidoreductases; Animals; Antigens; Male; Proteoglycans; Rats; Rats, Sprague-Dawley; Retinal Dehydrogenase; Spinal Cord Injuries; Tretinoin

To explore the feasibility of inducing rat bone mesenchymal stem cells (BMSC) to differentiate into neuron-like cells with the use of Vitamin A acid, zinc and rat injured spinal cord extracts in vitro.. The BMSC were isolated from rat, cultured for 4 passages, and were treated with 10 ng/ml basic fibroblast growth factor (bFGF) for 24 h before induction. Then the medium was replaced by an induction media containing Vitamin A acid, zinc and rat injured spinal cord extracts. The morphological changes of the cells were observed. At day 12 of induction, the cells were stained immunocytochemically with neuron-specific enolase (NSE), neurofilament (NF) and glial fibrillary acidic protein (GFAP) antibodies.. At day 12 of induction, a certain number of BMSC became neuron-like cells and showed NSE and NF expression. But the neuron-like cells did not express GFAP.. The BMSC can be induced to differentiate into neuron-like cells with the use of Vitamin A acid, zinc and rat injured spinal cord extracts.

Topics: Animals; Bone Marrow Cells; Cell Differentiation; Cell Division; Cells, Cultured; Glial Fibrillary Acidic Protein; Neurofilament Proteins; Neurons; Rats; Rats, Sprague-Dawley; Spinal Cord Injuries; Stem Cells; Tretinoin; Zinc

Recent advances in the isolation and characterization of neural precursor cells suggest that they have properties that would make them useful transplants for the treatment of central nervous system disorders. We demonstrate here that spinal cord cells isolated from embryonic day 14 Sprague-Dawley and Fischer 344 rats possess characteristics of precursor cells. They proliferate as undifferentiated neurospheres in the presence of EGF and bFGF and can be maintained in vitro or frozen, expanded and induced to differentiate into both neurons and glia. Exposure of these cells to serum in the absence of EGF and bFGF promotes differentiation into astrocytes; treatment with retinoic acid promotes differentiation into neurons. Spinal cord cells labeled with a nuclear dye or a recombinant adenovirus vector carrying the lacZ gene survive grafting into the injured spinal cord of immunosuppressed Sprague-Dawley rats and non-immunosuppressed Fischer 344 rats for up to 4 months following transplantation. In the presence of exogenously supplied BDNF, the grafted cells differentiate into both neurons and glia. These spinal cord cell grafts are permissive for growth by several populations of host axons, especially when combined with exogenous BDNF administration, as demonstrated by penetration into the graft of axons immunopositive for 5-HT and CGRP. Thus, precursor cells isolated from the embryonic spinal cord of rats, expanded in culture and genetically modified, are a promising type of transplant for repair of the injured spinal cord.

Topics: Animals; Axons; Cattle; Cell Differentiation; Cell Separation; Cell Survival; Fetal Blood; Fetal Tissue Transplantation; Growth Substances; Neurons; Phenotype; Preservation, Biological; Rats; Rats, Inbred F344; Rats, Sprague-Dawley; Spheroids, Cellular; Spinal Cord; Spinal Cord Injuries; Time Factors; Tretinoin