Tumors are the second most common cause of death in humans worldwide, second only to cardiovascular and cerebrovascular diseases. Although methods and techniques for the treatment of tumors continue to improve, the effect is not satisfactory. These may lack effective therapeutic targets. This study aimed to evaluate the value of SNHG12 as a biomarker in the prognosis and clinical characteristics of various cancer patients.We analyzed SNHG12 expression and plotted the survival curves of all cancer samples in the TCGA database using the GEPIA tool. Then, we searched for eligible papers up to April 1, 2019, in databases. Next, the data were extracted from studies examining SNHG12 expression, overall survival and clinicopathological features in patients with malignant tumors. We used Review Manager 5.3 and Stata 15 software to analyze the statistical data.P < 0.00001), high tumor stage , high grade , distant metastasis , tumor size , and lymph node metastasis .In the TCGA database, abnormally high expression of SNHG12 in tumor samples indicates that the patient has a poor prognosis. Results of meta-analysis is that SNHG12 high expression is related to low overall survival (HR = 2.72, 95% CI = 1.95–3.8, Our study confirmed that the high expression level of SNHG12 is closely related to the clinicopathological characteristics and prognosis of patients and is a new predictive biomarker for various cancer patients.The online version contains supplementary material available at 10.1186/s41016-021-00250-4. According to the Global Burden of Disease Study (GBD), in 2018, there were approximately 18.1 million newly diagnosed tumor patients and 9.6 million patient deaths associated with tumors; malignant tumors are the second most common killer of humans worldwide, second only to cardiovascular and cerebrovascular diseases .Long noncoding RNAs (lncRNAs) are currently a comparatively hot RNA. What is impressive is that it does not develop reading frames and coding capabilities. Compared with approximately 2% of the protein-coding genes, most lncRNAs play a negligible role in transcription and translation , 4. WithThe US government-sponsored database The Cancer Genome Atlas (TCGA) uses high-throughput genome sequencing, gene chip technology, and multidimensional data integration analysis to collect data on almost all human cancers  to provide a resource for the elucidation of the mechanisms of cancer occurrence and development. Based on this database, we used web tools to explore SNHG12 expression in different patient samples in the TCGA database and tried to generate a curve to show the relationship between SNHG12 expression and patient prognosis. In addition, this study provides new biomarkers for clinical diagnosis and treatment to detect tumors early and help select improved treatments.The long noncoding RNA SNHG12  was first reported to be upregulated in microarray data in endometrial carcinoma by Zhai. SNHG12 is located at 1p35.3 (chr1: -28905061 - -28909492) and includes 6 exons, which can be translated to generate 8 transcript variants with polyA tails . Liu et SNHG12 was shown to be an effective diagnostic biomarker in nasopharyngeal carcinoma with a multivariate Cox regression analysis . Furtherhttp://gepia.cancer-pku.cn/index.html).We analyzed 9497 samples with SNHG12 expression in the TCGA database through the GEPIA tool and plotted the survival curve  AND (“cancer” OR “tumor”). We also searched the references of the original articles and manually consulted the relevant supplementary results. We only examined articles written in English.This study will strictly abide by the PRISMA statement. First, we excluded duplicated articles. For the remaining articles, we first examined the title and abstract of the study. The exclusion criteria were as follows: (1) not related to the long noncoding RNA SNHG12, (2) only detected SNHG12’s composition and functions, (3) lacked clinical data, and (4) written in another language (not English). By contrast, the acceptance criteria were as follows: (1) the expression level of SNHG12 is described in tumors; (2) research objects must be grouped according to the SNHG12 expression levels; (3) a description of the clinical case characteristics, for example, clinical stage, lymphatic metastasis, distant metastasis (DM), tumor size, and overall survival (OS), was provided; and (4) the study was written in English. Finally, we identified ten papers about SNHG12 by the inclusion and exclusion criteria.Three people  independently scanned and extracted all useful data from the 10 papers: (1) paper title, paper authors, year, and area of the patients; (2) tumor type, sample, and size; (3) SNHG12 evaluation method; (4) cut-off values; (5) ORs of SNHG12 for the clinicopathological characteristics, including tumor stage, lymphatic metastasis, grade, depth, tumor size, and gender; and (6) follow-up time. Because the original literature did not provide survival information, according to the method described previously, we used graphs from Engauge Digitizer 4.1 software to estimate the prognosis of the survival curve of each article. If only a Kaplan-Meier curve was used, we extracted survival data from the papers and calculated the HR and 95% CI by previously published techniques , 24. TheP < 0.1). Otherwise, we used a fixed-effect model  [P < 0.05 as significant. We performed all statistical analyses using Review Manager 5.3 and Stata 15.Based on the studies documented in the literature, we collected the 95% confidence intervals and HRs associated with patient prognosis from articles identified based on our criteria. We used the O-E combined variance method to calculate the patient survival outcomes by Review Manager . Then, tP > 0.1) . The resP = 0.0012, HR = 1.1). High expression of SNHG12 in these tumors  was associated with poor prognosis  by Review Manager 5.3 software. We recalculated the data in another way through Stata software . These results indicate that high expression of SNHG12 can significantly reduce the overall survival of patients, suggesting that the prognosis of the patients is poor. The fixed model was adopted because there was no significant heterogeneity  , nervous system , respiratory system carcinomas , cancers of the reproductive system , and locomotor system . Next, according to the different cut-off scores from the articles, we conducted analysis between different groups, including the median , and other studies . Then, based on the sample size (≥ 60 or < 60), we divided the studies into two categories. In this analysis, regardless of whether the study had a large sample size  or a small sample size , high expression of SNHG12 was an obvious prognostic marker for poor OS. However, we divided the studies into categories based on the sample quality score based on a score of 80. Highly expressed SNHG12 predicts short overall survival for patients from studies with a high quality score , but the low quality score subgroup did not show significant results due to the combined effects .Nine papers, which included 639 patients, described the relationship between the expression of SNHG12 and OS; the pooled HR was determined to be 2.14 . In the same way, we also performed corresponding tests between the different subgroups. The large tumor sample size (Egger’s P = 0.326), high quality score (Egger’s P = 0.387), and median subgroups , lymphatic metastasis , high grade , tumor size , and distant metastasis . However, there was no significant difference in patient age  or gender .The clinical and pathological characteristics of the included research articles were statistically analyzed, and the results, including the ORs and 95% CIs, are shown in Tables 2 = 60%, P = 0.01) showed obvious statistical heterogeneity in 8 studies , the large sample size subgroup , and other the cut-off subgroups . According to the three subgroups, we found that the Jing Dong study  was in the above three subgroups and may be the main source of heterogeneity. No proof of statistical heterogeneity was found in lymphatic metastasis subgroup, the tumor stage subgroup and distant metastasis subgroup . However, Zhi-Biao Liu  showed no clinical significance or obvious heterogeneity by stratification analysis of the lymphatic quality subgroup. Further investigation of heterogeneity in distant metastasis revealed obvious heterogeneity in the low quality score  subgroups and tumor size .Tumor size  . Furthermore, the aggregated data showed that SNHG12 high expression was associated with tumor stage (P < 0.00001), poor cancer outcome (P = 0.01), positive lymph node metastases (P < 0.00001), distant metastases (P = 0.0006), and tumor size (P < 0.00001). However, whether using the TCGA database or meta-analyses, there are limitations that cannot be ignored. First, most of the sample sources in the TCGA database and research articles are geographically limited. There are many regions in the world, the geographical environment and customs are different, and the diversity of the samples cannot be ignored. Second, the lack of statistical analysis of the raw data in some articles resulted in heterogeneity in the HR and OR in the later statistical analyses. Third, because different articles have different classifications and qualitative characteristics of the original data, different standards are an important reason for the heterogeneity of the results. At last, in these studies, Liu et al. used univariate and multivariate methods to analyze the survival data of the NPC patients; others only used the univariate method to assess the survival data and did not discuss the relationship between multiple factors.Here, we wanted to observe whether the role of SNHG12 in tumors is the same, that is, to suppress or promote cancer. First, we found that SNHG12 can play a role in promoting tumors in different tumors via the GEPIA. Second, the meta-analysis showed that high SNHG12 expression may be considered a negative factor for prognosis of patients with various tumors (The current analysis shows that, whether in the TCGA database or in published articles, overexpression of SNHG12 is closely related to the poor prognosis of tumor patients. SNHG12 high expression is related to the OS, tumor stage, lymph node metastasis, tumor size, distant metastasis, and high tumor grade, especially in the Chinese population. Therefore, SNHG12 can be used as an effective biomarker to predict the prognosis and tumor progression of cancer patients. However, larger and well-designed studies are needed to confirm the results of this analysis.Additional file 1 : Supplementary Table S1. The cancer type of the 10 study papers.Additional file 2 : Supplementary Table S2. The clinicopathological characteristics of 9 study in this paper.Additional file 3 : Supplementary Table S3. The value of quality evaluation estimated on the REMARK guideline.Additional file 4 : Supplementary Table S4. The Egger’s test of different subgroup.Additional file 5 : Supplementary Table S5. The Egger’s test of different subgroup.Additional file 6 : Supplementary Figure 1. Sensitivity analysis of the effect of the individual subgroup on the pooled ORs.


Among older adults living in long-term nursing homes (LTNHs), maintaining an adequate functional status and independence is a challenge. Whilst a poor nutritional status is a potential risk factor for a decreased function in this population, its role is not fully understood. Here, using a transversal multicenter study of 105 older adults living in 13 LTNHs, we analyzed the associations between nutritional status, as measured by the Mini Nutritional Assessment (MNA), and the parameters of functional status, physical performance, physical activity, and frailty as well as comorbidity and body composition. The MNA scores were positively correlated with the Barthel Index, handgrip strength, Short Physical Performance Battery (SPPB) scores, absolute muscle power, and Assessment of Physical Activity in Frail Older People (APAFOP) scores and were negatively correlated with dynamic balance and frailty. In a multiple linear regression model controlling for gender and age, the APAFOP score (β = 0.386), BMI (β = 0.301), and Barthel Index (β = 0.220) explained 31% of the variance in the MNA score. Given the observed close relationship between the MNA score and functional status, physical performance and activity, and frailty, interventions should jointly target improvements in both the nutritional status and functional status of LTNH residents. Strategies designed and implemented by interdisciplinary professional teams may be the most successful in improving these parameters to lead to better health and quality of life. Given the aging of the global population, maintaining an adequate functional status and independence among older adults is a fundamental challenge. Although recent years have seen much progress in this area with extensive research in the field of frailty and quality of life in older individuals, many issues remain unresolved such as clear evidence of the effect of physical activity or nutrition in slowing the progression of frailty or functional deterioration in aging especially in older adults living in long-term nursing homes (LTNH). In particular, the role that nutritional status may play in functional status, physical performance and activity, and frailty remains unclear.According to the International Association of Gerontology and Geriatrics and the International Academy of Nutrition and Aging, the risk factors for malnutrition in older adults include being 85 years or older, a low nutrient intake due to a loss of the ability to eat independently, difficulty swallowing and chewing, becoming bedridden, pressure ulcers, a history of hip fracture, dementia, depressive symptoms, and suffering from two or more chronic illnesses ,2,3,4. ISeveral risk factors such as a lack of the ability to eat independently, being bedridden, pressure ulcers related to immobility, hip fractures, and even depressive conditions can be countered or prevented by physical activity ,10,11,12Institutionalized and community-dwelling older populations differ in their body composition, nutritional deficiencies, predisposition to malnutrition, cognitive impairment, and deterioration in physical performance ,16. WhilMalnutrition may also increase the risk of frailty. Two recent reviews highlighted the relative lack of studies conducted on frail patients, particularly the lack of nutritional intervention studies especially clinical trials ,25. ReflThus, despite evidence linking nutritional status with physical and functional status of the LTNH population, its effect on these variables remains unclear. Here, we analyzed the relationship between the nutritional status, as measured by the Mini Nutritional Assessment (MNA), and functional status, physical fitness and activity, and frailty in 105 older adults living in 13 LTNHs. Understanding this relationship can help inform strategies to promote health and increase the quality of life among institutionalized older adults.This transverse, multicenter study was a secondary analysis of a clinical trial of a multicomponent physical exercise program (registry number NCT04221724) of LTNH residents. Data were collected between September and December 2019 in the post-intervention stage. The nutritional status, body composition, functional status, physical performance, physical activity, and frailty parameters were measured. Each parameter was measured by the same researcher to avoid an inter-assessor bias. The participants were recruited from 13 LTNHs in Gipuzkoa . The inclusion criteria were as follows: aged ≥ 70 years, scored ≥ 50 on the Barthel Index, scored ≥ 20 on the Mini Examen Cognoscitivo (MEC-35) test , and ablThe nutritional status was assessed using the full version of the MNA . The MNAThe clinical data were collected from the databases of the participating LTNHs. The comorbidities were calculated and categorized according to an age-adjusted Charlson comorbidity index, where one point was added for each decade after the age of 50 years .−2). The BMI was classified according to the National Heart, Lung and Blood Institute guidelines: underweight (<18.5 kg/m2), desirable (18.5–24.9 kg/m2), overweight (25.0–29.9 kg/m2), obesity (30.0–39.9 kg/m2) and morbid obesity (≥40 kg/m2) [The anthropometric measures included height, weight, and body mass index (BMI) (kg·m0 kg/m2) .The functional status was assessed by the Barthel Index , which mThe physical performance was assessed using a Short Physical Performance Battery (SPPB), the best-known instrument for evaluating the physical performance of senior citizens . The SPPHabitual physical activity was measured using the Assessment of Physical Activity in Frail Older People (APAFOP) , an inteIn order to ensure a more holistic assessment of frailty and given that each instrument regards frailty from different approaches, three measurement instruments were used so that frailty was measured using Fried’s frailty phenotype , the Roct-tests. A series of linear regressions was used to separately analyze the relationships of functional status, physical fitness, physical activity, and frailty on the MNA, controlling for age and sex. The explanatory variables that were statistically significant in those regressions were included in a multiple linear regression model predicting the MNA, again controlling for age and sex. A stepwise backward elimination was used to select the final variables. The proportion of the variance in the MNA explained by the explanatory variables in the multiple regression was estimated by the coefficient of determination (R2). In all tests, the differences were considered significant at p < 0.05.A statistical analysis was performed using SPSS version 26.0. The continuous variables were expressed as a mean ± standard deviation (SD) and he categorical variables as frequencies and percentages. The normality was assessed using a Kolmogorov–Smirnov test and the non-parametric variables were square-root transformed. The group means were compared using Student´s Participant characteristics are summarized in p < 0.05), Barthel Index (p < 0.01), absolute muscle power (p < 0.05), and APAFOP score (p < 0.001) and a lower Fried’s frailty phenotype score (p < 0.01).p < 0.001), handgrip strength (p < 0.01), SPPB score (p < 0.01), absolute muscle power (p < 0.01), and APAFOP score (p < 0.001); they were negatively correlated with the TUG test score (p < 0.05), Fried’s frailty phenotype (p < 0.05), Rockwood Clinical Frailty Scale (p < 0.01), and Tilburg Frailty Indicator (p < 0.05) scores. The highest standardized regression coefficient (β) values were for the Barthel Index (β = 0.449) and APAFOP (β = 0.432) scores.p = 0.001), followed by the BMI  and the Barthel Index score . Together, these three variables explained 31% of the variance in the MNA.The explanatory variables included in the multiple linear regression model were the BMI, Barthel Index score, TUG score, SPPB score, handgrip strength, absolute muscle power, APAFOP score, and Fried’s frailty phenotype score . Among tDespite the evidence that the nutritional status of older adults may affect functional and physical parameters, its effect on older adults living in LTNHs has remained unclear. Our results showed that a better nutritional status, as indicated by the higher MNA scores, was associated with greater functionality, physical performance, and physical activity as well as with lower frailty. In addition, physical activity (as measured by APAFOP questionnaire), functional status (as measured by the Barthel Index), and BMI collectively predicted the MNA. To our knowledge, this is the first study to find a relationship between the nutritional status and physical activity levels of LTNH residents without a cognitive impairment.Our sample represented 1.75% of all older adults residing in LTNHs in Gipuzkoa . Notably, our mean participant age, 86.34 ± 6.77 years, was higher than all other study populations to date on institutionalized older adults including the 2012 European SHELTER study of 57 LTNHs from 7 EU countries and Israel where the mean age of the participants was 83.4 years ,46,47,48From this perspective, the mean BMI value of our sample fell in the overweight range and was notably higher than in previous studies ,43,46,51LTNH residents are disproportionately at risk of malnutrition, potentially making them more likely to develop functional problems, sarcopenia, and frailty in the near future . Our finPrevious studies also found that physical performance is closely related to the nutritional status of institutionalized older adults and has an impact on their functionality, dependence level, and risk of falls ,57. In aThe MNA provides important information for predicting frailty in older adults  and the In order to delve deeper into this bidirectional relationship and draw conclusions about causality, intervention programs are needed. To our knowledge, few intervention programs in institutionalized older populations have measured both the nutritional parameters and physical activity ,77,78,79Different studies have concluded that both nutrition and physical activity improve the functional status of, and prevent sarcopenia in, older adults ,82,83. NAlthough our study did not address causality, it did support the hypothesis that physical activity may possibly improve the nutritional status of institutionalized older adults ,85. TherTaking into account these data and given the close relationship we found between the MNA and functional status, physical performance and activity, and frailty, future approaches to improve these parameters in LTNH residents should not be focused on each variable individually but on all these variables together. Interdisciplinary professional teams  should participate in the design and implementation of strategies to simultaneously improve the nutritional and functional status and physical performance of LTNH residents to increase health and the quality of life. Our results reinforce the need to develop updated physical activity and nutrition routines in LTNHs and may play a fundamental role in the redesign of nursing homes for the better overall wellness of the residents.Finally, and based on our results, we consider it essential to implement preventive actions that allow the diagnosis and treatment as early as possible of the appearance of pathologies that directly affect the quality of life of LTNH residents. In the future, and with the aim of being able to analyze the causality that we hypothesized in the present study, it is necessary to develop further nutritional and physical activity interventions.One of the strengths of this study is the combined analysis of the relationships between nutritional status and functional status, physical performance, physical activity and frailty. To our knowledge, this is the first study on LTNH residents without a cognitive impairment to relate nutritional status with physical activity. Our results may help inform the design of future studies.Our study also had several limitations. Due to the difficulty in precisely measuring the body composition of an institutionalized population, we used the BMI, a relatively simple measurement. Analyzing the fat mass and skeletal muscle mass would provide more detailed information on the relationship between nutritional status and the measurements indicative of sarcopenia. Similarly, although the MNA is validated and widely used in the LTNH environment , a more Our study demonstrated that the nutritional status measured by the MNA was related to functional status, physical fitness and activity, and frailty in older adults living in LTNHs. The results of our study highlighted the importance of taking into account all these parameters as a whole in order to maintain and care for the quality of life of older adults living in LTNHs.


Vaccinium ssp.) is a perennial shrub belonging to the family Ericaceae, which is highly tolerant of acid soils and heavy metal pollution. In the present study, blueberry was subjected to cadmium (Cd) stress in simulated pot culture. The transcriptomics and rhizosphere fungal diversity of blueberry were analyzed, and the iron (Fe), manganese (Mn), copper (Cu), zinc (Zn) and cadmium (Cd) content of blueberry tissues, soil and DGT was determined. A correlation analysis was also performed. A total of 84 374 annotated genes were identified in the root, stem, leaf and fruit tissue of blueberry, of which 3370 were DEGs, and in stem tissue, of which 2521 were DEGs. The annotation data showed that these DEGs were mainly concentrated in a series of metabolic pathways related to signal transduction, defense and the plant–pathogen response. Blueberry transferred excess Cd from the root to the stem for storage, and the highest levels of Cd were found in stem tissue, consistent with the results of transcriptome analysis, while the lowest Cd concentration occurred in the fruit, Cd also inhibited the absorption of other metal elements by blueberry. A series of genes related to Cd regulation were screened by analyzing the correlation between heavy metal content and transcriptome results. The roots of blueberry rely on mycorrhiza to absorb nutrients from the soil. The presence of Cd has a significant effect on the microbial community composition of the blueberry rhizosphere. The fungal family Coniochaetaceae, which is extremely extremelytolerant, has gradually become the dominant population. The results of this study increase our understanding of the plant regulation mechanism for heavy metals, and suggest potential methods of soil remediation using blueberry.Blueberry ( Rorippa globulosa, Solanum nigrum, Sedum alfredii, Viola baoshanensis and Pteris vittata can enrich Cd and some other heavy metals in soil [Cadmium (Cd) is a toxic heavy metal that accumulates in soil as a result of air and water circulation, and acts on the human body through the food chain. The accumulation of Cd in the human body causes severe toxic reactions . The pol in soil . HoweverVaccinium ssp.) is a perennial shrub belonging to the family Ericaceae that thrives on acid soil. It was introduced and domesticated successfully in the early twentieth century. However, it is only within the last 20 years that, as the unique flavour and high nutritional value of this crop have led to growing demand for its production, its cultivation area has expanded rapidly worldwide, particularly in Europe, North America, South America and Asia, including China [V. myrtillus (bilberry) to heavy metals in three contaminated sites in Finland. Their results showed that V. myrtillus could adapt to the polluted soil [V. angustifolium and V. myrtilloides grow around the ore smelters in Sudbury, Canada. Both species can tolerate heavy-metal-contaminated soil and grow well on it. It is of paramount importance that the levels of heavy metal pollutants (Cu and Ni) in the fruits of these species do not exceed the internationally agreed safe values, so that they are safe for human consumption [. measured the heavy metal content of soil and fruit. Their results showed that although the heavy metal levels in soil exceeded the maximum safe value, the corresponding levels in fruit did not. Chen et al. studied blueberry plants that had been cultivated in Cd-contaminated soil, and found that the Cd content of the fruit was not significantly affected by soil pollution [Blueberry  can resist Cd toxicity by a combination of cell wall thickening, an increase in antioxidant activity and the formation of complexes with heavy metals under conditions of Cd stress, and there are also specific signal transduction pathways, such as the brassinosteroid and ethylene signalling pathways, in this species [Festuca arundinacea, mainly reflecting activity of the pathways related to glutathione metabolism, phenylpropanoid biosynthesis and nitrogen metabolism [Kandelia obovata under Cd or Zn stress, three phenolic metabolism pathways are involved in the heavy metal tolerance mechanism, and the heavy metal content of the leaves is positively correlated with their total phenol content [The absorption and detoxification of heavy metals in plants are complex regulatory processes. Unnecessary heavy metals seriously impair morphological and structural development, inhibit physiological processes, cause oxidative stress reactions, and ultimately result in death of the plant –20. Cott species . In resptabolism . Comparetabolism . In Kand content .Heavy metals present in the soil not only affect the plants growing in it, but also have a significant impact on the microorganisms in the soil. In previous studies it has been reported that soil microorganisms are much more sensitive to heavy metal stress than are either soil animals or plants growing in the same soil . ExcessiTo date there have been only a few reports on the physiological effects of heavy metals on blueberry cultivation. Blueberry can reduce the toxic effects of heavy metals by regulating its own antioxidant stress response. The toxic effects are related to the type of heavy metal and the duration of exposure. It has also been found that the levels of nitrogen, phosphorus and potassium in blueberry are positively correlated with the plant’s heavy metal content, and that the glutathione (GSH) content of blueberry decreases under excessive stress , 32. HowVaccinium corymbosum ‘Bluegold’, a late-maturing variety of blueberry grown on the horticultural farm of Jilin Agricultural Science and Technology University, was used as the experimental material for this study. In May 2017, 2-year-old blueberry seedlings were planted in a non-woven pot (50.0 cm × 50.0 cm × 50.0 cm). The planting medium was peat soil, and the soil pH was adjusted to 5.0 with sulfur. Blueberry plants were normally maintained in the pot for two growth cycles to ensure that they developed up to the high-yield stage. On 1 June 2020, blueberry plants with the same rate of growth, normal pollination and fruit setting were selected for Cd stress testing. In order to avoid the interference of Cl– ions with plant growth, the treatment group was treated with an aqueous solution of CdSO4 to give a soil Cd2+ concentration of 400 mg/kg , and the control group (CK) was potted without CdSO4. Each treatment was repeated three times. After the potted plants had been treated, they were all maintained in full sunlight in a greenhouse to prevent leaching by rainwater. Management consisted of irrigation of each basin with 3.0 L of deionized water every 2 days, and the leachate under the basin was collected for reinjection. Blueberry fruits were harvested on 25 August 2020, and roots, stems, leaves and soil samples were collected on 30 September 2020. All tissue samples were washed with deionized water three times and dried in the shade. They were then frozen in liquid nitrogen and stored in aseptic bags at –80℃ in a freezer. After the blueberry plant roots had been dug up and excess soil removed from them, they were shaken in sterile self-sealing bags so that the remaining soil attached to the roots was separated out. The roots were then frozen in liquid nitrogen and stored in a freezer at –80℃.Total RNA was extracted from the roots, stems, leaves and fruits using the Total RNA Purification Kit, TRK1001 , and was then subjected to agarose gel electrophoresis prior to testing. The quality and concentration of samples was checked using the Agilent 2100 Bioanalyzer . The O.D. A260/280 was above 1.70–2.10, and the concentration was between 0.03–0.23 µg/µL.http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). De novo assembly of the transcriptome was performed with Trinity 2.4.0. All the newly assembled gene sequences (UniGene) were compared with the NR database (http://www.ncbi.nlm.nih.gov/), GO (http://www.geneontology.org), SwissProt (http://www.expasy.ch/sprot/), KEGG (http://www.genome.jp/kegg/), Pfam(http://pfam.xfam.org/) and EggNOG (http://eggnogdb.embl.de/) Compare and annotate the database.Total RNA was extracted from roots, stems, leaves and fruits of the treatment group and the control group for transcriptome sequencing. The sequencing included three biological replicates, giving a total of 24 groups of samples. The sequencing work was undertaken by Hangzhou Lianchuan Bio Technologies Co., Ltd., and the NovaSeq 6000 System  was used for sequencing. In order to maximize the quality of data, the original data were filtered. The content of filtering includes: removing the splice sequence from the sequencing reads; The sequencing reads were scanned by window method, and the default scanning window was 6 bp, When the average quality value in the window is less than 20, the part of the read from the beginning of the window to the end of 3 ‘is cut off; Poly-A / T was removed; The truncated sequences less than 100 bp in length were removed; Remove the truncated sequence with more than 5% N content The sequence quality, including effective sequencing quantity, Q20, q30 and GC content, was estimated using FastQC  by R package edgeR [Salmon was used to perform expression level for Unigenes by calculating TPM. The differentially expressed Unigenes were selected with log2 (fold change) > 1 or log2 (fold change) < -1 and with statistical significance (ge edgeR –35.−ΔΔCT) method. The gene-specific primers for qRT PCR were designed using Primer Express Software v3.0, and the internal reference gene was blueberry glyceraldehyde-3-phosphate dehydrogenase . The qRT PCR specific primers and internal reference primers are listed in Table The transcription levels of differentially expressed genes (DEGs) in roots, stems, leaves and fruits were determined by quantitative real-time PCR (qRT PCR), which was performed using SYBR Premix Ex Taq . cDNA (20 times diluted) was used as the template for qRT PCR. The operation process and reaction conditions were as described in the manufacturer’s instructions, and the qRT PCR was repeated three times. The relative gene expression was calculated using the comparative CT . The specific operation mode was as described in 3) for 24 h [2+) content [DGT technology was used to simulate the absorption of heavy metals in soil by plant roots. A 200 g sample of soil taken from the middle of the basin was dried at 85℃ to constant weight, and was then passed through a 2 mm sieve to remove large particles. Deionized water was added to achieve 70% of maximum field capacity, and was mixed well with the soil. The soil was sealed with plastic film and placed in an incubator at 25℃ for 48 h to balance the soil. The balanced soil was then evenly smeared into the inner cavity of the DGT ring to ensure the same amount of soil was loaded into all of the DGT cavities. After loading, the soil was placed in an incubator at 25℃ for enrichment for 24 h before the recovery device. After the soil had been cleaned with deionized water the fixed membrane was removed, the front of the membrane was rolled into a 2.0 mL centrifuge tube containing 1.8 mL of 1.0 M HNOfor 24 h , and the content , and ICPhttp://unite.ut.ee/index.php). Finally, taxonomic annotation at the level of phylum, class, order, family, genus and species was performed. The species annotation, alpha diversity, Simpson and Shannon indices were analyzed using krona, Mothur v.1.30 (http://www.mothur.org/).Fungal DNA was extracted from the rhizosphere of blueberry using the Total TopTaq DNA Polymerase Kit , and total fungal DNA was then subjected to agarose gel electrophoresis prior to testing. The quality and concentration of DNA samples were checked using the NanoDrop 2000 spectrophotometer. The concentration was higher than 20 ng/ L, the total amount was more than 500 ng, and OD260/280 was in the range of 1.8 ~ 2.0. DNA samples were stored in a freezer at –80℃. After the total DNA of rhizosphere fungi had been extracted, the primers were designed according to the conserved region, and the sequencing connector was added at the end of the primers. After PCR amplification, the single band was cut and recovered for library quality inspection. The library was then sequenced using the Illumina MiSeq platform and a 2 × 250 bp paired-end sequencing strategy. For operational taxonomic unit (OTU) analysis and species annotation, UCLUST in QIIME2 (version 1.8.0) software was used , 39. AftSPSS 22.0 was used for data analysis, and Excel and R were used to plot the data.A total of 24 blueberry cDNA libraries were sequenced using the NovaSeq 6000 platform. After filtering and assembling the original data , of which 2521 DEGs (1644 up-regulated genes and 877 down-regulated genes) were present in stem tissue . There were 360 (10.68%) DEGs in fruits, 305 (9.05%) DEGs in leaves, and 184 (5.46%) DEGs in roots.The DEGs in different tissues of blueberry that had been treated with Cd are shown in Fig. In order to verify the reliability and reproducibility of RNA-Seq, 20 genes were randomly selected from blueberry root, stem, leaf and fruit tissue for qRT PCR detection, and the expression of these genes in different tissues under Cd stress was analyzed , Cu4 (2.17%), Zn2 (1.09%) and Fe1 (0.54%). Of these 37 DEGs, 16 DEGs were positively correlated and 21 DEGs were negatively correlated, and these genes included TRX2 and transcription factor myb26, among others. Of the 2521 DEGs in blueberry stem tissue, 1280 DEGs were screened, including Cd1230 (48.96%), Zn13 (0.52%), Fe5 (0.23%), Mn5 (0.23%) and Cu2 (0.09%). Of these 1255 DEGs, 762 DEGs were positively correlated and 493 DEGs were negatively correlated. including calmodulin-dependent protein kinase and L-ascorbate oxidase. Of the 305 DEGs in blueberry leaf tissue, 181 DEGs were screened, including Cu109 (35.74%), Cd48 (15.74%) and Mn24 (7.87%). Of these 181 DEGs, 100 DEGs were positively correlated and 81 DEGs were negatively correlated, including flavonoid 3-O-glucosyltransferase and peroxidase. Of the 360 DEGs in blueberry fruit tissue, 120 DEGs were screened, including Cu49 (13.61%), mn35 (9.72%), Zn17 (4.72%), Fe12 (3.33%) and Cd7 (1.94%). Of these 120 DEGs, 82 DEGs were positively correlated and 38 DEGs were negatively correlated, including rhamnosyltransferase and heavy-metal-associated isolated plant protein.The results of the correlation analysis of heavy metal content and DEGs in different tissues are presented in Table A total of 76 848 original data were obtained in the treatment group (M-Cd) and 78 259 original data were obtained in the control group (M-CK). After quality filtering, noise reduction, splicing and removal of chimeras, 64 661 sequences were obtained in M-Cd and 67 148 sequences were obtained in M-CK, accounting for 84.28 and 86.03% of the original sequences, respectively. For M-Cd and M-CK, 93.66 and 94.82% of the sequences, respectively, were 245–259 bp in length see Table According to the taxonomic statistics, M-Cd includes the annotated fungi, which are divided into 6 phyla, 15 classes, 31 orders, 55 families and 69 genera, with a total of 190 species. M-CK included 141 species of fungi, which were classified into 8 phyla, 14 classes, 34 orders, 44 families and 57 genera. M-Cd was stronger than M-CK with regard to family, genus and species classification, but weaker than M-CK with regard to phylum and order. The labeled M-Cd and M-CK samples included 67 common species at the level of species classification.P < 0.05 was used as the screening threshold of significant difference. At the phylum level, Ascomycota was the dominant microflora of M-Cd and M-CK, with richness index values of 0.9080 and 0.8039, respectively. Basidiomycota was also the dominant microflora of M-CK , six orders , seven families (e.g. Conioscyphaceae), 12 genera (e.g. Trichocladium) and 6 species (e.g. Trichocladium opacum) were positively correlated with soil Cd level.The results of the correlation analysis between soil Cd content and species richness are presented in Table Gossypium hirsutum) found that most DEGs were associated with catalytic activity and metal ion binding. Cotton responded to cadmium stress by increasing cell wall thickness, antioxidant activity and detoxification. It has been reported that the genes involved in preventing transport of Cd to rice grains include OsIRT1, OsNramp5, OsVIT2, OsNRT1.5a and OsABCC1 [Broussonetia papyrifera showed that Cd stress affected 47 KEGG pathways, mainly involving bHLH, MYB and other important transcription factors, plant–pathogen interactions, phenylpropanoid biosynthesis, plant hormone signal transduction, and other protein synthesis and metabolism processes [Panicum virgatum) involves activation of redox-related metabolism, and 21 different shock transcription factors (HSFs) and 22 heat shock proteins (HSPs) are differentially expressed [Some studies have suggested that blueberry, a plant that grows in acid soil, is the dominant species in acid soil that is contaminated with heavy metals, but there has been little research on the mechanism of heavy metal tolerance in this species at the molecular level. The present study has utilized high-throughput sequencing technology, which can provide information about the response of plants to heavy metal stress. A total of 84 374 annotated genes, which included 3370 DEGs, were obtained from the root, stem, leaf and fruit tissue of blueberry plants that had been subjected to Cd stress. The DEGs were mainly involved in plant hormone signal transduction, ABC transport, the MAPK signal transduction pathway, starch and sucrose metabolism, riboflavin metabolism, porphyrin and chlorophyll metabolism, mismatch repair, photosynthesis, flavonoid biosynthesis and sphingolipid metabolism. The metabolic pathways of DEG enrichment also differed according to the specific tissue in which they were evaluated. For example, the DEGs in root tissue were mainly involved in glycosaminoglycan degradation, glycosphingolipid biosynthesis, sphingolipid metabolism, amino sugar and nucleotide sugar metabolism, and other degradation pathways related to cell wall synthesis and breakdown. The DEGs in stem tissue were associated with various defense responses  and in signal transduction . The DEGs in leaf tissue were mainly involved in photosynthesis, diterpenoid biosynthesis, circadian rhythms, oxidative phosphorylation, RNA polymerase activity, plant hormone signal transduction and flavonoid biosynthesis. The DEGs in fruit tissue were mainly associated with diterpenoid biosynthesis, oxidative phosphorylation, galactose metabolism, starch and sucrose metabolism, phenylpropanoid biosynthesis and carotenoid biosynthesis. A study of cadmium stress in cotton  was planted in polluted sludge, the heavy metal content of the roots was reported to be much higher than that of the buds and fruits, and the plant could more readily absorb Cd than either Pb or Ni from the soil [Almost all plants accumulate essential heavy metals such as Fe, Mn, Cu and Zn in the body, and these heavy metals are also very important for the growth and development of plants. The absorption and accumulation of heavy metals by plants are dependent not only on the physical and chemical properties of the external environment, but also on the characteristics of the plant . It has the soil . These sVitis vinifera the Cd content of the fruits of plants grown in polluted soil was lower than that of any other tissues [In the study of heavy metal stress on blueberry, most of them are around the leaves and fruits , 48. In  tissues . However2+ interacts with other metal cations in plants and soils [2+. Mn was mainly absorbed by plants via active transport through the roots, and had an antagonistic effect on Cd accumulation [It has been widely reported that Cdnd soils . In the mulation . Externamulation , but the42–) can promote the absorption of Fe by increasing competition [Fe is an important element in chlorophyll synthesis, and an increase in Cd levels in leaves will lead to iron deficiency and chlorosis. An increase in Fe will inhibit the absorption of Cd , and sulpetition . Howeverpetition , but alsPicea abies [Cu has a role in the multi-channel oxidation process in plants, but there have been few studies on the interaction between Cu and Cd. It has been reported that the addition of excessive amounts of Cd to the culture medium can reduce the Cu and Ca content of the xylem in ea abies . Similarea abies . In the 2+ in soil has been proved to be a competing ion for Cd2+ [Lycopersicon esculentum) [Potentilla griffithii [Znfor Cd2+ . Zn has ulentum) , but to iffithii . The Zn It is clear that treatment with Cd affected the Mn, Fe, Cu and Zn content of blueberry tissues, which is consistent with the findings of previous studies . In blueConiochaeta fodinicola and Talaromyces helicus, which are considered to be the dominant fungi in acid, industrial- and mining-contaminated soils, and are used to remove soil pollutants [Soil microorganisms are an important component of the soil ecosystem, especially for plants such as blueberry, which has roots with a low absorption capacity and uses fungal mycorrhiza to improve nutrient uptake and support growth. The interaction between soil microorganisms and soil environmental factors is extremely complex. In heavy-metal-contaminated soil, some microorganisms can modify, degrade or precipitate heavy metal compounds, while soil environmental factors can screen microbial communities . In the llutants –68.After Cd entered the blueberry planting soil, it reflected the changes of the external environment of the root system. These changes altered the composition of the soil fungal community. The growth of several of the main mycorrhizal fungi of blueberry was inhibited, and some fungi that are resistant to extreme environmental conditions exhibited vigorous growth. At the same time, Cd altered the status of heavy metal absorption by the root system, which stimulated a series of reactions within the blueberry plant Fig. .Fig. 7DiTriticum aestivum) [PR-1, Xa21, PYl4 and MPK16 in the MAPK signaling pathway (map04016) and ABF2, PYl4, IAA, EBF2, XTH23 and SAPK1 in the hormone signaling pathway (map04075). At all levels of phosphorylation in the MAPK pathway the changes in MAPKK and MAPKKK led to the up-regulation of WRKY33 expression, which promotes an increase in PAD3, as has also been reported in Arabidopsis thaliana [CNGCs) have an important role in control of the plant–pathogen interaction pathway [CDPK), which affects the up-regulation of calmodulin (CaM/CML), RPM1, RPS2, HSP90 and a series of disease-resistance-related genes. These genes jointly cause blueberry allergy. In addition, differential gene expression associated with phenylpropanoid biosynthesis (map00940), flavonoid biosynthesis (map00941), ascorbate and alginate metabolism (map00053) and carotenoid biosynthesis (map00906) also played a role in the Cd defense response of blueberry.First, Cd was absorbed along with other metal cations by the roots. When it had penetrated the root tissue by strong passive diffusion, reactions were induced in the root cell wall and cell membrane, such as sphingolipid metabolism (map00600), amino sugar and nucleoside sugar metabolism (map00520), other glycan degradation (map00511), and galactose metabolism (map00052). It was found that bgal3, os03g0165400, at5g34940, at3g59480, chitinase, bxl2, xyl2 and chil3 were up-regulated, but only JKD was down-regulated. All of these results indicated that the cell wall and cell membrane of blueberry root are important sites for Cd signal sensing, which is consistent with previous findings in studies on wheat(estivum) . The upwthaliana . In addi pathway . The up-ABCC3 and ABCG5 [PSBC, PSBA, PSBD and BVRB), while all DEGs in the cytochrome P450 (CYPs) pathway of diterpenoid biosynthesis (map00904) were up-regulated. This indicated that the transfer of Cd to blueberry leaves had a marked effect on the photoperiod of the leaves, and the CYPs of blueberry leaves were involved in the regulation and detoxification of Cd. In the present study, the fruit was shown to be the tissue with the lowest Cd content. In the pathway of oxidative phosphorylation (map00190), Mtnd2, MT-CYB, COI and Mtatp6 were up-regulated, while diterpenoid biosynthesis (map00904), oxidative phosphorylation (map00190), galactose metabolism (map00052), and starch and sucrose metabolism (map00500) were up-regulated. In the pathways of phenylpropanoid biosynthesis (map00940) and carotenoid biosynthesis (map00906), most of the DEGs were down-regulated. However, further research is needed to confirm the mechanism whereby fruit tissue inhibits the entry of Cd.Several genes for ABC transporters (map02010) were up-regulated in blueberry stem, such as nd ABCG5 . ABCG-tyBlueberry, a perennial shrub that thrives in acid soil, is considered to be the dominant plant species in acid soils polluted with heavy metal compounds. However, the mechanisms of heavy metal absorption and metabolism in blueberry are unclear. Although high-throughput sequencing technology has enabled increasing numbers of studies on the mechanisms of plant regulation and metabolism of heavy metals, blueberry exhibits some unusual features. In response to Cd stress, the signal transduction, defense and detoxification mechanisms of this plant differ in each tissue type, reflecting the synergy and characteristics of each organization. In the present study, a total of 84 374 annotated genes were obtained from four tissue samples of blueberry by high-throughput sequencing technology, including 3370 DEGs, of which there were 184 DEGs in root tissue, 2521 DEGs in stem tissue, 360 DEGs in leaf tissue and 305 DEGs in fruit tissue. These DEGs were enriched in different metabolic pathways in different tissues. The Fe, Mn, Cu, Zn and Cd content of different tissues, soil and DGT of blueberry was determined by ICP-MS. It was found that the main Cd accumulation site was the stem, which was consistent with the sequencing results. The Cd content of stem tissue was higher than that of the other tissues, whereas the lowest Cd content was found in fruit tissue. Cd competed with other metal cations, and this severely affected the metabolism of other metal elements in blueberry tissues. Only through active absorption into blueberry roots, but also through a large number of passive diffusion into the roots. Cd in the soil also had a significant effect on the mycorrhizal fungi associated with blueberry. Growth of the dominant populations of mycorrhizal fungi, namely members of the Archaeorhizomycetes and Sebacinales, was inhibited, and members of the Coniochaetaceae, which can tolerate extreme environments, gradually became the dominant population. Thus, in the presence of the acid soil conditions that are necessary for growth of blueberry, Cd alters the growth environment of this plant and has toxic effects on its tissues. However, blueberry can resist the toxicity of Cd through its own metabolic regulatory mechanism.The results of the present study, which demonstrate excessive accumulation of Cd and abundant DEGs in blueberry stem under Cd stress, point to interesting new avenues for future research. However, the most pressing need is for studies to improve our understanding of the long-distance transport of heavy metals in blueberry, the inhibition of Cd transport into fruit, the effect of Cd on the composition of the community of attached fungi and endophytic fungi in blueberry roots, and the regulation of Cd by bacteria in blueberry roots.


Macroglial cell changes were analyzed by GFAP and vimentin signal intensity. Results: After ONC, MC density increased significantly from 5 to 21 dpl in the inner layers of injured retinas, remaining within intact values in the contralateral ones. However, in both retinas there was a significant and long-lasting increase of CD68+MCs. Constitutive CD206+MCs were rare and mostly found in the ciliary body and around the optic-nerve head. While in the injured retinas their number increased in the retina and ciliary body, in the contralateral retinas decreased. Astrocytes and Müller cells transiently hypertrophied in the injured retinas and to a lesser extent in the contralateral ones. Conclusions: Unilateral ONC triggers a bilateral and persistent activation of MCs and an opposed response of M2 MCs between both retinas. Macroglial hypertrophy is transient.Background: To analyze the course of microglial and macroglial activation in injured and contralateral retinas after unilateral optic nerve crush (ONC). Methods: The left optic nerve of adult pigmented C57Bl/6 female mice was intraorbitally crushed and injured, and contralateral retinas were analyzed from 1 to 45 days post-lesion (dpl) in cross-sections and flat mounts. As controls, intact retinas were studied. Iba1 The retina is part of the central nervous system (CNS) and is a widely used model to study the response of CNS neurons and glial cells to insults and potential neuroprotective therapies.Glial cells are pivotal in the maintenance of CNS homeostasis and neuronal well-being, so glial deregulation leads to neuronal disfunction and death ,2,3. In MCs are derived from myeloid progenitors of the primitive yolk sac and are the resident immune cells of the CNS . They acAs a result of an injury or infection, MCs change their morphology to an activated state, losing their ramifications and becoming ameboid ,16. WhenAstrocytes derive from the neuroectoderm and participate in the metabolic support of neurons ,27. DuriMüller cells are the predominant glial cells in the retina, and their body spans the entire retinal thickness, from the RNFL to the outer nuclear layer (ONL) ,31. MüllIn their resting state, astrocytes and Müller cells express low-medium levels of type III intermediate filaments, GFAP and vimentin, respectively. After an injury, both become hypertrophic and over-express these cytoskeletal proteins ,32. In aOptic nerve crush (ONC) is a very well defined lesion that results in retinal ganglion cell (RGC) degeneration. The temporal course of ONC-induced RGC degeneration in rats and mice has been described in depth by our group ,35,36,37Furthermore, these responses to ONC are not limited to the injured retina. Recently, it has been described that lesions do affect not only the injured retina but also the contralateral one—the so-called contralateral effect . As theoThe dual role of glial cells has acquired great importance in the understanding of neuro-inflammatory and degenerative diseases. It is not still clear how these phenotypes of microglia and astrocytes work together , whetherTherefore, the main purposes of this study are: (i) to characterise the two states of MCs in intact mouse retinas; (ii) to analyse the temporal course of MC activation and M2 appearance in injured and contralateral retinas after unilateral ONC; and (iii) to analyse astrocyte and Müller cell activation, in both injured and contralateral retinas.+ cells were Iba1+ but not vice versa. In addition, all CD206+MCs were CD68+ and Iba1+. Thus, changes of MC density were assessed with Iba1, whereas activation states were assessed with CD68 and CD206.All CD68+MCs were found in the RNFL, GCL, IPL and OPL with a ramified morphology, indicative of a resting/surveying state  there was a progressive and parallel increase of CD68m 21 dpl C. When wm 21 dpl C.+MCs increased significantly at day 3 and remained higher than control up to 45 dpl  and in the inner retina, there was a significant increase of Iba1e points A–C.+MCs increased significantly at 5 days, while the density of activated CD68+MCs was significantly higher from 1 to 45 days after ONC  than in the contralateral (1.6-fold at 5 days) retinas. The time course of increased MCs’ density in the injured retinas fits with the time window in which most RGCs die after axotomy . +MCs is explained by the fact that in both retinas there are RGCs dying for a long time after the lesion. In the injured retina, >90% RGCs die during the first 2–3 weeks, and the remaining ones die steadily but very slowly up to 3 months . In RGCs die , but theAlternative M2 activation refers to MCs that are phagocytic—to help clear the tissue and restore homeostasis, hence their high expression of CD68—and that secrete anti-inflammatory mediators, to dampen the acute inflammatory environment created by the insult. They express M2-specific antigens, such as CD206 ,26.To our knowledge, this is the first report describing M2 MCs in the retina. Their morphology and location are quite different from that of resting or classically activated MCs. In intact retinas they are few (~250/per retina) with large, slightly elongated and thick somas, few or no ramifications, and are found mainly in the ciliary body and around the optic-nerve head.Upon injury, they become thinner and longer, as if they were polarized because in the optic-nerve head they arrange radially along the RGC axons, while in the CB they are perpendicular to the retina. One day after the lesion, M2-MCs decrease around the optic-nerve head and increase in the CB, where they remain significantly higher up to day 21. In the GCL, their number increases 2.3–2.6 times at 5 and 9 days, and they are found throughout it. This increase occurs, again, during the phase of maximum RGC death. Then, they return to normal levels in the CB and GCL, but remain below normal levels in the optic-nerve head. In the contralateral retinas, their morphology does not change in the CB but it does in the optic-nerve head at day 5. Contrary to the dynamics observed in the injured retinas, their number decreases in the GCL and the optic-nerve head, and it is maintained in the CB after a transient increase.What is the meaning of this behaviour? Do they populate the injured retina through the optic nerve and the CB? Do they migrate from the contralateral retina through the optic chiasm to the injured retina?. And most importantly, what is their function in this model?In the spinal cord it has been shown, as we show here, that there is a quick and maintained response of classically activated MCs in the lesion site that overwhelms the comparatively smaller and transient M2 response, which promotes a regenerative growth in adult sensory axons . Thus, wSurprisingly, macroglial activation, although observed in both retinas and more so in the injured ones, was not very strong. It was mostly restricted to the GCL, indicating that astrocytes and Müller cells’ end feet are the main responders to RGC death.Müller cell hypertrophy assessed by expression of GFAP and overexpression of vimentin in their stalks, somas and distant processes, was very low, and only observed in the injured retina. The end feet of Müller cells form the inner basal lamina, which is essential for RGC survival . Thus, wHypertrophy of the entire Müller cells has been observed in both retinas in models of acute ocular hypertension . This isOptic-nerve axotomy is a clean lesion that specifically affects RGCs, and it is not then surprising that astrocytes, connected to RGC axons and vessels, hypertrophy when the RGCs are dying. In addition, the activation of MCs in the GCL may activate astrocytes and the inner region of Müller cells, as there is a crosstalk neuron-micro-macroglia. Therefore, it may be that astrocytes are more sensitive to RGC death and/or axonal degeneration, i.e., affectation of the RGC complex, while the whole Müller cell responds when there is loss of photoreceptors and probably INL neurons.For this study, adult pigmented C57Bl/6 female mice, averaged weight of 30 g, from the University of Murcia breeding colony were used. All animals were treated according to the European Union guidelines for Animal Care and Use for Scientific Purpose (Directive 2010/63/EU) and the guidelines from the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research. All procedures were approved by the Ethical and Animal Studies Committee of the University of Murcia, Spain .Before the surgery, animals were anesthetized by intraperitoneal injection of a mixture of xylazine  and ketamine . Analgesia was provided by subcutaneous administration of buprenorphine . To avoid corneal desiccation, the eyes were covered with Tobrex . Animals were sacrificed with an intraperitoneal injection of an overdose of sodium pentobarbital .In experimental animals, the left ON was intraorbitally crushed at 0.5 mm from the optic disc for 10 s using watchmaker’s forceps, following previously described methods ,35,42. AAnimals were perfused transcardially with 0.9% saline solution followed by 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer, and eyes were fixed one extra hour in 4% PFA. After that, retinas were dissected as flattened whole-mounts , leavingImmunodetection was carried out in whole-mounted and cross-sectioned retinas as previously reported ,35. PrimSecondary detection was carried out with the appropriate combination of Alexa-coupled secondary antibodies each diluted 1:500 : donkey anti-rabbit , donkey anti-goat , donkey anti-goat  and goat anti-rat .Images of whole-mounted and cross-sectioned retinas were acquired using an epifluorescence microscope  equipped with the following filters: dichroic filters for fluorescein , rhodamine , far red (BP 620/60/LP 700/75) and Dapi . Images were taken at 20x magnification, reconstructed from 154 individual images (11 × 14)  controllMCs were blind analyzed in cross-sections and whole-mounts, using Iba1 for detecting all MCs, CD68 for activated MCs and CD206 for M2-MCs.+ and CD68+MCs (cells/mm2) in the outer retina  and inner retina (RNFL to IPL) was quantified in 3 representative sections of each animal (n = 3/time-point). For that, outer and inner retinas were manually outlined with ImageJ software , and positive cells in each area were manually dotted. Finally, the mean density was calculated. The total MC density was inferred as the sum of both.In cross-sectioned retinas, mean density of Iba1+MC and CD68+MC density (cells/mm2) was manually quantified in 12 individual images of each sample (n = 4/time-point), 3 from each retinal quadrant  [+MCs were very few and they were distributed across the retina; thus, their total number was manually quantified. As most CD206+MCs were observed close to the ON and in retinal periphery, the whole number of CD206+MCs in the CB and in a ring centered on the ON of radius 283 µm (0.25 mm2) was analyzed separately.In whole-mounted retinas, photographs were taken with the retina upwards, focusing on the GCL. Iba1riphery) . CD206+M+MCs were manually dotted on the photomontages, quantified and their topography represented by neighbour maps that depict the number of neighbours around a given cell in a radius of 0.200 mm with a colour scale that goes from 0–2 neighbours (purple) to >21 neighbours (dark red).CD206Finally, macroglia cells were qualitatively analyzed in cross-sectioned retinas using GFAP staining for astrocytes and activated Müller cells (n = 3/group).p < 0.05.Data are presented as mean ± standard deviation (S.D.) and analyzed with GraphPad Prism v.7  using the nonparametric Kruskal–Wallis test. Differences among groups were considered significant when Axonal damage caused by trauma or disease results in immediate and irrevocable loss of function in mammals. To date, there are no therapies that work, leaving patients with lasting disability. Animal models are very useful for understanding the whys and hows of neuronal damage and rescue, but until now most research has focused on studying the fate of neurons in injured tissue. We now know that the effect of a unilateral injury extends to the uninjured contralateral area, and that glial cells play a very important role in this bilateral response.As we showed here, after unilateral optic nerve axotomy, microglial cells from both retinas invade the GCL at the expense of the outer retina, remaining chronically activated, while macroglial activation is transient and limited to the GCL. Thus, axotomy elicits a significant response in time and space, and we wonder whether chronic MC activation is caused by the continued loss of RGCs or whether microglial activation is the cause of the protracted RGC death.


Ossiculoplasty outcome parameter staging (OOPS) and middle ear risk index (MERI) are the most commonly used indices for predicting prognosis of patients with chronic otitis media (COM). This study aimed to verify the efficiency of OOPS and MERI scores in predicting outcomes of patients with COM who underwent tympanoplasty.We retrospectively reviewed the data of patients who underwent tympanoplasty (n = 526). OOPS, and MERI scores were collected. Hearing data were measured 1 day preoperatively, and 3 and 12 months postoperatively. Operation success was defined according to the Korean Society of Otology guidelines.For calculation of success, the ROC values of MERI were 0.551 at 12 months. ROC values of OOPS were 0.637 at 12 months. There were no significant differences in hearing variables among the three groups according to MERI. There were significantly favorable outcomes in hearing variables in the low-risk group in OOPS. The mean OOPS score was greater in patients with success than those with non-success. Otorrhea, ossicle status, and status of mucosa as variables in both indices were associated with success. The type of mastoidectomy as a variable in OOPS alone was associated with success. Absence of hypertension, presence of ossiculoplasty, and use of incus as ossiculoplasty material were associated with poor success rate.Compared with MERI, the OOPS index was more closely associated with the hearing outcomes, which may be due to the extent of inflammation in the OOPS index. Chronic otitis media (COM) is a well known ear disease globally. The prevalence of COM in the United Kingdom or South Korea is 4.1% and 3.8%, respectively . AlthougOssiculoplasty outcome parameter staging (OOPS) and middle ear risk index (MERI) are the most commonly used indices for predicting prognosis of patients with COM or ear operation. In 1994, Kartush et al. initially suggested a scoring system termed MERI, and Becvarovski and Kartush revised the final system, including the smoking status . HoweverWe retrospectively reviewed the clinical data of 614 patients who underwent tympanoplasty at Kyungpook National University Hospital between January 2017 and December 2018. In our study, the first date of the participant’s medical information was January 1, 2017, and the authors accessed medical records between September 1, 2019, and August 31, 2020. All the operations in our study were performed by three surgeons with more than 7 years of experience in performing tympanoplasty. We excluded patients aged < 18 years, those with incomplete data, those with < 1 year of follow-up duration, those with preoperative profound hearing loss, or those with cholesteatoma beyond the middle ear and the mastoid cavity, and 526 participants were included in the analysis.The following baseline demographic and clinical characteristics were collected: age at operation, sex, follow-up duration, presence of diabetes mellitus or hypertension, materials for ossiculoplasty, status of eustachian tube, OOPS, and MERI scores, pre- and post-operative speech discrimination test (SDT), postoperative air-bone gap (ABG), air gain (AG), pre- and post-operative air conduction (AC), ABG closure, and change of high tone bone conduction (△Ht-BC).In our center, tympanoplasty alone was performed in patients with perforation of tympanic membrane, no abnormalities in ossicle mobility, and no evidence of soft tissue density in the middle ear and the mastoid cavity using preoperative computed tomography. Tympanoplasty with ossiculoplasty was performed in patients with fixation or discontinuity in ossicle mobility and no evidence of soft tissue density in the mastoid using preoperative computed tomography. Tympanoplasty with mastoidectomy was performed in patients with evidence of soft tissue density in the middle ear and the mastoid using preoperative computed tomography and/or with purulent discharge and otorrhea for ≥ 3 months. The eustachian tube opening was directly inspected during the operation to check if it had a mucosal web or was obstructed; this was later recorded. The study was approved by the Kyungpook National University Hospital Institutional Review Board (KNUH 2020-06-058). The board waived the need for informed consent.In our center, we did not routinely calculate the OOPS and MERI scores; however, most data for the two scores were independently evaluated and recorded in medical charts. We retrospectively analyzed these data and calculated the two scores. All patients had data for the two scores and outcomes. Some did not have the data for these, but the patients were excluded in our study. The OOPS and MERI scores were calculated using a previously described method , 6. BrieHearing data were measured 1 day preoperatively, and 3 and 12 months postoperatively. The data included pure tone air and bone conduction thresholds at 0.5, 1, 2, 3, 4, 6, and 8 kHz. The data were calculated according to the 1995 American Academy of Otolaryngology–Head and Neck Surgery, Committee on Hearing and Equilibrium consensus guidelines [P-value < 0.05 was considered statistically significant.Data were analyzed using SPSS version 21 . Categorical data are expressed using frequencies and percentages, and continuous data are expressed as means ± standard deviations. Categorical data were compared using the chi-square test and continuous data using Student’s t-test. Correlations were analyzed to assess the strength of the associations between continuous variables. The receiver operating characteristic (ROC) curve was used to calculate the probability of predicting success, cutoff values, sensitivity, and specificity. MedCalc version 11.6.1.0.  was used for ROC analysis. A Our cohort predominantly comprised women, and the mean age was 52.3 ± 15.4 years . The numP = 0.895) at 3 months and 0.551  at 12 months. ROC values of OOPS were 0.645  at 3 months and 0.637  at 12 months.The number of successes at 3 and 12 months after surgery was 446 (84.8%) and 445 (84.6%), respectively. P = 0.949 for 3 months and P = 0.382 for 12 months). The number of successes in the mild, moderate, and severe OOPS groups was 238 (90.5%), 140 (79.5%), and 87 (78.2%), respectively, at 3 months and 236 (89.7%), 140 (79.5%), and 69 (79.3%), respectively, at 12 months (P = 0.001 for 3 months and P = 0.005 for 12 months). The number of cases with intact grafts in the mild, moderate, and severe MERI groups was 277 (97.9%), 147 (93.0%), and 83 (97.6%), respectively. Further, those in the low, intermediate, and high OOPS groups was 249 (94.7%), 173 (98.3%), and 85 (97.7%), respectively. There were no significant differences in six hearing variables  among the three groups according to MERI , 134 (84.8%), and 73 (85.9%), respectively, at 3 months and 234 (82.7%), 136 (86.1%), and 75 (88.2%), respectively, at 12 months ( to MERI . There wP = 0.808 at 3 months and P = 0.139 at 12 months). The mean OOPS score of patients with success or non-success was 3.2 ± 2.8 and 4.6 ± 2.4 at 3 months and 3.2 ± 2.8 and 4.5 ± 2.4 at 12 months, respectively (P < 0.001 at 3 months and P < 0.001 at 12 months).Correlation analysis showed that OOPS alone was positively associated with postoperative ABG, △Ht-BC, AG, and postoperative AC and inversely associated with postoperative SDT . The meaSmoking status as a variable in MERI alone was associated with success . OtorrheOur study showed that OOPS was a better predictor of success after tympanoplasty than MERI. OOPS scores also correlated with other hearing outcomes such as postoperative ABG, △Ht-BC, AG, postoperative AC, and postoperative SDT; however, MERI scores did not correlate with these outcomes. There was a significant difference in OOPS alone between patients with and without success. Among variables within two indices, smoking, otorrhea, ossicle status, status of mucosa, and presence/type of mastoidectomy were associated with success. Beyond two indices, presence of hypertension, absence of ossiculoplasty, and use of artificial material in ossiculoplasty were also associated with success.Previous studies have shown the usefulness of two indices to predict prognosis in patients who underwent tympanoplasty and/or ossiculoplasty; however, most studies considered only surgical aspects as outcomes or included small sample sizes. A study comparing the two indices may be insufficient to conclude due to the small sample size. Therefore, additional studies are needed to evaluate the superiority of prediction for prognosis between MERI and OOPS or identify the other possible factors that affect hearing outcomes.Our study included a relatively large sample size with long-term follow-up and evaluated comprehensive hearing data. In our study, almost all patients 96.4%) had an intact graft after surgery, which was a parameter for success in the surgical aspect. Therefore, we evaluated various hearing outcomes. The Korean Society of Otology recommended the requirement of the three indicators; postoperative ABG, AG, and postoperative AC to define success. However, the American Academy of Otolaryngology-Head and Neck Surgery consensus recommended the evaluation of postoperative ABG, ABG closure, and △Ht-BC [6.4% had The proportions of the three groups in the two indices were different, as shown in The type of graft and surgical techniques influenced the outcome of tympanoplasty, but the OOPS and MERI score systems did not include all the variables in the surgical approaches. Our study compared all surgical approaches and showed that tympanoplasty with ossiculoplasty (type 3 or 4) or mastoidectomy was associated with a poor success rate. The outcomes of columellization and interposition in ossiculoplasty were similar. The presence of mastoidectomy, in addition to tympanoplasty, was considered while calculating OOPS scores; however. The canal wall down type of mastoidectomy was associated with poorer outcomes than canal wall up mastoidectomy.The presence of a cholesteatoma influences the prognosis after tympanoplasty. Detailed information for cholesteatoma was not included in the OOPS and MERI scoring systems. MERI calculation considered the presence of a cholesteatoma without extension in the scoring system; however, our data showed that a cholesteatoma was not associated with a low success rate; the extension of cholesteatoma had more of an impact on the surgical outcome. Previous attempts to categorize cholesteatomas rely on extension, which depends on the selection of surgical approach and outcome after tympanoplasty. In the two scoring systems, ossicular status indirectly reflects the extension of cholesteatoma. In our study, cholesteatoma extension was restricted at the mastoid and the middle ear cavity. Patients with cholesteatoma beyond the middle ear cavity and the mastoid were excluded due to the high risk of postoperative complications and poor preoperative/postoperative hearing. Additionally, our study included data on the surgical site and the hearing result up to 12 months after surgery. The graft or recurrence of cholesteatoma was evaluated using endoscopy up to 12 months after the operation. No patient suffered from recurrent or remnant cholesteatoma at 12 months after the operation, however, this is a shorter duration than that required for the identification of recurrent or remnant cholesteatoma.Surgical time and the expertise of the operating surgeon also influence the prognosis of a patient after tympanoplasty. A previous study showed that surgery performed by experienced staff members is associated with better results . Our datIn our study, the presence of hypertension, absence of ossiculoplasty, and ossiculoplasty materials were also associated with success. We suggest that patients with hypertension on medications such as antihypertensives, dyslipidemic or anti-platelet agents, exhibit pleiotropy through anti-inflammatory effects. These may be associated with favorable results in such patients. Our study also evaluated the impact of diabetes mellitus and hypertension on the outcome of tympanoplasty; however, comorbidities beyond these two may influence the success rate of surgery. Most studies either excluded patients with other comorbidities, included a small proportion of these patients, or did not analyze the effect of comorbidities. These comorbidities can be associated with non-otologic complications, such as pulmonary and, cardiovascular diseases. However, there are little data regarding the association between comorbidities and outcomes after tympanoplasty. Further studies regarding the association between various comorbidities, including diabetes mellitus or hypertension, and their impact on hearing outcomes of tympanoplasty are needed to elucidate the role of comorbidities. In addition, there might be a difference in outcome between hypertensive patients who were on medication and hypertensive patients who were not. Furthermore, the classification of antihypertensive medications, such as inhibitors of the renin-angiotensin-aldosterone system or calcium channel blockers, or the achievement of the target blood pressure can influence the outcome. For diabetic patients, differences according to their characteristics, such as the achievement of target blood glucose levels and method of glucose control , may be greater. Although these issues are beyond the scope of our study, they could be another interesting issue regarding their impact on the outcome according to the presence of medication or classification in hypertensive or diabetic patients. Unfavorable results for incus compared with hydroxyapatite or titanium may be because of the small number of patients who underwent ossiculoplasty using incus (n = 28).As Becvarovski and Kartush introduced a revised version of the MERI scoring system that included smoking, some studies have validated its importance , 11–13. Cox et al. validated OOPS in 68 children and 126 adults who underwent ossiculoplasty and evaluated the changes in ABG (defined success as the ABG closure ≤ 20 dB) . They shA recent study compared the OOPS and MERI indices in 68 patients who underwent ossiculoplasty . They shOne of the major limitations of the scoring systems was that they calculated the same score for different conditions. Furthermore, these two scoring systems measure scores for each variable using simple dichromatic or trichromatic approaches. For example, dry perforation and the presence of cholesteatoma are scored as 1. However, the importance of these two conditions would be very different according to the specific type of COM or severity/nature of each variable. Moreover, the scoring systems did not consider variables that were important factors in determining the types of COM; they apply equally to each type of COM. Important risk factors for suppurative, cholesteatomatous, or adhesive otitis media are the duration of otorrhea and pathogen species, position and extension of cholesteatoma, and patency of the eustachian tube and severity of adhesion, respectively. However, these were not considered in the two scoring systems. Some centers may not routinely use these scoring systems because of these limitations. Our center also did not use the OOPS or MERI scoring systems, but various factors associated with prognosis were collected from medical charts. Before the operation, clinical factors, such as otorrhea, smoking status, or underlying comorbidities were described, and photographs of the tympanic membrane were captured using endoscopy. In addition, the status of the ossicle and the presence or absence of cholesteatoma were routinely described in the operation records. These are well-known traditional factors associated with postoperative hearing or prognosis in patients with COM. An individualized approach, according to the presence or absence of each indicator was performed at our center. We compared two scoring systems, and our results would be useful as a bridge study before developing more accurate scoring systems according to the specific type of COM.There were a few limitations to this study. First, this study had a retrospective design with a single center and ethnicity. The two scores were calculated using medical records that were independently collected; therefore, the score calculated using them may not completely reflect the status of each variable. Additionally, the type of surgery was selected based on clinical indication regardless of the scoring system. Additionally, in the comparison of some variables such as presence of diabetes mellitus and ossiculoplasty materials, the difference between sample size limited the analysis. Second, our study included a relatively large sample size with long-term follow-up. However, 12 months is insufficient to investigate the extrusion of artificial material. Therefore, a longer follow-up period with multi-center studies is warranted. Third, our study did not use high-frequency audiograms as outcome measurements. The mean age of the patients was 53.2 ± 15.4 years old. Relatively old patients may be prone to a increased hearing threshold at high frequencies, regardless of ear disease. Therefore, the change in hearing at high frequencies after the operation may be less clear than that at low frequencies. Furthermore, both the 1995 American Academy of Otolaryngology–Head and Neck Surgery, Committee on Hearing and Equilibrium consensus guidelines and the Korean Society of Otology guidelines recommend that the success of the surgery can be evaluated using air gain, change of high-tone bone conduction, air-bone gap closure, or postoperative air conduction without a high-frequency audiogram. Fourth, in our study, the graft was evaluated using endoscopy alone. A tympanogram is more useful for diagnosing complications such as re-perforation or adhesions, but this is not routinely evaluated in our center. In addition, the patency of the Eustachian tube was assessed using medical records and intraoperative microscopic findings. However, this is not an accurate method for evaluating the patency of the Eustachian tube, such as dynamic motion. At our center, physical examinations using endoscopy and pure-tone audiometry are routinely evaluated during the postoperative period. If middle ear conditions, such as effusion or inflammation, ossicle chain discontinuity, and tympanic membrane perforation are suspected upon physical examination or pure-tone audiometry, we performed tympanogram and temporal bone CT. This step-by-step approach alone could be sufficient to assess complications without routinely specific evaluations.In conclusion, compared with MERI, the OOPS index was more closely associated with the hearing outcomes, which may be due to the extent of inflammation in the OOPS index. The modified index, including smoking and comorbidities, among variables in OOPS, is considered to be a good scoring system for predicting hearing outcomes in patients who underwent tympanoplasty.S1 FigThe number of patients in the mild, moderate, and severe MERI groups was 283, 158, and 85, respectively. Regarding the mild group for MERI, the number of patients in the low, intermediate, and high OOPS groups was 163 (57.6%), 85 (30.0%), and 35 (12.4%), respectively. Regarding the moderate group for MERI, the number of patients in the low, intermediate, and high OOPS groups was 68 (43.0%), 65 (41.1%), and 25 (15.8%), respectively. Regarding the high group for MERI, the number of patients in the low, intermediate, and high OOPS groups was 32 (37.6%), 26 (30.6%), and 27 (31.8%), respectively. MERI, middle ear risk index; OOPS, ossiculoplasty outcome parameter staging.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


The femoral neck system (FNS) may be a valid alternative for treating femoral neck fractures, but few studies have compared the outcomes between FNS and cannulated cancellous screws. This study aimed to compare the clinical efficacy and complications of FNS with those of inverted cannulated cancellous screws (ICCS) for the treatment of femoral neck fractures in adults.This retrospective study evaluated patients with femoral neck fractures who underwent FNS or ICCS internal fixation between June 2019 and January 2020. The operative time, intraoperative blood loss, number of fluoroscopies, length of surgical incision, length of hospital stay, time to fracture healing, and other indicators were compared between the two groups. The incidence of complications such as nonunion, avascular necrosis of the femoral head, loosening of the internal fixation, and shortening of the femoral neck during follow-up was also assessed in both groups.P > 0.05). The incidence of complications such as fracture nonunion, avascular necrosis of the femoral head, and screw loosening was also comparable between the two groups. However, the number of fluoroscopies was significantly lower in the FNS group than in the ICCS group (P < 0.05). The fracture healing time was also significantly lower in the FNS group than in the ICCS group (P < 0.05). Furthermore, the femoral neck shortening and Harris hip score at the last follow-up were significantly better in the FNS group than in the ICCS group (P < 0.05).Among the 92 patients included, 47 and 45 patients were categorized into the FNS and ICCS groups, respectively. Follow-up ranged from 14 to 24 months. There were no significant between-group differences in sex, age, side of the injured limb, and type of fracture and in operative time, intraoperative blood loss, incision length, and length of hospital stay (all FNS for femoral neck fractures improves hip functional recovery and reduces the femoral neck shortening rate and fluoroscopy exposure postoperatively. Thus, FNS can be an alternative to ICCS for the treatment of femoral neck fractures. Considering the trends in population growth and ageing, the incidence of femoral neck fractures is projected to increase to 6.5 million by the year 2050, increasing the socio-economic burden and becoming a challenging problem for orthopaedic surgeons . FemoralOrthopaedic surgeons face a significant challenge in treating femoral neck fractures because they need to take into account the following five independent variables: bone quality, fragment geometry, reduction, implant, and implant placement . RobertsThe present study aimed to compare the short-term outcomes between the FNS group and ICCS group for femoral neck fractures. We hypothesized that the FNS group would yield superior outcomes compared to the ICCS group, with better perioperative status and improved postoperative function.After obtaining the Institutional Review Board approval, we performed a retrospective study, and informed consent was obtained from all including patients. The inclusion criteria were as follows: (1) the femoral neck system and inverted cannulated cancellous screw were used for surgical treatment respectively, (2) only unilateral fracture of the neck of femur, and (3) the follow-up time more than 12 months. The exclusion criteria included (1) pathological femoral neck fractures, (2) femoral neck fracture beyond 7 days, and (3) open fractures.After epidural or general anaesthesia, the patient was transferred to an orthopaedic traction bed. Open reduction was performed if the reduction did not meet the I and II levels under C-arm fluoroscopy, as assessed according to the Garden reduction index. A 4-cm incision parallel to the femoral shaft was made under the greater trochanter. In the ICCS group, three Kirschner wires forming an inverted triangle configuration, which were positioned approximately 5 mm beneath the surface of the femoral head cartilage, were inserted into the femoral head. After ensuring the proper location, 7.5-mm cannulated cancellous screws were inserted along the guide wires, and the Kirschner pins were removed. In the FNS group, a 2.5-mm guide pin was placed in the anterior superior part of the femoral neck to maintain fracture reduction. A 130° guide was inserted along the femoral neck. Under C-arm fluoroscopy, the needle insertion point and angle were adjusted by adjusting the guide so that the central guide was in the centre of the femoral neck in the anteroposterior and lateral positions, 5 mm from the subchondral bone. After reaming along the central guide, the bolt-and-plate assembly was inserted into the femoral head. Subsequently, the anti-rotation screw and locking screw were positioned in the proper location  was routinely injected until discharge to prevent deep vein thrombosis.Oral rivaroxaban and loxoprofen sodium tablets were prescribed at 10 mg/day and 180 mg/day (three times a day) for 3 weeks after discharge to prevent thrombosis and pain in a later stage. Partial weight-bearing and full weight-bearing exercises were gradually increased according to the rate of fracture healing on X-ray radiography. All patients were required to return to the outpatient clinic for follow-up at 1, 3, 6, 9, and 12 months postoperatively and then once every 6 months thereafter.All clinical data for operative time, incision size, surgical blood loss, length of stay, incidence of postoperative complications, Harris hip score (HHS), femoral neck shortening, and fracture healing time were assessed and recorded. Ischemic necrosis of the femoral head and nonunion were judged according to the criteria described by Dhar et al. and Slobogean et al. , 20. Thet test. The chi-square test was used to analyse categorical variables. Ranked data was tested by the Mann-Whitney U test. All statistical analyses were performed using SPSS 26.0 . P < 0.05 means the difference was statistically significant.Continuous variables were expressed as mean ± standard deviation (SD) and were analysed by Student’s P > 0.05).Finally, a total of 92 patients with femoral neck fractures were included in this retrospective study with following up 14–24 months. The FNS group and ICCS group, according to different internal fixation devices used, respectively included 47 patients and 45 patients. There are no statistically significant differences in terms of age, gender, injury sides, Garden classification, and Pauwels classification between the two groups (Table P > 0.05). However, there was a statistically significant difference in fluoroscopy frequency . There was a significant difference in fracture healing time between the FNS group and the ICCS group . In the FNS group, nonunion occurred in 2 (4%) patients, osteonecrosis occurred in 1 (2%) patient, and screw-out occurred in 3 (6%) patients. In the ICCS group, nonunion occurred in 4 (9%) patients, osteonecrosis occurred in 3 (7%) patients, and screw-out occurred in 5 (11%) patients. At the last follow-up by outpatient or telephone, the mean Harris hip score of the FNS group was significantly higher than that of the ICCS group   of 47 patients, moderate shortening occurred in 11 (23%) patients, and severe shortening occurred in 2 (5%) patients. In the ICCS group, no or mild femoral neck shortness occurred in 25 of 45 (56%) patients, moderate shortening occurred in 9 (20%) patients, and severe shortening occurred in 11 (24%) patients. A significant difference was found between no/mild and severe shortening  [P = 0.027) [Shortness of the femoral neck following internal fixation of femoral neck fractures is a known phenomenon, and clinicians who advocate for arthroplasty for the treatment of femoral neck fractures often use shortening as an argument against internal fixation of these fractures . In our < 0.001) . Similar= 0.027) . CollectWhether shortening of the femoral neck impairs hip function remains controversial. Zielinski observed that femoral neck shortening decreased gait velocity and impaired physical functioning in a multicentre clinical study of 75 patients . SlobogeAlthough femoral neck fracture reduction and fixation are performed in an attempt to restore vascular supply to the femoral head, 10–20% and 10–30% of patients develop complications of nonunion and avascular necrosis, respectively , 36. In A clinical study by Ma et al. reported a 9.4% rate for both nonunion and osteonecrosis . ConsideThe design and mechanism of FNS for fixing femoral neck fractures are very similar to these of the Targon FN system, both of which provide an angular and rotator stable construct and offer a unique sliding mechanism, allowing for controlled fracture impaction . HoweverWe acknowledge some limitations of the current study. First, the retrospective study design might have resulted in a selection bias. Second, the relatively small sample size and short follow-up time might have an impact on clinical outcomes. Therefore, our results should be confirmed in larger multicentre randomized controlled trials with a longer follow-up period.FNS as a treatment for femoral neck fractures has comparable efficacy to ICCS with respect to operation time, incision length, surgical blood loss, and incidence of perioperative and postoperative healing complications. Moreover, FNS achieves superior Harris score, fracture healing time, and femoral neck shortening. Thus, FNS is a feasible alternative to ICCS for the treatment of femoral neck fractures.