Lumbar cistern blockage is a common complication of continuous lumbar cistern drainage. This paper analyzes the risk factors for lumbar cistern blockage drainage due to various causes and proposes a series of prevention and intervention measures to reduce blockage or improve recanalization after blockage.The clinical data of 637 patients with various lesions who underwent lumbar cistern drainage in our hospital were retrospectively collected and analyzed. Perioperative clinical and imaging data were assessed. Variates were analyzed using univariate and multivariate logistic regression analyses.A total of 13.7% (87/637) of patients had lumbar cistern blockage. Multivariate analysis revealed that drainage time (≥7 days), CSF volume <200 (mL/d), CSF leakage, and abnormal CSF properties were predictors of lumbar cistern blockage. Reducing the probability of lumbar cistern blockage can be achieved by repeatedly flushing, increasing the drainage flow and shortening the drainage time. The recanalization rate after blockage was 67.8% (59/87). After the drainage tube was removed, no complications related to the drainage tube occurred during the 1-week follow-up.Lumbar cistern blockage is the main reason for poor drainage. Prevention or early intervention can effectively reduce the probability of blockage and achieve the purpose of drainage of cerebrospinal fluid. Lumbar cistern drainage (LCD) is used to treat intracranial infection, vasospasm after hemorrhage, intractable intracranial hypertension after craniocerebral trauma, cerebrospinal fluid leakage, and other related diseases . It is wThe study confirmed that the drainage volume of cerebrospinal fluid (250–300 ml/d) is safe and effective . InsuffiAlthough lumbar cistern drainage is a common and minimally invasive neurosurgery procedure, there is no systematic study on the risk factors for lumbar cistern blockage, and there are few reports on the methods of recanalization after blockage. This paper retrospectively analyzed 637 patients who required lumbar cisterna drainage after craniocerebral surgery for various reasons in our department, determined the risk factors for lumbar cisterna blockage and introduced the treatment experience of recanalization after blockage to provide guidance and reference values for clinical prediction.A retrospective study was performed on 637 patients who underwent lumbar drainage for various reasons in the Department of Neurosurgery, Tangdu Hospital of the Air Force Medical University  from July 2012 to November 2022. We collected patient data from radiology systems and electronic medical records. The criteria for inclusion were as follows: (1) Fisher grade 3 and 4 subarachnoid hemorrhage; (2) partial ventricular hemorrhage; (3) antimicrobial therapy for central nervous system infections; and (4) adjuvant treatment of cerebrospinal fluid leakage. Exclusion criteria were as follows: (1) severe increase in intracranial pressure; (2) puncture site lumbar deformity or bone destruction, resulting in lumbar puncture or catheterization difficulty; (3) dying individuals with severe systemic infections , shock or on the verge of shock, and unstable vital signs; (4) cerebrospinal fluid circulatory pathway incomplete obstruction; and (5) patients with restless or abnormal mental behavior who cannot cooperate with the diagnosis and treatment. All the study procedures were approved by the ethics committee of Tangdu Hospital and followed the guidelines of the Helsinki Declaration.via telephone interviews. Clinical data, such as age, sex, BMI primary diagnosis, causes of lumbar drainage, and complications of lumbar drainage, were retrieved. Drainage time was classified as <7 days and ≥7 days. CSF volume was divided into ≥200 ml/d and <200 ml/d. CSF properties were divided into normal and abnormal. Normal CSF was defined as CSF protein levels between 120–800 mg/L, colorless, and WBC of 0–50 × 106/L. Abnormal CSF was defined as CSF protein ≥800 mg/L, turbid/bloody/yellow, and WBC >50 × 106/L. The patient’s position after lumbar drainage is mainly prone or supine. Drainage tube choice manufacturer was either Medtronic or Branden.All patient data were collected from the hospital electronic medical records. Follow-up data were obtained p < 0.05. To detect risk factors associated with the incidence of lumbar drainage blockage, a univariate regression analysis was used. The risk factors with p < 0.05 in univariate logistic regression analysis were selected for further multiple regression analysis. The odds ratio (OR) and 95% confidence interval (CI) were calculated. All statistical analyses were performed using R software version 4.0 1.The categorical variables were expressed in numbers (percentages), and differences were evaluated using chi square tests or Fisher’s exact tests, the statistical significance level was set at This study analyzed the medical records of 637 patients who underwent lumbar drainage at our institution. Among the 87 drainage tube blockage cases, drainage was achieved in 59 cases after recanalization by reducing the drainage height, adjusting the body position, repeatedly flushing with normal saline, achieving suction under negative pressure and pulling out part of the drainage tube, and the recanalization rate was 67.8% (59/87). Thirteen patients underwent lumbar cistern drainage again. Sixty-seven cases of cerebrospinal fluid leakage were combined with drainage tube blockage, which was pressurized and sutured through local wounds. After the drainage tube was opened, 54 cases had no cerebrospinal fluid leakage. The drainage tube was removed, and the wound was sutured in 13 cases. Excessive drainage (13 cases) is not the total amount of cerebrospinal fluid drained daily, but there is no guarantee of a continuous average drainage volume of cerebrospinal fluid per hour (average drainage speed <10–15 ml/h) . In someLumbar drainage blockage was the most important complication of lumbar drainage , and possible risk factors for lumbar drainage blockage were analyzed.P = 0.015, OR 1.79, 95% CI 1.14–2.85). CSF volume <200 mL/d was significantly more often associated with lumbar drainage blockage than CSF volume ≥200 mL/d . Patients undergoing CSF leakage had lumbar drainage blockage significantly more often than those without CSF leakage . Patients with abnormal CSF properties had lumbar drainage blockage significantly more often than those with normal CSF properties   .p = 0.008, OR 1.92, 95% CI 1.18–3.11), CSF volume <200 mL/d , CSF leakage , and abnormal CSF properties  were identified as independent and significant predictors of lumbar drainage blockage , adjust the body position , pull out part of the drainage tube in the spinal canal (the side hole of the drainage tube is adsorbed to the arachnoid), and the drainage tube may recanalize.Lumbar cisterna drainage is a routine operation in neurosurgery. At the same time, it is also a double-edged sword that can be used to shorten the recovery time of patients. If used improperly, it will cause many adverse consequences. Therefore, after catheterization of the lumbar cisterna, the daily drainage volume (200–300/ml/day) and retention time (<7 days) should be maintained in strict accordance with the guidelines or existing research conclusions. This should be performed with a relatively sterile operation and careful disposal to prevent lumbar cisterna blockage and ensure continuous drainage and achieve the purpose of disease treatment.The 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 Tangdu Hospital Ethics Committee. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.MZ: conceptualization and writing—original draft. QT: methodology. XW, LL, and XD: writing—original draft. YQ: supervision. QC: writing—review and editing. All authors contributed to the article and approved the submitted version.


Ascending testis or acquired undescended testis develops in approximately 30% of cases of retractile testis, and orchiopexy is recommended for these cases. This study aimed at assessing the intraoperative anatomical findings of ascending testis and acquired undescended testis in search of better management for retractile testis.  We retrospectively collected data of patients with confirmed diagnosis of retractile testis between February 2012 and November 2021. Orchiopexy was performed for cases with ascending testis and for patients with increasing difference of right and left testicular volume. The site of gubernaculum attachment and patent processus vaginalis were evaluated during surgery.  A total of 119 testes in 71 patients with retractile testis were included in this study. Sixteen retractile testes in 12 patients (17%) underwent orchiopexy. The weight at birth was significantly higher, and bilateral retractile testes were significantly more common in the follow-up group than in the surgical intervention group. In the surgical intervention group, the abnormal site of gubernaculum attachment was found in 12 out of 16 testes (75%), and patent PV was found in nine out of sixteen testes (56%). Sites of gubernaculum attachment in testes with patent PV were significantly higher than in sites with closed processus vaginalis, and all testes with patent processus vaginalis had abnormal site of gubernaculum attachment.  Patients with ascending testis and acquired undescended testis have clinical features and intraoperative abnormal findings similar to a cryptorchidism. Therefore, our surgical indication for retractile testis is considered appropriate. Retractile testis (RT) is defined as the testis located in the upper scrotum or groin that can descend into the lower scrotum spontaneously or with manipulation and remain there for a period of time . RT is tWe investigated the natural course and outcome of RT, including spontaneous resolution and surgical intervention, at a single institution in Japan. The aim was to assess the anatomical anomalies and validity of surgical indication associated with RT with the intention of elucidating better management for RT.We retrospectively collected data of patients who had confirmed diagnosis of RT at Wakayama Medical University, Japan, between February 2012 and November 2021. All patients underwent physical examination and ultrasonography (US) by experienced urologists. RT was defined as the testis located in the upper scrotum or groin that could manually descend into the lower scrotum without tension and remain there for a period of time. These patients with RT were followed every six months or one year by the same urologist. Surgical indications were AT or increasing difference in testicular volume between right and left during the duration of follow-up. AT was defined as testis located in the upper scrotum or groin that could not descend into the lower scrotum and immediately return to its original position. Acquired UDT was defined as the testis that could descend to the bottom of the scrotum manually with smaller testicular volume compared with the contralateral testis and/or testicular volume decrease during the course of follow-up. The testicular volume was measured by ultrasound. The standard orchidopexy was performed under general anesthesia for all patients eligible for surgical intervention. The operation involved a groin skin incision and a scrotal incision with fixation of the testis in tunica dartos in all cases. There was no case that performed the testicular biopsy. The site of gubernaculum attachment was evaluated by pulling the testis under gentle traction during surgery. Furthermore, the presence of a patent PV was identified by dissection of the aponeurosis of external oblique muscle up to the inner inguinal ring. Resolution of RT was defined as the testis that had descended into the lower scrotum spontaneously and remained in the scrotum, following induction of cremasteric reflex.Patients were divided into two groups based on management as follow-up or surgical intervention. Patients in the follow-up group were further divided into two groups based on the outcome as follows: continuation of follow-up or spontaneous resolution. The clinical data were retrospectively reviewed, including patient characteristics, duration of follow-up, physical findings, and intraoperative findings. The duration of follow-up in the surgical intervention group was regarded as the period from the first diagnosis of RT to surgery. Data of the two groups were compared to assess the difference in the outcome of RT. This study was conducted with the approval from the Wakayama Medical University Institutional Review Board  and in accordance with the Declaration of Helsinki.P values <0.05 were considered significant.Statistical analysis was performed using JMP Pro 14 . The Wilcoxon signed rank test was used to compare continuous variables between the two groups, and the chi-square test and Fisher's exact test were used for categorical variables. P=0.706). Weight at birth in the surgical intervention group was significantly lower than that in the follow-up group. The bilateral RTs of the follow-up group were significantly more common than in the surgical intervention group. The mean (interquartile range) duration of follow-up was 28 (12–46) months in the follow-up group and 25 (6–38) months in the surgical intervention group (P=0.276).A total of 119 testes in 71 patients were diagnosed with RT and included in this study. Included in the follow-up group were 103 retractile testes (RTs) in 59 patients (83%), and surgical intervention was performed in 16 RTs in 12 patients (17%). Patient characteristics of the two groups are shown in In the follow-up group, 81 testes in 45 patients continued to have RT at the most recent examination and continued to be followed up . There wIn the surgical intervention group, eight testes in seven out of twelve patients were diagnosed with AT and eight testes in five patients had an increasing difference between the right and left testicular volume without AT as acquired UDT . The abnSome causes of RT have been reported, including an overactive cremasteric reflex . AlthougAT is defined as a previously descended testis that cannot be manipulated into the scrotum . In addiOne hypothesis suggests that a fibrous remnant of the normally closed PV remains tethered to the testis and peritoneum , 13. TheThis study has several limitations. Patient data were retrospectively collected from a single institution, and the sample size was small. Another limitation was the risk of bias in diagnosis of AT or acquired UDT and differentiating them from cryptorchidism. However, we have confidence in the diagnosis of AT and acquired UDT in this study because patients were examined by two urologists with a strict and consistent definition of RT. In addition, 45 out of 71 patients (63%) in this study still had RT without spontaneous resolution at the most recent visit. Further observation is therefore necessary to more accurately evaluate the distinctive features of RT to identify the predictive factors for spontaneous resolution, AT or acquired UDT. Furthermore, intraoperative findings including absorption into the parietal peritoneum and fibrous remnant of the PV should be recorded in detail to gain a deeper understanding of AT and acquired UDT from RT.In conclusion, although RTs are considered a variant of normal testes, surgical intervention is required in some cases with AT or acquired UDT. Patients with AT and acquired UDT have clinical features similar to a cryptorchidism, including weight at birth and laterality. In addition, intraoperative abnormal findings of AT and acquired UDT, including patent PV and abnormal site of the gubernaculum, seem to share similarity with cryptorchidism. Therefore, surgical indication for RT, including nondescending testis by manual and increasing differences in testicular volume, is considered appropriate in this study. To avoid misdiagnosis, screening failures, and unnecessary surgery, it is important to acquire the follow-up and skills for examinations to investigate AT and acquired UDT with a significant risk of anatomical anomalies. A better understanding of RT obtained by further investigation may lead to the development of better management.


The present study provides OAC is a subarachnoid space independent from the cisterna magna. Because of its distinctive anatomy, as well as theoretical and clinical significance, OAC deserves its own name.The cisterna magna has been defined as the space between the inferior margin of the cerebellar vermis to the level of the foramen magnum, while an enlarged dorsal subarachnoid space at the occipito-cervical junction extending from the foramen magnum to the upper border of the axis (C2) is still ignored. Recently, the myodural bridge complex is proved to drive the cerebral spinal fluid flowing via this region, we therefore introduce the “occipito-atlantal cistern (OAC)” to better describe the subarachnoid space and provide a detailed rationale. The present study utilized several methods, including MRI, gross anatomical dissection, P45 sheet plastination, and three-dimensional visualization. OAC was observed to be an enlarge subarachnoid space, extending from the foramen magnum to the level of the C2. In the median sagittal plane, OAC was a funnel shape and its anteroposterior dimensions were 15.92 ± 4.20 mm at the level of the C0, 4.49 ± 1.25 mm at the level of the posterior arch of the C1, and 2.88 ± 0.77 mm at the level of the arch of the C2, respectively. In the median sagittal plane, the spino-dural angle of the OAC was calculated to be 35.10 ± 6.91°, and the area of OAC was calculated to be 232.28 ± 71.02 mm However, the posterior cervical subarachnoid space from the level of the arch of the atlas (C1) to the foramen magnum has been considered as an aspect of the cisterna magna by some2. It’s unclear whether there is a subarachnoid space located at the occipito-cervical junction (OCJ) distinct from the cisterna magna.The cerebral spinal fluid (CSF) flows out of the fourth ventricle via the median and lateral apertures, and then drains into the posterior cerebellomedullary cistern (PCC). The PCC, also called the cisterna magna, is located between the dorsal surface of the medulla oblongata and the inferior aspect of the cerebellum. The upper horn of the PCC communicates with the fourth ventricle via the median and lateral apertures. The base of the PCC has previously been described as being at the level of the foramen magnum 10. Recently, Xu et al.11 found that the head rotation significantly changed CSF flow rate at the level of atlas. CSF located in the ventricles initially drains into the PCC and then spreads to the subarachnoid space over the brain and the spinal cord via a kinetic mechanism12. Therefore, future researches involving CSF circulation should consider the upper part of the cervical subarachnoid space.The OCJ is one of the most complicated and flexible portions of the human body. The circulation of cerebrospinal fluid within the PCC has been reported to be affected by motions of the head and neck. It also has been proposed that the myodural bridges (MDB) previously described in the suboccipital region can place tension on the upper part of the cervical spinal dura mater “pumping” the CSF flowing during head and neck movements17. These changes to CSF circulation near the OCJ may play an important role in the pathogenesis of Arnold-Chiari type 1 malformation. Currently, the PCC above the FM, and the subarachnoid space of the upper cervical spine below the FM, are considered as separate entities when considering treatments for pathologies of the head and neck18.In clinic, Arnold-Chiari type 1 malformation often results in abnormal CSF pressure and flow rate, which leads to changes in the circulation patterns of the CSF near the OCJ. And syringomyelia occurs when cerebrospinal fluid infiltrates or impacts the spinal cordIn the present study, the subarachnoid space in the upper cervical spine was observed and measured. We propose that the best name for this space is the “occipito-atlantal cistern”, which will hopefully assist anatomical, physiological, pathological research, and clinical studies in the region of the OCJ.This study was approved by the Ethics Committee for Research at the Basic Medical College of Dalian Medical University. All methods were performed in accordance with relevant guidelines and regulations.19.Sixty cases of head and neck MR images were analyzed , which were derived from patients without obvious head and neck lesions. Six adult cadaveric head and neck specimens fixed with 10% formalin in the present study were obtained from the Body and Organs Donation Center of Dalian Medical University. Before the donors involved in the current study passed away, written informed consent was obtained from them in compliance with the ethical committee's rules. Three specimens were utilized for gross anatomical dissection, and the other three were used for P45 sheet plastination studies, which were prepared at the Dalian Hoffen Preservation Technique Institution. In addition, one set of head and neck digital data was reconstructed, which was obtained from the Visible Korean project, an anatomical photographic digital databaseDuring dissection in the suboccipital region, the soft tissue, the posterior wall of the vertebral canal, and the basilar and lateral parts of the occipital bone were separated layer by layer to open the cranial cavity and the vertebral canal, to expose the cranial and spinal dura mater. And then the dura mater was incised longitudinally along the posterior median line to explore the arachnoid mater in the posterior cranial fossa to the third cervical vertebra. After that, a cruciform incision was made on the arachnoid mater to open and observe shape and contents of the subarachnoid space in the region of the OCJ.21. The slice thickness in the present study was 3 mm and the subarachnoid space adjacent to the OCJ was observed in situ.P45 sheet plastination of three head and neck specimens was carried out, to obtain sagittal, coronal, and horizontal sections. The procedures involved in the P45 sheet plastination, including slicing, bleaching, dehydration, forced impregnation, and curing have been described in current literatureAll MRI study underwent in the supine position. The subarachnoid space posterior to the medulla oblongata and the spinal cord were observed in the median sagittal head and neck MR images (Tesla parameter: 0.35 T). Adobe Bridge CC 2018 software was used to measure the horizontal distances of the dorsal subarachnoid space at the level of the FM (D-FM), the posterior arch of the atlas (D-C1), the arch of the axis (D-C2), and the arch of the third cervical vertebra (D-C3) respectively  observed posterior to the lower part of the medulla oblongata and upper part of the cervical portion of the spinal cord was visualized in the 3D images.Dissection of the three of head and neck specimens clearly evidenced the normal structures contained in the posterior cranial fossa and the upper cervical spine Figs. , 3. AfteStructures in the occipito-cervical junction, such as the occipital bone, the cervical vertebrae, the medulla oblongata, the cerebellum, the spinal cord, the dura mater, and the subarachnoid space were clearly observed in the P45 plastinated slices.In the median sections, an enlarged subarachnoid space (ESS) was observed below the FM, dorsal to both the lower part of the medulla oblongata and the upper part of the cervical spinal cord. The roof of the ESS was at the level of the FM and the floor was narrowed down to the level of axis (C2) Fig. . In the In addition, in the coronal head and neck sections through the posterior arch of the atlas (C1), a large and rhombic-shaped cistern was observed between the cerebellum and the atlas. Its upper portion, namely the PCC, was located above the FM, and the lower portion of the cistern was positioned within the vertebral canal and located between the FM and the atlas (C1) Fig. .Figure 6In the median sagittal head and neck sections, the ESS was located along the dorsal side of both the inferior portion of the medulla oblongata and the superior portion of the cervical spinal cord. This space extended downwards and narrowed gradually from the level of the C0 down to the level of the C2 Fig. .Figure 72. These morphological parameters showed no significant differences between males and females, with their frequency distribution being shown in Figs. On the base of MRI measurements of the 60 cases, and the anteroposterior extension parameter in the median horizontal plane of the ESS was measured 15.92 ± 4.20 mm at the level of the FM (D-FM), 4.49 ± 1.25 mm at the level of the atlas (D-C1), 2.88 ± 0.77 mm at the level of the axis (D-C2), and 2.62 ± 0.61 mm at the level of the third cervical vertebra (D-C3). In the median plane, the spino-dural angle (SDA) of the ESS was 35.10 ± 6.91°, and the median sagittal area (MSA) of this space from the FM to the upper edge of the arch of the axis was 232.28 ± 71.02 mmThe structures contained within the posterior cranial fossa and the upper cervical spine were reconstructed from the digital human dataset. The reconstructed structures included the tonsils of the cerebellum, the medulla oblongata, the spinal cord, the nerve roots, the vertebral arteries, and the dural sac Fig. . The durThe present study found that an enlarged subarachnoid space (ESS) located around the OCJ, whose upper aspect was communicated with the cisterna magna through the FM (C0), and whose ventral wall extended along the dorsal sides of the following structures: the C1-2 spinal segments, their spinal nerve roots, and the denticulate ligament. In the median sagittal section, the ESS appeared to be triangular in shape and was observed to be located dorsal to both the medulla oblongata and the upper portion of the cervical spinal cord. Its floor was narrowed down to the level of the axis. Furthermore, morphometric parameters of the ESS are also described in the present study.Based on the results obtained from the dissectional observation and MRI measurements, it was believed that there is a subarachnoid space located at the OCJ distinct from the cisterna magna, and the logical name for this space is the “occipito-atlantal cistern (OAC)”. In a result, the dorsal subarachnoid space surrounding the OCJ should now be considered as consisting of two portions, the cisterna magna and the OAC. The OAC includes a significant amount of the CSF in the OCJ. This new finding has theoretical and clinical significance.12. Some scholars have reported that head rotations changed the flow rate of CSF at the OCJ10. In 2014, Zheng et al. proposed a functional hypothesis that the myodural bridges (MDBs) may provide power for CSF circulation6. In the suboccipital region, the MDBs originate from the rectus capitis posterior minor (RCPmi), the retus capitis posterior major (RCPma), and the obliquus capitis inferior (OCI) muscles, and the nuchal ligament. All of these structures were found to connect to the spinal dura mater after passing through the posterior atlanto-occipital or atlanto-axial interspaces24. Thus, during head and neck movements the posterior wall of the upper cervical portion of the dural sac would be pumped via the MDBs. Recently, researchers proposed that the myodural bridge complex could provide power for the circulation of CSF during head movements24. The OCJ appears to be a critical anatomical and physiological region for CSF circulation. Therefore, it becomes important to consider the CSF both above and below the FM in future studies of CSF circulation. The OAC becomes a pivotal hub for the diffusion of CSF from the cisterna magna to the subarachnoid space of the upper cervical spine over the surface of brain and spinal cord.Considering CSF circulation, the OAC is an important relay center, accepting CSF from the cisterna magna, then releasing it slowly into the subarachnoid space of the vertebral canal17. At present, its main etiology is CSF hydrodynamics and CSF pressure separation in the subarachnoid space of the OCJ16. The reduction of the subarachnoid space at the OCJ is commonly associated with CIM. The abnormal development of the posterior cranial fossa and the downward movement of the tonsils of the cerebellum result in a disruption of cerebrospinal fluid circulation and potentially hydrocephalus. With this condition, the OAC becomes narrowed which leads to an abnormal increase in the velocity of the cerebrospinal fluid flow rate which may represent the pathogenic mechanism of syringomyelia17. Syringomyelia may worsen and develop superiorly to lead to syringobulbia. CIM is diagnosed by MRI findings of at least 5 mm of cerebellar ectopy below the foramen magnum. The position of the tonsils and brain stem, the presence of a syrinx and other associated imaging findings suggestive of symptomatic lesions are assessed25. Additionally, phase-contrast magnetic resonance imaging (PC-MRI) is a useful supplement to morphological imaging for postoperative evaluation and follow-up of CIM patients27. The analysis of CSF flow dynamics focused on the ventral space between the clivus and brain stem at the level of the foramen magnum, the central aqueduct and IV ventricle, and the posterior fossa space behind the cerebellum and tonsils. Our research points out that the OAC is a crucial area connecting the subarachnoid space of brain and spinal cord. The morphological changes of the OAC and the CSF flow dynamics at its upper and lower openings should be noted in future imaging examinations. Thus, we suggest that the newly described OAC is a clinically significant subarachnoid space independent of the cisterna magna. Increased knowledge of the subarachnoid space above the upper rim of C2 provides an important anatomical basis for future clinical studies of nervous system disorders.In clinical practice, Chiari I malformations (CIM) mainly occur at the OCJ, and syringomyelia is a common complication28. There are often a median sheet of arachnoid divides the cistern into sagittal halves, and two additional paramedian septi are formed at the level of C1/2 and extend to the level of T11-12, dividing the dorsal spinal subarachnoid space longitudinally. We propose the expanded space formed by the OAC and PCC at the OCJ may act as a buffer zone for CSF dynamics stability in both cranial cavity and spinal canal, and regulate the flow of CSF through those longitudinal spaces mentioned by Yasargil. Furthermore, the OAC and the terminal cistern are enlarged subarachnoid spaces in the spine at the cephalic and caudal ends, respectively, that create a dumbbell-shaped distribution of CSF. This volume distribution may help to prevent sudden changes in CSF pressure during movement.Previously, the cistern just below the cisterna magna was noticed by Yasargil, and it was described as “cervical subarachnoid space”The study has some limitations, the foremost being the use of limited research materials. This is a small sample study, based solely on a population of northern China and South Korea. We look forward to further research expanding the number of subjects and populations. Additionally, all MRI examinations were conducted in the supine position, leading to a smaller measurement of the OAC due to the effect of gravity. And there may be slight shrinkage of the P45 plastination samples, which potentially could affect the results of the P45 section studies. Moreover, the research methods employed in this study were gross and sectional dissection. Tissue micro-structure within the subarachnoid space could not be observed. Morphological studies of subarachnoid trabeculae in the OAC will be improved in the future, and their potential role in CSF flow will be investigated.At the OCJ, there is an enlarged subarachnoid space dorsal to both the medulla oblongata and the upper portion of the cervical spinal cord, extending from the foramen magnum to the upper border of the axis. This newly described broad subarachnoid space, which is named the “occipito-atlantal cistern”, becomes a hub for the diffusion of CSF from the cisterna magna to the subarachnoid space of the upper cervical spine. And the present study adds a knowledge base to the structural and functional studies of the occipito-cervical junction.


Aggregation of α-synuclein (α-syn) is a prominent feature of Parkinson’s disease (PD) and other synucleinopathies. Currently, α-syn seed amplification assays (SAAs) using cerebrospinal fluid (CSF) represent the most promising diagnostic tools for synucleinopathies. However, CSF itself contains several compounds that can modulate the aggregation of α-syn in a patient-dependent manner, potentially undermining unoptimized α-syn SAAs and preventing seed quantification.In this study, we characterized the inhibitory effect of CSF milieu on detection of α-syn aggregates by means of CSF fractionation, mass spectrometry, immunoassays, transmission electron microscopy, solution nuclear magnetic resonance spectroscopy, a highly accurate and standardized diagnostic SAA, and different in vitro aggregation conditions to evaluate spontaneous aggregation of α-syn.n = 31 SAA-negative control CSF samples spiked with preformed α-syn aggregates.We found the high-molecular weight fraction of CSF  to be highly inhibitory on α-syn aggregation and identified lipoproteins to be the main drivers of this effect. Direct interaction between lipoproteins and monomeric α-syn was not detected by solution nuclear magnetic resonance spectroscopy, on the other hand we observed lipoprotein-α-syn complexes by transmission electron microscopy. These observations are compatible with hypothesizing an interaction between lipoproteins and oligomeric/proto-fibrillary α-syn intermediates. We observed significantly slower amplification of α-syn seeds in PD CSF when lipoproteins were added to the reaction mix of diagnostic SAA. Additionally, we observed a decreased inhibition capacity of CSF on α-syn aggregation after immunodepleting ApoA1 and ApoE. Finally, we observed that CSF ApoA1 and ApoE levels significantly correlated with SAA kinetic parameters in Our results describe a novel interaction between lipoproteins and α-syn aggregates that inhibits the formation of α-syn fibrils and could have relevant implications. Indeed, the donor-specific inhibition of CSF on α-syn aggregation explains the lack of quantitative results from analysis of SAA-derived kinetic parameters to date. Furthermore, our data show that lipoproteins are the main inhibitory components of CSF, suggesting that lipoprotein concentration measurements could be incorporated into data analysis models to eliminate the confounding effects of CSF milieu on α-syn quantification efforts.The online version contains supplementary material available at 10.1186/s13024-023-00613-8. Parkinson’s disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA) are neurodegenerative diseases pathologically characterized by the presence of intracellular α-syn inclusions in vulnerable brain regions and are commonly referred to as synucleinopathies. Seed amplification assays (SAAs), known as protein misfolding cyclic amplification (PMCA)  and realIn this work, we comprehensively characterized the inhibitory effect of CSF on α-syn aggregation: we first identified a high-molecular weight (HMW) fraction of CSF exerting most of the inhibitory effect. Subsequently, we selected putative inhibitors based on their relative abundance in the CSF HMW fraction and specifically analysed their interactions with α-syn, their effect on α-syn aggregation, and possible impact on α-syn SAAs.In the current work we thoroughly characterized the inhibitory effect of human CSF on α-syn aggregation by means of several complementary techniques. The study design of the present work is summarised in Fig. Escherichia coli BL21 (DE3) Gold were transformed with a pT7-7 vector cloned with the gene encoding α-syn. The overnight preculture of transformed cells was diluted 100-fold in LB medium and induced at an OD600 value of 0.6–0.8 with 1 mM isopropyl-β-d-thiogalactoside; after 5 h of incubation at 37 °C, the cells were harvested at 4000 rpm . The extraction was carried out through osmotic shock using 100 mL of the buffer Tris 30 mM, EDTA 2 mM, and sucrose 40%, at pH 7.2, according to Shevchik et al. [k et al.  and Huank et al. .2 and then ultra-centrifuged a second time. Supernatants derived from these two centrifugation steps were combined and dialyzed against 4 L of 20 mM Tris/HCl buffer at pH 8.0. The protein was then loaded in the fast protein liquid chromatography system, and anion-exchange chromatography was carried out with 0–50% linear gradient 1 M NaCl . The collected fractions were lyophilized and resuspended in 10 mM Tris/HCl, 1 mM EDTA, and 8 M urea at pH 8.0 for chemical denaturation. To eliminate all of the protein that formed aggregates, two size-exclusion chromatographies (HiLoad 16/600 Superdex 75 pg Column) were performed with 20 mM phosphate and 0.5 mM EDTA at pH 8.0 as the elution buffer. Purified α-syn was dialyzed against Milli-Q water and lyophilized in batches for long-term storage. The Roche complete protease inhibitor cocktail was added only during the extraction step in the quantity suggested by the producer.The suspension was then ultra-centrifuged at 20 000 rpm  for 25 min, and the pellet was collected and resuspended with 90 mL precooled ultrapure water containing 38 μL of 1 M MgCl15N-labeled wild-type α-syn was expressed in Escherichia coli grown in M9 minimal medium supplemented with 15NH4Cl and purified as started for E. coli in LB medium.15N-labelled and unlabelled α-syn, protein expression and purification were performed as previously described [For escribed . 1H solu600 reached 0.6–0.8, then induced with 1 mM isopropyl β-D-1-thiogalactopyranoside. They were further grown at 37 °C overnight and then harvested by centrifugation at 6500 rpm (JA-10 Beckman Coulter) for 15 min at 4 °C. The pellet was suspended in 20 mM Tris–HCl, pH 8.5 (60 mL per litre of culture) and sonicated at 4 °C for 40 min. The suspension was centrifuged at 40,000 rpm (F15-6 × 100y Thermo Scientific) for 40 min and the pellet discarded. TTR was purified by anionic-exchange chromatography using a HiPrep Q FF 16/10 column . The protein was eluted in 20 mM Tris–HCl buffer at pH 8.6 with a linear 0–1 M NaCl gradient. Fractions containing pure TTR were identified by Coomassie staining SDS-PAGE gels, then joined and purified by Size Exclusion Chromatography using HiLoad Superdex 26/60 75 pg in 50 mM phosphate buffer at pH 7.5.For TTR, Escherichia coli BL21(DE3) RIPL PLysS cells were transformed with pET-28a( +) plasmid encoding TTR gene. The cells were cultured in LB Medium containing 0.1 mg/mL of Kanamycin, grown at 37 °C, until ODThe neurological control (NC) CSF samples used in this work had been previously collected and stored at -80 °C according to international guidelines . NC wereCSF from 19 different NC subjects  were pooled (CSF pool 1) reaching a total volume of 8 mL that was split in 2 aliquots of 3 mL and 10 aliquots of 0.2 mL.n = 10 NC subjects . This second pool was then split in 10 aliquots of 0.5 mL each, which were then used for ApoA1 and ApoE immunodepletion experiments and protein aggregation assays.A second CSF pool of 5 mL was prepared from different CSF samples collected form Two aliquots of 0.5 mL relative to 31 CSF samples collected from NC subjects , were selected for ApoA1 and ApoE ELISAs, total protein measurement, and SAA experiments.Biochemed Services  and were collected from 3 male and 3 female subjects (age = 26–39 y). With reference to the experiments summarised in Fig. Biochemed Services. All neat HC CSF samples tested negative in α-syn SAA. With reference to the same experiments, PD10, PD34, PD62 CSF samples  belonged to PD patients with an Hoehn and Yahr (H&Y) stage of 2 and were purchased from PrecisionMed . PD47  was instead purchased from BioIVT . All neat PD CSF samples tested positive in α-syn SAA.CSF collected from healthy control (HC) and PD subjects was used to perform initial α-syn seed spiking experiments and to test the impact of adding HDL, LDL, HSA, and TTR at Amprion Inc. . The HC1-6 samples used in α-syn seed spiking experiments shown in Fig. An aliquot of 3 mL of CSF pool 1 was resuspended in 1.5 mL of PBS 3 × in order to have 4.5 mL of human pooled CSF in PBS 1x. This volume was then subjected to a series of filtrations using Amicon® Ultra-4 molecular weight cut-off (MWCO) filters. The procedure used to fractionate human CSF is schematized in Fig. Preformed α-syn aggregates were generated by incubating 1 mg/mL of α-syn in PBS for one week at 37 °C under vigorous double orbital shaking (500 rpm) in a sealed 1.5 mL polypropylene vial. The final products were subjected to cycles of sonication  with an amplitude of 12 μm. The polypropylene vial had been immersed in ice for the whole duration of the sonication procedure. The aggregates were then diluted at 0.25, 2.5, 25, 250 and 2500 pg/μL, considering the initial monomer concentration as reference. The generated α-syn aggregates were then aliquoted and stored at -80 °C.3N4 bead (Tsubaki Nakashima). Beads were blocked with 1% BSA in 100 mM PIPES pH 6.50 for 1 h, washed twice with 100 mM PIPES pH 6.50, and dried overnight. This assay was performed in a BMG FLUOstar Omega shaker/reader at 37 °C, plates were shaken for 60 s every 30 min for 150 h. The assay outcomes of the assay are positive, inconclusive, or negative, based on a probabilistic algorithm that uses maximum fluorescence and kinetic parameters [Samples were analysed as previously reported , 20. Brirameters . With rerameters , 20.3 was also present in all the tested conditions. All reactions were performed at 37 °C in sealed plates. Each condition was tested at least in triplicate.The protein aggregation experiments used to characterize interaction between α-syn and CSF constituents were performed with a programmable BMG LABTECH ClarioStar® fluorometer in Greiner clear-bottom 96-well plates . The ThT fluorescence was read from the bottom using excitation and emission wavelengths of 450 and 480 nm, respectively. An incubation temperature of 37 °C was used for all the experiments. Slightly different gain values were used to avoid overflow of the analog-to-digital converter. In each experiment, lyophilized recombinant α-syn was thawed in 3 mM NaOH at the concentration of 3.5 mg/mL. The solution was brought to physiological pH by diluting it with concentrated PBS (4x) and distilled water. In all the experiments described, the final reaction volume was 200 μL, α-syn final concentration was 0.7 mg/mL and ThT final concentration was 10 μM. To avoid bacterial contamination, 0.1% NaN158 μL of the solution containing monomeric α-syn was poured in wells, each of them containing 6 glass beads of 1 mm diameter. Depending on the experiment, 40 μL of human pooled/NPH CSF, 40 μL of PBS or 40 μL of CSF fractions were added. In seeded experiments, 2 μL of PBS containing 0, 0.25, 2.5, 25, 250, and 2500 pg/μL of α-syn aggregates were added. Plates were sealed and subjected to cycles of shaking  inside the fluorometer.To compare the results with those of a previously published paper, the experiment was performed in the exact same way as it is described in Bellomo et al. . With re2O) of human serum HDL (Sigma-Aldrich LP3), human serum LDL (Sigma-Aldrich LP2), recombinant TTR were added. Each condition was replicated in 3 distinct wells in the presence and absence of α-syn. For the experiments in which we tested the effect of different concentrations of human serum HDL  the plate was sealed and subjected to cycles of 1 min double-orbital shaking at 500 rpm, 14 min rest. For the experiments in which we tested the effect of different concentrations of human serum HDL (0.3 mg/mL), LDL (0.03 and 0.3 mg/mL) and TTR  the shaking protocol was changed to 2 min double-orbital shaking at 500 rpm and 13 min rest at 37 °C.In these experiments 40 μL of solutions containing different dilutions  was calculated for each fitting. The α-syn SAA result of each CSF sample was determined using a probabilistic algorithm:Ppos is the probability for a replicate to be positive, A = -4.02, B = 2.98, C = 1.87, and Fmax5000 and Rsquare93 are binary values depending on a threshold. If the Fmax of a given replicate is above 5,000 RFU, then Fmax5000 = 1, else 0. If the R2 for the fitting of the 4-parameter model to the fluorescence data is above 0.93, then Rsquare93 = 1, else 0. The coefficients A, B, and C were estimated using a database of more than 900 aggregation curves. Among the PD and HC samples in the database, Fmax and the R2 showed statistically significant differences . If the probability for positivity (Ppos) is higher than 0.12, the replicate is determined positive, otherwise it is determined negative. Since CSF samples were analysed in triplicates, if all 3 replicates were positive the CSF sample was called positive. If none or one replicate was positive the CSF sample was called negative. If two replicates were positive and the average Fmax of the 3 replicates was less than 5,000 RFU (or a.u.) or the coefficient of variation (CV) was higher than 110, the sample was also called negative.During the ultrasensitive SAA experiments performed at Amprion Inc., fluorescence readings were collected every 30 min to estimate kinetic parameters with high accuracy. The following 4 parameter sigmoid was used to fit the raw fluorescence readings:2 was below 0.3.The average background fluorescence produced by three wells containing the analyte without α-syn was subtracted prior to the analysis from the ThT intensity profiles relative to the same analyte in the presence of α-syn. While analysing data relative to the sole α-syn the background fluorescence from well containing only the reaction buffer was subtracted. Each ThT kinetic trace was then fitted with a double sigmoid function using Origin Pro v9.0. In the fitting model, A2 fits the fluorescence value of the second plateau, A1 fits the fluorescence value of the first plateau and A0 fits the baseline fluorescence. The time parameters t1 and t2 fit the first and the second inflection points, respectively, while d1 and d2 represent the slopes of the sigmoids. In the non-linear fitting procedure used, the following bounds were applied: 0 < A0 < 1000, 500 < A1 < 5000, A2 > 2000, 0 < t1 < 100 h and t2 > 0. For some kinetic traces, a decrease in fluorescence was observed after reaching the second plateau. This known phenomenon is caused by the sequestration of ThT molecules by mature fibrils and by the sedimentation of HMW insoluble aggregates . In thesEqual amounts of assay products (volumes containing 2 µg of α-syn) were added with Laemmli’s sample buffer without sodium dodecyl sulphate (SDS), without boiling them to prevent solubilization of SDS-sensitive aggregates. Samples were separated through SDS-PAGE on 4–20% polyacrylamide gels (Bio-Rad) and transferred into PVDF membranes  by wet transfer at 100 V constant for 90 min using 25 mM Tris–HCl, 192 mM glycine, 20% methanol, and 0.015% SDS. Membranes were fixed with 4% PFA for 30 min prior to blocking with 5% non-fat milk in TBS-T (TBS with 0.1% Tween 20) for 1 h at room temperature. After blocking, filters were incubated with primary antibody against α-syn  O/N at 4 °C. The membranes were further incubated with a goat-anti-mouse IgG-HRP conjugate  secondary antibody for 1 h at RT, and signals were visualized using an ECL reaction. Densitometric analysis was performed with ImageJ software . Dot-blot images were inverted, and window/level adjusted prior to the analysis. A rectangular region of interest (ROI) was used to measure the average grey level intensity in each band relative to monomeric α-syn. The average grey level of an equivalent ROI not containing bands was subsequently subtracted. To estimate the approximate percentage of remaining α-syn monomer, the average intensity of the WB bands was divided by the one at t = 1 h.2 to estimate the integrated density. No significant grey level fluctuations were found while repeating the same procedure for control samples not containing α-syn.For dot blotting aliquots of products obtained in SAAs corresponding to 300 ng of monomeric α-syn in the initial reaction mixtures were spotted (2 μL/spot) on nitrocellulose membrane pre-equilibrated with TBS-T. Samples were dried at RT and fixed with PFA (0.4% in PBS) for 30 min, and then filters were blocked with 2% non-fat milk (in TBS-T). The membranes were incubated overnight at 4 °C with OC (1:1000) or A11 (1:1000) conformational antibodies  followed3 in Tris-buffered saline with 0.1% Tween®20  (TBST) with addition of a phenol red as a pH indicator. After the incubation, the membrane was washed with TBST and the secondary antibodies diluted 1:5000 in a blocking buffer were applied for 1 h at RT. Depending on the used primary antibodies, goat anti-mouse  (for MIA1404) or goat anti-rabbit IgG-HRP   were added. Signal development was performed with use of the enhanced chemiluminescence (ECL) solution .Immunoprecipitation (IP) was performed to deplete ApoA1 and ApoE from CSF pool sample. 50 µL of settled Immobilized Protein A/G  and 60 µg of anti-ApoA1 antibody  were combined in a 2-mL tube. The beads-antibody slurry was incubated for 4 h at 4 °C (constant rotation). The tube was centrifuged at 1,000 × g for 2 min at 4 °C and the supernatant was discarded. The bead pellet was washed with Phosphate Buffered Saline (PBS) twice. Similarly, 50 µL of settled Immobilized Protein A/G  and 20 µg of anti-ApoE antibody  were combined a 2-mL tube. The beads-antibody slurry was incubated for 4 h at 4 °C (constant rotation). The tube was centrifuged at 1,000 × g for 2 min at 4 °C and the supernatant was discarded. The bead pellet was washed with PBS twice. Subsequently, ApoA1-antibody-bound beads and ApoE-antibody-bound beads were combined in a single 2-mL tube. The solution was gently mixed and centrifuged at 1000 × g for 2 min at 4 °C. The supernatant was discarded and 400 µL of CSF pool was added to the tube with beads. The sample underwent overnight incubation at 4 °C (constant rotation). At the end of incubation, the CSF-beads slurry was centrifuged  and the supernatant and bead pellet were collected separately for further analysis. In parallel, a control sample was prepared by following the same procedure on beads without anti-ApoA1 or anti-ApoE antibody bound to IP. Briefly, 50 µL of neat resin slurry was transferred to 2-mL tube and washed with PBS twice. After the second wash cycle, PBS was removed and 400 µL of CSF pool was added instead. The sample underwent overnight incubation at 4 °C (constant rotation). At the end of incubation, the sample was centrifuged  and the supernatant and bead pellet were collected separately for further analysis. Performance of ApoA1 and ApoE IP was assessed by Western Blot. Briefly, all the samples , 3. bead pellet of IP CSF pool sample, 4. bead pellet of CSF pool sample undergoing procedure mimicking IP (without antibody), 5. neat CSF pool sample which did not undergo any treatment) were resuspended in a reducing loading buffer and boiled 5 min at 95 °C (thermoblock). Tubes with bead pellet (samples 3. and 4.) were centrifuged and the supernatant was collected and used for further analysis. A 12% polyacrylamide gel was prepared, and the samples were run in reducing conditions. All samples were transferred from the gel to a nitrocellulose blotting membrane . The quality of the transfer was evaluated by the reversible Ponceau S staining. The membrane was blocked and subsequently probed overnight at 4 °C (shaker) with the selected primary antibodies  diluted in 5% bovine serum albumin (BSA), 0.02% NaNn = 31 SAA-negative NC CSF samples. Both assays were performed according to the manufacturer protocol. All the standard curve points and CSF samples were run in duplicate. Optical density (OD) was read at 450 nm (wavelength correction 570 nm) by the Clariostar  plate reader. The 4-parameter logistic model was applied to generate a standard curve and interpolate concentrations of analysed CSF samples.CSF levels of ApoA1 and ApoE were assessed with use of the commercially available ELISA kits—Human Apolipoprotein AI ELISA Kit, ab108803 and Human Apolipoprotein E ELISA Kit, ab108813  in Total protein content was evaluated in the same samples with use of the Pierce™ 660 nm Protein Assay Reagent cat. 22,660 . The assay was performed according to the manufacturer protocol. A set of BSA dilutions of known concentrations served as the standard to calculate total protein content of CSF samples. All the standard curve points and CSF samples were run in duplicate.One-way analysis of variance (ANOVA) and Tukey post-hoc test for mean comparisons were applied while assessing differences among fitted kinetic parameters for different samples. Correlation between added seed mass and T2 parameters were computed according to Spearman. Two-tailed Student’s t-test was applied when comparing adjusted integrated densities of dot blot images. Standard error of mean (SEM) is reported in each image showing bar plots and/or average fluorescence profiles. In the analysis of mass spectrometry data, an false discovery rate (FDR) of 1% was imposed and the criterion used to accept protein identification included probabilistic score sorted by the software. Correlations among SAA parameters and Log2-transformed CSF levels of ApoA1, ApoE and total protein were computed by means of Pearson’s correlation coefficients. Ward linkage criterion was applied for hierarchical clustering of correlations.A detailed description of the study design (experiments type and workflow) is present in Sect. 2.1 of Material and Methods and graphically summarised in Fig. . [p <  < 0.001, see Supplementary material Fig. SCSF inhibition of α-syn aggregation in SAAs remains poorly characterized, although it has been already reported in the literature , 11, 12.. , 20. Alt1H solution high-field nuclear magnetic resonance (NMR) spectroscopy. The most apparent differences between the two spectra lay between 1.5 and 0 ppm, where methyl resonances are usually observed  with respect to NPH2 CSF  is usually available in large volumes and is often used for assay development or as negative controls in diagnostic SAAs. Although considered negative controls due to lack of detectable α-syn seeds, dilution of CSF components has been reported to be common in NPH patients  which coved Fig. B. Intere spectra , 25. Themax) for all 6 CSF-derived samples  filters   and serum purified human serum albumin  as control. Albumin levels were quite similar between CSF fractions, and we have already shown that HSA only partially reduces α-syn aggregation at plasma concentrations (43 mg/mL) [ion Fig. , there wAfter confirming that purified HDL at plasma concentration replicates the inhibition of α-syn aggregation shown by whole CSF and by the > 100 kDa CSF fraction, we evaluated if HDL could retain the same level of inhibition when tested within a range of concentrations from 1 to 0.003 mg/mL . Interestingly, we observed a dose-dependent partial inhibition of α-syn aggregation when adding 0.003 and 0.03 mg/mL, while 0.3 and 1 mg/mL completely blocked the formation of ThT-reactive aggregated species Fig. A. The pamax/A2 and > 200 h t2). TTR did not inhibit α-syn spontaneous aggregation when tested within the physiological concentration range (0.03 and 0.3 mg/mL)  –28, whicmL) Fig. A-B. Neven of TTR . These rWe have shown lipoprotein inhibition of α-syn aggregation by ThT, detection of oligomeric and amorphous species by conformational antibodies, and the measurement of monomeric α-syn by WB, but these techniques do not allow the observation of α-syn fibrillary species in the presence of lipoprotein. Thus, we used transmission electron-microscopy (TEM) to evaluate final products of aggregation reactions containing α-syn alone, α-syn with LDL or HDL, and HDL and LDL without α-syn Fig. C-H. In an = 4 PD-CSF and n = 4 HC-CSF SAA reactions that have been tested consistently positive and negative in the SAA, respectively, with different levels of HDL and LDL. Each SAA reaction contains CSF, thus, we considered them to carry 1X the CSF physiological concentration of these proteins. Thus, 1X reactions are neat CSF reactions, while 2X and 5X reactions were supplemented with the purified lipoproteins to reach 2 and 5 times their respective CSF physiological concentration  CSF , 19. We ion Fig. . HDL parion Fig. C. This iion Fig. D. LDL foTo further confirm the role of lipoproteins in the patient-dependent CSF inhibition on α-syn aggregation, we depleted ApoA1 and ApoE from a newly made pooled CSF . Interestingly, Log2-transfomed sum of ApoA1 and ApoE was the variable showing the highest correlation with T50 (r = 0.55) and TTT (r = 0.62), which is consistent with the fact that lipoproteins in general can modulate α-syn aggregation in SAAs.Lastly, we measured levels of the two most abundant CSF apolipoproteins (ApoA1 and ApoE) in al blood  and we eal blood , we prefInhibition of spontaneous and seeded fibrilization of α-syn by human CSF has been briefly reported in recent literature, but not yet characterized , 11–13. We used TEM to observe the ultrastructure of the α-syn fibrils in the presence of HDL and LDL and found both lipoproteins to colocalize with the fibrils, suggesting a direct interaction with α-syn aggregates. Although solution NMR has previously proven to be a suitable tool to identify proteins interacting with monomeric α-syn , 32, we We then evaluated the effect of lipoproteins on the amplification of α-syn seeds from human PD CSF samples in an ultrasensitive SAA. In these experiments, the reactions were supplemented with HDL and LDL to mimic CSF samples with 2X or 5X the physiological levels of HDL and LDL. We found that both HDL and LDL could significantly change the kinetic parameters of the amplification reaction starting from 2X concentration. At 2X LDL concentration, the SAA outcome of one of four PD samples changed from positive to inconclusive, while the same happened for two of four PD samples at both 5X HDL and LDL. Nevertheless, the likelihood of encountering CSF samples containing such high levels of lipoproteins is low, which is in line with the impressive sensitivity and specificity consistently reported by diagnostic SAAs . HoweverOur results describe a novel interaction between lipoproteins and α-syn that inhibits the formation of α-syn fibrils and could have relevant biological implications in vivo. Our findings also have direct and important implications for the future development and improvement of α-syn SAAs, which are currently the most promising diagnostic tool for synucleinopathies. The donor-specific inhibition of CSF on α-syn aggregation reported here offers an explanation for the lack of correlation seen between kinetic parameters and clinical progression. Moreover, our data reveals apolipoprotein as the main inhibitory components of CSF, suggesting that measurements of apolipoproteins and/or total protein content could be incorporated into data analysis models to eliminate confounding effects of CSF milieu on α-syn seed quantification efforts using kinetic parameters.Additional file 1:Fig. S1. Silver staining on α-syn used in Protein aggregation experiments. Two replicate silver staining experiments performed on a 4-20% SDS-PAGE gel with 1 μg of purified α-syn after one (lane 3) and two (lane 2) size-exclusion chromatography steps. Fig. S2. T50 measured in Six different human CSF samples spiked with 20 fg of seeds. The measured T50 parameters were globally different, as assessed by one-way analysis of variance (ANOVA) and Fisher’s LSD post-hoc test for mean comparisons. *0.01<p<0.05; **0.001<p<0.01: ***p<0.001. Fig. S3. Quantitative regression analysis of the seed masses in the absence of human CSF. Measured t2 parameters for the seeded experiment vs the quantity of seeds added; the horizontal axis is displayed in log10 scale. The t2 values displayed result from the average of three replicates, error bars reflect the standard deviation of the mean value. The data were fitted with a natural log function, the correlation between t2 and the added seed masses was assessed by means of Pearson’s correlation coefficient (r). Fig. S4. NMR titrations of α-syn with CSF fractions. Intensity decreases of the signals of two-dimensional (2D) 15N–1H HSQC experiments acquired at 950 MHz at T = 283 K on 15N labelled α-syn (100 μM) in PBS after the addition of: (A) whole pooled CSF in PBS, (B) < 3 kDa CSF fraction in PBS and (C) > 100 kDa CSF fraction in PBS. The residues experiencing the largest decreases in signal intensity  are highlighted in light blue. The intensity ratios corresponding to overlapping peaks are highlighted in red . Fig. S5. CSF pH drift. The pH change due to the exposure of CSF to air was monitored over time in 500 μL of undiluted pooled CSF (A) and in the presence of PBS (400 μL CSF + 200 μL PBS 3x) in polypropylene vials with a Thermo Scientific Orion pH-meter equipped with a glass 6 mm diameter pHenomenal MIC 220 Micro electrode. Right before each measurement, the sample was vortexed for 20 sec and left open to air for another 20 sec. Fig. S6. Different CSF fractions differently affect α-syn aggregation. Mean fitted t2 parameters of samples with 40 μl of PBS/CSF fractions. The values displayed result from the average of three replicates with error bars reflecting the SEM. For whole CSF and the >100 kDa fraction the total duration of the experiment is shown due to the absence of appreciable aggregation. Fig. S7. Raw images of the dot-blot assays. A-B) Native images of the dot-blot assay performed on α-syn alone replicates at different timepoints with OC (A) and A11 (B) conformational antibodies. C-D) Native image of the dot-blot assay performed on the HSA and HDL containing samples with OC (C) and A11 (D) conformational antibodies. Fig. S8. WB experiments to track α-syn aggregation in the presence of human HDL. A) α-Syn aggregation patterns in samples collected at different timepoints of the spontaneous aggregation process, was monitored by WB using Syn211 antibody . Monomeric α-syn decreases as t increases due to the formation of fibrils. B) In a similar way, a WB with Syn211 was performed on the reaction products obtained after 180 h, at different HDL concentrations with and without α-syn (exposure time 210 s). C) The experiment was then repeated by doubling the amount of sample loaded into the gel to better highlight the presence of oligomeric species (exposure time 30 s). Under these conditions, chosen to better visualize the signal at 150-200 kDa, the α-syn monomer bands at 1 and 0.3 mg/mL HDL may not quantitively reflect monomer concentration (overloaded lanes). Fig. S9. Representative TEM images of α-syn incubated with CSF. Representative TEM images obtained by analyzing samples obtained by the co-incubation of α-syn 0.7 mg/mL at 37 °C with pooled human CSF . Samples were subjected to cycles of incubation (13 min) and shaking  at 500 rpm. Fig. S10. NMR titrations of α-syn with HDL, LDL and TTR. A) Intensity decreases of the signals of two-dimensional (2D) 15N–1H HSQC experiments acquired at 950 MHz at T = 283 K on 15N labelled α-syn (100 μM) in PBS after the addition of 0.57 mg/mL serum-derived HDL. The intensity ratios corresponding to overlapping peaks are highlighted in red. B) Intensity decreases of the signals of two-dimensional (2D) 15N–1H HSQC experiments acquired at 950 MHz at T = 283 K on 15N labelled α-syn (100 μM) in PBS after the addition of 1 mg/mL serum-derived LDL. C) Intensity decreases of the signals of two-dimensional (2D) 15N–1H HSQC experiments acquired at 950 MHz at T = 283 K on 15N labelled α-syn (100 μM) in PBS after the addition of 3 mg/mL TTR. Fig. S11. WB experiments performed on immunodepleted CSF. WB experiments were performed with anti-ApoA1 (MIA1404) and anti-ApoE (PA5-27088) antibodies on neat CSF and supernatants  resulting from immunoprecipitation procedure performed using different quantities of the same antibodies (IP CSF samples), immunoprecipitation performed without antibodies (CSF IP no Ab). The conditions relative to IP CSF ApoA1 60 µg and IP CSF ApoE 20 µg were then selected for Protein aggregation assays. Table S1. Concentration factors and final volumes of the CSF fractions.


Herpesviridae including cytomegalovirus (CMV). We show in a murine model of HCT that macrophage colony‐stimulating factor (M‐CSF) promoted rapid antiviral activity and protection from viremia caused by murine CMV. M‐CSF given at transplantation stimulated sequential myeloid and natural killer (NK) cell differentiation culminating in increased NK cell numbers, production of granzyme B and interferon‐γ. This depended upon M‐CSF‐induced myelopoiesis leading to IL15Rα‐mediated presentation of IL‐15 on monocytes, augmented by type I interferons from plasmacytoid dendritic cells. Demonstrating relevance to human HCT, M‐CSF induced myelomonocytic IL15Rα expression and numbers of functional NK cells in G‐CSF‐mobilized hematopoietic stem and progenitor cells. Together, M‐CSF‐induced myelopoiesis triggered an integrated differentiation of myeloid and NK cells to protect HCT recipients from CMV. Thus, our results identify a rationale for the therapeutic use of M‐CSF to rapidly reconstitute antiviral activity in immunocompromised individuals, which may provide a general paradigm to boost innate antiviral immunocompetence using host‐directed therapies.Therapies reconstituting autologous antiviral immunocompetence may represent an important prophylaxis and treatment for immunosuppressed individuals. Following hematopoietic cell transplantation (HCT), patients are susceptible to  Herpesviridae like CMV are a major cause of morbidity and mortality in patients after HCT. Therapies reconstituting the host's antiviral immunocompetence for prophylaxis and treatment are an unmet medical need since licensed therapies are either insufficiently effective or have severe side effects. Herpesviridae family, in patients that have undergone hematopoietic stem cell transplantation (HCT) is a major cause of acute mortality at the leukopenic phase when donor stem cell reconstitution has yet to occur. Unfortunately, licensed drugs against CMV are either insufficiently effective and rely on the viral replication machinery (letermovir) or have severe side effects including bone marrow toxicity and risk of posttreatment mutagenesis (ganciclovir). Hence, further alternative well‐tolerated host‐directed therapies are needed that rapidly confer broad‐spectrum antimicrobial competence in severely immunocompromised patients following HCT.Viral infection or reactivation of lifelong dormant viruses in immunosuppressed patients accrues to a significant death toll. For example, reactivation of cytomegalovirus, an opportunistic virus of the The administration of M‐CSF around the time of transplantation revealed a novel mechanism to protect against lethal CMV infection in leukopenic mice following HCT. Here, M‐CSF stimulated hematopoietic stem cells to overcome leukopenia by committing to monopoiesis. We show that in mice M‐CSF engages critical host‐directed initial mechanisms to reconstitute antiviral immune responses after HCT, which are type I interferon and natural killer (NK) cell‐dependent. Mechanistically, NK cell‐mediated protection from lethal CMV infection arose from IL‐15 and type I interferon production by M‐CSF‐driven monopoiesis or plasmacytoid dendritic cell differentiation, respectively. Synergistically, these brought about NK cell differentiation and activity from recruited progenitors. As in mice, human G‐CSF‐mobilized stem and progenitor cells within PBMCs also responded to M‐CSF administration with elevated monopoiesis, IL‐15Rα expression, and functional NK cell generation. Lastly, we could show that M‐CSF did not confer any adverse effects on long‐term hematopoietic stem cell engraftment and hematopoietic lineage balance in the blood or acute post‐transplantation symptoms following allogenic HCT.Overall, our preclinical results support the efficacy and feasibility of M‐CSF administration as a prophylactic host‐directed therapy that confers rapid protection from lethal CMV infection. Moreover, our data suggests a paradigm by which M‐CSF boosts innate antimicrobial immunocompetence, which may constitute a well‐tolerated general host‐directed therapeutic in states of severe immunosuppression.et al, et al, et al, et al, et al, et al, et al, The first months after hematopoietic cell transplantation (HCT) are characterized by profound immunosuppression, which leaves patients at high risk of viral infection or reactivation of common opportunistic viruses such as cytomegalovirus (CMV). The infection itself but also its subsequent treatment is associated with significant morbidity and mortality 1, a key transcription factor for mounting IFN responses, is also required for the early control of MCMV infection and spleen pathology but does not affect viral clearance ‐dependent delivery of granzyme B (GrB) into infected cells , demonstrating that IL‐15 signaling from myeloid cells was required for NK cell support  alone or in combination with the multi‐lineage myeloid cytokine IL‐3 or with M‐CSF, respectively  as observed by increased expression of CD64 . tors Fig  and a coions Fig . Consistions Fig , in partGMPs Fig   at day 9 of culture  and GMPs alike in the BM 12 weeks after allogeneic HCT  granulocytes, mediating cytotoxic bacterial killing, (ii) monocytes and macrophages, capable of pathogen control by phagocytosis and reactive oxygen production, and (iii) dendritic cells with the strongest antigen presentation activity that alerts the adaptive immune system. In this study, we now show that M‐CSF also induced I‐IFN‐producing pDCs and indirectly stimulated NK cell differentiation and activation through induction of IL‐15‐producing monocytic cells, which together mediated strong antiviral activity.Several properties of M‐CSF make it an ideal candidate for accelerating immunocompetence recovery in HCT recipients and present key advantages over other myeloid cytokines used in clinical practice. We showed before in murine models that M‐CSF directly engages HSPCs and thus intervenes at the earliest point of the differentiation hierarchy to initiate the production of innate immune cells  and FACS sorting from 6 to 8‐week CD45.1+ bone marrow, were injected with 150,000 cKit−Ter119+ CD45.2+ carrier cells (Miltenyi Biotec) and murine (baculovirus expressed) or human recombinant M‐CSF (Chiron/Novartis) in 200 μl PBS retroorbitally into lethally irradiated  8–14 weeks sex‐matched CD45.2+ mice as described previously  from WT or IL15Rα‐KO or IFNAR1‐KO mice were FACS sorted and injected on day 10 after HCT. For consistency, all survival analyses using Kaplan–Meier estimates pertaining to MCMV‐infected mice are illustrated until 35 days post HSPC transplantation, although several mice were observed until day 50 without any further signs of morbidity and mortality.For reconstitution 3,000 c‐Kit/CD117+ recipient and C57BL/6j CD45.1+ donor mice were used. In brief, BALB/c CD45.2+ recipient mice were irradiated with 5 Gy, followed by allogeneic HCT after 24 h. Imminently before (1 h) or shortly after (5 and 20 h) allogeneic HCT with 2 × 105 Lin− HSPCs from C57BL76j CD45.1+ donors, the mice received PBS or 10 μg of baculoviral expressed human M‐CSF. Following alloHCT, scoring for disease activity was performed according to Lai et al  of MCMV K181 v70 in 200 μl PBS. Viral loads were measured by quantitative reverse transcription polymerase chain reaction (RT‐qPCR) of et al, et al, 5 cells (1 × 106 cells ml−1) of the CD3/CD15‐depleted G‐CSF‐mobilized PBMCs were subsequently transferred to 96U‐bottom ultralow adherence plates  to be cultured in StemPro34 serum‐free medium  with 1× penicillin/streptomycin  supplemented with stem cell factor (SCF)  ± the following cytokine compositions: (i) none, (ii) recombinant human IL‐3  or (iii) human M‐CSF recombinant protein . A partial medium change was performed every 48 h with 2× cytokine composites to replenish cytokines. Cell differentiation and viability were confirmed using cytospins on days 5 and 9.Human G‐CSF‐mobilized HSPCs were obtained from leukapheresis samples from the Department of Transfusion Medicine of the TU Dresden. On the day of donation, a Ficoll‐density gradient centrifugation step was performed as described previously  such as common lymphoid progenitors , common myeloid progenitors  or granulocyte‐macrophage progenitors  with mature GMPs additionally expressing HLA‐DR from mature myeloid cells (either CD11b+CD66b− or Lin+ ± CD14/CD16) whose IL15Rα expression was quantified. For NK cell abundance and activity, an optimized panel as published previously was used  or on days 5 and 9, respectively.For human samples, an antibody panel was used to distinguish progenitors of human HSPCs (LinFreshly frozen OCT embedded (Sakura Finetek). 8 μm sections (Leica CM3050 S cryostat) were fixed 10′ in 4°C acetone, blocked 30′ with PBS/2%BSA, stained with 1:100 directly coupled antibody  and specific gene expression  test were used in this study depending on the data type, and the details can be found in the figure legends: test used and exact value of n. For in vivo experiments, block randomization was used to establish identical or similar group sizes. No statistical methods were used to predetermine sample sizes. Veterinary pathologists assessing and scoring liver sections were blinded to sample identity for indicated parameters. No further blinding was performed. Experiments were performed independently at least twice.None of the mice or human samples were excluded from the experiments. Multiple statistical methods, including Student's t‐tests. All statistical analyses were performed using GraphPad Prism . All data were analyzed using the FlowJo software  and expressed as medians + individual data points or means ± SEM unless stated otherwise. P‐values less than 0.05 were considered significant.Data between two groups were analyzed with unpaired Student's Procedures involving animals and their care were conducted in accordance with institutional guidelines and internal laws and policies and approved by the Institutional Animal Care and Use Committee (APAFIS #17258‐2018102318448168‐v5 and APAFIS #36188‐2022032912082580‐v6). All mouse experiments were performed under specific pathogen‐free conditions and animals were monitored daily for signs of morbidity.The use of human samples was approved by the Ethical Review Committee of the TU Dresden  and all human research conformed to the Declaration of Helsinki and the Department of Health and Human Services Belmont Report. Informed consent was obtained from all participants.Prashanth K Kandalla: Conceptualization; data curation; formal analysis; validation; investigation; visualization; methodology; writing – original draft; project administration; writing – review and editing. Julien Subburayalu: Conceptualization; data curation; formal analysis; validation; investigation; visualization; methodology; writing – original draft; project administration; writing – review and editing. Clément Cocita: Resources; formal analysis; investigation; methodology. Bérengère de Laval: Resources; data curation; formal analysis; validation; investigation; visualization; methodology; writing – review and editing. Elena Tomasello: Resources; methodology. Johanna Iacono: Methodology. Jessica Nitsche: Investigation. Maria M Canali: Resources. Wilfried Cathou: Formal analysis; methodology. Gilles Bessou: Resources. Noushin Mossadegh‐Keller: Methodology. Caroline Huber: Methodology. Guy Mouchiroud: Resources. Roland P Bourette: Resources. Marie‐France Grasset: Resources. Martin Bornhäuser: Writing – review and editing. Sandrine Sarrazin: Formal analysis; methodology; project administration; writing – review and editing. Marc Dalod: Conceptualization; resources; formal analysis; methodology; writing – review and editing. Michael H Sieweke: Conceptualization; formal analysis; supervision; funding acquisition; validation; writing – original draft; project administration; writing – review and editing.MHS is a patent holder of WO2014167018A1 (Use of M‐CSF for preventing or treating myeloid cytopenia and related complications). The authors declare no further competing interests.https://tu‐dresden.de/cmcb/crtd/forschungsgruppen/crtd‐forschungsgruppen/sieweke & www.ciml.univ‐mrs.fr/science/lab‐michael‐sieweke/stem‐cell‐and‐macrophage‐biology.Authors' home website: https://www.cedars‐sinai.org/health‐library/diseases‐and‐conditions/c/cmv‐and‐transplant‐patients.html.Information on CMV in transplanted patients provided by Cedars Sinai: Expanded View Figures PDFClick here for additional data file.Table EV1Click here for additional data file.Table EV2Click here for additional data file.Table EV3Click here for additional data file.Source Data for Expanded ViewClick here for additional data file.PDF+Click here for additional data file.Source Data for Figure 1Click here for additional data file.Source Data for Figure 2Click here for additional data file.Source Data for Figure 3Click here for additional data file.Source Data for Figure 4Click here for additional data file.Source Data for Figure 5Click here for additional data file.Source Data for Figure 6Click here for additional data file.Source Data for Figure 7Click here for additional data file.Source Data for Figure 8Click here for additional data file.


DCD NRP technique is a viable method to expand the pediatric heart donor pool.The sole curative therapy for end-stage heart failure in pediatric patients is organ transplantation. Mechanical circulatory support in pediatrics is only a bridge to transplant or decision. In recent years, transplantation of hearts donated after determination of circulatory death (DCD) has been proposed as a novel method to increase the pool of donor allografts. In 2008, the first pediatric series of DCD implants used direct procurement and perfusion, followed by implantation in the recipient. In 2015, adult series of DCD donors with normothermic machine perfusion (NMP) from Australia as well as normothermic regional perfusion (NRP) from the United Kingdom with now 5-year follow-upIRB#21 to 001629 approval was obtained September 20, 2021. An 8-year-old 28-kg donor became available after being found unresponsive at home after a viral illness. The recipient was a 5-year-old redo heart transplant patient weighing 17 kg with left-sided pulmonary venous obstruction that had previously been stented. The recipient had decompensated due to coronary allograft vasculopathy requiring ECMO and Centrimag biventricular assist device complicated by a new stroke on computed tomography scan; however, the patient was responding to verbal stimuli and moving extremities. The recipient also was in renal failure on dialysis. Donor/recipient height ratio was 1.26 and weight ratio was 1.64. Informed consent for publication of data was obtained from the donor family for DCD donation as well as the recipient family. The donor was located 85 miles away from the accepting institution with a travel time of 20 minutes door to door via helicopter.2 and age normal mean arterial pressure was maintained. This was sufficient flow to correct acidosis because cerebral blood flow was eliminated. The donor heart recovered in sinus rhythm within a minute of reperfusion, and was weaned off in an hour  of pediatric heart transplants were from DCD. There was no significant difference in the survival at 1 year between donor after brain death and DCD infant recipients.


In lumbar spinal stabilization pedicle screws are used as standard. However, especially in osteoporosis, screw anchorage is a problem. Cortical bone trajectory (CBT) is an alternative technique designed to increase stability without the use of cement. In this regard, comparative studies showed biomechanical superiority of the MC  technique with longer cortical progression over the CBT technique. The aim of this biomechanical study was to comparatively investigate the MC technique against the not cemented pedicle screws (TT) in terms of their pullout forces and anchorage properties during sagittal cyclic loading according to the ASTM F1717 test.Five cadavers (L1 to L5), whose mean age was 83.3 ± 9.9 years and mean T Score of -3.92 ± 0.38, were dissected and the vertebral bodies embedded in polyurethane casting resin. Then, one screw was randomly inserted into each vertebra using a template according to the MC technique and a second one was inserted by freehand technique with traditional trajectory (TT). The screws were quasi-static extracted from vertebrae L1 and L3, while for L2, L4 and L5 they were first tested dynamically according to ASTM standard F1717  and then quasi-static extracted. In order to determine possible screw loosening, there movements were recorded during the dynamic tests using an optical measurement system.The pull-out tests show a higher pull-out strength for the MC technique of 555.4 ± 237.0 N compared to the TT technique 448.8 ± 303.2 N. During the dynamic tests , 8 out of the 15 TT screws became loose before completing 10,000 cycles. In contrast, all 15 MC screws did not exceed the termination criterion and were thus able to complete the full test procedure. For the runners, the optical measurement showed greater relative movement of the TT variant compared to the MC variant. The pull-out tests also revealed that the MC variant had a higher pull-out strength, measuring at766.7 ± 385.4 N, while the TT variant measured 637.4 ± 435.6 N.The highest pullout forces were achieved by the MC technique. The main difference between the techniques was observed in the dynamic measurements, where the MC technique exhibited superior primary stability compared to the conventional technique in terms of primary stability. Overall, the MC technique in combination with template-guided insertion represents the best alternative for anchoring screws in osteoporotic bone without cement. Pedicle screw instrumentation is the most common surgical technique in lumbar spine stabilization. Cement augmentation is commonly used in osteoporotic spine , 16, 41.The objective of this biomechanical study was to conduct a comparative investigation between the MC technique, utilizing patient specific placement guides and non-cemented pedicle screws implanted via freehand technique (TT) with regards of their pullout forces and anchorage properties under sagittal cyclic loading according to the ASTM F1717 test protocol.Like in our recent study  five humDuring dissection, all vertebrae were separated into single levels. Muscular and soft tissue was removed from each vertebra while preserving its anatomy. The specimens were stored at − 83 °C until testing. A dual-energy X-ray absorptiometry (DXA) analysis was performed to determine the bone mineral density . In addition, a low-dose computed tomography (CT) scan  of all specimens was taken for the exclusion of bone defects and for preoperative planning.The diameter and the lengths of TT screw was identified by an experienced surgeon using the CT scans. Planning for the screws used in the MC technique followed the same protocol as Jarvers et al. . An indiAll specimens were divided into two groups for static and dynamic examination, respectively.Thereupon the quasistatic tests group 1) were performed with L1 and L3 and the dynamic tests group 2) with L2, L4 and L5. Concerning the static testing , the scr were per with L2,For the dynamic tests using a servo-pneumatic uniaxial testing machine , a self-developed test set-up was used and the same procedure as in Jarvers et al. was chosen . The cycThe data were compared descriptively using Excel 2016 . Furthermore, the data were examined statistically with the Wilcoxon test and the use of SPSS 24.0 . The level of statistical significance was set at p < 0.05.The mean age of the body donors was 78.55 ± 11.74 years  with a mean BMD of 0,65 ± 0,04 g/cm² and T Score − 3.92 ± 0.38.In the static pull-out tests, all 20 vertebrae were successfully tested with both screws. The L1 and L3 vertebrae showed an average higher pull-out force with the MC screws (535.45 ± 249.85 N) than the TT screws (448.83 ± 319.66 N) (Fig. In the dynamic tests, no signs of failure or loosening were observed in the MC screws. In the TT technique, seven screws loosened before reaching 10,000 cycles, and loosening occurred between 99 and 6,300 cycles. The mean range of motion was only analysed of those screws that reached total 10,000 cycles. The optical measurement resulted in a lower range of motion of the MC screws compared to the TT screws Fig. . The subSpinal stabilizations in osteoporotic patients remain challenging with various instrumentation techniques available. According to the literature , 37, 39,Therefore, alternatives were evaluated. Santoni et al.  first deThe optimal size and length of the screws for use in CBT is a commonly debated topic. Matsukawa et al.  analyzedRecent studies have underlined that longer and deeper placement of CBT screws not only provides biomechanical advantages but also promotes bone fusion , 22, 29.Moreover, these advantages can be relevant in obese patients, as extensive paraspinal dissection can be limited or more cortical bone purchase is desirable such as in osteoporotic patients. Recently both static and dynamic biomechanical studies have validated the superior pullout strength of CBT and MC versus the traditional pedicle screws , 33, 40.In this biomechanical study, the pullout tests demonstrated that the MC screws have a higher pullout force compared to TT screws. Direct pairwise comparison of the two techniques for each vertebra shows advantage for MC technique. The two techniques show a higher pull-out force in 5 cases each, so that no direct advantage can be derived. The main difference was observed in the more practical dynamic tests, where only the TT technology loosened before reaching 10,000 cycles. Out of the 15 screws, 7 loosened between 99 and 6300 cycles. In addition, the successfully tested screws showed a lower range of motion of the MC screws compared to the TT screws, as well as a higher pull-out force of the MC screws. Additionally, dynamic testing provides a better indication of screw stability as it better reflects reality. This point can be illustrated by showing the wiper movement of the screw from the test.In comparison to the TT placement, achieving the correct trajectory in CBT or MC can be challenging for surgeons. The screw path has to be reached in the denser bone with fewer anatomical landmarks available. Intraoperative fluoroscopic support is needed to enhance accuracy and safety apart from high-level surgical skills. Optical navigation and robot-assisted systems are the most alternative advanced technologies achieving high accuracy of screw placement linked with minimal invasiveness. However, the additional time, the radiation exposure and the high cost for installation and maintenance, required space and personnel for operation, must be considered , 26, 32.However, there are some limitations to this study that need to be considered. Firstly, the study sample size is very limited and the varying sample sizes of the individual groups should be critically reviewed. Therefore, a more extensive evaluation using equal sample sizes is desirable to confirm these results. Moreover, the position of the embedded vertebrae is not physiological, but a standardized procedure regarding the literature. Finally, for biomechanical testing, only cadaveric specimens were used, and the results may not necessarily translate to in vivo settings.The highest pullout forces were achieved by the MC technique. The main difference between the techniques was shown in the dynamic measurements where the MC technique was clearly superior to the conventional technique in terms of primary stability. Overall, the MC technique in conjunction with insertion using a template seems to represent the best alternative for anchoring screws in osteoporotic bone without cement.