The influence of sustained dual-factor presentation on the expansion and differentiation of neural progenitors in affinity-binding alginate scaffolds.

Biomaterials capable of controlling the release of multiple growth factors (GFs) could potentially promote the integration of co-transplanted neural progenitor cells (NPCs) and stimulate the plasticity and regenerability of the lesioned spinal cord. As a first step towards the employment of such a vehicle for cell therapy, this study examined the capability of an alginate-sulphate/alginate scaffold, able to capture and rigorously control the release of GFs, to promote the expansion and lineage differentiation of NPCs in vitro. Epidermal growth factor (EGF) and fibroblast growth factor-2 (bFGF) were affinity-bound to alginate-sulphate (200 ng/scaffold) and the bioconjugates were mixed with partially calcium-crosslinked alginate. NPCs isolated from 18 day-old rat embryo brains and seeded into the scaffold during preparation were found to proliferate and differentiate within the vehicle. A continuous release of both bFGF and EGF was noted for a period of 21 days. The concentrations of released GFs were sufficient to promote extensive NPC proliferation at initial cultivation times; the number of neurospheres in the scaffold was twice the number found in the 2D cultures supplemented with 20 ng/ml each factor every 3 days. Between days 10-14, when the GF concentrations had substantially declined, extensive cell migration from the neurospheres as well as lineage differentiation were noted in the scaffold; immunocytochemical analyses confirmed the presence of neurons, astrocytes and oligodendrocytes.The scaffold has a potential to serve as cell delivery vehicle, with proven capability to promote cell retention and expansion, while enabling NPC lineage differentiation in situ.

Oral administration of inosine promotes recovery after experimental spinal cord injury in rat.

Inosine, a purine nucleoside, is one of the novel substances, which can preserve the neuronal and glial viability and stimulate intact neurons to extend axons. We, herein, evaluated the effect of oral inosine treatment on spinal cord injury (SCI) recovery by means of locomotor and bladder function, quantification of neurons and spinal cord tissue sparing. Rats after compression SCI were divided into groups-SCI-Aqua and SCI-Inosine (daily application of aqua for injection or inosine)-locomotion of hind limbs (BBB score) and urinary bladder function were evaluated from day 1 to 28 after SCI. The neuronal profile was determined by immunohistochemistry with NeuN antibodies and tissue sparing by Luxol fast blue staining method. SCI affected the functional movement of hind limbs in both groups with gradual improvement (increased BBB score) during survival. However, we found a significant difference in BBB score and recovery of bladder function between SCI-Aqua and SCI-Inosine groups during the second week of survival following SCI. In addition, the number of NeuN positive cells and percentage of tissue sparing was also significantly higher in SCI-Inosine group when compared with the SCI-Aqua group. Daily oral administration of inosine after SCI throughout the survival was beneficial for locomotion and micturition, neuronal survival and tissue sparing. This indicates that inosine may represent one of the co-stimulatory factors for treatment strategies to promote neuronal plasticity after SCI.

Alterations of protein composition along the rostro-caudal axis after spinal cord injury: proteomic, in vitro and in vivo analyses.

Based on proteomic analyses we investigated the differences of released molecules in the conditioned media (CM) from the spinal cord central lesion and adjacent rostral and caudal segments at 3, 7, and 10 days after spinal cord injury (SCI), in order to specify the molecular environment within greater extent of tissue damage. Proteins found in CM were analyzed by shot-gun MS using nanoLC coupled to an orbitrap. The results showed some specific proteins at each site of the lesion at 3days. Among the proteins from rostral and lesion segments, some are related to chemokines, cytokines or to neurogenesis factors. In contrast, proteins from caudal segments are more related to necrosis factors. The CM from each spinal segment were used in vitro, on microglial BV2 cell lines and DRGs explants, showing a lesion site-dependent impact on microglia activation and DRGs neurite outgrowth. In addition, while naive BV2 cells exhibited insignificant staining for CX3CR1 receptor, the level of CX3CR1 was strongly enhanced in some BV2 cells after their stimulation by CM collected from SCI. The molecular data might correlate with different polarization of activated microglia and macrophages along the rostro-caudal axis following acute injury. This was partially confirmed in vivo with CX3CR1 receptor, revealing higher expression in the rostral segment, with potential neuroprotective action. In addition, the neurotrophic factors released from rostral and lesion segments enhanced outgrowth of DRGs explants. Taken together these data suggest that regionalization in terms of inflammatory and neurotrophic responses may occur between rostral and caudal segments in acute SCI.

A comparative study of PKH67, DiI, and BrdU labeling techniques for tracing rat mesenchymal stem cells.

Mesenchymal stem cells (MSCs) have generated a great deal of promise as a potential source of cells for cell-based therapies. Various labeling techniques have been developed to trace MSC survival, migration, and behavior in vitro or in vivo. In the present study, we labeled MSCs derived from rat bone marrow (rMSCs) with florescent membrane dyes PKH67 and DiI, and with nuclear labeling using 5 µM BrdU and 10 µM BrdU. The cells were then cultured for 6 d or passaged (1-3 passages). The viability of rMSCs, efficacy of fluorescent expression, and transfer of the dyes were assessed. Intense fluorescence in rMSCs was found immediately after membrane labeling (99.3 ± 1.6% PKH67+ and 98.4 ± 1.7% DiI+) or after 2 d when tracing of nuclei was applied (91.2 ± 4.6% 10 µM BrdU+ and 77.6 ± 4.6% 5 µM BrdU+), which remained high for 6 d. Viability of labeled cells was 91 ± 3.8% PKH67+, 90 ± 1.5% DiI+, 91 ± 0.8% 5 µM BrdU+, and 76.9 ± 0.9% 10 µM BrdU+. The number of labeled rMSCs gradually decreased during the passages, with almost no BrdU+ nuclei left at final passage 3. Direct cocultures of labeled rMSCs (PKH67+ or DiI+) with unlabeled rMSCs revealed almost no dye transfer from donor to unlabeled recipient cells. Our results confirm that labeling of rMSCs with PKH67 or DiI represents a non-toxic, highly stable, and efficient method suitable for steady tracing of cells, while BrdU tracing is more appropriate for temporary labeling due to decreasing signal over time.

The effect of hypothermia on sensory-motor function and tissue sparing after spinal cord injury.

BACKGROUND CONTEXT: In recent years, hypothermia has been described as a therapeutic approach that leads to potential protective effects via minimization of secondary damage consequences, reduction of neurologic deficit, and increase of motor performance after spinal cord injury (SCI) in animal models and humans. PURPOSE: The objective of this study was to determine the therapeutic efficacy of hypothermia treatment on sensory-motor function and bladder activity outcome correlated with the white and gray matter sparing and neuronal survival after SCI in adult rats. STUDY DESIGN: A standardized animal model of compression SCI was used to test the hypothesis that hypothermia could have a neuroprotective effect on neural cell death and loss of white and/or gray matter. METHODS: Animals underwent spinal cord compression injury at the Th8-Th9 level followed by systemic hypothermia of 32.0°C with gradual re-warming to 37.0°C. Motor function of hind limbs (BBB score) and mechanical allodynia (von Frey hair filaments) together with function of urinary bladder was monitored in all experimental animals throughout the whole survival period. RESULTS: Present results showed that hypothermia had beneficial effects on urinary bladder activity and on locomotor function recovery at Days 7 and 14 post-injury. Furthermore, significant increase of NeuN-positive neuron survival within dorsal and ventral horns at Days 7, 14, and 21 were documented. CONCLUSIONS: Our conclusions suggest that hypothermia treatment may not only promote survival of neurons, which can have a significant impact on the improvement of motor and vegetative functions, but also induce mechanical allodynia.