EPO-releasing neural precursor cells promote axonal regeneration and recovery of function in spinal cord traumatic injury

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

Counteracting neuroinflammation in experimental Parkinson's disease favors recovery of function : effects of Er-NPCs administration

Background Parkinson\u2019s disease (PD) is the second most common neurodegenerative disease, presenting with midbrain dopaminergic neurons degeneration. A number of studies suggest that microglial activation may have a role in PD. It has emerged that inflammation-derived oxidative stress and cytokine-dependent toxicity may contribute to nigrostriatal pathway degeneration and exacerbate the progression of the disease in patients with idiopathic PD. Cell therapies have long been considered a feasible regenerative approach to compensate for the loss of specific cell populations such as the one that occurs in PD. We recently demonstrated that erythropoietin-releasing neural precursors cells (Er-NPCs) administered to MPTP-intoxicated animals survive after transplantation in the recipient\u2019s damaged brain, differentiate, and rescue degenerating striatal dopaminergic neurons. Here, we aimed to investigate the potential anti-inflammatory actions of Er-NPCs infused in an MPTP experimental model of PD. Methods The degeneration of dopaminergic neurons was caused by MPTP administration in C57BL/6 male mice. 2.5\u2009 7\u2009105 GFP-labeled Er-NPCs were administered by stereotaxic injection unilaterally in the left striatum. Functional recovery was assessed by two independent behavioral tests. Neuroinflammation was investigated measuring the mRNAs levels of pro-inflammatory and anti-inflammatory cytokines, and immunohistochemistry studies were performed to evaluate markers of inflammation and the potential rescue of tyrosine hydroxylase (TH) projections in the striatum of recipient mice. Results Er-NPC administration promoted a rapid anti-inflammatory effect that was already evident 24\u2009h after transplant with a decrease of pro-inflammatory and increase of anti-inflammatory cytokines mRNA expression levels. This effect was maintained until the end of the observational period, 2\u2009weeks post-transplant. Here, we show that Er-NPCs transplant reduces macrophage infiltration, directly counteracting the M1-like pro-inflammatory response of murine-activated microglia, which corresponds to the decrease of CD68 and CD86 markers, and induces M2-like pro-regeneration traits, as indicated by the increase of CD206 and IL-10 expression. Moreover, we also show that this activity is mediated by Er-NPCs-derived erythropoietin (EPO) since the co-injection of cells with anti-EPO antibodies neutralizes the anti-inflammatory effect of the Er-NPCs treatment. Conclusion This study shows the anti-inflammatory actions exerted by Er-NPCs, and we suggest that these cells may represent good candidates for cellular therapy to counteract neuroinflammation in neurodegenerative disorders

Counteracting neuroinflammation in experimental Parkinson’s disease favors recovery of function. Effects of Er-NPCs administration

INTRODUCTION: Parkinson's disease (PD) is the second most common neurodegenerative disease, caused by midbrain dopaminergic neurons degeneration. A growing number of study show that inflammation-derived oxidative stress and cytokine-dependent toxicity may contribute to PD pathogenesis. Cell therapies have long been considered a feasible regenerative approach to compensate for cellular loss. Erythropoietin-releasing Neural Precursors (Er-NPCs) are a subclass of SVZ-derived neural progenitors with high neural differentiation features and able to survive in an unfavorable microenvironment. Our aim was to characterize the therapeutic potential of Er-NPCs in PD. METHODS: The degeneration was obtained by the intraperitoneal administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in C57BL/6 male mice. 2.5x105 GFP-labeled Er-NPCs were administered by stereotaxic injection unilaterally in the left striatum. Functional recovery was assessed by two independent behavioral tests. Neuroinflammation was investigated measuring the mRNAs levels of pro-inflammatory and anti-inflammatory cytokines and immunohistochemistry studies were performed to evaluate markers of inflammation. RESULTS: Our results show that animals grafted with Er-NPCs present a remarkable behavioral improvement beginning the third day after transplantation. We found that transplanted Er-NPCs were vital, able to migrate ventrally and caudally from the injection site, and reach the Striatum and Substantia Nigra pars compacta. Morphological analyses revealed that Er-NPCs can differentiate into dopaminergic (40%), cholinergic (60%), and gabaergic neurons (20%). Furthermore, Er-NPCs administration promoted a rapid anti-inflammatory effect that was already evident 24h after transplant with a decreased expression of pro-inflammatory and increased anti-inflammatory cytokines. Er-NPCs transplant also reduces macrophages infiltration, directly counteracting the M1 pro-inflammatory response of primary murine activated microglia and inducing M2 pro-regeneration traits. Moreover, we show that Er-NPCs-derived Erythropoietin (EPO) mediates this activity, since the co-injection of precursors with an anti-EPO antibody neutralizes the effect of the Er-NPCs treatment. DISCUSSION & CONCLUSIONS: We suggest that Er-NPCs represent good candidates for cellular therapy in PD for their differentiation capabilities and their anti-inflammatory properties. ACKNOWLEDGEMENTS: The authors are deeply grateful to Professor Alfredo Gorio (University of Milan, Italy) for his scientific support and unswerving encouragement to the work. Dr. Zuzanna Gombalova participated in the research when she was visiting a student in the Laboratory of Pharmacology, Department of Health Sciences, University of Milan, supported by the Erasmus Program for PhD students. Dr. Federica Rey and Maria Carlotta F. Gorio are PhD student in the Nutritional Sciences, University of Milan. Federica Rey is supported by theFondazione F.lli Confalonieri

Counteracting neuroinflammation in experimental Parkinson’s disease favors recovery of function : effects of Er-NPCs administration

Parkinson\u2019s disease (PD) is the second most common neurodegenerative disease, presenting with midbrain dopaminergic neuronal degeneration. A growing number of studies suggest that microglial activation plays a role in PD. Indeed, inflammation-derived oxidative stress and cytokine-dependent toxicity may contribute to nigrostriatal pathway degeneration and exacerbate the progression of the disease in patients with idiopathic PD. Cell therapies have long been considered a feasible regenerative approach to compensate for the loss of specific cell populations such as the one that occurs in PD. We recently demonstrated that Erythropoietin-releasing Neural Precursors (Er-NPCs) administered to MPTP intoxicated animals survive after transplantation in the recipient damaged brain, differentiate and rescue degenerating striatal dopaminergic neurons. Here we aimed to investigate the potential anti-inflammatory actions of Er-NPCs infused in an MPTP experimental model of PD. The degeneration was obtained by the intraperitoneal administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (36 mg/kg) in C57BL/6 male mice. 2.5x105 GFP-labeled Er-NPCs were administered by stereotaxic injection unilaterally in the left striatum. Functional recovery was assessed by two independent behavioral tests. Neuroinflammation was investigated measuring the mRNAs levels of pro-inflammatory and anti-inflammatory cytokines and immunohistochemistry studies were performed to evaluate markers of inflammation and the potential rescue of endogenous TH positive projections in the recipient striatum. Our results show that animals grafted with Er-NPCs determined a remarkable improvement of behavior measured by means of horizontal, vertical and olfactory tests beginning the third day after transplantation. By means of immunofluorescence staining, we found that the majority of transplanted Er-NPCs were vital and promote the recovery of dopaminergic markers such as DAT and TH. Furthermore, Er-NPCs administration promoted a rapid anti-inflammatory effect that was already evident 24h after transplant with a decrease of pro-inflammatory and increase of anti-inflammatory cytokines mRNA expression levels. This effect was maintained during the complete observational period, two weeks post-transplant. We also show that Er-NPCs transplant reduces the macrophage infiltration, directly counteracting the M1 pro-inflammatory response of primary murine activated microglia, with the decrease of CD68 and CD86 markers, and inducing M2 pro-regeneration traits, as indicated by the increase of CD206 expression. Moreover, we also show that Er-NPCs-derived Erythropoietin (EPO) mediates this activity, since the co-injection of precursors with an anti-EPO antibody neutralizes the anti-inflammatory effect of the Er-NPCs treatment. This study shows the anti-inflammatory actions exerted by Er-NPCs and we suggest that these cells may represent good candidates for cellular therapy to counteract neuroinflammation in neurodegenerative disorders

Neural Stem Cells transplantation in pre-clinical experimental model of Parkinson’s: counteraction of neuroinflammation and promotion of functional recovery

Parkinson's disease (PD) is the second most common neurodegenerative disease, caused by midbrain dopaminergic neurons degeneration. Cell therapies have long been considered a feasible regenerative approach to compensate for cellular loss. Erythropoietin-releasing Neural Precursors (Er-NPCs) are a subclass of SVZ-derived neural progenitors with high neural differentiation features and able to survive in an unfavorable microenvironment. The aim of this work was to investigate the therapeutic potential of Er-NPCs in pre-clinical experimental model of PD, obtained by the intraperitoneal administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in C57BL/6 mice. 2.5x105 GFP-labeled Er-NPCs were infused unilaterally by stereotaxic injection in the left striatum. Functional recovery was assessed by two independent behavioral tests. The potential effects of Er-NPCs on neuroinflammation was assessed by measuring the expression of pro-inflammatory and anti-inflammatory cytokines and evaluating by immunohistochemistry approach the expression of specific markers. The fate of transplanted cells was also investigated. Our results show that animals grafted with Er-NPCs present a remarkable behavioral improvement beginning the third day after transplantation and increasing in the following observational period. Engrafted Er-NPCs are vital, do not form tumors, and migrate ventrally and caudally from the injection site within the striatum, reaching the Substantia Nigra pars compacta. Furthermore, Er-NPCs administration promotes a rapid anti-inflammatory effect that was already evident 24h after transplant, with a decreased expression of pro-inflammatory cytokines (IL-1alpha, 1beta, IL6, TNF-alpha) and an increased expression of anti-inflammatory cytokines such as IL10. Er-NPCs transplant also reduces macrophages infiltration, directly counteracting the M1 pro-inflammatory response of primary murine activated microglia and inducing M2 pro-regeneration traits. Moreover, we show that erythropoietin, physiologically released by Er-NPCs, mediates these activities, since the co-injection of precursors with anti-EPO/EPOR antibodies neutralizes the effect of the treatment. We suggest that Er-NPCs represent good candidates for cellular therapy in PD for their differentiation capabilities and their anti-inflammatory properties