The use of Augmented Reality (AR) in anatomical education has been promoted by numerous authors. Next to financial and ethical advantages, AR has been described to decrease cognitive load while increasing student motivation and engagement. Despite these advantages, the effects of AR on learning outcome varies in different studies and an overview and aggregated outcome on learning anatomy is lacking. Therefore, a meta-analysis on the effect of AR vs. traditional anatomical teaching methods on learning outcome was performed. Systematic database searches were conducted by two independent investigators using predefined inclusion and exclusion criteria. This yielded five papers for meta-analysis totaling 508 participants; 240 participants in the AR-groups and 268 participants in the control groups. . Meta-analysis showed no significant difference in anatomic test scores between the AR group and the control group (− 0.765 percentage-points (%-points); P = 0.732). Sub analysis on the use of AR vs. the use of traditional 2D teaching methods showed a significant disadvantage when using AR . Meta-regression analysis showed no significant co-relation between mean difference in test results and spatial abilities . Student motivation and/or engagement could not be included since studies used different assessment tools. This meta-analysis showed that insufficient evidence is present to conclude AR significantly impacts learning outcome and that outcomes are significantly impacted by students’ spatial abilities. However, only few papers were suitable for meta-analysis, indicating that there is a need for more well-designed, randomized-controlled trials on AR in anatomy education research. In line with this, the anatomical assessment is based on the ability to recall spatial relationships between structures, both in two-dimensions (2D) and three-dimensions (3D)2. However, with an increasingly cramped curriculum for medical students, anatomy educator have been searching for engaging and interactive teaching methods based on state-of-the-art technologies3. Augmented reality (AR) concerns such a new technology which is believed to hold great potential for anatomy education5. AR has been defined as a technique that allows the user to superimpose virtual objects onto physical objects in real space and allows individuals to interact with both simultaneously. An essential difference with virtual reality concerns that with AR, the user is not completely immersed in a digital environment, which enables the user to combine digital input and real world objects6. With regard to the use of AR in anatomy education, AR can offer a highly realistic situated learning experience which is supportive to complex medical learning situations7. An important advantage of AR over physical models and cross-sections in learning anatomy, is that AR offers the opportunity to study the anatomy of a structure thoroughly by virtually disassembling and reassembling anatomical parts. A possible disadvantage of AR concerns the absence of tactile feedback4. It has been reported that, on a meta-level, three-dimensional visualization technologies yielded significant better results with regard to acquisition of spatial knowledge as compared to other teaching methods 8.Anatomy education has historically been facilitated by cadavers, anatomical models and drawings in anatomical atlases9. Especially with regard to students’ motivation to study anatomy, various favorable reports have been published over the years12. The effects of AR on anatomy learning have also been investigated by various authors14. However, such studies are sparse and more evidence on a meta-study level is needed to investigate whether AR could effectively replace or supplement other anatomy teaching methods. Recently, three systematic reviews on the use of AR in anatomy learning were published17. However, two of the systematic reviews do not analyze pooled data17. In addition, the study of Moro et al., although published recently, does not only review the evidence on the use of AR in anatomy education, but also included study which investigated the use of AR in physiology education. Thereby, an unalloyed meta-analysis on the use of AR in anatomy education remains lacking in the recent scientific literature. This study therefore aimed to assess the effectiveness of AR in anatomy education. For that reason, we performed a systematic literature review and meta-analyzed the available quantitative evidence on the impact of AR on learning outcomes in anatomy education.Although the research concerning the implementation of AR in anatomical education is relatively limited, there are promising results regarding the teaching potential of AR18. To assess a wide number of eligible papers, exploratory searches were carried out to assess suitability of literature databases , PubMed, Sciencedirect, PsychINFO, Google Scholar). Exploratory searches were conducted until March 2020. Thereafter, an independent librarian was consulted to help the researchers to identify the most suitable database to obtain literature from and to construct suitable search strategies. Various databases  were then searched systematically. Searches were conducted until January 2021. Search strings per database are provided in the The present study focuses on the effectiveness of learning anatomy by students by use of AR and was conducted following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelinesThe first round of assessment of the obtained papers concerned screening title and/or abstract. The second round of assessment comprised full-text assessment and included whether these articles met the aforementioned inclusion criteria to be included. When in disagreement, a third investigator (G.d.J.) was contacted to make the final decision. The PRISMA flow diagram can be appreciated in Fig. After inclusion, data were extracted from the individual papers using a data extraction sheet by two authors independently (K.B. and D.H.). These data included: (1) type of AR used in the study, (2) type of anatomical education in the control group, (3) number of participants, (4) characteristics of the included participants , (5) type of anatomical test, (6) mean post-intervention anatomic test scores for the experimental (AR) group, (7) mean post-intervention anatomic test scores for the control group and (8) Mental Rotations Test (MRT) scores in percentages of each included group as this test assesses the spatial abilities of participants. When the design of the study was a multiple group comparison study, each individual group that was not using AR was considered a separate control group. All control groups were then included for the meta-analysis.19. Additionally, risk of bias was assessed by two authors independently (K.B. and D.H.). Discrepancies were resolved by discussion or reference to a third author (G.d.J.). Risks of biases which were assessed included: selection bias , performance bias , attrition bias , detection (or measurement) bias  and reporting bias (criterion 8). Also, the Kirkpatrick’s model of change of knowledge was assessed for each paper as well. This model evaluates the learning outcomes and classifies these in four levels: 1) reaction; 2A) learning (change in attitude); 2B) learning  behavior (change in behavior); 4A) results ; and 4B) results (improvement in learner performance)21. Each potential source of bias was graded as low, high, or unclear. Assessing the risk of bias was performed by the criteria presented in Table 19. In addition, the second version of the Cochrane risk-of-bias tool for randomized trials (RoB 2) was used to assess the risk of bias in the included randomized trials  according to the GRADE approach guidelines defined by The Cochrane Collaboration’s Handbookhttps://www.r-project.org; Metafor)22: OpenMeta[Analyst] software ). A forest-plot was created to graphically display the estimated differences in pre-intervention and post-intervention test results from the included studies, along with the overall results. Cohen’s d metric was used to assess the effect size. In addition, OpenMeta[Analyst] was used to assess heterogeneity. Heterogeneity in meta-analyses refers to the variation in outcomes between included studies. To measure heterogeneity, Cochran's Q was calculated as the weighted sum of squared differences between individual study effects and the pooled effect across studies. To improve interpretation, the heterogeneity index (I2), defined as the proportion of total variability explained by heterogeneity and refers to the percentage of variation across studies, was introduced23. I2 is independent from the number of studies included in the meta-analysis. Therefore, I2 highlights the inconsistency across studies and ranges from 0%  to 100% .The statistical package SPSS Statistics, version 25  was used for descriptive statistical analyses of the aggregated data. Descriptive statistical analyses were represented as mean with ± standard deviation (± SD). Meta-analysis with continuous random-effects was carried out by use of the visual front-end for the R-package  group. However, no difference between the conventional atlas group and the AR group was foundIn the study of Moro et al., 2017 adverse effects were reported for the VR study tool, which caused students to experience nausea, headaches and dizziness. No such symptoms and problems plagued the use of their AR tool. Discomfort was also experienced by students using GreyMapp, as they reported trouble with getting used to operating the application. In combination with taking notes during the lesson, some students assumed uncomfortable positions to multitask. This problem was easily solved by creating a bigger tablet interface. In the REFLECT study, it was reported that time on task increased significantly. In addition, students engagement was significantly higher in the AR group, causing the longer time on task.25. Engagement was gauged differently in the study of Barmaki et al., 2019, where they measured time on task has been suggested as an important marker for knowledge retention and student engagement. The time on task was significantly higher in the AR group, compared to controls (P = 0.01). Finally, a significant difference was found by Bogomolova et al. in the enjoyment during learning between 2D anatomical models and the AR intervention (P = 0.003)24. Table Henssen et al. reportedly did not find an increase in motivation when comparing the AR group to the conventional group. However, focus group interviews showed that students did find the concept novel and interesting. Additionally, some students expressed their disappointment with not being able to work with the program2 = 21.301; Q = 15.493; df = 7; I2 = 54.82%; P = 0.030). Based on the mean differences in anatomic test scores  between the AR groups and the control groups, a difference of -0.765%-points was estimated . This indicated that there was no significant advantage or disadvantage when learning anatomy with AR  in studies which showed a low interstudy heterogeneity , as seen in Fig. 25. Meta-regression showed no significant relation between mean difference in anatomic test results (%) and mean difference in MRT scores (%) between the AR- and control-groups (Omnibus P = 0.229), which can be appreciated in Fig. Meta-analysis showed a substantial heterogeneity in the included papers (TauAlthough cadavers are most commonly used for teaching.27). Therefore, various other teaching methods are merited, including AR. AR is explicitly useful in anatomical education as it presents the first consumer-grade technology that can depict realistic 3D models and concepts to students, which, at the same time, can be directed by a teacher28. However, the present meta-analysis showed that AR yields no significant learning benefits when compared to other forms of anatomical education. Moreover, a significant lower anatomic test score was observed when comparing the results from the AR-groups to groups that used 2D anatomical learning methods . The results from the present meta-analysis partially conflict with the results from the meta-analysis of Yammine and Violato (2015) in which it was found that three dimensional visualization techniques (1) resulted in higher factual knowledge, (2) yielded significant better resulted in spatial knowledge acquisition, and (3) produced significant increase in user satisfaction and in learners’ perception of the effectiveness of the learning tool8. However, these three dimensional visualization techniques included various 3D images, annotated radiological data and VR simulators and that did not include AR features. On the use of AR in anatomical education, two other recent meta-analyses have been published. The publication of Moro et al. (2020), although also integrating VR methods and non-anatomical education purposes , demonstrated that VR and AR can be used as delivery methods in medical education, without any adverse effects on student performance29. Although not supported by their analyses, Moro et al. also expressed that there is a chance that the use of these technologies may have a positive impact on students spatial understanding and 3D comprehension of anatomical structures29. A second meta-analysis, however focusing on VR, showed that VR may act as an efficient way to improve the learners’ level of anatomy knowledge30. The present meta-analysis partially contradicts the conclusions of the other studies, showing that AR can indeed worsen the learners’ performance when compared to 2D anatomy teaching methods. An explanation for these different outcomes can be explained by the fact that the present study maintained strict inclusion criteria and thereby only focused on the effects of AR in anatomy education. On the one hand, this could have purified the results, whereas on the other hand, this could cause an overestimation of the effects related to a limited sample size.Although the use of cadavers and/or prosections form the cornerstone of anatomical education for medical and biomedical sciences students, various limitations constrain their use . However, the use of cadavers is known to have practical and ethical drawbacks32 and previously validated by Vandenberg and Kuse (1978)33. The findings of three of the included studies that used MRT25 showed that an aptitude–treatment interaction caused by visual-spatial abilities needs to be considered when reviewing evidence of AR in anatomical learning. However, no significant correlation was found between the mean difference in anatomic test scores and the MRT scores of the different groups in this meta-analysis. This could be due to the fact that only limited data was available. On the contrary, previous studies which focused on spatial ability and the use of 3D visualization methods found that significant differences in pre-intervention spatial ability confounded the study results37. Still, various reports have shown that cognitive load decreases when students study anatomy by use of AR38. This could, however, not be incorporated into this meta-analysis as most of the included papers did not provide this information.One of the co-variates in most studies investigating AR concerns spatial ability. Most studies use the MRT to assess spatial ability of participants. The MRT assesses mental visualization and mental rotation, which are considered the main components of visual-spatial abilities. The MRT concerns a 24-item psychometric questionnaire designed in 197142. Literature has suggested that AR would be attractive to students, increasing their motivation to learn anatomy45. Several studies investigated various forms of student motivation with regard to learning anatomy. For example, Allen et al. (2016) reported that students felt confident that learning with 3D models, including AR 3D models, could help them to understand anatomical concepts. Also, the majority of the respondents would encourage the development of similar learning sources46. Kucuk et al. distilled from interviewing students that more permanent learning was achieved in a shorter time by using AR38. Such permanent learning, however, remains rather understudied in research on AR in anatomy education47. Another report by our group showed that students feel motivated to study neuroanatomy by use of AR, although men and women and students from different study directions have different attitudes towards learning with AR. As well, students expressed that they felt AR was especially beneficial to study structures that cannot be visualized properly by use of prosected cadavers 48. Although most of the included studies in the present meta-analysis included motivation as a (secondary) outcome measure26, there is still no validated method to measure students’ motivation for learning anatomy49. Therefore, this could not be included into this meta-analysis. Future research elucidating methods of gathering data on student motivation will therefore provide valuable insights. In addition, the novelty effect, which is defined as “a person’s subjective first response to (using) a technological innovation”, plays an important role in the studies that used AR as an anatomical teaching method50. Previous studies noted that as the novelty effect wears off, users discontinue their use of new technologies, indicating a loss of interest and motivation51. This could partially be explained by the law of diminishing returns, as novel technologies create inherent interest, which tapers off after students get familiarized with their new environments52.Numerous studies reported improvements in the learners’ motivation after implementation of AR in different fields of education53. Unfortunately, the lack of a statistical analyses partially limits the interpretation of this finding. Furthermore, whether this is also the case for the use of AR when studying three dimensional anatomy remains unclear. With regard to the most effective AR technique in anatomy education, much remains unknown. For example, Chytas et al. (2020) demonstrated in their review that AR technology has a remarkable teaching potential with encouraging learning outcomes. However, it was noted that these papers generally compared tablet-based AR features which could enhance anatomical images and/or text with the anatomical atlases and/or textbooks themselves. In the minority of papers, three-dimensional AR applications were studied. In addition, comparisons with cadavers remains a relatively understudied field5. Based on the findings of the present study, we could carefully suggest that virtual mirrors with AR capabilities could help students to learn anatomical relations in the most effective way. These mirrors allow users to see a reflection of themselves with virtual information superimposed on a large display which acts as a digital representation of a mirror. Thereby, they can interact with otherwise invisible anatomical structures in real-time whilst benefiting from the anatomical context of their own bodies.Within this review, different types of AR were investigated ranging from practical tablet based 3D models, virtual mirrors with AR capabilities and headset-based AR applications. However, much debate still remains with regard to the most optimal implementation of different AR techniques in the correct period of a course. For example, in the study of Kügelmann et al. (2018), students were presented with a mirror AR system by which they could explore radiological images in several anatomical intersection planes in an attempt to increase motivation to study anatomy. The participants were asked to fill in questionnaires with regard to the levels of motivation and teaching potentials of the studied AR system. The participants declared that the benefits of AR were enhanced as time passed during the course33 to assess spatial ability in the included studies. A limitation, on the other hand, is caused by the lack of validated tools to evaluate students’ engagement, motivation and cognitive load.One of the strengths of the present meta-analysis concerns the systematic search for available literature and the independent consideration of each paper prior to inclusion and the independent assessment of the risk of bias, level of change in education as defined by Kirkpatrick and the results. A limitation of the present meta-analysis concerns the heterogeneity of the included papers, especially with regard to the different teaching methods in the intervention group . However, to the authors knowledge, no studies exists which show that different AR modalities are cognitively processed in a different fashion. Also, no studies were found which investigated the cognitive load with regard to the used AR teaching methodology. Therefore, we cannot determine the effects of these different AR modalities on a meta-level with regard to percentage-point differences in anatomy tests. Another limitation of the current meta-analysis concerns the relatively limited amount of papers included. However, a strengths concerns the strict inclusion and exclusion criteria which resulted in a meta-analysis which focuses on AR technologies in anatomy education only. In addition, testing of anatomical knowledge was performed by using a combination of multiple-choice questions, matching questions and open-ended questions. One of the strengths of the meta-analysis is caused by the consequent use of a validated MRTThis meta-analysis suggested that AR has no significant beneficial or disadvantageous effects on students’ learning anatomy when compared with various traditional educational tools. For that reason, we concluded that AR could be a viable addition to traditional anatomy education in an increasingly technological world.Supplementary Information.


We found that patients who were younger , who were receiving inpatient care more than twice in 1 year before their CP diagnosis , and who have pneumonia , epilepsy  and dysphagia  have higher risk of mortality. Rehabilitation ≥ 6 times within 6 months has a potentially positive impact on pediatric CP survival. Besides having a younger age, being hospitalized more than twice within a year before diagnosis and having pneumonia, epilepsy and dysphagia were modifiable risk factors in clinical practice for these children.Though numerous studies demonstrated the positive effect of rehabilitation on cerebral palsy (CP) children, there was no literature addressing the role of rehabilitation on mortality among children with CP. Therefore, we aimed to evaluate the impact of rehabilitation intensity on mortality among children with moderate to severe CP. This retrospective cohort study was conducted by National Health Insurance Research Database in Taiwan. Children (<12 years) with newly diagnosed moderate to severe CP between 1 January 2000 and 31 December 2013 were included. All patients were followed up for 3 years after CP diagnosis or death or until 31 December 2013. The intensity of rehabilitation therapy within 6 months after CP diagnosis was categorized into <6 times and ≥6 times. The Cox proportional hazard analysis was used to determine the association between rehabilitation intensity and all-cause mortality after adjusting age, sex, other demographic factors and comorbidities. Among 3936 severe CP children, 164 (4.2%) died during the 3-year follow-up period. The mortality rate was higher among patients receiving rehabilitation < 6 times within 6 months than those ≥6 times within 6 months after adjusting demographic profile and comorbidities (adjust HR (aHR): 1.96, 95% CI 1.33–2.89,  Cerebral palsy (CP) has been recognized as damage to the immature brain that leads to poor motor function coordination, aberrant movement and posture development, and impaired perception. These worsen a child’s health outcome, quality of life and lifespan . A recenHealth or social caregivers and families who have CP children often pay special attention to CP-associated diseases, which can lead to hospitalization and be life-threatening. Most of the deaths among children with CP were attributed to modifiable risk factors, including respiratory problems , epilepsAccording to the recent CP management guideline , referriResearch has shown the positive effect of rehabilitation on mortality for conditions such as chronic obstructive pulmonary disease , cardiovTherefore, the present study aims to fill a gap in the literature by investigating the association between rehabilitation and mortality in children with severe CP. To this end, we assessed the impact of rehabilitation and modifiable factors associated with 3-year mortality among children with severe CP.Data for this retrospective cohort study were obtained from the National Health Insurance Research Database (NHIRD) from 1 January 2000 to 31 December 2013. Taiwan’s National Health Insurance (NHI) was established in 1995 and covered up to 99.99% of Taiwan’s population. The NHIRD was released for research purposes and provided thorough population-based evidence to support clinical decisions ,19. The This study was approved by the Institutional Review Board of Kaohsiung Veterans General Hospital, Kaohsiung in Taiwan (VGHKS15-CT12-01). Informed consent was not obtained due to all personal identifications being replaced with surrogate numbers.All patients younger than 12 years old with a new diagnosis of severe CP (identified by catastrophic illness) between 1 January 2000 and 31 December 2013 were enrolled from the NHI dataset and RCIPD. The index date was defined as the date of CP diagnosis since once CP is diagnosed, pediatricians arrange rehabilitation as soon as possible according to the public health policy. We excluded patients who died too soon (within 6 months rehabilitation period). Taiwan’s rehabilitation system typically allows patients to undergo it a maximum of three times a day; therefore, more than 540 rehabilitation times within six months would be deemed inconsistent with current medical conditions in Taiwan. It is not clear if there may be errors in the data input due to administrative or system problems, but these data were considered to be inaccurate, and they were deleted.Moderate to severe CP diagnoses in this study were based on the International Classification of Disease, Ninth Revision, Clinical Modification (ICD-9-CM) diagnosis code 343.X and an NHI-issued catastrophic illness certificate that was validated in previous research articles . CatastrPatients who had their CP catastrophic illness certificate approved when they were over age 12, whose data were incomplete, or who died within six months of their CP diagnosis were excluded.Only the patients who survived over 6 months after a CP diagnosis were included in the study. Those who received rehabilitation ≥6 times within 6 months were then regarded as the high-intensity rehabilitation cohort, and patients who never received rehabilitation or received <6 times within 6 months were assigned to the low-intensity rehabilitation cohort. We chose 6-months-rehabilitation on the basis of current clinical guidelines for pediatric care  and rehaThe amount of physical, occupational and speech therapy services after CP diagnosis was retrieved from outpatient clinic visits. Each rehabilitation time is 30 min.Patients’ sociodemographic characteristics, including CP diagnosis age (based on RCIPD), gender, hospital level , residential region  and urbanization , were obtained from enrolment data.Several coexisting morbidities that may associate with mortality were evaluated when we followed up for outcome. Inpatient care, more than two times within 1 year before CP diagnosis, was assessed and retrieved from inpatient claims and ambulatory data. Other comorbidities in the analysis were epilepsy (ICD-9-CM codes 345), congenital heart disease (ICD-9-CMcode 746.9), scoliosis , GERD , dysphagia with tube feeding , perinatal complications including preterm labor and small size for gestational age , traumatic brain injury  and intellectual disability . The ICD-CM codes occurring in the inpatient setting or in ≥3 ambulatory care claims for a chronic disease or ≥1 ambulatory care claims for the acute disease were enrolled.The main measured outcome is all-cause mortality, which is defined by disenrollment from the NHI due to death. The follow-up time was defined as the period from the index date to the death, or withdrawal from the NHI program, or 3 years, whichever came first.The chi-squared test was used for categorical variables and the t-test for continuous variables.p-value less than 0.05 was considered statistically significant.Univariate Cox proportional hazards models were applied to depict survival over time by each of the independent variables. Then we used the multivariable Cox proportional hazards models to assess independent variables associated with mortality. All recorded variables were considered in estimating hazard ratios (HRs) with a 95% confidence interval (CI). All variables were entered in a multivariate Cox proportional hazards regression model by the step-forward method. A The Kaplan–Meier method was used to estimate the cumulative survival between the groups during the follow-up period. The log-rank test was performed to compare the outcome distributions of two cohorts.All statistical analysis was conducted using Statistical Analysis Software (SAS) version 9.4  and SPSS .p < 0.001. The mean age in p < 0.001), hospital character (p < 0.001), residential region (p < 0.001), and comorbidities including pneumonia (p = 0.001), epilepsy (p < 0.001), perinatal complication (p < 0.001), GERD (p = 0.001), dysphagia (p = 0.006) and intellectual disability (p < 0.001) showed a significant difference between the high intensity rehabilitation cohort and the low intensity rehabilitation cohort  CP children received rehabilitation ≥6 times within 6 months, and 2725 (69.2%) severe CP children did not receive rehabilitation or received rehabilitation <6 times within 6 months.  Of thosen cohort .p < 0.001) and low rehabilitation intensity , requiring inpatient care more than two times within 1 year before a severe CP diagnosis , epilepsy , congenital heart disease , dysphagia  and intellectual disability . After adjustment for covariates by stepwise Cox regression analysis, we found that patients who received low intensity rehabilitation within 6 months  had a higher risk of mortality. Other factors that had higher mortality risk include younger age , receiving inpatient care more than twice within one year before a severe CP diagnosis , and comorbidities of pneumonia , epilepsy  and dysphagia  (= 0.024) .p < 0.001 ( < 0.001 .)To the best of our knowledge, this is the first cohort study using nationwide population-based data to assess the impact of rehabilitation intensity on pediatric CP mortality. Our study comprised strict inclusion criteria and longer follow-up periods. We found that ≥6 times rehabilitation within 6 months (approximately monthly frequency rehabilitation) have a positive association with mortality. Younger age, having been previously hospitalized more than twice within one year before a severe CP diagnosis, and with a history of pneumonia, epilepsy or dysphagia were significant predictors for mortality among children with severe CP.Though the rehabilitation intensity in our study was lower than the recent guideline suggested  , the preFurthermore, one randomized controlled trial conducted by Bower et al.  suggesteThere is a trend that younger moderate to severe CP patients have higher rehabilitation service utilization than older individuals in As in previous studies, younger age  carries We found that CP children have pneumonia history have a higher risk of mortality. Though global child mortality is reducing, children die because pneumonia remains high . As wellDysphagia is common in CP patients and exposes children to health problems such as food aspiration, malnutrition and respiratory infections . We founAs in previous research, epilepsy was associated with reduced life expectancy for CP patients . CP patiSome limitations should be addressed. First, we did not present Gross Motor Function Classification System level because the information was not present in NHIRD. Therefore, ambulation status cannot be measured. However, we used severe illness certificated criteria (including ambulation ability) to define. Second, we did not assess detailed rehabilitation times per day due to the feasibility of green cloud electronic medical records services since 2016. Third, we cannot obtain confounding factors that may affect the outcome, such as birth bodyweight, parental educational level, breastfeeding duration and household environment factors such as cigarette smoking.From a public health viewpoint, policymakers are encouraged to enforce prevention strategies focusing on this high mortality risk group. Additionally, applying integrated medical care and rehabilitation therapy is warranted.There is a lower rehabilitation services usage rate among CP children in Taiwan. For children with severe CP, high-intensity rehabilitation within 6 months after CP diagnosis was associated with a lower 3-year all-cause mortality risk than low-intensity therapy. Those who were young, have a history of inpatient care twice within one year and have pneumonia, epilepsy and dysphagia need to be more concerned population. Therefore, efforts to promote applying high-intensity rehabilitation therapy for these patients to improve the outcome are warranted.


Augmented reality (AR) applications are increasingly being used in various fields , as AR techniques help improve efficiency and reduce costs. Moreover, collaborative AR systems extend applicability, allowing for collaborative environments for different roles. In this paper, we propose a multi-user collaborative AR system ; it is composed of three ends—MUCStudio, MUCView, and MUCServer. MUCStudio aims to construct industrial content with CAD model transformation, simplification, database update, marker design, scene editing, and exportation, while MUCView contains sensor data analysis, real-time localization, scene loading, annotation editing, and virtual–real rendering. MUCServer—as the bridge between MUCStudio and MUCView—presents collaborative and database services. To achieve this, we implemented the algorithms of local map establishment, global map registration, optimization, and network synchronization. The system provides AR services for diverse industrial processes via three collaborative ways—remote support, collaborative annotation, and editing. According to the system, applications for cutting machines were presented to improve efficiency and reduce costs, covering cutting head designs, production line sales, and cutting machine inspections. Finally, a user study was performed to prove the usage experience of the system. Augmented reality (AR) provides a view composed of the real physical world and digital virtual elements, covering a set of technologies, such as localization, rendering, scene understanding, etc. In recent years, AR has trended in various areas, including in the entertainment and medical fields ,5,6,7,8.Limitations in devices and algorithms have constrained AR applications up until a few years ago. Nowadays, there is no lack of devices, due to the popularization of mobile devices, such as smartphones and tablets, as well as different types of AR glasses. Moreover, due to technological developments, we have achieved accurate localization and map establishments in general scenes, resulting in stable virtual–real fusion performances. Release libraries, such as ARCore and ARKit, contribute to a variety of applications, further lowering the development threshold.Pertaining to current research in the AR field—AR industrial systems are mainly applied toward design, maintenance, assembly, repair, training, and inspection processes, with the goal of improving performance and reducing costs and machine losses ,10. In tSimilar to the above request, the inspection process can be enhanced by AR technology. Industrial product creation is a complex procedure, it usually includes conception, design, and realization. It is necessary to check whether errors occur after product realization. To achieve this goal, the inspection procedure will involve an organized examination of a particular device. AR is regarded as a promising technology to set up the inspection process ,16,17,18Other AR research fields cover maintenance, repair, and assembly, with the goal of reducing time, costs, and the error rate ,20,21. OIn addition to the above, collaborative AR systems further satisfy industrial demands by providing cooperation among operators ,27,28. ICompared to previous AR systems, our MUCSys has the following peculiarities: (1) the developed system can support various industrial processes, such as design, inspection, assembly, sales, etc.; (2) three collaborative AR modes are presented to bridge interactions among different roles. In detail, remote support provides remote assistance between experts and workers  in actual scenes. Collaborative annotation offers asynchronous adding, deleting, or modifying operations for different workers. Meanwhile, multiple users can collectively complete target tasks  via collaborative editing in real-time.The whole system is composed of MUCStudio in PC, and MUCServer and MUCView in mobile devices. In detail, MUCStudio aims for industrial scene construction, while MUCView mainly provides AR experiences for users. Moreover, interlinking MUCStudio and MUCView, MUCServer presents database and collaborative services. To prove the effectiveness of the proposed system, laser cutting machine applications containing designs, sales, and inspections are provided.We propose a multi-user collaborative AR system for laser cutting machines, containing three collaborative modes—remote support, annotation, and editing. The system comprises MUCStudio for scene generation, MUCView for AR experience, and MUCServer for collaboration.To implement collaborative services, we designed several algorithms. Firstly, the local map was established with additional marker and QR code inputs based on ORB-SLAM3. Secondly, we propose a map registration pipeline by combining ORB match and point cloud registration. Global optimization was performed to promote the relative transformation accuracy. Finally, the network synchronization was exploited to improve the user experience.Based on the system, three applications for laser cutting machines were developed—cutting head design, production line sale, and cutting machine inspection.A user study was conducted to demonstrate the user experience of the system.In summary, compared to prior works, our work provides the following contributions.The remainder of the paper is organized as follows: related works are summarized in In the design process, AR systems can identify and avoid design errors in the early stages to reduce time, costs, and the number of physical prototypes. The CAD product model and factory layout design are two common application scenarios.In the context of the CAD product model, Jimeno et al.  implemenBesides the CAD product model, planning a suitable factory layout is also a challenge. Shariatzadeh et al. , focusinIn the laser cutting machine applications of our system, both the CAD product model and layout design can be verified through SMCStudio and SMCView. Through SMCStudio, the user designs the production line layout or the model position relative to the real environment by the marker. Then, SMCView superposes the virtual model on the real environment. Moreover, multiple users can cooperate through the SMCServer. With sensor data supporting, SMCView also simulates the trajectory and drives the virtual part to provide more visualization information for designers.In maintenance, repair, assembly, and training processes, AR technology can efficiently help operators understand the procedure of the tasks by means of overlaying the virtual instruction information onto the real environment ,32. The An early attempt to support technicians in simple maintenance procedures involved the intent-based illustration system proposed by Feiner et al. . Then, vAR-based industrial training tasks are always related to the repair, assembly, and maintenance processes, with the aim of improving learning efficiency and reducing device costs . VariousThrough our MUCSys, it is convenient to construct maintenance, assembly, repair, and training scenarios. However, in our applications for cutting machines, these kinds of demands are slight, as workers receive meticulous training and need to pass an examination before entering the workplace. Therefore, applications about maintenance, assembly, repair, and training are not provided.After the design and production phases, the product is realized. On account of the whole process complexity, it is essential to check whether errors and differences occurred during the phases, which can be achieved with AR technology .An AR-based reconfigurable framework for inspection was proposed by Ramakrishna et al. , prioritThe inspection pipeline introduced above mainly focuses on realizing inspections to ensure quality. In our paper, after analyzing the requirements of the laser cutting machine, we employed our system to the patrol inspection process, to judge if the device was under the normal operating conditions and to find the running problem in the early stage.As demonstrated in MUCStudio looks at “constructing” the industrial scene for the AR experience. On the one hand, an industrial scene always covers various products with different material warehouses, marking machines, transmissions, etc. Through MUCStudio, users can select their wanted products to generate the final scene. On the other hand, the designed virtual scene should be appropriately fused with the real environment. MUCStudio adopted the marker to solve this problem. By designing markers in virtual and real scenes, both coordinate systems are unified, achieving an ideal virtual–real fusion effect.The interface of MUCStudio is illustrated in The purpose of MUCView is to provide AR experiences for users. It is mainly composed of camera tracking, collision detection, object editing, data loader, annotation editing, and virtual object rendering.The camera tracking and object editing parts are implemented by Google ARCore, enabling the mobile device to understand and track its position relative to the world. Based on the obtained feature points and camera pose, the virtual object is rendered in the real environment by Unity. During the collaborative mode, the pose of the virtual object is then transformed by a relative matrix from MUCServer. After rendering the virtual object, users can edit its position, orientation, and scale. For the convenience of moving objects, MUCView presents a grouping function to bind different models. Moreover, to void collision detection during layout design, MUCView gives a reminder when virtual objects have collisions. After determining the model position, the data loader module loads sensor data to drive the model to perform the motion. Annotation editing offers the user addition, modification, and deletion of the virtual annotation, which will be illustrated in the next subsection on patrol inspection.Through the communication with MUCServer, MUCView achieves multiple collaborative operations covering remote support, collaborative editing, and annotation.The MUCServer mainly performs database and collaborative services. The database covers CAD, 3D model, annotation, and scene information that can be updated by MUCStudio and MUCView. The collaborative service provides three ways to satisfy different demands. When users need the help of remote experts, remote support presents virtual–real video transmissions and shared interactions, demonstrated in In the implementation, remote support technology relies on the video transmission realized by the FFmpeg library, while collaborative editing and annotation are based on the collaborative localization and map establishment among different devices illustrated in Local map. With the gathered image, IMU data, local camera pose by ARCore, feature point, detected marker, QR code, and annotation, the local map is established by ORB-SLAM3  , while the median result of SMEQ was 12 with IQR [8–15] . It is obvious that both SEQ and SMEQ performances show that completing the tasks were not hard. In particular, all participants received a simple training process before completion, and most had not used an AR system before. In actual application, after systemic training, users could have better performance.In this paper, focusing on industrial applications, we proposed a multi-user collaborative AR system composed of MUCStudio for content generation, MUCView for providing AR experiences, and MUCServer for database and collaborative services. On the one hand, the system could be applied to various industrial stages that contain designs, sales, inspections, etc. On the other hand, three collaborative ways are presented among the different roles, covering remote support, collaborative editing, and annotation. To achieve collaborative localization and improve user experience, we implemented the algorithms of local map establishment, global map registration, optimization, and network synchronization. Based on the proposed system, we develop three laser cutting machine applications, which include the cutting head design, production line sale, and patrol inspection of a cutting machine. Furthermore, to evaluate the system, we conducted a user study that covered three tasks through SEQ, SMEQ, and SUS, demonstrating use difficulty and user experience.


SOX9, SRY, NR0B1 (DAX1), NR5A1 (SF1), DHH, sex differentiation genes-AR, SRD5A2, MAMLD1, INSL3, and hypospadias-associated genes-FGF8, FGF10, BMP4, BMP7, ATF3, and MID1 were screened by using next generation sequencing. A total of 18 patients were found to have mutations identified by PCR and sequencing, including 11 cases of SRD5A2 genes, 6 cases of AR genes, and 1 case of MID1 gene, respectively. One novel missense mutation p.I817N was discovered in AR gene. Further molecular analysis found that subcellular localization of the ARI817N was the same as that of wild type ARWT in the absence or presence of hormone. But it led to 50% reduction in AR-induced transcriptional activity in the presence of either the synthetic androgen R1881 or the natural ligand dihydrotestosterone. Our results indicate that SRD5A2 and AR genes are two top candidate genes associated with 46, XY hypospadias in Chinese patients. Further epidemiological and genetic analysis are still needed to further clarify the pathogenesis of hypospadias in Han Chinese patients.It is estimated that around 10–20% of hypospadias are caused by genetic abnormalities worldwide although the spectrum of associated genes does vary across different ethnicities. The prevalence of hypospadias among the Chinese population has been increasing the last couple of decades. However, the pathogenesis underlying the disease and its associated genetic abnormality remains unclear. Here we performed a genetic analysis of 81 children with karyotype 46, XY and the hypospadias phenotype in order to characterize the genetic components that contribute to the development of hypospadias in Chinese patients. 15 candidate genes, including sex determination genes- Hypospadias is one of the most common congenital malformations of the penis . It is cAlthough the exact etiology of hypospadias is unknown, it is agreed upon that environment, endocrine hormones, and genetic factors all play a collective role in the pathogenesis . EnvironGenetic disruption of male external genital development and urethral growth contribute to 20% of the etiology of hypospadias . There aSRY), SRY-related high mobility group box gene 9 (SOX9), nuclear receptor subfamily 0 group B member 1 (NR0B1), nuclear receptor subfamily 5 group A member 1 (NR5A1), desert hedgehog (DHH) (2), sex differentiation genes: SRAD5, AR, mastermind-like domain-containing protein 1 gene (MAMLD1), Leydig cell insulin-like 3 (INSL3) (3), and hypospadias genes reported by other studies: bone morphogenetic protein gene 4 and 7 (BMP4 and 7), fibroblast growth factor 8 and 10 (FGF8 and 10), activating transcription factor 3 (ATF3), and midline 1 (MID1). Multiple mutations were found in these patients, but not in 50 healthy controls. One of 4 identified AR missense mutations was de novo and functional impairment of AR from this mutation was further explored in vitro.To explore the spectrum of genetic abnormality in Chinese hypospadias patients, we performed next generation sequencing (NGS) on 81 patients with hypospadias targeting 15 candidate genes that could potentially cause hypospadias including (1), sex determination genes: sex determining region of Y chromosome .Clinical assessment of these patients included the child’s birth history, family history, maternal history (exogenous sex hormone exposure or intake history during pregnancy), living environment and possible history of exposure to environmental pollutants. Age, gender, body weight, gonadal developmental status, degree of external genital malformation according to the external masculinization score (EMS), and type of hypospadias were recorded. The measurement of penis length and reference value of Chinese men were performed according to the published study by Fu Chao et al. in 2010 . The diaGnRH agonist testing was performed in some patients using intravenous administration of a standard dose of GnRH . Blood samples were collected at time points 0, 30, 60 and 90 min after stimulation. Normal test was defined as luteinizing hormone (LH) increases about 3–6 times within 30–45 min, and follicle stimulating hormone (FSH) increases by 20–50% after GnRH injection. Serum LH and FSH were measured by immunoradiometric assay . The hCG stimulation test which was used to evaluate the function of testicular synthetic androgens was performed in some patients with a daily injection of hCG  for 3 days. Serum testosterone (T) and dihydrotestosterone (DHT) were measured using a radioimmunoassay kit .1. PCRs were performed in a reaction volume of 50 μl containing 20 ng template DNA. A 5 μl aliquot of each PCR was loaded on a 2% agarose gel and visualized by ethidium bromide  staining to confirm the presence of an appropriately sized product. The PCR products were purified using a gel extraction kit  and sequenced in both sense and antisense directions on an Illumina-miseq sequencer . DNA fragments were 250–400 bp and the coverage of samples was 80x–3,000x. Positive and negative controls were added to the samples. Sequences generated from all patients were compared with the published reference sequences from the National Center for Biotechnology Information2. The mutation was annotated according to HGVS nomenclature3. For identified mutation from NGS, purified PCR products were further sequenced and verified by Sanger sequencing on an ABI 700 sequencer . The ACMG guidelines , according to the manufacturer’s instructions. 15 genes  according to literature search were selected as candidates for polymerase chain reaction (PCR) and sequencing. Primers targeting all exons of these genes were designed using online tool Primer3idelines  were useWT) (SC114220) was bought from Origene . It served as a template to construct the mutant p.I817N (ARI817N) using the QuikChange II site-directed mutagenesis kit . The primers were ARt2450aF: ccatccactggattaatgctgaagagtagcagtgct andARt2450aR: agcactgctactcttcagcattaatccagtggatgg. Positive clones were selected and sequenced to confirm the site-directed mutation (The wild type AR expression plasmid (ARmutation .The cultured cells were fixed and then followed by overnight incubation with primary antibody anti-AR (Invitrogen). Sections were washed and then incubated with Cy2-anti-mouse and Cy5 anti-rabbit . Nuclear stating was performed using 4′,6-diamidino-2-phenylindole . Confocal microscopy was performed using a Leica SP2AOBS system  in the Light Microscopy Core Center at the Shanghai Jiao Tong University.Cells harvested from cultures were homogenized in cell lysis buffer . Protein concentration determination and immunoblotting were performed as previously described. Briefly, twenty micrograms of protein were separated by SDS-PAGE, transferred to polyvinylidene difluoride membranes and blotted with primary antibodies and secondary antibodies . Quantification of the image was performed by scanning densitometry and using NIH Image J 1.54 software .WT or ARI817N) and the Cignal reporter plasmid  using transfection reagent Lipofectarmine 2000 . Cells were incubated overnight with dihydrotestosterone , methyltrienolone , and hydroxyflutamide  each in triplicate. For OHF treatment, 0.1 nM of R1881 was added 30 min before the addition of OHF to determine the competition binding. Then cells were lysed and luciferase activity assayed using Victor3 plate reader .The transactivation activity of the p.I817N mutated AR and wild-type AR were compared in a reporter gene assay as previously described . In briet-test for comparing between ARWT and ARI871N. The one-way ANOVA was used for comparing cellular responses after R1881 treatment. The clinical data description was not involved in the statistics. Calculations were performed using Graphpad Prism 9 software. Significance was determined using a threshold of P = 0.05. All values were reported as mean ± standard error of the mean (SEM) for three independent experiments.Statistical analyses were performed using an unpaired, two-tailed All 81 patients diagnosed with hypospadias exhibited other features of external genital malformations at variable degrees as listed in Forty-two cases received GnRH stimulation test as a follow up. Seven patients showed high gonadotrophin dysplasia and the remaining cases had normal response. Fifty-two patients underwent the hCG challenge test, and only five patients had a poor response . Other patients demonstrated a normal testicular response to hCG.G banding method was used for karyotype analysis and all children have 46, XY chromosomes. 15 candidate genes were selected for the initial screening using Illumina-miseq sequencing according to previous literatures. All mutations detected in the initial screening by NGS were confirmed by traditional Sanger sequencing. A total of 18 patients were found to have mutations, including 11 cases of SRD5A2 genes, 6 cases of AR genes, and 1 case of MID1 gene. According to ACMG guidelines, these mutations were assessed as pathogenic (P) or likely pathogenic (LP) . The pre∗60, and p.G203S/F219fs∗60) and 4 homozygous mutations . 10 of them were reported in the literature. Only p.Y136Ter was a newly identified mutation, where cytosine at position 408 of exon 2 of SRD5A2 gene mutated to adenine causing the stop codon TAA and terminating translation. According to ACMG guidelines, this mutation was assessed as pathogenic (P/PVS1 + PM2 + PM6 + PP3).There were 11 cases identified with SRD5A2 mutations. The age of diagnosis ranged from 4-month to 22-years old. The clinical characteristics are listed in There were 6 cases with an identified AR gene mutation, the clinical phenotype characteristics are shown in MID1 gene, p.P667L, which was located in exon 10 of the MID1 gene and was the last amino acid encoded by the gene. MID1 gene mutation is associated with X-linked Opitz G/BBB syndrome  of the AR, which may play a role in coactivator mediated activation of the transcription. Therefore, functional studies were performed to address whether the ARI871N affects the transcription activation potential of AR. The ARI871N or ARWT expression vector was co-transfected with Cignal reporter in luciferase assay. In dose-response curve, the mutated AR showed reduced ability to induce AR-mediated activation of a reporter gene, compared to wild-type AR assessed by the natural ligand DHT or R188. p.I817N caused a 20∼46% reduction in activity at doses between 1 and 10 nM DHT or 0.1–100 nM R1881  which is higher than reported in the literature which has previously suggested that 10–20% of hypospadias are caused by genetic abnormalities  is a multiple congenital anomaly disorder that shows a wide spectrum of severity and a highly variable expressivity (OMIM 300000) . In malehenotype . Hypospahenotype . The patignaling . Like thignaling . Howeverignaling .SRD5A2 gene encoded 5α-reductase is mainly expressed in male genital and prostate tissues, whose defects cause 46, XY DSD due to defects in testosterone metabolism. In this study, 13.6% (11/81) hypospadias is caused by SRAD5 mutation, which takes 61.1% (11/18) genetic etiology of hypospadias. A de novo mutation p.Y136Ter leads to a stop codon TAA and terminates the translation of SRD5A2 gene. This loci is assessed as pathogenic. The patient carries compound heterozygote p.Y136Ter/p.R227Q and exhibits type II hypospadias, with no external genital malformations except hypospadias. The EMS score was 10 and hCG stimulation tests showed non-response.The AR defects have been reported in the literature, presenting with a wide variety of phenotypic outcomes. The phenotypes of 46, XY individuals with AR gene mutations are categorized as complete androgen insensitivity syndrome (CAIS) or PAIS, respectively, and the more severe forms belong to disorders of sex determination. They range from a complete male-to-female sex reversal in CAIS patients to variable grades of ambiguous genitalia in PAIS patients. Milder forms of PAIS also include male phenotypes with gynecomastia, decreased fertility, or isolated hypospadias. PAIS could be a consequence of partial defect of AR structure or function caused by defects in AR gene. In this study, 7.4% (6/81) hypospadias was caused by AR mutation, which accounted for 33.3% (6/18) genetic abnormality of hypospadias.So far, more than 1000 cases of mutations with AR gene. The LBD domain consists of 12 contiguous alpha helices, most of which are hydrophobic amino acids that form a hydrophobic pocket which in turn binds the androgen ligand through its hydrophobic interaction. Functional impairment after mutation in this region weakens the binding of androgen to the AR protein in the target cell resulting in a decrease in the activity of androgen. It has been reported that p.A871V mutation was associated with a decrease in the binding affinity between AR and androgen by approximately 56%. p.R856H mutation led to changes in AR tertiary structure, resulting in decreased thermal stability and mutual N/C interactions. Large deletions in AR gene have been reported in patients with CAIS . p.I817N was a novel missense mutation. Other two patients have gross deletions of AR gene and both were not reported. All 4 missense mutations were located within the highly conserved carboxy-terminal LBD of the ith CAIS , but in AR gene. It is located in the LBD region of AR and encodes the first amino acid of exon 7, which is involved in the splicing of amino acid chains during transcription and translation. Conservative analysis of 10 mammalian proteins suggested that this region is highly conserved. The concentration of T in the laboratory test was 9.37 ng/ml, and it was 11.95 ng/ml after hCG challenge. It was significantly higher than the serum T level in boys of the same age. This also supports its molecular diagnosis. In vitro functional assay was further utilized and revealed that this mutation led to impaired AR transcriptional activity (20∼46%), decreased sensitivity for androgen ligand, and was responsible to hypospadias observed in the patient.The p.I817N mutation identified in our study changed thymine to adenine at position 2450 in the P > 0.05). This study used a candidate gene strategy, there may be unknown gene variation. We cannot rule out the association of these complications with other gene variations.To exclude the correlation between the identified mutations and clinical manifestations other than hypospadias, we performed Fisher’s exact test which was used to compare the incidences of inguinal hernia, SGA, ASD, and puberty breast development in children with and without candidate genes mutations. There were no statistical differences  repository, accession number CNP0001939. The data are also available on request from the corresponding author, ZD and XM to dzy831@126.com and 179788825@qq.com.The data presented in the study are deposited in the China National GeneBank (CNGB) Nucleotide Sequence Archive (CNSA: The studies involving human participants were reviewed and approved by the Ethics Committee of Shanghai Jiao Tong University School of Medicine. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.LC was responsible for the writing and editing of this manuscript. LC and JW conducted the experiments. WL, YX, JN, and WW elaborated clinical data. XM and ZD were responsible for the editing of this manuscript. All authors contributed to the article and approved the submitted version.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.


Therefore, natural products targeting Nrf2 (nuclear factor erythroid 2-related factor 2)-regulated antioxidant response might represent promising anti-photoaging candidates. Hesperetin (HSP) was suggested as a putative bioactive compound of the HRF, as previously shown by its chemical profiling using the liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (LC-QTOF-MS). In this study, we explored the anti-photoaging effects of HRF extracts and HSP on normal human dermal fibroblasts (NHDFs) and mouse skin exposed to UVA irradiation. Pretreatment of NHDFs with HRF extracts and HSP protected against UVA (8 J/cm2)-mediated cytotoxicity and reactive oxygen species (ROS) formation. The HRF and HSP pretreatment also attenuated the UVA-induced MMP-1 activity and collagen depletion concomitant with an upregulation of Nrf2 activity and its downstream genes (GST and NQO-1). Moreover, our findings provided the in vivo relevance to the in vitro anti-photoaging effects of HRF as topical application of the extracts  and HSP  1 h before UVA exposure 3 times per week for 2 weeks  mitigated MMP-1 upregulation, collagen loss in correlation with enhanced Nrf2 nuclear accumulation and its target protein GST and NQO-1 as well as reduced 8-hydroxy-2′-deoxyguanosine (8-OHdG) in irradiated mouse skin. Thus, our findings revealed that HRF extracts and HSP attenuated UVA-induced photoaging via upregulating Nrf2, together with their abilities to reduce ROS formation and oxidative damage. Our study concluded that the HRF and its bioactive ingredient HSP may represent potential candidates for preventing UVA-induced photoaging via restoration of redox balance.Polyherbal formulation combining multiple herbs is suggested to achieve enhanced therapeutic effects and reduce toxicity. Harak herbal formula (HRF) extracts were proposed to regulate skin responses to UVR through their ability to suppress UVA-induced matrix metalloproteinase-1 (MMP-1) and pigmentation via promoting antioxidant defenses in  Ficus racemosa L. (Moraceae), Capparis micracantha DC. (Capparidaceae), Clerodendrum petasites (Lour.) S. Moore (Lamiaceae), Harrisonia perforata (Blanco) Merr. (Rutaceae), and Tiliacora triandra (Colebr.) Diels (Menispermaceae). Various chemical constituents identified in the extracts of HRF and their components include alkaloids, coumarins, flavonoids, phenolic compounds, tannins, and triterpenoids  can cause premature aging of the skin through triggering stimulation of matrix metalloproteinase-1 (MMP-1) responsible for collagen degradation, a hallmark of photoaged skin . MoreoveNuclear factor E2-related factor 2 (Nrf2) is a master transcription factor regulating several phase II detoxification genes, such as glutathione S-transferase (GST) and NAD(P)H:quinone oxidoreductase-1 (NQO-1), involved in cellular defenses against oxidative stress and environmental insults including UVR . UpregulHRF powder was produced by Ayurved Siriraj Manufacturing Unit of Herbal Medicines and Products, Bangkok, Thailand (a GMP-certified manufacturer). Briefly, all ingredients were washed, dried, ground, and mixed to obtain a uniform batch of HRF powder. Then the powder was inspected for its quality, such as loss on drying, particle size distribution, powder flow, and density, to ensure that it conformed with the specification. HRF extract was prepared by mixing 50 g of HRF powder with 500 ml of 80% ethanol for 30 min, followed by 30 min ultrasonication. After filtration through a Whatman filter paper, the solvent was removed using a rotary evaporator and a freeze dryer. The dry extract was kept at room temperature until used.We previously performed the qualitative analysis of the HRF by LC-QTOF-MS in both positive and negative electrospray ionization (ESI) modes to investigate chemical profiles and structures. The chemical structure of 5,7-Dihydroxy-2-(3-hydroxy4-methoxyphenyl)-2,3-dihydro-4H-chromen-4-one or “hesperetin (HSP)” was identified in positive ESI mode at the retention time of 4.9435 and the negative ESI mode at the retention time of 4.9827 and was expressed in all HRF’s five herb components .2 (PCO2 = 40 Torr). The NHDFs were then assigned to experimental treatments after washing by phosphate-buffered saline (PBS). The HRF and HSP (CAS 69097: Sigma–Aldrich), the HRF’s putative bioactive compound, were dissolved in 80% ethanol. Therefore, the vehicle-sham or the vehicle control group was the NHDFs treated with 80% ethanol . The treatment groups were therefore compared with the vehicle-sham group with or without UVA exposure. In the in vitro study, three-different concentrations: HRF at the concentration of 7.5, 15, and 30 μg/ml and HSP at the concentration of 2.5, 5, and 10 μg/ml, were obtained to perform in the experiments for determination of cytotoxicity, ROS formation, photoaging parameters, and Nrf2-regulated antioxidant response.Full details are supplied in 2) was added to the cells then the cells were irradiated with UVA. The UV-meter  equipped with a UVA sensor (330–400 nm) was obtained to evaluate the constant intensity of UVA ray, 1 W/cm2 at a 21 cm distancing from the xenon arc UVA lamp . The irradiation of UVA (8 J/cm2) was performed after the cell treatment with each compound. Irradiated cells were immediately incubated with serum-free medium, which were then harvested at different time points; 12 h post-irradiation for cytotoxicity, 1 h post-irradiation for ROS formation, Nrf2 nuclear translocation  at 37°C for 30 min. Permeated H2DCFDA was deacetylated by intracellular esterases to non-fluorescent product (H2DCF). Stained cells were immediately analyzed by flow cytometry  as previously described  according to the manufacturer’s instructions . After the pre-treatment of the cells with compounds, the cells were irradiated with UVA 10 J/cms report . Data weThe conditioned media collected at 24 h after UVA exposure was used to detect MMP-1 activity using collagen zymography as previously described . CultureThe expressions of MMP-1 gene and Nrf2 target genes (GST and NQO-1) were determined at 12 and 2 h post-irradiation, respectively. Total RNA was isolated using the Illustra RNAspin Mini RNA Isolation Kit  and the cDNA was then synthesized from total RNA using Improm-II reverse transcriptase  following the manufacturer’s instructions. Primers for PCR were designed based on strict criteria using the Primer Express version 3.0 software  as listed in β-actin   for whole cell proteins or cytosol Nrf2 and collagen, and TATA binding protein (TBP)   for nuclear Nrf2. Data were expressed as a percentage of control .Treated cells were harvested at 1 h post-irradiation for the Nrf2 nuclear translocation, while, they were harvested at 24 h post-irradiation for the collagen type I protein expression. Cytosolic and nuclear extracts were carried out to detect nuclear localization of Nrf2  (1:2500), while whole-cell extracts were carried out to detect the collagen type I protein expression (C-18) Ab  (1:2500). For the nuclear extraction, cytosolic and nuclear extraction were prepared using a commercial kit according to the manufacturer's instructions (Sigma). The HRP-conjugated secondary antibodies  (1:2000) were used for the immunochemical detection. The immunoreactivity was then detected by the chemical reaction with the Bio-Rad Clarity western ECL (Bio-Rad). Protein bands were visualized using an ImageQuant LAS 4000 digital imaging system  and the integrated optical density of the bands was analyzed by the ImageJ software version 1.45 s . The protein expressions were normalized to the expression of loading controls; 2, HSP at the concentrations of 0.3, 1, and 3 mg/cm2 and sulforaphane (SFN) 0.1 mg/cm2 were obtained to perform in all biomarkers of the in vivo study. HRF, HSP, and SFN powders were dissolved in 20 μL of ethanol: acetone  and topically administered to each 1-cm2 site in the center area of the shaved dorsal skin 1 h prior to each UVA exposure. The mice were exposed to UVA irradiation for 10 min to achieve a single dose of 10 J/cm2, 3 times a week for 2 weeks . The dorsal skin flaps were then removed at different time points. Skin thickness was assessed by hematoxylin and eosin (H&E) and immunofluorescence (IF) staining. Photoaging markers MMP-1 and collagen, oxidative DNA damage (8-OHdG), as well as Nrf2 nuclear accumulation and the protein levels of its downstream target genes GST and NQO-1, were assessed by IF. The timeline of treatment, the UVA dose selection, procedures of IF staining protocol and all analysis of the protein expression data were described below following previous reports , SiACUP 023/2557. BALB/c mice were obtained from the National Laboratory Animal Center, Mahidol University and were housed under standard housing conditions with light: dark cycle at 12:12 h. BALB/c mice (4–5 weeks of age with an average body weight of 20 g) were anesthetized by intraperitoneal injection of a ketamine (80 mg/kg)/xylazine (10 mg/kg) cocktail. The dorsal surfaces were then shaved on using an electric hair clipper . The scarified area was cleansed with a cotton-tipped applicator saturated with sterile normal saline solution. The three-different concentrations of each treatment, HRF at the concentrations of 10, 30 and 100 mg/cm reports .Frozen tissues were sectioned by using Cryostat  for 8 μm per 1 section. Cryo-cut tissue sections were fixed in ice-cold acetone and air-dried for 30 min at room temperature. H&E staining was performed for histological evaluation of skin thickness as previously described in Dorsal skin tissue samples were collected at various time points following the final UVA irradiation; 1, and 6 h post-irradiation for Nrf2 and its target proteins, respectively; 1 h post-irradiation for oxidative DNA damage; 24 h post-irradiation for MMP-1 and collagen. Tissue sections were washed with PBS for 5 min/time (3 times) and blocked with phosphate-buffered saline (PBS) containing 2% BSA for 30 min. After removing excess blocking buffer, the slides were incubated with Nrf2 Ab , GST Ab , NQO1 Ab  (1:50), 8-OHdG [N45.1] Ab  (1:50), MMP-1 Ab  (1:50), collagen I (C-18) Ab  (1:50) for 1 h. The slides were then washed for 5 min/time (3 times) with a PBS solution and incubated for 1 h at room temperature with FITC-conjugated the secondary Ab (green) and with DAPI (blue) to counterstain the nuclei for detection of nuclear Nrf2, the secondary Ab Alexa Fluor 488 goat anti-rabbit (Abcam) for detection of MMP-1 and collagen levels. An inverted fluorescent microscope equipped with a Nikon Intensilight was used for the imaging of IF stainings (20X) which were quantified using ImageJ software.ImageJ software  was calculated using the following equation: CTCF = integrated density–(area of each region of interest (ROI) × mean fluorescence of background readings) and the data were presented as percentage of control. Quantitative fluorescence analysis from ImageJ was then imported into Microsoft Excel to create histograms for further analysis. All analysis of protein expression data was presented as a percentage of control. The Nrf2 nuclear localization was analyzed based on the ratio of fluorescence intensity in the nuclear (indicated by the DAPI staining) to cytoplasmic intensity of Nrf2 (N/C ratio). Background subtraction was done to correct the intensity from each compartment .n ≥ 3) were reported as a scientific value for both in vitro and in vivo studies. The significance analysis was evaluated by independent t test  or one-way analysis of variance followed by Tukey or Dunnett tests, where appropriate, using Prism  . A one-way analysis of variance followed by the appropriate post hoc analysis was used to determine individual group differences of the UVA-treated sham group vs. the groups treated with compounds and UVA exposure. The statistical significance was considered at a value of p < 0.05 and the significance level was defined in the figure legends.Means ± standard deviation from at least three biological replicates (in vitro part of our study (30 μg/ml and 10 μg/ml), each compound alone did not cause any significant cytotoxicity  inductions of cytotoxicity -irradiated cells. At 24 h post-UVA irradiation, MMP-1 protein expression and enzyme activity were examined by western blotting , one of the main enzymes responsible for the extracellular matrix (ECM) breakdown. We then investigated the two parameters, MMP-1 and collagen, of photoaging to explore the effects of HRF and HSP on these parameters. Our data showed that vehicle control using sham treatment of NHDFs had no significant effect on MMP-1 protein expression , MMP-1 eblotting  and collblotting , respectblotting . The UVAblotting , enzyme blotting , and mRNblotting  were sigblotting .Nrf2 is suggested to be a transcription regulator of the antioxidant defense system, contributing to the effective redox balance. The mRNA levels of Nrf2 transcriptional targets including GST and NQO-1 were then determined to study the activity of Nrf2. We thus investigated the effects of HRF and HSP in the modulation of the Nrf2-regulated cytoprotective genes including GST and NQO-1. Our findings revealed that UVA significantly reduced nuclear and cytoplasmic Nrf2 levels  and nuclin vitro study implied that HRF and HSP showed the antioxidant and anti-aging properties in NHDFs irradiated with UVA. This study thus further examined the effects of the HRF and HSP on UVA-induced photoaging in a relevant in vivo mouse model. Since we previously optimized the timeline of in vivo treatment and the irradiation dose of UVA (2). H&E and IF staining of skin tissue sections revealed that UVA treatment markedly induced epidermal thickness (2) and HPS (0.3–3 mg/cm2) led to the reduction of the epidermal thickness  (2 and 3 mg/cm2) (2) and HSP (3 mg/cm2) also markedly abrogated UVA-induced reduction of GST protein levels (2 and 0.3–3 mg/cm2) in mouse skin exposed to UVA  and HSP (0.3–3 mg/cm2) dose-dependently protected against UVA-induced oxidative DNA damage in the mouse skin (Mechanistic studies have been previously reported that Nrf2 is required for protection against UVA-stimulated MMP-1 upregulation . Our in  studies  supporte0 J/cm2)  and this mg/cm2) . The HRFn levels . The decd to UVA . To evaluse skin .in vitro and on mouse skin in vivo exposed to UVA in association with modulation of Nrf2-related antioxidant signaling.The development of botanical products including herbal formula as potential anti-photoaging products has gained attention in phytomedicine and dermatology research. Several plant extracts and their active ingredients have been investigated for their anti-photoaging effects against UVR-induced various MMPs in association with the antioxidative and anti-inflammatory actions in cultured keratinocytes and dermal fibroblasts as well as mouse skin . We and via the promotion of antioxidant defense capacity. To support further development of the HRF extracts as potential photoprotective products, this is the first time the protective effects of HRF extracts on the skin were tested using in vitro and in vivo model of UVA-induced photoaging in association with modulation of Nrf2-mediated redox balance. Indeed, both HRF extracts and HSP, its possible bioactive constituent, decreased UVA-induced cytotoxicity, oxidative stress, MMP-1 induction, and collagen degradation in a concentration-dependent manner, in association with concomitant rises in Nrf2 nuclear translocation and its target gene levels in a concentration-dependent manner. These findings were also reproduced in vivo and both HRF extracts and HSP dose-dependently protected against UVA-mediated MMP-1 induction, collagen loss, and oxidative DNA damage. The HRF extracts and HSP also enhanced a substantial Nrf2 activation in mouse skin, together with the upregulation of antioxidant defense enzymes GST and NQO-1. We and other studies have reported that activation of Nrf2 is involved in the mechanisms underlying the photoprotective effect of phytochemicals and botanical extracts against UVR-induced damage of the skin cells including melanocytes and keratinocytes -mediated signaling cascades including MAPK-AP-1/NF-κB, leading to transcriptional regulation of MMP-1  because treatment with the HRF and HSP alone for 30 min did not significantly promote Nrf2 nuclear translocation nor affect MMP-1 expression in non-irradiated mouse skin . TherefoAttempts have been made to develop Nrf2 activators as chemopreventive strategies including photoprotective agents for the prevention of photocarcinogenesis, although it should be taken into consideration that Nrf2 can be the double-edged sword in human cells and a harmful aspect of Nrf2 in the biology of skin cancer has been discussed . Overexpin vitro and mouse skin in vivo via suppressing oxidative damage and downregulating MMP-1-mediated collagen degradation through the regulation of cytoprotective Nrf2 activity. In this respect, the topical use of the HRF, a polyherbal formulation, may represent a complementary and alternative medicine strategy for preventing and/or delaying the skin photoaging through the restoration of redox homeostasis.In summary, we have demonstrated that HRF extracts and their potential bioactive compound HSP prevented UVA-induced photoaging of NHDFs


Bell’s palsy is a relatively rare neurologic disorder with a limited selection of helpful therapies. This case report describes the author’s initial three-month experience living with severe Bell’s palsy, including a detailed history and timeline of the initial development of symptoms, treatments pursued, and psychological stress during the disease progression. A particular focus is placed on the emotional burden Bell’s palsy can have, exploring possible avenues to improve physician to patient education on mental health and well-being during initial and delayed recovery. Initially described by Sir Charles Bell in 1821, unilateral Bell’s palsy, also known as “idiopathic facial palsy” or “acute facial palsy of unknown cause,” is a non-progressive neurological disorder of cranial nerve VII, commonly referred to as the “facial nerve” ,4.Bell’s palsy, often characterized by a sudden paralysis of the facial muscles innervated by the facial nerve, commonly results in the temporary weakness of the affected side. While symptoms generally present with unilateral facial weakness, rare occurrences of bilateral facial weakness have been reported. Surprisingly, the true cause of Bell’s palsy remains unclear. Several proposed causes have been examined, with the reactivation of an existing or dormant viral infection as the cause with the most popularity within the scientific community ,6. FortuThis case report describes the author’s initial experience living with Bell’s palsy, including detailed descriptions of symptom onset, adaptations, and the often-omitted impact on lifestyle and quality of life. While this account covers only three months, I hope this autobiographical case report provides those afflicted with this neurologic disorder with greater clarity as to what can possibly be expected, while also serving as an additional resource for colleagues managing those with Bell’s palsy. Throughout this case report, “I” will refer to myself, Kyril Cole.I was diagnosed with Bell’s palsy in September 2021, at the age of 28 years, with no significant past medical history. I presented to a neurosurgeon with a three-day history of right-sided post-auricular pain, a right-sided cervicogenic patterned headache, and right-sided neck pain initially thought to be occipital neuralgia, in addition to a six-hour history of right-sided facial weakness. On physical examination, I was found to have severe weakness in all facial expressions on the right side of my face, including movements in my forehead, nasal, periorbital, and perioral areas. Based on the acute onset of symptoms and physical findings, I was diagnosed with idiopathic facial palsy and started on prednisone the same day. Before initial symptoms, I took no medications and was current on all vaccinations, including my coronavirus disease 2019 (COVID-19) vaccination. Childhood illnesses were significant for only a varicella-zoster viral infection at the age of five years. I had no past surgical history, head trauma, medication allergies, or family history of Bell’s palsy or other neurological conditions. In terms of social history, at the time of diagnosis, I was a full-time MD/MBA student, married, enjoyed hikes and outdoor activities, and ate an entirely plant-based diet with appropriate vitamin supplementation. Over the past three months since my initial diagnosis, I have pursued all recommended treatments and suggested therapies; however, I have yet to regain any motor function to my right-sided facial muscles. Progression of symptomsMy symptoms began three days prior to the facial weakness, which began with a dull ache in and around my right ear, with sharp pain behind my ear, which would radiate down my neck. A headache accompanied this pain with a cervicogenic pattern on the right side of my head. After two days of this pain, I spoke with a colleague who gave a few possible explanations, with occipital neuralgia as the most likely diagnosis given my recent gym workouts. However, I noticed my soup at dinner had a dish-soap taste on the third day of discomfort, which did not agree with occipital neuralgia.The following day, I awoke to brush my teeth and use mouthwash, only to find myself spilling mouthwash all over the counter. Upon examination in the mirror, I realized I could no longer fully smile or purse my lips. Additionally, my ability to close my right eye had been severely reduced. During the same day, I confirmed suspicions of Bell’s palsy with a neurosurgeon. I received a prescription for a steroid, beginning within 72 hours of the onset of symptoms as clinically suggested . ThroughAfter 48 hours of the onset of symptoms, a complete understanding of my physical challenges became apparent. These included a rapid onset of right-sided facial paralysis , loss of taste to the front right two-thirds of my tongue, right eye dryness, continued right-sided postauricular pain, drooling in my sleep, paralysis of my right platysma and occipitofrontalis muscles, blockage in my left nasal passageway, and hyperacusis in my right ear.Treatments pursuedWhile no effective cure exists for new-onset Bell’s palsy, steroids have shown to be effective at increasing the probability of recovery of facial nerve function . Within After the prednisone, valacyclovir, and acupuncture therapy had ended, I still had no initial improvement in my symptoms. A magnetic resonance imaging (MRI) scan with and without intravenous contrast was pursued two months after my initial diagnosis to ensure no other pathology was present mimicking the presentation of Bell’s palsy. A T1 image with 15 ml of gadolinium contrast demonstrated enhancement at the right internal auditory canal (IAC) fundus Figure . Fluid-sImpact on physical and social activityPrior to the onset of Bell’s palsy, I was living a hectic yet active lifestyle. This included multiple hikes each week, daily hour-long workout sessions with my wife at the gym, academic and professional courses 60 hours each week, and everyday errands with the family. However, once the symptoms and facial palsy had solidified, I realized just how difficult each of these daily activities would be. Due to my right ear hyperacusis, any loud or high-pitched noise would cause significant pain to my ear. I could no longer go to the gym due to the volume of music, nor go to movies, football games, or bars with family and friends where sounds were far too loud. Even the clanging noises from stacking the cleaned dishes in the cupboard caused me a great deal of ear pain.Similarly, my right eye no longer produced the tears needed to lubricate my eye, nor could I close my eye sufficiently to keep the moisture in. Studying on the computer screen or watching TV became a significant challenge and strain on my eye, requiring constant artificial tear application. Enjoying a walk outside with even moderate sunlight or wind caused great discomfort. Eating became a significant challenge, as my mouth could not open enough to eat things like sandwiches, pasta, desserts, etc. without making a giant mess and causing me to feel embarrassed. Lastly, sleeping became a difficult challenge, as I would struggle to tape my eyelid closed well enough for a comfortable sleep each night, in addition to my nasal breathing largely blocked off. Through these and other challenges, my illness quickly became an isolating time in my life. Impact on psychological wellbeingMany of those afflicted by Bell’s palsy have significant improvement within three weeks of their initial symptoms, reaching a full recovery within three months . ReadingAnother feature of Bell’s palsy often overlooked is the impact the physical changes to the face can have on a patient’s psychological well-being. Initially, my new physical appearance was difficult for me, as I could not laugh or express my feelings through animated facial expressions. My appearance was difficult to contemplate, as any emotion was removed from one side of my face, and the other side overcompensated, causing me to look nothing like my original self. Personally, this made online meetings and courses challenging during the COVID-19 pandemic, as it is unprofessional not to show your face on camera. However, it is emotionally taxing to explain to each person what has happened to my face. The challenges of my physical appearance also transitioned to physical encounters, as many would stare and attempt to figure out what is wrong with my face, or they would ask if I had a stroke, dental work, or any facial trauma. These encounters made me want to remove myself and not interact with others, furthering the depression caused by my other social and physical limitations. However, I have become more comfortable with my appearance and understanding of how best to approach others with time.This case report describes many of the facial palsy-related challenges identified in the literature, including unilateral facial paralysis or increased weakness, hyperacusis, loss of taste, dry eyes with absent tear production, drooling, difficulty eating or drinking, loss of typical facial expressions, headaches, inability to close the eye on the involved side, absent corneal reflex, eye irritation, post-auricular pain, inner ear pain, in addition to unilateral occipitofrontalis and platysma muscle paralysis . While sStudied risk factors that put the patient at higher risk for developing Bell’s palsy include a family history of Bell’s palsy, current or recent pregnancy, Lyme disease, head trauma, diabetes, upper respiratory infections, or past viral infections (including herpes simplex virus type 1 and varicella-zoster virus) . InteresThough not curative, several treatment options have been studied to help relieve the symptoms associated with Bell’s palsy. Kim et al. performed a retrospective study on 1710 Bell’s palsy patients, comparing the effectiveness of steroids plus antiviral agents vs. steroids alone in treating severe Bell’s palsy patients over 40 years old. Subgroup analysis showed the combination therapy resulted in significantly higher recovery rates than steroids alone  . A randoMedical professionals are proficient at describing the pathophysiology of the neurologic disorder, current best treatments to reduce nerve inflammation, and the limitations in knowledge of the cause. However, in my experience, physicians do not mention the emotional toll that Bell’s palsy can have on one’s life, especially in cases of severe Bell’s palsy  lasting over three months. Sugiura et al. looked at the stress levels of Bell’s palsy patients from initial presentation through full recovery. They found the psychological stress response score of patients with facial palsy to be high on the first hospital visit, suggesting the importance for physicians to treat the psychological aspects of Bell’s palsy . To helpThe limitation of this case report is that I can report only the first three months of my experience with Bell’s palsy, given the timeline of the requested autobiographical reports and the recency of my Bell’s palsy onset. A longer timeline with a full recovery and reviewed response to physical therapy would benefit such a case series. A strength of this case report is my detailed description of each portion of my experience thus far, including an in-depth look at the timeline of symptom onset, medications and therapies utilized with their timelines, and the images demonstrating the physical changes in my facial paralysis. Another strength of this case report is the explanation of the emotional toll Bell’s palsy can have on everyday life, something that is often overlooked in medical reports -5.This autobiographical case report illustrates the typical initial progression of Bell’s palsy symptoms, with the addition of a detailed timeline and description of each step in the progression of my facial palsy. Apart from the various medical articles on Bell’s palsy, few pay particular attention to the emotional, physical, and social toll the disorder can put on the affected individual’s life. Specifically, those who suffer from severe Bell’s palsy, such as my case, would benefit significantly from initial explanations and recommendations on psychological well-being from their treating physician. Consultation with psychologists, psychiatrists, therapists, or other emotional guidance counselors should be encouraged for those affected by delayed recovery from Bell’s palsy. This approach could reduce overall stress from the disorder and potentially assist in hastening any delayed recovery.