Objective: Treadmill interventions have been shown to promote ‘normal’ walking patterns, as they facilitate the proper movement and timing of the lower limbs. However, prior reviews have not examined which intervention provides the most effective treatment of specific gait impairments in neurological populations. The objective of this systematic review was to review and quantify the changes in gait after treadmill interventions in adults with neurological disorders. Data Sources: A keyword search was performed in four databases: PubMed, CINAHL, Scopus, and Web of Science (January 2000–December 2021). We performed the search algorithm including all possible combinations of keywords. Full-text articles were examined further using forward/backward search methods. Study Selection: Studies were thoroughly screened using the following inclusion criteria: study design: Randomized Controlled Trial (RCT); adults ≥55 years old with a neurological disorder; treadmill intervention; spatiotemporal gait characteristics; and language: English. Data Extraction: A standardized data extraction form was used to collect the following methodological outcome variables from each of the included studies: author, year, population, age, sample size, and spatiotemporal gait parameters including stride length, stride time, step length, step width, step time, stance time, swing time, single support time, double support time, or cadence. Data Synthesis: We found a total of 32 studies to be included in our systematic review through keyword search, out of which 19 studies included adults with stroke and 13 studies included adults with PD. We included 22 out of 32 studies in our meta-analysis that examined gait in adults with neurological disorders, which only yielded studies including Parkinson’s disease (PD) and stroke patients. A meta-analysis was performed among trials presenting with similar characteristics, including study population and outcome measure. If heterogeneity was >50% (denoted by I2), random plot analysis was used, otherwise, a fixed plot analysis was performed. All analyses used effect sizes and standard errors and a p < 0.05 threshold was considered statistically significant (denoted by *). Overall, the effect of treadmill intervention on cadence  and step length  in adults with stroke was significant. We also found a significant effect of treadmill intervention on paretic step length  and stride length . For the active control group, including adults with PD, we found that overground physical therapy training had the largest effect on step width . Additionally, for PD adults in treadmill intervention studies, we found the largest significant effect was on step length  and stride length . Conclusion: Treadmill intervention with sensory stimulation and body weight support treadmill training were shown to have the largest effect on step length in adults with PD and stroke. In the United States, the number of adults ≥65 years of age is estimated to increase from 53 million in 2018 to 88 million in 2050. As this population increases in size, the financial burden on the healthcare system will also increase, and effective preventive and/or therapeutic approaches will be desperately needed to help diminish the increased burden associated with the increase in the number of older adults. Among the various age-related diseases, neurological disorders with or without concomitant cognitive decline are particularly relevant given their adverse impact on people’s quality of life [Stroke, one of the most common neurological disorders, is a leading cause of long-term disability in older adults. Brain damage due to stroke can lead to symptoms such as cognitive and motor impairments including pain, paralysis, poor balance, spasticity, muscle weakness, and ineffective gait patterns . More thAnother common neurological disorder is Parkinson’s disease (PD). PD is a common neurodegenerative disorder that occurs in about 1% of adults over the age of 60 years old . Adults Adults living with these conditions must undergo sustainable forms of short-term and long-term rehabilitation to regain function and perform the activities of daily living. Many studies have demonstrated that treadmill training is a common approach for improving mobility and gait. Treadmill training (TT) has been shown to promote ‘normal’ walking patterns, as it facilitates the proper movement and timing of the lower limbs, thus eliminating the need for compensatory gait mechanisms . It can Given the fact that TT is a widely accepted method of gait training, the purpose of this study was to investigate the effectiveness of TT in improving spatiotemporal gait parameters in older adults with neurological disorders. To date, there is no systematic review and meta-analysis which has been directed to summarize what is known from randomized controlled trials about the effectiveness of therapeutic interventions to address walking concerns associated with spatiotemporal gait characteristics among individuals with neurologic diseases. As such, it is difficult for physical therapists to provide the most effective, up-to-date, specific evidence-based intervention to individuals with neurologic diseases presenting with specific gait impairment. This systematic review aimed to: (1) quantify the effect of different treadmill interventions on spatiotemporal gait parameters of older adults with neurological disorders; and (2) evaluate each randomized controlled trial (RCT) based on the quality assessment criteria. While we tried to be inclusive of neurological disorders in this study, after applying the inclusion criteria, the two main neurological disorders were stroke and PD.Studies that met all the following criteria were included in the review: (1) study design: randomized controlled trial; (2) year: published after 2000; (3) population: older adults ≥55 years old with a neurological disorder; (4) intervention: any type of treadmill intervention; (5) main outcome measures: spatial and temporal gait characteristics including stride length, stride time, step length, step width, step time, stance time, swing time, single support time, double support time, or cadence; and (6) publication language: English. Studies were excluded from the review if they contained one or more of the following exclusion criteria: (1) non-randomized controlled trials; (2) studies published before 2000; (3) population under the age of 55 years; (4) experimental groups contained only healthy older adults; and (5) studies that did not include any spatial or temporal gait characteristics.The systematic review and meta-analysis described in the Preferred Reporting Items for Systematic Reviews and Meta-analysis process was adopted to guide the review process. A keyword search was performed in 4 databases: PubMed, Cumulative Index to Nursing and Allied Health Literature (CINAHL), Scopus, and Web of Science. The search retrieved articles published from January 2000 to December 2021. The search algorithm was formulated to include all possible combinations of keywords from the following 5 groups: (1) walk OR ambulatory OR mobility OR gait; (2) variability OR complexity OR unsteadiness OR inconsis* OR stability OR equilibrium OR dynamics OR balance OR ataxia; (3) “neurological disorder” OR “neurological pathology” OR “multiple sclerosis” OR “Parkinson’s disease” OR “Huntington’s disease” OR ALS OR “cerebellar ataxia” OR Alzheimer OR stroke; (4) intervention OR therapy OR treatment OR “best practices”; (5) older adults OR elderly OR aged OR elder OR older OR senior OR geriatric. Certain keywords were made to be excluded, such as musculoskeletal OR posture OR postural OR animal OR robot OR amputee OR trunk OR knee OR hip OR freezing of gait OR spasticity OR heart OR blood OR cardiac. The search algorithm for each database can be found in Titles and abstracts of the articles identified through the keyword search were screened for the study selection criteria. Two reviewers (A.B. and R.L.) independently performed title and abstract screening to determine eligibility. Interrater agreement was determined by Intraclass correlation coefficient value and authors showed a moderate correlation (ICC = 0.69). Any disagreements were resolved through discussion. Full-text articles were obtained after both authors agreed that the potential article satisfied all study selection criteria. A cited reference search  and reference list search  were performed based on the articles meeting all inclusion criteria identified from the keyword search. The articles that resulted from the forward and backward reference search were also screened and evaluated using the same study selection criteria. Three layers of forward and backward reference searches were completed until no additional relevant articles met the inclusion criteria.A standardized data extraction form was used to collect the following methodological outcome variables from each of the included studies: author(s), publication year, population, age, sample size, and outcome measures. Outcome measures include spatial gait characteristics  in centimeters, temporal gait characteristics including single-limb support time, double limb support time, cadence, stride cycle in either percentage or time (seconds) of the gait cycle, and cadence in steps/minute. If the characteristics were not provided in desired units, necessary conversions were performed.For treadmill training groups in our study, if the study had a pure treadmill training (TT) group with an active control group involving conventional PT, or overground walking, then our pure TT group was considered the intervention group. However, if in the study there was a group involving TT with either sensory feedback, perturbation, or bodyweight support, then that was considered the intervention group, and the pure TT was considered the active control group.For research that utilized the same population in more than one study, meta-analysis was performed to estimate the pooled effect size (pre-intervention mean—post-intervention mean) and the standard error of specific spatiotemporal gait parameters under specific therapeutic intervention for a specific neurological population. The equation used was:2 index. The level of heterogeneity represented by I2 was interpreted as modest (I2 < 25%), moderate (25% < I2 < 50%), substantial (50% < I2 < 75%) or considerable (I2 > 75%). A fixed model was estimated when modest to moderate heterogeneity was present, and a random-effect model was estimated when substantial to considerable heterogeneity was present. Publication bias was visually inspected using funnel plots and empirically tested using Egger’s tests. All statistical analyses were conducted using Stata 14.2 SE version . All analyses included 2-sided t-tests, where p-values < 0.05 were considered statistically significant.Secondary analysis was performed to examine the effect of dosage (times × week = minutes) and age on cadence after pure TT and the effect of body weight support load and dosage on step length after body weight support TT in a specific neurological population. Study heterogeneity was assessed by using the IWe used the National Institutes of Health’s (NIH) study quality assessment tool for intervention studies to assess the quality of all included studies . The folAs illustrated in Basic characteristics for the age and sample size of all included studies are summarized in p < 0.001, W = 25%). In addition, TT on incline surface (W = 53.76%) and pure TT (W = 40.60%) had the most significant effect and highest weightage on stride length in adults with stroke . Interestingly, step length in paretic leg also showed significant improvements after TT with visual cues (W = 29.48%) and pure TT (W = 60.40%) had the highest weightage  in comparison with step length on non-paretic length, which was not significant . Additionally, there was significant difference in step length after TT, including virtual reality (W = 28.41%) and pure TT (W = 36.53%), which had the highest weightage . Lastly, we had one group of non-treadmill interventions in people with stroke and found prosthetic walking to have the strongest effect on cadence, though it was not significant   .p < 0.05). In addition, pure TT, TT with auditory cues and body support TT also showed weightage and effect on cadence and step width in adults with PD, but this effect was not significant . In contrast, TT with either perturbation training (W = 29.35%) or pure TT (W = 49.09%) had the highest weightage and smallest effect on stride length in adults with PD   and body weight support treadmill training (W = 49.75%) had the highest weightage (W) and strongest effect on step length in adults with PD  .p = 0.001, W = 99.90%); other than that, all effects of non-treadmill interventions on cadence, step length, and stride length were not significant  or stride length   .Studies included in the current meta-analysis averaged a score of 16.55 out of 20 possible points. The distribution of the scores depended on the category of criteria . All theThis study systematically reviewed and quantitatively synthesized existing scientific evidence on TT intervention studies among adults with and without PD or stroke. Gait outcome measures, including cadence, step length, and stride length of adults with neurological disorders, were examined in 30 published studies. This systematic review explicitly targeted: (1) quantifying the different treadmill interventions effects on gait parameters in different populations; and (2) evaluating each study based on a pre-defined set of quality assessment criteria.Results from our meta-analysis show that overall TT interventions had the largest effect on cadence, step length, and stride length in adults with stroke and PD. Pure TT results are similar to previous reviews showing that task-specific TT had the greatest effect on spatiotemporal gait parameters, especially stride length and step length in people with PD . FurtherNeurorehabilitation TT programs are goal-based, repetitive, and include intensive motor learning components for adults with or without PD and stroke. From our systematic review and meta-analysis, it can be seen that pure TT increases stride length in adults with PD ,33,41, aSpeed-dependent treadmill training is a repetitive, intensive TT in which the belt speed is increased or decreased incrementally by 10% based on the individual’s performance. Incline-based TT is a repetitive, intensive training in which incline level is increased incrementally with the belt speed based on the performance of individuals. The results from our systematic review indicate that speed-dependent TT was more effective to increase stride length and gait speed in individuals with stroke in comparison with pure TT . Speed- Treadmill training with sensory feedback is a task involving training on a treadmill while providing sensory feedback such as auditory cues, visual cues, rhythmic cues, or a combination of these. From our systematic review, we found that auditory cues had the strongest effect on step width and step length in adults with PD . We alsoMoreover, in adults with stroke, we found that visual cues with treadmill training have the strongest effect on cadence, paretic step length, and non-paretic step length ,17. TherGiven that gait is a complex sensorimotor behavior that involves the coordination of neural networks, bones, muscles, and joints, it is not surprising that sensory information can aid and even influence gait performance . Work frBodyweight support TT is locomotor training on a treadmill with partial body weight supported (PBWS) with an overhead harness, a pelvic belt, and thigh straps. PBWS is effective in improving mobility outcomes in adults with stroke and spinal cord injuries . The loaFrom our systematic review, we found that TT with PBWS can create better gait kinematics, symmetry, velocity, and endurance ,28. PBWSCurved walking involves the coordination of whole-body movement and the complex integration of multiple sensory systems and motor output to respond to balance demands . Curved In adults with PD, TT improves functional capacity, balance, and quality of life after even short-term treadmill training . TT has 2 levels while lowering the energy cost needed for hemiplegic gait and further enhancing motor functions [In adults with stroke, TT has been shown to improve lower limb function and gait performance, which in turn promotes stroke recovery by inducing further brain plasticity through activation of the sensorimotor cortex, supplementary motor cortex, and cingulate motor area . Furtherunctions . Recent unctions . Lastly,unctions .Although we do see these changes at physiological and biomechanical levels in adults with stroke and PD, these changes depend on the intensity and duration of exercise in each population. Through this review, we tried to show the importance of treadmill training in different neurological populations by its ability to improve specific spatial–temporal gait parameters. We were successful in showing the impact of treadmill training on spatial–temporal gait parameters. We found that although treadmill training is effective on its own, the use of sensory stimulation in unison with TT provides a more effective therapy for neurological populations, such as PD and stroke.While the majority of TT studies did not report dropouts, 7/32 studies reported drop-outs due to personal reasons , while only a single study reported subjects dropping out due to leg pain, fear of falling, or subjective intolerance to training.We found changes in gait outcomes among older adults with and without neuro-logical disorders. Unfortunately, there were not enough healthy older adult studies to include them in this meta-analysis. Although no publication bias was detected using Egger’s test in adults with PD and stroke, the small number of included studies here does limit the power of the test to detect bias. The current meta-analyses could have benefited from the inclusion of additional studies to prove the results of the subgroup analysis of TT with BWS. Future work should examine the effects of these promising TT interventions on spatiotemporal gait parameters with larger sample sizes in more diverse neurological populations. Additionally, healthy older adult studies are needed to compare and improve patterns across older adults with and without neurological disorders.The current review and meta-analytic study provides comprehensive information on the effect of treadmill interventions such as TT with sensory feedback, bodyweight support TT, TT on an inclined surface, and pure TT on spatiotemporal gait characteristics. Among these interventions, TT with sensory feedback or bodyweight support had the largest effect on cadence, stride length, and step length in adults with PD. Furthermore, in adults with stroke, pure TT and TT with visual cues or incline were shown to have the largest effect on step length, stride length, and cadence. Overall, TT interventions had the largest effect on cadence, step length, and stride length among adults with stroke and PD. However, due to a lack of studies in other populations, we were unable to justify the effect of all interventions across a wider number of neurological disorders. Nonetheless, this study will aid clinicians in choosing tailored interventions based on the specific needs of patients, as it provides information regarding which type of treadmill intervention program will have the largest effect on a specific gait parameter in adults with stroke and PD.


Case Report. The 13-year-old girl presented with a right lateral neck swelling. In detailed history, she was found to be an old case of ventriculoperitoneal shunt. The clinical examination and radiological investigation revealed proximal dislodgment of the stent from the cranium, causing persistent cerebrospinal fluid (CSF) leak and pseudocyst formation in the neck.  Ventriculoperitoneal shunt is a common neurosurgical procedure, for the definitive management of hydrocephalus. Shunt failures may occur due to various causes but are usually due to infections in adults and catheter occlusion in the paediatric population.  The case highlights CSF pseudocyst formation as a rare differential for lateral neck swellings. Ventriculoperitoneal (VP) shunt is surgically placed in patients with hydrocephalus . Due to Pseudocysts get formed due to the collection of CSF fluid, usually accumulating around the distal end of the shunt and have a wall of fibrous tissue, thus devoid of the epithelium as seen in cyst . In CSF Here, we present a case of a very rare VP shunt complication, CSF pseudocyst in the neck. However, it is second in world English literature, but very first to report proximal end migration of VP shunt .The 13-year-old girl presented in our outpatient department with a complaint of right-sided neck swelling for four months. It was insidious in onset, painless, and was gradually progressive. There was no history of fever, restriction of head neck movement, or sudden change in size. Her parents gave a history of hydrocephalus at birth, for which she was operated on at 6 months of age. There was no history of headache, vertigo, or any neurological deficit. History of any head-neck trauma prior to the swelling was negative. The previous surgery details were not available, and thus, the knowledge of shunt material used or whether it was tied to the pericranium was not known. There was no history of any shunt revision surgery after the first operation.On examination of the patient, the swelling of 12 cm × 4 cm was observed on the right lateral neck Figures . The sweOn palpation, it had a normal temperature and was nontender. It was well defined, cystic, compressible but not reducible or fluctuant. A cord-like structure was felt running from the postauricular region to the swelling. The cord was hard in consistency not fixed to underlying tissue, as was mobile side to side. No pulsations were felt in and around the swelling. The transillumination test was positive with a brilliant glow of the swelling  with theThe ultrasonography (USG) of the neck revealed the intracervical part of the VP shunt with perishunt large fluid collection. Fine-needle aspiration was done in our outpatient department, under aseptic measures which revealed clear colourless thin aspirate. The fluid sent for biochemical analysis confirmed CSF with glucose: 73 mg/dL and proteins: 45 mg/dL. The fluid was also sent for culture and sensitivity, which gave no growth at 48 hours.The patient was advised for a computed tomography scan (CT) to look for the position of the shunt intracranially and possible changes in the brain due to increased intracranial pressure following shunt dysfunction. Because of the availability of CT, its high sensitivity in comparison with plain X-ray, and better delineation of neck mass, the plain X-ray (shunt series) was not asked for in our case. On the CT brain, there was no evidence of shunt intracranially, its proximal end lying just external to the cranium was present , and hydThe patient was then referred to the neurosurgery department for VP shunt revision surgery. After written informed consent was obtained from the parents, the shunt was replaced and successfully sutured to the pericranium on the same side in the posterior parietal part of the cranium and the peritoneal cavity. The pseudocyst resolved, with no recurrence in one-year follow-up.Hydrocephalus, as the name suggests, is a condition of the brain following excessive accumulation of CSF . This leIn our patient, despite a nonfunctioning shunt, no symptoms were seen, due to continuous trickle of CSF through the cranium opening into the neck subcutaneous plane. Lateral neck swellings in children are mostly thought to be reactive lymphadenopathy, lymphadenitis , lymphangioma, branchial cleft cyst, and vascular malformation .The use of VP shunt in hydrocephalus patients has almost replaced the ventriculoatrial (VA) shunt due to the fewer incidences of infection and clotting . The varBut, in our case, first one in literature, there was dislodgment of the ventricular end  of the shunt, which led to extravasation of CSF extracranially. This persistent fistula in the cranial vault of the child resulted in CSF pseudocyst of the neck. As there was free flow of CSF in the neck, our patient had no complaints of headache, vertigo, or vomiting.  CSF pseudocyst formation in the neck after VP shunt surgery is very rare  This case adds an important differential for lateral neck cystic swelling  The proximal end of VP shunt may also get dislodged, and on regular follow-up of the patient after VP shunt surgery, this point in mind may avoid such complicationIt is rare to see CSF pseudocyst in the neck after VP shunt surgery. The case presented here adds another differential for lateral neck swelling and further stresses the need for detailed history.


Research in the field of Chinese as a foreign language (CFL) education has been increasing in the past decades. However, the number of studies on CFL teacher identity is limited. To bridge the gap, this study employed a qualitative method to explore Chinese CFL teachers’ identity formation and reformation in Australian contexts. A Chinese-Australian language program was studied to examine the challenges, struggles and developments of Chinese CFL teachers who came to Australia to pursue professional growth. Five Master’s theses and three interview participants were included to paint a picture of how Chinese CFL teachers interact internally and externally with a new environment. Guided by Mead’s theory of self and other, we found that Chinese CFL teachers’ identity formation and reformation in Australian classrooms are deeply influenced by their self-identification and their integration with others in the community. Cultural connectedness is a key for organizational attitudes in the relationship of self and other. Chinese CFL teachers were found lacking the wholeness of self in Australian contexts, which led to obstacles in teacher identity construction. Insufficient communication between self and other resulted in their positioning crisis. Internationalization in education has brought an increase in global teacher mobility . ChineseChinese as a foreign language teachers play a vital role in the implementation of CFL curricula and improvement of the effectiveness of Chinese language teaching. Therefore, professional development should be a priority in CFL research . HoweverIn Australia, CFL teachers have been in continuous demand, because the number of language learners has increased more than five times in the last 50 years and language accessibility is increasingly regarded as a factor for all-around development . CommuniMead’s self and other theory is the theoretical foundation of this study. While examining self, In our mechanism, there is a tendency to view ourselves through others’ perspectives and to address ourselves like others do . As a coTo understand one’s position in a society, one needs to be viewed regarding both “I” and “me” . In prepIn the understanding of the development of self, several theories were proposed to approach different aspects of it. Positioning Theory  focused We take the attitudes of others in controlling our responses; thus, “The organized community or social group which gives to the individual his unity of self may be called ‘the generalized other”’ , p. 154.Targeting the understanding of cultural dialogue and its responsiveness toward social life, Moving to a foreign society, language teachers have to adapt to the new environment in which self and other collaboratively contribute to the formation of their new identity. By this theoretical guidance, we intend to explore the value of CFL teachers’ self-identification and factors that impact it. To understand who they are and how they work, the individual and social identities they are assigned have to be accounted for. Transformative as these identities are , negotiaTeacher identity has been widely regarded as an important factor that influences the way teachers view themselves and, to a large extent, how they interact with students. It is and could be shaped and reshaped under the impact of various factors and in different contexts . Global Administrative management and professional learning could also shape and reshape teacher identity. Identity could be shaped by recipients and contextual factors. Teacher identity, thus, is closely related to students and environments. From a sociological point of view, As for language teacher identity, Language teachers and students in any setting naturally represent a wide array of social and cultural roles and identities: as teachers or students, as gendered and cultured individuals, as expatriates or nationals, as native speakers or non-native speakers, as content-area or TESL/English language specialists, as individuals with political convictions, and as members of families, organizations, and society at large (p. 451).Positioning of self and other in a complex social and cultural context is crucial for language teachers, because it involves power relationships and mainstream discourses that determine the legitimacy of their languages and teaching. This is in agreement with Divergent, if not contradictory, findings have been revealed in the field of CFL teacher identity studies in cross-cultural contexts. Some find that CFL teachers are impacted strongly by Chinese educational culture situating in a cross-cultural context, and have reported that under the influence of Confucianism their professional identities tend to be authoritarian and teacher-centered . While oPedagogical and linguistic authority is an important theme in the study on CFL teacher identity. The interactions between native resources, such as Chinese textbooks and interests of new learners, reflect the negotiation of education systems . Old-fasTeacher identity was identified as one of the key factors impacting CFL pedagogy in a book concentrating on exploration of innovative CFL pedagogy edited by As a country of diverse cultural backgrounds, Australia has witnessed the complexity of teacher identity interaction and transformation. CFL teachers, as one of the groups, are situated in the context of multilateral negotiations of power relations and language ideologies . UnderstA Chinese-Australian bilateral provincial program is the focus of this study. Each year, a group of graduates (around 10) from Chinese universities  is recruited to come to Sydney pursuing a Master’s degree in Education. Research-based as it is, the 2-year study includes practices of teaching Chinese in Western Sydney primary and secondary schools. As a start of the program, the graduates, who predominantly majored in Teaching Chinese as a Foreign Language and English as a Foreign Language (the English degree in Chinese universities refers to learning English language and culture as a major), need to take up International English Language Test System (IELTS), designed by the University of Cambridge and measures a person’s ability to listen, speak, read, and write English at the level that is necessary to go to universities mainly in Britain, Australia, Canada, and New Zealand where English is the native language, to ensure a reasonable English proficiency. There is a series of workshops to inform the candidates of Australian culture and potential environments. After arriving at the university in Sydney, the CFL teacher candidates undertake another series of workshops where teaching and culture are specified regarding local schools. Allocation to schools was random, depending partially on accommodation distance and transportation availability.Eight participants of this study were from the above-mentioned program. Five of them are indirect participants, which means they did not participate in the study in person; instead, their reflective journals from their theses are resorted to as research data for this study. The other three participants, two males and one female, were involved in this study in person, from whom interview data were collected. One interviewee was enrolled in 2016 and the other two in 2017. They volunteered to participate in this study and liked to share their experiences. Formal consent forms were signed by the three interview participants.Data for this study cover both theses data and interview data, which contribute to data triangulation . As for The reason why there was a gap between interview and reflective journal data sources is that the CFL teachers who wrote the reflective journals in their theses had graduated and went back to China while the data for this study were being collected; the interview data came from another three participants from the same program who were conducting their CFL teaching and studying at the time and, thus, were available for interview participation. It is believed that this gap would not harm the reliability and validity of the study, because the same linguistic and cultural backgrounds they come from and the same linguistic and cultural contexts they enter into would bring them similar experiences and issues. The quotes demonstrated in the “Findings” section were both from reflective journals in their theses and interviews, with the interview data marked.As a source of data, all the self-reflective journals in the CFL teachers’ thesis between 2013 and 2017 were resorted first as a general pool for consistency and randomness. It is noteworthy that only a few of them mentioned their struggles extensively as they were not the focus of their study. Accounts from candidates C1 to C5 were organized according to relevance toward the Chinese self, Australian student, and Australian environment see . FurtherTo understand the dynamics related to the establishment of CFL teacher identity from the perspectives of self and other in Australian schools, this study targets the following research questions:(1)What role does CFL teachers’ Chinese self play in the formation of their Australian teacher identity?(2)How do social relations with Australian other affect CFL teacher identity?(3)How to negotiate between self and other in Australian contexts for CFL teachers to develop a favorable teacher identity?Based on categorizations and connections of data, the findings of this study were structured into three sections to display the meaning of self-other relationship to CFL teachers in Australian classrooms, variant factors that contribute to different identities, and implications as well as results of different approaches.Chinese self refers to the self developed in China over time as the CFL teachers experienced the social network there. “Both subject and object” , p. 137,I remember I had to work hard and listen to the teachers when I was a student. Therefore, it is to me that that should be the norm of the teacher-student relationship. It is in my culture. But I found students here wouldn’t care that much about you. In this culture, students do not listen to what you say. They do whatever they like and that causes conflicts. I feel I’m not a real teacher here .“Culture” was T1’s keyword in describing troubles and his teacher identity was severely compromised. Consistent with the theses data key ideas , different attitudes from Australian students have caused issues on self-communication. The self they acquired from China in understanding education was that students need to show respect to teachers and that teachers possess unconditional authority. Although the CFL teachers were informed about the cultural differences beforehand, no one expected the reality to be “so bad” (T3) and it “hurts” (T1). All the participants mentioned the title “teacher” should at least have a certain meaning to students rather than being treated as “nothing” (T2). T2 viewed the cultural clash in a positive sense and took it as a chance to learn Australian culture. He thought it was fine to start from “zero” and believed in his “adaptability.” T1 said he did not feel good being a teacher in Australia and would look for other professions. He was weary of “surviving” when “so much have been paid” to be a teacher. T3 did not feel as bad because she had “low expectations” for both herself and the students from the beginning. There was no need for Australian teacher identity and her aim was to “survive.”I might not be respected; so what? I don’t really care about it that much. This is a journey to experience something and I want things to be natural. My goal is to finish the degree. When I go back to China, students there will respect me anyway .Students were regarded as the key “problem” (T1) that “burned” (T2) the CFL teachers out. As a major determinant of the CFL teacher’s Australian “me,” they displayed different ways through which teacher identity could be shaped. While “no respect” was the main concern of all the CFL teachers, they have different views toward it. These data mostly revealed frustration from gauging students with Chinese standards .Another interesting issue discovered in the process of the interview was that mentor teachers/supervisors played an important role in the CFL teachers’ connection to Australian culture, especially about schooling. As mentioned once in these data, mentors seemed invisible in the CFL teachers’ life, so we focused on them during the interviews. While T3 said her mentor was a “happy person” and “pretty helpful” in giving her some advice, T1 and T2 believed their mentors were “not that helpful.” They viewed the mentors as their “teachers” and were hesitant to talk about their problems. They thought it was from their culture that “mentors are our teacher and should be respected.” “The respect means no questioning even when things are not that great” (T2). “Personality” was mentioned as a key that “makes difference” (T1).I know this sounds harsh but to be honest, we do not connect. She [my mentor]is very busy and I understand that, but since she is a mentor, doesn’t she think there’s a role to play? I feel that she was waiting for me to ask questions; I was too shy. Anyway, maybe I’m overthinking .Well, she doesn’t know much about Chinese, and I don’t know what’s going on here, so we don’t know what to talk about. There are conversations now and then but not that much .While intrapersonal and interpersonal factors were essential for the construct of CFL teacher identity, environmental elements also played in the self-other interaction. Both theses data and interviews revealed the forms of environment and corresponding influences. There was a mention about learning atmosphere as well as resource issues in theses data. “Not satisfying” was the keyword that indicated environmental challenges. Interviewees mentioned “lack of academic” (T1) and “low level of persistence” (T2). The overall learning culture was “unsatisfactory” (T2), and Chinese learning was “the worst” (T3).A student was silly, and I got angry. Instead of helping me to manage him, the class teacher said, “he’s low and sometimes acts like that to me too.” I didn’t argue with her, but definitely that’s an excuse. Well, others were not much better .I heard students say, “no one likes this subject” or “I’d rather stay home.” They don’t see the point of school .Lack of resources was another issue that reflected contextual challenges. T1 said he could not find anything from the school library for language learning and everything was designed from scratch. T3 mentioned the amount of money she spent on materials and equipment. While T2 supported the idea that “no textbook means flexibility for teachers,” he argued that “there should be a pool available to choose from.”I would give CFL teachers something and let them try and see what materials are contributing to what skills of students. Then we can build on that and make adjustments. Records should be kept of what is useful .Drawing on theses of 5 years and interviews of three participants of the CFL program, the foregoing part offered a picture of CFL teachers’ construction of teacher identity in Australian contexts. Although the sample size is limited, this study reveals a new angle to approach existing issues. On the whole, our findings support the existing literature e.g.,  that CFLIn searching for answers to our first research question, CFL teachers’ cultural identity stands out and poses questions of self-other interactions interculturally. First, CFL teachers are equipped with Chinese self that they acquired over the years from social networks in China. The self is a collection of social processes of Chinese characteristics. Viewing Australian students from the Chinese self, CFL teachers may think they are not respected when students do not greet them. There was a mention of “no greeting” (C1) and “ignoring” (C5) in theses data. Such desire for “automatic respect” (C1) comes from the Chinese self where Chinese students’ attitudes to teachers formed the “me” . AnotherMost CFL teachers choose to adapt to the Australian culture by changing the self, while T2 wants to change other as well. He believes students’ attitudes toward him as a CFL teacher are manageable and it is part of his “mission.” As a “significant symbol,” language (English) enables adjustments . T1 and Adding to previous studies on mentor teachers’ contribution to CFL teachers’ growth , we findThe second research question brings us to the functional side of social relationships. In order to have a “complete self,” CFL teachers need “various aspects of the structure of the social process as a whole” , p. 144.Finally, in searching for ways to negotiate between the Chinese self and the Australian other to develop teacher identity, we find that the CFL teachers struggle mostly with acquiring a new self to connect with the new environment. While English proficiency is a major challenge mentioned across the theses and interviews, “purposeful contextual improvement” (T2) seems more achievable and beneficial than trying to be “as local as class teachers” (T1), which may cause distress. Understanding what to use in specific situations allows CFL teachers’ appropriate me to respond to other. Instead of struggling to change accent and fluency, the focus should be on the practical function of English in expressing the Chinese self. Using appropriate English for the communication of Chinese cultural identity is crucial for the construct of teacher identity. Therefore, although English proficiency is key for communication, a specific selection of areas to improve defines the progress of self-identification. CFL teachers’ mindset is also integral for the shaping of their identity , as the This study examines new CFL teacher identity transformation in Australian contexts and factors that lead to various forms of self-other relationships. Coming from Chinese culture and social relationships, the CFL teachers’ Chinese self dominates the way they respond to the new environment. When looking for the Chinese type of respect from Australian students, the Chinese self is not satisfied, and discomfort arises. To connect with the new other, understanding of self is a prerequisite to having proper positioning. Culture shock in this sense is the result of self-other mispositioning. The desire for “unconditional respect” (T3) from the Chinese self is adjusted to “earned respect” (T2), and Australian students’ attitudes toward teachers reshape the “me” of the Chinese self. Furthermore, the relationship between other teachers and students also contributes to the self of CFL teachers, as “me” is a collection of organizational attitudes. Lack of communication with other teachers has not only given the CFL teachers a sense of disconnectedness but has also led to lack of information on how students are viewed and treated in specific contexts.When approaching Australian other, English was a challenge to the CFL teachers, although they had an English-related degree and sat English tests for the program. Lack of practical use of English leads to confidence issues such as “don’t know what to do” and “afraid of giving instruction” (theses data). Mentor’s help and CFL teacher’s growth-mindset are important in facing this challenge, which may benefit both CFL teacher’s English skills and student’s learning. Another finding is that CFL teachers are shy to seek help even when they know that it is needed. This cultural presentation of Chinese self limits CFL teacher identity development. On this point, we suggest different sessions for programs and the like to explore intercultural communication needs both inside and outside of classrooms and find ways to promote proactiveness of all parties.Limitations of this study should be highlighted on two fronts: first, the small-scale research focuses on the need of new CFL teachers in Australian schools for identity construction based on a self-other relationship without consideration of personality, which could be a factor influencing self; second, there is a gap between the theses data and interview data, because identity issue is rarely discussed in depth in the former. We suggest that future research should have more CFL teachers included and take their individualities into account for a more comprehensive picture of this issue. Moreover, it is also advisable to look into CFL teachers’ personalities in the future regarding their identity formation.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.The studies involving human participants were reviewed and approved by Western Sydney University Ethics Committee. The patients/participants provided their written informed consent to participate in this study.Both authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication.The 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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Homology-directed repair occurs primarily in the S/G2 phases of the cell cycle, whereas long-term engrafting hematopoietic stem cells are typically quiescent. Alternative methods for a targeted integration have been proposed including homology-independent targeted integration and precise integration into target chromosome, which utilize non-homologous end joining and microhomology-mediated end joining, respectively. Non-homologous end joining occurs throughout the cell cycle, while microhomology-mediated end joining occurs predominantly in the S phase. We compared these pathways for the integration of a corrective DNA cassette at the Bruton’s tyrosine kinase gene for the treatment of X-linked agammaglobulinemia. Homology-directed repair generated the most integration in K562 cells; however, synchronizing cells into G1 resulted in the highest integration rates with homology-independent targeted integration. Only homology-directed repair produced seamless junctions, making it optimal for targets where insertions and deletions are impermissible. Bulk CD34+ cells were best edited by homology-directed repair and precise integration into the target chromosome, while sorted hematopoietic stem cells contained similar integration rates using all corrective donors.Gene editing utilizing homology-directed repair has advanced significantly for many monogenic diseases of the hematopoietic system in recent years but has also been hindered by decreases between   There are alternative methods for nuclease-mediated targeted gene integration, including homology-independent targeted integration (HITI) and precise integration into target chromosome (PITCh), which utilize non-homologous end joining and microhomology-mediated end joining, respectively. These DNA repair pathways can be utilized in both cell lines and primary hematopoietic stem cells. However, murine studies of HDR-based gene editing in hematopoietic stem and progenitor cells (HSPCs) by many groups have come upon a common hurdle: the rates of editing via the HDR pathway in human HSPCs often drop substantially over time in murine xenograft models, while NHEJ-mediated editing typically persists at stable rates.2 phases of the cell cycle, while NHEJ is active throughout all phases of the cell cycle.0/G1 improved the ratio of HDR to NHEJ events.0/G1 and may be inherently less capable of HDR than of actively dividing progenitor cells.,1/S phases due to its reliance on MMEJ.,In this article, each of these three methods of DNA integration—HDR, HITI, and PITCh—was assessed for their potential to achieve a permanent gene-therapy-based cure for X-linked agammaglobulinemia (XLA) through gene editing of autologous HSPCs. XLA is a primary immunodeficiency resulting from a block in the pre-B cell phase of early B-lymphocyte development, which leads to a lack of mature B-lymphocytes and of antibody production in most patients.BTK) gene, which encodes a cytoplasmic signaling protein found in most hematopoietic lineages.BTK gene to patient HSPCs has produced promising results,,BTK is a tightly regulated gene with evidence showing that both under- and overexpression can reduce protein efficacy.BTK at its endogenous locus in the genome and presents an attractive way to sidestep the potential problems associated with viral vectors. Site-specific integration should retain most of the endogenous control of the BTK gene, leading to a regulated expression near wild-type levels. Furthermore, using a DSB to guide integration with a highly specific endonuclease should nearly eliminate all off-target integration events and drastically reduce the risk of insertional oncogenesis.Patients with XLA have loss-of-function mutations in the Bruton’s tyrosine kinase .While some genes can be repaired by replacing only specific bases or even small regions of the gene, neither of these approaches are feasible for XLA, as pathogenic mutations span the entire locus without any noteworthy hotspots. Instead, a universal strategy that addresses almost all known disease-causing mutations is to integrate a nearly full-length BTK cDNA donor at the start of the endogenous BTK was delivered along with Cas9 either from an expression plasmid, as in vitro-transcribed mRNA, or was pre-complexed to Cas9 protein as a ribonucleoprotein (RNP) to introduce a targeted DSB  sgRNA cut sites. The small regions of homology guide the MMEJ repair process, although this donor may also integrate via NHEJ through end-trapping of the donor. Three PITCh donors were assembled: the first (“PITCh-1”) had Cas9 binding sites positioned to minimize the length of residual DNA in the donor template after cleavage, the second (“PITCh-2”) had both Cas9 binding sites positioned in the same orientation, and finally, a third template (“PITCh-0c”) lacked cut sites entirely.BTK intron 1. Four days post-electroporation, gene integration rates of each donor were quantified using droplet digital PCR (ddPCR). The HDR donor achieved the highest integration rates of 18.2% ± 5.6%, while 11.4% ± 0.1% integration was observed using the HDR ΔPAM donor (Each donor plasmid was delivered to K562 human erythroleukemia cells via electroporation in conjunction with a Cas9 expression plasmid (pX330) encoding a sgRNA specific to AM donor E. The HIAM donor F. These To characterize the fidelity of the integration junctions using each of the donors, PCR products produced by primers that amplified on-target cDNA integration events in proper orientation were analyzed by Sanger sequencing (n = 30–44). The HDR ΔPAM integration events all displayed seamless integration junctions, whereas the HDR donor, which contains an intact PAM sequence, yielded no perfect clones A and 2B.BTK locus. There was also some concatemerized cDNA product detected using the HDR donor, likely resulting from sequential integration events due to an intact PAM/sgRNA cut site.To describe the integration patterns of the different donors, In/Out PCR was performed using primer pairs designed to detect integration and the orientation of the cDNA cassette or plasmid backbone at the 5′ and 3′ junctions A. Each p0, whereas K562s are rapidly cycling and have a much higher prevalence of cells in S/G2. To evaluate whether these donors may be effective in HSCs, K562 cells were reversibly synchronized to the G1 phase of the cell cycle using hydroxyurea, an inhibitor of ribonucleotide reductase most active during the S phase. Cells were first treated with hydroxyurea for 24 h and analyzed by flow cytometry to identify changes in the cell cycle  gRNA with Cas9 mRNA, and chemically modified gRNA pre-complexed to Cas9 as RNPs  were tested to maximize gene editing while minimizing toxicity of the donor template . These conditions also produced 7.6% ± 2.1% editing with the HDR donor  and progenitors (CD34+/CD38+) D. After BTK locus in cell lines and primary HSPCs. In this study, nine donor variations allowing different DNA repair pathways  were evaluated for integration efficiency and integration junction fidelity. In cell lines, HDR-based methods led to the highest levels of integration, likely due to the frequently cycling nature of these cells. HDR, and particularly the HDR donor with a PAM mutation, led to the fewest undesired integration events detected by PCR. The same donor led to 100% base perfect sequence junctions. HITI donors at the same target site had the second highest rates of integration. The addition of only one cut site on the 5′ end of the donor achieved the highest integration rates. Notably, the addition of a second cut site on the 3′ end of the donor led to a drop in integration by about 50%. The addition of a second cut site divides the donor into two similarly sized fragments (one being the corrective cassette and the other being the plasmid backbone), which compete to integrate into available nuclease-mediated DSBs. In this scenario, each fragment would integrate approximately half of the time, thereby decreasing the rate of desirable integrations.The results here offer an unbiased comparison of three different methods of targeted integration at the When examining the fidelity of the 5′ integration junctions, some donor variants led to higher rates of precise integration. Notably, the HDR ΔPAM donor yielded only seamless junctions, while the almost identical HDR donor that lacked a PAM had no sequence-perfect junctions. There are likely two different mechanisms that explain the discrepancy that results from only a 2 bp change in these donors. First, the presence of an intact binding site for the gRNA/Cas9 in the donor plasmid made the donor capable of end-capture integration via NHEJ, similar to the HITI donors, resulting in indels in the junction. The second more common mechanism is that even a perfect integration of the HDR donor restores the Cas9 binding site in the genomic DNA, allowing for the endonuclease to re-cleave an already integrated product. In this case, NHEJ could occur following the desired HDR event to produce indels at the junction. For HITI donors, even a “perfect” integration event with no additional indels will leave a scar by design. As expected, all of the HITI donors produced high levels of aberrant integration events via qualitative PCR screening, and the HITI-2c donor had very few base perfect junctions identified via sequencing. Surprisingly, many of the sequenced junctions had a deletion of a region of microhomology, suggesting that they may have integrated via the MMEJ pathway in some cases rather than the NHEJ pathway alone. PITCh donors consistently resulted in the lowest integration rates, although the majority of integration junctions had base perfect sequences.HDR donors are ideal for use in rapidly cycling cell lines or in genomic loci that are intolerant of indels. However, in instances where homology arms cannot be practically added, such as large payloads approaching the maximum capacity of a delivery vector, using a HITI donor may be an acceptable replacement. It is important to note that these HITI donors leave DNA scarring in the form of duplicated portions of the protospacer or PAM sequences. Therefore, 1 phase of the cell cycle with hydroxyurea significantly altered the dynamics of donor integration, reflecting the different mechanisms by which integration can occur based on the cell type and its characteristics. HDR and PITCh donor integration rates both dropped to nearly 0%, while rates of HITI integration approximately doubled. Notably, rates of integration using the HDR donor, which contains an intact PAM site, only dropped by half compared with unsynchronized cells. This may be due to the ability of this particular donor to integrate via a HITI-like pathway upon cleavage of the donor; this is in contrast to the HDR ΔPAM donor, which lacks an intact cut site and can only integrate via HDR. Hydroxyurea synchronization of PBSCs also yielded similar results, reducing rates of HDR while increasing rates of HITI-mediated gene modification. Together, these experiments support the cell-cycle dependencies of each pathway and are consistent with the initial hypothesis that HITI may be an ideal mechanism for targeted integration for non-replicating cells.Synchronizing K562 cells into the GIn primary human PBSCs, the optimized donor for each integration method led to predicted integration rates in bulk cells. HDR donors achieved the highest overall integration rates, likely due to the cycling, larger proportion of more differentiated progenitor cells that compose the bulk population. Gene correction in cycling progenitor cells possesses less therapeutic relevance because these cells are less likely to engraft long term and self-renew like true HSCs. While HDR vastly outperformed the two other pathways in bulk cells, all three donors led to similar levels of targeted integration in immunophenotypic HSCs. In fact, the percentage of gene integration was either maintained or significantly higher in sorted CD34+CD38–CD90+CD45RA– HSCs compared with bulk-edited PBSCs across all donor types. The ability of the PITCh donor to achieve similar rates of integration in HSCs was unexpected due to MMEJ’s dependence on the S phase of the cell cycle. One possible explanation for this finding is that the PITCh donor can also integrate via HITI due to the presence of flanking cut sites just beyond the microhomology arms.In all, these findings suggest that HDR-, HITI-, and PITCh-mediated targeted integrations can be effective for editing in primary human HSCs. While levels of bulk editing vary substantially, integration rates are nearly equivalent in immunophenotypic HSCs. Integration into an exon or sequence that requires a seamless junction is likely best approached with an HDR donor. However, integration into non-coding regions may tolerate all three approaches. HITI and PITCh donors do not require the full homology arms present in an HDR donor, which can conserve the sequence length in larger donors that are nearing the size limits for viral packaging. At the BTK locus, HITI donors achieved similar or better results than PITCh donors in each experiment, making it the preferable alternative to HDR. However, the ideal length of microhomology arms is also not known for MMEJ-mediated integration and may have required additional optimization for BTK. While there may be some locus dependent variables that impact the efficiency of the different repair pathways, the data presented here support the potential for all three pathways to lead to functional integration in HSCs and to act as potential avenues for clinically relevant gene therapies.BTK intron 1.sgRNAs targeting BTK intron 1 were identified using the design algorithm from CRISPRscan.BTK cDNAs from exons 2 to 19 that were codon-optimized using the GeneOptimizer online tool . BTK exon 2 has a small portion of an untranslated sequence that was included in each donor but was not codon-optimized, as there are no coding regions in this sequence. The donor template contains the 3′ splice site from the end of BTK intron 1, the exon 2 portion of the 5′ UTR, codon-optimized exon 2–19 BTK cDNA, three C-terminal HA tags, a stop codon, the BTK 3′ UTR, and a short sequence to improve polyadenylation.HDR donors contained 5 K562 cells per condition were electroporated with 500 ng of sgRNA/Cas9 expression plasmid and 3 ug of the respective donor plasmid using the Lonza 4D Nucleofector X Unit  in 20 uL of Lonza SF buffer . After electroporation, cells were plated in 500 uL of R10 media  supplemented with 1× penicillin/streptomycin/glutamine  and 10% fetal bovine serum . For cell-cycle synchronization, K562 cells were pre-treated with 100 ug/mL of hydroxyurea for 1 day before nucleofection . Cell-cycle analysis was performed using Hoechst dye  following manufacturer protocol. Three days after electroporation, treated cells were washed and re-suspended in R10 without hydroxyurea. Genomic DNA was harvested from the cells using the Invitrogen PureLink Genomic DNA Mini Kit . Protein lysates from 2 × 106 cells per condition were harvested 1 to 3 weeks post-nucleofection using Denaturing Cell Extraction Buffer  + 1× Halt Protease Inhibitor . Lysate concentrations were quantified via bicinchoninic acid (BCA) assay .2 × 10BTK specific primers and probes are listed in Integration rates were measured by ddPCR. Equal amounts of protein lysates per sample were loaded in each well of 4%–12% Bis-Tris gel . A primary antibody for HA  was used to detect integrated protein, while beta actin was used as a loading control . Secondary antibodies were conjugated with horseradish peroxidase  for the HA secondary and Alexa Fluor 647 for the actin secondary  at dilution factors of 1:750 for HA and 1:1,000 for actin.5 cells/mL in X-Vivo 15 medium  supplemented with 1× penicillin/streptomycin/glutamine , 50 ng/uL recombinant human (rh) stem cell factor , 50 ng/uL rh Flt-3 ligand , and 50 ng/uL rh thrombopoietin . After 2 days of pre-stimulation, cells were electroporated using the BTX system . 2 × 105 cells/condition were electroporated in 100 uL of BTX buffer . For RNP samples, 9 μg of chemically modified sgRNA  were incubated with 200 pmol of Cas9 protein for 20 min at room temperature to make RNP complexes. For Cas9 mRNA electroporations, 5 μg of the chemically modified sgRNA was introduced with 5 μg of SpCas9 mRNA. Following electroporation, samples were transduced with the relevant AAV6 donor vector . One day post-electroporation, cells were resuspended in 500 uL of outgrowth media: IMDM , 10% fetal bovine serum, 1× penicillin/streptomycin/glutamine , 5 ng/mL of rhIL-3, 10 ng/mL of rhIL-6, and 25 ng/mL of rhSCF . Four days post-electroporation, cells were harvested for analysis. In experiments involving cell-cycle synchronization, PBSCs were cultured in 0.03 mg/mL hydroxyurea for 24 h pre- and post-electroporation.Human CD34+ granulocyte colony-stimulating factor (G-CSF) mobilized peripheral blood stem and progenitor cells were thawed 2 days prior to electroporation. Cells were pre-stimulated at a density of 5 × 10Cells were sorted into two populations: HSC  and progenitors  . Three to ten days post-sort, gDNA was extracted from each condition for analysis.


Streptococcus suis-infected pigs. The animals were anesthetized over a seven-hour period with isoflurane in air/oxygen at physiologic arterial partial pressure of oxygen. Oxygen levels in CSF of anesthetized pigs were compared to euthanized pigs. The detected partial pressure of oxygen in the CSF remained constant in a range of 47–63 mmHg, independent of the infection status . In contrast, the pH value showed a slight drop during infection, which correlated with cell and bacterial number in CSF. We present physiologic oxygen and pH values in CSF during the onset of bacterial meningitis.During infection and inflammation, a reduced oxygen level clearly affects cellular functions. Oxygen levels during CNS infections are unknown. Here we established and evaluated an in vivo measurement system to characterize the oxygen level in parallel with bacterial numbers (CFU/mL), the cell number and pH level inside the CSF of healthy compared to The online version contains supplementary material available at 10.1186/s12868-021-00648-x. The paO2/FiO2 values decreased slightly for this pig, with values one hour after induction, 4 h after induction and 7 h after induction of 402, 309 and 317 mmHg, respectively.The status of all animals was during the anesthesia frequently checked for abnormalities. Only minor adjustments in ventilatory settings and fraction of inspired OS. suis was reisolated from an inner organ or from serosa or from joint fluid, indicating an undergoing infection  [The infection site during meningitis is the CSF, which is produced by the choroid plexus cells, circulating around the brain, inside the ventricles and around the spinal cord. This compartment is highly protected by the brain barriers and the entrance or diffusion of cells, ions and substrates are strictly regulated by these barriers . The dif.8 mmHg) . Interes.8 mmHg) . The pre.8 mmHg) . The com.8 mmHg) –44. TherNevertheless, whether or not oxygen is permanently regulated inside this compartment during infections with pathophysiological increased oxygen consumption  inside the CSF is unknown.2, pH, cells/mL and CFU/mL as well as to collect CSF samples for later analysis of deep-frozen CSF samples, we established the described system  and a slightly lower value was detected inside the cisterna (58 mmHg), decreasing further inside the sagittal sinus [2 values in CSF from lumbar puncture in humans (breathing in room air) vary highly depending on the used methods from 39.0 ± 6.9 mmHg to 77 ± 8.7 mmHg [cisterna cerebellomedullaris at the atlantooccipital joint as the puncture localization for the CSF compartment the. This was a compromise for an easily accessible point compared to ventricles and a close localization to the host–pathogen interaction region during meningitis (meninges and blood brain barriers). One limitation of this puncture localization in the pig is that several blood vessels can be injured during the process of gaining access to the CSF compartment. This is a critical point in the whole method, which can be avoided by training. If a blood vessel is affected during puncturing of the CSF compartment, blood cells could contaminate the CSF compartment. In case blood cell contamination occurs at the first measurement point, and the bleeding has stopped, the turnover rate of CSF [To determine values in CSF of pOtem Fig. . This syal sinus . The pO28.7 mmHg , 45. We e of CSF  may resuS. suis can induce CSF acidification.Finally, the negative correlation of the pH level with cell number and CFU is of interest  and included females and males. The pigs were bought and the management of the farm agreed to use the animals in animal experiments in this study.post mortem. The pigs were three to five months old and had a body weight of approximately 70 ± 20 kg.Part A: Initially, measurement trials in cerebrospinal fluid (CSF) taps of healthy euthanized pigs were conducted post mortem measurement in CSF the pigs were housed in groups. The pigs had an average age of eight weeks and a body weight of 17 ± 2 kg at the day of infection in the case of the in vivo measurement. The health status of the pigs was checked, and the animals fed twice per day. Water was available ad libitum.Part B: For the in vivo measurement in CSF as well as part of the S. suis infection rate depend on the age of the animals. The lowest antibody level against S. suis can be observed between weeks 6 and 8, presumably corresponding to a decrease in maternal immunity. A marked increase can be seen at 10 weeks of age, shortly after the onset of clinical signs in the herd during an outbreak [It was important to use in this study animals from a specific age, as outbreak . The avaThe in vivo experiment was divided into three runs with run 1 = 3, run 2 = 4 and run 3 = 5 animals, respectively. In total there were eight females, two males and two castrated male pigs used in the experiment. Six pigs were used as uninfected control and six pigs were infected as described below.2. To calculate the number of animals the software GraphPad StatMate 2.00 was used.The sample size in part A was not statistically calculated, as it was only used for technical adjustment. The sample size in part B was calculated with 6 animals per group and two animals for replacement. For statistical calculation, the following parameters were used: Error 1st type, α = 5%, error 2nd type, β = 20% (power 80%), relevant difference to be detected: 2% pOThe total number of animals used in this study was 20.If it was impossible to get an excess to CSF, the experiment was stopped and the animal euthanized due to animal well fare reasons .In order to establish good puncture results as fast as possible regarding the CSF at the atlantooccipital joint in living animals, the procedure of finding the correct puncture site was trained on half-carcasses at the Institute for Food Quality and Food Safety  and on dead pigs with a body weight of approximately 13 kg. The position of an inserted epidural catheter inside the subarachnoid space  was documented by computed tomography  scan. A picture and video were generated with standard settings in the CT software.Articulatio atlantooccipitalis . Equally important are the correct stitch angle and stitch depth as well as hitting the center of the back. The goal of the exercises was to obtain a sterile, blood-free CSF sample in the shortest possible time.The training of the personnel was carried out in previously published animal trial . At leas−1 body weight (BW), Stresnil ad us. vet., Elanco Tiergesundheit AG, Basel, Switzerland) and ketamine-hydrochloride  intramuscularly. The injection was carried out in the Musculus (M.) biventer cervicis near the base of the ear with a 21 G cannula . The pig was separated and therefore protected from the other pigs to allow a gentle start of the anesthesia. The depth of anesthesia was proven by observation of a trained veterinarian. Then the pig was transported in a separate room for further examination and euthanasia. The neck area was shaved around the Articulatio atlantooccipitalis . By dorsoventral movement of the neck, the point of most flexibility was identified and marked with a laterolateral transverse line with a permanent marker. Furthermore, a median sagittal line was drawn. With this, an intersection point was found  with T 61®  during anesthesia. The death of the pig was determined by a trained veterinarian and confirmed by absence of heartbeat and reflexes. The procedure of euthanasia followed the recommendations for euthanasia of experimental animals [The pigs were anesthetized with azaperone , a Spinocan® epidural needle  was inserted in a rostral direction approximately 1 cm caudal of the intersection point. After detecting a slight resistance by penetrating the arachnoid membrane, the stylet was removed. Clear CSF dripped out of the needle hub if the subarachnoid cavity was punctured. If no CSF was observed or in case of blood contamination, the needle was moved slightly until clear CSF dripped out. After clear CSF had been observed, the needle was immediately connected to the 10-cm-long tube of a three-way valve . The FTC oxygen sensor  was then connected in series to this three-way valve. A second three-way valve with a syringe was connected to this combination. Fresh CSF was drawn into the entire system via suction from the syringe. Fresh CSF aspirates were drawn after five and ten minutes to determine the pOpost mortem. The value one-minute after taking CSF over the sensor was used for statistical analysis to achieve one-step sensor equilibration in the case of the first taken value. After five minutes, fresh CSF was drawn over the sensor and the value one minute later was used for statistical purposes (two-step sensor equilibration). Since the ambient pressure and the temperature of the medium are essential parameters to calculate the oxygen level in liquids, the current ambient pressure of the room was determined  and used to calculate the oxygen level. Since an exact temperature measurement of the CSF proved impossible, the rectal body temperature was measured and taken as the basis for the oxygen calculation.The measuring sensors were covered with aluminium foil to keep the temperature constant and to protect the sensors from light. The sample was measured at intervals over a time-span of maximum ten minutes Streptococcus (S.) suis cps type 2 strain 10 (S. suis) was used. This strain has been shown to be highly virulent in experimental infections of pigs [S. suis was grown on Columbia agar plate with 7% sheep blood  and incubated for 20–24 h at 37 °C. To prepare the infection inoculum, 10 mL Tryptic Soy Broth without dextrose (TSB)  were filled in T405-Cultubes™  with air exchange and two freshly grown S. suis colonies were added. The culture was incubated for ten hours (37 °C / 5% CO2). A 1:100 dilution was created in an Erlenmeyer flask with preheated TSB and incubated for three-four hours (37 °C / 5% CO2) to the late exponential growth phase reflected by an OD600nm of 0.3 ± 0.02. Then, 40 mL of the bacterial culture were transferred to a 50 mL Falcon Tube  and was centrifuged at 4816 g for ten minutes at room temperature. The supernatant was discarded, and the pellet was resuspended in 3 mL sterile phosphate-buffered saline (PBS). From this suspension a 1:10 dilution was made, and 1 mL thereof was intravenously injected into pigs as described below. The exact infection dose for each piglet was determined by plating serious dilutions on blood agar plates and counting colonies after 20-24 h incubation at 37 °C.In this study,  of pigs –52. S. sVena auricularis) inoculated with 1 mL PBS containing 2.5–3.7 × 108 CFU of S. suis infection inoculum (as described above) or 1 mL PBS in case of the control animals.Seven-to nine-week-old German Landrace pigs (weaned at four to five weeks of age) were anesthetized as described above with azaperone and ketamine-hydrochloride intramuscularly and were intravenously , azaperone (2 mg kg−1 BW) and atropine .After the infection, the animals were housed separately for the next eight-12 h. Using a scoring system, eight hours −1 BW; Narcofol® 10 mg/mL BW, CP Pharma, Handelsgesellschaft mbH, Burgdorf, Germany) was administered to achieve endotracheal intubation. After topical anesthesia with tetracaine spray , the pigs were endotracheally intubated under visual control with a cuffed endotracheal tube .Venous access was established by an indwelling venous catheter  either in an ear vein, the cephalic or lateral saphenous vein. Propofol  and volume controlled mechanical ventilation  with a tidal volume of 10–16 mL kg2 as needed to maintain an arterial partial pressure of oxygen of 80 to 110 mmHg. A constant infusion rate of ketamine  and dexmedetomidine  was administered for MAC-sparing effects. To facilitate mechanical ventilation, 0.15 mg kg−1 levomethadone/fenpipramide  was injected intravenously immediately after intubation and repeated after four hours of anesthesia. For cardiovascular support, a balanced, lactate buffered electrolyte solution  was administered at a rate of 5 mL kg−1 h−1 throughout the procedure. Additional file Anesthesia was maintained with isoflurane  in air. To avoid hyperoxia and hypoxia, oxygen was supplemented to adjust FiO2) via a transmission pulse oximetry probe placed at the tail or a claw and the rectal temperature were continuously monitored via an anesthesia multiparameter monitor . For invasive blood pressure measurement, access was made to the femoral artery by an arterial catheter . The pressure was recorded via a calibrated pressure transducer  levelled and zeroed to ambient pressure at the base of the heart. End-tidal CO2, inspired and expired isoflurane concentrations as well as the inspired and expired (FeO2) fraction of oxygen were monitored via a respiratory gas monitor .The depth of anesthesia was monitored by testing muscle relaxation, the eye reflexes, the interdigital withdrawal reflexes and skin stimulation with a Kelly type artery clamp. The ECG, heart rate, arterial blood pressure, peripheral oxygen saturation  before each pOThe temperature of the animals were maintained by means of red-light heat lamps and adjustable electrical heating mats during the period of anesthesia. The initial temperature was the temperature at the time of inducting anesthesia.After completing the measurements, the animals were euthanized by a trained veterinarian during isoflurane anesthesia, for further examination. Therefore, the pigs were euthanized intravenously via one of the already existing venous access points (described above) via the indwelling venous catheter  either in an ear vein, the cephalic or lateral saphenous vein with T 61®  during isoflurane anesthesia. The death of the pig was determined by a trained veterinarian and confirmed by absence of heartbeat and reflexes. The procedure of euthanasia followed the recommendations for euthanasia of experimental animals .After endotracheal intubation, the pigs were positioned, and the CSF access carried out as described above. The needle was connected to the 10-cm-long tube of a three-way valve . One side was connected to a pressure transducer  and the other side to the measurement system. Therefore, the FTC oxygen sensor  and the pH sensor  were then connected in series to the leftover connection. At the open side of the pH sensor, it was possible to collect CSF samples with a syringe by means of a second three-way valve.2 and the pH level inside the CSF, a six minute two-step sensor equilibration was conducted. For this purpose, the three-way valves were opened so that 1.8 mL of fresh CSF was drawn into a 2 mL syringe via the oxygen and pH sensors. Thereafter, the three-way valves were reversed so that CSF was trapped over the sensors without any air contact. For the following five minutes, the sensor probes equilibrated to the CSF (first step). The solution in the syringe was disposed of (CSF in the syringe (13 h p.i.) and the CSF- sodium chloride mixture due to rinsing procedures beforehand (16 h p.i. / 19 h p.i.)), respectively. After this first equilibration, and rinsing out the leftover sodium chloride solution, 1 mL fresh CSF was drawn into a sterile syringe as previously described before and the second one-minute equilibration step of the sensors was already performed with measuring values. During a ten-minute period, values were collected every 30 s leading to a results curve. The five-minute adaptation (from sodium chloride to CSF over the sensor) and the following one-minute equilibration (fresh CSF over the sensor for temperature and pressure compensation) were termed as the two-step equilibration. All values of pO2 and pH presented in main figures were taken after this two-step equilibration. All other values were analyzed and summarized in Additional file. After these measurements, the system was rinsed with 0.9% sodium chloride solution using method “III” as described in Additional file post infection), four hours (16 h post infection) and seven hours (19 h post infection). For every measurement, this two-step equilibration was performed. The sensors were covered with aluminium foil to keep the temperature constant and to protect the sensors from light. After each measurement, the system was rinsed with an outflow of 0.9% sodium chloride solution from the pressure transducer.After installing the measurement set-up, the three-way valve was opened in such a way that the pressure of the CSF (pCSF) was monitored permanently with the transducer. Before measuring Oair) of the room (~ 21% O2)  with the CSF pressure that had been directly measured before the measurement set-up was opened for the measurement (no pressure drop caused by opening the measuring system). Since the CSF pressure (pCSF) also influences the oxygen calculation, the sum of both pressures (pAir + pCSF) was used as the final value for the pressure compensation. The rectally measured body temperature was taken as the basis for the temperature compensation in the oxygen calculation and pH determination.For oxygen determination in liquids, the ambient pressure and the temperature of the medium are needed. Here, we combined the prevailing air pressure (pAt each time point (13–16-19 h p.i.), the CSF sample in the syringe was transported on ice to the laboratory for further examinations.A change in the measuring system during the experiment was not possible. Therefore, a rinsing process to decontaminate the measuring system between the measurement points was established as described in the Additional file.Vena jugularis or Vena cava cranialis pre-infection and from the Arteria femoralis, at defined time-points  in lithium–heparin monovettes (SARSTEDT AG & Co. KG). Serial dilutions were plated on Columbia blood agar to determine the CFU/mL after incubation at 37 °C for 20–24 h.Blood samples were taken from the S. suis.During the first anesthesia, a tonsillar swab  was taken for bacterial examination to exclude bacterial colonization of pigs with post mortem oxygen measurement in CSF, a necropsy was conducted. Swabs of the brain surface, the mitral valve, the pleura, the pericard and the peritoneum and organ samples of the liver, spleen, lung and tonsils as well as liquid from the carpal and tarsal joints were collected for bacteriologic examination.After euthanasia and S. suis were analyzed by multiplex PCR as previously described [All colonies suspected of escribed  for the In the CSF samples, at each time-point, the following parameters were determined: 1. Serial dilutions were plated on Columbia blood agar to determine the CFU/mL after incubation at 37 °C for 20–24 h, 2. Counting of neutrophil numbers in the Neubauer chamber, 3. Counting of neutrophils after fixation  analyzed by flow cytometry using the Attune® NxT Acoustic Focusing Flow Cytometer (FACS). The analysis was based on Forward Scatter  and Side Scatter  and analyzed using FlowJo software version (v)10.S. suis was identified by colony morphology on and afterwards by multiplex PCR identified as described previously [During section swabs of the brain surface, the mitral valve, the pleura, the pericard and the peritoneum were taken for bacteriology. Furthermore, for bacteriology and histology, organ samples of the brain, liver, spleen, tonsils, heart, lung, pleura, pericard and peritoneum were collected. Samples for histology were stored immediately in 10% buffered formalin and after latest 72 h embedded in paraffin and cut into 2–4 µm sections for hematoxylin–eosin (HE) staining and histological examination. The histological screenings were scored as described  and furteviously , 53.2 in the CSF was simulated by cardiac arrest by stopping the blood circulation, leading to an undersupply of oxygen to the organism. For this purpose, the oxygen level in the CSF was measured in a living animal directly before cardiac arrest. The animal was then euthanized. Ten minutes after verification of death, the oxygen level in the CSF was measured again.The influence of a possible hypo-oxygenation on the pO2% was calculated based on 38 °C and 1000 hPa (= 750 mmHg) from the measured mmHg values. The values are presented in each figure and in the text with ≜.The oxygen OP < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). Detailed information can be found in the figure legend.Data were analyzed using Excel 2010 and 2016 (Microsoft) and GraphPad Prism 8.1 (GraphPad Software). Normal distribution of data was verified by the Kolmogorov–Smirnov normality test (GraphPad software) prior to statistical analysis. Differences between groups were analyzed as described in the figure legends (*Additional file 1. CT video of epidural catheter location.Additional file 2. Additional figures.Additional file 3. Table S1. Raw data of Fig. Additional file 4. Table S2. Raw data of Fig. Additional file 5. Table S3. Raw data Fig. Additional file 6. Table S4. Analysis of various measurement cut-off's from all data in Fig Additional file 7. Table S5. Individual pO2 values in CSF and blood and mean values per group.Additional file 8. Table S6. Reisolation of the challenge strain from piglets after intravenous challenge with S. suis serotype 2.Additional file 9. Table S6. Overview of drugs used in the animal experiment under isoflurane anesthesia.


In the present study, we aimed to elucidate the molecular coupling between inflammatory markers and development of iNPH and determine whether inflammation-induced hyperactivity of the choroidal NaLumbar CSF samples from 20 iNPH patients  and 20 elderly control subjects were analyzed with the novel proximity extension assay technique for presence of 92 different inflammatory markers. RNA-sequencing was employed to delineate choroidal abundance of the receptors for the inflammatory markers found elevated in the CSF from iNPH patients. The ability of the elevated inflammatory markers to modulate choroidal NKCC1 activity was determined by addition of combinations of rat version of these in ex vivo experiments on rat choroid plexus.11 inflammatory markers were significantly elevated in the CSF from iNPH patients compared to elderly control subjects: CCL28, CCL23, CCL3, OPG, CXCL1, IL-18, IL-8, OSM, 4E-BP1, CXCL6, and Flt3L. One inflammatory marker, CDCP1, was significantly decreased in iNPH patients compared to control subjects. None of the inflammatory markers differed significantly when comparing iNPH patients with and without clinical improvement upon CSF shunting. All receptors for the elevated inflammatory markers were expressed in the rat and human choroid plexus, except CCR4 and CXCR1, which were absent from the rat choroid plexus. None of the elevated inflammatory markers found in the CSF from iNPH patients modulated the choroidal NKCC1 activity in ex vivo experiments on rat choroid plexus.The CSF from iNPH patients contains elevated levels of a subset of inflammatory markers. Although the corresponding inflammatory receptors are, in general, expressed in the choroid plexus of rats and humans, their activation did not modulate the NKCC1-mediated fraction of choroidal CSF secretion ex vivo. The molecular mechanisms underlying ventriculomegaly in iNPH, and the possible connection to inflammation, therefore remains to be elucidated.The online version contains supplementary material available at 10.1186/s12987-021-00289-6. However, since reference ICP values in healthy individuals remain sparse and pseudo-healthy patients are often used instead [+/K+/2Cl− (NKCC1) transport mechanism, illustrated to sustain approximately 50% of the CSF secretion by the choroid plexus [Idiopathic normal pressure hydrocephalus (iNPH) is a potentially reversible neurological condition first described by Hakim and Adams in 1965 , 3 and t instead  it is st instead , 17 some instead , 18, 19  instead –24. This instead , 25–29 b instead –32 and a instead  and has  instead . Altered instead . Elevate instead –38, whil instead , 40. We d plexus , 41, 42,Lumbar CSF samples from 20 iNPH patients  and 20 elderly control subjects of similar age and sex distribution  were provided retrospectively by the Danish Dementia Biobank at the Danish Dementia Research Centre . The patients were diagnosed in accordance with the international iNPH guidelines ; ventricg for 10 min, the supernatant was aliquoted into cryo tubes and stored at − 80 °C within two hours from sampling according to previous recommendations [CSF samples were obtained by lumbar puncture between 9 a.m. and 1 p.m. The first 4 ml were collected for routine investigations and the following 8–12 ml were collected in polypropylene tubes, centrifuged at 2000×ndations . The CSFBioXpedia A/S  analyzed CSF samples for presence of 92 different inflammatory markers  using the novel proximity extension assay (PEA) technique  . In brieAll procedures involving experimental animals conformed to the legislations for animal protection and care provided in the European Community Council Directive (2010/63/EU) and complied with the ARRIVE guidelines . Nine-we2, 1.3 MgSO4, 1 NaH2PO4, 10 glucose, 17 Na-HEPES, pH 7.4. Rat brains were kept in ice-cold aCSF for 10 min followed by separation of the two hemispheres and isolation of the choroid plexus from the lateral ventricles. The acutely isolated lateral choroid plexus was placed in Dulbecco’s Modified Eagle’s Medium  supplemented with penicillin–streptomycin , rat epidermal growth factor , and fetal bovine serum  (DMEM growth medium). As inflammatory markers may exert their effect on cells and tissue on a longer time scale, the choroid plexus was incubated for 16 h at 37 °C, 5% CO2, in presence of select inflammatory markers (or vehicle). To verify choroid plexus viability during the 16 h culturing ex vivo, we performed calcein acetoxymethyl  staining and evaluated the tissue by microscopy [2 prior to incubation in aCSF containing calcein-AM (16.7 µM) for 10 min at room temperature. An acutely isolated choroid plexus was employed as a positive control and the negative control consisted of an acutely isolated choroid plexus placed in distilled water (dH2O) for 16 h prior to calcein-AM staining. Images were acquired using Zeiss Axioplan 2 microscope equipped with epifluorescence and interference filters with a 702 moni AxioCam, and using Zeiss Zen Black software.Rats were anesthetized with an intraperitoneal injection of ketamine and xylazine  and quickly decapitated. Isolated rat brains were immersed in ice-cold artificial CSF (aCSF) containing (in mM): 120 NaCl, 2.5 KCl, 2.5 CaClcroscopy . Viable 86Rb+  and 3H-mannitol . 3H-mannitol does not enter the choroid plexus and serves as an extracellular marker [86Rb+ activity was corrected for extracellular background using 3H-mannitol. Data are shown as the natural logarithm of the 86Rb+ activity at time each point (AT) normalized to the initial 86Rb+ activity (A0) as a function of time. The slope from linear regression analysis was used to determine the 86Rb+ efflux rate constant (min−1) in absence (including vehicle) and presence of bumetanide  or select inflammatory markers [Following 16 h of incubation, the choroid plexus was allowed to recover for 10 min in 37 °C aCSF prior to 10 min of incubation in isotope solution containing: r marker . After i markers , 51.https://github.com/Sorennorge/MacAulayLab-RNAseq1 [® Ultra™ RNA Library Prep Kit  prior to RNA sequencing  on an Illumina NovaSeq 6000 . Novogene performed QC and removed low quality reads and adapters. The 150 base paired-end reads were mapped to Reference genome Rnor_6.0 (Rattus_norvegicus) using Spliced Transcripts Alignment to a Reference (STAR) RNA-seq aligner (v 2.7.2a) [https://mygene.info [https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE137619 [http://www.bioinformatics.babraham.ac.uk/projects/fastqc [STAR-RNA and RSEM parameter settings for the library build, mapping and quantification, together with all scripts for the gene annotation and analysis are available online at -RNAseq1 . Rat cho 2.7.2a) . The map 2.7.2a)  with RSE 2.7.2a) . Gene in 2.7.2a) , 57, httene.info . The humSE137619 . The datRat interleukin-18  was purchased from R&D Systems (UK). Rat C–C motif chemokine ligand 28 , rat CCL6 (cat. no. chm-268), rat CCL3 (cat. no. chm-343), and rat oncostatin-M  were purchased from ProSpec (Israel). Rat C-X-C motif chemokine ligand 1  was purchased from Sigma (USA). All chemicals were dissolved in sterile water or phosphate buffered saline supplemented with 0.1% bovine serum albumin  to a stock concentration of 100 µg/ml and kept frozen in aliquots at − 20 °C until use to avoid repeated freeze–thaw cycles. Final concentrations are indicated in the Figure legends.Inflammatory markers in CSF: The limit of detection (LOD) was calculated for each inflammatory marker as the background signal plus two times the standard deviation of the inflammatory marker. Inflammatory markers were excluded from further analysis if > 35% of the samples were below the LOD. This criterion led to the exclusion of 33 inflammatory markers. None of the excluded inflammatory markers showed any tendency towards preferential presence in one of the study groups. The remaining 59 inflammatory markers were included in the statistical analysis. Additional file 86Rb+efflux experiments: Data are presented as mean ± standard deviation and n corresponds to the number of individual animals. Data were analyzed with an unpaired two-tailed t-test and P < 0.05 was considered statistically significant.Data analysis and statistical tests were conducted in GraphPad Prism version 8 (GraphPad Software). To determine if iNPH patients displayed altered CSF distribution of inflammatory markers, we compared lumbar CSF from 20 iNPH patients and 20 elderly control subjects. The clinical characteristics of the study groups are summarized in Table The CSF level of one inflammatory marker, CUB domain-containing protein 1 (CDCP1), a transmembrane glycoprotein chemotactic for T-cells , was sigINPH patients often improve clinically upon VP shunt insertion –11. HoweOf the 11 inflammatory markers significantly elevated in the CSF from iNPH patients, eight were selected as candidates for further ex vivo investigation of their ability to modulate the transport activity in choroid plexus: CCL28, CCL23, CCL3, CXCL1, IL-18, IL-8, OSM and CXCL6. The remaining inflammatory markers OPG, 4E-BP1, and Flt3L were not investigated further as they serve physiological roles which make them less likely to directly influence choroidal CSF secretion.https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE137619 [The ability of the eight candidate inflammatory markers to alter choroidal transporter activity requires that their respective receptors are expressed in choroid plexus Fig. a. Such vSE137619 , Fig. 1b+/K+/2Cl− cotransporter NKCC1, a key contributor to choroidal CSF secretion [86Rb+) efflux experiments, in which 86Rb+ replaces K+ at its binding site and acts as a tracer for the K+ transport through NKCC1. Inclusion of the NKCC1-specific inhibitor bumetanide (20 µM) diminished the choroidal 86Rb+ efflux by ~ 56%  from the rat choroid plexus was not significantly altered by exposure to cocktail 1  were not investigated experimentally, as they were unlikely to directly influence choroidal CSF secretion: OPG acts as a decoy receptor , 4E-BP1 CCL28 is a chemokine constitutively expressed by epithelial cells at mucosal sites , which pThe chemokines CXCL1, CXCL6, and IL-8 exert their actions through the receptors CXCR1 and CXCR2 . CXCL1 iSeveral studies have investigated the proinflammatory chemokine IL-8 in relation to hydrocephalus. While some studies report elevated IL-8 in CSF from patients with different hydrocephalic conditions , 98, 99,OSM is a pleiotropic interleukin-6-type cytokine that utilizes two receptor complexes, IL-6ST/LIFR and IL-6ST/OSMR, to mediate a broad range of homeostatic activities, some of which include hematopoiesis, liver regeneration, and homeostasis of neural precursor cells , 107. OvOne inflammatory marker, CDCP1, was significantly decreased in the CSF obtained from iNPH patients compared to elderly control subjects. CDCP1 is a transmembrane glycoprotein chemotactic for T-cells  that has86Rb+ efflux following the 16 h incubation time, prolonged presence of the inflammatory markers may have initiated oxidative stress, apoptosis, or other cellular mechanisms that could have compromised potential effects of the inflammatory markers on choroidal NKCC1-mediated ion transport. However, other research groups have reported successful exposure of choroid plexus epithelial cell lines to inflammatory stimuli for longer time periods [Inflammatory markers such as cytokines, interleukins, and chemokines signal through various intracellular pathways and are capable of acting synergistically or antagonistically . In the  periods .The inability of the inflammatory markers to modulate choroidal NKCC1 transport activity ex vivo and thus potentially contribute to development of iNPH leaves the molecular coupling between inflammatory markers and iNPH unresolved. Elevated levels of inflammatory markers in CSF from iNPH patients indicate presence of an ongoing inflammatory condition. However, whether this inflammatory condition promotes iNPH development or arises secondarily to the pathological changes observed in the iNPH brain remains uncertain. Some studies demonstrate that CSF levels of inflammatory markers decrease following CSF shunting in parallel with clinical improvement , 117 sugThe present study is associated with certain limitations. Although all included iNPH patients were in moderate to severe stage of disease see “”, the exHere, we demonstrate that the CSF from iNPH patients contains elevated levels of a subset of inflammatory markers. Although the corresponding inflammatory receptors are, in general, expressed in the choroid plexus of rats and humans, their activation did not modulate the activity of the NKCC1 transport mechanism that is key in the CSF-secreting molecular machinery. Inflammation may possibly modulate other CSF-secreting transport mechanisms expressed in choroid plexus or other proposed CSF-secreting barriers, i.e. the endothelium or the ependymal cell layer. Unravelling of the molecular mechanisms underlying the pathological CSF accumulation seen in iNPH patients, and its relation to the inflammatory marker profile observed in this patient group, therefore awaits future research efforts.Additional file 1: Table S1. Inflammatory markers from Olink’s inflammation panel (Art. No. 95301). This table provides an overview of the inflammatory markers included in Olink’s inflammation panel and highlights the inflammatory markers excluded from the statistical analysis of the CSF samplesAdditional file 2: Table S2. Statistical tests and results for the comparison of inflammatory markers levels iNPH patients and elderly control subjects.Additional file 3: Table S3. Statistical tests and results for the comparison of inflammatory markers levels in iNPH responders and iNPH non-responders.