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Sparing of myelin in the injured cord. The images and the relative quantification show the protective action of PM-NPCs on the myelin tracts in the injured cord. After animal perfusion, spinal cords were dissected, postfixed, and coronally sectioned by means of a cryostat. Myelin was quantified by means of Fluoromyelin™ staining (green) performed in sections at the lesion epicenter and 0.5 mm and 1.9 mm caudal to the lesion site through quantitative confocal analysis of ventral and lateral white matter as indicated in the graphs. The confocal microscope images for the saline and PM-NPC-treated mice were obtained using the same intensity, pinhole, wavelength, and thickness of the acquisition. As reference, we used sections close to the ones analyzed and not treated with fluoromyelin. For the quantification we considered sections from at least three animals per group. Values represent the mean ± SEM. We determined the statistical differences by means of ANOVA test followed by Tukey's posttest. ○○○p < 0.001, ○○p < 0.05, ○p < 0.01 versus PBS; ***p < 0.001, **p < 0.05, *p < 0.01 versus LAM.
Source publication
Spinal cord injury (SCI) is a debilitating clinical condition, characterized by a complex of neurological dysfunctions. Neural stem cells from the subventricular zone of the forebrain have been considered a potential tool for cell replacement therapies. We recently isolated a subclass
of neural progenitors from the cadaver of mouse donors. These ce...
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Purpose:
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Citations
... NPCs expressing green fluorescent protein (GFP) were isolated 6 h post-mortem from adult C57BL/6-Tg (UBC-GFP) 30Scha/J mice weighing 25-30 g (Charles River) as previously described [38][39][40][41][42]. All animals' procedures conform to the European Communities Directive of September 2010 (2010/63/UE) and have been approved by the Review Committee of the University of Milan. ...
... This work aims at characterizing the deregulation of non-coding RNAs expression in NPCs expanded inside the Nichoid with respect to standard floating conditions. NPCs were grown for seven days both in standard floating conditions [39,41,64] and inside the Nichoid [32]. Nichoid-grown NPCs expanded themselves in the niches showing different morphology to that of the typical spheroids' conformation ( Figure 1A). ...
Non-coding RNAs show relevant implications in various biological and pathological processes. Thus, understanding the biological implications of these molecules in stem cell biology still represents a major challenge. The aim of this work is to study the transcriptional dysregulation of 357 non-coding genes, found through RNA-Seq approach, in murine neural precursor cells expanded inside the 3D micro-scaffold Nichoid versus standard culture conditions. Through weighted co-expression network analysis and functional enrichment, we highlight the role of non-coding RNAs in altering the expression of coding genes involved in mechanotransduction, stemness, and neural differentiation. Moreover, as non-coding RNAs are poorly conserved between species, we focus on those with human homologue sequences, performing further computational characterization. Lastly, we looked for isoform switching as possible mechanism in altering coding and non-coding gene expression. Our results provide a comprehensive dissection of the 3D scaffold Nichoid’s influence on the biological and genetic response of neural precursor cells. These findings shed light on the possible role of non-coding RNAs in 3D cell growth, indicating that also non-coding RNAs are implicated in cellular response to mechanical stimuli.
... Here, we investigated the Nichoid's effect on neural precursors stem cells (NPCs), already investigated for their therapeutic efficacy in experimental animal models of neurodegenerative diseases such as Parkinson's disease (PD) and in traumatic spinal cord injury (SCI) [28]. Indeed, transplantation of NPCs in preclinical models is feasible and safe [29][30][31][32][33][34][35]. NPCs, isolated from SVZ after the donor's death, intrastriatally infused in a preclinical experimental model of PD promoted a rapid improvement of both animal motility performances and the expression of dopaminergic markers associated to a potent anti-inflammatory effect [29,30,32]. ...
... NPCs, isolated from SVZ after the donor's death, intrastriatally infused in a preclinical experimental model of PD promoted a rapid improvement of both animal motility performances and the expression of dopaminergic markers associated to a potent anti-inflammatory effect [29,30,32]. Moreover, the intravenous administration of NPCs in a preclinical animal model of traumatic SCI improved hind limb functional recovery, axons regenerations, and differentiation into cholinergic neuron cells [31,[33][34][35]. The safety and efficacy of NPCs as cell therapy was investigated not only in preclinical animal models but also in human patients [36]. ...
... NPCs were grown for 7 days both in standard floating conditions [28,31,33] and inside the Nichoid. At the end of this period, cells presented a different 3D organization and morphology ( Figure 1A,B). ...
3D cell cultures are becoming more and more important in the field of regenerative medicine due to their ability to mimic the cellular physiological microenvironment. Among the different types of 3D scaffolds, we focus on the Nichoid, a miniaturized scaffold with a structure inspired by the natural staminal niche. The Nichoid can activate cellular responses simply by subjecting the cells to mechanical stimuli. This kind of influence results in different cellular morphology and organization, but the molecular bases of these changes remain largely unknown. Through RNA-Seq approach on murine neural precursors stem cells expanded inside the Nichoid, we investigated the deregulated genes and pathways showing that the Nichoid causes alteration in genes strongly connected to mechanobiological functions. Moreover, we fully dissected this mechanism highlighting how the changes start at a membrane level, with subsequent alterations in the cytoskeleton, signaling pathways, and metabolism, all leading to a final alteration in gene expression. The results shown here demonstrate that the Nichoid influences the biological and genetic response of stem cells thorough specific alterations of cellular signaling. The characterization of these pathways elucidates the role of mechanical manipulation on stem cells, with possible implications in regenerative medicine applications.
... The use of acute erythropoietin was suggested, and recent small studies are suggestive of possible positive outcomes [11][12][13]. Many different approaches had been developed with unfortunately poor clinical outcomes [14], and even cellular and embryonic tissue transplantation were suggested and studied for a long time with the aim to improve recovery of function through the partial replacement of neural tissue loss or the creation of a favorable environment for survival to injury and regrowth [13][14][15][16][17][18][19]. Up to now, no favorable clinical results are known and long-term safety remains a significantly open unmet medical need [16][17][18]20,21]. ...
... In the present study, we thus aimed to investigate the regenerative potential of the mechanically activated lipoaspirate adipose tissue (MALS) transplanted into the lesion epicenter in a murine experimental model of SCI, immediately after the contusion [13,15,34]. We show that MALS results engrafted two weeks after transplantation and promotes recovery of motor function, which is already evident at 24 hours after the insertion in the lesion site. ...
... Thirty-five days after transplant animals were sacrificed and cords were dissected for subsequence gene expression analysis (n = 6 mice for each group). Tissue homogenates were derived from five regions: the lesion epicenter and 1 cm and 2 mm both rostral and caudal to the lesion site [13,15]. The distance from the lesion was evaluated using a square ruler in order to always have a reproducible distance from the lesion epicenter and an analogous thickness of the tissue homogenate. ...
Spinal cord injury (SCI) is a devastating disease, which leads to paralysis and is associated to substantially high costs for the individual and society. At present, no effective therapies are available. Here, the use of mechanically-activated lipoaspirate adipose tissue (MALS) in a murine experimental model of SCI is presented. Our results show that, following acute intraspinal MALS transplantation, there is an engraftment at injury site with the acute powerful inhibition of the posttraumatic inflammatory response, followed by a significant progressive improvement in recovery of function. This is accompanied by spinal cord tissue preservation at the lesion site with the promotion of endogenous neurogenesis as indicated by the significant increase of Nestin-positive cells in perilesional areas. Cells originated from MALS infiltrate profoundly the recipient cord, while the extra-dural fat transplant is gradually impoverished in stromal cells. Altogether, these novel results suggest the potential of MALS application in the promotion of recovery in SCI.
... We had shown that intravenous administration of erythropoietin-releasing adult neural precursors cells, isolated from SVZ six hours after donor death (Er-NPCs; formerly called post mortem-neural precursor cells; PM-NPCs; Marfia et al., 2011), improve hind limb functional recovery. Er-NPCs accumulate at the lesion site, where they differentiate mostly into cholinergic neuron cells, favouring preservation of myelin (Carelli et al., 2014a;Carelli et al., 2014b;Carelli et al., 2015). Acute traumatic SCI is followed by vascular changes with loss of neurons, oligodendroglia, and astrocytes, neuroinflammation quickly follows with consequent invasion of the injury by a variety of inflammatory cells. ...
... Er-NPCs were obtained from 6 weeks old CD-1 albino mice; their isolation, growth and characterization were performed by following methods described in previously published papers (Marfia et al., 2011;Carelli et al., 2014a;Carelli et al., 2014b;Carelli et al., 2015) and set up in the past by Gritti and co-workers (Gritti et al., 2002). Briefly, cells were isolated from the SVZ 6 hours after sacrifice by cervical dislocation. ...
... After this time, the medium was changed and cells were exposed to the same medium containing foetal bovine serum (1% vol/vol; Euroclone) and depleted of bFGF. This incubation lasted for the following 5 days (Marfia et al. 2011;Carelli et al. 2014b, Carelli et al. 2015. Then, the extent of differentiation was investigated by immunocytochemical staining (Marfia et al., 2011). ...
Background
Spinal cord injury (SCI) is a debilitating condition characterized by a complex of neurological dysfunctions ranging from loss of sensation to partial or complete limb paralysis. Recently, we reported that intravenous administration of neural precursors physiologically releasing erythropoietin (namely Er-NPCs) enhances functional recovery in animals following contusive spinal cord injury through the counteraction of secondary degeneration. Er-NPCs reached and accumulated at the lesion edges, where they survived throughout the prolonged period of observation and differentiated mostly into cholinergic neuron-like cells.
Objective
The aim of this study was to investigate the potential reparative and regenerative properties of Er-NPCs in a mouse experimental model of traumatic spinal cord injury.
Methods and Results
We report that Er-NPCs favoured the preservation of axonal myelin and strongly promoted the regrowth across the lesion site of monoaminergic and chatecolaminergic fibers that reached the distal portions of the injured cord. The use of an anterograde tracer transported by the regenerating axons allowed us to assess the extent of such a process. We show that axonal fluoro-ruby labelling was practically absent in saline-treated mice, while it resulted very significant in Er-NPCs transplanted animals.
Conclusion
Our study shows that Er-NPCs promoted recovery of function after spinal cord injury, and that this is accompanied by preservation of myelination and strong re-innervation of the distal cord. Thus, regenerated axons may have contributed to the enhanced recovery of function after SCI.
... Immunofluorescence analysis was performed as previously reported [11]. Briefly, cryostat coronal sections (15 µm) were collected onto glass slides and processed for immunocytochemistry. ...
Aims:
Hyperoxic breathing might lead to redox imbalance and signaling changes that affect cerebral function. Paradoxically, hypoxic breathing is also believed to cause oxidative stress. Our aim is to dissect the cerebral tissue responses to altered O2 fractions in breathed air by assessing the redox imbalance and the recruitment of the hypoxia signaling pathways.
Results:
Mice were exposed to mild hypoxia (10%O2), normoxia (21%O2) or mild hyperoxia (30%O2) for 28 days, sacrificed and brain tissue excised and analyzed. Although one might expect linear responses to %O2, only few of the examined variables exhibited this pattern, including neuroprotective phospho- protein kinase B and the erythropoietin receptor. The major reactive oxygen species (ROS) source in brain, NADPH oxidase subunit 4 increased in hypoxia but not in hyperoxia, whereas neither affected nuclear factor (erythroid-derived 2)-like 2, a transcription factor that regulates the expression of antioxidant proteins. As a result of the delicate equilibrium between ROS generation and antioxidant defense, neuron apoptosis and cerebral tissue hydroperoxides increased in both 10%O2 and 30%O2, as compared with 21%O2. Remarkably, the expression level of hypoxia-inducible factor (HIF)-2α (but not HIF-1α) was higher in both 10%O2 and 30%O2 with respect to 21%O2 INNOVATION: Comparing the in vivo effects driven by mild hypoxia with those driven by mild hyperoxia helps addressing whether clinically relevant situations of O2 excess and scarcity are toxic for the organism.
Conclusion:
Prolonged mild hyperoxia leads to persistent cerebral damage, comparable to that inferred by prolonged mild hypoxia. The underlying mechanism appears related to a model whereby the imbalance between ROS generation and anti-ROS defense is similar, but occurs at higher levels in hypoxia than in hyperoxia.
Traumatic spinal cord injury (SCI) is a leading cause of lifelong disabilities. Permanent sensory, motor and autonomic impairments after SCI are substantially attributed to degeneration of spinal cord neurons and axons, and disintegration of neural network. To date, minimal regenerative treatments are available for SCI with an unmet need for new therapies to reconstruct the damaged spinal cord neuron-glia network and restore connectivity with the supraspinal pathways.
Multipotent neural precursor cells (NPCs) have a unique capacity to generate neurons, oligodendrocytes and astrocytes. Due to this capacity, NPCs have been an attractive cell source for cellular therapies for SCI. Transplantation of NPCs has been extensively tested in preclinical models of SCI in the past two decades. These studies have identified opportunities and challenges associated with NPC therapies. While NPCs have the potential to promote neuroregeneration through various mechanisms, their low long-term survival and integration within the host injured spinal cord limit the functional benefits of NPC-based therapies for SCI.
To address this challenge, combinatorial strategies have been developed to optimize the outcomes of NPC therapies by enriching SCI microenvironment through biomaterials, genetic and pharmacological therapies. In this review, we will provide an in-depth discussion on recent advances in preclinical NPC-based therapies for SCI. We will discuss modes of actions and mechanism by which engrafted NPCs contribute to the repair process and functional recovery. We will also provide an update on current clinical trials and new technologies that have facilitated preparation of medical-grade human NPCs suitable for transplantation in clinical studies.
In this review, we describe a new avenue that involves the therapeutic use of human adipose tissue. In the past two decades, thousands of papers have described the potential clinical use of human fat and adipose tissue. Moreover, mesenchymal stem cells have been a source of great enthusiasm in clinical studies, and these have generated curiosity at academic levels. On the other hand, they have created considerable commercial business opportunities. High expectations have emerged for curing some recalcitrant diseases or reconstructing anatomically defective human body parts, but several concerns have been raised by generating criticism on the clinical practice that have not been substantiated by rigorous scientific evidence. However, in general, the consensus is that human adipose-derived mesenchymal stem cells inhibit the production of inflammatory cytokines and stimulate the production of anti-inflammatory cytokines. Here, we show that the application of a mechanical elliptical force for several minutes to human abdominal fat activates anti-inflammatory properties and gene-related expression. This may pave the way for new unexpected clinical developments.
Neurodegenerative diseases, including Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), multiple sclerosis (MS), spinal cord injury (SCI), and amyotrophic lateral sclerosis (ALS), are characterized by acute or chronic progressive loss of one or several neuronal subtypes. However, despite their increasing prevalence, little progress has been made in successfully treating these diseases. Research has recently focused on neurotrophic factors (NTFs) as potential regenerative therapy for neurodegenerative diseases. Here, we discuss the current state of knowledge, challenges, and future perspectives of NTFs with a direct regenerative effect in chronic inflammatory and degenerative disorders. Various systems for delivery of NTFs, such as stem and immune cells, viral vectors, and biomaterials, have been applied to deliver exogenous NTFs to the central nervous system, with promising results. The challenges that currently need to be overcome include the amount of NTFs delivered, the invasiveness of the delivery route, the blood–brain barrier permeability, and the occurrence of side effects. Nevertheless, it is important to continue research and develop standards for clinical applications. In addition to the use of single NTFs, the complexity of chronic inflammatory and degenerative diseases may require combination therapies targeting multiple pathways or other possibilities using smaller molecules, such as NTF mimetics, for effective treatment.
Spinal cord injury (SCI) is a distressing incident with abrupt onset of the motor as well as sensory dysfunction, and most often, the injury occurs as result of high-energy or velocity accidents as well as contact sports and falls in the elderly. The key challenges associated with nerve repair are the lack of self-repair as well as neurotrophic factors and primary and secondary neuronal apoptosis, as well as factors that prevent the regeneration of axons locally. Neurons that survive the initial traumatic damage may be lost due to pathogenic activities like neuroinflammation and apoptosis. Implanted stem cells are capable of differentiating into neural cells that replace injured cells as well as offer local neurotrophic factors that aid neuroprotection, immunomodulation, axonal sprouting, axonal regeneration, and remyelination. At the microenvironment of SCI, stem cells are capable of producing growth factors like brain-derived neurotrophic factor and nerve growth factor which triggers neuronal survival as well as axonal regrowth. Although stem cells have proven to be of therapeutic value in SCI, the major disadvantage of some of the cell types is the risk for tumorigenicity due to the contamination of undifferentiated cells prior to transplantation. Local administration of stem cells via either direct cellular injection into the spinal cord parenchyma or intrathecal administration into the subarachnoid space is currently the best transplantation modality for stem cells during SCI.
Background
The secondary injury plays a vital role in the development of Spinal cord injury (SCI), which characterized by the occurrence of oxidative stress, neuronal apoptosis, and inflammatory response. Notoginsenoside R1 (NGR1) has been involved in the modulation of anti-oxidative stress and anti-inflammatory response. However, its roles in SCI-induced injury are still unknown.
Methods
Sprague-Dawley rats were subjected to SCI through a weight-drop method. the therapeutic effect of NGR1 and its underlying mechanism after SCI were explored by using behavioral, biochemical, and immunohistochemical techniques.
Results
The administration of NGR1 after SCI enhanced the neurological function, mitigated tissue damage, and motor neuron loss than those in SCI + vehicle group. Meanwhile, significantly increased expression of Nrf2 protein and HO-1 protein was found in the SCI + NGR1 group compared with those in the SCI + vehicle group. In addition, the inhibitory effects of oxidative stress, apoptotic neuron ratio, and neuronal inflammation in the SCI + NGR1 group can be partially reversed when the Nrf2/HO-1 signaling pathway was inhibited by ML385.
Conclusions
The administration of NGR1 after SCI can attenuate oxidative stress, neuronal apoptosis, and inflammation by activating the Nrf2/HO-1 signaling pathway after SCI, thereby improving neurological function.
