Traumatic brain injury represents physical damage to the brain tissue that induces transitory or permanent neurological disabilities. The traumatic injury activates an important inflammatory response, followed by a cascade of events that lead to neuronal loss and further brain damage. Maintaining proper ventilation, a normal level of oxygenation, and adequate blood pressure are the main therapeutic strategies performed after injury. Surgery is often necessary for patients with more serious injuries. However, to date, there are no therapies that completely resolve the brain damage suffered following the trauma. Stem cells, due to their capacity to differentiate into neuronal cells and through releasing neurotrophic factors, seem to be a valid strategy to use in the treatment of traumatic brain injury. The purpose of this review is to provide an overview of clinical trials, aimed to evaluate the use of stem cell-based therapy in traumatic brain injury. These studies aim to assess the safety and efficacy of stem cells in this disease. The results available so far are few; therefore, future studies need in order to evaluate the safety and efficacy of stem cell transplantation in traumatic brain injury. Traumatic brain injury (TBI) is the main cause of death in young people living in industrialized countries . TBI is The severity of the injury is clinically identified by the Glasgow Coma Scale (GCS). The GCS is a neurological rating scale used to assess patient’s level of consciousness after a head injury. This scale allows evaluating visual, verbal, and motor functions. The scoring system is based on three types of response to stimuli: best eye-opening (maximum 4 points), appropriate and consistent verbal response (maximum 5 points), and best motor response (maximum 6 points). The sum of these scores gives a score between 3 and 15 . AccordiThe TBI is characterized by a primary lesion directly related to the traumatic event that causes impaired brain function. Within a few hours of the traumatic event, a complex cascade of cellular and molecular events that characterize the secondary lesion is triggered, responsible for further death/necrosis of brain tissue .Today, pharmacological treatments of TBI is somewhat complex. Researchers are still studying new therapeutic strategies in order to reduce neurological damage . RecentlThis review aims to provide an overview of the effectiveness of stem cells in the management of TBI. For this purpose, we have collected the clinical trials and the studies approved by Local Ethics Committees that use stem cell transplantation as a therapeutic treatment in patients with TBI.TBI is defined as an impairment of brain function caused by mechanical damage. It represents one of the main causes of death and disability in individuals aged between 1 and 45 years. The most common causes of TBI include falls, car accidents, assaults, and sports-related injuries . AccordiThe severity of the injury is commonly classified using the GCS score. TBI is defined as mild when patients retain good neurologic function and sequelae can be resolved completely without any treatment. Conversely, moderate or severe TBI can be fatal or cause severe disability which makes the quality of life negligible. Moreover, the injuries are typically classified as open or closed injuries. The open injuries involve a fracture of the cranial theca caused by a foreign object. While the closed injuries are marked by brain damage due to indirect impact without the entry of any foreign object into the brain. Most of the TBIs observed are of the closed type; however, a small percentage is represented by open TBI, generally experienced by gunshot injuries .2+), potassium and sodium [2+ is responsible for activating a series of enzymes such as caspases and calpases that lead to cell death directly or indirectly through the activation of the apoptotic process. Additionally, an increase in the flow of Ca2+ in the mitochondria is also responsible for the production of reactive oxygen species (ROS) [The TBI is characterized by two phases. The primary phase occurs at the time of exposure to external force and causes a mechanical breakdown of brain tissue. While the secondary phase has its onset hours after the traumatic event and represents the main cause of the worsening in the evolution of the TBI ,16. In Td sodium . The inces (ROS) . Oxidaties (ROS) ,21,22. MTherefore, the pathophysiology of TBI is complex, thus management, regardless of gravity, represents a great challenge. The treatment tools currently available for TBI are still not effective, with the exception of some therapies that are limited only to the reduction of secondary symptoms. Therefore, new therapeutic approaches are needed for the treatment of TBI.Currently, interventions to improve the lives of people with TBI in use—including drug treatments, surgeries, and rehabilitation therapy—provide poor outcomes . The useNSCs derived from the lateral ventricle, the dentate gyrus of the hippocampus . MoreoveThe MSCs are multipotent stromal cells that can be easily harvested from a variety of tissue sources such as bone marrow, umbilical cord, adipose tissue, placenta, and oral cavity . Also, MThe BM-MSCs derive from the bone marrow and showed the ability to differentiate into several cell lineage including neurons and glial cells . It was The UC-MSCs are obtained easily by treating the umbilical cord or the cord blood. The UM-MSCs compared to other stem cells showed several advantages such as a low immunogenicity power, less risk of rejection after transplantation, easy harvesting, and no ethical controversy . These cThe AD-MSCs are easily isolated from adipose tissue . These cTo date, studies report that the principally used stem cell routes of administration are the intravenous route and local administration by stereotactic injections . The inthttp://clinicaltrial.gov and the clinical trials approved by Local Ethics Committees.In recent decades, several studies have shown the use of SCs as potential therapies in the treatment of neurological diseases such as TBI. This review provides an overview of the studies recorded on clinicaltrial.gov we carried out the research using the terms “head injury” and “stem cells”. This research led us to find 18 clinical studies. We have considered only clinical trials involving the use of stem cells as a therapeutic treatment for patients with TBI. We excluded clinical studies involving the use of drugs such as erythropoietin that stimulate the production of stem cells or other therapeutic interventions and clinical studies that involved the use of stem cells in diseases other than TBI. Additionally, on PubMed, we looked for studies approved by local ethics committees that evaluated the use of stem cells as a therapeutic treatment in patients with TBI. The keywords used for this research were “head injury”, “stem cells”, “clinical trial”, and “human”.In All the clinical studies  reported below have evaluated the safety and/or efficacy and tolerability of the administration from SCs in the TBI . Most of6 mononuclear cells/kg body weight in the 36 h after the lesions. The safety of the study was determined by monitoring cerebral and systemic hemodynamic both in the collection and transplant phase. Moreover, neurological events—such as seizures, alterations in GCS, stroke, as well as secondary organ injury—in the 12 h following the transplantation and up to 21 days after treatment were evaluated, in order to assess the efficacy of treatment. The results have not yet been posted.A completed phase 1 clinical trial NCT00254722 was performed on 10 patients (aged 5–14 years) with acute TBI (within 24 h) diagnosed by GCS between 5 and 8. The aim of the study was to evaluate both the safety and efficacy of autologous bone marrow progenitor cell (BMPCs) transplantation in children with isolated TBI. The bone marrow samples were collected between 12- and 30-h post-injury at a dose of 3 mL/kg of body weight. The transplantation was performed by a single intravenous infusion of BMPCs at a dose by 6 × 106, 5 × 106, or 10 × 106 SB623 cells. The SB623 were transplanted thought stereotactic intracranial injection. The primary outcome was evaluating the motor functions by using the Fugl–Meyer Motor Scale (FMMS) from baseline to 6 months in SB623 patients compared to the control group. Besides, it was assessed both changes in the Disability Rating Scale (DRS) and gait velocity, as well as an upper limb function from baseline at 6 months. The results of the study showed a significant improvement in the motor function by changes in FMMS at week 24 in patients treated with SB623 versus the control group. Besides, five severe side events were reported in patients treated with SB623 and three in patients in the control group. In contrast, there were no differences in the rate of side events occurred during the treatment in all experimental groups. Finally, the results of this study showed that intracranial SB623 transplantation was safe and effective, thanks to motor function improvements observed 24 weeks after treatment [The double-blind, controlled phase 2 study NCT02416492 has enrolled 61 male and female subjects (aged 18 years to 75 years) with chronic motor deficits secondary to TBI. The aim of the study was to evaluate the effectiveness, safety as well as tolerability in the stereotactic intracranial implantation of allogeneic modified bone marrow-derived mesenchymal stem cells (SB623). The SB623 cells are adult bone marrow cells that are transfected transitory way with a plasmid construct encoding the intracellular domain of human Notch-1. The participants were randomly divided into four groups: patients that had not received the surgical intervention (control group), and patients that received 2.5 × 10reatment .6 cells/mL/kg, 9 × 106 cells/mL/kg, or 12 × 106 cells/mL/kg in 0.9% saline containing 5% of human serum albumin were infused by intravenous injections. Twenty-five participants were assigned in 5 arms: 10 patients received no interventional surgical, 5 were included in control group 1 and 5 in control group 2; this additionally control group was included in order to include participants across the time spectrum of the trials. Five patients received 6 × 106 cells/kg (low dose group), 5 patients received 9 × 106 cells/kg (medium-dose group) and 5 subjects received 12 × 106 cells/kg (high dose group). Neurological events such as seizures, changes in GCS score, cerebral vascular accident (CVA) were assessed both 12 h and 21 days after the infusion. Moreover, were assessed infectious morbidity up to 21 days after infusion and also the global functional status to assess both functional status and overall functioning in the 6 months post-injury. Additionally, inflammatory cytokines were measured in the plasma at baseline and 1 and 6 months after treatment. None of the patients manifested severe side events after collected bone marrow or after BMMNCs transplantation. After cell therapy, no serious pulmonary toxicity occurred in patients treated with a high dose of cells, but these events were not clinically significant. The cell therapy showed structural preservation of critical regions of interest brain tissue. The cytokine biomarker data showed a dose-dependent downregulation of the pro-inflammatory. Specifically, the IL-1β, interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α) pathway show a statistically significant decrease in concentrations compared to the baseline. These results demonstrated that cell therapy, reduce the inflammatory response to injury. Finally, the autologous BMMNCs transplantation in patients with serious TBI is safe and feasible. This BMMNCs administration showed its efficacy in terms of maintenance of critical regions of central nervous tissue, probably due to a reduction of pro-inflammatory signaling [The cohort study NCT01575470 of 1/2 phase has enrolled 25 male and female patients (aged 18 years to 55 years) with acute serious TBI (GCS score 5–8) that showed injury at least 24 h before study start. This study was authorized by the University of Texas Health Sciences Center at Houston Committee for the Protection of Human Subjects and by the Memorial Hermann Office of Research. The goal of this trial was to assess the safety, feasibility, and efficacy of bone marrow mononuclear cells (BMMNCs) transplantation at the escalated doses. After 36 h of injury, from each patient were collected 3 to kg of bone marrow from anterior iliac crests. Before transplantation BMMNCs were tested for quality control including cell viability, nucleated cell count, gram stain, aerobic and anaerobic bacterial and fungal cultures, endotoxin and mycoplasma presence, colony-forming unit test, and multi-parameter flow cytometry analysis for cells characterization. The final doses of 6 × 10ignaling .6 cells/kg body weight followed by a higher dose of 9 × 106 cells/kg of body weight. In contrast, 22 control patients will be undergoing a fake collection of bone marrow, as well as treated with a placebo. The primary outcome is to evaluate the macrostructural and microstructural properties both of gray matter and white matter, as well as the integrity of the regions in the corpus callosum, following the BMMNC autologous transplantation. The subjects will be evaluated by using magnetic resonance imaging (MRI) within 7–10 days at baseline, as well as to 1 and 6 months after lesions. The secondary outcome is to assess whether BMMNCs transplantation improves both functional and neurocognitive deficits, as well as the neuroinflammatory response following TBI. The subjects will be undergoing an evaluation of both size and splenic blood flow, also will be evaluated both inflammatory cytokines and infusion-related toxicity, as well as long-term safety. The toxicity and any complications associated with the infusion will be monitored within 14 days post-transplantation. Besides, both the safety and the results regarding the transplant will be evaluated after 1, 6, and 12 months from the injury. The results of this trial  will make it possible to clarify the efficacy of BMMNCs transplantation.The phase 2 interventional study NCT02525432 expected to recruit 55 male and female patients (aged 18–55 years) with acute TBI diagnosed by GCS between 5 and 8. The aim of this trial will evaluate the efficacy of intravenous BMMNCs transplantation. The patients will be divided into two groups; 33 subjects will be treated with autologous intravenous transplantation of BMMNCs within 48 post-TBI. The transplantation will be started with the lowest dose of 6 × 10The non-randomized study NCT02795052 aims to recruit 300 male and female participants (aged over 18 years) with damage to the central or peripheral nervous system documented by least 6 months, including TBI. The aim of the study is to show if the autologous BM-MSCs transplantation by intravenous and intranasal administration will provide an improvement in neurological functions in patients with some neurological conditions. The participants will be divided into two experimental groups. Patients in group 1 will be treated with BM-MSCs intravenous route. While the participants in group 2 will receive BM-MSCs intravenous and intranasal  route. The primary outcome will be to evaluate daily activities at 3, 6, and 12 months after the administration. The secondary outcome aims to assess deficits of neurologic function. This study is still active and the results have not been published yet.6 cells/kg or 10 × 106 cells/kg weight in the 48 h after lesions. Participants in the placebo group will undergo a fictitious collection of bone marrow and, subsequently, a single intravenous infusion of 0.9% sodium chloride. The primary outcome will evaluate by diffusion tensor MRI the conservation of both white and gray matter in the groups of patients treated and untreated after the trauma. The secondary outcome will measure the conservation of both the white matter and the gray matter in the CNS, as well as the improvement of functional and neurocognitive deficits. Furthermore, will be assessed the safety in the 7 days after treatment. For this trial, no result is available today.The multicenter, randomized, blinded, placebo-controlled study NCT01851083 of phase 1/2 aims to recruit 50 male and female patients (aged 5–17 years) with severe TBI diagnosed by GCS between 3 and 8. The purpose of the study is to evaluate the effects of intravenous infusion of autologous BMMNCs on nervous tissue, as well as to evaluate the efficacy of cell therapy in neurocognitive and neurological function in children with severe TBI. The patients will be divided into two groups. The experimental group will be subjected to the collection of bone marrow and administration of BMMNCs in a single intravenous dose of 6 × 108 of AD-MSCs will be administered three times for 6 weeks. Furthermore, each infusion will be performed 14 days from the other. The primary outcomes will be evaluated by blood tests at baseline, as well as to 6 months and one year after infusion. The secondary outcomes involve an assessment of the macro and microstructural properties of the brain by MRI at baseline and 6 months post-treatment. Moreover, several tests will be performed for both functional and neuropsychological assessments at baseline, after 6 months and 1 year post-transplantation. Finally, will be assessed the inflammatory cytokines by cytometry analysis and monitoring the changes at 6 months and one year from baseline. The data from this trial will be available soon, as the final data collection is expected by August 2020.The single-arm non-randomized study NCT04063215 of phase 1/2 hopes to recruit 24 male and female patients (aged 18–55 years) with sub-acute or chronic TBI (GCS score> 2 and ≤ 6) at least of 6 months. The study aims to evaluate both the safety and the efficacy of autologous AD-MSCs transplantation; moreover, it will evaluate the neurocognitive and neurological function and neuroinflammatory response after treatment. In adult patients, 2 × 10The randomized phase 1/2 study NCT02959294 aims to recruit 200 male and female participants (16–70 years of age) with mild TBI or concussion syndrome. The aim of the study will be to evaluate both the safety and efficacy of adipose-derived stromal vascular fraction (AD-cSVF) in patients with TBI and concussion syndrome. The patients enrolled in the intervention arm will undergo a microcannula harvest of AD-cSVF. The primary outcome referring to the measurement of side events at baseline and 6 months after treatment. Besides, will be assessed the clinical symptoms associated with TBI and concussion such as recurrent headaches, amnesia, behavioral change, cognitive impairment, and recurrent sleep disturbances. The secondary outcomes will be evaluated through Beck’s questionnaire, in order to evaluate depression conditions. Furthermore, will be evaluated attention deficit and brain changes by MRI at baseline, 3 years, and 5 years after treatment. The results expected from this study will allow observing if the administration of AD-cSVF will be efficacy and safe for the long-term.The phase 1 study NCT02742857 will be recruiting 20 male and female patients (15 years to 65 years) which present brain death caused by TBI with axonal injury shown by MRI. The study was designed using different therapeutic interventions including MSCs transplantation, infusion of intra-thecal bioactive peptides, transcranial laser therapy, and median nerve stimulation. The aim of the study is to show the possibility of cell inversion in the case of brain death induced by TBI with diffuse axonal injury. The primary outcome will be documented by clinical examination or electroencephalogram in the 15 days’ frame time. The secondary outcome is to provide a cerebrospinal fluid analysis, as well as the count and microbial evaluation. Furthermore, by using the MRI examination, both the presence of aseptic or bacterial meningitis, as well as any changes will be shown. Also, will be assessed blood pressure, pulse rate, oxygen saturation, as well as changes in breathing in the 15-day time frame. To date, no superior information is yet available. Final data collection is expected by June 2022.In this section, clinical trials published in indexed journals have been described. Many of these studies report the results obtained on treatment with stem cell therapy and provide important efficacy and safety data.7 SCs, via lumbar puncture in the lumbar 3–4 or 4–5 intervertebral space. This surgical intervention was performed four times in 5–7 days. To evaluate the efficacy of the treatment, the motor function of the limbs, the sensation, the balance and the ability to live independently, were assessed at the baseline and after 6 months of treatment . Prior to treatment, the two groups of participants showed equivalent baseline scores. After six months of treatment compared to the control group, patients treated with UC-MSCs showed a statistically significant improvement in motor function, sensitivity, and balance. The transplants patients, compared to the control group, also showed an improvement in mobility, locomotion, communication and ability to live independently. Four patients 48 h after transplantation experienced low intracranial pressure reactions with symptoms such as mild dizziness and headache. These adverse events resolved after lying down in bed or after being treated with intravenous saline infusions. In addition, during transplantation, clinical values—such as body temperature, oxygen saturation, blood pressure, and respiratory and heart rate—were monitored in all patients. No obvious anomalies were found in any subject. In conclusion, the results of this study demonstrate that the transplantation of UC-MSCs can be considered safe and can significantly improve several neurological parameters.The study conducted by S. Wang et al.  aimed toThe retrospective cohort study conducted by G.P. Liao et al.  aimed to6 cells/mL were administered by lumbar puncture between the lumbar vertebrae L3/L4 or L4/L5. In order to evaluate the efficacy of transplantation, 40 days after surgical intervention, the participants were followed-up for persistent vegetative state assessment and motor function analysis. None of the participants experienced severe side events. After two days of transplantation, 5 subjects showed transient fever and 2 participants perceived a slight headache. After 14 days of BM-MSCs transplantation. Thirty-eight of 97 participants showed enhancement of the TBI condition. Eleven of 24 participants before transplantation showed a vegetative state after treatment manifested enhancements in consciousness. Twenty-seven of 73 patients with motor dysfunction showed an enhancement in motor activity. Moreover, the age of participants affected the finding of cell transplantation, demonstrated that the young subjects enhancements the TBI state more than older patients. In conclusion, this study demonstrated that BM-MSCs transplantation is safe and effective in the management of serious TBI; furthermore, the early intervention in the subacute phase of this disorder showed better outcomes.This interventional cohort study conducted by C. Tian et al.  aimed toTBI is a very complex disease. Nowadays, there are no effective treatments able to reduce the effects of the primary injury, but only therapies capable of blocking their progression. In recent decades, NSCs and MSCs , have demonstrated to be a useful tool that can reduce the effects of post-traumatic brain injury. In several of the clinical studies described, BM-MSCs administrated via intravenous and via lumbar puncture, shown improvement in damaged brain areas. The same results were obtained using the UC-MSCs transplantation; however, the number of studies is fewer. Therefore, the available results encourage the use of both SCs and therapies described as useful for the treatment of TBI. The awaited outcomes and future studies will be necessary to exploit the use of cell transplantation for management TBI.


Stimulation produced stimulus-locked motor responses but did not elicit behavioral seizures either during or after stimulation. After as little as 10 min of hfrTMS, immunostaining using phospho-specific antibodies for the phosphorylated form of ribosomal protein S6 (rpS6) revealed robust induction of rpS6 phosphorylation in large numbers of neurons in the cortex, especially the piriform cortex, and also in thalamic relay nuclei. Quantification revealed that the extent of the increased immunostaining depended on the number of trains and stimulus intensity. Of note, immunostaining for the immediate early genes Arc and c-fos revealed strong induction of IEG expression in many of the same populations of neurons throughout the cortex, but not the thalamus. These results indicate that hfrTMS can robustly activate molecular pathways critical for plasticity, which may contribute to the beneficial effects of TMS on recovery following brain and spinal cord injury and symptom amelioration in human psychiatric disorders. These molecular processes may be a useful surrogate marker to allow optimization of TMS parameters for maximal therapeutic benefit.Patterns of neuronal activity that induce synaptic plasticity and memory storage activate kinase cascades in neurons that are thought to be part of the mechanism for synaptic modification. One such cascade involves induction of phosphorylation of ribosomal protein S6 in neurons due to synaptic activation of AKT/mTOR and via a different pathway, activation of MAP kinase/ERK1/2. Here, we show that phosphorylation of ribosomal protein S6 can also be strongly activated by high frequency repetitive transcranial magnetic stimulation (hfrTMS). HfrTMS was delivered to lightly anesthetized rats using a stimulation protocol that is a standard for inducing LTP in the perforant path  Transcranial magnetic stimulation (TMS) is increasingly being used as a therapeutic intervention to enhance recovery following brain and spinal cord injury . TMS is To further explore the idea that the therapeutic benefits of TMS are due to enhancement of synaptic plasticity, one approach is to assess whether TMS can activate molecular pathways that have been implicated in activity-dependent plasticity. In this regard, signaling pathways involving kinase cascades are of particular interest because they can be assessed using immunocytochemical markers that identify neurons in which molecular cascades are activated. For example, recent studies have revealed that induction of LTP and learning experiences robustly activate phosphorylation of ribosomal protein S6 in neurons . AlthougImmunostaining with phospho-specific antibodies provides a convenient tool to identify the populations of neurons in which S6 phosphorylation is activated. S6 phosphorylation is activated within minutes after a triggering event and can persist for hours . BecauseDifferent antibodies are available that recognize different phosphorylation sites on S6 (Ser235-236 vs. Ser240-244). This provides an important tool to begin to identify upstream kinase pathways that are driving S6 phosphorylation. For example, phosphorylation at Ser240-244 is thought to be via mTOR-dependent signaling whereas phosphorylation of Ser235-236 can be via multiple signaling pathways including MAPK/ERK, PI3 kinase and mTOR .Based on the rationale supported by these previous data, the present study defines the pattern of activation of S6 phosphorylation as a consequence of high frequency TMS in rats. To link to previous mechanistic studies, we use the same pattern of stimulation that we have used in previous studies of synaptically-driven S6 phosphorylation in the dentate gyrus. Using phospho-specific antibodies for different phosphorylation sites (Ser235/236 and Ser240/244) for immunocytochemistry, we show that high frequency repetitive TMS (hfrTMS) at 400 Hz strongly activates S6 phosphorylation at both Ser235/236 and Ser240/244 in select populations of neurons in the cortex, especially the piriform cortex, as well as in thalamic relay nuclei. The extent of activation by hfrTMS is comparable to what can be achieved by direct electrical stimulation of the cortex at the same frequencies. Of note, comparisons of patterns of immunostaining for the immediate early genes Arc and c-fos reveal that both S6 phosphorylation and IEG expression are activated in many of the same populations of cortical neurons whereas thalamic neurons exhibit robust S6 phosphorylation with minimal if any induction of IEG expression. Taken together, our results expand understanding of the neuronal populations that are affected by TMS and document that TMS does activate kinase cascades that are strongly implicated in activity-dependent synaptic plasticity.Experiments involving hfrTMS were carried out at Oita University and were approved by the Oita University Ethical Review Committee. Experiments involving electrical stimulation of the cortex were done at the University of California Irvine (UCI) and were approved by the Institutional Animal Care and Use Committee (IACUC) at UCI.ad libitum food and water. The entire study was composed of 2 separate experiments involving 64 animals  that were housed at controlled room temperature (24.5–25.0°C) with a 12/12 h light/dark cycle and To deliver hfrTMS, rats were lightly anesthetized with urethane  10 min before starting the hfrTMS as previously described in n = 8) were anesthetized and placed in a stereotactic frame (n = 3) were prepared similarly and received direct electrical stimulation of the motor cortex. For this, a craniectomy was done over the motor cortex and stimulating electrodes spaced 1 mm apart were positioned at different locations in the motor cortex. Electric stimulation yielded mMEPs from the forelimb muscle when the motor cortex was stimulated 2 mm anterior, 2–3 mm lateral to bregma.Preliminary studies were undertaken to test three different size coils  delivering magnetic pulses at 1.2 the motor threshold (MT) of the motor evoked potentials (MEPs). MT was determined by decreasing stimulator output by 1% machine output until MEPs disappeared and then increasing the output in 1% increments until six MEPs of 50 μV peak-to-peak were elicited out of every 12 trains of single monophasic wave pulses. For this study, rats  connected with a specially designed combining module  at 10 s intervals for 60 or 180 hfrTMS trains. We chose this stimulation paradigm so as to link to our previous studies documenting activation of S6 phosphorylation in the dentate gyrus, which dissected signaling pathways and explored molecular mechanisms , 2017. In = 3). Rats were perfused with 4% paraformaldehyde, brains were sectioned in the coronal plane, and sections were stored in buffer prior to immunostaining.To evaluate the time course of increases in rpS6 expression, rats that had received 60 (10 min) stimulus trains were killed humanely by anesthetic overdose 15, 30, 60, 120, 180, and 360 min after the initiation of the stimulation (3 rats per condition). Rats that received 180 (30 min) stimulus trains were killed 30, 60, 120, 180, and 360 min after the initiation of the stimulation. Measures were compared with controls that received no stimulation  were anesthetized and positioned on the same stereotactic frame as hfrTMS animals but the stimulation coil was positioned 8 cm above the rat’s head so that no stimulation was delivered, but the click sound associated with each pulse was still present. Rats were euthanized 30 min post-sham stimulation.Rats in the sham stimulation-treated control group  of stimulus trains at different stimulator output 0, 18.75, 25, 50, 75, and 100% of machine output were killed humanely by anesthetic overdose 30 min post-stimulation , and prepared for immunohistochemistry for rpS6.In vivo physiological experiments involved adult Fischer rats of both sexes. Rats were anesthetized with urethane and placed in a stereotaxic apparatus. The scalp was incised and machine screws were placed in burr holes at 2.0A, 2.0L and 4.0P, 3.0L from bregma. Leads from a stimulus isolation unit were attached to the screws, stimulus intensity was set so that single pulses elicited forepaw twitch on the side contralateral to the stimulation, and then 400 Hz trains (8 pulses per train) were delivered every 10 s for 10 min (480 pulses in 60 trains). Rats were allowed to survive for 20, 30, or 40 min after the initiation of stimulation, and then received Fatal Plus and were perfused with 4% paraformaldehyde. Brains were prepared for IHC as below. Data from these animals have also been included in another study  for 1–2 h followed by washes and incubation in ABC-HRP . For the mouse monoclonal antibody, the secondary antibody was a horse anti-mouse IgG used at a dilution of 1:100 in 5% normal horse serum. For the rabbit polyclonal antibody against c-fos, the secondary antibody was donkey ant-rabbit IgG, was used at a dilution of 1:100 in normal goat serum. Sections were then washed with TBS, mounted on 0.5% gelatin subbed slides and cover slipped with Vectashield®.For immunofluorescence, free-floating sections underwent the same antigen retrieval procedure described above except that following incubation in ABC-HRP, sections were stained by catalyzed reporter deposition (CARD) amplification using tyramide-Cy3 as the substrate. Primary antibodies were phospho-S6 ribosomal protein-ser235/236 , Arc antibody , Secondary antibodies were as above. Following the amplification of Cy3 by CARD, sections were washed in TBS, mounted on 0.5% gelatin subbed slides and cover slipped with VectashieldFor quantitative assessment of immunostaining, optical density (OD) measurements were taken across the cortex using an M4 Microcomputer Imaging Device (MCID), Imaging Research. Digital images were collected at 400×. The light intensity was adjusted so that areas exhibiting background levels of labeling  were just above threshold, whereas areas exhibiting maximal levels of labeling  were within the measuring range. Then, a series of OD measurements were taken across all layers of the cortex from the cortical surface to the white matter with a 20 μm × 20 μm measuring frame see . A row opost hoc analysis . Experiments with three or more groups were analyzed with a two-way ANOVA, followed by the post hoc Bonferroni-Dunn test. Differences were considered significant at P ≤ 0.05.All data are represented as mean ± standard error of the mean (SEM). Different groups of animals were compared using one-way (two-way for time course) ANOVA with the Student-Newman-Keul Previous studies have documented that S6 phosphorylation is activity- and experience-dependent. For example, S6 phosphorylation is robustly induced with strong synaptic activation as with induction of perforant path LTP and by behavioral experience , 2017. PIn rats perfused after anesthesia and sham stimulation, overall levels of immunostaining for rpS6 at p-Ser235/236 were low. There were relatively few p-Ser235/236 positive neurons in most brain regions including the cerebral cortex . The oveNevertheless, certain populations of neurons did exhibit moderate to high levels of immunostaining under anesthesia . For exaAs described elsewhere , some neThe pattern of immunostaining was somewhat different for p-Ser-240/244 although many of the same populations of neurons were labeled . For exaWe note here only some aspects of differential labeling under resting conditions.The selective staining of certain populations of neurons is of course of interest, but full description would require a manuscript of its own. In addition, as we and others have reported elsewhere, there is activation of phosphorylation of different populations of neurons as a consequence of behavioral experience, including simply removing the animal from its home cage. Thus, full descriptions of differential staining would have to include animals that were killed under different conditions.To provide data against which to compare results of TMS, we first assessed consequences of unilateral electrical stimulation of the cortex using patterns of stimulation that are typically used to LTP in perforant path projections to the dentate gyrus . We chose this paradigm to link to our previous studies documenting that this pattern of stimulation induces robust phosphorylation of ribosomal protein S6 in target neurons in the dentate gyrus , 2017. RActivation of S6 phosphorylation was completely blocked in rats that were anesthetized with ketamine/xylazine that received 400 Hz electrical stimulation (data not shown). This strongly suggests that activation of phosphorylation is due to synaptic activity and NMDA-receptor activation consistent with previous findings for activation of S6 phosphorylation in dentate granule cells with HFS of the perforant path .Our previous studies of activity-dependent S6 phosphorylation in the dentate gyrus have shown that intense synaptic activity induces immediate early gene (IEG) expression in many of the same neurons in which S6 phosphorylation is activated . To asseSurprisingly, the pattern of induction of IEG expression was different than what was seen with pS6 activation, and also differed between the two IEGs. Unilateral cortical stimulation induced c-fos expression in virtually all areas of the cortex on the side of the stimulation including areas in the dorsal cortex such as the cingulate cortex and sensorimotor cortex  and ventAlthough c-fos was induced in large numbers of neurons in cortical layers II-IV and layer VI, there were very few c-fos positive neurons in layer V . To docuThe pattern of induction of Arc expression was similar to that of c-fos with a few exceptions. As with c-fos, there was robust induction of Arc protein throughout the cortex on the side of the stimulation with large numbers of neurons in cortical layers II-IV showing induced expression. In contrast to c-fos, there were also numerous Arc-positive neurons in layer V . Higher During high frequency magnetic stimulation, there were visible muscle contractions associated with each stimulus train . It is likely that the effective depth of stimulation with hfrTMS may vary across cases leading to variability in activation in deep brain structures like the hippocampus. There were no obvious areas of damage in any brain region, although sections were not stained for H&E.Notably, there were also increases in immunostaining for p-Ser-S6-235/236 in small cells in layer I of the cortex with the morphological appearance of astrocytes . At 6 h, immunostaining of astrocytes was no longer evident . In thisGenerally, the results seen when immunostaining for p-Ser-240/244 were similar to what was seen with p-Ser-235/236. The main exception was that astrocytes were not obviously labeled for p-Ser-240/244 .To quantify increases in immunostaining for rpS6 p-Ser-235/236 over time and in response to different stimulation paradigms, we first quantified levels of immunostaining across cortical layers by assessing optical density (OD). P = 0.0003). Post hoc comparisons by SPSS  revealed that levels were significantly higher than unstimulated control at all time points except 6 h . Activation was less dramatic in rats that received stimulation at less than 50% output . Thus, there appeared to be a sharp change in the extent of rpS6 phosphorylation between 25 and 50% stimulus intensity.We assessed whether activation could be increased by increasing stimulus intensity. With direct cortical stimulation, the pattern of rpS6 in individual neurons is different from what is seen with IEG transcription (c-fos). To assess whether this differential pattern is also seen with hfrTMS, we compared the pattern of labeling for c-fos and Arc protein with what is seen for rpS6 (p-Ser-235/236) . We focuOur results demonstrate that non-invasive hfrTMS strongly induces phosphorylation of rpS6 and activates IEG expression in widespread areas of the cortex. The fact that the pattern of activation is similar to what is seen with high frequency electrical stimulation of the dorsal surface of the cortex suggests that S6 phosphorylation is triggered by propagated synaptic activity rather than directly by the currents generated by the stimulation. In what follows, we discuss the approach and possible mechanisms and functional significance.in vivo. Previous studies document that magnetic stimulation can indirectly modulate neuronal gene expression  and ventral regions . A similar pattern was seen with direct electrical stimulation of the dorsal surface of the cortex. In the case of direct electrical stimulation, widespread activation throughout the cortex ipsilateral to the stimulation suggests that activation is not driven directly by currents generated at the site of stimulation. The most likely interpretation is that HFS triggers bursts of synaptic activation that propagate through the cortex. This interpretation is supported by previous studies that documented that HFS at 400 Hz that induces perforant path LTP induces rapid and prolonged phosphorylation of rpS6 in dentate granule cells that are completely blocked by local delivery of NMDA receptor antagonists . Also, oThe important implication of our results is that even though TMS causes focal neuronal activation related to the exact pattern of current flow, there can be widespread molecular consequences, presumably driven by propagated synaptic activity. This provides a provocative explanation for the fact that functional consequences of hfrTMS that outlast the time of stimulation and may involve activation of plasticity-related signaling pathways at long distances from the actual areas of magnetic current flow.Increases in immunostaining for pS6 indicate activation of phosphorylation of S6 protein, but there may also be increases in levels of ribosomal protein S6 (rpS6) itself due to the stimulation. For example, it has been reported that induction of LTP in hippocampal slices via delivery of brief 100 Hz trains is accompanied by protein synthesis-dependent increases in levels of rpS6 protein 30 min post-stimulation . In contOne caveat for our interpretations is that the actual patterns of magnetic current flow and resulting eddy currents are un-defined. Also, the patterns of neuronal activity that are induced are unknown, and may involve differential activation of neurons of different size or structure. Indeed, hfrTMS could activate inhibitory as well as excitatory circuits, so selective patterns of phosphorylation in different neuron groups could reflect the interplay between excitation and inhibition. Determining patterns of activation is technically challenging because with the current coils, the magnetic current flow occurs over a wide area, which interferes with physiological recording during the stimulation. In this regard, using activity-dependent markers such as S6 phosphorylation and IEG induction may actually provide a useful proxy measure.Following hfrTMS, small cells in layer I of the cortex with the morphology of astrocytes also stained for rpS6-235/236 but not rpS6240-244. This is noteworthy, because in our previous study involving HFS of the perforant path, there was no activation of rpS6 phosphorylation in astrocytes even when HFS was delivered for 2 h. In contrast, there was striking activation of S6 phosphorylation in astrocytes by a learning experience . ImportaThe mechanisms underlying rpS6 phosphorylation in astrocytes remain to be defined. One possibility is direct activation of ionic currents in astrocytes at layer I because eddy currents triggered by TMS may be preferentially induced in superficial layers of the cortex. Another is that hfrTMS-triggered neuronal activity leads to the release of neurotransmitters that activate astrocytes via different intercellular signaling cascades than are activated by HFS of the perforant path. Phosphorylation at ser235/236 is activated by multiple signaling pathways including MAPK/ERK  and PKA HfrTMS also strongly induced expression of the immediate early genes c-fos and Arc in cortical neurons. Arc appeared to be induced in the same populations of neurons in which rpS6 phosphorylation was induced. However, c-fos was expressed in neurons with a somewhat different intracortical distribution. Further studies will be required to fully define differences in patterns of activation.With hfrTMS, activation of S6 phosphorylation is especially prominent in large neurons in layer V in the sensorimotor cortex, which are the cells of origin of the corticospinal tract (CST). The degree of activation of rpS6 phosphorylation is comparable to that seen with conditional deletion of PTEN, which enables cortical motoneurons to regenerate their axons after SCI . With PTc-mediated caspase 9 activation in vitro  enhances regenerative growth of corticospinal tract (CST) axons in the spinal cord and recovery of motor function . The mecAlthough direct electrical stimulation of the brain to promote motor recovery after SCI and other injuries to the motor system is potentially translatable, it would require neurosurgical implantation of stimulating electrodes. From the point of view of translatability, it would be better if it was possible to use non-invasive techniques such as hfrTMS.One limitation, however, is that optimal parameters for TMS delivery are unknown. To assess TMS or any other intervention, it would be extremely valuable to have surrogate markers for the beneficial effects of functional stimulation. Surrogate markers are important because it would be extremely inefficient to test different stimulation paradigms by measuring regeneration or behavioral recovery because assessing either regeneration or recovery requires months to complete. To understand the underlying mechanisms and to verify compatibility with human and animal results, further experiments involving other patterns of stimulation such as theta burst stimulation will be informative. Safety may be a limitation because to our knowledge, 400 Hz-hfrTMS has never been employed in humans . CompariImmunocytochemical studies of phosphorylation of S6 and activation of IEG expression may offer considerable advantages for future studies because the assays are straightforward and these are molecular pathways that are involved in synaptic plasticity and regenerative axonal growth. S6 phosphorylation may be especially relevant because it is a downstream marker for activation of multiple signaling pathways in neurons including mTOR, MAPK/ERK, and PI3 kinase . The preThe degree of activation of rpS6 phosphorylation shown here is similar to what is seen with PTEN deletion, which enables neurons to regenerate their axons after spinal cord injury . In thisAll datasets generated for this study are included in the article/The animal study was reviewed and approved by Ethical Committee of the School of Medicine, Oita University.MF and OS designed the research and wrote the manuscript. MF, OS, and KY performed the research. MF analyzed the data.OS was a co-founder and has economic interests in the company “Axonis,” which holds a license on patents relating to PTEN deletion and axon regeneration. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Cryocrystallography, which is commonly used in macromolecular crystallography, may sometimes reduce the quality of diffraction data and the visibility of crystals owing to frost adhesion. A device has been developed to remove frost by drizzling liquid nitrogen over the crystals, which enabled noise reduction of diffraction images and the centering of crystals with low visibility owing to frost adhesion. Cryocrystallography is a technique that is used more often than room-temperature data collection in macromolecular crystallography. One of its advantages is the significant reduction in radiation damage, which is especially useful in synchrotron experiments. Another advantage is that cryopreservation provides simple storage of crystals and easy transportation to a synchrotron. However, this technique sometimes results in the undesirable adhesion of frost to mounted crystals. The frost produces noisy diffraction images and reduces the optical visibility of crystals, which is crucial for aligning the crystal position with the incident X-ray position. To resolve these issues, a computer-controlled device has been developed that drizzles liquid nitrogen over a crystal to remove frost. It was confirmed that the device works properly, reduces noise from ice rings in diffraction images and enables the centering of crystals with low visibility owing to frost adhesion. The dewar tank is used to store LN2 and is equipped with a heater unit and a solenoid valve. To blow out LN2, the heater is turned on and the valve is closed by the increasing gas pressure inside the dewar . The dewar also has upper- and lower-limit fluid-level sensors that are used when refilling the LN2. The LN2 is blown out through an insulated transfer tube and is transferred to an LN2–gas separator. The blowout contains nitrogen gas, which causes intermittent LN2 flow. Therefore, the nitrogen gas should be discarded using the separator, which is made of a cylindrical aluminium tube with a filter plate with four 0.7 mm diameter holes . Only LN2 flows through these holes and passes down to the next LN2 drizzling nozzle, and the gas flowing out from the LN2 passes through the top hole of the separator tube . The gas is then completely removed by the funnel-shaped top of the LN2-drizzling nozzle  and LN2 is delivered to the sample position. The distance from the tip of the LN2-drizzling nozzle to the sample position is 15 mm.The device consists of four major components, as shown in Fig. 1ar Fig. 1b. The des Fig. 1c. Only be Fig. 1d. The ge).This device is designed to be compact for installation without interfering with the sample position, diffractometer and other beamline devices such as the cryostream nozzle, beam stopper and collimator Fig. 1e.In the development of this device, it was important to avoid blowing nitrogen gas, which can cause a temperature increase at the sample position. As mentioned previously, only LN2 is able to drizzle onto the sample by completely relieving pressure using the two stages of the LN2–gas separator and LN2-drizzling nozzle. The LN2 supply parts  were procured from Taiyo Nippon Sanso Corp., Tokyo, Japan. The LN2–gas separator and LN2-drizzling nozzle are both standard metalwork products and cost a total of approximately 1000 dollars.2.2.BSS . This type of frost, powder-like frost with dimensions of approximately a few micrometres, often occurs during long-term storage in dry shippers and might also occur at low temperatures and lower supersaturation conditions according to the Nakaya snow-crystal morphology diagram .To evaluate the effect of frost adhesion in data collection, diffraction data were collected from the same crystal before and after frost removal using the device. The measurements were conducted under the same conditions as those reported for beamline BL26B2 at SPring-8 . This was performed inside a cool workbench, which was operated at 277 K and located beside the beamline experimental hutch (Baba c).The diffraction images were taken with the same oscillation range in both data sets, as shown in Fig. 3XDS  changed from 17.66 to 18.25 in the overall statistics  there is no diffraction from the attached frost and/or dust, improving the experimental data, and (ii) the visibility in sample photographs is improved. It is also suggested that the device can contribute to the success rate of crystal centering during automatic measurements.Furthermore, the computer-controlled automation of LN2 drizzling (i) improves experimental throughput by shortening the operation time of drizzling and (ii) enables the implementation of LN2 drizzling in remote experiments.et al., 2013We started the development of this device in 2016, and it is currently available on SPring-8 MX beamlines BL26B2, BL41XU (Hasegawa Even though some types of frost may not be completely removed, as stated above, the challenge for the future is to apply more pressure in the LN2 dewar to increase the flow rate of LN2. For this purpose, it will be necessary to ensure the safety of pressurization, to prevent dew condensation in the flow path and to prevent nitrogen gas from being blown out of the drizzling nozzle.


Impaired cerebrovascular function is an early biomarker for cerebral amyloid angiopathy (CAA), a neurovascular disease characterized by amyloid-β accumulation in the cerebral vasculature, leading to stroke and dementia. The transgenic Swedish Dutch Iowa (Tg-SwDI) mouse model develops cerebral microvascular amyloid-β deposits, but whether this leads to similar functional impairments is incompletely understood. We assessed cerebrovascular function longitudinally in Tg-SwDI mice with arterial spin labeling (ASL)-magnetic resonance imaging (MRI) and laser Doppler flowmetry (LDF) over the course of amyloid-β deposition. Unexpectedly, Tg-SwDI mice showed similar baseline perfusion and cerebrovascular reactivity estimates as age-matched wild-type control mice, irrespective of modality (ASL or LDF) or anesthesia . Hemodynamic changes were, however, observed as an effect of age and anesthesia. Our findings contradict earlier results obtained in the same model and question to what extent microvascular amyloidosis as seen in Tg-SwDI mice is representative of cerebrovascular dysfunction observed in CAA patients. Cerebral amyloid angiopathy (CAA) is a neurovascular disease characterized by accumulation of the amyloid-β peptide in the brain vasculature, which ultimately leads to stroke and cognitive decline . In bothMouse models of cerebral amyloidosis are invaluable tools for testing safety and effectiveness of greatly needed novel therapies for amyloid-β-related diseases, including CAA. However, for the results to be translatable to the clinic, it is essential that the amyloidosis model shows similar structural and functional phenotypes as the patient. The transgenic Swedish Dutch Iowa (Tg-SwDI) mouse model is an amyloidosis model expressing low levels of the human amyloid-β precursor protein (APP) gene with three familial mutations, of which the Dutch and Iowa mutations are located in the amyloid-β coding region of APP . The neuHere, we therefore used the noninvasive arterial spin labeling (ASL)-magnetic resonance imaging (MRI) technique to study the cerebrovascular function in the Tg-SwDI brain. With ASL, arterial blood is magnetically labeled and used as endogenous tracer flowing into the tissue of interest, which is most often the brain. The distribution of the label over the different brain regions reflects local tissue perfusion and can be converted into absolute cerebral blood flow (CBF) values, expressed as mL/100 g/min. When combined with a hypercapnic challenge, both CBF and CVR can be determined for different brain regions. A known unfavorable characteristic of ASL is a possible underestimation of CBF in case of slow flow. In that case, a delayed arterial transit time (ATT) – the time that it takes for the label to travel from the labeling plane to the brain tissue – could be misinterpreted as decreased CBF. ATT evaluation is therefore valuable to prevent a potential underestimation of the ASL-based CBF estimates, as well as indicative in itself of vascular pathology .The noninvasive nature of ASL was fully exploited in our study with the use of a longitudinal study design in which CBF and CVR were repeatedly measured during increasing amyloid-β accumulation in the brain vasculature of Tg-SwDI mice. As it is conceivable that high microvascular amyloid burden could lead to delayed ATT in Tg-SwDI mice, an ATT measurement was added to the protocol by means of a modified ASL sequence optimized to capture the inflow of the tracer into the brain tissue . To allo2(3)=13.00, p=0.005 and χ2(3)=8.49, p=0.037, respectively. Post hoc analysis indicated that this was attributable to a decrease in baseline CBF between the ages of 3 and 6 months. Between these time points, median (iqr) CBF significantly decreased from 155 (143–159) to 121 (112–124) mL/100 g/min in WT mice, p=0.008. In Tg-SwDI mice, a similar trend was observed, from 147 (133–151) to 126 (103–135) mL/100 g/min, p=0.036, but this was not significant . From 6 months of age, the baseline CBF remained stable inside each group. Age also had a significant effect on CVR in WT mice, χ2(3)=8.33, p=0.040. No differences were observed with post hoc analysis, however, besides a trend toward increased CVR between the ages of 3 and 6 months, from 13 (7–17) to 31 (23–37) %, p=0.038. Age had no significant effect on CVR in Tg-SwDI mice, χ2(3)=6.94, p=0.074. Additional analysis in cortical and thalamic areas did not reveal any difference between WT and Tg-SwDI mice either  baseline ATT values in the mid-brain were also similar for both WT and Tg-SwDI mice, i.e. 206 (186-240) milliseconds (ms) and 223 (202–246) ms respectively at 12 months of age . The hyp2 absorption, as the tc-pCO2 responses to the hypercapnia challenges only increased from an average of 15 mmHG during isoflurane to an average of 19 mmHG during U and A  anesthesia. This was done at 12.3 months, 10 days after the last MRI measurement under isoflurane. The change of anesthesia protocol resulted in profound hemodynamic changes: baseline CBF was markedly reduced and the hypercapnic response was higher in amplitude, but also slower , and mor U and A .An additional smaller second cohort of mice was used to cross-validate our MRI findings with the previously used LDF readout as imaging modality to assess cerebrovascular function in this mouse model . After aLastly, the brain tissue was stained for amyloid-β to assess the degree of pathological burden. All Tg-SwDI mice developed extensive amyloid-β plaque pathology by the end of the experiment, with mainly diffuse parenchymal plaques in the cortex and microvascular plaques in the hippocampus and thalamus, but none of the WT mice displayed any amyloid-β deposition .2 challenge, allowing to measure absolute CBF as well as CVR in the entire brain. The ASL-MRI perfusion data were acquired longitudinally using a low-level isoflurane anesthesia protocol to capture the dynamics of decreasing cerebrovascular function over increasing microvascular amyloid-β loads. This study setup was substantially different from literature studies, where relative CBF measurements performed using LDF under a terminal anesthesia protocol with urethane and α-chloralose (U and A), after removal of the skull, showed impaired hemodynamics in the Tg-SwDI mouse model , it could be argued that local hematocrit changes during hypercapnia could explain this difference. Indeed, hematocrit has been reported to decrease during hypercapnia in the rat brain , but witaneously  and poinLastly, another finding in our study that is of interest is the observation of edema and the local reduction in CBF and CVR with MRI after craniotomy. This was observed in all animals, while the brain tissue was normal by visual inspection. Normal appearance of brain tissue is therefore not enough to conclude that the brain tissue is healthy when working with acutely prepared cranial windows.In conclusion, this study shows that cerebrovascular function in the Tg-SwDI mouse model of microvascular amyloidosis is preserved up to 12 months of age, despite the high amyloid-β burden observed at that age. This observation was confirmed using two different anesthesia protocols and two different imaging modalities. These observations call into question to what extent microvascular amyloidosis as seen in the Tg-SwDI mouse model is a correct model for cerebrovascular dysfunction as observed in CAA patients, and calls for further research to clear up the discrepancy in results.All the experiments were approved by the local ethics committee  and the national ethical committees  under OZP PE.18.029.002 of AVD116002017859, and the experiments have been reported in compliance with the ARRIVE guidelines .Two cohorts of homozygous Tg-SwDI mice and age- and gender-matched WT controls on a C57Bl/6J background were ordered from the Jackson Laboratory . The first cohort was used for a hypothesis-driven study. Based on a previous report by 2) was monitored  with a neonate probe attached to the previously shaved skin on the right flank of the mouse  were performed in all three directions for consistent planning across animals of the subsequent pseudo-continuous ASL (pCASL) scans. The coronal slice package with 21 slices was additionally used for quantification of the brain volume. The pCASL scan protocol was similar as in 2 resolution and 1.5 mm slice thickness (no slice gap). 7.5% CO2 was administered to the mouse from minute 7 till minute 14. For the last imaging session (12.3 months) of the mice in the first cohort, the number of dynamics of the pCASL scan was extended to 767 (scan duration of 90 min) to also capture the effect of switching from isoflurane to U and A anesthesia on CBF. Specifically, the following steps were performed during the 1.5 hr ASL scan: at 5 min, U and A was injected, between minutes 40 and 50 isoflurane was decreased from 1.75% till 0% and between minutes 72 and 79, 7.5% CO2 was administered. At the fourth scan session (12 months), two time-encoded pCASL (te-pCASL) sequences were additionally acquired per mouse for measuring the ATT. The second te-pCASL sequence was performed while administering 7.5% CO2 to the mouse, which allowed measuring the effect of arterial pCO2 elevation on the ATT. The scan parameters of the te-pCASL sequence were the same as in 2 to increase the signal-to-noise ratio (SNR). A three slice SE-EPI readout was used, with the same slice orientation as slices 1, 3, and 5 of the standard pCASL scan (thus with a slice gap of 1.5 mm). During every imaging session, for CBF quantification purposes, the T1 of the tissue (T1t) and the tissue magnetization (M0t) were estimated by collecting an additional inversion recovery scan with the same five slice SE-EPI readout as the pCASL scans. Furthermore, a pCASL flow-compensated FLASH was acquired at the level of the carotids, 3 mm downstream of the labeling plane, to measure the labeling efficiency (α). An additional T2-weighted (T2W) RARE anatomical sequence was acquired with the same slice orientation as that of the pCASL scan for registration purposes.MRI acquisition – a 7 T Pharmascan MRI scanner  with a 23 mm transmit-receive volume coil was used. After proper placement of the mouse head in the bore was confirmed with a scout scan, three standard Bruker T2-weighted RARE scans . Both probes were placed at the somatosensory cortex, one directly on the exposed skull above the left hemisphere, the other approximately 0.5 mm above the exposed brain tissue of the right hemisphere . Extraction of the LDF signal profile was done through DASYLab software .After the last MRI measurement, a subgroup of mice (6 Tg-SwDI and 6 WT) was i.v. injected with 200 µL of DyLight-594-coupled lectin  in the tail vein for staining of the endothelium. This was followed 3 min later by an i.p. overdose of pentobarbital after which the mouse was transcardially perfused with 20 mL of ice-cold PBS and 20 mL of ice-cold 4% PFA. The brain was isolated and fixed overnight in 4% PFA. The tissue was thereafter preserved at 4°C in PBS with 0.02% sodium azide until further processing. The other mice followed the exact same steps, but without the i.v. lectin injection.T2W processing – One full-brain volume of interest (VOI) was manually drawn on one of the 21-slice T2W RARE scans, after which the VOI was propagated to the T2W RARE scans of the other data sets (both to other mice and to other time points) using the EVolution algorithm . To deri1b is the longitudinal relaxation time of blood, that is, 2230 ms at 7 T .The fixed brains were embedded in paraffin, and subsequently cut in sections of 5 µm. After deparaffinization with xylene and rehydration through graded ethanol series, the slides were cooked for 20 min in citrate buffer for antigen retrieval. Slides were then stained overnight at 4°C with anti-amyloid-β antibody  followed by a 1 hr room temperature incubation with biotinylated secondary antibody . Immunodetection was visualized using an Avidin-Biotin Complex kit , and sections were counterstained with haematoxylin before mounting. The slides were digitized with an automatic bright field microscope  and assessed by one examiner (LPM) for positivity for amyloid-β.2, Mann–Whitney U-tests were performed. For the longitudinal cohort, Friedman tests were performed to test the effect of age on CBF and CVR, post-hoc followed by Wilcoxon signed-rank tests to determine which of the individual age groups differed from each other. Only consecutive age groups were compared to each other to restrict the stringency of the Bonferroni correction for multiple comparisons. Wilcoxon signed-rank tests were used to test the effect of anesthesia on CBF and CVR and to test the effect of CO2 on the ATT. No statistical testing was performed in cohort 2, given the small group size. All tests were performed in the SPSS statistics software package, version 26 . The results of all tests are summarized in To test the effect of genotype on CBF, CVR, ATT, and tc-pCO In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:In this longitudinal experimental study using the Tg-SwDI mouse model of Alzheimer's disease which develop cerebral amyloid angiopathy, the authors employed arterial spin labeling-MRI longitudinally and laser Doppler flowmetry under anesthesia to measure whether amyloid-β deposits impact the hemodynamic response under hypercapnic challenge. In contrast to the previous reports, Tg-SwDI AD mice and age-matched controls show similar baseline perfusion and cerebrovascular reactivity, with differences in hemodynamic responses demonstrated for age and anesthesia. This work demonstrates that cerebrovascular function remains unaffected by microvascular amyloidosis in the Tg-SwDI AD model and raises the question whether this model is representative of cerebrovascular dysfunction observed in patients with Alzheimer's disease and cerebral amyloid angiopathy.Decision letter after peer review:eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by a Reviewing Editor and a Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Philipp Boehm-Sturm (Reviewer #2).Thank you for submitting your article "Cerebral Blood Flow and Cerebrovascular Reactivity Are Preserved in a Mouse Model of Cerebral Microvascular Amyloidosis" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.eLife papers without additional data, even if they feel that they would make the manuscript stronger. Thus the revisions requested below only address clarity and presentation.We would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). Specifically, we are asking editors to accept without delay manuscripts, like yours, that they judge can stand as Summary:In their manuscript, Derieppe and co-authors report on a longitudinal study of the Tg-SwDI mouse model of cerebral microvascular amyloid-β deposits. The authors employ ASL longitudinally under two regimes of anesthesia  as well as LDF in one time point under isoflurane to measure whether deposits impact the hemodynamic response with CBF and CVR under hypercapnic challenge. In contrast to previous reports, Tg-SwDI and age-matched controls show similar baseline perfusion and cerebrovascular reactivity, with difference in hemodynamic responses demonstrated for age and anesthesia. The authors carefully carried out a comprehensive study to estimate the impact of amyloid-β deposits and the lack of difference between the experimental groups suggests that perhaps the Tg-SwDI may not be an effective model of CAA. While raising possible causes for the discrepancy between the current results and prior reports, the failure to replicate remains unresolved. A strong aspect of the work is the characterization of cerebrovascular reactivity as a function of age using ASL as well as the comparison of anesthesia regimes and ASL to LDF.Essential revisions:1) The wild type group demonstrates variability that seems to be greater relative to Tg-SwDI and bimodal at 12 months of age . This raises the question of whether indeed the control group is homogenous relative to the transgenic group. Was the physiological state of the animals at 12 months assessed? What were the weights of the animals?2) Did the authors acquire whole-brain T2W scans? If yes, it will useful to quantify brain volumes as a function of age in both groups to help rule out issues with the control group.3) Regional effects: The authors report in Figure 2—figure supplement 1 two ROIs which do not show a difference. Is this lack of difference also shown in other ages? It will be useful to present all ages. Further, it is unclear that these ROIs provide enough granulation. Did the authors attempt a direct group-wise voxel-by-voxel comparison of the aligned images to see whether any differences are present?4) Statistics: A priori sample size calculations were not performed. Although the effect sizes from Park et al. seem extraordinarily high , the present study has low n and is underpowered for a hypothesis-driven study. The statistical analyses, exclusion criteria etc. are well described but were not preregistered and strategies to reduce bias (randomization/blinding) neglected. Please state your hypotheses in the Introduction more clearly. Clearly label your study as exploratory or hypothesis-driven.5) U&A anesthesia: Animals under U&A anesthesia have extremely low CBF, the animals seem to be in a non-physiological state and all experiments under U&A remain with a question mark. Although this is an important finding also for the previous investigations of Park and others, this presents a major limitation of the study.6) Craniotomy: Instead of LDF, laser speckle without craniotomy may have been the method of choice to corroborate the MRI results. The craniotomy is correctly discussed as a major confounder. This puts all the results of cohort 2 into question and weakens the study's main finding. Almost any manipulation on the dura induces a spreading depolarization (SD) in mice if you are not very well aware of it (i.e. measure it) and highly trained. Most importantly, the first SD leads to a massive reduction of cortical CBF >40% for >2 h which is well described and very specific to mice . The problem is, that after this baseline drop, the vascular response to SD looks normal in the mouse but essentially isn't. The implications of the phenomenon for a CO2 challenge/CVR have unfortunately not yet been investigated. For a craniotomy on the right side, the phenomenon is observed on the right hemisphere.Is this lateral effect present in the data? It is unfortunate, that the two confounding factors (different anesthesia and craniotomy) cannot fully be unmixed, the missing experiment is LDF under isoflurane . Essential revisions:1) The wild type group demonstrates variability that seems to be greater relative to Tg-SwDI and bimodal at 12 months of age . This raises the question of whether indeed the control group is homogenous relative to the transgenic group. Was the physiological state of the animals at 12 months assessed? What were the weights of the animals?We thank the reviewer for the observation of the greater variability and bimodality of the perfusion in the wild type animals at 12 months of age. We agree that an explanation for this phenomenon would make the data stronger. For that reason, as well as to further support the data in general, we decided to add data on body weight, brain volume and respiration rate to the manuscript . However, when looking at the added curves, there is no clear indication that either body weight, brain volume or respiration could explain the bimodality in the cerebral blood flow (CBF) of the wild type mice at 12 months of age. To be sure, we performed a correlation analysis of the CBF in wild type mice at 12 months of age, with body weight, brain volume and respiration.The correlation analysis illustrates that indeed, there is no indication that either of the three parameters could explain the bimodality in the CBF of the wild type mice. We considered the correlation analysis too detailed for the manuscript, so we have not added this to the manuscript. However, we explicitly mentioned the greater variability in the WT group in the Results section.2) Did the authors acquire whole-brain T2W scans? If yes, it will useful to quantify brain volumes as a function of age in both groups to help rule out issues with the control group.We did acquire T2W scans, and we agree with the reviewer that adding brain volume data would make the data stronger. In the Materials and methods section, we have therefore added the following description of the processing of the T2W scans:“T2W processing – One full-brain volume of interest (VOI) was manually drawn on one of the 21-slice T2W RARE scans, after which the VOI was propagated to the T2W RARE scans of the other datasets (both to other mice and to other time points) using the EVolution algorithm . To derive the brain volume, the number of voxels in the VOI was multiplied by the voxel volume.”The results of the brain volume calculation have been added in Figure 3—figure supplement 1 . As illustrated in 3) Regional effects: The authors report in Figure 2—figure supplement 1 two ROIs which do not show a difference. Is this lack of difference also shown in other ages? It will be useful to present all ages. Further, it is unclear that these ROIs provide enough granulation. Did the authors attempt a direct group-wise voxel-by-voxel comparison of the aligned images to see whether any differences are present?We agree with the reviewer that more information on the regional analysis is needed. Therefore, as suggested, we added the information on the regional analysis for the other time points in Figure 3—figure supplement 2. Note that, to not overcrowd this figure, we decided to only show the boxplot analysis, not the CBF time-profiles.We also agree with the reviewer that using the aligned images to add group-wise maps for more granulation would make the data stronger. We therefore decided to do this for all time points. Because we were positively surprised about the quality of the group-wise aligned CBF and CVR maps, we decided to add a new figure, which fully focuses on these new maps . However, no differences were observed between the wild type and transgenic CBF and CVR maps. Because of the much better quality of the group average maps versus the example maps, we decided to also replace the example maps in Figure 4 and Figure 5 with group average maps (not added in this response letter). Furthermore, we realized that in Figure 4, we had previously used a different slice than in the other figures (posterior slice instead of mid-brain slice), so we updated Figure 4 with mid‑brain data so it is in line with the rest of the data. Please also note that, because of the addition of the new Figure 2, the maps in former Figure 2 became redundant. Therefore, we decided to merge the former Figure 2 (without the maps) and former Figure 3 together in a new Figure 3.4) Statistics: A priori sample size calculations were not performed. Although the effect sizes from Park et al. seem extraordinarily high , the present study has low n and is underpowered for a hypothesis-driven study. The statistical analyses, exclusion criteria etc. are well described but were not preregistered and strategies to reduce bias (randomization/blinding) neglected. Please state your hypotheses in the Introduction more clearly. Clearly label your study as exploratory or hypothesis-driven.Indeed, we did not report our a priori sample size calculations, and agree with the reviewer that they contain valuable information and therefore should be added. We had already performed these calculations when applying for the animal experimentation license and now added these calculations with a description in the manuscript:“The first cohort was used for a hypothesis-driven study. Based on a previous report by Park et al., 2014, we expected an effect size of 30 percentage points difference – in cortical CBF response to a hypercapnia challenge – between Tg-SwDI and wild type mice, and a standard deviation of 12 % in both groups. […] However, we would also like to point out that during analysis, blinding was performed, and randomization is not applicable to this study, as the animals did not undergo treatment.”5) U&A anesthesia: Animals under U&A anesthesia have extremely low CBF, the animals seem to be in a non-physiological state and all experiments under U&A remain with a question mark. Although this is an important finding also for the previous investigations of Park and others, this presents a major limitation of the study.We agree with the reviewer that the low CBF during U&A anesthesia is questionable, and represents a non-physiological state. We therefore emphasize this in the Discussion:“The low CBF during U&A is non-physiological and thus forms a limitation of this study.”6) Craniotomy: Instead of LDF, laser speckle without craniotomy may have been the method of choice to corroborate the MRI results. The craniotomy is correctly discussed as a major confounder. This puts all the results of cohort 2 into question and weakens the study's main finding. Almost any manipulation on the dura induces a spreading depolarization (SD) in mice if you are not very well aware of it (i.e. measure it) and highly trained. Most importantly, the first SD leads to a massive reduction of cortical CBF >40% for >2 h which is well described and very specific to mice . The problem is, that after this baseline drop, the vascular response to SD looks normal in the mouse but essentially isn't. The implications of the phenomenon for a CO2 challenge/CVR have unfortunately not yet been investigated. For a craniotomy on the right side, the phenomenon is observed on the right hemisphere.Is this lateral effect present in the data? It is unfortunate, that the two confounding factors (different anesthesia and craniotomy) cannot fully be unmixed, the missing experiment is LDF under isoflurane .We thank the reviewer for addressing the possible confounding effect of a spreading depolarization in the mouse cortex after skull removal. We agree with the reviewer that this is a possible explanation for the altered CBF and CVR we observed after craniotomy, and we have added and referenced this idea in the Discussion:“However, even though the brain surface in these animals was visually normal  after the craniotomy, edema was observed with MRI, and with both imaging modalities marked reductions in CBF and CVR were measured in the underlying brain regions in both WT and Tg‑SwDI mice. […] Hence, it is of interest to elucidate in future research if our surgical procedure indeed triggers a CSD, and whether Tg‑SwDI mice are more susceptible to CSDs.”Because with ASL-MRI, we observe a significant reduction in baseline cortical blood flow in the hemisphere where the craniotomy was performed, our data confirms the notion of the reviewer . Furthermore, we also observe a profound reduction in CVR to hypercapnia in the same brain region, which can be seen in the same figure.Because we used two LDF probes – one of the probes measured CBF through the skull in the untouched hemisphere – we could see, as the reviewer points out, a lateral effect in the data . Furthermore, this lateral effect was confirmed with ASL-MRI (see same figure). We are therefore convinced that the impaired response after craniotomy is not an effect of anesthesia, but an effect of the craniotomy. In addition, our ASL-MRI data in the unaffected hemisphere corresponded well with our measurements under U&A anesthesia in cohort 1, thus further supporting this notion. Therefore, we did not acquire additional data with LDF under isoflurane. Because we did not write this clearly before, we did now emphasize in the Results and in the figure legends, that we measured LDF with two probes in two hemispheres, both through the skull and after craniotomy.


To evaluate the impact of a training programme for arthrocentesis on procedural skills enhancement and self-confidence in medical students.P-values, 95% confidence intervals about the mean, standard error of the mean, and standard deviations of the differences were calculated.Participants were provided a structured workshop on injection models. A self-confidence questionnaire and medical knowledge assessment were performed. Retention of knowledge and skills were assessed at a later time point during a formal OSCE examination and compared to participants who had not attended a lecture and clinical attachments only. p < 0.05).All participants gained self-confidence, and improvement of their skills was significant. The mean self-confidence with performing an arthrocentesis procedure increased from 1.3 pre- to 5.9 points post-workshop . The knee was the joint students felt most confident with (1.3 to 6.5 points). Knowledge on the selection of corticosteroid preparations (1.2 to 5.8 points) gained substantially, as well as confidence in providing post-injection advice (1.9 to 6.6 points). Upon the OSCE examination, attendance to the workshop resulted in a significant higher total score . Disorders of the musculoskeletal system are estimated to cause one in four consultations in primary care according to the World Health Organisation, ranking musculoskeletal problems first among the most common reasons to seek medical care . Pain anHence, international efforts have been focused on improving medical student musculoskeletal basis skills to adequately address the need , and a sAs clinical opportunities to perform arthrocentesis are limited, a half-day arthrocentesis workshop for fourth year medical students was developed with the aim of introducing the basic concepts of arthrocentesis and increasing self-confidence in the performance of these procedures along with an increased understanding of the indications, injection options and possible complications. The objective of this study was to evaluate the impact of the hands-on workshop for arthrocentesis procedures on procedural skills enhancement and self-confidence in fourth year medical students.The bachelor of medicine / bachelor of surgery (MBBS) course within the University is a six year course. The majority of clinical teaching begins in the 4th year with approximately 150 students per year. After graduation students will enter a specialty-specific intern period during their year with some undertaking a musculoskeletal (MSK) or emergency medicine internship for 3 months. The arthrocentesis workshop was held for medical students during their 4th year of training. As the program was introduced during a running year, 15 students missed the opportunity to participate, because the program was only introduced after those students had absolved their musculoskeletal module. After its implementation, the program was offered as a non-compulsory training opportunity for the students absolving the musculoskeletal module. There was no formal recruitment process. Apart from the workshop, all students had access to online learning material, a one-hour lecture on dislocation management and aspirations and injections as well as access and training to the injection models for 20 min per model over 3 weeks as part of their weekly anatomy sessions.The workshop was designed as a half-day program. The goal of the program was to introduce the basic concepts of arthrocentesis and increase self-confidence in the performance of these procedures. Trainees received both didactic and hands-on instruction. There was a one-hour lecture, during which general principles related to the arthrocentesis procedure were outlined. The students were familiarized with indications and contraindications to the procedure, and the equipment required for safe performance of an arthrocentesis. Topics such as the consent process, risks of corticosteroid injection and site-specific anatomy including approaches were evenly analyzed. The models available and the approaches for arthrocentesis discussed are shown in Table To ensure the workshop was meeting its objectives, a self-assessment questionnaire and medical knowledge test were administered before and after the session. The workshop finished with an impact survey that was completed optionally by the participants.A total of four knee models, one shoulder, one elbow and two spine models were available. All models were “Limbs and Things” . The models allowed for palpation of anatomic landmarks and featured skin like coverings that tolerated multiple needle sticks without showing former aspiration sites. The knee models were filled with artificial synovial fluid that could be aspirated upon proper positioning of the aspirating needle. The shoulder and elbow models featured electronic feedback indicating correct and incorrect positioning of the needle. The spine models allowed for fluid aspiration from the spinal canal to simulate a lumbar puncture.The questionnaire included 19 self-assessment questions and was distributed to all participants prior to the lecture and immediately after termination of the hands-on workshop .The questions of all surveys  were designed by the investigators to align with the established learning objectives of the MSK module. An internal validation of the questions was performed. Expert consent among the lecturers of the musculoskeletal module was used to achieve face/content validity.All 4th year medical students undergo a summative yearly skills assessment test (OSCE examination). Performance of a knee joint aspiration was among the possible scenario’s students could be assigned to during this evaluation. Thus, the aspiration scenario could be undertaken by students who had attended the workshop. Students assigned to the scenario were asked to perform an aspiration of the knee on a synthetic model after taking consent from the examiner. After successful arthrocentesis, laboratory results from a knee aspirate were shown to the examinee, and they was asked to report possible diagnoses and initial management options based on the findings. Each examinee was evaluated for professionalism (0 (min) to 6 (max) points), clinical skills (0 (min) to 9 (max) points) and reasoning (0 (min) to 5 (max) points) by a senior staff member of the University of Adelaide. The raters were blinded towards the participation status of the examinees. A total test score (0 (min) to 20 (max) points) was calculated summarizing all three subsections of the evaluation. The scores for those attending the workshop were compared to those who did not. This study was approved by the Ethics committee of the University of Adelaide .P-values, 95% confidence intervals about the mean, standard error of the mean, and standard deviations of the differences were also calculated. All statistical analyses were performed with R .As the data were not normally distributed, a Wilcoxon signed rank test was used to evaluate whether or not there were significant changes in the scores given before and after the workshop. The null hypothesis was that the mean difference in ranks (corresponding to score shift) between the paired observations was zero, whereas the alternative hypothesis was that the mean difference between the paired observations was not equal to zero. Test significance was determined at the alpha level of 0.05. A positive mean difference indicated that the workshop was effective in increasing confidence and a negative mean difference indicated that the workshop lowered confidence. A mean difference near zero indicated that the workshop had little effect. A total of sixty-nine students participated in the arthrocentesis workshop, while twenty-one (23%) students did not. Three students out of the 69 attending had performed one to five arthrocentesis procedures prior to the workshop, whilst the other 66 students did not have practical exposure towards the procedure up to that date.The pre-workshop confidence towards performing an arthrocentesis in general averaged 1.2 on the 10-point Likert scale. People were most confident performing an arthrocentesis of the acromioclavicular (AC) joint (1.5), as opposed to the glenohumeral joint, subacromial space or spine (1.4 each). The students were least confident performing an arthrocentesis of the knee or elbow (1.3 each). Confidence in the knowledge of the relevant anatomy was generally higher, reaching values between 4.4 (elbow) and 5.0 points (spine) respectively. Interestingly, students felt the highest confidence with knowledge of the anatomy of the spine but at the same time less confidence to perform an infiltration on it. In spite of being less confidence performing an arthrocentesis, participants overall felt quite comfortable to obtain consent for the procedure, with a mean of 5.5 points. Risks of arthrocentesis and indications for corticosteroids showed mean values of 3.9 and 4.0 in the 10-point Likert scale, whilst contraindications for corticosteroids scored 3.2 point on average. According to the answers provided, the students were not confident regarding the choice of the preparation, dosage or frequency of corticosteroid infiltrations as well as the provision of post-injection advice to patients. A detailed description of the self-assessment questionnaire results is given in Table p < 0.001 for all subsections of the questionnaire). The mean confidence levels range from 4.8 (performance of an arthrocentesis at the spine) to 6.9 points (obtaining consent for the procedure) on the 10-point Likert scale. The mean change in confidence when comparing each pre- and post-workshop answer showed values between 0.9  and 5.3 points . In general, the gain in self-confidence was less in the fields were the participants already felt quite comfortable prior to the workshop , whilst self-confidence increased substantially with performing an arthrocentesis procedure in general (+ 4.7 points / + 369%), knowledge on the selection (+ 4.6 points / + 382%), quantity (+ 5.3 points / + 763%) and frequency of corticosteroids in use (+ 4.4 points / + 303%) as well as providing post-injection advice (+ 4.7 points / + 247%). Students felt most confident in performing an aspiration of the knee (6.5 points), followed by the elbow (5.9 points) and glenohumeral joint (5.8 points). In contrast, confidence was less with procedures involving the spine (4.8 points).Overall, students gained significant benefit from the workshop in terms of confidence towards the procedure (p < 0.001). All students correctly agreed on performance of a cell count and a synovial fluid culture as a diagnostic tool for synovial fluid analysis. Overall, 75.0% of the answers in the section on diagnostic synovial fluid analysis were completed correctly prior to the workshop, while 93.1% correct answers were obtained after the workshop . The lowest proportion of correct answers prior to the workshop were achieved with the questions to corticosteroid preparations (18.4% correct answers), followed by the question regarding incidence of infective complications after arthrocentesis (26.1% correct answers). Questions relating to corticosteroid preparations were answered correctly by 53.1% of students after the workshop , and 85.5% of all participants ticked the right answer when asked about the incidence of infection after arthrocentesis . Knowledge about contraindications and adverse effects of corticosteroid injections also improved after the workshop, but there was a high proportion of correct responses prior to the workshop. A summary of all of the answers is presented in Table All participants showed profound knowledge of the anatomy relevant to the arthrocentesis procedure. The students achieved a mean correct score of 17.9  in the anatomical section of the pre-test. The result was even better in the post-workshop knowledge assessment, where the mean number of correct answers increased to 19.1 out of 21 . This was an improvement of 7%  on the 10-point Likert scale on average. The quality of the equipment provided scored 6.8 points on average (4–9), while the mean value for quality of the lecture was 8.1 points (5–9). Students were also given the opportunity to provide feedback as free text. The students praised the quality of the presentation and the didactic value of the course, but wished improvements to the models provided. Many students appreciated the possibility to aspirate fluid upon correct needle positioning as with the knee models, and wished the same feature with the other models as well.p < 0.05), whilst a tendency towards a better performance among workshop attendees was found in the subsections of the evaluation , elbow and shoulder (one each) were lower. The chance to practice on some of the models was therefore potentially limited and could have had an impact on the student’s confidence with the procedure. However, the aim of the study was not to compare the suitability of different injection models, and there are other factors that explain the differing confidence to the procedure on different locations as stated above.s [With any self-reported questionnaire social desirability bias is present and is a limitation. However, we proactively attempted to limit this by having some unstructured questions and stressing to the students that the answers were anonymised and purely used to provide feedback on ways we can improve the course in future. Furthermore, we did a second proxy questionnaire at the end of the MSK module asking students about many elements of the course with many forced choice questions where students highlighted the joint injection workshops as one of the highlights. Although this will have not completely mitigated social desirability bias we followed Nederhofs  methods A single session, half-day hands-on workshop for arthrocentesis procedures is well suited to increase procedural skills and self-confidence towards the procedure in fourth year medical students. The participants developed important baseline knowledge necessary to perform arthrocentesis procedures safely. Workshops for medical students, in conjunction with other learning activities, not only help learners to gain confidence and to practice invasive techniques without risk to a patient, they allow students to gain knowledge, comprehension and application with time for focused learning in a more structured way, possibly enabling students to progress up Bloom’s taxonomy faster and more efficiently.Additional file 1. Practical skills development form (Knee)Additional file 2.  Self-assessment questionnaireAdditional file 3.  Medical knowledge checkAdditional file 4. Impact survey


Marker gene surveys have a wide variety of applications in species identification, population genetics, and molecular epidemiology. As these methods expand to new types of organisms and additional markers beyond 16S and 18S rRNA genes, comprehensive databases are a critical requirement for proper analysis of these data.Here we present an ITS2 rDNA database for marker gene surveys of both free-living and parasitic nematode populations and the software used to build the database. This is currently the most complete and up-to-date ITS2 database for nematodes and is able to reproduce previous analysis that used a smaller database.This database is an important resource for researchers working on nematodes and also provides a tool to create ITS2 databases for any given taxonomy. The internal transcribed spacer 2 (ITS2) rDNA locus has been widely used as a marker for species identification in both free-living and parasitic nematodes for many years –3. NematThe nematode ITS2 database was constructed using markerDB, which we have provided as an open-source tool to quickly and reliably construct an ITS2 database for any NCBI taxonomic level. This tool is made available to facilitate reproducibility and transparency, and to provide users with the option to construct their own databases. markerDB is implemented in the R programming language  and run Potential ITS2 sequences are retrieved from NCBI using the rentrez R package  based onMany of the ITS2 annotated sequences also contain the partial or full upstream and downstream 5.8S and 28S genes and so trimming to the ITS2 region is required. However, no good sequence models exist that capture a wide range of diversity due to the divergence of this region. For this step Infernal,   is used Finally, sequences too long or too short  are discarded. The final sequence set contains a fair bit of redundancy and so a non-redundant version of the database with unique sequences only is returned. If an alignment is required an option to align the sequences using MAFFT  is also www.nemabiome.ca). A simple shiny app is also provided that allows users to work with the database interactively, filtering taxonomic groups as needed and downloading the filtered data in any of the above formats.The output of markerDB is a fasta file with the final sequences and a corresponding tab-delimited text file with the taxonomy, linked by Genbank accession number. The pipeline also provides function to write out the database in formats used with popular taxonomy assignment methods including dada2 , the rRDThe database (version 1.0.0 at the time of writing) currently contains 8630 non-redundant sequences with a median length of 263 bp and standard deviation of 97 bp. There are 1429 species and 325 genera and across the taxonomic ranks we were able to obtain good quality, non-redundant sequences for approximately 30% of the taxa in the NCBI database in the Nematoda phylum (see Fig. We have also provided a simple web app, which allows users to search and filter the database and create versions customized to their research area of interest. Further, the database will be updated every 3-6 months, feasible due the automation and reproducibility of the database construction using markerDB. Rapid updates allow researchers to generate analysis that reflect the most current sequences in Genbank.In conclusion, we provide a database of nematode ITS2 sequences that greatly expands the range of sequences suitable to study both parasitic and free-living nematode communities allowing a broader selection of hosts and environments to be studied. We have also provided open source software to easily and reproduceably build ITS2 databases for any taxonomy of interest.Project name: markerDBProject home page:https://github.com/ucvm/markerDBOperating system: Unix-based, i.e. MacOS or LinuxProgramming languange: R and PythonOther requirements: BiocondaLicense: MITAny resitriction ot use by non-academics: NoneAdditional file 1 Supplementary figure 1.