Postoperative morphine consumption was similar among groups. However, intraoperative fentanyl consumption was significantly lower in the Parasternal group . Parasternal group showed shorter times to extubation  and better performance at incentive spirometer with a median (IQR) of 2 raised balls (1–2) vs. 1 (1–2) after awakening (p = 0.04). Ultrasound guided parasternal block provided an optimal perioperative analgesia with a significant reduction in intraoperative opioid consumption, time to extubation and a better postoperative performance at spirometry when compared to the Control group.Ultrasound guided parasternal block is a regional anaesthesia technique targeting the anterior branches of intercostal nerves, which supply the anterior thoracic wall. The aim of this prospective study is to assess the efficacy of parasternal block to manage postoperative analgesia and reduce opioid consumption in patients undergoing cardiac surgery throughout sternotomy. A total of 126 consecutive patients were allocated to two different groups, receiving  or not (Control group) preoperative ultrasound guided bilateral parasternal block with 20 mL of 0.5% ropivacaine per side. The following data were recorded: postoperative pain expressed by a 0–10 numeric rating scale (NRS), intraoperative fentanyl consumption, postoperative morphine consumption, time to extubation and perioperative pulmonary performance at incentive spirometry. Postoperative NRS was not significantly different between Parasternal and Control groups with a median (IQR) of 2 (0–4.5) vs. 3 (0–6) upon awakening ( Due to the improvement in life expectancy, heart surgery has become increasingly common among elderly population. However, due to the frailty and comorbidities of these patients, perioperative complications are frequent. Moreover, the impact of anaesthetics and analgesic drugs play an important role on the perioperative course . Post-operative pain is mainly related to median surgical sternotomy; it is intense and difficult to control, especially in the immediate few hours after surgery . It requRecently, many thoracic fascial plane blocks have been proven to be a valid option in controlling pain in cardiac, thoracic and breast surgery ,5,6,7. The sternal region is innervated by the anterior branches of intercostal nerves, which arise from the anterior branches of the spinal nerves from T1 to T11 . ParasteSeveral studies have investigated the parasternal block in cardiac patients trying to understand its impact on intra- and post- operative pain ,11,12,13The aim of this prospective study is to assess the efficacy of parasternal block to manage postoperative analgesia, reduce opioid consumption and improve respiratory function in patients undergoing cardiac surgery.This study was approved by Campus BioMedico Hospital Ethical Committee (protocol number 20/20 PAR ComEt CBM) and was registered on clinicaltrias.gov (NCT04319588).A total of 126 patients who underwent elective cardiac surgery were enrolled between November 2019 and March 2020. Written informed consent was obtained from all the subjects. Inclusion criteria to participate included: patients aged 18 years or older, ASA physical status I-IV and candidates for elective cardiac surgery with median sternotomy. Exclusion criteria included: allergy to local anaesthetics, site of puncture infection, weight < 30 Kg, impaired cerebral function and patient’s disapproval.All patients received general anaesthesia conducted with sevoflurane 2%, fentanyl 3–5 mcg/kg, remifentanil continuous infusion 0.1 mcg/kg/min , rocuronium 0.8–1 mg/kg and propofol 2% continuous infusion . The administration of adjunctive boluses of fentanyl (1 mcg/kg) intraoperatively was decided by the anaesthetists based on the hemodynamic parameter variations suggesting pain, such as a rapid increase in blood pressure and heart rate in the absence of fluid loading or amine infusion.Parasternal” group received ultrasound guided bilateral parasternal block after general anaesthesia induction and an infiltration with local anaesthetic of the surgical drainage sites at the end of surgery.The “Control” group received just the infiltration with local anaesthetic of the surgical drainage sites at the end of surgery.The “Patients were freely allocated in two groups: Drainages sites’ infiltration was provided since the parasternal block does not target the sub-phrenic area where the drainages exit the skin. Moreover, we performed it in both groups to eliminate potential bias generated by patients’ upper abdominal pain perception. Both groups received the same protocol of multimodal perioperative analgesia, which included: dexamethasone 0.1 mg/kg i.v. intraoperative; ketorolac 60 mg/24 h i.v. and paracetamol 1 g every 8 h i.v., postoperatively. In addition, morphine 2 mg i.v. was administered in the case of postoperative NRS pain scores ≥ 6.®, a device which consist of three air columns with different weights and coloured balls that move up when the patient inhales. A spirometry evaluation was performed both immediately before surgery and in the postoperative period with the aim to evaluate respiratory function after the surgical intervention and to compare to baseline values. This evaluation was performed with the TriFlo Inspiratory ExerciserAt the end of the operation, all patients were transferred to the intensive care unit (ICU) for weaning from invasive mechanical ventilation and immediate postoperative monitoring. Data collected: anthropometric data; chronic opiate medications; diabetes mellitus; pre-existing pulmonary conditions; type of surgery—aortocoronary bypass, off pump aortocoronary bypass, valvular surgery and ascending aortic surgery; length of surgery; postoperative pain at extubation and after 6–12–24 h on a 0–10 numeric rating scale (NRS) expressed as maximum pain experienced during that period of time; postoperative opiates consumption: time to first morphine bolus administration (from the patient awakening); total morphine consumption during the first 24 h after surgery; intraoperative opiate consumption ; time to extubation; side effects—nausea and vomiting; delirium ; and respiratory performance with the TriFlo expressed as the number of balls moved up before and after the surgery. All the data in the ICU  were collected by the ICU nurses specialized in the management of cardiac surgery patients.The patients in the Parasternal group received bilateral ultrasound guided parasternal block immediately after induction of general anaesthesia and intubation . The bloAt the end of the surgery, both groups received a local infiltration of the surgical drainages with ropivacaine 0.25% 20 mL performed by the surgeon.To calculate a sample size, we focused on our primary hypothesis that perioperative analgesia is improved with the parasternal block. We estimated the density of pain scores  based on published data regarding the use of parasternal block for cardiac surgery . To simuStatistical analysis and graphical presentation were obtained thanks to the use of the GraphPad Prism 8 software .The values of continuous quantitative variables are expressed as mean ± standard deviation (SD); the values of discrete variables are expressed as median and interquartile range (IQR). Qualitative variables are expressed as number of observations and percentage of distribution.The parametric distribution of numerical variables was evaluated using the Shapiro–Wilk normality test. Difference between groups was assessed by T-Student test for continuous parametric variables, while Wilcoxon–Mann–Whitney U test was used when appropriate. Bonferroni–Dunn correction has been applied to multiple repeated measures in order to reduce the risk of type 1 error.2 test. The level of statistical significance was set for p value < 0.05. Categorical variables were compared with Pearson’s χ2 ± 2.1 in the Parasternal group and 27.8 kg/m2 ± 1.1 in the Control group.A total of 126 patients were enrolled in this study. In the Parasternal group, the median age was 67 ± 10 years, while in the Control group was 70 ± 10; both groups had a higher prevalence of males, and the mean body mass index (BMI) was 26.5 kg/mAortocoronary bypass in extracorporeal circulation (CEC) was the most performed intervention overall , followed by valvular surgery ; less frequent were off-pump aortocoronary bypasses, combined bypasses plus valvular surgery and thoracic aorta aneurysm. Surgical average duration was 230.7 ± 53.5 min in the Parasternal group, and 213 ± 40.8 min in the Control group .p-value < 0.001). Intraoperative remifentanil administration was not statistically significative between the two groups.Intraoperative fentanyl consumption emerged to be significantly higher in the Control group with a mean of 864.3 mcg compared to 406.3 mcg in the Parasternal group (p = 0.07); in the next 6 h it was 0 (0–3) in the Parasternal and 2 (0–4) in the Control group, while it was 0 in both groups during the following hours. However, pain was always under 4 at 48 h after extubation (mild pain) in both groups. Median (IQR) postoperative pain at awakening, expressed as maximum NRS scale value (range 0–10), was 2 (0–4.5) in the Parasternal and 3 (0–6) in the Control group  consumption of postoperative morphine during the first 24 h (0 (0–2) mg). Furthermore, the median (IQR) time to first opioid administration was similar (30 (10–45) mg for parasternal group vs. 30 (11–60) mg for control group) .Mean extubation time from admission in ICU was 191 ± 48 min in the Parasternal group and 305 ± 62 min in the Control group .Respiratory performance was evaluated by the TriFlo Inspiratory Exerciser and was expressed in the number of balls moved up during inspiration one hour after extubation. The median number of balls moved up was 2 (1–2) in the Parasternal group versus 1 (1–2) in the Control group. Side effects, such as nausea, vomiting and delirium, were present in a small percentage of patients (2–3%) in both groups. Data regarding respiratory performance and side effects are reassumed in This study was conducted on an extremely heterogeneous population, including every patient who underwent cardiac surgery; we tried to confront two different groups with different analgesic strategies in terms of pain management, recovery capacity and postoperative outcome.Due to the increased life expectancy, heart surgery has become frequent in the elderly population; in our study, while the mean age of our patients was 70 years old, we faced many patients aged 80 years or more and were affected by many comorbidities. To best manage this frail population, it is mandatory to devise an anaesthetic plan aimed at minimizing the impact on the patient’s vital functions and homeostasis . PeriopePain, in fact, plays a central role in this therapeutic process; it reduces thoracic excursion and influences respiratory functional recovery, finally promoting pulmonary complications, such as pneumoniae, pleural effusion and lung atelectasis. Different regional anaesthetic protocols have been proposed to better control sternal pain after cardiac surgery. Starting from neuraxial techniques, different approaches have been developed to target the thoracic fascial plane where intercostal nerves from T1 to T11 lay, such as the pectoral blocks ,19, the The parasternal block is one of the more promising fascial blocks to manage sternal pain as described in the recent studies conducted by Sepolvere et al. ,25. In our study, post-operative pain, expressed with an NRS scale from 0 to 10, was very low in both groups in absence of a significant statistical difference . These rAdditionally, postoperative opiate consumption was low in both groups without any significant statistical difference, with a mean morphine consumption of 1 mg overall. This result could be correlated to the long and complex type of surgery and the timing of the block execution. In fact, we observed a mean surgical time between 3 and 4 h; moreover, a phase of recovery from anaesthesia and weaning from mechanical ventilation in ICU before weaking up was always needed. Therefore, extubation often happened several hours after the critical phase of acute postoperative pain, which is particularly intense in the immediate period after the end of the surgery, as shown in the study of Zubrzycki et al. . MoreoveAnother interesting result regards intraoperative fentanyl consumption: intraoperative administration in the Control group was twice the dose used in the Parasternal group. This result is in line with a recent study conducted in 2020 , which cMoreover, considering the low NRS scores and the successful postoperative pain control in both groups, the analgesic intraoperative regimen adopted probably had an important influence on the postoperative period. Multimodal pre-emptive analgesia with opiates, FANS, regional anaesthesia, corticosteroids, and paracetamol plays a fundamental role in preventing the development of surgical pain . Notably, time to extubation could be affected by several other factors, such as preoperative conditions, i.e., pre-existing pulmonary and neurologic disorders; surgical factors, such as intraoperative and postoperative bleeding; and postoperative conditions, such as arrhythmia or bleeding. Nevertheless, we did not register significant differences in patients’ characteristics and perioperative conditions that could have affected or delayed time to extubation. It must be said that other parameters, such as intraoperative heart rate and pressure values, use of vasopressor drugs, conscious state at awakening and respiratory complications, have not been evaluated in our study.Pulmonary performance is another interesting result to underline. Postoperative chest pain is usually evaluated with the patient in a supine position in the absence of movement. Respiratory evaluation could be a way to evaluate parasternal block efficacy when the thorax is moving. Every patient underwent the TriFlo Inspiratory Exerciser test immediately after extubation and weaning from mechanical ventilation, which was compared to the baseline test performed immediately before surgery. Data showed that patients in the parasternal block moved up a mean of one ball more than the Control group . This result may suggest a better analgesic effect of the parasternal block during thoracic excursions and consequently a better respiratory performance in the postoperative period; this implies better blood oxygenation and oxygen saturation, better airway clearance and a lower risk of postoperative pneumonia. Moreover, earlier extubation and less mechanical ventilation time could also have improved respiratory performance in the Parasternal group.This study has several limitations. Firstly, it is difficult to show a clinically meaningful result for postoperative pain, which was one of our primary outcomes, when baseline postoperative pain scores are already low. However, these results confirm those of Lee et al. .Secondly, we did not record pain on movement, although performance at spirometer may be considered an inversely proportional indirect index of chest pain.Moreover, we did not investigate the correlation between perioperative analgesia and other outcomes, such as, days of ICU stay, total hospitalization time and incidence of chronic postoperative pain. At the same time, we did not correlate time to extubation and postoperative spirometer performance with the incidence of respiratory complications, leading, for example, to supplemental oxygen therapy or mechanical ventilation. We expect several future studies focus in depth on these aspects.Another limit of the study is broad inclusion and exclusion criteria without reporting more baseline characteristics that could have influenced the primary outcome, such as pre-existing chronic pain and neuropathy, although the incidence of chronic opioid medications was similar among groups. Nevertheless, some important pre-existing conditions have been investigated and discussed.Lastly, postoperative multimodal analgesia included on-demand administration of i.v. morphine, although recommendations indicate the oral route is preferable for opioid intake. However, this is not always possible as patients undergoing cardiothoracic surgery may have dysphagia and an increased aspiration risk in the first postoperative period . In this regard, a solution could be represented by the administration of sublingual sufentanil through a patient-controlled analgesia (PCA) system, which has already proved to be effective and safe in thoracic surgery .Ultrasound guided parasternal block seems to be an effective, safe and easy to perform technique in patients undergoing cardiac surgery under sternotomy. Although it did not significantly affect postoperative analgesia, it showed a relevant reduction in intraoperative opioid consumption and a better postoperative performance at spirometry compared to the Control group. Further studies are expected to confirm these findings and explore the medium- and long-term impact of this technique on postoperative morbidity.


Heterosis, also known as hybrid vigor, is the basis of modern maize production. The effect of heterosis on maize phenotypes has been studied for decades, but its effect on the maize-associated microbiome is much less characterized. To determine the effect of heterosis on the maize microbiome, we sequenced and compared the bacterial communities of inbred, open pollinated, and hybrid maize. Samples covered three tissue types  in two field experiments and one greenhouse experiment. Bacterial diversity was more affected by location and tissue type than genetic background for both within-sample  and between-sample (beta) diversity. PERMANOVA analysis similarly showed that tissue type and location had significant effects on the overall community structure, whereas the intraspecies genetic background and individual plant genotypes did not. Differential abundance analysis identified only 25 bacterial ASVs that significantly differed between inbred and hybrid maize. Predicted metagenome content was inferred with Picrust2, and it also showed a significantly larger effect of tissue and location than genetic background. Overall, these results indicate that the bacterial communities of inbred and hybrid maize are often more similar than they are different and that non-genetic effects are generally the largest influences on the maize microbiome. All plants coexist with communities of fungi and bacteria in, on, and around them ,2. TheseAn active research area in plant–microbe interactions is determining the extent to which plant genetic variation alters the microbial community ,29,30,31Maize has been a model crop for plant genetics for over 100 years , due, inF1 hybrids show increased vigor and yield relative to their parents , an effeRecently, it was found that field-grown maize displays heterosis in bacterial rhizosphere communities, as well as fungal communities in the rhizosphere and phyllosphere . In addiThese previous studies focused on the exterior communities of the plant (the rhizosphere and phyllosphere). In this study, we sought to characterize how inbreeding and heterosis affect both the interior and exterior bacterial communities of maize by looking at the bacteria of the rhizosphere, root endosphere, and stalk endosphere communities in three differently inbred maize groups . Our primary goals were to (1) characterize the bacterial communities in each compartment for each group, (2) determine aspects of the community that were consistent across them, (3) determine differences in the communities that could be linked to heterosis, and (4) test the hypothesis that hybrid maize may select superior microbial communities.Fields were planted in the summer of 2018 (Year 1) and 2019 (Year 2) at different locations within the Iron Horse Research Farm in Watkinsville, Georgia. Plants were grown via standard agronomic practices for the state of Georgia . Our firA single greenhouse experiment was carried out in 2019, looking at rhizosphere, root, and stalk samples within the same plant. For each pot, four seeds were planted in a 5-gallon pot with 90% Fafard 3B/Metro-Mix 830 professional growing mix (Sungro Horticulture) and 10% vermiculite. Upon emergence, pots were thinned down to one plant per pot. Three pots per genotype were grown, and pots were arranged in a randomized block design, with each table in the greenhouse consisting of a block containing all 14 genotypes. .In total, we had 248 samples across the three experiments. Not every genotype was used in all 3 experiments. Plants were harvested in a single day to avoid batch effects. A 10 cm section of stalk was cut from the plant 20 cm off the ground using sterilized razor blades and gloves. Plants were dug up around the roots, and roots were removed from the center of the root ball and placed into a clean falcon tube for root and rhizosphere samples.g for 10 min in preparation for DNA extraction. Roots were then thoroughly cleaned with deionized water to remove any residual rhizosphere. Then, 2–3 cm of roots were chopped up with a sterile razor blade and loaded into a 2 mL conical tube for GenoGrinding . The outer portions of stalks were removed with a sterile razor blade, and the inner tissues  were cut into 1–3 mm pieces and loaded into a 2 mL conical tube for GenoGrinding (SPEX SamplePrep). Root samples were vortexed at the max setting for 15 s in deionized water to separate the rhizosphere from the root. This wash was then centrifuged at 4500× DNA was extracted with a Quick-DNA Fecal/Soil Microbe 96 Kit  following the manufacturer’s instructions, and 16 s rDNA gene amplification was performed using Earth Microbiome Project 515F  and 806RFive μL of the first PCR product for each sample was used in the second PCR amplification. The reaction mix consisted of 5 μL of the first PCR product, 5 μL Nextera i5 and i7 Barcode Primers, 12.5 μL 2 × Taq DNA polymerase master mix, and 2.5 μL PNA mix. The second amplification reaction was conducted at 95 °C for 45 s; 25 cycles of 95 °C for 15 s, 78 °C for 10 s, 60 °C for 45 s, 72 °C for 45 s; and finally, 68 °C for 5 min, followed by holding at 4°C. The second PCR products were purified using AMPure beads, and the cleaned products were eluted in 27uL of sterile water and stored at −20 °C until sequencing. Three blanks were used in DNA extraction and library prep. Libraries were sequenced at the Georgia Genomics and Bioinformatics Core on an Illumina MiSeq instrument  using one paired-end 250 flow cell. The raw data are available in the NCBI Sequence Read Archive under accession PRJNA924784.Sequence processing and quality filtering was completed within the QIIME2 version 2019.1 toolbox . Cutadaphttps://github.com/wallacelab/paper-schultz-microbiome-2023, accessed on 20 January 2023.First, we compared alpha diversity based on genetic background, tissue, and location. We used observed ASVs, as well as Shannon and Simpson indices, from the phyloseq package . PairwisMaize lines B73 and Mo17 and their F1 hybrid seeds were sterilized via our previously established method . Seeds w2O, then shaken 50 times and vortexed at max speed for 10 s. Then, 30 mLs of liquid was decanted into another falcon tube, using the tube cap to exclude large debris. The microbe suspension was centrifuged for 2 min at 3500 RPM to pellet plant debris, 20 mLs which was filtered through a 2 μm Whatman filter. Root samples were placed into a falcon tube without drill tip pulverization, and the same method was used to extract rhizosphere microbiomes.Microbiome extraction was modified from ,72. StalSterilized MiniMaize seeds were planted in 2.7 L sterilized pots with autoclave media mixture (same as above). Two sterilized MiniMaize seeds were planted in each pot. Pots were inoculated with 10 mLs of either B73, Mo17, or F1 combined stalk and rhizosphere microbiomes, with 8 pots per treatment. Autoclaved tin foil was placed over the pots for 4 days to ensure no outside microbes were introduced; then, plants were thinned to one plant per pot. Plants were allowed to grow for 5 weeks. Shoots were cut at the soil line and placed in brown paper bags. Roots were gently washed of soil and bagged. Above- and belowground samples were dried and weighed.We grew hybrid, inbred, and open pollinated maize lines in two field experiments (2018 and 2019) and one greenhouse experiment. Bacterial microbiomes were extracted from stalks, roots, and rhizospheres of the plants and quantified with QIIME2 and deblur. High-quality reads were retained and classified with the SILVA taxonomy classifier. Low-abundance amplicon sequence variants (ASVs), as well as ASVs associated with mitochondria, chloroplasts, and our blanks, were filtered out. Our final dataset consisted of 241 samples and 10922 ASVs, 938 (9%) of which were present across all three experiments.Throughout the three experiments, we found that stalk tissue had lower read depth and fewer associated ASVs compared to the rhizosphere and roots. The rhizosphere had the largest number of ASVs, with a majority of these also found in the roots A. Only aTo investigate which taxa were shared by groups of samples, we plotted intersections of common ASVs collapsed at the genus level . We defip < 0.001). Similarly, the root and rhizosphere samples had higher alpha diversity than stalk samples (p < 0.001). Post hoc tests show that there were no significant differences in alpha diversity between inbred, hybrid, and open pollinated samples across experiments   . GeneticBurkholderiaceae and Rhizobiaceae families.To identify the microbes that are most different between samples, we analyzed differentially abundant microbes with DESeq2 , using tPrevious work has shown that metabolic functions provide a better characterization of microbial communities than 16s-based taxonomy ,75,76. Wp = 0.048) and the F1 hybrid (p = 0.037) resulted in a small but significant decrease in shoot biomass relative to the control , energy production, and photosynthesis/carbon metabolism. Several pathways related to energy production and storage were found to be increased in hybrid maize. These included oxidative phosphorylation, heme uptake proteins, and electron transport. Outside of these energy-related pathways, a number of energy and protein pathways related to phototrophic capabilities were differentially abundant in hybrid maize root bacterial communities. These include plastocyanin , cytochrWhen we inoculated mini maize with the filtered bacterial microbiome from B73, Mo17, and their F1 hybrid, we saw minor differences in biomass aboveground but not belowground . SpecifiOur results are somewhat similar to those reported by Wagner et al., who showed significant differences in inbred and hybrid maize microbial communities in the field  and thatOur primary goals were to (1) characterize the bacterial communities in each compartment for each group, (2) determine aspects of the community that were consistent across groups, (3) determine differences in the communities that could be linked to heterosis, and (4) test the hypothesis that hybrid maize may select superior microbial communities.We found that (1) bacterial communities in the roots and rhizospheres were very similar to each other and that stalk communities only have a small portion of these bacteria; (2) common bacteria were present across compartments, regardless of inbreeding; (3) intraspecies heterosis played a much smaller role in microbial community diversity, composition, and function than tissue compartment or location; and (4) in a small greenhouse experiment, hybrid microbiomes failed to benefit inoculated mini maize.Modern maize breeding is based on heterosis, and with many new biologicals coming to market, it is paramount to understand how these microbes interact with intraspecies plant genetics. The literature has shown that host species play a large role in shaping niche microbiome, and our data show that inbreeding has small and significant effects on taxa and function in maize microbial communities. However, these effects pale in comparison to the effects of environment and tissue type on community composition. With the environment playing such a large role in shaping bacterial communities, investigating the extent to which maize can recruit specific taxa/functions would shed light on the potential to use the plant itself to alter its microbiome. Further work is needed to examine how maize genetic diversity and the environment shape community function. Understanding how the microbiome interacts with maize genetic diversity will allow breeders and scientists to make better use of microbial communities for more sustainable crop production.


Despite current clinical guidelines recommending suboccipital decompressive craniectomy (SDC) in cerebellar infarction when patients present with neurological deterioration, the precise definition of neurological deterioration remains unclear and accurate timing of SDC can be challenging. The current study aimed at characterizing whether clinical outcomes can be predicted by the GCS score immediately prior to SDC and whether higher GCS scores are associated with better clinical outcomes.In a single-center, retrospective analysis of 51 patients treated with SDC for space-occupying cerebellar infarction, clinical and imaging data were evaluated at the time points of symptom onset, hospital admission, and preoperatively. Clinical outcomes were measured by the mRS. Preoperative GCS scores were stratified into three groups . Univariate and multivariate Cox regression analyses were performed using clinical and radiological parameters as predictors of clinical outcomes.3, tonsillar herniation, brainstem compression, and a preoperative GCS score of 3–8 .In cox regression analysis GCS scores of 12–15 at surgery were significant predictors of positive clinical outcomes . For GCS scores of 3–8 and 9–11, no significant increase in proportional hazard ratios was observed. Negative clinical outcomes  were associated with infarct volume above 6.0 cm3 and with GCS between 12 and 15, as these patients may show better long-term outcomes than those in whom surgery is delayed until a GCS score below 11.Our preliminary findings suggest that SDC should be considered in patients with infarct volumes above 6.0 cm Space-occupying cerebellar infarctions constitute only 1–4% of all ischemic strokes –7. TreatPrevious studies have found that 24–40% of patients with space-occupying cerebellar infarction will undergo surgical treatment by suboccipital decompressive craniectomy , 9. The Despite evidence pointing toward the benefit of surgical therapy in selected patients, clear clinical or radiological criteria based on which the decision to perform SDC can be made are lacking. Daily management of patients with space-occupying cerebellar infarction is often interdisciplinary so parameters used to evaluate “neurological deterioration” should be standardized and easily applicable by the team of clinicians involved in patient care.To this end, the Glasgow Coma Scale (GCS) score is a well-established clinical grading system that requires an evaluation of the verbal response, eye opening, and motor response to evaluate the level of consciousness. The use of the GCS score in the management of space-occupying cerebellar lesions has shown that SDC is usually performed in patients with a GCS score between 8 and 10 , 16. CliIn this retrospective analysis of 51 adult patients undergoing SDC for space-occupying cerebellar infarction, GCS scores were stratified into three categories  to examine whether clinical outcomes can be predicted by the GCS score immediately prior to SDC and, if so, to examine whether higher GCS scores are associated with better clinical outcomes. Furthermore, clinical and radiological characteristics among patients with space-occupying cerebellar infarction were analyzed as additional predictors of clinical outcomes.This study was conducted as a single-center retrospective analysis, which was approved by the local ethics committee (Nr. 2022-825) and was conducted in accordance with the Declaration of Helsinki.We retrospectively identified all patients admitted to our hospital for space-occupying cerebellar infarction between January 2010 and June 2022. Electronic medical records were used to screen which patients underwent SDC vs. standard medical therapy, thus leaving 51 patients who were then further analyzed in detail.Electronic medical records and archived imaging data (computed tomography) were used to gather data on patient demographics, clinical history pertaining to symptoms at onset, time of clinical admission, imaging data , lysis or thrombectomy performed prior to SDC, the last follow-up, the mRS score at the last follow-up, and GCS scores. Imaging data and GCS scores were collected for each of the following time points: at symptom onset, at hospital admission, and at the surgery.Indications for performing SDC were considered signs of neurological deterioration and the judgment of these were at the discretion of the managing physicians. Most commonly, these included deterioration in terms of GCS score, signs of hydrocephalus, brainstem compression, dilated pupils, or anisocoria.Surgical techniques included preoperative insertion of an external ventricular drain (EVD), craniectomy of the affected hemisphere, strokectomy, and primary dural closure.GCS scores were categorized into three separate groups: 3–8 points, 9–11 points, and as we aimed to examine whether patients with higher GCS scores than those previously described may also profit from SDC, a third group GCS, 12–15 points) was defined. The separation of these groups was chosen to reflect clinically distinct states of neurological deterioration and values previously used in the literature  points w.All analyses were performed on non-contrast computer tomographic data with a 0.2-mm slice thickness.3) = A × B × C/2  and its largest perpendicular diameter (B) in an axial image. The vertical diameter (C) was determined by summing the number of slices in which the lesion is visibly multiplied by the slice thickness . Infarct volume was then calculated according to the formula: Volume .Quantitative values are presented as median values with a range unless otherwise noted. Group comparisons were performed using the Mann–Whitney We analyzed a total of 51 patients who underwent suboccipital decompressive craniectomy in our institution between 2010 and 2022. The median age of patients was 62 years old (ranging from 37 to 88 years) consisting of 31 (60%) male patients and 20 (40%) female patients. The most affected vascular territory was the PICA territory (47%), followed by the vertebrobasilar territory (31%), the SCA territory (6%), and AICA and SUCA territories (2% each). In 12% of cases, more than one territory was affected. In total, 35% of patients suffered a left-sided infarct, 37% a right-sided infarct, and 28% a bilateral infarct. In 78% of patients, the etiology was determined to be thromboembolic, 14% of patients were classified as having idiopathic cerebellar infarction, and 8% of patients had vascular dissection .The most common symptom at onset was ataxia (30 patients), followed by dizziness (28 patients), nausea and vomiting (25 patients), headache and reduced consciousness (11 patients), and dysarthria (10 patients), respectively .In six patients (12%), emergency thrombectomy was performed prior to surgery. A total of nine patients (17%) received intravenous lysis therapy prior to surgical treatment .The mean follow-up time was 87 days following surgery (standard deviation of 200 days). At the last follow-up, two patients (4%) had an mRS of 1, five patients (10%) an mRS of 2, three patients (6%) an mRS of 3, 16 patients (31%) an mRS of 4, and 21 patients (41%) an mRS of 5. A total of four patients died during treatment . The mean time from symptom onset to surgery was 69 h and from hospital admission to surgery was 52 h . The avep = 0.001). A total of three patients (6%) had a GCS score of 9–11 at admission which increased to 14 patients (27%) at the time of surgery (p = 0.001). A total of nine patients (18%) had a GCS score of 3–8 at admission, with 29 patients (57%) displaying this score at the time of surgery (p = 0.005)  had a GCS score of 12–15, whereas immediately preceding surgery, only eight patients (16%) remained at a GCS score of 12–15 (= 0.005) .n = 40 patients at admission and n = 46 patients immediately before surgery. Median infarct volume at admission was 1.89 cm3 (ranging from 0.3 to 7.8 cm3) which increased to 6.20 cm3 (ranging from 2.2 to 10.8 cm3) at the time of surgery (p = 0.001). At admission, 12 patients (30%) showed radiological signs of occlusive hydrocephalus. Among these 12 patients, three (25%) received an EVD and were monitored until neurological deterioration occurred. Occlusive hydrocephalus at the time of surgery increased to 44 patients (83%) (p = 0.001). Tonsillar herniation was seen in one patient (2.5%) at admission and in 38 patients (83%) at the time of surgery (p = 0.001). Brain stem compression was found in 12 patients (30%) at admission and in 45 patients (98%) at the time of surgery (p = 0.001). Clear brainstem infarction was only observed in one patient following SDC. In three patients (8%), the hemorrhagic transformation was observed at admission and was seen in nine patients (20%) at the time of surgery (p = 0.001)  .p-value of 0.031 for GCS scores of 12–15 at the surgery. This group contained five of the overall seven patients with an mRS score of 1–2. For lower GCS scores of 3–8  and 9–11 , no significant increase in proportional hazard ratios was observed  of 6.581  and a observed . In a mu= 0.045] .3 led to a significant increase in the proportional hazard ratio of 2.473 ; p = 0.013. Tonsillar herniation , brainstem compression , and a preoperative GCS score of 3–8  were found to be significantly associated with negative clinical outcomes  following SDC was analyzed using a univariate Cox regression analysis. In this study, we found that at the time of surgery, infarct volume above 6.0 cmoutcomes . GCS scop = 0.993). Among patients with GCS scores of 12–15, surgery was indicated based on the worsening of imaging findings in three patients. In the remaining five patients, the dynamics of clinical deterioration from a GCS score of 15 at admission to a GCS score of 12 or 13 combined with extensive signs of infarct volume, brainstem compression, or tonsillar herniation were the trigger for surgery.Subgroup analysis comparing the distribution of clinical and radiological characteristics among patients with a preoperative GCS score of 12–15 and all other GCS scores revealed no significant difference from those patients with lower GCS scores . The folStudies examining the efficacy of surgical treatment vs. the best medical care in space-occupying cerebellar infarction have shown that SDC provides better clinical outcomes than conservative therapy alone in patients with neurological deterioration –13. DespIn the current study, we, therefore, sought to stratify patients into three categories of preoperative GCS scores in order to pragmatically define the term “neurological deterioration” and to examine whether SDC performed at time points at which the GCS score is comparatively higher than previous studies reporting values between 8 and 10 may improve clinical outcomes , 16.Our findings show that patients with higher GCS scores from 12 to 15 significantly benefitted from SDC vs. patients with GCS scores of 11 or below, independent of infarct volume. It is important to note that in our cohort, we had no patients with a GCS score of 15, rather three with GCS scores of 12 and 13, respectively, and two patients with a GCS score of 14. Surgery was indicated in these cases due to increased infarct volume in control scans performed within 72 h after hospital admission. Furthermore, we observed that preoperative GCS scores between 3 and 8 are significantly associated with poor clinical outcomes, as has been previously described , 13. AmoWhereas, our analysis shows that patients with GCS scores of 12 and higher preceding SDC show better clinical outcomes than those with GCS scores of 11 and lower, the concept of “preventative” SDC has been evaluated in a retrospective matched case–control analysis among selected patients in a single center with GCS scores of 9 or higher . Among pA GCS-based evaluation of surgical candidates was also examined by a prospective German–Austrian series of 84 adult patients with space-occupying cerebellar infarction, which found that the overall risk for poor outcomes depended on the level of consciousness after clinical deterioration (OR 2.8) . AlthougIn addition to neurological evaluation of patients with space-occupying cerebellar infarction, radiological criteria may also be considered as surrogate parameters of infarct severity and may also guide the decision process in performing SDC. The pathophysiology of neurological deterioration and fluctuating levels of consciousness secondary to space-occupying cerebellar infarction can be traced back to brainstem compression/infarction, tonsillar herniation, and hydrocephalus . Brainst3 were positively associated with negative clinical outcomes in our cohort, with infarct volume showing the largest effect (HR = 2.47).In our cohort, 30% of patients showed radiological signs of brainstem compression at admission which then increased to 98% immediately preceding surgery. As brainstem compression was also observed in all patients with GCS scores of 12–15 preceding surgery, radiological criteria alone may not be sufficient to predict neurological deterioration or indicate SDC. Furthermore, MRI imaging studies of the brainstem have confirmed that brainstem compression alone cannot reliably predict neurological deterioration  so clinip = 0.408) but rather the preoperative GCS score. Although patients with GCS scores of 12–15 underwent surgery at an earlier time point than those with GCS scores of 3–11, there was no statistically significant difference between the two (21 vs. 50 h). These findings underscore the importance of clinical observation and GCS assessment when determining the need for SDC regardless of the time point at which the patient may present after symptom onset.An additional aspect of the practical management of patients with space-occupying cerebellar infarction is the time at which critical swelling and possible neurological deterioration may occur. A commonly observed period during which radiological progression of cerebellar swelling and clinical deterioration occurs is within 72 h following ictus , 21, 23.Although this study focuses on the role of SDC in space-occupying cerebellar infarction, the use of CSF drainage via an external ventricular drain (EVD) as an isolated treatment of occlusive hydrocephalus over SDC has been proposed . The res3, brainstem compression, and tonsillar herniation immediately preceding surgery were significantly associated with negative clinical outcomes (mRS ≥ 3), hydrocephalus was not associated with negative outcomes. We, therefore, conclude that in patients with any of the abovementioned accompanying radiological signs, management using EVD alone is not sufficient, whereas SDC more adequately addresses brainstem compression and herniation.We therefore also examined the effect of occlusive hydrocephalus among patients in our cohort prior to surgical treatment in relationship to mRS at the last follow-up. Whereas, infarct volume above 6.0 cmRegarding surgical strategies in SDC, no clinical standard exists to date; however, previous studies including patients with different surgical treatments have revealed SDC with strokectomy to be a more commonly performed procedure vs. SDC alone . All patA further important aspect of clinical management in space-occupying cerebellar infarction is patients' age and whether older patients may profit from surgical intervention in relation to age-related perioperative risks needs to be identified. A Swedish series of 32 patients with unilateral space-occupying cerebellar infarction found that advanced age was not associated with poor outcomes , two addFinally, it has been proposed that space-occupying cerebellar infarction affecting certain vascular territories is associated with increased mortality. In contrast to a previous study in which infarcts in the PICA territory were found to be associated with negative clinical prognosis, our analysis found no significant predictive value of infarct territory and clinical outcomes . As has The major limitation of the current study is the monocentric, retrospective design among a small study population. Due to the comparatively low frequency of space-occupying cerebellar infarction, multicentric cooperation to further the implementation of clinical standards regarding surgical intervention is needed. Furthermore, the use of CT as a standard imaging method was chosen due to its uniform availability in clinical practice and our cohort. However, we cannot rule out that brainstem infarction, which was not detectable on CT imaging, may have been present in some patients if MRI had been performed, therefore possibly contributing to worse clinical outcomes.3 with GCS scores higher than previously described in the literature as these patients may show better long-term outcomes than those in whom surgery is delayed until a GCS score of 11 or lower. Further studies are necessary to support these recommendations in clinical practice.Our preliminary results highlight the relevance of the preoperative GCS score as a simple clinical tool to determine when SDC may be performed in patients with space-occupying cerebellar infarction. Based on our findings, we propose that surgical intervention (SDC) should be considered in patients with infarct volumes above 6.0 cmThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by University Hospital Frankfurt. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.KL and MC conceptualized the study. KL collected and analyzed patient data, wrote the manuscript including figure and table production, and performed statistical analysis. SR, EH, HS, VS, and MC reviewed and approved the final manuscript. All authors contributed to the manuscript and approved the final submitted version.


Purpose: This study quantifies the gait characteristics of patients with degenerative lumbar scoliosis (DLS) and patients with simple lumbar spinal stenosis (LSS) by means of a three-dimensional gait analysis system, aiming to determine the image of spinal deformity on gait and the correlation between spinal–pelvic parameters and gait characteristics in patients with DLS to assist clinical work. Methods: From June 2020 to December 2021, a total of 50 subjects were enrolled in this study, of whom 20 patients with DLS served as the case group and 30 middle-aged and elderly patients with LSS were selected as the control group according to the general conditions  of the case group. Spinal–pelvic parameters were measured by full-length frontal and lateral spine films one week before surgery, and kinematics were recorded on the same day using a gait analysis system. Results: Compared to the control group, DLS patients exhibited significantly reduced velocity and cadence; gait variability and symmetry of both lower limbs were notably better in the LSS group than in the DLS group; joint ROM (range of motion) across multiple dimensions was also lower in the DLS group; and correlation analysis revealed that patients with a larger Cobb angle, T1PA, and higher CSVA tended to walk more slowly, and those with a larger PI, PT, and LL usually had smaller stride lengths. The greater the PI-SS mismatch, the longer the patient stayed in the support phase. Furthermore, a larger Cobb angle correlated with worse coronal hip mobility. Conclusions: DLS patients demonstrate distinctive gait abnormalities and reduced hip mobility compared to LSS patients. Significant correlations between crucial spinopelvic parameters and these gait changes underline their potential influence on gait disturbances in DLS. Our study identifies a Cobb angle cut-off of 16.1 as a key predictor for gait abnormalities. These insights can guide personalized treatment and intervention strategies, ultimately improving the quality of life for DLS patients. Degenerative lumbar scoliosis (DLS) is a common disease in the elderly, characterized by coronal malformations of the lumbar or thoracolumbar spine due to degeneration in adulthood, affecting 32% to 68% of population over 65 years old . The iniSurgical treatment for patients with DLS has two main goals: releasing spinal stenosis through decompression fusion and correcting spinal deformity through long-segment fixation. Therefore, detailed physical examination and appropriate imaging studies, such as magnetic resonance imaging (MRI), should be performed on patients with DLS . HoweverDiebo et al. have emphasized the importance of the dynamic aspects of alignment when managing spinal deformities , and botFollowing approval from the institutional review board committee, we prospectively recruited patients with DLS who presented to our clinic and were deemed surgical candidates.This single-center prospective study was conducted from June 2020 to December 2021 following approval from the institutional review board committee. Participants diagnosed with DLS (degenerative lumbar scoliosis) and LSS  who were deemed surgical candidates were recruited from our institution.Control Group Clarification: LSS patients requiring surgery but without spinal deformity were selected as the control group. Importantly, all LSS patients in this study exhibited symptoms of intermittent claudication, thereby serving as a suitable control to counteract the effect of intermittent claudication observed in the DLS group.All participants provided informed consent prior to enrollment, and the study was approved by the ethics committee at our institution.Inclusion criteria are (1) DLS group are patients clinically diagnosed thoracolumbar deformity, with a Cobb angle of ≥15 degrees; (2) patient age  ≥  55 years; (3) surgical intervention is recommended for symptoms; (4) had complete and clear preoperative X-ray of the whole standing spine and thoracolumbar or lumbar MRI; and (5) are able to ambulate without assistance. (6) All patients were receiving conservative treatment at study entry, including general physiotherapy or pain control through analgesics. Exclusion criteria are (1) diagnosis of idiopathic scoliosis, congenital scoliosis or other spinal deformities; (2) patients with lumbar spine fracture, tumor, infection or spondylolisthesis; and (3) those who have undergone thoracolumbar surgery or major lower extremity surgery that affect ambulation.The standard radiographs were undertaken using a long-cassette standing radiographs of the spine generating anterior/posterior as well as lateral views, before-surgery, including spinal parameters and pelvic parameters.T1PA: the sum of the pelvic tilt and the angle between the line from the hip axis to the center of Tl and the plumb line from the hip axis;SVA: offset horizontally from a plumb line dropped from C7 to the posterosuperior corner of the sacrum;CSVA: offset horizontally from a plumb line dropped from C7 midline to the center of the sacrum;TK: the sagittal angle between the superior endplate of T5 and the inferior endplate of T12, which was a positive value in kyphosis patients;TLK: the sagittal angle between the superior endplate of T10 and the inferior endplate of L2;LL: the sagittal angle between the superior endplate of L1 and the inferior endplate of S1, which was a positive value in lordosis patients.PT: the angle between the line from the center of the femoral head to the upper endplate of S1 and the plumb line;PI: Draw a vertical line through the midpoint of the upper endplate of S1. The angle between the vertical line and the midpoint of the upper endplate of S1 and the femoral head center.SS: the angle between the upper endplate of S1 and the horizontal line;These radiographic parameters were then measured on the PACS client software  by 2 investigators independently.The test subjects were all fitted with a total of 7 external reflective sensors . One at The figure demonstrates the positioning of the external reflective sensors used for gait analysis. Reflective sensors were placed at the waist and three lower limb joints on one side of the body. These sensors allowed for precise measurement of kinematic data during walking, enabling the assessment of gait characteristics and mobility parameters in patients with degenerative lumbar scoliosis (DLS) and lumbar spinal stenosis (LSS).t-test was used to compare parameters between DLS patients and LSS patients. For the analysis of the relationship among spinopelvic radiographic parameters and gait characters in patients with DLS, lasso regression and Pearson correlation analysis were used. The lasso regression model was used to identify the most important radiographic parameters associated with gait characters. Pearson correlation analysis was used to determine the correlation between spinopelvic radiographic parameters and gait characters, and the strength of the correlation. To investigate the relationship among spinopelvic radiographic parameters, range of motion variables, and gait variables in patients with DLS, mediation analysis was conducted to determine if the effect of spinal variables on gait variables is mediated by ROM variables. ROC analysis was added to determine cut-off values for key spinopelvic parameters like the Cobb angle, assessing their diagnostic performance through AUC, sensitivity, and specificity.Descriptive statistics were calculated for all variables, and the Shapiro–Wilk test was used to test for normal distribution. Continuous variables were expressed as means ± standard deviation (SD). An independent sample p-value < 0.05 was considered statistically significant.All statistical analyses were conducted using R version 4.1.1 . A two-sided p > 0.05)  .Apart from Cobb angle, when comparing parameters between DLS and LSS patients, there was a significant difference in the CSVA, T1PA, LL, PT, SS, and PI-LL. The CSVA was significantly larger in the DLS group (27.73 ± 5.58) than in the LSS group (3.52 ± 5.59). The T1PA was twice as large in the DLS group (32.29 ± 9.26) than in the LSS group (16.55 ± 7.61). The LL was twice as small in the DLS group (21.76 ± 5.09) than in the LSS group (44 ± 4.02). The PT and SS were also significantly different, which led to a significant difference in the PI-LL between the DLS group (32.2 ± 4.77) and the LSS group (12.71 ± 1.13) .During walking, the DLS group exhibited significantly reduced hip ROM in the sagittal , coronal , and axial planes  compared to the LSS group. On the other hand, the sagittal ROM of the knee was significantly larger in patients with LSS (55.91 ± 5.14) than in DLS patients (46.91 ± 9.35). Pelvic anteversion was also significantly increased in patients with DLS (6.10 ± 2.44) compared to LSS patients (3 ± 1.82). No differences in ROM were found for the lumbar spine, knees, or ankles, except for a few indicators .Preoperative gait ability was significantly worse in patients with DLS compared to the LSS group. During gait, both groups demonstrated similar stride length , total stance phase , total swing phase , and double stance phase . However, there was a significant difference in cadence between the DLS (87.05 ± 2.61) and LSS (102.95 ± 13.98) groups. Additionally, the velocity of the DLS group were significantly lower than those of the LSS group, indicating the impact of spinal deformity on gait.We observed different gait parameters in the comparison of bilateral lower extremities between the two groups. A similar gait variability was found in the LSS (3.45%) group, but a higher gait variability was observed in the DLS group (9.53%). Furthermore, for gait symmetry, there was little difference in bilateral lower extremities (4%) in the LSS group compared to the DLS group (8%) .Lasso regression is a biased estimation regression used to solve covariance problems: parameter λ = 0.364 , the varThe figure visualizes the process of selecting the optimal λ value using cross-validation. The vertical axis represents the model mean squared error, while the horizontal axis represents the logarithmic values of λ. The plot provides a visual representation of the relationship between λ and the model’s performance, aiding in the determination of the most suitable regularization parameter for the analysis.The figure illustrates the changes in model coefficients as the logarithmic values of λ vary.p = 0.015)The Cobb angle was correlated with velocity The CSVA was correlated with velocity The T1PA was correlated with velocity The PT was correlated with velocity The sagittal ROM of the hip as correlated with velocity Velocity = y = 1.138 − 0.005 × Cobb − 0.005 × T1PA − 0.007 × PT , the mediation analysis revealed that waist coronal ROM was fully media)The SS was correlated with cadences The sagittal ROM of the hip was correlated with cadence The coronal ROM of the waist was correlated with cadence Cadence = 145.88 + 0.48 × SS − 1.251 × sagittal ROM of the hip − 3.041 × The coronal ROM of the waist ; the mediation analysis revealed that waist coronal ROM fully mediated the relationship between the Cobb angle and cadence.The Cobb angle was correlated with waist coronal ROM The PT was correlated with stride length The LL was correlated with stride length The sagittal ROM of the hip was correlated with stride length The axial ROM of the hip was correlated with stride length stride length = 0.569 − 0.017 × PT + 0.016 × sagittal ROM of the hip + 0.01 × axial ROM of the hip The LL was correlated with total stance phase The PI-LL was correlated with total stance phase The sagittal ROM of the hip was correlated with total stance phase  during walking.The Cobb angle was correlated with gait symmetry .The T1PA was correlated with gait variability  analysis was performed to identify the predictive value of the Cobb angle for gait abnormalities, specifically for walking speeds lower than 0.8 m/s, in DLS patients .The area under the curve (AUC) for the Cobb angle was 0.751, suggesting a good ability to discriminate between patients with and without gait abnormalities. Importantly, the optimal cut-off value for the Cobb angle was identified as 16.1. This cut-off value demonstrated a sensitivity of 0.692 and a specificity of 0.800, indicating its robustness as a diagnostic criterion. The cut-off value of 16.1 for the Cobb angle serves as a critical threshold for clinicians. Patients with a Cobb angle greater than this value are at a higher risk for gait abnormalities and may require more aggressive intervention strategies. This cut-off can be instrumental in early identification and targeted treatment, thereby potentially improving the quality of life for DLS patients .Degenerative lumbar scoliosis (DLS) is a highly prevalent disorder in middle-aged and elderly populations , and itsGait-motion analysis can provide clinicians with a wealth of data and can help in identifying functional deficits in DLS patients . While sPrevious study shows sagittal spine alignment parameters, rather than coronal Cobb angle, have a greater impact on disability and quality of life, as confirmed by several studies ,22.The present study’s findings on sagittal imbalance are supported by previous studies, which add credibility to the next step gait study. The study found that patients with degenerative lumbar scoliosis (DLS) had only half the lumbar lordosis (LL) of the control group. This reduction in LL is a common finding in patients with adult spinal deformity (ASD), which results in forward trunk movement, leading to an increased sagittal vertical axis (CSVA) ,24. The The most clinically relevant and strongly correlated radiological parameters with HRQOL were PT, CSVA, and PI-LL, as supported by previous studies on adult spinal deformities . In thisWhen it comes to dynamic function, the results of the study show that the preoperative gait ability of the DLS group was worse compared to the LSS group.p = 0.015; CSVA r = −0.522, p = 0.046; T1PA r = −0.636, p = 0.011), which showed that both coronal and sagittal positions have an effect on velocity. indicating that coronal and sagittal positions together have an effect on velocity. Sagittal imbalance, as studied by Semaan et al. on ASD, has been found to be negatively correlated with velocity (CSVA PT), further supporting the association between gait velocity and sagittal imbalance [The slower gait velocity (0.65 ± 0.15 m/s vs. 0.77 ± 0.23 m/s) observed in the DLS group compared to the LSS group indicates the impact of spinal deformity on gait, as suggested by the results of the Lasso regression analysis   [While Miura et al. showed that gait balance in the spine with sagittal imbalance deteriorated significantly with trunk advancement, compared with coronal changes, that did not affect spine balance and gait ; howeverubjects) . The gluubjects) . The quaubjects) . This map = 0.049). The compensatory changes caused by the pelvis rotating posteriorly around the femoral head can result in the sacrum tilting posteriorly, which in turn can affect gait cadence. The SVA, which is a measure of sagittal balance, has also been associated with a decrease in gait cadence [Gait cadence, which refers to the number of steps taken per minute, is an important aspect of gait that has been found to be strongly correlated with gait velocity . Despite cadence . DLS patGait variability and asymmetry were significantly higher in the DLS group than in the LSS group, This also explains to some extent the poorer gait of DLS, because a large variability or asymmetry means extra muscles do work .p = 0.037), Similar studies have confirmed its relationship with sagittal imbalance, in a study by Mickle et al., a higher SVA was found to be associated with increased stride time variability in older adults [Gait variability refers to the fluctuation of gait parameters between consecutive steps during walking. We found the T1PA was correlated with gait variability . Although no other studies have been conducted on gait symmetry in patients with DLS, previous research on patients with adolescent idiopathic scoliosis (AIS) provides some insights. A study by Yang et al. found that the scoliosis group had an asymmetrical gait compared to the control group [On the other hand, gait symmetry, which is the consistency of step length and duration between the left and right sides of the body during walking was found to be correlated with the Cobb angle in the study  patients have a shorter total stance phase and lower gait velocities compared to healthy controls .Similarly, older adults tend to have slower gait velocities and longer total stance phases than younger adults due to age-related changes in muscle strength and joint mobility . Howeverp = 0.045) and double stance phase . This finding is consistent with previous research that has demonstrated a relationship between sagittal imbalance and gait parameters [Nonetheless, we identified a significant correlation between the pelvic incidence minus sacral slope (PI-SS) and the total stance phase , suggesting that the alteration of hip sagittal ROM may be related to compensatory mechanisms to maintain balance and compensate for spinal deformities such as DLS.Several gait parameters are associated with sagittal hip ROM. Lumbar spinal stenosis (LSS) and spinal deformities such as adolescent idiopathic scoliosis (AIS) can significantly affect hip sagittal ROM, leading to impaired gait patterns and increased risk of falls. A study by Hasegawa et al. found that LSS patients had significantly lower hip flexion and extension ROM compared to healthy controls, and this was associated with lower walking speed and stride length . Similarp = 0.015), indicating that altered hip sagittal ROM may be related to compensatory mechanisms to maintain balance and compensate for scoliosis [Moreover, Monticone et al. found that AIS patients had reduced hip flexion and extension ROM compared to healthy controls, suggesting that scoliosis can also affect hip sagittal ROM . Our stucoliosis . Kawkabacoliosis .However, our study has some limitations that should be considered for future research. An increased number of groups, including different severities of DLS and other spinal deformities, could be included to better understand the impact of spinal parameters on gait characteristics. And we did not directly measure quality of life, particularly concerning gait.Additionally, electromyographic (EMG) studies may be useful to explore muscle activation patterns and their contributions to gait alterations in patients with DLS and scoliosis. By addressing these limitations in future studies, we can better comprehend the complex relationship between spinal parameters, ROM, and gait characteristics, ultimately improving treatment and management strategies for patients with spinal deformities.This study illuminates significant differences in gait characteristics and hip mobility between patients with degenerative lumbar scoliosis (DLS) and those with lumbar spinal stenosis (LSS), thereby highlighting the unique challenges associated with managing DLS. Importantly, our findings pave the way for personalized treatment strategies. We identified a Cobb angle cut-off of 16.1 as a critical threshold for predicting gait abnormalities, particularly for walking speeds below 0.8 m/s. Patients exceeding this threshold may require targeted interventions, including focused attention on lower limb conditions, to improve their quality of life.Furthermore, our study elucidates the specific sagittal and coronal spinal parameters, such as Cobb angle, T1PA, CSVA, PI, PT, LL, and PI-SS, that significantly influence gait in DLS patients. These correlations not only offer insights into the underlying biomechanical and neurological mechanisms affecting gait but also provide clinicians with actionable data for surgical planningFurthermore, our study elucidates the specific sagittal and coronal spinal parameters, such as Cobb angle, T1PA, CSVA, PI, PT, LL, and PI-SS, that significantly influence gait in DLS patients. These correlations not only offer insights into the underlying biomechanical and neurological mechanisms affecting gait but also provide clinicians with actionable data for surgical planning.These findings underscore the need for comprehensive assessments and personalized interventions in managing spinal disorders. Future research should expand the study population and include electromyographic assessments to deepen our understanding of the underlying mechanisms, thereby facilitating more effective treatment strategies.


The aim of this study is to identifying post treatment recurrence rates in pneumothorax patients under 35 and without any comorbidities according to the treatment types, gender, and age categories based on nationwide population data. Clinical information of pneumothorax patients was extracted from the Korean National Health Insurance Service (NHIS) database between January 2002 and December 2020. Enrolled patients were categorized into two groups; (1) Group I, those who underwent conservative management including pain relief, oxygen therapy, and closed thoracostomy, and (2) Group II, surgical intervention. Recurrence rates were compared according to age, gender, and type of treatment. Surgical intervention was performed in 25.6% patients as first treatment. The overall recurrence rate was 20.3%. Male patients showed a higher 5-year recurrence rate than female . Those with conservative management showed lower 5-year recurrence rates than those with surgical treatment . The 5-year recurrence rates of patients aged 14≤, and < 20 was higher than other age groups . Surgical intervention, male gender and aged under 20 showed association with higher recurrence rates. The term ‘primary’ can be added when no underlying pulmonary pathologies are found, which is common in the first peak population with reported annual incidence of 7.4 men and 1.2 women per 100,000 individuals4.Spontaneous pneumothorax is defined as air collection in pleural space without any traumatic forces. Bimodal curve was noted in age distributions, one was between 15 to 34, and the other was aged over 55–658, no consideration relating with age differences have been reflect. Current treatment process for young SP patients otherwise healthy tended to be largely relied on clinician’s customization10. Methodologies for estimating severity of pneumothorax such as calculating the amount of collected air in the thorax or the size of bullae on the pleural surface were mostly derived from imaging studies of adults11. Therapeutic algorithms suggest non-surgical management including observation, oxygen inhalation, needle aspiration, and drainage using a small- or large-bore catheter for first episodes8, however, surgical intervention would be justified considering high recurrence rates reported up to 50%5.Although there are well established treatment guidelines8, initial surgical intervention for correcting the pathologic status directly may be attractive. This would remove the potential risk factors of air leakage or recurrence, such as bullae or blebs, thereby significant reduction in recurrence rates and hospital stays12.Because the main purposes of PSP treatment are re-expansion of the collapsed lung and recurrence prevention15. Age under 18 was often referred as one of risk factors for recurrence after surgery. Other studies recommended Video-assisted thoracoscopic surgery (VATS) to be first-line therapies for children10, occasionally confined to cases that predisposing factors exist9.Nevertheless, surgical outcomes in young PSP patients seemed to be controversial. Several researches proposed that the prevalence of recurrence tends to increase in younger patients despite surgical treatment16; but this hypothesis has have never been proven8. Moreover, the ELCs are not always detectable17. A unique pleural fibrosis pattern with fibroblastic foci at the margin of pleura and parenchyma in young pneumothorax patients was noted18. There is a possibility of diffuse air passage through invisible pores lining a weak area of visceral pleura19.The ambiguity in PSP treatment could be due to the poor understanding of precise pathogenesis. The visible emphysema-like changes (ELCs), called bullae or blebs, have been considered to be causes of intrapleural air collection through a rupture site20. Delayed puberty was frequently observed in young PSP patients to which the risk of recurrence after the surgical intervention can be attributed21. Because puberty is associated with early acceleration and late deceleration of growth velocity22, surgery can induce the vulnerable environment for recurrence by modifying the premature lung parenchyma. recent studies have suggested the possibility of changing paradigms to minimize treatment invasiveness24The incomplete growth process can be one reason for the increase in recurrence rate. The rapid longitudinal growth during puberty was reported to induce inflating pressure and, therefore, to reduce complianceBased on nationwide population data, we aimed to estimate the recurrence rate after surgery in young PSP patients according to age distribution; children, adolescence, or young adult, and gender Comparison with recurrence tendency of patients who did not undergo surgery was also performed. The aim of this study is to understand trends of recurrence pattern in young PSP patients who were in difference developing periods according to the gender and treatment types.https://nhiss.nhis.or.kr/bd/ab/bdaba032eng.do). Health insurance claim data from this system is stored and managed by classifying a single claim statement into several tables such as general statement details, disease details, treatment details, and medical institution prescription details, and it includes the statement identification number information as a common variable, enabling linkage between tables. The general statement detail table includes basic information of the patient , disease (codes of major diagnosis/first auxiliary diagnosis codes), treatment information, and information on medical expenses.For a nationwide population-based study, we extracted data from the Korean National Health Insurance Service (NHIS) cooperation system between January 2002 and December 2020 (customized data number: NHIS-2021-1-808). The customized data of this study was extracted and provided according to the workflow of the National Health Insurance System Sharing Service . The therapeutic records were detected using specific treatment relating diagnosed diseases.Patient registry who were given ICD-10 codes relating with spontaneous pneumothorax Korean NHIS between January 2002 and December 2020. Associating ICD codes were J93 (pneumothorax), J93.0 (spontaneous tension pneumothorax), J93.1 (other spontaneous pneumothorax), J93.8 (other pneumothorax) and J93.9 (pneumothorax unspecified). Pneumothorax relating with trauma (S27.x) was not involved.3. Primary spontaneous pneumothorax generally affects young healthy population, therefore we enrolled first peak of age-distribution in this study. Patients who were (1) aged ≥ 35 when pneumothorax was first diagnosed, or assigned with other codes associated with pulmonary diseases before the first diagnosis of pneumothorax or simultaneously, such as (2) with congenital pulmonary disease, (3) diagnosed as having pneumonia, (4) tuberculosis, and (5) with cancers were not included for excluding any secondary causes. Relating ICD codes  were used  of the Korea University Anam Hospital (IRB number: 2021AN0334) and was performed in a manner consistent with the institution’s ethical policies and standards.Healthcare Insurance Claim Data contains unique identifiers, enabling patient differentiation and facilitating the creation of patient follow-up information. Recurrence of pneumothorax was defined when the diagnostic codes  were assigned after the first treatment until the data extraction time (2022.05) due to: (1) readmission with pneumothorax diagnostic codes or (2) more than three outpatient visits for pneumothorax within two months in any time of follow-up periods.We identified therapeutic options, including simple observation, pain relief, oxygen inhalation, closed thoracostomy, and surgical intervention. Surgical intervention included video-assisted thoracoscopic surgery, usually wedge resection for bullae. Treatment modalities other than surgery were classified as conservative management. Since Health insurance claim data assign specific codes to each treatment action and prescription for payment, data were extracted using those. For identifying treatment, codes for oxygen therapy (C1000021), closed thoracostomy (T006405), Pleurodesis , and surgery (T006217 and T006219). Patients who were assigned by codes for surgery were classified as Group II, and others as Group I.25, and age 20 is the border between adolescence and adulthood in the WHO classification26. Patient distribution according to age categories is described in Table Patients were divided into three age categories: (1) age under 14 years, 2) age ≥ 14 years and < 20 years, and (3) ≥ 20 years. Age 14 is considered to be the age of the last growth spurt in Korean children age ≥ 14www.r-project.org).The cumulative incidences of recurrence were calculated using the competing risk method regarding any death event as a competing risk. Gray's test estimated the differences in cumulative incidences between groups. The comparison among groups was performed using the chi-squared test for categorical variables and the student t-test for continuous variables. All statistical analyses were performed using R 3.0.2 software (www.nhis.or.kr), the study received a waiver of Ethical approval from Yonsei University Health System, Severance Hospital, Institutional Review Board . Moreover, the informed consent was waived as the data is provided by NHIS. The waiver was approved based as Enforcement Rules Of The Bioethics and Safety Act, Republic of Korea .This study was approved by the Institutional Review Board (IRB) of the Korea University Anam Hospital (IRB number: 2021AN0334). The necessity for acquiring written informed consent from the patients included in this study was waived because this was retrospective study and any individual information was not identifiable in the text. The study used de-identified secondary health insurance claim data collected and distributed by NHIS under the government Ministry of Health and Welfare. As the data is available upon request to NHIS , diagnosed as having pneumonia or tuberculosis before the attack of pneumothorax , and with cancers (N = 2190) were excluded . A flowchart for patient categorization is shown in Fig. A total of 170,846 patients were enrolled in this study. Men comprised 146,480 (85.7%) and women, 24,366 (14.3%). There were significantly more men in Group II than Group I , and the 5-year recurrence rates of Group I patients were significantly lower than those of Group II patients . According to the age categories, patients aged ≤ 14 years, and > 20 years showed significantly higher recurrence rates compared with the other age groups  .The cumulative recurrence rates were compared between Group I and Group II Fig.  accordinP < 0.001). Adjusted hazard ratio (HR) of recurrence according to the age categories relating with gender and treatment groups were described in Table Table P = 0.097), and Group II ages ≥ 20 . Adjusted hazard ratio (HR) of recurrence according to treatment group and gender were described in Table Comparison of cumulative recurrence rates between male and female was performed according to the age categories and treatment groups , interstitial lung diseases or other pulmonary abnormalities therefore treatment process including prognosis could be differed; Preference of conservative treatment rather than surgery, and longer hospital stay as well as chest tube indwelling periods27. The goal of treatment in young spontaneous pneumothorax is to evacuate of air and if necessary, to prevent recurrence8. Surgery could be considered as first line treatment28.The incidence trends of spontaneous pneumothorax showed bimodal age-distribution, first peak at age between 15 to 34, and the other age beyond 60 s. Primary spontaneous pneumothorax usually involves young healthy population without pre-existing pulmonary diseases20. Surgical interventions are often recommended for prevention12, especially in cases in which bullae or blebs have been demonstrated on chest CT29. As a potential prevention, surgery may differ from the other therapeutic modalities that include observation, oxygen therapy, air aspiration, and catheter or chest tube insertion. These other modalities we describe as conservative management.A substantial proportion of PSP patients suffer from recurrence. In the relevant literature, there are reports of up to 50% recurrence after first events, mainly appearing within two to 24 months30. Previous studies have reported that surgery could be a risk factor for recurrence, not improving prognosis31. The recurrence rates in the young population who underwent surgery tended to increase14. In our study, with a few exceptions, PSP patients with surgical intervention experienced increased risk over those who had not been treated surgically. Only male patients aged ≥ 20 years showed no statistical significance between conservative and surgical management.However, the protective effect of surgery has remained a controversial issueNotably, patients under 14 had a large discrepancy in recurrence rates between conventional and surgical treatment groups, regardless of gender. The overall 5-year recurrence rates were significantly higher in the ≥ 14 years, and < 20 years categories, and those of Group I showed similar trends. In Group II patients, those under 14 showed up to a 50% recurrence rate, nearly double that of other age categories.32. The maximal growth velocity has tended to be faster25; therefore, 14 would be a better cutoff for Korean children to distinguish ages in volumetric growth from maturation periods. Age under 20 could suggest late physical maturation and end of adolescence. We assumed that patients under the growth spurt age were likely to be more susceptible than in maturation periods.A recent Korean study showed that the plateau of the pubertal growth spurt was at 14 years in girls and at 16 in boys20. Lower BMI was often observed in children prior to the pubertal growth spurt; children experienced increases in height earlier than weight gain. Delayed puberty was related to a postponed growth spurt because the acceleration of skeletal growth velocity was observed in the early stage of puberty22. In male patients aged ≤ 20, the recurrence rate was not significantly different. This finding was consistent with our hypothesis.Several investigations have suggested that younger age, lower BMI, and delayed puberty could be risk factors for recurrence, and these findings are consistent with the results of this study33. The exclusion of combined lung disease patients could have influenced our results.Our study revealed that the recurrence rates of female patients were significantly lower than those of males in all age categories . This does not guarantee that that surgery is a negative prognostic factor for recurrence because of significant possibility in data skewedness, however, increasing interest in less invasive treatment strategies for primary spontaneous pneumothoraxHigher recurrence rates after 1st episodes of PSP were significantly associated with adolescence, male and surgery. Patients of children and preadolescence in surgery group showed over 50% of recurrence rates. Adolescent patient showed higher recurrence both in conservative and surgical groups, however, the latter was significantly higher than the former. Incompleteness of physical growth could be related with higher recurrence of pneumothorax. The differences in recurrence rates in surgical group seemed to be decrease when the patients are in adult periods. Enrolled data from NHIS did not contain precious individual differences such as BMI, the size of detected bullae or necessity for surgical intervention, results should be considered carefully.Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.Supplementary Information 4.


One way to conserve biodiversity is to cryobank good quality semen. Unsurprisingly, analysing multiple parameters improves semen quality and fertility evaluation. Semen membrane integrity, which indicates sperm cell viability and acrosomal status that reflects sperm-oocyte fusion capacity, are some of the basic parameters evaluated, among many others. Specific fluorescent markers (fluorochromes) that emit mostly green and red light routinely assess these parameters. Color overlapping limits simultaneous evaluation of multiple parameters. Small volume ejaculates, which naturally occur in roosters, small dogs and honeybees, will preclude repetition of analysis, thus interfering with fertility estimation. The use of fluorescent semen quality markers emitting in different light channels is the most appropriate for multiple parameter analysis. The purpose of this experiment is to establish Calcein Violet, a blue fluorochrome, as a marker of viability and acrosomal status in domestic animals in order to free the red and green channels for concomitant analysis of other parameters. Our serial analyses validate Calcein Violet for evaluation of membrane integrity and acrosomal status. Its use in multiple parameters analysis should therefore be recommended.Apis mellifera). The purpose of this experiment is to establish Calcein Violet (CaV), a blue fluorochrome, as a marker of viability and acrosomal integrity in domestic animals in order to free the red and green channels. SYBR®14/Propidium Iodide (PI) was used as reference dye, heat-treated samples as negative controls, serial staining combination for validation and epifluorescence microscopy for observation. Dead spermatozoa marked in red with PI showed no blue fluorescence either from the head or the tail. Live spermatozoa showed a decreasing blue emission from head to tail when single stained with CaV. Unreacted acrosomes showed intense blue fluorescence irrespective of plasma membrane integrity. This needs to be further confirmed for species with small and difficult to observe heads. Establishment of CaV as a marker of membrane integrity by fluorescence microscopy is a decisive first step towards further technical development and use with flow cytometry.Many fluorochromes routinely used in semen quality analysis emit in the green and red channels, limiting their possible combination for multiple parameter analysis. The use of fluorophores emitting in different light channels broadens the possibilities of combination to expand the range of simultaneously evaluated criteria. This is of great interest in cases of small ejaculated volumes, such as those naturally occurring in roosters, small dog breeds and drones ( Fertility prognosis is of great interest for breeding decision and semen cryobanking in general, but most particularly in the context of biodiversity preservation. In vitro quality parameters of fresh and post-thaw semen of mammals are commonly used to predict fertility rates in veterinary medicine. Semen concentration, motility, morphology, viability, acrosomal status, peroxidation of membrane lipids and DNA fragmentation are among the evaluated criteria. Assessment of semen concentration, motility and viability is routinely performed either manually or with computer-assisted sperm analysis (CASA) systems. Additional parameters, such as acrosomal status, DNA fragmentation and lipid peroxidation, are further evaluated in cases of clinical infertility, in artificial insemination centers  and in rSemen evaluation must be run on multiple parameters to develop reliable predictive models of fertility ,6. An ob®14 (SYBR) and propidium iodide (PI) [Spermatozoa viability, a core parameter, can be grossly assessed by their motility, which, in turn can be used as a predictive marker of fertility in domestic animals ,10,11,12ide (PI) ,14,15.Calcein violet (CaV) is another fluorochrome that has been suggested as a potential candidate for semen evaluation , and hasAcrosomal integrity evaluation with peanut agglutinin (PNA) conjugated to a fluorescent marker fluorescein isothiocyanate (FITC-PNA) or phycoerythrin (PE-PNA) is validated for domestic mammals ,25,26,27The fact that CaV emits in shorter wavelength and is detected in the common blue channel used in microscopy or flow cytometry should be seen as an opportunity for multiple colour staining and avoid spectral overlapping. Thus, validating its use for the evaluation of semen viability would prove highly valuable as an alternative to commonly used dyes. The aim of this paper is to investigate, characterize and validate the use of CaV for viability assessment of semen and acrosome membrane integrity from different species of domestic animals  using fluorescence microscopy, as it is the recommended method to validate new stains . To the For bull, horse, dog and boar, five individual semen samples were used: two for Experiment 1 and three for Experiment 2. Bull and stallion semen samples were obtained from frozen thawed straws. Straws were plunged in water at 37 °C for 30 s for thawing. Donors of the other species were all hosted at the Veterinary Faculty of the University of Liège . Drone semen was collected from 100 individuals one month prior to analysis and kept in the dark after pooling in a sealed straw at 18 °C as commonly described [®, Germany) for all species with a solution of 10 µL of semen diluted in 390 µL of formaldehyde 2%. Heads of spermatozoa were counted in 10 squares of 0.2 mm under optical microscope at ×400. The concentration of the undiluted semen sample was calculated following the formula: (number of heads/0.04) × 40 × 1000 = concentration in millions of spermatozoa/mL.The same operator evaluated all samples. Semen concentration was evaluated using a haemocytometer  [®  was used [To facilitate individual observation of spermatozoa under epifluorescence microscopy, raw semen was diluted to a final concentration of 1–10 × 10tozoa/mL . Semen e France) . For dog France)  and catt France) , homemadwas used . For roowas used  was usedwas used  and dronwas used  were preEach diluted semen sample was divided into two aliquots. One was not heat-treated and was kept at 37 °C for horses  and bullThis part of the experiment was run on species whose large sperm heads ,43,44,456/mL), were either triply stained (CaV + SYBR+ PI), double stained (CaV + PI) or single stained (CaV). Three controls were not heat-treated and were either single stained (SYBR or PI) or left unstained, as a negative control allowing for autofluorescence assessment.For all species, two samples (heat-treated or not) of diluted semen  was added to 50 µL of diluted semen and incubated in the dark at room temperature for 10 min. A volume of 4 µL of a 93 µM CaV in PBS solution  was added to 50 µL of diluted semen and incubated in the dark at room temperature for 10 min. Finally, 1.5 µL of 2.4 mM propidium iodide solution in water  were added to 50 µL of diluted semen and incubated in the dark at room temperature for 7 min. For double staining (CaV + PI), PI was added to the sample following the minimum incubation time of CaV (10 min) and the sample was further incubated for 7 min. For triple staining (CaV + SYBR + PI), CaV and SYBR were added at the same time while PI was added 10 min later and the sample was further incubated for 7 min.Working solutions of the three fluorochromes were prepared as recommended by their manufacturer. A volume of 1.5 µL of a 20 µM SYBR®14 and propidium iodide (ThermoFisher) and the excitation and emission filters of this setting are illustrated in Fluorescent signals were visualised under a fluorescent microscope  at final magnification X 630  in oil  with the Leica original cube filter A . The setting allowed direct observation of the spermatozoa and visualisation of the resultant emitted fluorescence from the 3 fluorochromes without creation of an overlay. The fluorescence spectra of Calcein Violet, SYBRAs detailed in Three ejaculates per species were evaluated. Each sample, after dilution see , was divAs detailed in The images of the spermatozoa were taken with an Observer 7 widefield fluorescent microscope , equipped with a 40 X/1.1 NA oil immersion objective. The samples were illuminated with a 385 nm, 475 nm, 555 nm and brightfield respectively for CaV, SYBR, PE and brightfield. Emission wavelength filters were 430–470 nm, 500–550 nm, 570–640 nm and none, respectively, for CaV, SYBR, PE-PNA and brightfield. Images were acquired using Axiocam 506 monochrome  and Zen Blue Program  automatically created overlay images. Blue fluorescence intensity emitted from the acrosome was measured as a relative value to the background blue auto-fluorescence using Zen Blue program  in triply stained samples. All samples were pooled by species. Intensity was compared between cells with both intact plasma membrane integrity and acrosome membrane (Mb+/Acr+), cells with impaired plasma membrane integrity and intact acrosome membrane (Mb-/Acr+), cells with intact plasm membrane integrity and impaired acrosome membrane (Mb+/Acr-) and, finally, cells with both impaired membrane and acrosome integrity (Mb-/Acr-).®  and statistical significance was established at p < 0.05.Statistical analysis was run on triply stained samples (SYBR/PI/CaV) and dual staining (CaV/PE-PNA) using Graphpad PrismThe associations between Acrosome evaluation by CaV and PE-PNA, between plasma membrane integrity evaluation by CaV and SYBR/PI, and between plasma membrane integrity evaluated by SYBR/PI and acrosome membrane integrity evaluated by CaV, were all tested using an exact Fischer test.Parameters of routine semen evaluation evaluated prior to any staining or heat treatment are summarized in Observation of spermatozoa under brightfield and fluorescence microscopy was possible in all samples, and all species confirmed that fluorescence was emitted from a sperm cell and not from debris. In the negative control groups, where no dye was used, spermatozoa did not show any intrinsic fluorescence. Single stained and unstained samples were used as controls for cross positivity in fluorescence detection and analysed in each specific channel, confirming the absence of cross positivity and autofluorescence. Overall, heat-treated samples, used as dead controls, showed more than 90% of dead spermatozoa. These were marked in red when PI was used but did not show blue fluorescence within the head and the tail when CaV was used alone.p < 0.0001) between the evaluation by CaV and SYBR/PI used as reference dye.In all species, all samples, both heat-treated and not, displayed expected fluorescence of the tail and head when stained for positive (CaV) and/or negative (PI) marking of membrane integrity. In heat-treated samples, spermatozoa stained with PI fluoresced in red, demonstrating the loss of their membrane integrity, whereas those stained with CaV did not show blue fluorescence within the head or the tail, as illustrated in Subjectively, the intensity of the blue coloration of CaV seemed weaker and appeared to fade faster than the green and red ones of SYBR and PI, respectively. Sperm heads from samples with SYBR/CaV appeared green, as the more intense signal of SYBR produced with our settings masked the blue fluorescence of CaV in live spermatozoa, as shown in p < 0.0001 for boar, stallion and bull and p = 0.0003 for dog).As illustrated in p < 0.0001) between membrane integrity (Mb+) evaluated by the reference dye (SYBR/PI) and acrosome membrane integrity (Acr+) evaluated by CaV in species where the acrosome could be evaluated . p < 0.001). Interestingly and surprisingly, a significant difference in fluorescence intensity measured from the acrosome was found between Mb+/Acr+ and Mb-/Acr+ for the bull (p < 0.05) and a tendency for the dog (p = 0.056). No spermatozoa with Mb+/Acr- could be found in dog and in only one stallion sample, and very low numbers could be found in the boar and bull samples. No spermatozoa with Mb-/Acr+ were found in boar samples. In our samples, more than 30% of stallion sperm cells had an impaired plasma membrane with an intact acrosome membrane (Mb-/Acr+).As illustrated in Objective validation of a dye requires individual examination of the marked cells, precluding the use of automated sperm cells counting, such as fluorescence-based CASA or flow cytometry , and was®14, even when used alone. As shown in ®14, as no wavelength above 380 nm should reach the sample, and yet SYBR®14, which is normally excited by wavelengths 400 nm and above, showed fluorescence. This may probably be explained by the low specificity of excitation wavelengths of the SYBR fluorochromes family. Interestingly, the fluorescence spectrum provided by the manufacturer for SYBRTM Green I , another fluorochrome from the same family whose fluorescence is supposed to be similar to that of SYBR®14, shows two supplementary low excitation peaks at 300 nm and 375 nm [®14 displays a yet undocumented similar excitation pattern as SYBRTM Green I, which explains the unexpected excitation of SYBR®14 we observed.Surprisingly, during evaluation of membrane integrity in Experiment 1, we observed emitted fluorescence by SYBRd 375 nm . It may p < 0.0001) confirming the efficacy of CaV to evaluate sperm cell integrity. Although recently mentioned in the literature for bovine semen evaluation [Motility has been shown to correlate with viability in equine semen, and a similar relationship has been reported in bovine andrology ,51,52. Taluation ,54,55, tEsterases have been described to be generally distributed in the cell ,19. InteBecause esterases are present in both the cytoplasm and the acrosome, the intensity of fluorescence of live spermatozoa with unreacted acrosome (Mb+/Acr+) could be expected to be higher than that with reacted acrosome (Mb+/Acr-), thus allowing differentiation between these two subpopulations of live spermatozoa. However, this subpopulation of live spermatozoa with a reacted acrosome (Mb+/Acr-) is of rare natural occurrence , which wMotility is a predictive parameter of fertility in domestic animals ,10,11,12CaV fluoresces in blue, which frees the green channel spectrum or both the green and red light spectrum channels. This broadens the possibilities of combinations between fluorochromes to expand the range of parameters simultaneously evaluated in a multiple parameters analysis of semen. This is obviously particularly beneficial for species or donors with small volume of ejaculate, such as drones, and for wildlife animals where semen collection is challenging and usually requires general anaesthesia.Although its use had previously been suggested for that purpose, this is the first report that thoroughly tests and validates CaV as a marker of sperm membrane integrity and thus viability. Our results also demonstrate for the first time that CaV can be used to assess acrosomal integrity in horses, bulls, dogs, boars. Establishing CaV as a marker of membrane integrity through fluorescence microscopy is a milestone that leads the way to its further study and application with flow cytometry.