Rachelle Franzen

Spontaneous longitudinally orientated axonal regeneration is associated with the Schwann cell framework within the lesion site following spinal cord compression injury of the rat.

Spontaneous cellular reorganisation at the lesion site has been investigated following massive spinal cord compression injury in adult rats. By 2 days post operation (p.o.), haemorrhagic necrosis, widespread axonal degeneration, and infiltration by polymorphnuclear granulocytes and OX42‐positive macrophages were observed in the lesion site. By 7 days p.o., low affinity nerve growth factor receptor‐positive Schwann cells, from activated spinal roots, were identified as they migrated far into the lesion. Between 7 and 14 days p.o., the overlapping processes of Schwann cells within the macrophage‐filled lesion formed a glial framework which was associated with extensive longitudinally orientated ingrowth by many neurofilament‐positive axons. Relatively few of these axons were calcitonin gene‐related peptide (CGRP)‐, substance P (SP)‐, or serotonin (5HT)‐positive; however, many were glycinergic or gamma aminobutyric acid (GABA)ergic. At 21 and 28 days p.o. (the longest survival times studied), a reduced but still substantial amount of orientated Schwann cells and axons could be detected at distances of up to 5 mm within the lesion. Glial fibrillary acidic protein (GFAP) immunoreactivity demonstrated the slow formation of astrocytic scarring which only became apparent at the lesion interface between 21 and 28 days p.o. The current data suggest the possibility of developing future therapeutic strategies designed to maintain or even enhance these spontaneous and orientated regenerative events. J. Neurosci. Res. 53:51–65, 1998.

Effects of macrophage transplantation in the injured adult rat spinal cord: A combined immunocytochemical and biochemical study

Early and robust invasion by macrophages may be one of the reasons why axonal regeneration is more effective in the PNS than in the CNS. Therefore, we have grafted autologous peritoneal macrophages labeled with fluorescent latex microspheres into spinal cord compression lesions. At various survival times, we have studied their effect on the expression of neuronal (neurofilaments [NF], calcitonin gene‐related peptide [CGRP], 5‐hydroxytryptamine [5‐HT]) and nonneuronal markers (myelin‐associated glycoprotein [MAG], glial fibrillary acidic protein [GFAP], laminin) by using semiquantitative Western blot and immunohistochemical techniques. After 1 month, we observed a significant decrease of the expression of MAG as well as an important invasion of the lesion site by neurites, chiefly peptidergic axons of presumed dorsal root origin, in macrophage‐grafted animals compared with controls. In addition, angiogenesis and Schwann cell infiltration were more pronounced after macrophage grafts, providing an increase in laminin, a favorable substrate for axonal regrowth. By using reverse transcription‐polymerase chain reaction (RT‐PCR), mRNAs for tumor necrosis factor‐alpha (TNF‐α) were detected in the transplanted cells, whereas results were negative for nerve growth factor (NGF), neurotrophin‐3 (NT‐3), brain‐derived neurotrophic factor (BDNF), or acidic fibroblast growth factor (aFGF) and basic fibroblast growth factor (bFGF). Thus, macrophage grafts may represent an interesting strategy to promote axonal regeneration in the CNS. Our study suggests that they may exert their beneficial effects by degrading myelin products, which inhibit axonal regrowth, and by promoting a permissive extracellular matrix containing notably laminin. No evidence for a direct synthesis of neurotrophic factors by the transplanted macrophages was found in this study, but resident glial cells could secrete such factors as a result of stimulation by macrophage‐released cytokines. J. Neurosci. Res. 51:316–327, 1998.

Peripheral nerve regeneration using bioresorbable macroporous polylactide scaffolds

The ability of DRG-derived neurons to survive and attach onto macroporous polylactide (PLA) foams was assessed in vitro. The foams were fabricated using a thermally induced polymer-solvent phase separation. Two types of pore structures, namely oriented or interconnected pores, can be produced, depending on the mechanism of phase separation, which in turn can be predicted by the thermodynamics of the polymer-solvent pair. Coating of the porous foams with polyvinylalcohol (PVA) considerably improved the wettability of the foams and allowed for cell culture. The in vitro biocompatibility of the PVA-coated supports was demonstrated by measuring cell viability and neuritogenesis. Microscopic observations of the cells seeded onto the polymer foams showed that the interconnected pore networks were more favorable to cell attachment than the anisotropic ones. The capacity of highly oriented foams to support in vivo peripheral nerve regeneration was studied in rats. A sciatic nerve gap of 5-mm length was bridged with a polymer implant showing macrotubes of 100 microm diameter. At 4 weeks postoperatively, the polymer implant was still present. It was well integrated and had restored an anatomic continuity. An abundant cell migration was observed at the outer surface of the polymer implant, but not within the macrotubes. This dense cellular microenvironment was found to be favorable for axogenesis.

Neural differentiation potential of human bone marrow-derived mesenchymal stromal cells: misleading marker gene expression.

BackgroundIn contrast to pluripotent embryonic stem cells, adult stem cells have been considered to be multipotent, being somewhat more restricted in their differentiation capacity and only giving rise to cell types related to their tissue of origin. Several studies, however, have reported that bone marrow-derived mesenchymal stromal cells (MSCs) are capable of transdifferentiating to neural cell types, effectively crossing normal lineage restriction boundaries. Such reports have been based on the detection of neural-related proteins by the differentiated MSCs. In order to assess the potential of human adult MSCs to undergo true differentiation to a neural lineage and to determine the degree of homogeneity between donor samples, we have used RT-PCR and immunocytochemistry to investigate the basal expression of a range of neural related mRNAs and proteins in populations of non-differentiated MSCs obtained from 4 donors.ResultsThe expression analysis revealed that several of the commonly used marker genes from other studies like nestin, Enolase2 and microtubule associated protein 1b (MAP1b) are already expressed by undifferentiated human MSCs. Furthermore, mRNA for some of the neural-related transcription factors, e.g. Engrailed-1 and Nurr1 were also strongly expressed. However, several other neural-related mRNAs (e.g. DRD2, enolase2, NFL and MBP) could be identified, but not in all donor samples. Similarly, synaptic vesicle-related mRNA, STX1A could only be detected in 2 of the 4 undifferentiated donor hMSC samples. More significantly, each donor sample revealed a unique expression pattern, demonstrating a significant variation of marker expression.ConclusionThe present study highlights the existence of an inter-donor variability of expression of neural-related markers in human MSC samples that has not previously been described. This donor-related heterogeneity might influence the reproducibility of transdifferentiation protocols as well as contributing to the ongoing controversy about differentiation capacities of MSCs. Therefore, further studies need to consider the differences between donor samples prior to any treatment as well as the possibility of harvesting donor cells that may be inappropriate for transplantation strategies.

Effects of Schwann Cell Transplantation in a Contusion Model of Rat Spinal Cord Injury

Cultured Schwann cells were transplanted at various delays into a spinal cord contusion injury performed at low thoracic level in adult female rats. The Schwann cells were purified from the dorsal root ganglia of adult syngeneic animals. The transplants were well tolerated, and the transplanted Schwann cells invaded the injured spinal cord. As quantified using video image analysis, the survival and growth of the transplanted cells were poor when the grafting procedure was performed 3–4 days after injury and very good when performed immediately or 10 days after injury, in which cases post‐traumatic micro‐ and macrocavitation were strongly reduced. In animals grafted immediately after injury but not in animals grafted after 10 days, post‐traumatic astrogliosis was much reduced. The Schwann cells transplanted area was invaded by numerous regenerating axons, the vast majority of which were, based on the neurotransmitter (CGRP and SP) profile, originating from dorsal root ganglion. No regeneration of the cortico‐spinal tract as assessed after anterograde tracing or of descending aminergic fibers could be demonstrated.

Delayed GM-CSF treatment stimulates axonal regeneration and functional recovery in paraplegic rats via an increased BDNF expression by endogenous macrophages

Macrophages (monocytes/microglia) could play a critical role in central nervous system repair. We have previously found a synchronism between the regression of spontaneous axonal regeneration and the deactivation of macrophages 3–4 wk after a compression‐injury of rat spinal cord. To explore whether reactivation of endogenous macrophages might be beneficial for spinal cord repair, we have studied the effects of granulocyte‐macrophage colony stimulating factor (GM‐CSF) in the same paraplegia model and in cell cultures. There was a significant, though transient, improvement of locomotor recovery after a single delayed intraperitoneal injection of 2 µg GM‐CSF, which also increased significantly the expression of Cr3 and brain‐derived neurotrophic factor (BDNF) by macrophages at the lesion site. At longer survival delays, axonal regeneration was significantly enhanced in GM‐CSF‐treated rats. In vitro, BV2 microglial cells expressed higher levels of BDNF in the presence of GM‐CSF and neurons cocultured with microglial cells activated by GM‐CSF generated more neurites, an effect blocked by a BDNF antibody. These experiments suggest that GM‐CSF could be an interesting treatment option for spinal cord injury and that its beneficial effects might be mediated by BDNF.—Bouhy, D., Malgrange, B., Multon, S., Poirrier, A. L., Scholtes, F., Schoenen, J., Franzen, R. Delayed GM‐CSF treatment stimulates axonal regeneration and functional recovery in paraplegic rats via an increased BDNF expression by endogenous macrophages. FASEB J. 20, E493–E502 (2006)

Poly(D,L-lactide) foams modified by poly(ethylene oxide)-block-poly(D,L-lactide) copolymers and a-FGF: in vitro and in vivo evaluation for spinal cord regeneration.

The first goal of this study was to examine the influence that poly(ethylene oxide)-block-poly(D,L-lactide) (PELA) copolymer can have on the wettability, the in vitro controlled delivery capability, and the degradation of poly(D,L-lactide) (PDLLA) foams. These foams were prepared by freeze-drying and contain micropores (10 microm) in addition of macropores (100 microm) organized longitudinally. Weight loss, water absorption, changes in molecular weight, polymolecularity (Mw/Mn) and glass transition temperature (Tg) of PDLLA foams mixed with various amounts of PELA were followed with time. It was found that 10wt% of PELA increased the wettability and the degradation rate of the polymer foams. The release of sulforhodamine (SR) was compared for PDLLA and PDLLA-PELA foams in relation with the foam porosity. An initial burst release was observed only in the case of the 90:10 PDLLA/PELA foam. The ability of the foam of this composition to be integrated and to promote tissue repair and axonal regeneration in the transected rat spinal cord was investigated. After implantation of ca. 20 polymer rods assembled with fibrin-glue, the polymer construct was able to bridge the cord stumps by forming a permissive support for cellular migration, angiogenesis and axonal regrowth.

Conditioned medium from bone marrow-derived mesenchymal stem cells improves recovery after spinal cord injury in rats: an original strategy to avoid cell transplantation.

Spinal cord injury triggers irreversible loss of motor and sensory functions. Numerous strategies aiming at repairing the injured spinal cord have been studied. Among them, the use of bone marrow-derived mesenchymal stem cells (BMSCs) is promising. Indeed, these cells possess interesting properties to modulate CNS environment and allow axon regeneration and functional recovery. Unfortunately, BMSC survival and differentiation within the host spinal cord remain poor, and these cells have been found to have various adverse effects when grafted in other pathological contexts. Moreover, paracrine-mediated actions have been proposed to explain the beneficial effects of BMSC transplantation after spinal cord injury. We thus decided to deliver BMSC-released factors to spinal cord injured rats and to study, in parallel, their properties in vitro. We show that, in vitro, BMSC-conditioned medium (BMSC-CM) protects neurons from apoptosis, activates macrophages and is pro-angiogenic. In vivo, BMSC-CM administered after spinal cord contusion improves motor recovery. Histological analysis confirms the pro-angiogenic action of BMSC-CM, as well as a tissue protection effect. Finally, the characterization of BMSC-CM by cytokine array and ELISA identified trophic factors as well as cytokines likely involved in the beneficial observed effects. In conclusion, our results support the paracrine-mediated mode of action of BMSCs and raise the possibility to develop a cell-free therapeutic approach.

The effect of treadmill training on motor recovery after a partial spinal cord compression-injury in the adult rat.

Locomotor training on a treadmill is a therapeutic strategy used for several years in human paraplegics in whom it was shown to improve functional recovery mainly after incomplete spinal cord lesions. The precise mechanisms underlying its effects are not known. Experimental studies in adult animals were chiefly performed after complete spinal transections. The objective of this experiment was to assess the effects of early treadmill training on recovery of spontaneous walking capacity after a partial spinal cord lesion in adult rats. Following a compression-injury by a subdurally inflated microballoon, seven rats were trained daily on a treadmill with a body weight support system, whereas six other animals were used as controls and only handled. Spontaneous walking ability in an open field was compared weekly between both groups by two blinded observers, using the Basso, Beattie and Bresnahan (BBB) locomotor rating scale. Mean BBB score during 12 weeks was globally significantly greater in the treadmill-trained animals than in the control group, the benefit of training appearing as early as the 2nd week. At week 7, locomotor recovery reached a plateau in both animal groups, but remained superior in trained rats. Daily treadmill training started early after a partial spinal cord lesion in adult rats, which accelerates recovery of locomotion and produces a long-term benefit. These findings in an animal model mimicking the closed spinal cord injury occurring in most human paraplegics are useful for future studies of optimal locomotor training programs, their neurobiologic mechanisms, and their combination with other treatment strategies.

Microtubule-associated protein 1B a neuronal binding partner for myelin-associated glycoprotein

Myelin-associated glycoprotein (MAG) is expressed in periaxonal membranes of myelinating glia where it is believed to function in glia–axon interactions by binding to a component of the axolemma. Experiments involving Western blot overlay and coimmunoprecipitation demonstrated that MAG binds to a phosphorylated neuronal isoform of microtubule-associated protein 1B (MAP1B) expressed in dorsal root ganglion neurons (DRGNs) and axolemma-enriched fractions from myelinated axons of brain, but not to the isoform of MAP1B expressed by glial cells. The expression of some MAP1B as a neuronal plasma membrane glycoprotein (Tanner, S.L., R. Franzen, H. Jaffe, and R.H. Quarles. 2000. J. Neurochem. 75:553–562.), further documented here by its immunostaining without cell permeabilization, is consistent with it being a binding partner for MAG on the axonal surface. Binding sites for a MAG-Fc chimera on DRGNs colocalized with MAP1B on neuronal varicosities, and MAG and MAP1B also colocalized in the periaxonal region of myelinated axons. In addition, expression of the phosphorylated isoform of MAP1B was increased significantly when DRGNs were cocultured with MAG-transfected COS cells. The interaction of MAG with MAP1B is relevant to the known role of MAG in affecting the cytoskeletal structure and stability of myelinated axons.