The present pilot study aimed at investigating the feasibility of a leukemia-derived exosome enrichment approach followed by exosomal dsDNA target re-sequencing for adult Acute Myeloid Leukemias (AML) marker detection. To our knowledge, this is the first time that a proof-of-concept combining a leukemia-derived exosome enrichment strategy based on a commercial CE-IVD kit and next-generation sequencing was applied in a cohort of adult AML patients. The reported approach is easy, quick and user friendly and gives the possibility of obtaining a good quantity of exosomal dsDNA  suitable for further analysis. The time-effective procedure opens up future effective clinical applications. This pilot study presents the potential of a proof-of-concept based on exosome analysis to be applied in clinical practice, as well as the feasibility of this kind of investigations using a certified kit, avoiding many additional analyses. It may encourage further studies regarding extracellular vesicles in myeloid neoplasia.Exosomes are extracellular vesicles playing a pivotal role in the intercellular communication. They shuttle different cargoes, including nucleic acids from their cell of origin. For this reason, they have been studied as carriers of tumor markers in different liquid biopsy approaches, in particular for solid tumors. Few data are available concerning exosomes as markers of myeloid neoplasia. To better understand their real potential and the best approach to investigate leukemic exosomes, we present the results of a pilot feasibility study evaluating the application of next-generation sequencing analysis of dsDNA derived from exosomes isolated in 14 adult patients affected by acute myeloid leukemias. In particular, leukemia-derived exosome fractions have been analyzed. The concentration of dsDNA co-extracted with exosomes and the number and types of mutations detected were considered and compared with ones identified in the Bone Marrow (BM) and Peripheral Blood (PB) cells. Exosomal DNA concentration, both considering the cargo and the DNA surrounding the lipid membrane resulted in a linear correlation with leukemic burden. Moreover, exosomal DNA mutation status presented 86.5% of homology with BM and 75% with PB. The results of this pilot study confirmed the feasibility of a leukemia-derived exosome enrichment approach followed by exosomal dsDNA NGS analysis for AML biomarker detection. These data point to the use of liquid biopsy in myeloid neoplasia for the detection of active leukemic cells resident in the BM via a painless procedure. Extracellular Vesicles (EVs) are membrane vesicles of 30–1000 nm in size and are essential mediators of cell-to-cell communication . Up to nAcute Myeloid Leukemias (AMLs) are ones of the most challenging myeloid disorders. AML are clonal disorders of the hematopoietic stem cells committed to the myeloid lineage that most frequently occur in older people (60–65 years), and their incidence increases progressively with age ,8. AMLs In the present pilot study, we investigated the feasibility of a leukemia-derived exosomes enrichment approach in adult AML patients at different disease stages. The exosomal enrichment and the contemporary dsDNA extraction have been performed by a commercial kit certified both for the endosomal derivation of the enriched EVs (exosomes) and for diagnostic application. The present pilot study combined leukemic exosome enrichment with exosomal dsDNA Next-Generation Sequencing (NGS) analysis . In ordeFourteen patients affected by AML at different disease statuses afferent to the Blood Disease and Stem Cells Transplantation Unit at ASST Spedali Civili of Brescia  were enrolled in the clinical trial NP 4344, a monocentric non-pharmacological pilot clinical trial approved by the Ethics Committee of Province of Brescia  in 2020. One healthy 46 year-old male, one healthy 50 year-old female, and one healthy 53 year-old female without familial history for hematological malignancies served as healthy controls. The study was conducted according to the guidelines of the Declaration of Helsinki and all the enrolled subjects gave their written informed consent. The main characteristics of the patients are reported in A total of 101 samples were analyzed . No BM specimens were sampled from healthy controls  because of ethical consideration. Patients’ disease statuses at the moment of each specimen are summarized in At each time point, 10 mL of PB and BM were sampled in EDTA tubes (5 mL for each tube) for all enrolled patients. Only PB samples were obtained by the healthy controls. A total of 101 samples were collected and analyzed. Fresh PB and BM samples were treated within 4 h from sampling.PB samples were immediately centrifuged at 2800 rpm for 15′ to remove the supernatant containing the plasma. The plasma was stored. The buffy coat layer, with some red blood cells, was then removed and transferred to a new 15 mL tube. A lysing buffer solution was prepared  for the lysis of red blood cells. The procedure was grouped inro two lysis steps: 10′ at room temperature (RT) followed by a centrifuge at 2800 rpm for 10′. A second lysis was performed at RT for 5′ followed by a centrifuge at 2800 rpm for 5′. Then, two washing steps in PBS buffer (centrifuge at 2000 rpm for 10′) concluded the procedure. BM samples were treated with Ficoll–Paque density gradient centrifugation. Briefly, the collected BM samples, supplemented with anticoagulants and EDTA, were diluted 1:1 with PBS buffer. Then, they were carefully laired onto the required Ficoll–Paque volume. A centrifugation step was performed at 2000 rpm for 30′ without braking. The upper layer was discarded and the mononuclear cell layer was transferred to a new 15 mL centrifuge tube. Finally, two washing steps were performed in PBS buffer (centrifuge at 2000 rpm for 10′). After the cell count, the dried cell pellets were stored at −80 °C. The isolated cells and plasma were stored at −80 °C and −20 °C upon analysis, respectively.6 cells. Firstly, the cells were resuspended in 200 µL of PBS and 20 µL of Qiagen Protease and 200 µL Buffer AL was added. Samples were pulsed, vortexed and incubated at 56 °C for 10′. Then, 200 µL of 99% ethanol was added and the mixes were transferred to the QIAmp Mini spin column and centrifuged at 8000 rpm for 1′. Secondly, two washes were performed in Buffer AW1 (centrifuge at 8000 rpm for 1′) and Buffer AW2 . After an incubation with 50 µL Buffer AE and centrifugation at 8000 rpm for 1′, the purified DNA was finally eluted and stored at −20 °C.Cellular genomic DNA was manually extracted by a commercial kit QIAamp DNA Mini kit , according to the manufacturer’s Instructions. The input content of the cells was overall 5 × 10In order to select the best isolation kit for our purpose, 4 samples were treated in parallel with a commercial kit for total plasmatic exosomes isolation (Invitrogen) and with a commercial kit providing an enrichment in exosomes, derived from malignant cells .g for 30′ to eliminate any cellular debris. The isolation of exosomes was accomplished with proteinase treatment in order to achieve a better purification. The exosomes’ pellets were resuspended in 200 µL PBS buffer and stored at 4 °C overnight. Then, the extraction of exosomal dsDNA was assessed according to QIAamp DNA Mini kit  protocol starting from 200 µL of exosomes resuspended in 200 µL of PBS, as described above for cellular DNA extraction. Total exosomes’ isolation was performed by Total Exosome Isolation reagent (Invitrogen) following the manufacturer’s instruction. Briefly, 1 mL of plasma was centrifuged at 2000× g at RT. The supernatant containing the reagents was discharged and 1 mL of 1 × isolation buffer was added to the pellet and subsequently spinned at 7000 g for 7′ at RT twice. Proteinase K and lysis buffer were added in 1:10 ratio. The samples were incubated at 56 °C for 1 h. Then, 200 µL of 99% ethanol was added to each sample, the mixture was transferred on a new column and then was centrifuged at 10,000× g for 1′. The samples were then washed with different washing buffers, following the manufacturer’s instructions. The exosomal dsDNA was evaluated for quality and quantity and stored at −20 °C until analysis.The exosome fraction enriched for vesicles derived from malignant cells was isolated from 1 mL of plasma by the SeleCTEV™ Low Input DNA Enrichment kit . The SeleCTEV™ Low Input DNA Enrichment kit is CE-IVD certified and all the tests on the type and quality of enriched EVs and on exosomal dsDNA are declared by the manufacturer. The kit allows for direct exosomal dsDNA (exoDNA) extraction and the isolation is based on an affinity method, taking advantage of a proprietary peptide . BrieflyAfter the dsDNA quality control was performed based on quantification of the 230/260 and 260/280 ratio, the SeleCTEV™ Enrichment kit was used and the experiment described hereinafter refers to exosomal dsDNA extracted by enrichment. The exosomal dsDNA quality control results are reported in Exosomal dsDNA was quantified by the fluorometric method using the Quant-iT PicoGreen dsDNA Assay kit .® Single Cell Kit following the standard protocol provided by the manufacturer. This kit was chosen based on the previously reported efficiency and suitability for further NGS investigation [Exosomal dsDNA was amplified by the REPLI-gtigation . BrieflyThe Amp-ex DNA was loaded onto a 1% E-Gel Sybr-safe electrophoresis system  for quality control.ASXL1, BCOR, NRAS, TP53, RUNX1, CEBPA, FLT3, EZH2, IDH1, IDH2, NPM1, DNMT3A, TET2, CBL, KRAS, ETV6, SF3B1, SRSF2, U2AF1, ZRSR2, GATA2, TERT, TERC, SRP72, and ANKRD26. The protocol for library generation was performed following the manufacturer’s instructions, as previously reported, with 500ng of DNA as an input [Cellular and Amp-exDNA was analyzed by NGS using a custom panel for target resequencing based on Nimblegen technology . The custom panel included the coding and regulatory sequences of the following genes, genes known to be involved in myeloid leukemogenesis and suggested to be investigated by the recent international guidelines and by experts in the field: an input . DNA libA total of 5 NGS sequencing runs were performed and bioinformatic analysis was conducted on .vcf files automatically generated by the MiniSeq instrument by wAnnovar (Wang Genomics) . DiffereNovel mutations have been confirmed by a second target resequencing analysis by Sophia Genetic Myeloid Solution  following the manufacturers’ instruction. DNA libraries were sequenced by the MiSeq Illumina NGS platform with 2 × 150 sequencing by V3 for 600 cycles. Bioinformatic analysis on .Fastq files was conducted by Sophia DDM, an artificial intelligence-, machine learning-, and cloud-based software. It automatically queries a wide ranges of databases and performs an algorithm-supported variant classification with OncoPortal™. Sophia Genetic Myeloid Solution with bioinformatic analysis by Sophia DDM is a CE-IVD-certified diagnostic workflow for AML.Standard descriptive statistics were used to summarize the samples’ characteristics . CategorWe firstly evaluated the correlation between exosomal dsDNA concentration and the leukemia burden. A linear regression (R = 0.849) was observed considering the quantification performed by Quant-iT PicoGreen on exosomal dsDNA extracted by the peptide affinity method and the leukemia burden assessed by routine cytofluorimetric analysis, considering cells presenting with an aberrant immunophenotype . The cytThis evidence has suggested an increased release of exosomes related with the increment of leukemic blasts.NGS analysis allowed for the detection of variants in all sequenced samples. Variants known as polymorphisms were excluded from analysis. In healthy controls’ samples, only variants classified as polymorphisms were detected.We compared the mutations detected in PB and BM cells with those identified in exosomal dsDNA. The number of identified mutations in the different sequenced samples is reported in Considering the detected non-unique mutations, the highest number of mutations was detected on exosomal dsDNA (69 non-unique mutations), while the lowest was in PB cellular DNA (44 non-unique mutations). When filtering for unique mutations, BM and exosomal dsDNA presented 86.5% of homology A, higherThe types of mutations were also considered. The complete list of the detected mutations is reported in The variants were divided into Nonsynonymus Single Nucleotide Variants (SNV), Stopgain mutations and Frameshift mutations. Nonsynonymus SNV was the most represented type both in cells (65%) and in exosomes (67%), while Stopgain mutations were the less frequent ones . The frequency of each mutation type is graphically represented in We finally evaluated the types of genes mutated. Genes were divided into different categories based on their main function: signal transduction, epigenetic and transcription regulators, metabolism, splicing, polymerase activity, and other functions. Both in cells and in exosomes, the epigenetic and transcription regulators resulted to be the most mutated genes . Conversely, genes involved in metabolism and with polymerase activity were the less mutated ones in both the sequenced materials . The detailed frequency of mutated gene types is graphically represented in No statistically significant difference resulted from comparing the frequency of the type of mutations or the function of the mutated genes.Extracellular vesicles, exosomes in particular, have been identified as components of liquid biopsy approaches for many diseases, both oncological and non-oncological. In oncohematology, exosomes have been mainly investigated in lymphoid malignancies, while the majority of the data about myeloid neoplasia are derived from limited in vitro studies. Little is known about real exosomes’ capability to serve as leukemic markers shuttling detectable mutated nucleic acids . We prevIn the present pilot phase of a feasibility study, we have taken advantage of the synergy between a leukemia-derived exosome enrichment strategy, based on a commercial kit previously described , and a sThe correlation between the concentration of leukemic originated exosomal dsDNA and leukemia burden was firstly evaluated. A linear regression was identified (R = 0.849), and this confirmed the preliminary results previously presented . SimilarThe NGS analysis of exosomal and cellular DNA allowed for a high-resolution profiling of the genomic landscape of our patients and for the representation of the blast DNA within the shed exosomal compartment. The observed homology is similar with what reported in solid tumors  and in oThe EVs, as non-cellular copies of the cells of origin, have garnered clinical interest, and could be used when the cell of origin is neoplastic and it is not directly detectable. This is particularly evident when considering the samples, both BM and PB, collected at the moment of CR . The mutIn conclusion, these results reinforce what described both in vivo and in vitro for AML and other myeloid leukemias ,57 aboutThe selection of subsets of patients better stratified by small EVs analysis will be interesting, together with the study of additional exosomal markers, such as transcripts. Further studies will also clarify the best timing of samples analysis. In the era of personalized and precise medicine , the com


The high strength of 300M steel originates from the heat treatment process after forging, but how hot deformation affects the heat-treated microstructure and mechanical properties is unclear. In this study, compression tests under different hot deformation parameters and post-deformation heat treatment experiments were carried out, and the martensite transformation process was investigated using in situ observation. The results show that the grain size of the specimen deformed at low temperature and high strain rate is smaller, and annealing twins will be formed. Both austenite grain boundaries and twin boundaries hinder the growth of martensite blocks, reducing the size of martensite units after heat treatment and thus resulting in higher yield strength. Besides, the mathematical models were established to describe the relationship between hot deformation parameters and grain size after deformation, martensite packet size and martensite block width, respectively, after heat treatment. The relationship between yield strength and hot deformation parameters was also analyzed. According to the results and models, the hot deformation parameters would be optimized more reasonably to improve the final mechanical properties of 300M steel forgings. As one of the ultra-high strength steels, 300M steel has good fracture toughness, excellent stress corrosion resistance and fatigue resistance, and is widely used for key parts in the aviation industry and other fields, such as aircraft landing gear, pressure vessels, fasteners, etc. ,2,3. Gen−1 and found that temperature and strain rate significantly affect the microstructure evolution. Luo et al. [−1. Liu et al. [−1 and found that the dynamic recrystallization mechanism of 300M steel was closely related to the strain rate.Several scholars have studied the hot deformation and heat treatment process of 300M steel. For the hot deformation process, scholars mainly focus on the rheological and recrystallization behavior under different deformation parameters. Skubisz et al.  investigo et al.  studied u et al.  investigu et al.  investigSince the phase transformation during heat treatment determines the final mechanical properties of 300M steel, the research on heat treatment has focused on phase transformation and its effect on mechanical properties. Liu et al.  investigIn this study, through the hot compression and heat treatment experiments of 300M steel, the effect of hot deformation parameters on heat-treated microstructure and mechanical properties was studied. The martensite transformation during the cooling process of heat treatment was analyzed by in situ observation, and the influence mechanism of hot deformation parameters on the heat-treated microstructure was clarified. In addition, the relationship models between hot deformation parameters and grain size after deformation, martensite packet size and martensite block width after heat treatment were established. The relationship between yield strength and hot deformation parameters was also analyzed.−1, 0.1 s−1, 1 s−1 and 10 s−1 and the compression amount was 50% (corresponding to the true strain of 0.69). During the compression experiments, the specimens were heated to the deformation temperature at a heating rate of 5 °C·s−1, held for 4 min, then compressed isothermally and air-cooled to room temperature. The compressed specimens were subsequently heat treated. Firstly, the specimens were austenitized at 870 °C for 1 h, then oil quenched and twice tempered at 300 °C for 2 h.The material used in this study was commercial 300M steel, and its chemical composition (wt.%) is shown in After hot deformation and heat treatment, the specimens were cut along the axial direction and polished. The hot deformation specimens were etched by saturated picric acid solution at room temperature, and the grain structure was observed by a metallographic microscope (OLYMPUS BX61M). The heat-treated specimens were etched with a 4% nitric acid alcohol solution, and the microstructure was observed by scanning electron microscope (SEM). The distribution of martensite units and residual austenite were obtained by electron backscattered diffraction (EBSD) with a scanning step of 0.1 μm. In order to visualize the microstructure evolution during the quenching process of heat treatment, an in situ laser confocal microscope (VL2000DX) was used to observe the martensite transformation during cooling, with an image acquisition frequency of 5 images/s.A sample of φ 4 mm×6 mm was taken from the heat-treated specimens by wire cutting, and a compression test was performed at room temperature using an AC-IC 100 kN mechanical tester to obtain the yield strength.Z) parameter. The Z parameter is the strain rate coefficient coupled with the temperature effect, with the expression Q is the deformation activation energy (381.34 kJ·mol−1) [R is the gas constant (8.314 J·(mol·K)−1) and T is the thermodynamic temperature. The initial state of 300M steel is annealed, and its metallographic structure is shown in  expQ/RT , where QJ·mol−1) , R is thgraind), martensite packet size (packetd) and martensite block width (blockd) under different hot deformation parameters, as shown in graind, ln packetd and ln blockd decreased with the increase of ln Z. Among them, the relationship between ln graind and ln Z is linear, and the relationship model between recrystallized grain size after hot deformation and Z parameters was established by linear fitting, as shown in Equation (1). In Z > 36, the decrease tendency of ln packetd and ln blockd increased with the increase of ln Z. The relationship models between the martensite unit size after heat treatment and Z parameters were established respectively by data fitting, as shown in Equations (2) and (3). The reasons for this phenomenon will be analyzed in Based on the microstructure and EBSD results, the linear intercept method  was used−1 (ln Z = 37). Austenite grains were nearly equiaxed. In addition, austenite grain boundaries and twin boundaries can be clearly seen in The martensite transformation during the cooling process of heat treatment is shown in Z parameter increased, the grain sizes decreased during the hot deformation process. It is generally believed that distortion energy can promote the formation of annealing twins [Z parameter increased further (ln Z > 36), the distortion energy of the sample increased greatly, which led to the formation of annealing twins during the holding process. The twin boundaries and austenite grain boundaries simultaneously hindered the growth of martensite blocks during heat treatment, resulting in an increasing tendency to reduce the martensite packet size and block width.In addition, as shown in ng twins . As the Z parameter, the recrystallized grain size decreases, as shown in Based on the above analysis, the mechanism of hot deformation parameters on the martensite unit size after heat treatment is proposed in yσ) and Z parameters. Based on previous studies, yσ can be described by the following equation [σ0 is the friction stress for pure iron, pσ is the precipitation hardening, sσ is the solid solution hardening, ρσ is the hardening of dislocations and HPdk−1/2 is the grain boundary strengthening . In the heat treatment process, the deformed sample was held for a long time above the recrystallization temperature, making the dislocation density greatly decrease and remain stable. In addition, the cooling rate was sufficient to make almost all austenite transform into martensite [blockd. As discussed in Z parameter. Therefore, the yield strength increased with the increase of the Z parameter. In addition, when ln Z > 36, annealing twins were formed during the heat treatment. The twin and austenite grain boundaries simultaneously hindered the growth of the martensite block, which had a stronger inhibition on the growth of martensite. Accordingly, the increasing tendency of yield strength increased, as shown by the blue dotted line in The compression curves of the heat-treated specimens at room temperature are shown in equation ,23:σy = rtensite . Meanwhirtensite ,25. TherIn this study, the effect of hot deformation parameters on heat-treated microstructure and mechanical properties was investigated through hot compression and heat treatment tests of 300M steel. In addition, in situ observation of the martensite transformation during the cooling process of heat treatment was carried out. The following conclusions were obtained.(1)Z parameter, the recrystallization grain size and the martensite unit size decreased. In addition, the decreasing tendency of the martensite block width and the martensite packet size also increased.Dynamic recrystallization occurred after hot deformation, and the microstructure after heat treatment was mainly martensite. With the increase of the (2)Z parameter, annealing twins were formed during the heat treatment. The twin boundaries hinder the growth of martensite, making the decreasing tendency of the martensite unit size increase.With the increase of the (3)Z parameter. When ln Z > 36, annealing twins were formed and the increasing tendency of yield strength increased.The yield strength was mainly affected by the martensite unit size and increased with the increase of the


There is still a lack of consensus on which internal fixation method can better maintain the stability of femoral neck fractures (FNF), promote fracture healing, and reduce postoperative complications such as femoral head necrosis and nonunion. Therefore, the purpose of this study was to evaluate the clinical efficacy of the novel dynamic compression locking system (DCLS) versus dynamic hip screw (DHS) in the treatment of FNF.Fifty cases of FNF from July 2018 to February 2020 were retrospectively analyzed. According to different treatment methods, they were divided into DCLS group (26 cases) and DHS group (24 cases). Baseline data, intraoperative and postoperative clinical data, reoperation rate, and Harris score were collected to evaluate the clinical efficacy.P > 0.05). There weren’t significant differences in the length of hospital stay and mobility after two years postoperatively between the two groups (P > 0.05). The operation time, blood loss, incision length, fluoroscopy times and the degree of femoral neck shortening after two years postoperatively in the DCLS group were significantly less than those in the DHS group . Harris score after two years postoperatively in the DCLS group was significantly higher than that in the DHS group (P < 0.05). Although the reoperation rate in the DHS group was slightly higher than that in the DCLS group, it wasn’t statistical significance (P > 0.05).All patients were followed up for 24 months. All “fractures” were caused by fall. The baseline data of the two groups were comparable (Compared with DHS, DCLS in the treatment of FNF had less surgical trauma, shorter incision length, shorter operation time, lower radiation dose and higher Harris scores. Although the reoperation rate in the DHS group was slightly higher than that in the DCLS group, it wasn’t statistical difference. Further research is needed. Femoral neck fractures (FNF) accounts for about 57% of hip fractures, and has become one of the major health problems with increasing incidence and economic burden year by year , 2. DysfThe optimal fixation strategy for FNF is still controversial . At presThere is still a lack of consensus on which implant can better maintain the stability of fracture ends, promote fracture healing, and reduce postoperative femoral head necrosis and internal fixation failure . TherefoInclusion criteria were as follows: 1 A clear history of trauma; 2 A Garden II-III fracture of FNF; 3 Two years of follow-up after surgery; 4 Unilateral fractures; 5 The informed consents of the surgery and implant signed by these patients; 6 The applied implants of DCLS or DHS. 7 Closed reduction and internal fixation for the fracture treatment. Exclusion criteria were as follows: 1 A history of hip trauma at the same hip in the past; 2 Pathological fractures other than osteoporosis; 3 Senile dementia and other diseases with poor compliance and uncooperative treatment; 4 Concomitant fractures in other parts; 5 Concomitant acetabular fracture; 6 Serious medical disease interferes with clinical efficacy, such as paralysis or hemiplegia after cerebral hemorrhage or cerebral infarction, hepatic failure, renal failure, and serious cardiopulmonary dysfunction.The cases of FNF in our hospital from July 2018 to February 2020 were retrospectively collected. A total of 50 cases of FNF treated by DCLS   was used for statistical analysis. Count data were expressed in absolute logarithm and analyzed by χP > 0.05). The operation time, blood loss, incision length, times of fluoroscopy and the degree of femoral neck shortening in the DCLS group were significantly less than those in the DHS group . The Harris score of DCLS group was significantly higher than that of DHS group (P < 0.05). Femoral head necrosis occurred in 1 case and nonunion occurred in 1 case in the DCLS group, and all of them were re-operated later. In the DHS group, 3 cases of femoral head necrosis and 1 case of nonunion occurred, and all of them were re-operated later. Although the reoperation rate in the DHS group was slightly higher than that in the DCLS group, it wasn’t statistical significance (P > 0.05). For patients with osteonecrosis of the femoral head and nonunion, they would initially experience hip discomfort. Especially when the lower limb was weight-bearing, the hip discomfort symptoms would be aggravated, often accompanied by limited walking activity. Later these patients needed to use a walking aid for walking activities. Patients with osteonecrosis of the femoral head and nonunion had similar early symptoms and were indistinguishable by symptoms alone. But these patients can be confirmed by further CT and MRI examination of the hip. In the DCLS group, 1 case of postoperative mobility decreased by 1 grade and 1 case decreased by 3 grades, and the rest were the same as the preoperative mobility (Tables P > 0.05).All patients were followed up for 2 years . The Harris score in the DCLS group was significantly higher than that in the DHS group, but it hasn’t significant change in postoperative mobility between the two groups. A previous study found that about 11.3% of the cases of FNF in the DHS group suffered femoral head necrosis, and 9.4% of the cases suffered nonunion [In this study, 1 patient (3.8%) in the DCLS group suffered femoral head necrosis and 1 patient (3.8%) suffered nonunion. In the DHS group, 3 cases (12.5%) suffered femoral head necrosis and 1 case (4.2%) suffered nonunion. Although the complications in the DHS group were slightly higher than those in the DCLS group, it wasn’t statistical difference (nonunion . The ratnonunion , 23. DHSnonunion . The DCLIn this study, the rate of re-operation in the DCLS group was lower than that in the DHS group, but it hasn’t statistical difference, which might be limited and related with the small number of cases in these groups. So further large sample research is needed. The retrospective analysis is prone to data bias. Moreover, this study was a preliminary single-center study of this system. In order to further verify the clinical efficacy of DCLS, further multi-center randomized, controlled, and double-blind clinical trials are required.Compared with DHS, DCLS in the treatment of FNF has less surgical trauma, shorter incision, shorter operation time, lower radiation dose and higher Harris scores. Although the reoperation rate in the DHS group was slightly higher than that in the DCLS group, it wasn’t statistical difference. Further research is needed.


The diagnosis and treatment of pulmonary embolism have multi-modal approach based on specificity, sensitivity, availability of the machine, and associated risks of imaging modalities.This review aimed to provide shreds of evidence that improve perioperative diagnosis and management of suspected pulmonary embolism.The study was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guideline 2020. After a clear criteria has been established an electronic searching database was conducted using PubMed, Google Scholar, Cochrane library, and Cumulative Index of Nursing and Allied Health Literature (CINAHL), with Key search terms included:, were used to draw the evidence.The quality of literatures were categorized based on WHO 2011 level of evidence and degree of recommendation, in addition, the study is registered with research registry unique identifying number (UIN) of reviewregistry1318.” and has high quality based on AMSTAR2 assessment criteria.A totally of 27 articles were included [guidelines (n = 3), Cochrane (=5), systemic reviews (n = 7), meta-analyses (=2), RCT (n = 4), cohort studies (n = 3), and cross-sectional study (n = 3) and illegible articles identified from searches of the electronic databases were imported into the ENDNOTE software version X7.1 and duplicates were removed.Currently divergent and contradictory approaches are implemented in diagnosis and management for patients suspected of pulmonary embolism..All perioperative patients, especially trauma victims, prostate or orthopedic surgery, malignancy, immobility, and obesity; smokers; and oral contraceptive users, antipsychotic medications are at increased risk of venous thromboembolism and need special caution during surgery and anesthesia •Currently divergent and contradictory approaches are implemented in diagnosis and management for patients suspected of pulmonary embolism.•This study improves perioperative diagnosis and management of suspected pulmonary embolism patients.•Patients having trauma, orthopedic surgery, malignancy, immobility; and being smoker; are at risk of VTE and need special caution during surgery. Anatomical obstruction and hypoxic vasoconstriction in the affected lung area lead to an increase in PVR and a proportional decrease in arterial compliance, 9. HeliThe initial management of pulmonary embolism may be started before a definitive diagnosis is established, started with supportive treatment followed by vasopressors aimed at stabilizing the patient and minimizing the effect of the embolic occlusion to improve right ventricular function and contract the systemic vasculature to maintain blood pressure respectively, 10.2Pulmonary embolism is a potentially life-threatening condition that needs immediate diagnosis and management , since SEven if Pulmonary angiography is the standard for establishing the presence of pulmonary embolism, a negative pulmonary angiogram doesn't rule out pulmonary embolism due to its insufficient sensitivity to detect small emboli ,9,12. In33.1To improve perioperative diagnosis and management of suspected pulmonary embolism patients.3.2To provide a working framework for diagnosis of pulmonary embolism.To prepare pulmonary embolism management protocol.4The study is conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guideline 2020  as shownSelection of studies was detailed using PRISMA 2020 flow diagram(14) as shown .4.1https://www.researchregistry.com/browse-the-registry#registryofsystematicreviewsmeta-analyses/with a unique identifying number (UIN) of 1318 and the study has high quality based on AMSTAR 2 quality assessment checklist/https://amstar.ca/Amstar_Checklist.php.Before inclusion each study in the review of the literature, the quality of each study was assessed by all authors independently using the Critical Appraisal Skills Programme (CASP) checklists ,16,and a5A summary of the included studies in the review of the literature can be seen in . Totally6Pulmonary embolism (PE) is a life-threatening condition in which a clot travels from deep veins of the lower extremity to the circulation and lodges into the lungs. ClinicaHuman immunodeficiency virus (HIV) increases the risk of PE two-to ten fold as compared with the general population, major surgery, Hip or knee replacement, and General anesthesia when compared with epidural ,19.Computed Tomography Pulmonary Angiography (CTPA) has greatly improved the diagnostic approach to patients with suspected PE and is considered to be the reference imaging test, but should be used, with caution in some patients, such as patients with severe renal insufficiency, those with known allergy to contrast media, and pregnant women. AdditioIf the patient presents with at least three parameters out of the five most common signs and symptoms of PE  with the inclusion of X-ray and echocardiography results it is satisfactory pieces of evidence to make high suspicions of acute pulmonary embolism that requires diagnosis and management at bedside within a few minutes, but if the patient is hemodynamically stable, CTPA can be performed to confirm the diagnosis12, .Acute pulmonary embolism requires anticoagulation to prevent early death and recurrent symptomatic fatal venous thromboembolism. The standard duration of anticoagulation cover at least 3 months and parenteral anticoagulation  over the first 5–10 days should be given to treat acute PE ,24,26,27Hypoxaemia is one feature of severe PE and resulted from the mismatch between ventilation and perfusion, so supplemental oxygen is required in patients' level of SpaO2 <90%. Patients with right ventricular failure are highly susceptible to the development of severe hypotension during induction of anesthesia, intubation, and positive-pressure ventilation . ThromboHemodynamically deteriorating suspected pulmonary embolism patients require rescue thrombolytic therapy, in addition, surgical embolectomy and catheter-directed treatment are alternatives to the treatment of pulmonary embolism to rescue thrombolytic therapy ,9,19,24.stable and instable patients are detailed as shown in (Deep venous thrombosis (DVT) can be prevented through non-pharmacologic prophylaxis , ambulation, and vena cava filter) and pharmacologic intervention, which is through the use of blood-thinning medications, 11, 27.shown in  respecti7.All perioperative patients, especially trauma victims, prostate or orthopedic surgery, malignancy, immobility, and obesity; smokers; and oral contraceptive users, antipsychotic medications are at increased risk of venous thromboembolism and need special caution during surgery and anesthesia•Lamesgen Geta Abate = study concept or design, litreture review, data cleaning, and data interpretation.•Samuel Debas Bayable = litreture review, data cleaning, and data interpretation, manuscript wrting•Melaku Bantie Fetene = Check grammer and plagiarism,corrected flow of ideas.Research studies.1. Name of the registry: 2. Unique Identifying number or registration ID: 1318.3. Hyperlink to your specific registration (must be publicly accessible and will be checked): researchregistry1318”.Samuel Debas Bayable, and Lamesgen Geta Abate.Ethical approval was granted with Debre Markos University School of Medicine.Data and materials are fully available without restriction and present upon request..The authors received no financial support for the research authorship/or publication of this articleNot required.All authors were equally participated to address Comments and suplimented in the manuscript before sending the revised form for publication.Not commissioned, externally peer, reviewer.kokeb.desta@gmail.com helps us in method of reviewing and overall writings in spelling, grammar and punctuation.Kokeb Desta  The authors declare that there is no conflict of interest.


Owing to intermittent/acute exposure to hypobaric hypoxia, highland miners may often suffer, the physiological characteristics between highland and lowland miners, however, are rarely reported. The objective of this study was to compare the physiological characteristics of coal miners working at disparate altitudes.Twenty-three male coal mining workers acclimating to high altitude for 30 ± 6 days in Tibet , and 22 male coal mining workers in Hebei  were recruited. Tests were conducted to compare ventilatory parameters, circulation parameters, resting metabolic rate (RMR), and heart rate variability (HRV) indices between the two groups in resting state.p = 0.000). In the meanwhile, O2 intake per heart beat (VO2/HR) was strikingly decreased . Resting metabolic rate relevant to body surface area (RMR/BSA) was found no significant difference between the two groups. Evident reduction in standard deviation of NN intervals (SDNN) and remarkable increase in ratio of low- and high- frequency bands (LF/HF) were manifest in highland miners compared to that of lowland ones .Ventilation volume per minute (VE) of the highland group was markedly raised compared to that of the lowland group (11.70 ± 1.57 vs. 8.94 ± 1.97 L/min, These results implicate that long-term intermittent exposure to high altitude can lead miners to an intensified respiration, a compromised circulation and a profound sympathetic-parasympathetic imbalance, whereas the RMR in highland miners does not distinctly decline. The number of mining workers exploiting kinds of mineral resources at altitudes has been increasing. Highland miners are commonly subjected to periodically alternating workplaces between altitude and lowland, characterized of being exposed to hypobaric hypoxia intermittently and acutely.The Tibet Plateau has an average altitude of 4,500 m above sea level. The Qinghai-Tibet Plateau, is the highest altitude in the world, known as the “roof of the world”. Environment at high altitudes is characterized of thin air, low atmospheric pressure, strong ultraviolet light, dry and windy climate .Individual who sustains an acute exposure to the hypobaric hypoxic environment can elicit a systemic and extensive stress responses . The res2 intake per heart beat  are non-invasive, sensitive and effective indicators as to reflect cardiac status and the general situation of O2 consumption.Highland exposure may also cause the reduction of arterial oxygen partial pressure and the decreased blood oxygen saturation, which leads to insufficient systemic oxygen supply  . The stBasal metabolic rate (BMR) is the minimum rate of energy expenditure or the compulsory energy cost to keep an individual himself alive. In adult humans, a large proportion  of the energy budget is allocated to the BMR to maintain body functions such as blood flow, breathing and tissue activities . As is eHear rate variability (HRV) refers to the temporal changes in the beat-to-beat intervals in the heart, which is subject to the autonomic nervous system (ANS) and reflects the competition for the predominance between sympathetic and parasympathetic nerves . HRV is At present, it is relatively sparse that investigations, aimed at the specific population of miners, into physiological responses to highland familiarization involve cardiorespiratory function, RMR and HRV collectively. The purpose of this project is to compare the physiological parameters of highland and lowland miners using portable cardiopulmonary function meter and 72-h single-lead ECG recording module. We intend to clarify the mechanism of altitude impairments on highland miners. The results from this study would provide some quantitative clues for clinical practice.A total of 45 male miners was recruited in the study: 23 in Tibet , and 22 in Tangshan, Hebei Province , respectively. Their baseline demographic characteristics are summarized in 2) and RMR simultaneously in a complete quiescent state of the participants. The device was able to link heart rate belt  via Bluetooth so as to collect HR. The variables mentioned above were averaged from 5-minute episode out of a total 25-minute recorded duration for each participant through the gas metabolism analyzer exclusive software. Gas metabolism analyzer was calibrated thoroughly prior to measurements. In order to obtain the derivative parameter RMR/BSA, body surface area was calculated with the Mosteller body surface area equation (2) = sqrt {[height (cm) * weight (kg)] / 3600}.Neither exhausting exercise within 24 h, nor food consumption within 12 h prior to the RMR measurement was allowed , but norequation : BSA . After Preparing the skin with an alcohol cotton swab, the electrodes  were pasted at the positions following the apparatus’ manual . The discharge course of heart was recorded and stored into the receiving module put on the chest through electrodes and leads. This equipment could continuously record data for 72 h. Each group of the participants collected 72 h of continuous data, excluding the data with too many artifacts. Time course segments on each day selected for different phases of HRV calculations  were 6:00–12:00 and 0:00–6:00. Exclusive analysis software facilitated the HRV calculations.p < 0.05.SPSS version 26 mathematical statistics software was employed. The data were expressed as Mean ± SD (mean ± standard deviation). The Levene’s test was applied for equality of variances. Except that BF and LF/HF did not satisfy the equality of variances, the other reported variables did. The differences in the mean between the two groups were assessed by 2-tailed independent sample T test. The degrees of freedom for BF and LF/HF were 31.38 and 24.75, respectively, and the degree of freedom for other reported variables was 43. The significant level was set at p = 0.002 and 11.70 ± 1.57 vs. 8.94 ± 1.97 L/min, p = 0.000; respectively), yet tidal volume (VT) of highland miners showed no statistically difference in comparison with that of lowland ones , as presented in The age and height showed no significant difference between highland and lowland miners, but significant differences existed in the body weight and BMI shown in . Both BFp = 0.028), as shown in 2/HR was strikingly decreased compared to that of the lowland group , as shown in HR was significantly higher in the highland group compared to that of the lowland group , p = 0.178), albeit the prominently less amount of RMR in the highland group , which were summarized below in RMR/BSA in highland miners presented no significant difference in contrast to that of lowland ones . Furthermore, LF/HF in highland miners manifested a significant increase . Nevertheless, HF of the highland group was far less in contrast to that of the lowland group, but the difference had not yet reached the statistical significance (show in RMSSD (asleep state) and SDNN in highland miners were found considerably lower than that of lowland ones . Thus, highland miners might have a tendency towards higher VT in resting state, but further investigation is needed before a viable conclusion can be drawn. Associated the particular environment of mining with hyperventilation, it can be inferred that highland miners are potentially more vulnerable to pulmonary diseases. It was reported that the development and progression of pneumoconiosis were accelerated at altitudes , the initial and immediate consequence is the reduced arterial oxygen partial pressure resulting in arterial carbon dioxide and Hthe body . Respirathe body . It was ltitudes , and thi2) to some extent  reflects the energy needed by the body to maintain the basic blood circulation, breathing, body temperature, and internal environmental homeostasis . In our The hypobaric hypoxic stress simultaneously activates both hypothalamic-pituitary-adrenal (HPA) and sympathetic-adrenal medulla (SAM) , of whicSeveral shortcomings of this study need to be acknowledged: (1)Relatively small sample size. We had to compromise the sample size due to the difficulty in recruiting participants and the restraint of logistics in the context of such a high altitude. It merits greater input of human and material resources so as to enlarge the sample size in the further study. (2)Significant body weight and BMI differences between the two groups. These directly led to a prominent inter-group difference in absolute RMR, which in our study was corrected via normalized variable RMR/BSA that made the results comparable. (3)We failed to execute the blood gas analysis of our great interest in this study.(I) Prolonged intermittent exposure to high altitude can lead the highland miners to alterations of ventilation, cardiovascular system and HRV.(II) Chronic intermittent altitude exposure, however, does not sufficiently alter RMR/BSA in present study, at least no noteworthy reduction being observed.(III) Chronic intermittent altitude exposure renders miners enhanced sympathetic and weakened parasympathetic nervous tones.(IV) The exacerbated sympathovagal imbalance may be more likely to predispose high altitude miners to cardiovascular diseases and psychological disorders.(V) What’s more, hyperventilation respiratory pattern probably involves in the high incidence of pulmonary diseases among altitude miners.Further healthcare and occupational protection are worthy of taking into consideration.10.7717/peerj.13899/supp-1Data S1Click here for additional data file.


This review was conducted to investigate the association between serum vitamin B12 levels as well as folic acid/vitamin B12 during pregnancy and the risk of gestational diabetes mellitus (GDM).A comprehensive search of electronic databases  was performed. The odds ratios (ORs) with 95% confidence intervals (CIs) of GDM risk were summarized using a random effects model. We also performed subgroup analyses to explore the source of heterogeneity.2: 59.0%). Subgroup analysis indicated that this association might differ based on sample size and geographical distribution. Elevated vitamin B12 levels may decrease the risk of GDM by 23%. The role of excess folic acid and low vitamin B12 levels in the occurrence of GDM is also controversial.A total of 10 studies, including 10,595 pregnant women were assessed. Women with vitamin B12 deficiency were at higher risk for developing GDM when compared with those who were vitamin B12 sufficient (OR, 1.46; 95% CI 1.21–1.79; IIn summary, vitamin B12 deficiency is associated with increased risk of GDM, it is necessary to pay more attention to the balance of vitamin B12 and folic acid. However, more in-depth studies across multiple populations are needed to verify these results.The online version contains supplementary material available at 10.1186/s12884-022-04911-9. Gestational diabetes mellitus (GDM) is one of the most prevalent complications of pregnancy. In a joint statement released in 2018, the International Federation of Obstetrics and Gynecology (FIGO) and the International Diabetes Federation (IDF) stated that high blood glucose levels during pregnancy have an impact on maternal, newborn, and child health and may contribute to the global burden of type 2 diabetes mellitus and cardiovascular metabolic disorders in the short and long term. This poses a critical public health challenge as well as a substantial financial burden  OR “Cobamides”[MeSH Terms] OR “Vitamin B 12 Deficiency”[MeSH Terms]) AND  in PubMed; Topic =  AND Topic  in Web of Science; and TX  AND TX  in Embase. The reference lists of retrieved articles and relevant reviews were also manually searched. The search was restricted to articles published in English.All of the published articles included in the analysis were matched to the following criteria:⑴Patients with clinically diagnosed GDM during pregnancy; ⑵ observational studies evaluated the association between vitamin B12 status or folic acid/vitamin B12 and prevalence or incidence of GDM, serum vitamin B12 concentration ≤ 220 pmol/L is considered vitamin B12 insufficiency; ⑶ the study reported effect estimate  and corresponding 95% confidence interval (CI) for comparisons of sufficient vitamin B12 and insufficient vitamin B12 levels. Studies were excluded if ⑴ the article did not provide full text or contained insufficient information for assessment; ⑵ extra supplements of vitamin B12 before GDM diagnosis; ⑶ conference paper;(4) papers that reported the same data from the same study populations. The selection process and reasons for exclusion are presented in Fig. Two reviewers (Jin He and Dongmei Jiang) independently screened titles and abstracts for full-text review, duplicate entries were removed, and any disagreements between the two authors were settled by group discussion until a consensus was reached. The collection of the data included author, year, country, study design, sample size, assessment of vitamin B12 and folic acid/vitamin B12 levels, GDM diagnostic criteria and time, and the effect estimate with 95% CI. Study quality was assessed according to the Newcastle–Ottawa Scale (NOS) , 32. NOS2 statistics. If the I2 ≥ 50%, heterogeneity is considered high and a random-effects model is used. Sensitivity analysis was also performed by excluding each study one by one to evaluate the credibility of pooled results. To identify sources of heterogeneity, we also used stratified analysis, including geographic location, trimester, GDM diagnostic criteria, gestational weeks for detecting serum vitamin B12 and folic acid levels, and the number of samples.Statistical analysis was performed on Stata software (Version 16.0), and the OR and 95% CI were used for the dichotomous variable . From thThe certainty of evidence from cohort studies was assessed using NutriGrade shown in the Supplementary Table Figure The baseline characteristics of the eligible studies are presented in Table 2 = 59%, P = 0.063; Fig. 2 = 81.6%, P < 0.001; Fig. To explore the link between vitamin B12 and GDM, 4 prospective cohort studies , 25, 35 P = 0.009). However, Li et al. [P = 0.002), Lai et al. [P = 0.034), Krishnaveni et al. [P = 0.01), and Sukumar et al. [P = 0.003) reported inverse results. Finally, the result of cohort studies showed that insufficient vitamin B12 with excess folic acid could increase the risk of GDM by 60% . When stratified by GDM diagnosis and the trimester of vitamin B12 measurement, in the first trimester, vitamin B12 deficiency was associated with an increased risk of GDM diagnosed by IADPSGC . Unsurprisingly, in the subgroup analysis of the cross-sectional study and the number of samples, the results showed that high levels of vitamin B12 in serum are protective factors for GDM .To explore the sources of high heterogeneity in the included studies, several subgroup analyses were carried out which contained geographic location, diagnosis of GDM, levels of vitamin B12, gestational weeks for detecting serum vitamin B12 and folic acid levels, and the number of samples. The results of all stratified analyses by study characteristics are shown in Table The NutriGrade scoring system had a score of 6.25, which indicated a low level of meta-analysis evidence . But the result of cross-sectional studies and case controls displayed that there was no distinct association between vitamin B12 levels and the risk of GDM . Additionally, we also found that the imbalance of serum folic acid and vitamin B12 (folic acid/vitamin B12) was not significantly associated with GDM. This contradicts previous studies showing that an imbalance of serum folic acid and vitamin B12 increases the incidence of GDM.The previous meta-analysis suggested vitamin B12 deficiency could increase the risk of GDM which was consistent with our findings . HoweverA large number of research not only allowed us to determine the relationship between vitamin B12 and the risk of GDM but also carry out huge heterogeneity which needs stratify data and sensitivity analyses to evaluate. Based on subgroup analyses, a significant association was observed between deficiency in vitamin B12 levels and an increased risk of GDM, sufficient vitamin B12 concentration may decrease GDM risk by 23% . As regards geographical differences, India showed a high rate of vitamin B12 deficiency in pregnant women, which increases the risk of GDM by 95% . Folic acid and vitamin B12 interweave during carbon metabolism, we also found that the existing studies focused on vitamin B12 and folic acid imbalance, therefore, the association between the folic acid/vitamin B12 ratio and GDM was analyzed and found that there was no significant association in either type of study. In light of the above findings, the relationship between nutrients and GDM is still controversial, it is necessary to further study the role of a balanced relationship between folic acid and vitamin B12 in disease.Our main findings were consistent with the major results of previously published studies. Krishnaveni et al.  conducteConsidering that the sample size and the diagnostic criteria affect the overall combined results, based on the subgroup analyses, we found sufficient vitamin B12 levels were related to protecting the effect on the decreased occurrence of GDM when come to numerous sample sizes. Given the regional differences in diet and physical properties of vitamins, pregnant women in countries like India, which mostly includes plant-based foods and less meat than is typical in European diets, because vitamin B12 widely occurs in animal-based food products people are vegetarian may be more likely to develop GDM. Moreover, it has been demonstrated that serum vitamin B12 content is dynamic throughout pregnancy, with the highest level in the first trimester decreasing gradually to the lowest level in the third trimester , 38. As Although an imbalance of folic acid and vitamin B12 was not observed to be associated with GDM, epidemiological studies have shown that the combination of high folic acid and low vitamin B12 is associated with the onset of GDM and adverse outcomes in offspring , potentiDespite the heterogeneity of our results, the quality of evidence is not very excellent, we just gave a relative result and provided a synthetic and comprehensive review. However, vitamin B12 still plays a significant role in the development of pregnancy-related disorders from a clinically significant perspective. As an essential microelement involved in DNA methylation, amino acid, nucleic acid, and lipid synthesis, a growing number of cohort studies have found that B12 insufficiency is associated with sleep disturbances , increasAdvantages of our study include those 10 studies were included, all OR values were combined which is more than in previous studies, and the subgroup analysis by time to detect serum vitamin content, along with an investigation of the relationship between folic acid/vitamin B12 and the risk of GDM was carried out for the first time.However, we still need to acknowledge that this review has several limitations, which means that our results should be interpreted with caution. First, the potential confounding factors in several studies could not be completely ruled out and such differences could have influenced the outcomes of individual studies. Second, data on dietary vitamin B12 intake, the use of additional vitamin B12 supplements, and nutritional habits of the participants were not available or reported by most of the included studies which may affect the concentration of vitamin B12. Third, the different diagnostic criteria used may also affect the prevalence of GDM to some extent, this was one cause of the large heterogeneity among articles. Last, half of the studies were cross-control and case reports, the analysis used pooled data , which restricted us from performing a more detailed relevant analysis and obtaining more comprehensive results. Therefore, more high-quality cohort studies are needed to verify the relationship between vitamin B12 as well as folic acid/vitamin B12 ratio and GDM.In conclusion, vitamin B12 deficiency plays an important role in the risk of GDM, especially during the first trimester in South Asian women. Folic acid/vitamin B12 has the potential ability to be a sensitive index to evaluate the relationship between nutrients and GDM. We recommend routine screening of pregnant women for vitamin B12 deficiency and folic acid excess to determine whether supplements of vitamin B12 and folic acid should be administered to ensure the nutritional status and to improve the health of the maternal. There is a need for high-quality research involving different geographical areas and ethnic groups to add credibility to the available evidence.Additional file 1:Supplementary Table. 1-1 Newcastle–Ottawa Scale of cohort studies.Additional file 2: Supplementary Table. 2. Assessment of the Meta-Evidence of Cohort Studies for NutriGrade.Additional file 3: Supplementary Table. 3 PICOS framework.


The study aimed to investigate the difference in refractive status and ocular parameters between ptotic and fellow eyes in patients with unilateral congenital ptosis.Thirty patients  with unilateral congenital ptosis diagnosed and treated at the First Affiliated Hospital of Sun-yat Sen University were enrolled and underwent detailed refractive examinations from March 2019 to February 2022. Ocular biometric measurements were performed by an IOL Master 700 biometer. The differences in refractive error characteristics, best-corrected visual acuity (BCVA), and ocular parameters including axial length (AL), central corneal thickness (CCT), aqueous depth (AQD), anterior chamber depth (ACD), lens thickness (LT), and keratometry values between ptotic and fellow eyes were analysed.P = 0.009) and a higher incidence of amblyopia (n (%), 7(23%), P = 0.016) were observed in ptotic eyes. The CCT of ptotic eyes was greater than that of fellow eyes . The keratometry values at the flat axis (K1) and mean corneal power (Km) were smaller in ptotic eyes . There was no significant difference in AL between ptotic and fellow eyes.A lower BCVA , 0.00 , Congenital ptosis influences ocular parameters, mainly causing a thicker and flatter cornea. Patients with unilateral congenital ptosis might have lower BCVA in the ptotic eyes.The online version contains supplementary material available at 10.1186/s12886-022-02511-x. Congenital ptosis is a relatively normal ocular abnormality caused by a drooping eyelid within the first year of life, characterized as a narrow palpebral fissure and more coverage by the upper eyelid on the cornea. Several studies have reported that congenital ptosis can increase the incidence of myopia, hyperopia, astigmatism, anisometropia and amblyopia –5. PreviRecently, the IOL Master 700 , a highly advanced, noncontact biometer, was introduced, allowing accurate ocular parameter testing on patients with congenital ptosis . It is bIn this study, we focus on both refractive status and the ocular biological parameters of unilateral congenital ptosis to compare the refractive error between ptotic eyes and fellow eyes and to investigate the influence of congenital ptosis on the ocular structure during eyeball growth.We enrolled 30 patients with unilateral congenital ptosis who were diagnosed and treated at the Ophthalmology Department of the First Affiliated Hospital of Sun Yat-sen University between March 2019 and February 2022. This study was approved by the Ethics Committee of the First Affiliated Hospital of Sun Yat-sen University in China ([2021]544) and complied with the tenets of the Declaration of Helsinki for biomedical research involving human subjects.The normal position of the upper lid covers the cornea by less than 2 mm, and ptosis can be diagnosed when the upper lid is below its normal position. The severity of ptosis can be classified as mild (1–2 mm), when the upper lid does not cover the pupil, moderate (3–4 mm), when the upper lid partially covers the pupil, and severe (more than 4 mm), when the pupil is completely covered by the eyelid .The inclusion criteria included unilateral congenital ptosis, age greater than 5 years, absence of other ocular pathology, and reliable measurement results. The exclusion criteria were acquired ptosis, patients younger than 5 years and inability to cooperate with the examination, a history of eye trauma, previous ocular surgery (including ptosis correction surgery), corneal opacity, wearing of an eye contact lens in the last 3 months, and other ophthalmic or systemic disorders.All patients were examined by the same trained examiner. Routine ophthalmic examinations were performed for all patients, including best-corrected visual acuity (BCVA) measurements using a Snellen chart, detailed slit-lamp examination of the anterior segment, fundus examination with dilated pupils, and measurement of intraocular pressure . Complete evaluation of ptosis, including measurement of the height of the palpebral fissure (HPF) and levator functions (LF), was performed by the lid excursion method, involving measurement of the upper lid from extreme downgaze to extreme upgaze with the frontalis muscle fixed.Cycloplegic refraction was performed for all patients. Children younger than 10 years were given 1% atropine ointment once a day for 3 days and were examined on the fourth day. Patients older than 10 years were examined following administration of combined 0.5% phenylephrine hydrochloride and 0.5% tropicamide eye drops three times every 5 minutes. We calculated the refraction by the spherical equivalent refraction (SER) and defined myopia as an SER of at least − 0.50 D and hyperopia as an SER of + 2.00 D or more. Astigmatism was defined as 0.75 DC or more. Anisometropia was defined as at least a 1.00 D difference with the opposite eye. We defined amblyopia as a visual acuity of 6/9 or less and a difference of more than two Snellen lines between the fellow and ptotic eyes, excluding any other ocular abnormalities .Biometric measurements were performed using an IOL Master 700 three times by the same well-trained technician before pupillary dilatation in all patients. Each time before the examination, the device was recalibrated. During the measurement, the examinees were asked to position their heads on a headrest and look at a fixation point for foveal scans. Poor-quality results were deleted, and new measurements were obtained until the readings were reliable.The axial length (AL), central corneal thickness (CCT), aqueous depth (AQD), anterior chamber depth (ACD), lens thickness (LT), keratometry values at the flat (K1) and steep (K2) axis, and mean corneal power (Km) were obtained. AL was defined as the distance from the tear film to the retinal pigment epithelium (RPE) of the fovea. CCT was defined as the distance from the anterior to the posterior cornea . AQD wasP value less than 0.05 was considered statistically significant.Statistical analysis was performed by using SPSS version 22.0. The Snellen VA was converted to the logarithm of the minimum angle of resolution (logMAR) value. The Shapiro–Wilk test was used to examine whether the measured values followed a normal distribution. Descriptive results for continuous variables are expressed as the mean ± standard deviation (SD) or median and interquartile range (IQR) depending on the normality of the distribution. Differences were compared using paired t tests for data with a normal distribution and Wilcoxon rank-sum tests for those with a nonnormal distribution. McNemar’s tests were used to compare the frequencies of refractive types between the fellow and ptotic eyes. A In total, 30 eyes of 30 patients with unilateral congenital ptosis in this study were analysed, and the 30 fellow eyes without ptosis served as controls. The distribution of sex was 16 (53%) males and 14 (47%) females. The average age was 22.00 ± 11.41 years, ranging from 5 to 46 years old. Out of 30 patients, 8 (27%) had mild ptosis, 15 (50%) had moderate ptosis, and 7 (23%) had severe ptosis. Fifteen (50%) had the right eye affected, while 15 (50%) had the left eye affected. Table p = 0.009) than that of the fellow eyes. The incidence of myopia and astigmatism did not differ significantly between ptotic eyes and fellow eyes. However, the prevalence of amblyopia in the ptotic eyes (n (%), 7(23%), p = 0.016) was higher than that in the fellow eyes (n (%), 0). All 7 amblyopic eyes were ptotic, 3 severe and 4 moderate. The results are shown in Table The BCVA of the ptotic eyes was significantly lower , 0.00 , (n (%), 73%, p = 0p < 0.001). The K1  and SE  of ptotic eyes were lower than those of fellow eyes, which means that the corneas of the former were thicker and flatter. The parameters AL, AQD, ACD, LT, K2 and IOP were not significantly different between the ptotic and fellow eyes.Ocular biometric parameters in eyes with ptosis were compared with those of the fellow normal eyes and are summarized in Table It has been established that congenital ptosis may increase the incidence of refractive errors, including myopia, hyperopia, and astigmatism, as well as the incidence of amblyopia and anisometropia , 19, 20.In the present study, the incidence of amblyopia in patients with congenital ptosis was 23%, similar to previous reports –23. In aIn normal human eyes, the upper eyelid covers 1–2 mm of the cornea, while the lower eyelid covers none of it. A previous study showed that the superior cornea is thicker than the inferior cornea . Under tIn our study, among the 30 patients with unilateral congenital ptosis, no difference in axial length between ptotic eyes and fellow normal eyes was observed, which is consistent with the reports individually by von Noorden et al. and Takahashi et al. , 29 One A previous study showed that the pressure from the upper eyelids may influence the shape of the cornea, causing it to steepen . In anotSeveral limitations of this research need to be considered. First, the average age of these patients (22.00 ± 11.41 years) is relatively high, since people at these ages in China typically have myopia. It is possible that different results regarding the refractive error and axial length could be obtained from younger patients. The second limitation is that the number of patients included in this study was small despite our having calculated the minimum required sample size in advance. Future research needs to be done to determine additional abnormalities induced by congenital ptosis, providing more useful information for treatment. Third, the ocular parameters in this study seem to be limited, and more parameter testing and more accurate techniques should be applied for patients with congenital ptosis. Finally, in order to better control the study findings, only patients with unilateral congenital ptosis were enrolled, and ocular changes caused by acquired ptosis remain unknown; therefore, further studies are needed.In summary, we confirmed that congenital ptosis may not only have an influence on refractive error and increase the prevalence of amblyopia but also cause the cornea to become thicker and flatter. The impact of congenital ptosis on axial length seems to be minimal in humans.Additional file 1.


Oxaliplatin is a widely used chemotherapeutic agent. Despite its many beneficial aspects in fighting many malignancies, it shares an aversive effect of neuropathy. Many substances have been used to limit this oxaliplatin-driven neuropathy in patients. This study evaluates the neuroprotective role of a grape pomace extract (GPE) into an oxaliplatin induced neuropathy in rats. For this reason, following the delivery of the substance into the animals prior to or simultaneously with oxaliplatin, their performance was evaluated by behavioral tests. Blood tests were also performed for the antioxidant activity of the extract, along with a histological and pathological evaluation of dorsal root ganglion (DRG) cells as the major components of the neuropathy. All behavioral tests were corrected following the use of the grape pomace. Oxidative stressors were also limited with the use of the extract. Additionally, the morphometrical analysis of the DRG cells and their immunohistochemical phenotype revealed the fidelity of the animal model and the changes into the parvalbumin and GFAP concentration indicative of the neuroprotective role of the pomace. In conclusion, the grape pomace extract with its antioxidant properties alleviates the harmful effects of the oxaliplatin induced chronic neuropathy in rats. Oxaliplatin  is a highly active, third-generation platinum chemotherapeutic agent, widely used against advanced colorectal cancer and as an alternative option to cisplatin in ovarian  1,2–diam,4, breasOxaliplatin-based chemotherapy leads to severe neurotoxicity characterized clinically by two different types of symptoms . The fir2), without interfering with total DRG neuronal cell number [Up until now, several researchers have reported that severe damage in Dorsal Root Ganglion (DRG) neurons leads to the development of PN ,18,19,20l number .+ driving to excess Ca2+ influx [V1.6 resurgent [Although DRG neurons are widely accepted to be the primary drug-target, several hypotheses have been proposed as possible pathogenetic mechanisms leading to PN. These include high levels of platinum-DNA adducts in DRG neurons compared to other cell tissues  prolonge+ influx  or sodiuesurgent  and neuresurgent . Howeveresurgent , suggestesurgent  leading Curcuma longa [Silybum marianum) [Use of antioxidant compounds, such as vitamin E ,29, glutma longa ,36 and carianum)  were alsIn this study, we evaluated the efficacy of a polyphenol-rich grape extract obtained from pomace for prevention of oxaliplatin associated PN in rats. The extract’s compounds, such as catechin and epicatechin and gallic acid have been proven to have potent neuroprotective , chemoprad libitum access to standard rodent food 4RF25 from Mucedola  and water. The general condition of the animals was evaluated daily, and body weight was recorded twice a week, before each oxaliplatin administration. Animals’ health was monitored according to FELASA recommendations using IDEXX GmbH  testing. Access to the facility was controlled. Room temperature of 20–22 °C, and humidity of 45–55%, were checked daily. A stable temperature was ensured by a HVAC system controlled by a thermostat. Lights were less than 325 lux and were programmed for 12 h:12 h light to dark. Noise was kept <85 db and there was no ultrasonic noise or vibration. The sanitation was undertaken every week by experienced personnel. An ear identification method was used in all animals.A total of 30 adult four-month-old female Wistar rats from our own colony, weighing between 160 and 260 gr at the start of experimentation were used in this study. During the experiment, animals were housed in spacious open cages with three animals per cage at the animal research facility of the Laboratory of Anatomy, Histology and Embryology, School of Veterinary Medicine, Faculty of Health Sciences, Aristotle University of Thessaloniki (EL-54-BIOexp-23). Experimentation received the approval of the Veterinary Directorate of Thessaloniki, and all experimental procedures and protocols were in accordance with the European Directive 86/609/EEC on the protection of animals used for scientific purposes since the new one, the Directive 2010/63/EU on the protection of animals used for scientific purposes EU, was adopted on 22 September 2010 and the experiments took place in 2008–2009. All animals had n = 5) were used as dextrose control group (125 μL dextrose 5% i.p. twice a week) while group B (n = 5), C and D animals received oxaliplatin treatment at a dose of 3 mg/kg i.p. biweekly for eight weeks. Groups C and D were provided with GPE in water at a level of 75 mg per day per animal for eleven weeks  or eight weeks   .Oxaliplatin  was made up for injection in 5% dextrose to obtain a solution of 5 mg/mL and was aliquoted in eppendorfs of 125 μL, to obtain a stock solution. Eppendorfs were kept frozen at −12 °C until use.Grape pomace was supplied by Union of Santorini Cooperatives, SantoWines. The raw material was dried in a shady, well-ventilated place and extracted with water 100% at 45 °C for 4 h. In continuation, the water extract was treated with absorption resin chromatography  to remove sugars and to obtain a fraction rich in phenolic compounds. The polyphenolic rich fraction was recovered using analytical grade isopropanol . The fraction was dried in a rotary evaporator and lyophilized for complete solvent removal.UPLC-HRMS analysis was performed on an AQUITY system (Waters) connected to an LTQ-OrbitrapR XL hybrid mass spectrometer  equipped with an electrospray ionization (ESI) source and operated in negative mode. A UPLC separation gradient was developed to efficiently resolve all compounds for a qualitative analysis. The flow rate was set at 0.4 mL/min and the solvent system was: (A) water 0.1% formic acid and (B) acetonitrile. The elution program was: 2% B for 2 min; 100% B in 18 min; and hold for 2 min. After returning to 2% B in 1 min, column equilibration was performed for 4 min at the end of the run. The injection volume was set to 10 μL and the sample was injected at 0.3 mg/mL in water-acetonitrile solution (1:1) on a Supelco Ascentis Express C18 . The HRMS and HRMS/MS data were acquired in negative mode over 100–1000 m/z range. The MS profile was recorded in full scan mode (scan time = 1 micro scans and maximum inject time = 500 ms). The ESI conditions were as follows: capillary temperature 320 °C; capillary voltage −40 V; tube lens −120 V; ESI voltage 2.7 kV. Nitrogen was used as sheath gas (40 Au) and auxiliary gas (8 Au). Chromatographic and spectrometric features were used for identification of extracts constituents such as retention time (Rt), polarity, accurate m/z, proposed elemental composition (EC), ring double bond equivalent (RDBeq) values as well as HRMS/MS spectra and derived fragmentation motifs. The raw data were acquired and processed with XCalibur 2.2.4 software from Thermo Scientific.The extract was found to be rich in catechin and epicatechin, phenolic acids, such as gallic acid and caffeic acid and flavonoids .GPE was orally administered through drinking water at a dose of 75 mg per day per animal (approximately 280 mg/Kg) for eleven weeks  or eight weeks .home-cage measurements , ii. hand-held observations , iii. open field activity , iv. sensorimotor reflexes , including responses with exceptional significance such as the righting reflex [sensorimotor tasks  was g reflex , the cong reflex  and the g reflex , as theyg reflex , v. senslay task , grip stRighting reflex describes the animal’s reaction after it has been dropped from a height of 30 cm with its belly up. Normal rats should turn upside down and land on their four limbs, while animals with sensorimotor deficits may land on their side or even on their back. When the dorsal surface of the paw of the hindlimbs of a suspended animal slightly touches the edge of an object, the animal lifts the tested paws and places them on the surface of the object. This reaction is described as the contact placing response. Furthermore, the crossed extensor reflex is elicited when in a suspended animal the one hind limb is pinched; as a response, the irritated hind limb is flexed, and the contralateral hind limb is extended.The details of the examined sensorimotor reflexes and tasks are: Landing foot splay is the measured distance between the imprints of the heels of the two hind limbs, validated after dropping the animal from 30 cm high on to a surface, covered with white recording paper. Several studies prove that animals with sensorimotor deficits are present with increased hind limb spread in the landing foot splay task [lay task .Grip strength can accurately demonstrate sensorimotor peripheral neuropathy [uropathy . It is auropathy . Animalssticky paper test was applied to evaluate the animal’s reaction to light touch stimuli. A self-adhesive paper (14 × 23 mm) was placed onto the paw of the right hind limb and the latency of the rat’s first reaction  was recorded at each study point. There was a cut off time of 120 s.The Von Frey hair pinch test is widely used to examine “pain-like” behavior including changes in mechanical thresholds in rodents [The  rodents . In this rodents  beginninFor the gait (footprint) analysis, rats were forced to walk on an 80 cm long and 10 cm wide runway covered with white paper after having their hind paws inked with non-toxic colors. Marks of the hind paws of five sequential steps were used to evaluate animal’s stride width, stride length, interpedal distance and rotation of the right and left hind limb . InterpeTests were performed in all animals at four different time points; prior to any treatment, at the middle of the oxaliplatin treatment period, at the end of the oxaliplatin treatment period and finally at three weeks after the treatment period. The landing foot splay, grip strength and sticky paper tests were performed three times at each tested period and the average score was presented. Tests were practiced at three sequential days each time with a time interval of 30 min between the tests, blindly by the same persons each time.An extensive description of the FOB tests used and the scoring scale used to express the data have been published elsewhere ,55.g for 10 min at 4 °C). Acquired plasma samples (centrifugation’s supernatant) were collected and stored at −80 °C until used.At the end of experimentation, animals were injected i.p. with a ketamine/xylazine mixture . Blood samples were collected from all animals to estimate total antioxidant capacity (TAC) values and to perform the thiobarbituric acid-reactive substances (TBARS) assay. Blood was collected after cardiac puncture and was properly centrifuged  by antioxidants of plasma.Total antioxidant capacity determination was based on the method of Janaszewska and Bartosz . Brieflyg for 3 min, and the optical density of the supernatant was determined at 530 nm. TBARS are expressed in terms of malondialdehyde (MDA) equivalents. The molar coefficient of MDA is 155 × 103 mol/L.Thiobarbituric acid-reactive substances assay was used for the determination of lipid peroxidation. TBARS were determined according to a slightly modified assay of Keles et al. (2001) . SpecifiFollowing blood collection, all animals were perfused with 100 mL of 0.9% sodium chloride followed by 300 mL of 4% paraformaldehyde. Sensory neurons that innervate rat’s hind limbs, clinically evaluated for the presence of PN, are located at L3 to L6 lumbar spinal cord level , and so To assess certain morphometrical and histological characteristics of untreated and oxaliplatin and/or GPE treated DRG sensory neurons, one of ten serial sections of each DRG was stained with H&E. An average of ten sections per animal were stained and evaluated afterwards.Double immunofluorescence staining against parvalbumin and Glial Fibrillary Acidic Protein (GFAP) was performed in order to evaluate the presence of large DRG sensory neurons along with the satellite cells excitation in response to evoked pathology ,59,60. A2), medium (500 μm2–1000 μm2)and large (>1000 μm2) DRG sensory neurons [Hematoxylin-eosin (H&E) stained sections were examined under a Nikon optical microscope  equipped with a Nikon D-Eclipse C1 camera. Morphometrical analysis data were obtained with the Image Analysis Pro-Plus 6.3 Program for Windows . Certain morphometrical parameters of DRGs of different experimental groups such as the mean cell area, mean diameter and perimeter and the ratio of small (<500 μm neurons  were evac.f. =50). Then the previous and the next cuts were checked to see if measured neurons were present synchronously and with a second nucleolus and the total nucleolus number (Nc.f.) of these fifty neurons was recorded. Then c.f. was calculated as follows: c.f.= Nc.f./nc.f., and was multiplied with the total DRG neuron number, found after measuring neurons in every tenth cut, giving us the final total number of the DRG neurons.In order to estimate total DRG neuron cell number, the number of neurons with nucleolus present in every tenth cut of each ganglion was measured and the sum of these measurements was multiplied by the number 10 . It has Immunofluorescence sections were examined with a Nikon D-eclipse C1 confocal laser scanning microscope (CLSM) and image recordings were captured with appropriate software (EZ-C1 3.20) and presented as z-Stacks. Laser beam gain was set at a specific point, which was stable for all studied sections of different experimental groups.p < 0.05.All data were analyzed using the SPSS 23.0 statistical software. Behavioral testing parameters were ranked , measured or validated as present/absent. Ranked and descriptive data were analyzed using a Chi-square test. Measured FOB variables, body weight measurements and oxidative stress marker score were examined using a one-way ANOVA  and independent samples t-test were used for comparisons between groups. Homogeneity of variances was tested using the Levene’s test and when violated, the non-parametric two-tailed Kruskal–Wallis was assessed for multiple comparisons, followed by the Mann–Whitney U test (two-tailed) for two-by-two comparisons. All measured data are expressed as mean ± S.E.M. Significance was set at During the experimental period, no fatigue, abdominal bloating, alopecia or kyphosis of treated animals were observed at any studied time point. As revealed by home-cage measurements, no statistically significant differences at body position, respiration rate and palpebral closure were found between experimental groups. Furthermore, reactivity, handling and palpebral closure of tested animals were found to be normal at all oxaliplatin and GPE treated rats.Open field test is widely used to measure locomotor activity and anxiety-like behavior of an animal when placed in a novel environment ,64. GaitBody weight of experimental animals was recorded twice a week, on drug or dextrose administration days, and data are presented in All experimental animals were present with normal approach and sound response at all studied time points. A normal eyelid and righting reflex were also evident for all animals. There were some differences observed in the following tests:Tail pinch response (TPR): while at the start of experimentation all tested animals were present with a normal response, at the end of oxaliplatin treatment 60%, 16.67% and 22.22% of group B, C and D animals, respectively, showed no reaction after having the base of their tail squeezed with tweezers. Statistical evaluation revealed that group B values differed significantly (p ≤ 0.05) from control values, whereas no statistical difference between control and group C and D responses was found. Interestingly, following the three weeks after-treatment period, all group C and D animals were present with a normal response, whereas 20% of group B animals were still lacking the tail pinch response.Contact placing response (CPR): With the use of this reflex, severely affected proprioception by oxaliplatin administration, can be clinically evaluated. Just from the middle of oxaliplatin treatment, 60% of group B animals had a normal reflex in only one hind limb, showing a statistically significant difference from controls (p ≤ 0.05), group C (p ≤ 0.05) and group D (p ≤ 0.1) where only 12.5% and 11.11%, respectively, of treated animals showed a decreased response. At the end of the after-treatment period, all group C and D animals had a normal score, while group B animals significantly differed (p ≤ 0.05) as 40% were still present with a decreased response.Crossed extensor reflex (CER): Amazingly, at the end of the oxaliplatin treatment, all group B animals showed no crossed extensor reflex, whereas 55.56% of group D animals were present with a normal reflex, and this difference was considered as statistically significant (p ≤ 0.05). A recovery was seen at the end of the after-treatment period, with 40% of group B and all group D animals having normal reflexes.In the latter three tested reflexes, an oxaliplatin induced significant decrease was evident. Groups receiving GPE had a better score in these three tested reflexes at all studied time points and also showed an increased repairing ability in the post-treatment period. All above data suggest that GPE has a prophylactic effect on the nervous system.As mentioned earlier we evaluated animal’s stride width, stride length, interpedal distance and rotation of the right and left hind limb  during tFoot rotation: All oxaliplatin treated groups were present with an increased rotation of the R and L hind limb compared to control animals, although this was not evaluated as statistically significant. Foot rotation is indicative of the sciatic nerve function, possibly affected by oxaliplatin administration.Stride length: Although there are some slight differences between the animal groups regarding the stride length of both feet, there is no statistical significance between them. This leads to the observation that ataxia, coordination and locomotion, which are mirrored into the stride length, have not been altered.Stride width: All oxaliplatin treated groups showed decreased stride width values. The observed difference was not found to be of statistical significance. Thus, the locomotion and the base of support, two characteristics implied by the measurement of the stride width, despite showing a decreasing tendency among the untreated and the oxaliplatin treated animals, is not indicative of motor neuropathy.Interpedal distance: as in previous measurements, there are some slight differences between the animal groups regarding the interpedal distance. No statistical significance was evident between them.landing foot splay task between the imprints of the hind feet was increased in group B, C and D animals  compared to untreated control animals where measured distance was 3.99 ± 0.5 cm. However, no statistically significant differences among oxaliplatin treated groups exists.Along with gait analysis, four more sensorimotor tasks were performed on all animals and gave us an informative overall characterization of the altered neuromuscular function evoked by oxaliplatin administration. Animals receiving GPE along with oxaliplatin treatment were present with mean task values closer to the untreated control values, whereas in group B animals’ severity of motor and sensory impairments was greater. As expected, the measured distance during the hind limb force between tested animals were also evident. A statistically significant difference in hind limb muscle strength between group B and group C (p ≤ 0.05) and D (p ≤ 0.01) animals was indicated. Group C and D animals were present with increased grip strength values  whereas group B animals showed exceptionally reduced values (2.807 ± 0.178 N).Significant alterations in measured sticky paper test. At the end of oxaliplatin treatment, mean latency of control animals was 157.68 ± 23.98 sec whereas group B and C animals showed a quicker reaction at 124.92 ± 24.31 and 126.77 ± 18.99 sec, respectively , medium (500–1000 μm2) and large (≥1000 μm2) DRG sensory neurons were estimated. The ratio of the large DRG cell population was found to be greater in group C and D compared to group B animals  and D (428.87 ± 2.82 μm2) compared to group B, where mean area was found to be decreased at 390.94 ± 2.48 μm2.Higher large DRG cell proportions in group C and D animals lead to increased values of mean somatic area as opposed to group B animals. Mean somatic area of DRG sensory neurons was higher in group C  and GFAP (red) immunohistochemistry staining in all experimental groups  revealedOxaliplatin has been widely used over the last few decades for the therapy of many malignant manifestations, such as colorectal, ovarian, lung, prostate and pancreatic cancer . DespiteMany attempts are being made by researchers to overcome this obstacle of the very beneficial oxaliplatin. Among the substances evaluated for neuroprotection from the oxaliplatin induced chronic neuropathy are anti-oxidants, since it is well known that platinum compounds, such as oxaliplatin, induce oxidative stress through mechanisms involving nuclear and mitochondrial DNA dysfunction, as well as mitochondrial oxidative damage and depletion of non-enzymatic antioxidants ,67.Grape pomace extract is rich in antioxidant molecules. It possesses high quantities of catechins, epicatechin, procyanidins, phenolic acids  and flavonoids  that areIn our study, we used a grape extract obtained from pomace in oxaliplatin treated rats to investigate its possible advantageous effect on oxaliplatin induced PN. Along with the exploration of TAC activity into the blood of all animals, we applied different behavioral tests to estimate the produced neuropathy and used histology and immunohistochemistry to evaluate the morphometric data of the affected neurons.To our knowledge, this is the first time that the FOB assessment tool is used to explore oxaliplatin induced chronic neuropathy in rats.The first tests of the FOB neurobehavioral assessment tool that we practiced on the animals, consisted of home-cage measurements, hand-held observations and the open field activity . We obseBody weight of experimental animals was recorded twice a week, on drug or dextrose administration days. No statistically significant differences in mean body weights were found to exist at any examined treatment period among the different experimental groups . Animals71. In our study we speculated the usefulness of FOB to understanding the type of toxicity induced by oxaliplatin and to evaluate the sensorimotor benefits of GPE administration.The functional observational battery (FOB) is a noninvasive procedure designed to detect sensorimotor and gross functional deficits in young adult rats following their exposure to chemicals and to better quantify the evoked neurotoxic effects crossed extensor reflex, all oxaliplatin treated animals felt the pain and withdrew the infected hindlimb, possibly due to the evoked paresthesia (increased sensitivity of nociceptors). On the contrary, they did not extend the contralateral hindlimb, which may be due to the histological damage that did not allow the stimulus to go on the contralateral side of the spinal cord.All oxaliplatin-receiving animals presented a normal approach and sound response at all studied time points. Normal eyelid and righting reflexes were also evident for all animals . On the contact placing response and oxaliplatin treatment was shown to significantly hamper proprioception of treated animal. This may be due to damage in large DRG neurons, which is evident in the morphometrical analysis of the DGR neurons, in accordance with numerous other publications [Proprioception of experimental animals was also tested through the ications ,21,73,74GPE treated animals presented higher percentages of normal proprioception at all studied periods. Results from examined responses and reflexes reflect the oxaliplatin-induced damage, mainly to corticospinal reflexes, and the prophylactic effect of the GPE.Following the reflexes, we tested the gait of the experimental animals and we evaluated animal’s stride width, stride length, interpedal distance (the mean of five measurements) and rotation of the right and left hind limb .All the observed differences were found to be without statistical significance , evidencIn order to better quantify the oxaliplatin-evoked damage in sensorimotor tasks and to test the possible benefits from GPE administration, we also performed the landing foot splay task, the grip strength task, the sticky paper task and the Von Frey hair pinch test.landing foot splay task values compared to untreated control animals and GPE administration did not exert any beneficial effect  showed GFAP in abundancy. GFAP staining encircled the large DRG, which is indicative of a long-lasting increase in oxidative stressors in the DRGs due to oxaliplatin. Abundance of satellite cells is generally indicative of inflammation in the region  . Xu et aIn conclusion, this study has shown the beneficial effect of a grape pomace extract, as an antioxidant regimen due to its compounds’ antioxidant effects to an oxaliplatin induced chronic neuropathic rat model. Rodent models have played an important role in the confrontation of the undesirable effect of oxaliplatin. Strain, sex and dose differences should be evaluated in the future for an integrated therapy of the chronic neuropathy.