Lucia Slovinska

Repetitive Intrathecal Catheter Delivery of Bone Marrow Mesenchymal Stromal Cells Improves Functional Recovery in a Rat Model of Contusive Spinal Cord Injury

Transplantation of bone marrow mesenchymal stromal cells (MSCs) has been shown to improve the functional recovery in various models of spinal cord injury (SCI). However, the issues of the optimal dose, timing, and route of MSC application are crucial factors in achieving beneficial therapeutic outcomes. The objective of this study was to standardize the intrathecal (IT) catheter delivery of rat MSCs after SCI in adult rats. MSCs labeled with PKH-67 were administered by IT delivery to rats at 3 or 7 days after SCI as one of the following treatment regimens: (1) a single injection (5×10(5) MSCs/rat), or (2) as three daily injections (5×10(5) MSCs/rat/d for a total of 1.5×10(6) MSCs/rat over 3 days, injected on days 3, 4, and 5, or days 7, 8, and 9 following SCI. The animals were behaviorally tested for 4 weeks using the Basso, Beattie, and Bresnahan (BBB) locomotor rating scale, and histologically assessed for MSC survival, distribution, and engraftment properties after 28 days. Rats treated with a single injection of MSCs at 3 or 7 days post-injury showed a modest, non-significant improvement in function and low survival of grafted MSCs, which were found attached to the pia mater or accumulated around the anterior spinal artery. In contrast, rats treated with three daily injections of MSCs at days 7, 8, and 9, but not on days 3, 4, and 5, showed significantly higher motor function recovery (BBB score 16.8±1.7) at 14-28 days post-injury. Transplanted PKH-67 MSCs were able to migrate and incorporate into the central lesion. However, only a limited number of surviving MSCs, ranging from 24,128±1170 to 116,258±8568 cells per graft, were observed within the damaged white matter. These results suggest that repetitive IT transplantation, which imposes a minimal burden on the animals, may improve behavioral function when the dose, timing, and targeted IT delivery of MSCs towards the lesion cavity are optimized.

Delivery of Alginate Scaffold Releasing Two Trophic Factors for Spinal Cord Injury Repair.

Spinal cord injury (SCI) has been implicated in neural cell loss and consequently functional motor and sensory impairment. In this study, we propose an alginate -based neurobridge enriched with/without trophic growth factors (GFs) that can be utilized as a therapeutic approach for spinal cord repair. The bioavailability of key GFs, such as Epidermal Growth factor (EGF) and basic Fibroblast Growth Factor (bFGF) released from injected alginate biomaterial to the central lesion site significantly enhanced the sparing of spinal cord tissue and increased the number of surviving neurons (choline acetyltransferase positive motoneurons) and sensory fibres. In addition, we document enhanced outgrowth of corticospinal tract axons and presence of blood vessels at the central lesion. Tissue proteomics was performed at 3, 7 and 10 days after SCI in rats indicated the presence of anti-inflammatory factors in segments above the central lesion site, whereas in segments below, neurite outgrowth factors, inflammatory cytokines and chondroitin sulfate proteoglycan of the lectican protein family were overexpressed. Collectively, based on our data, we confirm that functional recovery was significantly improved in SCI groups receiving alginate scaffold with affinity-bound growth factors (ALG +GFs), compared to SCI animals without biomaterial treatment.

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.

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.

Proteomic analysis of the spatio-temporal based molecular kinetics of acute spinal cord injury identifies a time- and segment-specific window for effective tissue repair

Spinal cord injury (SCI) represents a major debilitating health issue with a direct socioeconomic burden on the public and private sectors worldwide. Although several studies have been conducted to identify the molecular progression of injury sequel due from the lesion site, still the exact underlying mechanisms and pathways of injury development have not been fully elucidated. In this work, based on OMICs, 3D matrix-assisted laser desorption ionization (MALDI) imaging, cytokines arrays, confocal imaging we established for the first time that molecular and cellular processes occurring after SCI are altered between the lesion proximity, i.e. rostral and caudal segments nearby the lesion (R1-C1) whereas segments distant from R1-C1, i.e. R2-C2 and R3-C3 levels coexpressed factors implicated in neurogenesis. Delay in T regulators recruitment between R1 and C1 favor discrepancies between the two segments. This is also reinforced by presence of neurites outgrowth inhibitors in C1, absent in R1. Moreover, the presence of immunoglobulins (IgGs) in neurons at the lesion site at 3 days, validated by mass spectrometry, may present additional factor that contributes to limited regeneration. Treatment in vivo with anti-CD20 one hour after SCI did not improve locomotor function and decrease IgG expression. These results open the door of a novel view of the SCI treatment by considering the C1 as the therapeutic target.

Modulation properties of factors released by bone marrow stromal cells on activated microglia: an in vitro study

In the present paper we develop a new non-cell based (cell-free) therapeutic approach applied to BV2 microglial cells and spinal cord derived primary microglia (PM) using conditioned media from rat bone marrow stromal cells (BMSCs-CM). First we collected conditioned media (CM) from either naive or injured rat spinal cord tissue (SCI-CM, inflammatory stimulation agent) and from rat bone marrow stromal cells (BMSCs-CM, therapeutic immunomodulation agent). They were both subsequently checked for the presence of chemokines and growth, neurotrophic and neural migration factors using proteomics analysis. The data clearly showed that rat BMSCs-CM contain in vitro growth factors, neural migration factors, osteogenic factors, differentiating factors and immunomodulators, whereas SCI-CM contain chemokines, chemoattractant factors and neurotrophic factors. Afterwards we determined whether the BMSCs-CM affect chemotactic activity, NO production, morphological and pro-apoptotic changes of either BV2 or PM cells once activated with SCI-CM. Our results confirm the anti-migratory and NO-inhibitory effects of BMSCs-CM on SCI-CM-activated microglia with higher impact on primary microglia. The cytotoxic effect of BMSCs-CM occurred only on SCI-CM-stimulated BV2 cells and PM, not on naive BV2 cells, nor on PM. Taken together, the molecular cocktail found in BMSCs-CM is favorable for immunomodulatory properties.

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. Copyright

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.

The neuroprotective effect of rat adipose tissue-derived mesenchymal stem cell-conditioned medium on cortical neurons using an in vitro model of SCI inflammation

Abstract Objectives In this study, a new approach was used with an in vitro model in which neural cells were exposed to conditioned media from the injured spinal cord (SCI-CM) mimicking a local inflammatory microenvironment . Subsequently, the neuroprotective effect of rat adipose tissue-derived msesenchymal stem cell-conditioned media (ATMSC-CM) was investigated through a cell-free based therapy, which was used to treat cortical neurons and astrocytes under inflammation. Methods Primary cell cultures isolated from postnatal day (P6) Wistar rat brain cortex were exposed to SCI-CM derived from the central lesion, rostral and caudal segments of injured spinal cord. After 48 h incubation, the SCI-CM was replaced and primary cultures were cultivated either in DMEM media alone or in ATMSC-CM for 72 h. The impact of ATMSC-CM on the viability of neurons and astrocytes was assessed using a CyQUANT® Direct Cell Proliferation Assay Kit as well as immunocytochemistry analysis. Results Immunocytochemical analysis revealed significant decrease in the number of MAP2 positive neurons exposed to SCI-CM compared to Control. Protection by ATMSC-CM was associated with increased survival of neurons compared to primary culture cultivated in DMEM media alone. The ATMSC-CM effect on astrocytes was more variable and without any significant impact. Conclusion The results demonstrate that SCI-CM mimicking inflammation can reduce cortical neuron survival, and subsequent exposure to ATMSC-CM can stabilize the neuronal population most likely via released neuroprotective and trophic factors. In addition, astrogliosis was not affected by ATMSC-CM.

In Vitro Models of Spinal Cord Injury

Living organisms are extremely complex functional systems. At present, there are many in vivo models of spinal cord injury (SCI) that allow the modeling of any type of central nervous system (CNS) injury, however, with some disadvantages. The production of injury models can be a highly invasive and time‐consuming process and requires high technical requirements, and costly financial issues should also be taken into account. Of course, a large number of animals have been used to obtain the relevant data of statistical significance. All of these aspects can be reduced by carrying out experiments in in vitro conditions. The primary advantage of in vitro method is that it simplifies the system under study. There are two major groups of in vitro model in use: cell culture and organotypic slice (OTS) culture. OTS is an intermediate system of the screening of in vitro cell culture and animal models and represents the in vitro system preserving the basic tissue architecture that able to closely mimic the cellular and physiological characteristics in vivo. In vitro models are the preferred methods for the study of acute or subacute pathophysiology after a trauma stimulus, enabling precise control on the extracellular environment, easy and repeatable access to the cells.