Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that results in the loss of motor function in the central nervous system (CNS) and ultimately death. The mechanisms underlying ALS pathogenesis have not yet been fully elucidated, and ALS cannot be treated effectively. Most studies have applied animal or single-gene intervention cell lines as ALS disease models, but they cannot accurately reflect the pathological characteristics of ALS. Induced pluripotent stem cells (iPSCs) can be reprogrammed from somatic cells, possessing the ability to self-renew and differentiate into a variety of cells. iPSCs can be obtained from ALS patients with different genotypes and phenotypes, and the genetic background of the donor cells remains unchanged during reprogramming. iPSCs can differentiate into neurons and glial cells related to ALS. Therefore, iPSCs provide an excellent method to evaluate the impact of diseases on ALS patients. Moreover, patient-derived iPSCs are obtained from their own somatic cells, avoiding ethical concerns and posing only a low risk of immune rejection. The iPSC technology creates new hope for ALS treatment. Here, we review recent studies on iPSCs and their applications in disease modeling, drug screening and cell therapy in ALS, with a particular focus on the potential for ALS treatment. Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease of the central nervous system (CNS) that can lead to progressive degeneration and loss of motor function, and eventually to paralysis. In recent years, it has been found that in addition to traditional motor disorders, its clinical presentation includes behavioral changes, cognitive dysfunction and non-motor symptoms ,2. BecauSOD1) and expanded GGGGCC repeats in chromosome nine open reading frame 72 (C9orf72). It was first found that SOD1 caused fALS in 1993 [SOD1 leads to the accumulation of misfolded SOD1 protein, neuronal excitability, mitochondrial dysfunction and oxidative stress, all of which may increase cell death [C9orf72 had a large hexanucleotide (GGGGCC) repeat expansion on the affected haplotype [TARDBP), fused in sarcoma (FUS) [The overall crude worldwide ALS prevalence and incidence is between 1.59 and 4.42 per 100,000 person-years , though ll death ,9. In 20aplotype . Other cma (FUS) ,13. In ama (FUS) ,15. Intema (FUS) . Other sma (FUS) ,18,19. ADue to the uncertainty of pathogenesis and the diversity of clinical symptoms, early-stage ALS can be difficult to detect. At present, the diagnosis of ALS is based on comprehensive methods, including clinical history, physical examination and confirmatory tests . To faciGenerally, patients with ALS die of respiratory dysfunction within 3 to 5 years after diagnosis . ALS canUsing four transcription factors , Takahashi et al. reprogrammed mouse embryonic or adult fibroblasts to form induced pluripotent stem cells (iPSCs) ,29. ThesWe know that iPSCs can be reprogrammed from the three germ cells, such as from fibroblasts (mesoderm-derived), hepatocytes (endoderm-derived) and keratinocytes (ectoderm-derived). Fibroblasts are still the most commonly used somatic cell type for iPSCs generation. However, because skin biopsy remains invasive, patients prefer non-invasive and less invasive procedures, such as those requiring keratinocytes, urine cells and blood cells . These cReprogramming strategies for somatic cells have undergone much development. The first generation delivered genes to fibroblasts based on integrated retroviral vectors. Later, people developed strategies to avoid the genetic integration of foreign DNA, including nonintegrated transfer systems using viral or nonviral vectors, e.g., retrovirus, lentivirus, adenovirus, conventional plasmids, recombinant protein, RNA and chemical molecular compounds ,35,36,37To sum up, iPSCs have broad application prospects in disease modeling, cell therapy and drug screening  because There are a few reviews on iPSCs and ALS. For example, Lee et al. focus on disease modeling . They diThe advent of iPSCs accelerated ALS modeling in vitro because iPSCs can produce disease-related cell types with the same genetic background of patients with diseases and can summarize the key performance of diseases in in vitro research. It has been demonstrated that multiple cell types are related to the pathogenesis and progression of ALS, such as MNs, astrocytes, oligodendrocytes and microglia. With iPSC technology, MNs and other types of nerve cells derived from ALS patients can be obtained almost unlimitedly. At present, these cell types are successfully generated from somatic cells of sALS and fALS patients, using reprogrammed iPSCs. ALS modeling from human iPSCs (hiPSCs) in vitro is a valuable addition to animal models, and summarizing the effects of these models will help us better understand the pathogenesis of ALS . Here, wTraditionally ALS is thought of as related to the progressive loss of motor function, and the most important cells affected during its progression are MNs. iPSCs are becoming a new source of MNs.Direct differentiation of iPSCs into MNs is the first necessary step toward their subsequent applications. The differentiation protocols for spinal MNs include induction with small molecules, expression of lineage-specific transcription factors and 2-dimensional and 3-dimensional cultures, as well as the implementation of microfluidics devices and co-cultures with other cell types, including skeletal muscle . All of SOD1 mutation successfully generated MNs and glial cells, which produced a mild disease phenotype [In 2008, it was first reported that iPSCs derived from an ALS patient with a henotype . Howeverhenotype . Recentlhenotype . The reshenotype . TherefoSOD1 MNs recapitulated ALS phenotypes. In SOD1 mutant human iPSCs-MNs, the following features have been observed. One study demonstrated that iPSCs came from patients with SOD1 gene mutations differentiated into spinal MNs, which showed neurofilament aggregation and neurite degeneration that was closely related to abnormal neurofilament regulation [+/A4VSOD1 MNs exhibited disorders in mitochondria, morphology and movement, and exhibited endoplasmic reticulum stress induction. Further, +/A4VSOD1 hiPSCs-MNs showed shorter cell survival that could be saved through gene correction [SOD1 proteins in iPSCs-MNs was confirmed. The proteins played a pathological part in SOD1 mutant ALS, and the survival rate of ALS MNs decreased, indicating that ALS MNs were more prone to cell death than control MNs or mutation-corrected isogenic ALS MNs [SOD1 is an antioxidant enzyme that can protect cells from the influence of reactive oxygen species (ROS) and reduce levels of superoxide . It was gulation . Anotherrrection . Afterwa ALS MNs .SOD1, C9orf72 was identified as an ALS-related gene in 2011. European ALS populations are most likely to have this gene mutation [SOD1, C9orf72 mutation has been studied more in hiPSCs. In 2013, Donnelly et al. confirmed that C9orf72 ALS iPSCs exhibited toxic RNA foci and repeat-associated non-ATG (RAN) translation pathology; C9orf72 ALS iPSC neurons were highly susceptible to glutamate-mediated excitotoxicity [C9orf72 mutations. These neurons showed significantly elevated p62 levels and increased sensitivity to cellular stress induced by autophagy inhibitors [C9orf72-ALS/FTD could be driven by neuronal excitation and stress in patient-derived spinal MNs [GGGGCC repeat expansion compromises nucleocytoplasmic transport in hiPSC-derived neurons with C9orf72 mutation [C9orf72 and TARDBP in iPSCs-MNs from patients with ALS/FTD [C9orf72-associated ALS and FTD. Neurotoxic C9orf72-derived DPRs disrupted the VAPB–PTPIP51 interaction to perturb IP3 receptor-mediated delivery of Ca2+ from ER stores to mitochondria, a key ER-mitochondria signaling function. The findings described a new molecular target for C9orf72-mediated toxicity [C9orf72 has a variety of pathological ALS phenotypes, including RNA foci formation, DPRs generation, excitotoxicity, SG formation and endoplasmic reticulum mitochondrial destruction. In the iPSCs-MNs of ALS patients carrying C9orf72 hexanucleotide amplification, researchers studied DNA, RNA, epigenetics and proteins, and then identified novel and known dysfunctional pathways, hoping to find new targeted molecular pathways and target proteins to affect disease progress [After mutation . Comparetoxicity . Almeidahibitors . It sugginal MNs . Dipeptiinal MNs . Poly GRinal MNs . GGGGCC mutation . The exp ALS/FTD . Recent toxicity . On the TARDBP encodes TDP-43, which is the main protein component of cytoplasmic inclusion bodies found in sALS, and TDP-43 deposition has been proven to have toxic effects on motor neurons. In 2012, the establishment of iPSCs with TDP-43 M337V was reported [TARDBP hiPSCs-MNs show TDP-43 aggregates in cytoplasm, shorter neurites, mitochondrial calcium buffer junction damage, neuron loss and pathological neurofilament abnormalities, but the neuron loss was not related to TDP-43 mislocation or aggregation [2+ uptake disorder is a common characteristic of ALS caused by C9orf72 and TARDBP mutations. In addition, a study of iPSCs-MNs and TDP-43 mutant mice determined that cycloguanylic acid (GMP)–AMP synthase (cGAS), a cytoplasmic DNA sensor, caused upregulation of the cytokine profile of neuritis when TDP-43 entered the mitochondria and released DNA through the permeability transition pore [Mutations of other genes are also studied using iPSC models. reported . TARDBP regation ,64,65. Aion pore .FUS-P525L mutation disrupts FUS protein transport into the nucleus because of a lack of interaction between transportin and the nuclear localization site [FUS mutation following hyperosmolar stress or irradiation. The amount of FUS mislocation was positively correlated with the onset of human diseases  and the maturation status of motor neurons [FUS mutation [FUS is an RNA/DNA binding protein, which is mainly located in the nucleus and plays a role in many processes, such as transcription, splicing and DNA repair . In ALS-ion site . Higelin neurons . Other smutation ,72,73. Rmutation .However, representing only the pathophysiology of a few patients with ALS (fALS) is a common limitation of these models, which helps explain why drugs that perform well in preclinical studies continue to fail in clinical trials. Therefore, it is urgent to establish a representative model of ALS with less well-understood genetic backgrounds, such as sporadic ALS, since it affects many more patients. Currently the establishment of sporadic patient-specific iPSCs lines is flourishing . ResearcC9orf72 and TDP43 mutant patients, but not in SOD1 ones, indicating that different genotypes may cause MNs degeneration through different mechanisms. One interesting study indicated that hiPSCs-MNs derived from some SOD1, FUS and C9orf72 all exhibited hyperexcitability associated with decreased cell survival, despite differences in firing patterns [Based on the above findings, we explored the relationship between the genotype and phenotype of ALS disease and discovered that nucleocytoplasmic transport defects were detected in hiPSCs-MNs of patterns , which sIn conclusion, the development of ALS modeling with patient-iPSCs provides an exciting opportunity to probe molecular phenotypes underlying disease pathology and mechanisms of ALS within human cells. What is more, it provides a new broader scope for drug screening. This technology can model not only familial, but also sporadic ALS, expanding the scope of study and offering personalized or targeted treatment methods. These have greatly broadened the research vision of ALS.In 1996, glutamate transport defects were found in the spinal cord and motor cortex of ALS patients. This defect seems to be due to the selective deletion of astrocyte-specific glutamate transporter EAAT2 , which cThe method of differentiating astrocytes from hiPSCs usually consists of four main phases, as described by Tyzack et al. . Unlike C9orf72 or SOD1 reduced the survival of MNs. Dysfunction and neurotoxic factors released by astrocytes may cause their toxicity [C9orf73 mutant ALS. In addition, during co-culture, the output of action potentials by MNs was damaged, which could be reversed by the repeated amplification of excision C9orf73 mediated by CRISPR-Cas-9 [L39R-linked ALS patients. In MNs cultured with astrocyte-conditioned medium, the expression of the SG marker protein G3BP1 was increased, the expression of caspase 3/7 was upregulated, and autophagy was overactivated. The secretome of astrocytes from patient iPSCs reflected significant oxidative stress in MNs [Astrocytes are the most abundant type of glial cells in CNS and play roles in the development and function of neurons. Their dysfunction is related to several nervous system diseases (such as ALS). In 2017, researchers observed a greater accumulation of p62 in MNs treated with patient astrocyte-conditioned medium, along with increased expression of SOD1. These results suggested that patient astrocytes might regulate the death of MNs by impairing autophagy mechanisms . In 2019toxicity . One stuPR-Cas-9 . iPSCs-Ms in MNs . In 2022s in MNs . These fP525L mutation caused FUS protein to be mislocated from the nucleus to the cytoplasm. Homozygous P525L mutation disrupted the transcriptome, and many differentially expressed genes were related to the function of microglia [Microglia are immune cells of the CNS. They play a role in various aspects of brain development, plasticity and stability through specific recognition, phagocytosis and degradation. A recent report indicates that microglia affect neurofilament deposition in ALS iPSCs-MNs. Using iPSCs derived from a group of identical twins with ALS, astrocytes and microglia were evaluated for their effects on neurofilament protein expression and accumulation in MNs. It was found that the transcriptional level of three neurofilament subtypes of MNs increased. It was further seen that astrocyte-conditioned medium and microglial-conditioned medium influenced the neurofilament deposition of MNs. These results suggest that neurofilament pathology might be modified by glial-secreted factors in ALS iPSCs-MNs . Anothericroglia . Whole gicroglia . The aboOligodendrocytes wrap the axons of neurons to form a thick myelin sheath, which enables quick transmission of electrical signals. Oligodendrocytes communicate with other cell types of the CNS in a variety of ways, including by secreting nutrient factors to regulate the size of neuronal axons and the distribution of ion channels in axons . AlthougBesides acting on neurons, glial cells also interact with each other. Activated microglia secrete Il-1 α, TNF and C1q to induce type A1 astrocytes (a subtype of reactive astrocytes), causing them to lose the capacity to improve neuronal survival, growth and synaptogenesis, finally leading to the death of neurons . GainingThe study of hiPSCs-derived glial cells has improved our comprehension of the pathology and mechanisms of ALS. ALS affects the entire neural network as a multi-factorial disease, including MNs and glial cells. Although interactions among glial cells have been demonstrated in ALS, it is a puzzling problem what induces neuroinflammation and how these changes lead to neurodegeneration. We suggest that ALS treatment strategies must target MNs and glial cells to eliminate nervous system damage. It will be essential to systematically investigate glia across different genotypes. The control of neuroinflammation by regulating the communication between MNs and glial cells is currently an active area of study regarding therapeutic strategies for ALS.In summary, the modeling of ALS using iPSCs and research into changes in disease-associated cells not only lay a sound theoretical basis for pathogenesis, but also facilitate drug screening and cellular treatment of ALS. Continuing to expand the collection of phenotypes of related cell types from iPSCs in ALS patients, tracking the progress of the disease and summarizing physiological and pathological manifestations will all help achieve valuable insights into the physiological and pathological characteristics of ALS, and open up prospects for identifying new mechanisms and provide new perspectives for determining new treatment methods.Given its low endogenous repair ability, degeneration and damage of the CNS are difficult to cure. iPSCs have unlimited self-renewal ability and can differentiate into ectodermal cells, providing a feasible source of nerve cells for transplantation, and the transplantation of neural derivatives of iPSCs is a promising approach for clinical applications of cell therapy for ALS.Because MN loss is one of the main features of ALS, replacing damaged MNs is a tempting strategy. However, cultured MNs are generated mainly for modeling ALS diseases; few studies have directly transplanted cultured MNs to replace damaged MNs. The reasons may include that transplanted MNs no longer divide and must form neuromuscular synapses by precise long-distance axon pathfinding to the target muscle and become involved in the formation of physiologically functional synapses. Thus, direct replacement of degenerating MNs with iPSCs-MNs may not be a feasible treatment strategy for ALS.There is mounting evidence that the curative effects of cell therapy are based on paracrine effects, rather than the survival of transplanted cells . Causes iPSCs can differentiate into NPCs or neural stem cells (NSCs) under different transformation conditions. Then, NPCs or NSCs can differentiate into neurons, astrocytes or microglia. In theory, transplanted iPSC-derived NPCs (iPSCs-NPCs) or iPSC-derived NSCs (iPSCs-NSCs) could directly replace dead MNs in the host and reestablish interconnections to resume motor control of voluntary muscles in ALS patients. Transplanting human iPSCs-NPCs or iPSCs-NSCs into the brain or spinal cord to take the place of lost cells, regulating the injurious environment to protect and regenerate host neurons, is a potentially significant therapeutic strategy for ALS. In 2013, it was proved that NPCs derived from human iPSCs survived and differentiated upon transplantation into a rat model of ALS . Monica In summary, which type of cell product  is applied to cell replacement therapy will vary according to the situation of the disease. For instance, purified single-cell suspensions of dopamine neurons are most suitable for use in Parkinson’s disease. However, aggregates of NSCs/NPCs may be more available for transplantation in ALS. Studies have shown that iPSCs-NSCs and iPSCs-NPCs can effectively bind to host tissue parenchyma after transplantation, differentiate into glial cells and neurons, and exert positive effects through the release of growth factors and immune regulation . HoweverHowever, how to transfer cell therapy from laboratory and experimental animals to clinical application is still a difficult problem that requires considerable efforts. In order to obtain accurate and repeatable data in future preclinical and clinical trials, it is necessary to establish a standardized cell preparation and transplantation scheme  105,106106. AlthSome iPSC-based cell therapies are entering clinical trials, but these therapies are still in their early stages, and safety and effectiveness remain critical issues. In order to carry out this research in a safe and responsible manner, it is crucial to understand the developmental mechanisms underlying cell differentiation, multicellular cell interaction, tissue homeostasis and repair in each case. In conclusion, continuous clinical trials, a great quantity of patients and long-term follow-up should be properly adjusted to obtain significant insights into the efficacy, safety and feasibility of stem cell therapy for ALS in the future. Much work must be undertaken to achieve the maximum benefit of stem cell therapy for ALS.It has been 15 years since the advent of iPSCs technology, and its applications continue to expand. Given the progress of cell therapies using iPSC derivatives and the use of disease-specific iPSCs for pathological analysis, the safety and efficacy of drug development using iPSCs is booming. Using iPSCs from patients’ somatic cells, researchers can directly obtain pathogenic cell types with the same disease mechanisms from patients for drug development. Several groups have undertaken human iPSC-based drug development and drug repositioning for ALS , which hSOD1 mutations. Using this phenotypic assay, they performed a high-throughput screen of 1416 compounds. It was found that Src/c-Abl inhibitors ameliorated the degeneration of MNs in ALS. Src and c-Abl are ubiquitous nonreceptor tyrosine kinases that directly block targets of Src/c-Abl, like bosutinib. Results showed that the positive effect of bosutinib might be related to an increase of autophagy, and bosutinib treatment may reduce the amount of misfolded SOD1 protein in MNs. Among iPSCs-MNs from 11 patients with fALS or sALS, bosutinib treatment increased ALS MNs survival in all patients with fALS and in some with sALS [Many current ALS drug-screening platforms were built on iPSC technology. Imamura K et al. generated iPSCs-MNs from ALS patients with ith sALS . Bosutinith sALS ,117.SOD1 mutation. The mechanism of action of ROPI could be (1) to inhibit oxidative stress, (2) to inhibit TDP-43 and FUS aggregation or (3) to improve mitochondrial function [Fujimori et al. developed a case clustering system capable of subdivision according to the in vitro characteristics of heterogeneous sALS models. They evaluated multiple-phenotype rescue using drug screening and considered ropinirole (ROPI) a candidate . ROPI isfunction . Considefunction .SOD1, C9orf72 and FUS mutations, using multielectrode arrays and patch-clamp recordings. When tested in SOD1-mutated ALS cases, the Kv7 channel activator not only blocked hyperexcitability, but also improved motor neuron survival in vitro [+)-channel. These observations confirmed that ritigabine slows K+-channel gating [Brian et al. found that the hyperexcitability detected in ALS patients was reproduced in iPSC-MNs with in vitro . Retigabl gating . Recentll gating . FurtherP525LFUS mutation, HDAC inhibition restored the DNA repair reaction, pointing out a variety of neuroprotective mechanisms of HDAC inhibitors and arimoclomol [In addition to the above three drugs, iPSC-derived cells also provide tools for the study of other drugs or small molecular compounds. Experiments using iPSCs technology to extract compounds for pathological analysis are focused on the treatment of ALS. Tsuburaya et al. screened about 160,000 compounds and identified some low-molecular-weight compounds that inhibit the interaction of 122 SOD1mut and SOD1-Derlin-1, greatly improving the pathology of patient-induced multipotent stem-cell-derived MN and ALS in model mice. This indicated that the SOD1-Derlin-1 interaction contributes to the pathogenesis of ALS and is a prospective drug target for ALS . Fang etmoclomol . Choi etmoclomol .There is still the need to clarify whether the mechanisms of action of these drugs and compounds share a common pathway and how much the responder populations overlap for each drug. To this end, it is crucial to subdivide ALS according to clinical features, biomarkers and epigenomic information and to confirm the most appropriate drugs for each subcategory.In summary, because discovery of new drugs is time-consuming and costly, and the success rate very low, while traditional transgenic animal models and immortalized human cell lines cannot offer an accurate system to comprehensively evaluate the exact impact of drugs on ALS, the emergence of iPSCs technology provides a new modality with which to address these problems. iPSC modeling provides a platform for the screening of ALS-related phenotypes, which will also accelerate ALS drug screening and translation to clinical trials. Precision medicine approaches using iPSCs have already been applied to ALS, and further development of iPSCs as validation tools for disease modeling and drug discovery will require prediction of patient efficacy from phenotype-saving drugs in iPSC-derived models to allow stratification of patients on the basis of drug response rather than clinical characteristics.As technologies continue to develop, iPSC applications are entering a new phase. Combined with iPSC-based models, a considerable number of new strategies have been expanded to accelerate disease modeling, cell transplantation and drug screening in ALS, including three-dimensional (3D) culture technology, microfluidic technology, single-cell RNA sequencing, genome editing and artificial intelligence (AI).SOD1 iPSCs-MNs and demonstrated the utility of single-cell transcriptomes to map disease-related gene regulatory networks [SOD1 and FUS in hiPSCs, proving that the edited cells could effectively differentiate into corresponding MNs. This appears to offer great potential to develop human ALS models in culture dishes [In addition to single-cell culture and differentiation, multicellular and 3D culture compositions are now also used to study the interactions of disease-related cells. The 3D neuromuscular junction (NMJ) organ-chip model was used to co-culture the musculoskeletal tract and the hiPSCs-MNs of patients with sALS, which verified that bosutinib combined with rapamycin could reduce muscle contraction in sALS cultures. The ability to combine microfluidics and iPSCs technology opens up a new path to study molecular and cellular ALS phenotypes in vitro. Microfluidic devices can simulate NMJ and provide different spaces for different cell types, enhancing control of the cellular microenvironment. In addition, they can be used with 3D cell culture, which improves the function and maturity of NMJ . Namboornetworks . CRISPR e dishes . To assie dishes .In summary, advances in technology continue to promote the research and development of ALS diagnosis and treatment. In the future, automated systems will be used for cell culture and high-throughput screening of candidate compounds for large-scale preparation, and deep learning techniques may be helpful in combining phenotypic imaging and computation with microarray methods for large-scale screening.In conclusion, iPSCs provide an excellent model to assess the effects of pathogenic factors on ALS-related neurons and glial cells due to their easy access and retention of the genetic background. iPSC technology provides an opportunity to obtain disease-specific and patient-specific iPSCs for human disease modeling, drug screening and cell therapy. It brings new hope for the treatment of ALS.Although iPSCs technology is a powerful tool, it continuously faces various challenges. (1) Despite the efforts of researchers, the low reprogramming efficiency of iPSCs has limited their future application. Finding a simple and effective method to improve the efficiency of reprogramming is a problem worthy of serious consideration and urgent solution. (2) ALS is a neurodegenerative disease related to age. Since hiPSCs derivatives are more similar to the fetal stage than the adult stage, hiPSCs-derived cells may not capture the correct phenotype of ALS disease. We suggest finding a method to promote aging and applying it to iPSCs-derived cells from ALS patients, including genetic approaches and the upregulation of key drivers of aging regulators. (3) It is time-consuming and expensive to collect patient samples, establish iPSCs culture and induce specific neuron and glial cell differentiation. We propose to prepare cell banks of iPSC derivatives to promote stem cell clinical applications and future commercialization. Of course, attention should be paid to selecting the appropriate cell type, ensuring the quality of cell products and formulating quality standards at various stages of the manufacturing process. (4) While many reports have shown that iPSCs-derived neural cells can replicate the phenotype of ALS, most of these cases come from familial cases related to specific gene mutations. For sALS, it is still challenging to produce cytopathic cells with disease-related phenotypes. We suggest that sALS should be studied using epigenomic analysis and clustering including advanced mathematical and scientific methods and AI techniques to discover and fully identify relevant disease markers. Research conducted to date with aggregated sporadic cases can provide reliable data for further drug discovery and the formulation of treatment strategies.


The effects of adding two toughening agents on the mechanical and interlayer properties of GF/EP laminates were studied. The results showed that adding the two kinds of particles improved the mechanical properties of the epoxy matrix. When the additional amount of flexible CTBN rubber particles was 8 wt%, and the rigid nano-SiO2 was 0.5 wt%, the fracture toughness of the matrix resin was increased by 215.8%, and the tensile strength was only decreased by 2.3% compared with the pure epoxy resin. On this basis, the effects of two kinds of particles on the interlayer properties of GF/EP composites were studied. Compared with the unmodified GF/EP laminates, the interlayer shear strength and mode I interlayer fracture toughness is significantly improved by a toughening agent, and the energy release rate ICG of interlayer shear strength and interlayer fracture toughness is increased by 109.2%, and 86.8%, respectively. The flexible CTBN rubber particles and rigid nano-SiO2 improve the interfacial adhesion between GF and EP. The cavitation of the two particles and the plastic deformation of the matrix is the toughening mechanism of the interlayer properties of the composite. Such excellent interlaminar mechanical properties make it possible for GF/EP laminates to be widely used as engineering materials in various industries .Poor interlaminar fracture toughness has been a major issue in glass fiber-reinforced epoxy resin (GF/EP) laminate composites. In this paper, soft carboxy-terminated nitrile (CTBN) rubber particles and rigid nano-SiO Glass fiber-reinforced epoxy resin (GF/EP) laminates have the advantages of high mechanical strength, stable size, electrical properties, strong designability, and low price. Thus, they are widely used in construction, electrical engineering, rail transit, and other fields ,2. HowevAccording to the principle of material mechanics, there are two ideal modes of interface adhesive failure and cohesion failure in the layered failure process of fiber-reinforced composite materials , in whic2 particles to study the influence of matrix modulus changes on the interface bonding properties of high-modulus carbon fiber-reinforced epoxy resin composites. When the modulus of the epoxy matrix is increased by 30.6–34.4%, the interlaminar shear strength of its composite material is increased by 39.8–61.8%, and its failure mode changes from interface adhesive failure to matrix cohesion failure [2) with a particle size of 17 nm to the epoxy matrix [The modulus difference between the fiber and the matrix resin is significant, and the stress concentration at the interface can easily cause interface damage to the composite . Li Gang failure ,14. Simiy matrix . Then, tFor matrix toughening, the most prevalent toughening method is incorporating nano-sized or micro-sized fillers into epoxies. According to the different structures of the toughening modifier, it can be divided into polymer toughening  and rigiFor interlaminar toughening, it has been demonstrated that rubber frequently encourages inherent and extrinsic energy dissipation mechanisms like cavitation and matrix yielding that prevent the interlaminar crack from propagating. About 5~20 wt% rubber can increase the interlaminar toughness of fiber-reinforced epoxy resin by 52~85% ,27,28,292 with the epoxy matrix can significantly increase the matrix modulus [2 to epoxy resin can increase the resin’s elastic modulus and fracture toughness by 5–40% and 10–52%, respectively. However, due to the huge specific surface area of nanomaterials, the viscosity of the epoxy matrix will increase, which is not conducive to the processing performance of composite materials [Rigid inorganic particles with high modulus and chemical stability are commonly used as fillers in epoxy composites . The com modulus ,35,36 an modulus . Studiesaterials .2 can improve the strength and modulus of epoxy resin and then enhance the interface failure strength by reducing the difference in the modulus of the GF/EP interface [2 as co-toughening agents to modify the epoxy matrix. Then, the co-toughened epoxy matrix was hot-pressed with glass fiber to prepare GF/EP laminates. The effects of rigid and soft nanoparticles on the interlayer properties of laminates were studied. The fracture process of GF/EP-laminated composites was investigated using sectional analysis. In this study, the correlation between matrix modulus and interlaminar damage process is confirmed. The modulus of the matrix is preserved while the matrix is effectively toughened thanks to the synergistic action of soft and hard particles. The GF/EP laminates’ interlaminar characteristics were greatly enhanced. The materials used in the study are all industrialized and easy to industrialize mass production. This article provides theoretical and technical guidance for the industrial manufacturing of high-performance GF/EP laminates.In summary, the soft CTBN rubber particles are conducive to the improvement of the fracture toughness of the epoxy resin matrix. Nevertheless, the flexibility of the rubber also brings about the problem of reducing the modulus and heat resistance of the epoxy resin, which affects the interface strength of GF/EP. However, rigid nano-SiOnterface . Based o3, purity > 95%), purchased from Guangzhou Nasun Chemical Co., Ltd., Guangzhou, China. Methyl-tetrahydro phthalic anhydride , purchased from Kunshan Lvdun Chemical Co., Ltd., Suzhou, China. N, N-dimethylbenzylamine (purity > 98%) purchased from Shanghai Sinopsin Group Chemical Reagent Co., Ltd., Shanghai, China. CTBN  was purchased from Yuexin Chemical Plastics firm, Tianjin, China. Nano-SiO2  purchased from Boas Nanotechnology (Ningbo) Co., Ltd., Ningbo, China.Bisphenol A type (DGEBA) epoxy resin  to be stirred and defoamed.The stirred liquid was slowly poured into the mold and put in the oven at 60 °C for vacuum defoaming for 30 min.The mold is placed into the oven for curing, and the procedure is conducted at 80 °C for 4 h, and then at 140 °C for 8 h.CTBN rubber is added to E-51 epoxy resin in proportion, with MTHPA as the curing agent and N, N-dimethylbenzylamine as the curing accelerator. The ratios described in this paper are all by weight. The specific steps are as follows:2 with the additional amount of 0.25 wt%, 0.5 wt%, 0.75 wt%, and 1.0 wt% were added to tetrahydrofuran. Ultrasonic dispersion was carried out for 30 min.Based on epoxy resin, CTBN with the optimal ratio was added, and nano-SiO2 solution and stirred for 5 min.Corresponding quality epoxy resin was added into the tetrahydrofuran nano-SiOThe tetrahydrofuran in the resin was removed successively by rotating the evaporation apparatus and vacuum oven. The curing agent and accelerator were added at the ratio of epoxy resin:MTHPA:N, N-dimethylbenzylamine = 100:90:2. For the following steps, refer to The preparation requirements of the specimens for testing the fracture toughness and shear strength of mode I are different, and the preparation process is slightly different. The preparation of GF/EP laminates with mode I fracture toughness was taken as an example. The specific operation steps are described below and schematically shown in The glass fiber cloth was cut into a rectangular fabric of the required size. The glass fiber cloth was washed with ethanol absolute (ethanol concentration ≥ 99.5%) and put into the oven for drying.2 and CTBN is prepared.According to the configuration of the resin matrix, the epoxy resin matrix with different components of nano-SiOUse a brush dipped in a small amount of glue to coat the glass fiber cloth, use a scraper to scrape off excess resin after coating, and then lay the 2nd~12th layer in turn.After the 12th layer is laid, spread polyimide with a thickness of 25 μm in the width direction as a prefab crack , and continue to apply the adhesive liquid on the remaining glass fiber cloth. The layering of the 13th to 24th layers is the same as layers 2nd to 12.After the 24th layer is covered, the system is put into a vacuum bag to vacuum and remove bubbles for 30 min. Put the system into the mold and use the hot press for hot pressing. The curing procedure is 80 °C 4 h and 140 °C 8 h. The thickness of the laminates was about 3 mm, with the fiber volume fraction of 60~65%.B (mm) and width W (mm), crack length a (mm), and maximum pressure maxP (N) of the spline were recorded after the test. The critical stress intensity factor  of the sample was calculated according to the following formula:x = a/W (0 < x < 1), f(x) is the geometric correction factor related to crack length, calculated using Equation (2):The fracture toughness of the epoxy bulk materials was tested based on the single-edge-notch bend (SENB) sample following ASTMD 5045-14 . Standarb) were 150 mm and 20 mm, respectively. The prefabricated crack length was about 35 mm. Smooth each edge of the cut spline, and measure the actual width of the spline b (mm). The hinges were glued on both sides of the spline and placed at room temperature for 24 h. Specimens were stored and tested at a standard laboratory atmosphere of 23 ± 3 °C and 50 ± 10% relative humidity. The universal testing machine program was set to the tensile mode, with a 2 mm/min loading rate. When the sample was peeled 50 mm from the start of the prefabricated crack, the displacement δ (mm) and the load P (N) were recorded for each 5 mm crack length a (mm) extended. When the sample was peeled entirely, the test ended. The relationship between the energy release rate of interlayer peel toughness and the crack length can be obtained through the load–displacement curve, and the crack length is read synchronously online. The propagation interlaminar fracture energy  was the average ICG value when the crack growth (Δa) was increased from 15 to 65 mm. Then, the IC,ProG value of the mode I fracture toughness of the laminated composite with or without CTBN and nano-SiO2 can be obtained. Five samples were tested for each material system and the average result was recorded.According to the standard ASTMD 5528 , the douAccording to GB/T 2567-2021 , the unmAfter the fracture toughness and tensile test of the resin spline, the sample was fixed on the copper sample table with conductive adhesive. Platinum was sprayed twice under a vacuum for observation, and the accelerated voltage was 5 KV.b (mm) and thickness h (mm) were recorded. The universal testing machine performed a three-point bending test on the spline. The span was 15 mm, and the loading rate was 1 mm/min. The maximum load F (N) carried by the sample was recorded. The interlaminar shear strength of the laminated composite was calculated according to Equation (3). The specimens were stored and tested at a standard laboratory atmosphere of 23 ± 3 °C and 50 ± 10% relative humidity. At least three samples were tested for each material system and the average result was recorded.According to standard JC/T773 , the lamICK value showed an increasing trend. The ICK value of pure epoxy resin was 0.38 MPa.m1/2, and after adding 5 wt%, 8 wt%, and 10 wt% CTBN, the ICK value was 0.65 MPa.m1/2, 0.95 MPa.m1/2, and 0.90 MPa.m1/2, respectively, which showed an increase in value by 71%, 150%, and 137%, respectively, compared with pure epoxy resin. It can be seen that the addition of the CTBN toughening agent can improve the toughness of epoxy resin. When the amount added is 8 wt%, the fracture toughness of the epoxy matrix is the best, and the continued addition will lead to the slight decline of toughness. Since the toughening mechanism of epoxy by submicron rubber particles is mainly to produce holes and matrix plastic shear, which weakens the stress field at the crack tip, when the number of particles inside the material reaches the peak, the addition of CTBN cannot produce more holes or matrix plastic yield and also reduces the efficiency of internal stress transfer. Therefore, as the CTBN loading continues to increase, the fracture toughness of the composites begins to decrease.The mode I fracture toughness and tensile properties of epoxy resin were tested, respectively. As shown in 1/2), but its tensile strength is declining by 11.8%. The modulus reduction upon adding the CTBN was expected since the particles contained soft polymer polybutadiene, and its modulus was considerably lower than that of the epoxy resins [As shown in 2 nanoparticles were added to the epoxy resin toughened by 8 wt% CTBN for collaborative toughening. As shown in IC value of the material first increased and then reached the plateau with the loading of nano-SiO2 up to 0.5~1.0 wt%. When nano-SiO2 is not added, the ICK value of 8 wt% CTBN epoxy resin is 0.95 MPa.m1/2. When 0.25 wt%, 0.5 wt%, 0.75 wt%, and 1 wt% nano-SiO2 were added, the ICK values of the modified epoxy matrix were 0.99 MPa.m1/2, 1.20 MPa.m1/2, 1.26 MPa.m1/2, and 1.22 MPa.m1/2, respectively. Compared with the matrix modified by 8 wt% CTBN, the increases were 4.2%, 26.3%, 32.6%, and 28.4%, respectively. Adding nano-SiO2 further improves the fracture toughness of the epoxy resin matrix. When 0.75 wt% nano-SiO2 is added, the toughness of the epoxy resin matrix is the best, and the fracture toughness is 131.6% higher than that of the unmodified epoxy matrix.The authors tried to reduce the influence of rubber particles on the strength of the epoxy matrix. SiO2 on the tensile strength of 8 wt% CTBN-modified matrix. As shown from the figure, the addition of nano-SiO2 causes the tensile strength of the epoxy matrix modified by 8 wt% CTBN to first increase and then decrease. The tensile strength of the epoxy matrix modified by 8 wt% CTBN is 72.6 MPa, and the addition of 0.25 wt%, 0.5 wt%, 0.75 wt%, and 1 wt% nano-SiO2. The tensile strength of the epoxy matrix modified by two-component particles is 73.3 MPa, 80.4 MPa, 76.6 MPa, and 73.8 MPa, respectively. The nano-SiO2 concentration was found to be optimal at 0.5 wt%, resulting in a 10.7% increase in tensile strength relative to the one-component-modified matrix. Compared with the unmodified epoxy matrix, the fracture toughness of the matrix is only 2.3% lower, and the fracture toughness is 215.8% higher than that of the unmodified epoxy matrix. Meanwhile, it can be seen that the introduction of nano-SiO2 dramatically improved the matrix in stiffness. For the 8 wt% CTBN-modified epoxy, the Young’s modulus is 1.09 GPa. At 0.25 wt%, 0.5 wt%, 0.75 wt%, and 1 wt% nano-SiO2, the Young’s modulus of matrix is 1.18 GPa, 1.25 GPa, 1.33 GPa, and 1.35 GPa, corresponding to the increases of 8.2%, 14.7%, 22%, and 23.9%, respectively. The Young’s modulus of epoxy matrix slightly reduces as the concentration of nano-SiO2 reaches 0.5 wt% (1.25 GPa) comparing with neat epoxy (1.35 GPa).2 and 8 wt% CTBN rubber particles has a balanced strengthening and toughening effect on the epoxy matrix.Considering the matrix’s fracture toughness and tensile properties, the synergistic modification of 0.5 wt% nano-SiO2. As can be seen from 2 added. It can be seen that the load–displacement curve (black line) of the DCB specimen of unmodified GF/EP laminate composite material has two significant steps. The rest of the curve is relatively smooth. We analyze the reasons as follows. Under mechanical stress, the brittle epoxy cannot adequately dissipate the mechanical energy, resulting in crack propagation. These cracks propagate easily and quickly to a certain point, leading to a load drop. When 8 wt% CTBN is added, the load–displacement curve (red line) of the DCB specimen is in zigzags, meaning that the cavitation of rubber particles and the plastic deformation of the matrix material are obstructed in the crack propagation, and the displacement load and total displacement of the initial crack increase significantly. On this basis, when 0.5 wt% SiO2 is added, the total displacement of the load exceeds 85 mm, and the area under the load–displacement curve is much larger than that of the GF/EP composite DCB specimen without the modifier.(1) Interlayer fracture toughness: ICGPro, value of the mode I fracture toughness of the laminated composite with or without CTBN and nano-SiO2 added can be obtained  Delamination in fiber-reinforced resin composites is mostly caused by interlaminar shear forces. ICGPro, value is low, only 0.76 kJ/m2.The fracture surface analysis is helpful to analyze the fracture behavior of materials . Figure ICGPro, value increases to 1.18 kJ/m2. It is worth noting that due to the negative effect of CTBN rubber particles on the modulus of the epoxy matrix, the stress transfer efficiency between the fiber and the matrix decreases, and part of the interface debonding also occurs under load (the exposed glass fiber and blue dotted elliptic crack in The coexisting failure mode of cohesion failure and interface adhesive failure and the increased matrix plastic deformation makes the crack propagation in GF/EP laminate composite increase in load consumption, wherein the interlaminar fracture toughness is significantly improved, and the 2 was added to the epoxy matrix modified by 8 wt% CTBN, the glass fiber surface of the DCB specimen of GF/EP composite was covered with the matrix (black and purple arrows in 2 improves the modulus of the matrix, thereby improving the stress transfer efficiency between the matrix and the fiber and avoiding interface adhesive failure with low energy consumption. At this time, cracks propagate in the matrix between the layers. The interlaminar toughness of the composite is mainly determined by the fracture toughness of the matrix, and the fracture toughness of the matrix is higher [ICGPro, value of the composite further increases to 1.42 kJ/m2, which is 88.2% higher than that of the unmodified composite.After 0.5 wt% nano-SiOs higher , so the 2 particle-toughened epoxy resin, and their GF/EP composite laminates. Mode I fracture toughness and toughening mechanism were identified:The epoxy resin can be successfully toughened by CTBN. The epoxy resin’s mode I fracture toughness increases by 150% when CTBN concentration is 8 wt%, but its strength and modulus drop by 11.8% and 19.3%, respectively.2 are used as synergistic toughening agents to toughen the epoxy resin base, when 8 wt% CTBN and 0.5 wt% nano-SiO2 are added to the resin, the fracture toughness of epoxy resin is increased by 215.8%. The tensile strength is only decreased by 2.3%, showing the best comprehensive performance.When flexible CTBN rubber particles and nano-SiO2 increased the ICGPro, value of the GF/EP laminate composite by 86.8% and the interlaminate shear strength by 109.2%.The synergistic toughening effect of 8 wt% CTBN and 0.5 wt% nano-SiO2 makes the interface adhesive failure of GF/EP-laminated composites change to matrix cohesion failure. When cracks expand in the interlayer matrix, the cavitation of the two particles and the plastic deformation of the matrix is the toughening mechanism of the interlayer properties of the composite.The cross-section analysis of GF/EP-laminated composites shows that the addition of flexible CTBN rubber particles and rigid nano-SiOThis study experimentally investigated the fracture toughness of CTBN-toughened epoxy resin, CTBN + nano-SiO


There is lack of an internal fixation following resection of a dumbbell tumor by hemi‐laminectomy and facetectomy that achieves adequate stability with less trauma. Unilateral fixation and reconstruction  may be an ideal technique to address this problem. A biomechanical comparison and a case report were designed to evaluate its spinal stability and clinical effect.Seven fresh‐frozen human subcervical specimens were used for the biomechanical testing. The conditions tested were: (1) intact; (2) injured (single‐level hemi‐laminectomy and facetectomy); (3) unilateral pedicle screw (UPS) fixation; (4) UPS fixation combined with lateral mass (LM) reconstruction (UPS + LM); (5) UPS fixation and contralateral lamina screw fixation (UPS + CLS); (6) UPS + CLS + LM; (7) UPS fixation and contralateral transarticular screw fixation (UPS + CTAS); (8) bilateral pedicle screw (BPS) fixation. Range of motion (ROM) and neutral zone (NZ) were obtained at C5‐C7 segment under eight conditions. In addition, we report the case of a patient with a C7‐T1 dumbbell tumor that was treated by UPS + CLS + LM technique.Except left/right lateral bending and right axial rotation , ROM of UPS + CLS + LM condition in other directions was similar to that of BPS condition . There was no significant difference between UPS + CLS + LM and the UPS + CTAS condition in other directions of ROM , except in left/right axial rotation . Compared to UPS + CLS condition, left/right lateral bending ROM of UPS + CLS + LM condition were significantly reduced . UPS + CLS + LM condition significantly reduced ROM in all directions compared to UPS and UPS + LM condition . Similarly, except lateral bending (p < 0.05), there was no difference in NZ in other directions between UPS + CLS + LM and BPS condition . There was no significant difference between UPS + CLS + LM and UPS + CTAS condition in NZ in all directions . Axial rotation NZ of UPS + CLS + LM condition was significantly reduced compared to UPS + CLS condition (p < 0.05). Compared to UPS and UPS + LM condition, NZ of UPS + CLS + LM condition was significantly reduced in all directions . The patient's imaging examination at 3 months postoperatively indicated that the internal fixation did not move and the graft bone was seen with fusion.After resection of a dumbbell tumor in the cervical spine, UPS + CLS + LM technique is a reliable internal fixation method to provide sufficient immediate stability and promote postoperative bone fusion. After resection of a dumbbell tumor in the cervical spine, unilateral pedicle screw and contralateral lamina screw fixation combined with lateral mass reconstruction (UPS + CLS + LM fixation) is a reliable internal fixation method to provide sufficient immediate stability in the biomechanical study and promote postoperative bone fusion in the case study. A dumbbell tumor is a lesion that connects two or more separate regions of the spine, such as the intradural, epidural, intervertebral foraminal, and paravertebral regions, and accounts for approximately 18% of all spinal tumors.There are three methods for short segment spinal fixation, pedicle screw fixation, lateral mass screw fixation, and transarticular screw fixation, and each has its own limitations. Bilateral pedicle screw fixation is the gold standard for spine surgery instrumentation. However, with increased use and longer follow‐up time disadvantages of bilateral pedicle screw (BPS) fixation such as excessive soft tissue dissection, high risk of injury to neurovascular structures, stress shielding caused by strong fixation, and the potential for accelerating adjacent segment degeneration are being recognized.Bone graft fusion is an important method for internal fixation of the spine. Lateral mass (LM) reconstruction by graft bone can adequately transfer load‐bearing forces from adjacent segments to reduces the stress on screw and rod systems.During removal of a dumbbell tumor via a unilateral posterior approach, the damage to the posterior stabilizing structures, which include the lamina, facet joints, and posterior ligaments,This study was designed: (i) to compare the biomechanical stability of different instrumentation techniques after single‐level hemi‐laminectomy and facetectomy of the cervical spine; (ii) to report the results of a patient with a cervical dumbbell tumor who was treated with the UPS + CLS + LM technique.The study was approved by Medical Ethics Committee of Nanfang Hospital of Southern Medical University. The specimens were obtained from the Department of Anatomy, Southern Medical University. Seven fresh‐frozen human cadaveric cervical spine specimens from C4 to T1 were used in this study. The donors were all male with an average age of 44.3 years . Lateral and anterior/posterior radiographs of the spine specimens were taken to exclude obvious neoplastic, traumatic, congenital conditions, and prior spine surgery. Bone mineral density (BMD) of the lumbar region of the specimens was measured using dual‐energy X‐ray absorptiometry. The average BMD was 1.031 g/cm,Each injured specimen after right C6 hemi‐laminectomy and ipsilateral total C6 facetectomy served to simulate the bony defect after cervical dumbbell tumor resection through a unilateral posterior approach.For pedicle screw placement, anatomical landmarks were used to locate the pedicle entry points and then a high‐speed drill was used to open an entry point to the cortex. The pedicle screws were then carefully inserted. The optimal screw length such that the screws were close to the anterior cortex of the vertebral body was determined in the preoperative planning of the screw trajectories. The screws were 26–32 mm long.For lamina screw placement, the screw entry point was located at the beginning of the lamina from the spinous process at the midpoint of the dorsal arch. A high‐speed drill was used to enter the cortical window. Using a thin pedicle finder, the contralateral lamina was carefully drilled along its length with the pedicle finder aimed at the lateral mass. Procedures were performed under oblique cervical fluoroscopy  to confirm that the pedicle finder did not violate the inner cortex of the lamina. Typically, the tip of the screw should not pass the medial margin of the lateral mass, and its orientation should be parallel to the slope of the lamina under anterior–posterior fluoroscopy. The length of screws was 18–22 mmTransarticular screws were inserted using the Dal Canto technique. The ideal starting point was starting 2 mm caudal to the midpoint of the lateral mass and traveled 40° caudal and 20° lateral. Holes were prepared with a 2.5 mm drill and a 3.2 mm tap. The quadcortical transarticular screws were inserted through preserved channels into the C5/6 and C6/7 zygapophyseal joints. Screw lengths ranged from 20–22 mm.The iliac bone block for lateral mass reconstruction was harvested, shaped to the required size, and then inserted into the space between the C5 and C7 lateral masses on the affected side.All instrumentations  were made of titanium alloy (Ti‐6Al‐4 V). Hybrid fixations were inserted into each vertebra from C5 to C7 according to the experimental protocol of different instrumentation conditions, followed by the attachment of the rod for the pedicle screws and the lamina screws.Intact condition.Injured condition.Unilateral pedicle screws (UPS) fixation.UPS fixation combined with lateral mass reconstruction (UPS + LM).UPS fixation and contralateral lamina screw fixation (UPS + CLS).UPS + CLS fixation combined with lateral mass reconstruction (UPS + CLS + LM).UPS fixation and contralateral transarticular screw fixation (UPS + CTAS).Bilateral pedicle screw fixation (BPS).The eight conditions of each specimen were shown in Figure A custom‐designed and fabricated spine testing machine was used to apply a pure moment of ±2.0 Nm to the top vertebra, while allowing the specimen to move freely. The loading arm applied a continuous pure moment through two U‐joints and one linear bearing at a rate of 1°/s in all three primary loading directions: flexion‐extension, left/right lateral bending, and left/right axial rotation. The load was applied for three complete loading cycles. The first two cycles were used for preconditioning, and results of the third cycle were used for analysis. During flexibility test, the position of each vertebra was recorded by monitoring infrared light‐emitting diodes rigidly attached to each vertebra. A probe with four infrared light emitting diodes on the base of the spine machine defined a general anatomic specimen coordinate system. An optoelectronic camera system  was used to measure the three‐dimensional coordinates of the markers at a sampling frequency of 20 Hz. The range of motion (ROM) and neutral zone (NZ) between C5 and C7 in flexion‐extension, left/right lateral bending, and left/right axial rotation were calculated.The motion tests were also randomized, and the nail trajectory was strengthened in order to minimize any deterioration of the specimens. All eight conditions of each specimen test were completed on the same day.A 27‐year‐old female was admitted to the hospital after a C7/T1 spinal canal tumor was identified. She denied any prior medical problems, and stated she was in overall good health. She did not complain of any discomfort. Physical examination revealed normal cervical lordosis, no neck pain, no numbness of the limbs, normal muscle strength of the limbs, and a bilateral positive Babinski sign. Magnetic resonance imaging (MRI) showed a tumor the right intervertebral foramen of C7/T1, and its appearance suggested a benign lesion, most likely a neurogenic tumor  test. Statistical analysis was performed with SPSS 25.0 software . Values of p < 0.05 were considered to indicate statistical significance.No screw loosening or fixation failure of any specimen was observed during the testing. The ROM and NZ between the C5 to C7 segments are shown in Figure The ROM measurements of the C5‐C7 segments under eight conditions are shown in Table Left lateral bending, right lateral bending, and right axial rotation ROM of the UPS + CLS + LM condition were significantly larger than that of the BPS condition . However, flexion, extension, and left axial rotation ROM were similar between these two instrumentation conditions . Except for left axial rotation and right axial rotation ROM , there were no significant difference in flexion, extension, left lateral bending, and right lateral bending ROM between the UPS + CLS + LM condition and UPS + CTAS condition . Flexion, extension, left axial rotation, and right axial rotation ROM were similar between the UPS + CLS + LM condition and the UPS + CLS condition . However, left lateral bending and right lateral bending ROM of the UPS + CLS + LM condition were significantly reduced compared to the UPS + CLS condition . The UPS + CLS + LM condition significantly reduced ROM in all directions compared to UPS condition and UPS + LM condition .There were no significant differences in ROM in all directions between the BPS condition and the UPS + CTAS condition . Except for flexion direction (p > 0.05), the ROM in all other directions of the UPS + LM condition were significantly less than that of the UPS condition .The NZ measurements of the C5‐C7 segments for eight conditions are shown in Table The lateral bending NZ was significantly increased in the UPS + CLS + LM condition than in the BPS condition (p < 0.05), but there was no difference in the flexion‐extension and axial rotation NZ between the UPS + CLS + LM condition and BPS condition . The NZ of the UPS + CLS + LM condition was the same as that of the UPS + CTAS condition in all directions . In comparison to the UPS condition and UPS + LM condition, the NZs of the UPS + CLS + LM condition were significantly reduced in all directions .The NZ in the axial rotation direction of the UPS + CLS + LM condition was significantly smaller than that of the UPS + CLS condition p < 0.05), but similar to that of the UPS + CLS condition in the flexion‐extension and lateral bending direction . There were no significant differences in NZ in all directions between the BPS condition and the UPS + CTAS condition . The NZ in all directions of the UPS + LM condition were similar to those of the UPS condition   compared to the UPS condition, the UPS + LM condition, and the UPS + CLS condition. However, the contralateral pedicle screw of the BPS condition fixed the three columns to have a longer force arm. The contralateral transarticular screw of the UPS + CTAS condition also fixed the facet column on healthy side by penetrating four layers of cortical bone. Therefore, the UPS + CLS + LM condition was not the most stable of the eight conditions in the right axial rotation.Lateral mass reconstruction was found to improve lateral bending stability by comparing the stability of the UPS + CLS + LM condition with the UPS + CLS condition in our single‐level hemi‐laminectomy and facetectomy model. Because lateral mass reconstruction supported the middle column damaged by the facetectomy, the lateral bending ROM of the affected segment was limited. However, Ji et al. compared the stability of the unilateral pedicle and lamina screws fixation combined with graft bone implant condition and the unilateral pedicle and lamina screws fixation condition in a two‐level hemi‐laminectomy and facetectomy model in a biomechanical test, and found that the addition of a lateral mass improved anti‐rotation stabilityThe use of lateral mass reconstruction in addition to fixation with a rod‐screw system is worth considering when the middle column and posterior column of the injured segment are incomplete. Since the risk of screw loosening, fracture, or displacement is increased by the load transmitted over the adjacent segment through the rod‐screw system, lateral mass reconstruction reduces the load on the rod‐screw system and therefore reduces the occurrence of cervical instability when used to repair a cervical vertebral injury.Posterior internal fixation improves the fusion rate and eliminates the need for strict external fixation and as such pedicle screws and lateral mass screws are widely used to treat spinal instability as an alternative to posterior external fixationUnilateral pedicle screw and lamina screw fixation with lateral mass reconstruction was used to reconstruct C7‐T1 stability in our patient, and satisfactory bone fusion was achieved at 3 months after surgery without any significant complications.The hemi‐laminectomy and facetectomy destroy the posterior structure of the cervical spine, which may lead to the progression of cervical instability and cervical kyphosis, thus internal fixation and fusion are sometimes required. We not only carried out a biomechanical experiment but also reported a case about the short‐term clinical effects of this technique, so that the conclusions of this fixation technique drawn from the study can provide convincing references for clinical treatment. What's more, six instrument conditions have been tested on the same specimens in the biomechanical experiment to provide an exhaustive comparison and provide an important reference value for relevant fixation techniques.There are limitations to this study that need to be considered. The same specimen needs to be repeatedly loaded and unloaded, and the fixation method needs to be changed several times during the biomechanical testing. However, in this study the order of testing the different fixation methods was randomized, and the nail path was strengthened when changing fixation methods to reduce the influence on the test results. In this study we used isolated spine specimens to simulate ideal bone and ligament structure and ignored the role of muscle tissue for biomechanical stability testing; thus, the results only reflect the effect of fixation in the short term after surgery. Further study to examine the long‐term effects, such as fatigue testing, are needed. A minimally invasive approach combining strong fixation with a unilateral pedicle rod system and elastic fixation with contralateral pedicle screws should theoretically result in good bone fusion, reduced contralateral tissue damage, and delayed degeneration of the adjacent segment. However, we only reported the results of one actual case, and no comparative study with BPS was performed. Thus, the value of this fixation technique with respect to promoting bone fusion and delaying degeneration of adjacent segments needs further evaluation.After resection of a unilateral dumbbell tumor in the cervical spine, unilateral pedicle screw and lamina screws fixation with lateral mass reconstruction is a reliable method that provides sufficient immediate stability and promotes bone fusion. The method also reduces surgical trauma and complications.Yongqiang Zeng: Methodology, Investigation, Formal Analysis, Writing—original draft. Zhiping Huang: Methodology, Validation, Investigation. Zucheng Huang and Yongquan Cheng: Validation, Investigation; Qing'an Zhu: Conceptualization, Methodology; Hui Jiang and Wei Ji: Conceptualization, Writing—Review & Editing, Supervision, Project Administration. All authors listed meet the authorship criteria according to the latest guidelines of the International Committee of Medical Journal Editors. And all authors are in agreement with the manuscript.The authors declare that they have no personal, financial, or institutional interest and ethical/legal conflicts involved in this article.NA.


The field emission scanning electron microscopy and transmission electron microscope results show the nanostructured morphological characteristics of the blends. This study achieved the highest fracture toughness, with a fracture toughness in the form of critical stress intensity factors (ICK) value of 2.54 MPa m1/2, in epoxy/block copolymer blends compared to previous works in the field. The impact strength also increased by 116% compared to neat epoxy. This is a major advancement in epoxy toughening due to the use of a single secondary phase. The resulting highly tough and impact-resistant material is a promising candidate for coating applications in industries such as flooring, building, aerospace, and automobiles.We present an efficient and effective method for preparing a novel self-assembled nanostructured material with high toughness and impact strength from a blend of di-glycidyl ether of bisphenol-A (DGEBA) and epoxidized poly(styrene-block-butadiene-block-styrene) (eSBS Hence, over the last 50 years, increasing the fracture toughness of epoxy resins without losing desirable qualities such as modulus and glass transition temperature has been a serious problem [Toughening of epoxy-based thermoset has seen a remarkable surge in research interest during the past several decades ,2. The d problem ,17,18.Self-assembled amphiphilic block copolymers have been used as an effective and intensive modifier to toughen the epoxy resin without sacrificing modulus and glass transition temperature. Amphiphilic block copolymer in epoxy means that one block is “miscible with epoxy” and another block is “immiscible with epoxy. It has been proved that amphiphilic block copolymers have the ability to generate various ordered and disordered nanostructured morphologies via reaction-induced phase separation, and these core-shell nanodomains will be acting as a key factor to fabricate a highly toughened material ,41,42,431/2 [1/2 [In evaluating the toughening mechanism in epoxy block copolymer blends and hybrid blends, various factors, such as morphology, void growth, plasticization effect, fiber-matrix interaction, rubbery cavitation, etc., have been reported to greatly affect the toughening mechanism with maximum toughness that is below 1.5 MPa m1/2 ,49,50. I1/2 [1/2 . Hence, ICK value, that is >2.5 MPa m1/2, called highest toughness in epoxy blends. Furthermore, the combination of possible fracture mechanisms such as plasticization, shear yielding, crack deflection, crack pinning, energy dissipation, and others occurred in this system has been explained. We have demonstrated further how the highest epoxidation degree, thickness of rubbery shell, reaction-induced phase separation, and nanostructured core-shell morphology amplify and correlate with fracture toughness and impact characteristics. We believe that the newly synthesized highly toughened material will be a helpful and novel approach towards the development of toughened materials for coating applications such as flooring, various surfaces and woods, vehicles, aircrafts, and other engineering applications.In our previous studies, we have demonstrated the synthesis of highly epoxidized SBS (55 mol wt%) triblock copolymer and explained the reaction-induced phase separation, nanostructured morphology, wetting studies, viscoelastic, and thermal studies of blends in detail ,18. High−1 and the styrene weight percentage of 29.5%) was supplied by Kraton-D1102 AS, Mumbai, India. MCPBA (metachloroperbenzoic acid) and THF (Tetrahydrofuran −99%) were supplied by Sigma-Aldrich, India.The Lapox L12, diglycidyl ether of bisphenol-A (DGEBA) epoxy resin , Lapox-K5, and 4,4-diaminodiphenylmethane (DDM) were supplied by Atul Ltd. Pala, Kottayam, Kerala, India. The SBS (poly(styrene-block-butadiene-block-styrene) tri block linear copolymer  method in an inert atmosphere at 0 °C using Hukj as solvent according to our previous report ,18. A to55% and epoxy resin were dissolved in THF and heated at 80 °C in an oil bath for the complete removal of the solvent. Stoichiometric amount of DDM hardener was added at 85–90 °C under slow magnetic stirring for 10 min. Finally, the mixture was immediately poured into the pre-heated hot silicon mold at room temperature and cured at 90 °C for 3 h and post-cured at 170 °C for 3 h in an oven. The sample was then allowed to cool naturally to room temperature for further studies. The procedure for the preparation for Epoxy/eSBS-0, 5, 10, and 20 phr -DDM blends (phr means parts per hundred) by using the solvent casting method that was described in detail in our previous papers  In brief55-DDM 10 and 20 phr blends were obtained with a JEOL JEM −2100 microscope applying an acceleration voltage of 100 kV. Prior to the analysis, the specimens were prepared using a LEICA Ultracut ultramicrotome. Thin sections of about 70 nm were cut with a diamond knife at room temperature and deposited on a 300 mesh Cu grid, and analyzed without staining. ImageJ software was used to analyze the particle size and distribution.Transmission Electron Microscopy (TEM): TEM images of epoxy/eSBS55-DDM blends were performed by using a Tinius Olsen universal testing machine. The single edge-notched three-point bending test was selected for the evaluation of fracture toughness of the samples. The samples were tested according to standard method ASTM D-5045 with a crosshead speed of 10 mm/min. Rectangular specimens of dimensions 35 × 8 × 4 mm3 parallel piped bars were considered, with central V-shaped notches of around 4 mm and a razor-sharp crack tip initiated with a fresh razorblade.Fracture Toughness: The fracture toughness tests of neat and epoxy/eSBSICK) was evaluated according to the equation:maxF’ is the maximum load at failure, ‘W’ is the width of the specimen, ‘B’ is the thickness of the specimen, ‘a’ is the cracklength, and f(a/W) is the factor of geometry described in ASTM standard D-5045.Fracture toughness in the form of critical stress intensity factors  mode by applying 5.00 kV accelerating voltage and field-free lens mode. The samples were treated with liquid nitrogen to make them fragile and became easily breakable by hand. A small portion of the sample was then taken for analysis.Field Emission Scanning Electron Microscopy (FE-SEM): FE-SEM micrographs of epoxy/eSBSThe addition of the highest epoxidized SBS (55 mol wt%) that is compatible with epoxy resin leads to viscoelastic phase separation and plasticization effect. The detailed mechanism behind the generation of nanostructured core-shell morphology in epoxy/eSBS55 blend is explained in our previous studies ,18. Brie55 block copolymer has fulfilled these criteria for selective compatibilization of blocks in epoxy and led to reaction-induced phase separation (RIPS). This reaction-induced phase separation leads to nucleation and growth (NG) and self-assembly process, which play a major role in creating nanostructured morphology via microphase separation and offer an avenue for excellent mechanical properties in epoxy thermoset. The TEM images in To design a highly tough material by using epoxy, initial requirement is the choice of block copolymer and its good compatibility with epoxy resin. In this study, eSBSThe above-mentioned mechanisms and evolution of the highly ordered nanostructured spherical core-shell morphology system are clearly depicted in the schematic representation in ICK value of 1.4 MPa m1/2 in their study. Despite employing a hybrid technique in their system, they are unable to achieve high toughness. Functionalized block copolymers can offer better toughness than any other combination, according to current research. George et al. have reported excellent fracture toughness and impact strength by varying epoxidation degree in SBS block copolymer and reported maximum fracture toughness value (1.74 MPa m1/2) in 20 phr epoxy/eSBS blends with 47 mol wt% epoxidation degree [The toughness measurements and impact properties are the major mechanical properties of epoxy resin. Recent toughening strategies in epoxy include the addition of block copolymer alone, mixed block copolymers, and a hybrid approach combining block copolymer with rubber or nanoparticles, among others. Perez et al. claim to have discovered a toughening process in epoxy using the PEP-PEO/PS-PEO modifier . Dean etn degree ,53. As a55 content increased the toughness gradually. The most remarkable and highest improvement came with the blend with 10 phr eSBS55. In this work, fracture toughness values were measured as a critical stress intensity factor, ‘ICK’, and the topmost value obtained in 10 phr blend was 2.54 MPa m1/2. It is noteworthy that other research works in epoxy with amphiphilic block copolymers and other second phase inclusions only reported maximum of 2.2 MPa m1/2 toughness value (ICK) [The effects of various concentrations of eSBS with 55 mol wt% on the fracture toughness of the epoxy blends were investigated a. Increaue (KIC) ,52,53,5455 blend system also correlate with nanostructured core-shell morphology. Neat epoxy is transparent and its ‘ICK’ is 1.54 MPa m1/2. The addition of unmodified SBS lowered the toughness value of epoxy and showed opaqueness because of macrophase separation and poor interfacial interaction between epoxy and SBS [ICK value increased gradually from 1.54 MPa m1/2 (epoxy) to 2.54 MPa m1/2 in 10 phr blend via 1.62 MPa m1/2 (5 phr blend). This is due to the change in morphology from the microlevel to nanolevel . A careful examination of 10 phr and 20 phr blends shows that the nano domains are uniformly distributed in the continuous epoxy matrix. The hydrogen bonding interactions between the hydroxyl groups of the growing epoxy matrix and epoxy groups of the epoxidized block are more pronounced in the 10 phr and 20 phr blends and lead to good adhesion between the epoxy and nano dispersed domains. This factor is a key point for the highest improvement in critical stress intensity factor. Hence, the load applied is effectively transferred to all nanodomains from the major plane of epoxy phase. From the following SEM images, (55-modified epoxies with nanostructured spherical core-shell morphology could be attributed to the combined effect of crack pinning, crack deflection, shear yielding, nanocavitation, energy dissipation, plastic deformation, interfacial debonding of spherical domains, and matrix void expansion (We noticed that the neat epoxy system exhibits smooth morphology, and large cracks are propagated regularly and freely and also oriented in the direction of load in SEM images a. Theref images, b–e, we h images, . Nanodomxpansion . This le55 blends with different concentrations of eSBS55. The results show that the impact strength of neat epoxy has been significantly improved by the addition of eSBS55 and showed maximum value at 10 phr blend. Neat epoxy showed the impact strength value of 2590 J/m2, and after adding 5 phr eSBS55 block copolymer, a 41% improvement in impact strength was observed. The impact strength was enhanced effectively in a 10 phr blend, which shows a 116% increase in impact strength as compared to neat epoxy system. Further addition of epoxidized block copolymer decreases the impact strength. This could be attributed to the inefficient dispersion of eSBS55 as a result of the high viscosity. The 20 phr blend showed an impact strength of 4149 J/m2, which is 60% higher than the impact strength of neat epoxy. Impact strength and impact energy values of neat epoxy and epoxy/eSBS55 block copolymer blends with various concentrations are shown in 55-5, 10, and 20 phr blends. The fracture image of cured neat epoxy in 55-10 and 20 phr blends (r blends d–g. The 2 for impact strength, a 116% increase compared to the neat epoxy system. The high toughness was attributed to various mechanisms, such as crack pinning, crack deflection, shear yielding, nanocavitation, energy dissipation by plastic deformation, inter-facial debonding of nano domains, and matrix void expansion. The results of our study demonstrate that the highly epoxidized SBS block copolymer is an effective modifier for producing a super-toughened and high impact strength material in epoxy systems.In this study, we have successfully fabricated a highly toughened self-assembled nanostructured material in an epoxy thermoset by incorporating an amphiphilic block copolymer, the highly epoxidized SBS triblock copolymer, through the solvent casting method. TEM microscopy studies showed the formation of well-ordered core-shell spherical nanodomain morphologies in the epoxy/eSBS55 blends. The 10 phr blend exhibited exceptional improvement in impact strength and fracture toughness, with a value of 5597 J/m


Polymorphic variants of CX3CR1, I249/M280hCX3CR1, found in 25% of the human population, result in a receptor with lower binding affinity for FKN. Furthermore, disrupted CX3CR1-FKN signaling in CX3CR1-KO and FKN-KO mice leads to exacerbated microglial activation, robust neuronal cell loss and substantial vascular damage in the diabetic retina. Thus, studies to characterize the effects of I249/M280hCX3CR1-expression in microglia-mediated inflammation in the diseased retina are relevant to identify mechanisms by which microglia contribute to disease progression. Our results show that I249/M280hCX3CR1 mice are significantly more susceptible to microgliosis and production of Cxcl10 and TNFα under acute inflammatory conditions. Inflammation is exacerbated under diabetic conditions and coincides with robust neuronal loss in comparison to CX3CR1-WT mice. Therefore, to further investigate the role of I249/M280hCX3CR1-expression in microglial responses, we pharmacologically depleted microglia using PLX-5622, a CSF-1R antagonist. PLX-5622 treatment led to a robust (~70%) reduction in Iba1+ microglia in all non-diabetic and diabetic mice. CSF-1R antagonism in diabetic CX3CR1-WT prevented TUJ1+ axonal loss, angiogenesis and fibrinogen deposition. In contrast, PLX-5622 microglia depletion in CX3CR1-KO and I249/M280hCX3CR1 mice did not alleviate TUJ1+ axonal loss or angiogenesis. Interestingly, PLX-5622 treatment reduced fibrinogen deposition in CX3CR1-KO mice but not in I249/M280hCX3CR1 mice, suggesting that I249/M280hCX3CR1 expressing microglia influences vascular pathology differently compared to CX3CR1-KO microglia. Currently CX3CR1-KO mice are the most commonly used strain to investigate CX3CR1-FKN signaling effects on microglia-mediated inflammation and the results in this study indicate that I249/M280hCX3CR1 receptor variants may serve as a complementary model to study dysregulated CX3CR1-FKN signaling. In summary, the protective effects of microglia depletion is CX3CR1-dependent as microglia depletion in CX3CR1-KO and I249/M280hCX3CR1 mice did not alleviate retinal degeneration nor microglial morphological activation as observed in CX3CR1-WT mice.Diabetic retinopathy, a microvascular disease characterized by irreparable vascular damage, neurodegeneration and neuroinflammation, is a leading complication of diabetes mellitus. There is no cure for DR, and medical interventions marginally slow the progression of disease. Microglia-mediated inflammation in the diabetic retina is regulated  Diabetic retinopathy (DR), an incurable eye disease caused by prolonged high glucose levels is a leading complication of diabetes mellitus and a common cause of blindness amongst working age adults . DR has CX3CR1 gene encodes a protein with amino acid substitutions at residues 249  and 280 (threonine substituted for methionine), I249/M280hCX3CR1, in ~25% of the population and produces an adhesive-defective receptor with decreased binding affinity for FKN  associated with increased cerebellar neuronal cell loss and demyelination , a chemokine expressed uniquely in neurons, mitigates microglia mediated inflammation, often but not always tempering the severity of the reaction –16. A po for FKN , 18. In  for FKN , 19. Intlination . In the ntiation . Transcrneration , 21. It CreER:R26iDTR, in which microglia become susceptible to the effects of diphtheria toxin (DTx), we recently showed that depletion and repopulation of microglia correlated with decreased neuronal cell loss and vascular damage in the diabetic retina  were bred and maintained as previously described  injected once daily for five days with 60 mg/Kg of streptozotocin (STZ) to induce hyperglycemia  , 33. AgeI249/M280, mCX3CR1I250 and mCX3CR1M281 mice were fed 0.12% PLX-5622  chow  for 2-wks, until 10-wks of hyperglycemia, the time point of tissue collection.Eight-wks following STZ-induced hyperglycemia, CX3CR1-WT, CX3CR1-KO, hCX3CR14, 10 g PVP-40, 300 g sucrose and 300 mL ethylene glycol) at -20°C. Brain tissues were dissected from the skull and fixed in 4% PFA overnight at 4°C followed by cryoprotection overnight at 4°C. Cryoprotected brains were sectioned at 30µm using a freezing microtome and free floating brain sections were placed in cryostorage solution at -20°C.Mice were transcardially perfused with cold 1x Hanks’s Balanced Salt Solution (HBSS). Eyes were enucleated and placed in 4% paraformaldehyde (PFA) for 20 minutes. Next, retinas were dissected out of the globe of the eye and placed in 1% PFA for 1 hour. Fixed retinas were then place in cryoprotection solution  overnight at 4°C, and the following day placed in cryostorage solution  and an automatic threshold was applied. Data was normalized by volume based on X, Y and Z coordinates (i.e. 212µm *212 µm *Z stack thickness) to account for changes in confocal Z-stack thickness and images size . We analyzed microglial morphological changes by determining the transformation index (TI) of microglia. To measure TI, 40x images were converted to 32-bit in ImageJ Fiji analysis software (NIH) and individual microglial cells were traced to determine the perimeter and area of a microglia cell. TI was calculated using the equation: perimeter2/4π × area2 . Six images were obtained per ¼ retinal leaflet, 2 images at the central retina nearest the optic nerve, 2 images in the middle of the leaflet and 2 images in the outer leaflet, per mouse ( × area2 . The TI Rn18s (18s) and Actb (β-actin) and is presented as the fold change relative to genotype-specific naïve controls. The following primers were used: 18s (Accession number: NR_003278.3): CGGCTACCACATCCAAGGAA (forward),Retinal isolates from enucleated retinas were homogenized in Trizol  followed by RNA isolation using the Qiagen RNeasy Kit . To generate cDNA, 500 ng of total RNA was reverse-transcribed using the High-capacity cDNA Reverser Transcription Kit . The 7900 HT Fast Real-Time PCR system was used to run RT-qPCR reactions were ran in a 384-well plate in triplicates using SYBR Green PCR master mix , 250 nM forward and reverse primers and 20ng of cDNA template. Results were analyzed as previously described using the comparative Ct method . Data waβ-actin (Accession number: NM_007393) 5’-CTCTGGCTCCTAGCACCATGAAGA-3’ (forward), 5’-GTAAAACGCAGCTCAGTAACAGTCCG-3’ (reverse); Cxcl10 (Accession number: NM_021274.2): 5’-TGCTGCCGTCATTTTCTG-3’ (forward), 5’-GCTCGCAGGGATGATTTCAAG-3’ (reverse); Il1β (Accession number: NM_008361.3): 5’-GTGTGGATCCAAAGCAATAC-3’ (forward), 5’-GTCTGCTCATTCATGACAAG-3’ (reverse); Nos2 (Accession number:NM_010927.4): 5’-GGCAGCCTGTGAGACCTTTG-3’ (forward), 5’-TGCATTGGAAGTGAAGCGTTT-3’ (reverse); Tnfα (Accession number: NM_013639.3): 5’- GGTGCCTATGTCTCAGCCTCTT-3’ (forward), 5’- GCCATAGAACTGATGAGAGGGAG-3’ (Reverse); Il6 (Accession number: NM_031168.1): 5’- TACCACTTCACAAGTCGGAGGC-3’ (forward), 5’- CTGCAAGTGCATCATCGTTGTTC-3’ (reverse); Il10 (Accession number: NM_010548.1): 5’- CGGGAAGACAATAACTGCACCC-3’ (forward), 5’- CGGTTAGCAGTATGTTGTCCAGC-3’ (reverse).GTCGGAAATACCGCGGTC (reverse); P value <0.05 was considered statistically significant. Statistical significance is denoted as *P value <0.05, **P value <0.01, ***P value <0.001 and ****P value <0.0001. Statistical tests performed included a two-tailed parametric unpaired student’s t test with Welch’s correction when comparing two groups  in comparison to naïve controls (11028 ± 3657)  and GFAP+ astrogliosis  in comparison to PBS naive controls  , CD31  and GFAP (astrocytes) Figure 1+ neuronal densities in diabetic CX3CR1-WT and I249/M280hCX3CR1 mice in comparison to the non-diabetic CX3CR1-WT (3646 ± 811.7) control group  compared to diabetic CX3CR1-WT mice   and GFAP+ percent immunoreactive area  compared to diabetic CX3CR1-WT mice   or expression of the human variant alleles of CX3CR1 (I249/M280hCX3CR1), in the diabetic retina, mice were treated for 2-wks with PLX-5622 (+ cells and transformation index (TI), respectively, in PLX-5622 treated and normal chow mice , CX3CR1-KO  and I249/M280hCX3CR1  in comparison to their respective ND genotype-matched controls, CX3CR1-WT (26733.516± 1888.129), CX3CR1-KO (30248.529 ± 3004.598) and I249/M280hCX3CR1 (29796.14 ± 3240.632) (+ cells in the retina and there were no differences between the degree of depletion across the CX3CR1-WT (6994.483 ± 7421.66), CX3CR1-KO (4537.517 ± 5401.353) and I249/M280hCX3CR1 (9155.333 ± 3713.293) diabetic groups , CX3CR1-KO (53.73 ± 17.67) and I249/M280hCX3CR1 (30.398 ± 18.472) in PLX-treated ND mice, compared to ND normal chow CX3CR1-WT (33.607 ± 14.709) CX3CR1-KO (36.01 ± 14.22) and I249/M280hCX3CR1 (29.711 ± 13.76) controls  and I249/M280hCX3CR1  mice were significantly more susceptible to TUJ1+ axonal loss in comparison to diabetic CX3CR1-WT (32.078 ± 3.225) mice  in comparison to the diabetic CX3CR1-WT normal chow (32.078 ± 3.25) control, closely mirroring the levels of TUJ1+ percent immunoreactivity found in the ND normal chow control group (35.953 ± 2.703)  and CX3CR1-KO  mice in comparison to ND controls,  and CX3CR1-KO  in comparison to their diabetic CX3CR1-WT (47.639 ± 5.179) and CX3CR1-KO (49.601 ± 4.794) controls, respectively, closely resembling non-diabetic normal chow, CX3CR1-WT (29.969 ± 3.127) and CX3CR1-KO (30.325 ± 2.976) mice, respectively , CX3CR1-KO (20.751 ± 3.962) and I249/M280hCX3CR1 (16.373 ± 4.219) mice compared to ND normal chow CX3CR1-WT (16.51 ± 2.565), CX3CR1-KO (14.017 ± 1.168) and I249/M280hCX3CR1 (17.842 ± 2.977) controls, respectively  and hCX3CR1I249/M280  mice were more susceptible to an increase in CD31+ percent immunoreactive area indicative of more angiogenesis and vascular damage (CX3CR1-WT mice (14.098 ± 3.115), with a ~25% reduction in CD31+ percent immunoreactive area compared to diabetic normal chow CX3CR1-WT mice (18.926 ± 7.27), closely resembling their ND, normal chow CX3CR1-WT (16.51 ± 2.565) controls  and I249/M280hCX3CR1 (26.305 ± 3.417) retina, compared to ND CX3CR1-KO (16.51 ± 2.565) and I249/M280hCX3CR1 (17.842 ± 2.977) controls, respectively  and I249/M280hCX3CR1  mice had significantly higher CD31+ percent immunoreactive areas in comparison to CX3CR1-WT (14.098 ± 3.115) mice  mice and a ~40% reduction in CX3CR1-KO  mice with respect to their diabetic, normal chow, CX3CR1-WT (14.42 ± 9.626) and CX3CR1-KO (16.998 ± 6.096) controls, respectively  did not significantly alter the percent of fibrinogen deposition in comparison to their diabetic, normal chow I249/M280hCX3CR1 (8.913 ± 4.469) control  maintenance. As notably plastic cells, the various roles microglia perform are largely governed by their activation state  in I249/M280hCX3CR1 expressing mice, revealed increased angiogenesis and abundant microglia clustering around the vasculature  and a decrease in neuronal derived CX3CL1 , creating a loop of inflammation perpetuated by microglia pro-inflammatory cytokine production , 19, 44.reatment . We receCX3CR1 deficiency in diabetic mice is associated with enhanced neuronal and axonal loss  or M281 (M281/WTmCX3CR1) polymorphism in the mouse CX3CR1 loci in the diabetic murine retina (I250/WTmCX3CR1 and M281/WTmCX3CR1) closely resembled the phenotype of the humanized model I249/M280hCX3CR1 mice. Surprisingly, PLX-5622 treatment in diabetic mI250/WTCX3CR1 and mM281/WTCX3CR1 led to a 100% depletion of Iba1+ cells in the retina (To further validate the findings in CX3CR1-WT mice are CX3CR1-dependent as microglia depletion in CX3CR1-KO and I249/M280hCX3CR1 mice did not alleviate retinal degeneration. We characterized the humanized I249/M280hCX3CR1-expressing mice for the first time in the murine STZ model of diabetes. Our data revealed that PLX-5622 treated CX3CR1-KO microglial vascular responses diverge from I249/M280hCX3CR1, mI250/WTCX3CR1 and mM281/WTCX3CR1 microglial responses. These findings indicate that utilizing I249/M280hCX3CR1 mice in studies assessing CX3CR1-FKN signaling effects on the vasculature, represent a complementary model to study dysregulated CX3CR1-FKN signaling. Strategies to alter microglial densities in the human population may not be beneficial as we did not detect neuro- or vasculo-protection in PLX-5622 treated I249/M280hCX3CR1, I250/WTmCX3CR1 and M281/WTmCX3CR1 mice. Future studies to analyze the transcriptional changes of homeostatic, and repopulated microglia from CX3CR1-WT, CX3CR1-KO, FKN-KO and I249/M280hCX3CR1 mice will be valuable to further define the mechanisms by which FKN exerts its regulatory roles in the diabetic retina.Overall, this study reveals that the protective effects of microglia depletion in The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.The animal study was reviewed and approved by University of Texas at San Antonio Institutional Animal Care and Use Committee. Written informed consent was obtained from the owners for the participation of their animals in this study.AEC developed the concept of the study. AEC and KC designed experiments, analyzed and interpreted data and wrote the manuscript. Research development and data acquisition was performed by KC, DR, AM, DV, IG, IT, AA, PV, and SC. SG, AC, RR, and TK interpreted data and advised in manuscript preparation. All authors contributed to the article and approved the submitted version.