Efficacy of the immediate adipose-derived stromal vascular fraction autograft on functional sensorimotor recovery after spinal cord contusion in rats.

BACKGROUND: Spinal cord injuries (SCI) lead to functional alteration with important consequences such as motor and sensory disorders. The repair strategies developed to date remain ineffective. The adipose tissue-derived stromal vascular fraction (SVF) is composed of a cocktail of cells with trophic, pro-angiogenic and immunomodulatory effects. Numerous therapeutic benefits were shown for tissue reconstitution, peripheral neuropathy and for the improvement of neurodegenerative diseases. Here, the therapeutic efficacy of SVF on sensorimotor recovery after an acute thoracic spinal cord contusion in adult rats was determined. METHOD: Male Sprague Dawley rats (n = 45) were divided into 3 groups: SHAM (without SCI and treatment), NaCl (animals with a spinal lesion and receiving a saline injection through the dura mater) and SVF (animals with a spinal lesion and receiving a fraction of fat removed from adipocytes through the dura mater). Some animals were sacrificed 14 days after the start of the experiment to determine the inflammatory reaction by measuring the interleukin-1ß, interleukin-6 and Tumor Necrosis Factor-α in the lesion area. Other animals were followed once a week for 12 weeks to assess functional recovery (postural and locomotor activities, sensorimotor coordination). At the end of this period, spinal reflexivity (rate-dependent depression of the H-reflex) and physiological adjustments (ventilatory response to metabosensitive muscle activation following muscle fatigue) were measured with electrophysiological tools. RESULTS: Compared to non-treated animals, results indicated that the SVF reduced the endogenous inflammation and increased the behavioral recovery in treated animals. Moreover, H-reflex depression and ventilatory adjustments to muscle fatigue were found to be comparable between SHAM and SVF groups. CONCLUSION: Our results highlight the effectiveness of SVF and its high therapeutic potential to improve sensorimotor functions and to restore the segmental sensorimotor loop and the communication between supra- and sub-lesional spinal cord regions after traumatic contusion.

Blockade of Glial Connexin 43 Hemichannels Reduces Food Intake.

The metabolic syndrome, which comprises obesity and diabetes, is a major public health problem and the awareness of energy homeostasis control remains an important worldwide issue. The energy balance is finely regulated by the central nervous system (CNS), notably through neuronal networks, located in the hypothalamus and the dorsal vagal complex (DVC), which integrate nutritional, humoral and nervous information from the periphery. The glial cells' contribution to these processes emerged few year ago. However, its underlying mechanism remains unclear. Glial connexin 43 hemichannels (Cx43 HCs) enable direct exchange with the extracellular space and can regulate neuronal network activity. In the present study, we sought to determine the possible involvement of glial Cx43 HCs in energy balance regulation. We here show that Cx43 is strongly expressed in the hypothalamus and DVC and is associated with glial cells. Remarkably, we observed a close apposition of Cx43 with synaptic elements in both the hypothalamus and DVC. Moreover, the expression of hypothalamic Cx43 mRNA and protein is modulated in response to fasting and diet-induced obesity. Functionally, we found that Cx43 HCs are largely open in the arcuate nucleus (ARC) from acute mice hypothalamic slices under basal condition, and significantly inhibited by TAT-GAP19, a mimetic peptide that specifically blocks Cx43 HCs activity. Moreover, intracerebroventricular (i.c.v.) TAT-GAP19 injection strongly decreased food intake, without further alteration of glycaemia, energy expenditures or locomotor activity. Using the immediate early gene c-Fos expression, we found that i.c.v. TAT-GAP19 injection induced neuronal activation in hypothalamic and brainstem nuclei dedicated to food intake regulation. Altogether, these results suggest a tonic delivery of orexigenic molecules associated with glial Cx43 HCs activity and a possible modulation of this tonus during fasting and obesity.

Motor and sensitive recovery after injection of a physically cross-linked PNIPAAm-g-PEG hydrogel in rat hemisectioned spinal cord.

In line with experiments showing that implanted hydrogels are promising tools, we designed and injected, after a C2 spinal cord hemisection, a thermoresponsive and thermoreversible physically cross-linked poly(N-isopropylacrylamide)-poly(ethylene glycol) copolymer in order to reduce functional deficits and provide a favorable environment to axotomized axons. Nasal olfactory ecto-mesenchymal stem cells were cultured on the hydrogel in order to verify its biocompatibility. Then, inflammatory reaction (Interleukin-1ß and 6, Tumor Necrosis Factor-α) was examined 15 days post-hydrogel injection. Functional recovery (postural and locomotor activities, muscle strength and tactile sensitivity) was assessed once a week, during 12 weeks. Finally, at 12 weeks post-injection, spinal reflexivity and ventilatory adjustments were measured, and the presence of glial cells and regenerated axons were determined in the injured area. Our results indicate that cells survived and proliferated on the hydrogel which, itself, did not induce an enhanced inflammation. Furthermore, we observed significant motor and sensitive improvements in hydrogel-injected animals. Hydrogel also induced H-reflex recovery close to control animals but no improved ventilatory adjustment to electrically-evoked isometric contractions. Finally, regrowing axons were visualized within the hydrogel with no glial cells colonization. Our results emphasize the effectiveness of our copolymer and its high therapeutic potential to repair the spinal cord after injury.

Engraftment of Human Stem Cell-Derived Otic Progenitors in the Damaged Cochlea.

Most cases of sensorineural deafness are caused by degeneration of hair cells. Although stem/progenitor cell therapy is becoming a promising treatment strategy in a variety of organ systems, cell engraftment in the adult mammalian cochlea has not yet been demonstrated. In this study, we generated human otic progenitor cells (hOPCs) from induced pluripotent stem cells (iPSCs) in vitro and identified these cells by the expression of known otic markers. We showed successful cell transplantation of iPSC-derived-hOPCs in an in vivo adult guinea pig model of ototoxicity. The delivered hOPCs migrated throughout the cochlea, engrafted in non-sensory regions, and survived up to 4 weeks post-transplantation. Some of the engrafted hOPCs responded to environmental cues within the cochlear sensory epithelium and displayed molecular features of early sensory differentiation. We confirmed these results with hair cell progenitors derived from Atoh1-GFP mice as donor cells. These mouse otic progenitors transplanted using the same in vivo delivery system migrated into damaged cochlear sensory epithelium and adopted a partial sensory cell fate. This is the first report of the survival and differentiation of hOPCs in ototoxic-injured mature cochlear epithelium, and it should stimulate further research into cell-based therapies for treatment of deafness.

Using MRI to predict the fate of excitotoxic lesions in rats.

Excitotoxic lesions are frequently used to assess the role of cerebral structures in cognitive processes in rodents. However, the precise site and extent of these lesions remain unknown without histological verifications. Using a 7-Teslas MRI system and a T2-weighted turbo-RARE sequence, MR images were acquired at several time points following NMDA lesions (1h, 6h, 24h, 48h, 1 week and 2 weeks). NMDA infusions into the parenchyma induced a clear and delineable hyperintense signal from 1h up to 1-week post-surgery. Hyperintensity volumes were compared with NeuN and Cresyl violet histological quantifications of the lesion magnitude. NMDA-induced hypersignal is observed as soon as 1h post-injection and is a reliable estimate of the presence (or absence) of a lesion. Compared to NeuN, Cresyl violet staining underestimates the extent of the lesion in significant proportions. The MRI hyperintensity generated by NMDA instillation into the parenchyma can be used as a powerful tool to confirm the diffusion of the drug into the cerebral tissue, to ascertain the locus of injection and predict with a high success rate the fate of NMDA lesions as soon as 1h post-surgery. This approach could be very useful in a large variety of lesion studies in rodents.

Acute granulocyte macrophage-colony stimulating factor treatment modulates neuroinflammatory processes and promotes tactile recovery after spinal cord injury.

Neuroinflammation is known to play a key role in the prognosis of functional recovery after spinal cord injury (SCI). The involvement of microglial and mast cells in early and late stages of inflammation has been receiving increasing attention. This study was aimed at determining the influence of a pro-inflammatory cytokine, the granulocyte macrophage-colony stimulating factor (GM-CSF), on microglia and mast cell activation, glial scar formation and functional recovery following SCI. Rats were randomly injected with saline or GM-CSF one hour after a C4-C5 medio-lateral hemisection. To assess functional impairment and recovery, the rats were subjected to sensorimotor tasks for one month. Then, responses evoked by forepaw stimulation in the primary somatosensory cortex were recorded. We also quantified the changes in GM-CSF, IL-1ß, IL-6 and BDNF levels, the gliosis and lesion volume as well as microglial and mast cell density, and mast cell surface. Our findings show that GM-CSF promotes cortical reactivation and recovery of tactile abilities, whereas it does not influence motor performances. A transient decrease in pro-inflammatory cytokines after GM-CSF treatment was also observed, whereas the endogenous GM-CSF level was unchanged. While the beneficial role of GM-CSF in reducing glial scar is confirmed, our findings reveal that neuroinflammatory events mediated by microglial and mast cells as well as the alteration of IL-1ß and IL-6 levels are paralleled with an improvement in tactile recovery. These mechanisms could limit the duration and intensity of homeostatic imbalance and promote the plasticity of spared tissues.

Culture conditions have an impact on the maturation of traceable, transplantable mouse embryonic stem cell-derived otic progenitor cells.

The generation of replacement inner ear hair cells (HCs) remains a challenge and stem cell therapy holds the potential for developing therapeutic solutions to hearing and balance disorders. Recent developments have made significant strides in producing mouse otic progenitors using cell culture techniques to initiate HC differentiation. However, no consensus has been reached as to efficiency and therefore current methods remain unsatisfactory. In order to address these issues, we compare the generation of otic and HC progenitors from embryonic stem (ES) cells in two cell culture systems: suspension vs. adherent conditions. In the present study, an ES cell line derived from an Atoh1-green fluorescent protein (GFP) transgenic mouse was used to track the generation of otic progenitors, initial HCs and to compare these two differentiation systems. We used a two-step short-term differentiation method involving an induction period of 5 days during which ES cells were cultured in the presence of Wnt/transforming growth factor TGF-ß inhibitors and insulin-like growth factor IGF-1 to suppress mesoderm and reinforce presumptive ectoderm and otic lineages. The generated embryoid bodies were then differentiated in medium containing basic fibroblast growth factor (bFGF) for an additional 5 days using either suspension or adherent culture methods. Upon completion of differentiation, quantitative polymerase chain reaction analysis and immunostaining monitored the expression of otic/HC progenitor lineage markers. The results indicate that cells differentiated in suspension cultures produced cells expressing otic progenitor/HC markers at a higher efficiency compared with the production of these cell types within adherent cultures. Furthermore, we demonstrated that a fraction of these cells can incorporate into ototoxin-injured mouse postnatal cochlea explants and express MYO7A after transplantation. Copyright © 2016 John Wiley & Sons, Ltd.

Dynamic expression of the polysialyltransferase in adult rat hippocampus performing an olfactory associative task.

Neural cell adhesion molecule (NCAM) is associated with polysialic acid (PSA), and its function is highly dependent on the extent of polysialylation through the activity of two polysialyltransferases, sialyltransferase-X (STX) and polysialyltransferase (PST). PSA-NCAM plays an important role in synaptic plasticity in the hippocampus. The involvement of STX and PST during mnesic processes was assessed in the adult rat hippocampus. We investigated whether different levels in learning and memory using an olfactory associative task influenced STX and PST gene expression in the hippocampus using semiquantitative transcription-polymerase chain reaction. Then, NCAM polysialylation and cell proliferation were quantified in the dentate gyrus of a "Learning and Memory" group using immunohistochemistry. We found that only the expression level of PST mRNA increased with learning performance and returned to an initial level when learned associations were consolidated in long-term memory, while STX mRNA levels remained unchanged. This phenomenon was accompanied by an increase in PSA on NCAM but not by cell proliferation in the dentate gyrus. Our results suggest a different involvement for STX and PST in neural plasticity: while STX is probably involved in the proliferation of neural progenitor cells, PST could play a key role in synaptic plasticity of mature neural networks. The expression of the STX and PST genes could, therefore, be useful markers of neurobiological plasticity in the brain, allowing to follow chronological events in limbic and cortical structures related first to learning and memory processes (for PST) and, second, to adult neurogenesis processes (for STX).