Neuroimmune Modulating And Energy Supporting Nanozyme-mimic Scaffold Synergistically Promotes Axon Regeneration After Spinal Cord Injury

Background:The spinal cord is an integral part of the central nervous system,playing a vital role in transmitting information between the brain and effector tissues,as well as coordinating reflexes.Unfortunately,traumatic spinal cord injury（SCI）remains a prevalent and debilitating condition,with limited treatment options available.Each year,approximately27 million individuals worldwide suffer from long-term paralysis and mental disorders following SCI.However,current clinical therapies often fail to provide significant functional recovery.This is largely due to the formation of excessive glial scar tissue and impaired axon growth.After SCI,the harsh microenvironment triggers the accumulation and activation of pro-inflammatory macrophages（M1 macrophages）,which contribute to the formation of glial scar tissue.CGRP,a neuropeptide widely expressed in the dorsal horn of spinal cords,possesses immunotolerant and immunomodulatory properties,which can promote macrophages to transfer to M2 phenotype,and CGRP has been shown to protect the CNS against the deleterious effects of inflammation,blood-brain barrier injury,brain oedema,and cognitive decline that occur from cerebral ischemic injury.After SCI,additionally,injured axons meet severe energy-supporting deficits,due to mitochondrial damage in neurons.Therefore,exploring the role of neuroimmune and mitochondria energy-supporting microenvironment remodeling after SCI is of great significance for understanding the pathogenesis of SCI.Objective:1.Demonstrate that the immune microenvironment shifts to pro-inflammatory after SCI;2.Demonstrate that mitochondrial damage leads to insufficient energy supply after SCI;3.Demonstrate that CGRP+ nerve fibers grow into the injured area after SCI.Methods:Construct a rat spinal cord injury（Hemi-section）model.Spinal cord specimens from the modeled segment were collected at the time point of 8 weeks（8W）after surgery.H&E staining was used for histological evaluation,and immunofluorescence staining（IF）was used to assess the infiltration and phenotype（M1/M2 phenotype）of macrophages and CGRP+ nerve fiber ingrowth at the injury site,thus comprehensively evaluating the changes in the neural-immune microenvironment after SCI.Transmission electron microscopy（TEM）was used to observe the mitochondrial structure damage in axons.Results:1.A large number of M1 macrophages were recruited to the lesion site after SCI;2.Mitochondrial structures were severely damaged in axons after SCI;3.CGRP+ nerve fibers grew into the lesion area after SCI.Conclusion:This chapter clarifies the pro-inflammatory macrophage infiltration to the lesion site and mitochondrial structure damage after SCI and demonstrates that the CGRP+ nerve fibers grew into the lesion area,thus demonstrating a clear pathological change and providing new ideas and targets for effective treatment of SCI.Part 2: Preparation and characterization of NS@COP scaffoldBackground:In recent years,inorganic nanomaterials with a variety of enzyme-mimic activities,called nanozymes,have attracted significant attention in biomedicine.Compared with natural enzymes,nanozymes have the advantages of sustained activity,controllable release,and multifunctional activity.It has demonstrated excellent therapeutic effects in various disease models such as the nervous system,digestive system,and skeletal muscle system,and has therefore attracted significant attention in the biomedical field.Among them,cerium dioxide nanoparticles（COPs）,as classic nanozymes,have a variety of highly efficient enzyme-like activities,which make them potentially anti-inflammatory,stabilize the oxidative respiratory chain,and protect mitochondria.Therefore,cerium dioxide nanoparticles were stably loaded into a degradable polycaprolactone（PCL） porous scaffold（NS@COP）through electrospinning technology,and COPs were directly and continuously delivered to the spinal cord injury area with the help of the porous scaffold.From a bionics perspective,the biodegradability of the scaffold and the porosity of the simulated extracellular matrix structure can be used to provide growth for neuron axons.This study aims to provide a new approach as well as a theoretical basis for the treatment of SCI.Objective:To explore the preparation methods of NS@COP scaffold.To conduct a comprehensive characterization analysis of NS@COP scaffold to verify the successful preparation of NS@COP scaffold.Methods:The dynamic electrospinning technique was used to fabricate the enzyme mimicry nanoparticle-cerium oxide（COPs）into nanofibers to form NS@COP scaffold.The morphology of NS@COP scaffold and the structure of COPs were observed by Scanning electron microscopy（SEM）and Transmission electron microscope（TEM）.Mechanical instruments,X-ray diffraction（XRD）,and X-ray photoelectron spectroscopy（XPS）were used to characterize the properties of NS@COP.The superoxide dismutase（SOD）kit and catalase kit（CAT）were used to characterize the enzyme-like activity of COPs nanoparticles.Inductively coupled plasma mass spectrometry（ICP-MS）was used to verify the sustained release performance of NS@COP.Live/dead cell staining and CCK-8experiments were used to evaluate the cytocompatibility of NS@COP.Results:The NS@COP scaffolds were successfully prepared with stable physical and chemical properties,and a series of experiments verified that the NS@COP nanofiber scaffold has good biocompatibility for cells at 0.5 and 1.0（wt%）.In cell phagocytosis experiments,TEM observed that COPs nanoparticles could be successfully taken up into cells by neuronal cells and RAW264.7 macrophages.COPs nanoparticles had good SOD-mimetic and CAT enzyme activities and exhibited efficient ROS scavenging capabilities.Conclusion:NS@COP scaffolds were successfully constructed through electrospinning technology and characterized.The diameter distribution of the microscopic fiber morphology on the surface was uniform,and the pore ratio was appropriate.NS@COP scaffolds also had a sustained-release drug delivery property.In addition,the COPs nanoparticles had efficient SOD and CAT enzyme-like activities and could efficiently remove ROS.Therefore,NS@COP scaffold provides a promising treatment strategy for spinal cord injury.Part 3: NS@COP scaffolds regulation of neuroimmune axis and mitochondrial energy-supporting in vitroBackground:Sensory nerves play a crucial role in maintaining tissue homeostasis and promoting tissue regeneration by secreting sensory neuropeptide calcitonin gene-related peptide（CGRP）,which is known to modulate macrophage anti-inflammatory function.And previous studies showed that CGRP positive neuronal fibers undergo sprouting following SCI.Recent studies have uncovered that sensory nerve-immune communications are the key microenvironmental cues in driving macrophage fate toward a pro-resolution phenotype,which may orchestrate axon repair after SCI through the release of protective cytokines such as IL-4 and IL-10.Oxidative stress causes mitochondrial dysfunction and morphological changes,leading to increased ROS production and mitochondrial destruction.This vicious cycle results in excessive ROS accumulation,triggering secondary damage in SCI.Objective:1.To identify the response of the macrophage to neuropeptide was regulated by NS@COP via the key neuropeptide receptors on macrophage under ROS microenvironment and verify the macrophage fate commitment to an anti-inflammatory pro-resolution M2 phenotype;2.To verify the protective effect of NS@COP on mitochondria under oxidative stress microenvironment;3.To verify the protective effect of NS@COP on the growth of neuronal axons under oxidative stress microenvironment.Methods:DRGs were treated with H₂O₂（25 μM）for 4h to construct a ROS-mimic microenvironment in vitro,and then CGRP was detected by enzyme Linked immunosorbent assay（ELISA）.The co-culture system of RAW264.7,CGRP,H₂O₂,and NS@COP was applied to assess the NS@COP on the regulation of communication between macrophage and sensory nerve cytokines（CGRP）.The regulation effect of NS@COP on macrophage phenotype was assessed by immunofluorescence（i NOS&ARG1）.The protein expression levels of RAMP1 in different treatments were detected by Western Blot.The expression levels of inflammatory cytokines IL-1β,IL-10,and TNF-α were detected by ELISA.Co-culture of NS@COP and primary cortical neuron cells under ROS microenvironment was applied to assess the protective effect of NS@COP on the axonal repairing ability of neural cells by immunofluorescence（MAP2）.DCFH-DA probe was used to detect ROS levels.JC-1 and Mito-Tracker Green/Red Cmx Ros were used to detect the mitochondrial membrane potential of neurons.The ultra structure of mitochondria was observed and analyzed by TEM.Results:In in vitro experiments,Western Blot and RT-PCR demonstrated that the NS@COP restored the communication between the macrophage and neuropeptide of sensory nerves to promote the transition of pro-inflammatory macrophage into an anti-inflammatory pro-resolution M2 phenotype via CGRP/RAMP1/AKT axis.Owing to the superior ability of NS@COP to scavenge ROS,it stabilized mitochondrial membrane potential and mitochondrial ultra-structure under H₂O₂-induced microenvironment of excessive oxidative stress via JC-1,Mito-Tracker Green/Red Cmx Ros,and TEM.In addition,NS@COP effectively promoted the axonal elongation and branch spouting under ROS environment via immunofluorescence staining of MAP2.Conclusion:At the cellular level,NS@COP exhibits good neuroimmune regulatory effects under oxidative stress conditions,prompting macrophages to actively respond to CGRP and then polarize toward the anti-inflammatory M2 type.At the same time,NS@COP has good ROS scavenging ability and SOD and CAT enzyme activity;protective effects on mitochondrial membrane potential ΔΨm and ultrastructure;protective effect on neuron axon growth.It provides a good theoretical basis at the cellular level for subsequent in vivo experiments.Part 4: NS@COP scaffolds reduce injury-related cavity size and promote axon regeneration after SCIBackground:CGRP-positive neuronal fibers undergo sprouting following SCI.The RAMP1,a vital component of the CGRP receptor,is abundantly expressed in macrophages,which makes the macrophages able to effectively sense neuronal regulation under physiological conditions.However,macrophages lose their response to neuromodulation in the oxidative stress environment,as evidenced by a shift in the majority of macrophages toward a pro-inflammatory phenotype.Therefore,we hypothesized that excessive ROS hijacks the CGRP/RAMP1 axis-dependent cross-talk between sensory nerves and macrophages,leading to a switch in macrophage fate towards pro-fibrotic commitment and facilitating glial scar formation.Inorganic nanomaterials have received much attention recently due to their various biological-mimic functions.In this part of the study,we propose to prepare an intelligent ROS-responsive nanoenzyme scaffold.For the treatment of spinal cord injury by reconstructing the neuroimmune cross-talk to regulate macrophage fate determination toward an M2 anti-inflammatory phenotype and maintained the mitochondrial energy supply,leading to significantly enhanced neuroprotection and axonal regeneration.Objective:To explore the effect of NS@COP on the axonal regeneration after SCI and to test its biosafety based on in vivo experiments.Methods:In the in vivo experimental part,female SD rats were selected to prepare the model of SCI.The T9 hemi-transection SCI model was applied in all groups（Control group,NS group,0.5%NS@COP group,and 1.0%NS@COP group）.The efficacy of the NS@COP scaffolds for the treatment of SCI was evaluated by taking samples at 4 and 8 weeks postoperatively.IF staining was used to observe the changes in macrophage type,axonal regeneration,scar formation,and myelin sheath regeneration in the injured area after transplantation of NS@COP scaffolds,respectively.RT-PCR was used to explore the expression of inflammatory cytokines IL-1β,IL-4,IL-10,and TNF-α in each group.The ultra structure of mitochondria and the formation of myelin sheath were observed and analyzed by TEM.In addition,H&E staining of major organs（heart,liver,lung,spleen,kidney）was applied to assess the in vivo biosafety of each group of materials.The therapeutic effects of NS@COP scaffolds on the motor function of rats were assessed by BBB scoring,footstep imprinting experiment,and toe spreading level of hind paws.Results:In in vivo experiments,treatment by NS@COP implantation was able to promote recovery of motor function in rats with a significantly increased score of BBB,as well as footprint analysis.Following the macroscopic observation and H&E histological analysis of spinal cord injury tissue from rats,the NS@COP-treated group showed a significantly reduced lesion cavity compared to the control group.Immunofluorescence staining of RAMP1,CD68,CD86,and CD206 demonstrated that NS@COP improved the expression level of RAMP1 on macrophages to promote the conversion of M1 macrophages to M2 phenotype via regulating the response to CGRP.NF200 immunofluorescence staining demonstrated that NS@COP improved the axonal regeneration at the injured site.MBP immunofluorescence staining and TEM demonstrated that NS@COP improved the remyelination of myelin sheath at the injured site.GFAP immunofluorescence staining demonstrated that NS@COP significantly reduced the formation of astrocyte scars.We used TEM to visualize mitochondrial ultrastructure and found the mitochondria in the NS@COP group returned to nearly normal morphology,without swelling or membrane distortion compared with the control group.In addition,the H&E staining showed the primary function organs,including the heart,liver,spleen,lung,and kidney,exhibited no structural abnormality at 8 weeks post-implantation of different NS@COP scaffolds.Conclusion:In this study,we constructed an intelligent ROS-responsive nanomaterial for COPs delivery.In both in vitro and in vivo,NS@COP effectively reconstructed the neuroimmune cross-talk to regulate macrophage fate determination toward an M2anti-inflammatory phenotype and maintained the mitochondrial energy supplying,leading to significantly enhanced neuroprotection and axonal regeneration.The locomotor function of hindlimbs of SCI rats was remarkedly improved following the implantation of the NS@COP scaffold.Last,we hope that our work will contribute to the development of new and effective therapies for patients suffering from spinal cord injuries.