Improved Efficacy of Delayed Treatment with Human Bone Marrow-Derived Stromal Cells Evaluated in Rats with Spinal Cord Injury.

The treatment of spinal cord injury (SCI) with uncultivated human bone marrow-derived stromal cells (bmSCs) prepared by negative selection has been proposed to be therapeutically superior to treatment with stem cells that were expanded in vitro. To explore their use in clinical trials, we studied the functional effects of delayed application at 7 days after SCI by testing different doses of bmSCs. Spinal cord contusion injury was induced in adult male Wistar rats at the thoracic level T9. Human bmSCs were prepared by negative selection without expansion in vitro (NeuroCellsTM). Treatment consisted of one 150 µL injection into the cisterna magna containing 0.5 or 2.5 million fresh bmSCs or 2.5 million bmSCs. The recovery of motor functions was evaluated during a surveillance period of six weeks (6 W), during which spinal cords were assessed histologically. Treatment resulted in a significant, dose-dependent therapeutic effect on the recovery of motor performance. The histological analysis revealed a lower degree of axonal degeneration and better survival of neurons and oligodendrocytes in bmSCs treated rats. Our results support delayed intrathecal application of bmSCs prepared by negative selection without expansion in vitro as a treatment of SCI.

Bile acids attenuate PKM2 pathway activation in proinflammatory microglia.

Glycolysis is the metabolic pathway that converts glucose into pyruvate. Central nervous system (CNS) pathologies, such as spinal cord injury (SCI) and ischemia, are accompanied by an increase of the glycolytic pathway in the damaged areas as part of the inflammatory response. Pyruvate kinase is a key glycolytic enzyme that converts phosphoenolpyruvate and ADP to pyruvate and ATP. The protein has two isoforms, PKM1 and PKM2, originated from the same gene. As a homodimer, PKM2 loses the pyruvate kinase activity and acts as a transcription factor that regulates the expression of target genes involved in glycolysis and inflammation. After SCI, resident microglia and hematogenous macrophages are key inducers of the inflammatory response with deleterious effects. Activation of the bile acid receptor TGR5 inhibits the pro-inflammatory NFκB pathway in microglia and macrophages. In the present study we have investigated whether bile acids affect the expression of glycolytic enzymes and their regulation by PKM2. Bacterial lipopolysaccharide (LPS) induced the expression of PKM1, PKM2 and its target genes in primary cultures of microglial and Raw264.7 macrophage cells. SCI caused an increase of PKM2 immunoreactivity in macrophages after SCI. Pretreatment with tauroursodeoxycholic acid (TUDCA) or taurolithocholic acid (TLCA) reduced the expression of PKM2 and its target genes in cell cultures. Similarly, after SCI, TUDCA treatment reduced the expression of PKM2 in the lesion center. These results confirm the importance of PKM2 in the inflammatory response in CNS pathologies and indicate a new mechanism of bile acids as regulators of PKM2 pathway.

Taurolithocholic acid but not tauroursodeoxycholic acid rescues phagocytosis activity of bone marrow-derived macrophages under inflammatory stress.

Spinal cord injury (SCI) causes cell death and consequently the breakdown of axons and myelin. The accumulation of myelin debris at the lesion site induces inflammation and blocks axonal regeneration. Hematogenous macrophages contribute to the removal of myelin debris. In this study, we asked how the inflammatory state of macrophages affects their ability to phagocytose myelin. Bone marrow-derived macrophages (BMDM) and Raw264.7 cells were stimulated with lipopolysaccharides (LPS) or interferon gamma (IFNγ), which induce inflammatory stress, and the endocytosis of myelin was examined. We found that activation of the TLR4-NFκB pathway reduced myelin uptake by BMDM, while IFNγ-Jak/STAT1 signaling did not. Since bile acids regulate lipid metabolism and in some cases reduce inflammation, our second objective was to investigate whether myelin clearance could be improved with taurolithocholic acid (TLCA), tauroursodeoxycholic acid or hyodeoxycholic acid. In BMDM only TLCA rescued myelin phagocytosis, when this activity was suppressed by LPS. Inhibition of protein kinase A blocked the effect of TLCA, while an agonist of the farnesoid X receptor did not rescue phagocytosis, implicating TGR5-PKA signaling in the effect of TLCA. To shed light on the mechanism, we measured whether TLCA affected the expression of CD36, triggering receptor on myeloid cells-2 (TREM2), and Gas6, which are known to be involved in phagocytosis and affected by inflammatory stimuli. Concomitant with an increase in expression of tumour necrosis factor alpha, LPS reduced expression of TREM2 and Gas6 in BMDM, and TLCA significantly diminished this downregulation. These findings suggest that activation of bile acid receptors may be used to improve myelin clearance in neuropathologies.

Retinoic acid increases phagocytosis of myelin by macrophages.

Traumatic injuries of the central nervous system (CNS) are followed by the accumulation of cellular debris including proteins and lipids from myelinated fiber tracts. Insufficient phagocytic clearance of myelin debris influences the pathological process because it induces inflammation and blocks axonal regeneration. We investigated whether ligands of nuclear receptor families retinoic acid receptors (RARs), retinoid X receptors, peroxisome proliferator-activated receptors, lipid X receptors, and farnesoid X receptors increase myelin phagocytosis by murine bone marrow-derived macrophages and Raw264.7 cells. Using in vitro assays with 3,3'-dioctadecyloxacarbocyanine perchlorate- and pHrodo-labeled myelin we found that the transcriptional activator all-trans retinoic acid (RA)enhanced endocytosis of myelin involving the induction of tissue transglutaminase-2. The RAR-dependent increase of phagocytosis was not associated with changes in gene expression of receptors FcγR1, FcγR2b, FcγR3, TREM2, DAP12, CR3, or MerTK. The combination of RA and myelin exposure significantly reduced the expression of M1 marker genes inducible nitric oxide synthase and interleukin-1ß and increased expression of transmembrane proteins CD36 and ABC-A1, which are involved in lipid transport and metabolism. The present results suggest an additional mechanism for therapeutic applications of RA after CNS trauma. It remains to be studied whether endogenous RA-signaling regulates phagocytosis in vivo.

Treatment of rats with spinal cord injury using human bone marrow-derived stromal cells prepared by negative selection.

BACKGROUND: Spinal cord injury (SCI) is a highly debilitating pathology without curative treatment. One of the most promising disease modifying strategies consists in the implantation of stem cells to reduce inflammation and promote neural regeneration. In the present study we tested a new human bone marrow-derived stromal cell preparation (bmSC) as a therapy of SCI. METHODS: Spinal cord contusion injury was induced in adult male rats at thoracic level T9/T10 using the Infinite Horizon impactor. One hour after lesion the animals were treated with a sub-occipital injection of human bmSC into the cisterna magna. No immune suppression was used. One dose of bmSC consisted, on average, of 2.3 million non-manipulated cells in 100 µL suspension, which was processed out of fresh human bone marrow from the iliac crest of healthy volunteers. Treatment efficacy was compared with intraperitoneal injections of methylprednisolone (MP) and saline. The recovery of motor functions was assessed during a surveillance period of nine weeks. Adverse events as well as general health, weight and urodynamic functions were monitored daily. After this time, the animals were perfused, and the spinal cord tissue was investigated histologically. RESULTS: Rats treated with bmSC did not reject the human implants and showed no sign of sickness behavior or neuropathic pain. Compared to MP treatment, animals displayed better recovery of their SCI-induced motor deficits. There were no significant differences in the recovery of bladder control between groups. Histological analysis at ten weeks after SCI revealed no differences in tissue sparing and astrogliosis, however, bmSC treatment was accompanied with reduced axonal degeneration in the dorsal ascending fiber tracts, lower Iba1-immunoreactivity (IR) close to the lesion site and reduced apoptosis in the ventral grey matter. Neuroinflammation, as evidenced by CD68-IR, was significantly reduced in the MP-treated group. CONCLUSIONS: Human bmSC that were prepared by negative selection without expansion in culture have neuroprotective properties after SCI. Given the effect size on motor function, implantation in the acute phase was not sufficient to induce spinal cord repair. Due to their immune modulatory properties, allogeneic implants of bmSC can be used in combinatorial therapies of SCI.

Three-dimensional configuration of orientated fibers as guidance structures for cell migration and axonal growth.

Peripheral nerve injuries can be surgically repaired by suturing the transected nerve stumps or, in case of larger lesions, by the transplantation of an autologous nerve graft. To avoid donor site morbidity, the development of artificial implants is desired. Clinically, hollow conduits have been used for this purpose but are inferior to the autograft because they lack internal guidance cues for Schwann cells and regenerating axons. In this article, we describe the design of a three-dimensional (3D) scaffold consisting of parallel fibers embedded in a collagen matrix. For this purpose, an electrospinning device was developed to produce and manipulate a 3D array of aligned poly(ɛ-caprolactone) (PCL) microfibers. This fiber array was then incorporated into biodegradable PCL tubes to serve as artificial nerve bridges. Using primary cultures of embryonic chicken dorsal root ganglia, we show that PCL microfibers in the 3D matrix of our composite scaffold guide the direction of Schwann cell migration and axonal growth.

Functionalization of electrospun poly(ε-caprolactone) fibers with the extracellular matrix-derived peptide GRGDS improves guidance of schwann cell migration and axonal growth.

The best available treatment of peripheral nerve lesions involves transplantation of an autologous nerve. This approach, however, entails sensory deficits at the donor site and requires additional surgery. Such limitations have motivated the search for a bioengineering solution to design artificial implants. For this purpose we are producing orientated biodegradable microfibers of poly(ε-caprolactone) (PCL) with electrospinning. The present study describes the functionalization of these electrospun fibers with biologically active peptides to produce guidance structures for Schwann cell migration and axonal regeneration. For the chemical modification PCL was blended with star-shaped NCO-poly(ethylene glycol)-stat-poly(propylene glycol) (PCL/sPEG) as a covalent linker for the peptide GRGDS, derived from extracellular matrix proteins. To test biological functions of electrospun fibers, Schwann cell migration and axonal growth from dorsal root ganglia explants were investigated with time lapse video microscopy. Migrating Schwann cells as well as growing sensory axons closely followed the electrospun fibers with occasional leaps between adjacent fibers. Cell migration was characterized by frequent changes in velocity and direction reversals. Comparison of substrates showed that functionalized fibers caused more Schwann cells to move out of the explants, supported faster cell migration and axonal growth than the nonfunctional fibers. Using inhibitors of intracellular signaling kinases, we found that these biological effects required activation of the phosphatidyl inositol-3-kinase pathway. Since sPEG-containing fibers also showed low levels of nonspecific protein adsorption, which is desirable in the context of artificial implant design, the peptide modification of fibers appears to provide good substrates for nerve repair.

Inflammatory cytokine release of astrocytes in vitro is reduced by all-trans retinoic acid.

In the central nervous system inflammation is mediated by microglia and astrocytes. To investigate its regulation, murine astrocyte cultures were treated with bacterial lipopolysaccharides (LPS) and analyzed with Affymetrix gene array, qRT-PCR and ELISA. Cells responded to LPS with a strong upregulation of pro-inflammatory cytokines and chemokines. Treatment with the transcriptional activator retinoic acid (RA) suppressed mRNA expression and protein release of several important cytokines (IL-1ß 4%, IL-6 21%, TNFα 30%, IL-12p40 42%, and IL-12p35/p40 27%; p<0.01). The data are consistent with the hypothesis that all-trans RA takes part in endogenous anti-inflammatory feedback loops in the CNS.

Repetitive intrathecal VEGF(165) treatment has limited therapeutic effects after spinal cord injury in the rat.

Neuropathic pain and motor deficits are detrimental consequences of injury to the spinal cord. In experimental settings, numerous neuroprotective agents are being explored for their therapeutic benefits. Vascular endothelial growth factor (VEGF) is an interesting candidate molecule in this respect since it is not only associated with angiogenesis, but also with neuroprotection and neurite growth. Other investigators have reported improved motor outcomes following intraparenchymal VEGF treatment. Here we demonstrate the therapeutic effects of daily intrathecal treatment of the contused thoracic rat spinal cord with the 165-isoform of VEGF during the first week after injury. We show that VEGF treatment resulted in a statistically significant attenuation of mechanical, but not thermal, hypersensitivity of the hindpaws, while motor deficits remained unaffected. Tissue sparing was also unchanged by VEGF treatment. Microglial responses at the lumbar spinal cord, which have been linked with spinal cord injury-induced hypersensitivity, were found to be unaffected by VEGF treatment. We conclude that repetitive intrathecal VEGF delivery has limited therapeutic effects on spinal cord injury outcome.

Functionalization of electrospun fibers of poly(epsilon-caprolactone) with star shaped NCO-poly(ethylene glycol)-stat-poly(propylene glycol) for neuronal cell guidance.

Microfibers produced with electrospinning have recently been used in tissue engineering. In the development of artificial implants for nerve regeneration they are of particular interest as guidance structures for cell migration and axonal growth. Using electrospinning we produced parallel-orientated biocompatible fibers in the submicron range consisting of poly(epsilon-caprolactone) (PCL) and star shaped NCO-poly(ethylene glycol)-stat-poly(propylene glycol) (sPEG). Addition of the bioactive peptide sequence glycine-arginine-glycine-aspartate-serine (GRGDS) or the extracellular matrix protein fibronectin to the electrospinning solution resulted in functionalized fibers. Surface characteristics and biological properties of functionalized and non-functionalised fibers were investigated. Polymer solutions and electrospinning process parameters were varied to obtain high quality orientated fibers. A polymer mixture containing high molecular weight PCL, PCL-diol, and sPEG permitted a chemical reaction between hydroxyl groups of the diol and isocyanante groups of the sPEG. Surface analysis demonstrated that sPEG at the fiber surface minimized protein adhesion. In vitro experiments using dorsal root ganglia explants showed that the cell repellent property of pure PCL/sPEG fibers was overcome by functionalization either with GRGDS peptide or fibronectin. In this way cell migration and axonal outgrowth along fibers were significantly increased. Thus, functionalized electrospun PCL/sPEG fibers, while preventing non-specific protein adsorption, are a suitable substrate for biological and medical applications.

Human neural cell interactions with orientated electrospun nanofibers in vitro.

AIM: Electrospun nanofibers represent potent guidance substrates for nervous tissue repair. Development of nanofiber-based scaffolds for CNS repair requires, as a first step, an understanding of appropriate neural cell type-substrate interactions. MATERIALS & METHODS: Astrocyte-nanofiber interactions (e.g., adhesion, proliferation, process extension and migration) were studied by comparing human neural progenitor-derived astrocytes (hNP-ACs) and a human astrocytoma cell line (U373) with aligned polycaprolactone (PCL) nanofibers or blended (25% type I collagen/75% PCL) nanofibers. Neuron-nanofiber interactions were assessed using a differentiated human neuroblastoma cell line (SH-SY5Y). RESULTS & DISCUSSION: U373 cells and hNP-AC showed similar process alignment and length when associated with PCL or Type I collagen/PCL nanofibers. Cell adhesion and migration by hNP-AC were clearly improved by functionalization of nanofiber surfaces with type I collagen. Functionalized nanofibers had no such effect on U373 cells. Another clear difference between the U373 cells and hNP-AC interactions with the nanofiber substrate was proliferation; the cell line demonstrating strong proliferation, whereas the hNP-AC line showed no proliferation on either type of nanofiber. Long axonal growth (up to 600 microm in length) of SH-SY5Y neurons followed the orientation of both types of nanofibers even though adhesion of the processes to the fibers was poor. CONCLUSION: The use of cell lines is of only limited predictive value when studying cell-substrate interactions but both morphology and alignment of human astrocytes were affected profoundly by nanofibers. Nanofiber surface functionalization with collagen significantly improved hNP-AC adhesion and migration. Alternative forms of functionalization may be required for optimal axon-nanofiber interactions.

Time of transplantation and cell preparation determine neural stem cell survival in a mouse model of Huntington's disease.

Cell replacement therapies for neurodegenerative diseases, using multipotent neural stem cells (NSCs), require above all, a good survival of the graft. In this study, we unilaterally injected quinolinic acid (QA) into the striatum of adult mice and transplanted syngeneic NSCs of enhanced green fluorescent protein-transgenic mice into the lesioned striatum. The injection of QA leads to an excitotoxic lesion with selective cell death of the medium sized spiny neurons, the same cells that are affected in Huntington's disease. In order to investigate the best timing of transplantation for the survival of donor cells, we transplanted the stem cells at 2, 7 and 14 days after injury. In addition, the influence of graft preparation prior to transplantation, i.e., intact neurospheres versus dissociated cell suspension on graft survival was investigated. By far the best survival was found with the combination of early transplantation (i.e., 2 days after QA-lesion) with the use of neurospheres instead of dissociated cell suspension. This might be due to the different states of host's astrocytic and microglia activation which we found to be moderate at 2, but pronounced at 7 and 14 days after QA-lesion. We also investigated brain derived neurotrophic factor (BDNF)-expression in the striatum after QA-lesion and found no significant change in BDNF protein-level. We conclude that already the method of graft preparation of NSCs for transplantation, as well as the timing of the transplantation procedure strongly affects the survival of the donor cells when grafted into the QA-lesioned striatum of adult mice.

Effects of inflammatory cytokines IL-1beta, IL-6, and TNFalpha on the intracellular localization of retinoid receptors in Schwann cells.

It was investigated whether retinoic acid (RA) and the proinflammatory cytokines IL-1beta, IL-6, and TNFalpha influence the intracellular distribution of retinoic acid receptors (RAR) and retinoid X receptors (RXR) in Schwann cells. This question arose because nuclear translocation of RARalpha, RXRalpha, and RXRbeta was observed after nerve injury, and because mutual interactions exist between the signal transduction pathways of RA and proinflammatory cytokines. Schwann cell primary cultures from the rat sciatic nerve were incubated with IL-1beta, IL-6, and TNFalpha, with all-trans RA and with a combination of IL-1beta and RA. After incubation periods ranging from 5 min to 5 h, the intracellular distributions of RARalpha, RARbeta, RXRalpha, and RXRbeta were analyzed. All three cytokines caused a shift of RARalpha from the cytosolic compartments into the cell nuclei. This was also observed with RA, and combining RA with IL-1beta produced an additive effect. IL-1beta and IL-6 also affected the distribution of RARbeta, although immunoreactivity of this receptor always remained stronger in the cytosol. No effect of the cytokines on RXRalpha or RXRbeta was observed, whereas RA treatment caused a stronger nuclear signal of both receptors. Effects on the subcellular localization of retinoid receptors may provide a link in a feedback loop between RA/RAR and cytokines.

Macrophages and neurons are targets of retinoic acid signaling after spinal cord contusion injury.

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.

Retinoic acid downregulates the expression of ciliary neurotrophic factor in rat Schwann cells.

Neuropoietic cytokines, which serve as mediators in neuroglial interactions, are differentially regulated after peripheral nerve injury. In Schwann cells, the expression of ciliary neurotrophic factor (CNTF) decreases. Pursuing the hypothesis that retinoic acid (RA) serves as a regulator of lesion-induced cytokine signaling we found that all RA receptors and retinoid X receptors are expressed in Schwann cell primary cultures. Using quantitative reverse transcription-polymerase chain reaction, we have investigated the effect of RA on the expression of CNTF in these cells. After treatment with 10 nM all-trans RA for 22 h the concentration of CNTF mRNA was reduced to 63% of the control level, reminiscent of the regulation after nerve injury in vivo. In addition to CNTF, the mRNAs of leukemia inhibitory factor, interleukin-6, ciliary neurotrophic factor receptor component alpha and gp130 were detected in the Schwann cells.

Retinoic acid enhances leukemia inhibitory factor expression in OLN-93 oligodendrocytes.

Brain injuries trigger physiological reactions which are mediated by a number of cytokines including interleukin-6 (IL-6), ciliary neurotrophic factor (CNTF), and leukemia inhibitory factor (LIF). Astrocytes and microglia, the protagonists in these traumatic responses, are known to secrete a variety of paracrine signals. Oligodendrocytes are involved as well and constitute another possible source of cytokines. Here we show the expression of IL-6, CNTF, and LIF in OLN-93 cells, derived from rat oligodendrocyte primary cultures. While differential gene transcription after injury has been described for many cytokines, the regulation of these physiological responses is unknown in many instances. Recent experiments indicate that the transcriptional activator retinoic acid (RA) plays a role in peripheral nerve regeneration. Transcripts of the retinoic acid receptors and the retinoid X receptors were also detected in OLN-93 oligodendrocytes. Using quantitative RT-PCR, we have therefore investigated the effect of RA on the expression of neuropoietic cytokines in these cells. Treatment with 1 microM all- trans RA for 24 h increased the mRNA concentration of LIF by a factor of 3.1 ( P<0.01). In contrast, RA had no significant effect on the expression of CNTF. The results suggest RA as a possible regulator of cytokine signaling in the CNS.