The partial least squares structural equation modeling (PLS-SEM) provides researchers with an analysis tool for prediction theory. As the coronavirus disease 2019 (COVID-19) brings risks to teaching and learning, students have been forced to switch from classroom learning to online learning and most subjects have chosen massive open online courses (MOOCs) for online learning in China. This study examines whether MOOCs can replace traditional classroom education and explores the factors that influence the intentions of switching of the students from offline to online. We sequenced the PLS-SEM analysis of data with 397 students from a university in Zhejiang province of China, testing the model parameters, and discussing the push-pull-mooring (PPM) theory. Our data demonstrate that security risk is a push factor, switching costs are a mooring factor, and perceived usefulness and task–technology fit are pull factors that pull students from traditional, offline learning to MOOCs. In addition, the PPM model of the analysis results provides a more specific understanding of the importance–performance analysis of each factor. Our findings suggest that to constantly improve the switching intention to address unexpected challenges in the future, teachers should establish an effective emergency management measures, including curriculum design, to be consistent with their needs. Massive open online courses (MOOCs) represent essential technological advances that have taken place in higher education over the past 10 years. Smart devices and the Internet can be used to participate anytime and anywhere in open and massive online courses designed and provided by accredited higher education institutions and organizations, resolving the educational inequality in traditional teaching. Moreover, the massive online learning resources of such courses help to diverse groups, including elementary and middle school students, college graduates, and professionals, to discover and develop their technological potential, thereby enabling them to respond more favorably to market needs . EducatoMoreover, the adoption of the online teaching model has increased due to the coronavirus disease 2019 (COVID-19) pandemic, which has led to the shutdown of educational institutions worldwide. In response to rapid spread of the COVID-19, the Ministry of Education (MOE) of China announced the launch of online teaching in colleges and universities as a measure to reduce infections by reducing contact. The ministry also implemented the policy of “stopping class without stopping learning” to shift the teaching mode from traditional classroom education to online teaching . With thHowever, the implementation of MOOCs in online teaching in China is not optimal. Massive open online courses can replace traditional teaching styles that have become a matter of controversy, particularly during the pandemic when individuals have had to adjust previous teaching forms owing to the COVID-19. Specifically, many studies conducted during this period examined whether a positive attitude toward continuous learning (and considering it conducive to user development in the face of the pandemic) also plays an important role in the continuous promotion of the online learning mode . The rapBased on the above background, the following speculation can be obtained: Environment, technology, and learning intentions of the students might be the motivating factors for people to suddenly use MOOCs on a large scale. The effects of these factors were explored by PPM to obtain the conclusion that, if there is no pandemic in the future, information technology is not improved as in the past, and offline learning motivation of the students is greater than the online learning motivation, the users of MOOCs may be greatly reduced. Subsequently, the previous large-scale promotion and investment of our country may be wasted.The partial least squares structural equation modeling (PLS-SEM) helps researchers to understand the factors that predict the intention of the students to use MOOCs. This study extends the concept of population migration and investigates the factors affecting switching intention of the students . By switching learning modes, variables such as perceived usefulness and switching cost proposed by Push factors are factors that push individuals away from their original positions. Security risk was characterized by H1: The higher the perception of the impact of the university students on safety risks, the lower their acceptance of learning in physical courses and the higher their intention to switch to MOOCs.Pull factors attract individuals to a new role. For example, factors promoting students to switch from the traditional learning model to the model based on MOOC learning platforms are considered as pull factors. This study focused on the TTF model proposed by H2. The higher the perceived switching costs of university students, the lower their intention to switch from physical courses to MOOC learning.status quo to a new situation  following a semester-long experience of using MOOCs. As the researcher adopted convenience sampling to ensure the effective distribution of the questionnaire, respondents were mainly students from a university in Zhejiang province. The questionnaire was distributed to a teacher group on WeChat and group members were asked to help forward the questionnaire to students participating in MOOCs and to encourage the students to answer voluntarily. The main objective of the research questions was to examine the switching behavior of university students from courses in traditional physical classrooms to MOOCs due to the COVID-19 pandemic. A total of 403 valid questionnaires were returned and 6 invalid questionnaires were excluded by removing invalid samples  or uniform responses for all the items]; the number of valid questionnaires was 397.Therefore, data from the formal questionnaire were gathered through Wenjuanxing.The study population comprised 111 male (27.96%) university students and 286 female (72.04%) university students. Students who had never used MOOCs before the COVID-19 pandemic accounted for 57.49%, whereas those who had used MOOCs to study before the COVID-19 pandemic accounted for 42.51%. Finally, in terms of the distribution of respondents by colleges, 173 respondents were from the school of business/school of management, accounting for 43.56% of all the respondents, followed by 91 (22.37%) respondents from the school of humanities and the school of law, 81 (20.84%) respondents from the school of science and engineering, and 53 (13.23%) respondents from the school of education.First, the measurement model analysis included the Cronbach’s alpha validity, convergence validity, and discriminant validity, which verified the reliability and validity of the scale measurement. Second, the PLS was used to evaluate the model. The PLS-SEM was chosen for analysis, as it was more suitable than SEM based on covariance and was mainly adopted to conduct an exploratory study of theoretical development and the potential variable scores were required for subsequent analysis in this study . In addiThe common method variance (CMV) has two solutions. First, in the questionnaire design, the questionnaire items were paged to reduce the weariness of the respondents caused by long content when answering the questionnaire and to reduce the influence of CMV by continuous answering. Second, the Harman single factor test was used to check whether CMV was available. Principal component analysis (PCA) was adopted in the experiment and through the test results. It could be determined that there was no single factor accounting for more than 50% of the impact and the factor explained shows that the value is 0.385. Based on the test results, no CMV occurred in this study and all the results were in line with the values recommended in a previous study . Finallyp < 0.05)  . Based oDiscriminant validity refers to the degree to which an indicator of a potential variable differs from other indicators of potential variables that form part of the model . Two crip < 0.05, t = 2.040) had a negative influence on switching intention. Second, based on the H3 result, the pull factor  had a significant positive impact on switching intention. Third, the switching cost of H2  also had a significantly negative relationship with the switching intention. Finally, in terms of the influence of the adjustment effect, the results showed that neither H4  nor H5  habits have an adjustment effect on the push/pull factors. The results of this research model are summarized in The H1–H5 parts were mainly analyzed in this study based on the results. According to the statistical analysis results, the overall explanatory value of this study was as high as 0.63, which implies a high overall explanatory value in this study. Based on H1 results, perceived security risk (β = –0.094, The empirical results revealed that security risk was the push factor that transferred students from traditional, physical classrooms to MOOC platforms. During the COVID-19 pandemic, students felt insecure and preferred not to attend classes in physical classrooms, which corresponded with the switching intention demonstrated by previous PPM research . In termIn terms of pull factors, the use of MOOC platforms during the pandemic was affected by two essential subdimensions, namely perceived usefulness and TTF. Studies have indicated that TTF is the key dimension influencing intention of the students to engage in online learning . StudentAccording to the research results, the cost of switching traditional, physical courses into MOOCs is low, which is consistent with the conclusions of previous studies . During This study makes several significant contributions. First, efforts have been made to build barrier-free and high-quality education for MOOCs, which differs from other e-learning courses in universities . When unThis study had some limitations. First, it used a questionnaire survey and the number of recovered samples was limited. In particular, the samples were not obtained from universities in all the provinces in China and a single, specific university was surveyed. Therefore, subsequent studies should examine whether, given various regional characteristics, the COVID-19 pandemic affected the promotion of MOOC-based learning and the switching intentions of the students. Second, this was a cross-sectional study. However, under the continuous evolution of the COVID-19 pandemic, longitudinal studies should be conducted to explore and observe the factors of interest at different time points, which can further clarify whether the learning mode that emerged during the COVID-19 pandemic accelerated the subsequent promotion of MOOC platforms in universities. This study also did not discuss whether the basic information data had an adjustment effect on the structure of this study, which remains for further analysis in the future. Finally, this study is still in its initial stage and is unable to obtain a date for a normal environment. However, when possible, we suggest extracting data in a non-epidemic environment in the future.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.All authors contributed equally to the conception of the idea, implementing, analyzing the experimental results, writing the manuscript, read, and approved the final manuscript.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. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Stereotaxic surgery techniques are commonly used today in research laboratories by a range of students, technicians, and researchers. Over the past twenty years, technical and scientific progress has been made in neurosurgery to meet the evolving requirements imposed by international legislation, and to promote the implementation of 3R rules. These improvements were motivated by a greater awareness of animal welfare and the necessary effort in the reduction of the number of animals used in experiments. The data presented in the present study show that technical and methodological improvements brought to our surgical procedures from 1992 resulted in reproducible stereotaxic neurosurgeries and in a significant reduction in experimental errors and animal morbidity. The effects of these improvements include a decrease in the final number of animals used in our experiments as well as better management of pain during and after surgery and the use of appropriate aseptic techniques. Correct stereotaxic surgical approaches are precisely described throughout the text.Techniques of stereotaxic surgery are commonly used in research laboratories by a range of students, technicians, and researchers. To meet the evolving requirements imposed by international legislation, and to promote the implementation of 3R rules  by reducing experimental error, animal morbidity, and mortality, it is essential that standard operating procedures and proper conduct following such complex surgeries be precisely described and respected. The present report shows how refinements of our own neurosurgical techniques over decades, have significantly reduced the number of animals (rats) used in experiments and improved the animals’ well-being during the post-surgical recovery period. The current pre-, per-, and post-surgical procedures used in our laboratory are detailed. We describe the practical aspects of stereotaxic neurosurgery that have been refined in our laboratory since 1992 and that cover various areas including appropriate anesthesia and pain management during and after surgery, methods to determine the stereotaxic coordinates, and the best approach to the target brain structure. The application of these optimal surgical methods that combine reliable and reproducible results with an acute awareness of ethics and animal welfare leads to a significant reduction in the number of animals included in experimental research in accordance with ethical and regulatory rules as required by the European Directive on laboratory animal welfare. Stereotaxic neurosurgery is used in combination with other diverse techniques necessary for addressing the specific objectives of a proposed neuroscience study. These techniques include in vivo experimental procedures for activating or inactivating brain structures or transmitter systems, permanent selective lesioning, functional neuroanatomy using tracers, and acute or chronic measurements or recordings. With the Directive 2010/63/EU for the protection of laboratory animals 2010 [While legal frameworks applicable to animal research have considerably evolved since the early 1980s, animal care and use guidelines and recommendations have also been provided and regularly updated by expert groups or entities  , CanadiaAnimals, especially rodents, are widely used in neuroscience research in order to study normal and abnormal brain functions . Since 1The present work describes the techniques based on common standard procedures of stereotaxic surgery, during which material can be inserted into the brain in order to microinfuse drugs in animals performing a task. Based on the data published or collected in laboratory notebooks, reports, and theses from Masters and PhD-students under our supervision, we provide a detailed report on the changes that were introduced over time in our experimental procedures. The reasons or causes that motivated the improvement made to our procedures throughout our research experience were based on research evidence. Systematic post-mortem observations of lesion or cannula placement and implementation of end-point assessment sheets, precisely highlighting the reasons why some animals were discarded from the final experimental groups, made it possible to target the areas in which techniques needed to be improved in order to reduce the number of animals discarded from the final experimental groups. In particular, this analysis showed that progress could be made in terms of asepsis and postoperative recovery. The lack of precision in reaching the target structure motivated the use of pilot surgery in which animals that had already been used in an experiment were reused under anesthesia in non-survival surgeries to improve the accuracy of the coordinates of the approach to the target structure. Finally, the growing literature on the benefits of new methods in analgesia, anesthesia, and asepsis also advanced our methods and refinements.The analysis of the proportion of animals that were finally excluded from the experimental groups throughout our research clearly showed a positive impact of all these changes on the wellbeing of the animals, the reduction in the number of animals used and the quality of the experimental data. The description of the general methodology includes the chronological list of the refinements  that have been introduced in stereotaxic procedures during the period that covers our research activity from 1992 to 2018.Aseptic techniques have received special attention since we started our experiments in 1992 and sterilization of the surgical equipment was a first step implemented in our procedures. To this end, surgical tools  were sterilized by placing them for 30 min at 170 °C.The ear and incisor bars, the hand piece of the dental drill, and the stereotaxic frame were simply cleaned with disinfectant wipes.From 1994 on, a change was introduced in the procedure and the cannulas were no longer sterilized by heat. Instead, they were put in a bath of hexamidine solution and the inside was systematically infused by a solution of hexamidine and rinsed with a sterile NaCl solution.From 2005, we implemented, from the beginning to the end of surgery, a sequence of steps under the go-forward principle in order to limit contact between soiled and sterile instruments or materials and therefore to keep a high level of asepsis. The organization of space was included in this process with the delineation of two distinct areas, one called a “dirty” area for the preparation of the animal (see below), and the other, a “clean” zone for the surgery.From 2005 on, an assistant was always present and helped the experimenter to conform with the go-forward principle and to prepare the surgical set-up. After a thorough surgical handwashing, the surgeon is helped for gowning and gloving, with a sterilized gown and mask as well as sterile gloves prepared in advance. Once the surgeon is equipped, the assistant places the instrument box on a sterile drape next to the stereotaxic frame.®, Vetoquinol, Magny-Vernois, France), rinsed with sterile water, and disinfected with iodine solution . Alternatively, chlorhexidine-based soap and solution  are used. The surgeon then lets the antiseptic solution dry out and checks the position of the head.A clinical examination is made to ensure that each animal has a good health status before proceeding. Importantly, none of the rats is subject to food restriction before surgery. The animals’ weight is carefully measured for the adjustment of anesthesia dosage and this is kept as reference for the post-surgical follow-up. An animal is first anesthetized and prepared  in the “dirty” area. Second, the paws and tail of the animal are gently cleaned with an iodine or hexamidine scrub solution. The animal is then brought by the assistant to the “clean” zone. From 1994 on, a thermostatically controlled heating blanket with a rectal probe has been used to ensure an optimal control of internal body temperature. At this point, the surgeon takes charge, checks the depth of anesthesia and installs the animal into the stereotaxic frame by fixing its head between ear- and nose-bars. Blunt tip ear bars are used and care is taken for their insertion: first, the observation of a blink of the eyelids ensures an accurate positioning at the entrance of the external auditory canal and second, the scale on the bars is systematically used as an index of their progression and position. An ophthalmic ointment is applied on the eyes to protect the cornea from desiccation. The top of the animal’s head is then scrubbed with an iodine foaming solution  and ¼ part of the alpha-2 agonist xylazine hydrochloride  [From 1992 to 1999, anesthesia was induced by an intraperitoneal (i.p.) injection of 5 mg/kg diazepam followed by 100 mg/kg ketamine ,8. From 0 mg/kg) ,21,22. A®, Aguettant, Lyon, France) at the incision site once deeply anesthetized  0.4 mg/kg and later on buprenorphine  0.05 mg/kg in order to reduce pain during surgery [From 1992, the animals received a subcutaneous injection of lidocaine . The dorso-ventral (DV) coordinates of both landmarks are checked and the inclination of the head is adjusted whenever the difference between both DV coordinates exceeds 0.1 mm. Sterile anchor screws  are then inserted to the skull to strengthen the fixation of the implant. To avoid damage to the underlying pia, only short-length screws are used.From 1995, the “flat-skull” position was verified once. From 2004, the flat position of the skull was checked twice before and after the insertion of the anchor screws.Then, the ML and AP coordinates of the target structure are calculated relative to the appropriate reference (bregma or lambda) with support of a reference atlas and its updated versions ,27,28. IFrom 1995, the verification of the vertical position of the holders was also checked after having positioned the carrier on the micromanipulator on the stereotaxic frame.From 2004, the reference point for the calculation of the final DV coordinates was taken from the dura surface. Indeed, our observations showed marked differences in skull thickness as a function of age, occasionally resulting in shifts in the DV position of cannulas across animals when using to the bone surface as a reference. The cannula, or injection needle, was installed on the object carrier after the dura was resected. This step was repeated in the case of bilateral insertion.Then, the cannula or injection needle is carefully lowered according to the DV coordinates, with a speed of about 1 millimeter per minute.In 2012, we introduced a new holder with grooves aligned at fixed intervals to maintain two cannulas strictly in parallel for bilateral implantations. This tool allowed the simultaneous insertion of two parallel guide cannulas in a single move of the holder. From this period on, the axial position of the skull was also systematically verified before inserting the material.®, Sun Medical Co., Moriyama-shi, Japan) is applied [® powder and solvent, GC America Inc., Alsip, IL, USA). The cement is applied using the brush-dip technique which consists of forming a ball of powder/liquid at the tip of a brush, first by dipping the tip in the liquid and then touching the polymer powder. Compared to the bulk-mixing technique, this mode of application results in a high powder/liquid ratio that limits the thermal effect of polymerization, fastens the curing, and shortens the working time. The edges of the implant are made as smooth as possible. When the cement has completely dried, the object carrier is slowly raised leaving the guide cannula in place and visible on the skull. A sterilized obturator (stylet) is then inserted into the guide to avoid clogging during the post-operative recovery period. Depending upon the size of the skin incision and the volume of the implant, the wound is sutured at the end of the procedure. For this purpose, sterile suture material is used. Single stiches are done using resorbable suture wire on a 3/8 curved needle. At the end of the procedure, the wound is cleaned with a compress soaked with a 1:10 dilution of chlorhexidine in sterile water. Any trace of cement, hair, or blood clots or other debris that can interfere with wound healing is removed. Before the animal fully recovers from anesthesia, the nails of all paws are trimmed to minimize any impact of the animal’s normal scratching and grooming behavior on the healing process. The return of the palpebral reflex is checked using a cotton-tip applied on the eye’s corner.When the material has been inserted correctly in the brain, the skull is dried and a self-curing adhesive resin .Toe- or tail-pinch withdrawal reflexes, breathing rhythm and amplitude, color of the mucous membranes, body temperature, and possible dehydration of the animal are checked throughout the surgery. This monitoring has been applied since 1992. From 1994, in addition to the visual control of the animal’s breathing and the color of its mucous membranes, internal temperature has been systematically monitored and maintained throughout the surgery.At the end of the surgical procedure, the animal is temporarily placed in the recovery zone with an infrared spot above the cage. Importantly, the light is placed such that an animal can move away from the hot spot once having recovered its righting reflex. Post-operative care procedures such as manual feeding and facilitated access to drinking source (by lowering the drinking spout of the water bottle) are provided until an animal recovers its autonomy and a complete restoration of normal behavior. Each animal returns to the housing facilities only after complete awakening from anesthesia and is placed back in its home cage with its cage mates.From 1992 to 1994 systematic i.p. injection of 5 mL of NaCl and heating of the animal at the end of surgery were used. From that period, animal facility staff were involved in daily post-surgical care and nursing. Individual follow-up sheets were filled for each animal and listed all the substances an animal had received pre- per-, and post-surgery. In some rare circumstances of severe complication, the involvement of the technical staff enabled rapid euthanasia according to predefined endpoints, thus avoiding unnecessary suffering. Also, from 1992 to 2000, a prophylactic antibiotic treatment  was used in order to complete our aseptic techniques ,8,9,33.®, Zoetis France, Malakoff, France; 12–24 h duration of action). An evaluation of signs of distress, pain, and discomfort  was done daily and observations were written on the individual’s follow-up sheets [From 2012, i.p. injections of 5 mL of glucose Ringer lactate 5% replaced the use of NaCl at the end of the surgery and all animals received a subcutaneous injection of a nonsteroidal anti-inflammatory drug . In addition, the inside of the tubing was infused with a 70% alcoholic solution before each microinfusion. From 1994 on, the animal care staff was involved in the follow-up of the animals and would inform the experimenter whenever an abnormal or unexpected situation occurred.After completion of behavioral and pharmacological testing, each rat was euthanized for brain removal and histological examination of the injection sites. Euthanasia was performed with an overdose of anesthetic.All analyses were performed with Prism . For normalization purpose, a ratio of the “total number of animals discarded from the statistical analyses/total number of animals used in experimental research in our group from 1992 to 2018” was calculated and analyzed. The Agostino Pearson’s analysis was used to test the normality of the distribution and this was followed by Pearson’s parametric correlation test.n = 348 Wistar; n = 1880 Long–Evans, and n = 1000 Sprague-Dawley strains). The number of animals finally discarded in each study is compiled and presented as a proportion for normalization and inter-study comparison purposes. No effect of strain was ever observed during our experiments, whatever the refinement considered.Data were obtained from a total of 113 studies and reports in our research group from 1992 to 2018. A total of 3228 animals, weighing 250–300 g at surgical period, were involved in the present study  and welfare  refinements in our procedures from 1992. Statistical analyses confirmed these observation and Pearson rank correlation revealed a significant relationship between the proportion of animals discarded from the analyses and the refinement in the different techniques throughout the successive periods of research  and after a surgery relies strongly on the choice of sedation, anesthetic and analgesic compounds, and an appropriate rehydration procedure when needed, but also on the monitoring procedure. Depth of anesthesia, and vital signs such as respiratory rhythm and amplitude, should frequently be checked throughout surgery in order to react rapidly. General anesthesia causes a dose-dependent impairment of thermoregulation, a depression of cardiovascular and respiratory systems ,38, a loSurgical asepsis refers to the absence of infectious microorganisms during an invasive procedure. Such organisms are naturally present in the environment, in animals and humans, and on all foreign material that can be introduced in a living tissue (biofilm). Aseptic techniques are thus essential to prevent infection of the surgical site and post-surgical complications and to minimize the risk of mortality.When determining stereotaxic coordinates of a target structure one should consider its anatomical and functional characteristics, its spatial coordinates as given by the atlas, the organization of the cerebral vascular system, and the age or state of development of the animal. In order to ensure a high precision and reproducibility of stereotaxic surgeries, these should all inform the implantation of materials such as cannulas, electrodes, or microdialysis probes.In this regard, from 1992, a series of improvements have been brought to our stereotaxic calculation of the brain target coordinates by a systematic verification of the adequacy between the theoretical position of the target structure found in the atlases ,27,28, aAs shown in The second important improvement was observed during the 2002–2004 period when cannulas of a smaller diameter were introduced (from 23 to 24 gauge); less invasive, thinner cannulas have a better tissue tolerance and allow a more precise positioning into the brain. Also, from that period, due to the imprecision observed in some cases between the theoretical DV coordinate of the target structure and the position of the canula observed post-mortem , the final D/V coordinate of the target structure was calculated from the dura. In addition, from 2002, the flat position of the skull was verified before and after the screw anchoring.The third significant progress in the method appeared in the 2010–12 period and resulted from the development of custom-made holders maintaining a given distance and a strict parallelism between cannulas for bilateral implantations in symmetric areas. Such holders were manufactured with parallel groves to fit exactly the M/L distance between each structure targeted. Also, the M/L axial flat skull position was systematically verified in order to ensure perfect vertical position and correct M/L position of the cannulas. To this aim, the D/V coordinates of the point above the target structure was bilaterally checked for correspondence.As shown in the figure, these refinements led to a steady decline of the number of animals discarded from the analyses due to misplacement of lesion or injection sites. Our method has improved in accuracy and reproducibility so that only 1/166 subject was excluded from the final analysis in the final period compared to 12/109 during the first period.®) that does not cross the blood–brain barrier or choose another anesthesia method that does not cause excessive secretions. The choice of a drug should also take into account the potential delayed effect, such as anxiety signs, that may need a specific test to be objectivated.The refinements introduced in our research are somewhat diverse and might not apply to all stereotaxic surgeries. Stereotaxic surgeries can indeed be used in a variety of research projects; while general recommendations can apply, such as the systematic use of aseptic techniques in survival procedures, others need to be considered on a case-by-case basis. This seems particularly true when considering the nature of anesthetic and the analgesic and other drugs to administer to an animal. Dosage and administration route are obvious parameters, but others such as half-life and potential side-effects also deserve attention, especially in neuroscience research. Some drugs acting on the central nervous system can have delayed effects that can impact the animal’s behavior and well-being. For example, compounds like buprenorphine  or fentaRegarding anesthesia in our own research, we observed a significant decrease in mortality following the refinement in the method. As an alternative to fixed anesthesia, we have introduced inhalational anesthesia, since it has been shown to be safer and more effective than parenteral anesthetics ,46. IsofBecause of its poor analgesic properties and its rapid elimination, switching to isoflurane anesthesia prompted us to reconsider our analgesia protocols. Since pain can be much more difficult to control once the animal has become conscious of it, we systematically use local anesthetic drugs , and opioids  for surgery and a post-operative 48 h treatment with NSAIDs (carprofen) to provide good analgesic levels and comfort for the animals.Any significant change in a procedure should therefore be thoroughly considered before being implemented. A cost/benefit approach might be appropriate in this matter. Such methodology implies a careful assessment of many parameters, not only during the surgery itself, but also during the immediate and long-term periods that follow. These parameters can strongly differ between research studies. When targeting a brain area involved in motor control, the follow-up of an animal’s well-being should include signs of motor deficits or handicap. Such signs can be extremely subtle, or even somehow compensated, but they should always be anticipated. Animal caretakers are the first-line actors, because of their presence in animal facilities, but refining a complete procedure will also benefit from close interactions between experimenters, the animal welfare body involved in the follow-up of projects, and the designated veterinarian. If required by the ethical oversight body or institutional animal care and use committee, a retrospective assessment can also provide guidance and suggestions for future improvements.Although the proportion of animals excluded from our experiment today has reached a low level, we continue our efforts to further improve our procedure and the welfare of the animals without forgetting the well-being of the experimenter and facility staff, bearing in mind that good working conditions  will contribute to high-quality care to the animals and limit compassion fatigue. For this purpose, we regularly update and enrich our training program for experimenters and caretakers in our animal facility on stereotaxic surgery, nursing skills, and ethical requirements. Such training includes surgery planning, stereotaxic apparatus handling, determination of stereotaxic origin, coordinate calculations, and device placement. We also consider implementing technical innovations such as digital stereotaxic rulers and motorized arms which can contribute to improve targeting accuracy ,50.Over the past twenty years, technical and scientific progress has been made in neurosurgery to meet higher ethical standards. These improvements of pre- per-, and post-surgical approaches were motivated by a greater awareness of animal welfare and the necessity to reduce the number of animals used in experiments. We identified three major domains where improvements allowed us to achieve reliable and reproducible stereotaxic neurosurgeries with a lower number of subject rejections. These domains include (1) management of pain during and after surgery, (2) appropriate aseptic techniques, and (3) correct determination of stereotaxic coordinates and surgical approaches.The data presented in the present study show that refinements made in our procedures from 1992 to 2018 resulted in a significant reduction in the number of animals used in experimental research through continuous decrease in experimental errors and animal morbidity over time. Resulting from the refinement brought to our procedures, the implementation and completion of postoperative scoring grids revealed a decrease in the number of post-surgical reinjections of NDAIS, a decrease in the use of euthanasia after passing the endpoints, and a faster recovery of normal weight. These observations suggest the refinements probably impacted the well-being and the survival of the animals. These technical and methodological improvements were guided by growing ethical and animal welfare considerations which prompted us to evaluate and implement new practices and technologies found in the literature and throughout numerous exchanges within the neuroscience community.


Infants who are born prematurely are at significant risk of apnoea. In addition to the short-term consequences such as hypoxia, apnoea of prematurity has been associated with long-term morbidity, including poor neurodevelopmental outcomes. Clinical trials have illustrated the importance of methylxanthine drugs, in particular caffeine, in reducing the risk of long term adverse neurodevelopmental outcomes. However, the extent to which apnoea is causative of this secondary neurodevelopmental delay or is just associated in a background of other sequelae of prematurity remains unclear. In this review, we first discuss the pathophysiology of apnoea of prematurity, previous studies investigating the relationship between apnoea and neurodevelopmental delay, and treatment of apnoea with caffeine therapy. We propose a need for better methods of measuring apnoea, along with improved understanding of the neonatal brain's response to consequent hypoxia. Only then can we start to disentangle the effects of apnoea on neurodevelopment in preterm infants. Moreover, by better identifying those infants who are at risk of apnoea, and neurodevelopmental delay, we can work toward a risk stratification system for these infants that is clinically actionable, for example, with doses of caffeine tailored to the individual. Optimising treatment of apnoea for individual infants will improve neonatal care and long-term outcomes for this population. Apnoea of Prematurity (AOP) is frequently defined as a pause in breathing lasting more than 20 s, or more than 10 s with accompanying bradycardia and/ or oxygen desaturations in an infant born before 37 weeks' gestation . Along wApnoeas are classified as central or obstructive, in that they either derive from insufficient drive from the respiratory centres of the brain, or that the airway itself is obstructed. “Mixed” apnoeas are a combination of the two. AOP is thought to be a physiological consequence of immaturity, with insufficient central respiratory drive to maintain ventilation across the lung , 12. SubCentral control of breathing pertains to the role of the central nervous system in regulating rhythmicity and expansion of the chest in respiration. Normal inspiratory rhythm at rest is directed by the medullary dorsal respiratory group of neurons, with the pneumotaxic centre of the pons acting to slow inspiration. Expiration is the result of reduced dorsal respiratory group output and elastic recoil of the chest wall. The ventral respiratory group of the medulla is primarily involved in active expiration and tachypnoea, usually in exercise, in response to stimulation by peripheral chemoreceptors, and the pre-Bötzinger complex within the ventral respiratory group is an intrinsic site of rhythmogenesis  –16.In utero, foetal breathing movements are only intermittent, and work to facilitate lung bud growth through mechanical stretching, rather than for gas exchange (2) in the medulla is the primary controller of respiratory drive  and vagus (X) nerves, respectively. This normally triggers homeostatic breathing responses such as an increase in ventilation in response to hypercapnia through greater stimulation of the ventral respiratory group  sleep , 39, whiApnoea in preterm infants is frequently associated with bradycardia and oxygen desaturations. These events are precipitated by the arterial chemoreflex in response to hypoxaemia –44, and AOP ordinarily resolves with time and increasing maturity of the infant's respiratory systems. However, apnoea and the consequential chronic intermittent hypoxia, may have long-term effects including increased risk of infant mortality , neurodeWhilst mechanical respiratory support and non-invasive ventilation such as continuous positive airway pressure (CPAP) play an important role in supporting infants with AOP, pharmacological interventions are now a mainstay of treatment . Methylx1 and A2A, with A1 being the primary inhibitory receptor of postsynaptic neuron activation in response to hypoxic stress  of the variability of these data  . NAVA alWith better measurement of AOP and respiratory dynamics we will be able to develop techniques to improve our treatment for apnoea. Computational techniques, such as machine learning algorithms, have the potential to predict apnoeic and hypoxemic events in individual infants, leading to earlier interventions . OptimisStudies to date which have investigated a possible role of apnoea on later life neurodevelopmental outcomes have shown a correlative rather than causative relationship , 106. ToNumerous MRI studies have investigated differences in the brain structure in preterm infants as they develop –110. HowFor this purpose, techniques such as electroencephalography (EEG) can be used at the cot-side and have excellent temporal resolution, enabling the investigation of brain function before, during and after apnoeic episodes. Whilst a number of EEG studies have described seizure-related apnoeic episodes in infants, changes in the EEG related to non-seizure apnoeic episodes have not been described in detail –114. LowThere have been vast improvements in the understanding and treatment of AOP over the last 40 years, and the CAP trial is illustrative of the positive impact this understanding can have on the neurodevelopmental outcomes of children born prematurely. However, questions remain about the exact role of AOP in contributing to the worse neurodevelopmental outcomes faced by premature-born babies. To better understand the impact that apnoea has on the long-term outcomes of preterm infants, we must work toward more sophisticated measurements of apnoea and investigate further the brain's response to hypoxia. Improved measurement of AOP and respiratory dynamics in infants will allow us to stratify infants based on the risk of developing AOP and the risk of poor neurodevelopmental outcomes. Doses of pharmacological interventions and ventilatory support could then be tailored to the individual, optimising the balance between the benefits and risks of these interventions. With these measures in place, we may be able to move closer to a scenario where no infant born prematurely is at risk of adverse neurodevelopmental outcomes because of AOP.MW wrote the first draft of the manuscript, with supervision from CH. All authors critically reviewed and revised the manuscript.CH is funded by the Wellcome Trust and Royal Society through a Sir Henry Dale Fellowship (Grant Number: 213486/Z/18/Z).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. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Hymenoptera stings are generally well-tolerated and usually cause limited local reactions, characterized by self-resolving erythema and edema associated with pain. However, Hymenoptera stings can induce immediate and delayed hypersensitivity reactions. In addition to these manifestations, unusual reactions to Hymenoptera stings have been reported. The latter are defined as unusual because of their atypical characteristics. They may differ from classical hypersensitivity reactions due to the stings' particular localization and the unusual involvement of one or more specific organs. Although unusual reactions to Hymenoptera stings are infrequent, it is essential for clinicians to know the possible related clinical manifestations. Here, we review the available literature and propose a diagnostic and management algorithm. At present, there are no defined guidelines for most of the unusual reactions to Hymenoptera stings, which should be managed in a tailored way according to the specifical clinical manifestations presented by the patients. Further studies are needed to better define these conditions and the underlying pathogenetic mechanisms to improve the diagnostic and therapeutic approach. Precise epidemiologic data are not available, and differences between adults and children are unclear  local reactions following unusual localization of the sting and (ii) reactions with atypical signs and symptoms  5, 6). , 6. 5, 6case reports, including a case of a 3-year-old child  should be performed to assess whether the reaction may depend on an IgE-mediated allergic mechanism . If alleThe use of glucocorticoids with anti-inflammatory and immunomodulatory effects has been shown to be effective in case reports and case series, especially when the nervous system is involved. Toxic reactions after massive stings represent a medical emergency. Hymenoptera venom has no antidote. The primary principles of Hymenoptera massive sting management are (i) correcting hypovolemia to prevent renal ischemia; (ii) enhancing the clearance of heme proteins, toxins, or toxic wastes out of the systemic or renal circulation; and (iii) reducing the risk of direct venom toxicity, toxic waste, electrolyte imbalance, and heme protein in the kidney and other organs . In thisIn conclusion, although unusual reactions to Hymenoptera stings are infrequent, it is essential for clinicians to know the possible related clinical manifestations. Given the limited evidence available in the literature, it is unclear whether these reactions differ in severity and organ involvement between adults and children. However, since the child is an evolving organism and presents peculiar physiological characteristics distinct from adults, a specialized approach by pediatricians with experience in this field is essential .Further studies are needed to better define these conditions and the underlying pathogenetic mechanisms to improve the diagnostic and therapeutic approach.EN conceived the study and supervised it. RC wrote the manuscript. All the authors performed the research and the selection of the sources, critically revised the manuscript, and accepted the final version of the manuscript.The publication fee was financed by the Italian Society of Pediatric Allergy and Immunology. However, no significant funding source could have influenced the outcomes of this work.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. The reviewer AL declared a shared affiliation, with two of the authors RC and GM to the handling editor at the time of the review.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. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


On 20 October 2020, the Working Group “Exposure Models” of the Europe Regional Chapter of the International Society of Exposure Science (ISES Europe) organised an online workshop to discuss the theoretical background of models for the assessment of occupational exposure to chemicals. In this report, participants of the workshop with an active role before and during the workshop summarise the most relevant discussion points and conclusions of this well-attended workshop. ISES Europe has identified exposure modelling as one priority area for the strategic development of exposure science in Europe in the coming years. This specific workshop aimed to discuss the main challenges in developing, validating, and using occupational-exposure models for regulatory purposes. The theoretical background, application domain, and limitations of different modelling approaches were presented and discussed, focusing on empirical “modifying-factor” or “mass-balance-based” approaches. During the discussions, these approaches were compared and analysed. Possibilities to address the discussed challenges could be a validation study involving alternative modelling approaches. The wider discussion touched upon the close relationship between modelling and monitoring and the need for better linkage of the methods and the need for common monitoring databases that include data on model parameters. For some challenges, an attempt was made to draw a roadmap towards future exposure-modelling initiatives. To accomplish this, the workshop was characterised by mutual trust and respect for the competence and work of all the scientists, regulators, industry representatives, and other participants.During the Workshop, the challenges described in The Working Group organised the workshop as a virtual meeting, with the patronage of ISES Europe. It was open and free for anybody with an interest in exposure modelling and about 70 participants engaged in discussing the applicability of the currently available modelling approaches for workplace exposure assessment.The agenda and scientific information that was distributed to the workshop participants in advance facilitated the discussions; the presentations given during the workshop gave a clear understanding of the various exposure-modelling approaches.Scientists involved in the development of the models introduced different approaches for occupational exposure modelling and the theoretical background on which these approaches are based. In addition, some other participants were particularly active in the preparation of and the discussion during the workshop due to their substantial knowledge and experience with exposure modelling. These scholars attended the workshop to hear first-hand the debate and to contribute to the discussion and were also involved in the dissemination of workshop results. The workshop was organised and moderated by Natalie von Goetz and Urs Schlüter.All participants acted in an individual capacity and not as representatives of any organization or committee to which they are currently or were previously associated.How can the ISES Europe Working Group promote validation exercises for occupational-exposure models?How can the ISES Europe Working Group further develop occupational-exposure models for regulatory purpose?For what specific regulatory questions can different stakeholders use occupational-exposure models?What has to happen to improve occupational-exposure models in the future?After each presentation, all participants briefly discussed the respective topics in a plenary session with all experts and participants. After all presentations, experts and participants discussed all the topics in a plenary discussion collecting impressions, opinions, criticisms, and proposals. The Working Group had prepared the following key questions to structure this plenary discussion:The most interesting aspects of the discussion were collected by the moderators, who, together with additional active participants, prepared this workshop report to present the collective views of all the experts.® and ART are based on a modelling approach published by Cherrie et al. in 1996 ).For the moment, it would be a significant step forward if all available information, different modelling approaches, and measurements could be properly taken into account for regulatory decisions. That means that a combination of modelling and measurements could be a solution . AdditioBetween-user variability in the use of models was identified as relevant for modelling by different authors in the past e.g., ,40). In . In 40])During model development, subjectivity shall be eliminated as much as possible, but may have an important influence, as the derivation of exposure modifying factors or simplifications to model structures require, to some extent, expert judgement.Participants of the workshop explained that developing mass-balance-based exposure-modelling approaches for thousands of scenarios for chemical regulations  is currently not possible for practical reasons. Both modifying-factor approaches and mass-balance-based exposure models have been applied for decades in regulatory occupational e.g., ,42) and  and 42])For the moment, both modelling approaches are necessary and need to be used following the principles of a tiered approach. Some participants pointed out that mass-balance-based exposure-modelling approaches can follow a tiered approach, reducing model uncertainty as model complexity increases . ExposurThe workshop on the theoretical background of occupational-exposure models organised by the ISES Europe Working Group “Exposure Models”, aimed, among the objectives, to describe basic features of mass-balance-based and modifying-factor modelling approaches, identifying advantages and disadvantages of both approaches and classifying the reliability of the results. The general aim of the workshop was to discuss different opinions in a shared scientific context: this might help and support any further discussion that will arise in the future and base such discussion on a common ground. Highlights of the extensive and thorough experts’ discussion have been presented in this paper. However, the discussion , which could improve estimation of occupational exposure for regulatory purposes. As a first step for common validation activities, data-exchange templates for exposure measurements in order to arrive at a common minimum set of variables and quality criteria would be beneficial. Only then, a common framework for the evaluation of models and tools comprising the models themselves, the model documentation, and user-friendliness of the tools is possible. An interesting research project could be the comparison of the tools in question to suitable mass-balance approaches. However, good parameterization is needed, which could be achieved by the above-mentioned data-exchange templates.The workshop participants agreed that further model development and improvement requires international collaboration to meet global needs of chemical safety assessment. Since in Europe the chemical safety assessment and the improvement of exposure descriptions (modelling and measurements) are the responsibility of the manufacturer or importer of chemicals, funding by industry should be explored. The workshop participants generally observed that more systematic funding for exposure science is missing, but would be needed, and that this is especially the case for exposure modelling. If exposure science is funded, usually monitoring programmes are in the focus of funding bodies.Ideally, a common platform for hosting and funding of models should be developed on a global level. The private  and the public  sectors may both play a role in this effort. It should be noted that funding opportunities will not be emerging as long as the current state of the art of exposure assessment via modelling is deemed to be acceptable.However, it is the conclusion of this workshop that exposure modelling in the service of regulatory decisions should be improved in many aspects. The ISES Europe Working Group “Exposure Models” is asked to look deeper into funding possibilities and promote exposure science and modelling with different stakeholders and funding agencies.


The lymphatic system is involved in tissue homeostasis, immune processes as well as transport of lipids, proteins and pathogens. Aging affects all physiological systems. However, it is not well studied how aging affects the lymphatic vasculature. Therefore, this review aims at investigating how senescence could lead to changes in the structure and function of the lymphatic vessels. We report that lymphatic senescence is associated with alterations in lymphatic muscles and nerve fibers, lymphatic endothelial cells membrane dysfunction, as well as changes in lymphatic pump, acute inflammation responses and immune function.Lymphatic flow is necessary for maintenance of vital physiological functions in humans and animals. To carry out optimal lymphatic flow, adequate contractile activity of the lymphatic collectors is necessary. Like in all body systems, aging has also an effect on the lymphatic system. However, limited knowledge is available on how aging directly affects the lymphatic system anatomy, physiology and function. We investigated how senescence leads to alterations in morphology and function of the lymphatic vessels. We used the strategy of a review to summarize the scientific literature of studies that have been published in the area of lymphatic senescence. Searches were carried out on PubMed and Web of Science using predefined search queries. We obtained an initial set of 1060 publications. They were filtered to 114 publications based on strict inclusion and exclusion criteria. Finally, the most appropriate 57 studies that specifically addressed lymphatic senescence have been selected for the preparation of this review. Analysis of the literature showed that lymphatic senescence is associated with alterations in lymphatic muscles and nerve fibers, lymphatic glycocalyx function of lymphatic endothelial cells, effects of chronic ultraviolet light exposure and oxidative stress as well as changes in lymphatic pump, acute inflammation responses and immune function. The current review underscores the relevance of the understudied area of lymphatic senescence. Continued research on the impact of aging on the structure and function of the lymphatic vasculature is needed to provide further insights to develop innovative clinical diagnostic—and treatment—modalities as well as to reduce the morbidity associated with diseases related to the lymphatic system. An optimal and functioning lymphatic transport system is necessary to transport lipids absorbed in the gut, immune cells and to maintain fluid and macromolecular homeostasis ,2. ThereLymphatic function can be influenced by intrinsic and extrinsic factors, including active and passive lymph pumps and hydrodynamic factors like shear and pressure . The lymAging has been associated with alterations in muscle strength and function , with changes in the cardiovascular response to perturbations  as well as changes in bone tissue, upon others ,9. ThoseThe current literature on the subject of “lymphatic senescence” was systematically reviewed and summarized in a review. Both primary and secondary sources of literature on the topic were examined. The systematic approach for the current review is shown in PubMed and Web of Science served as search engines and were used to access relevant literature. The first approach in the search of this thematic yielded in a total number of 1060 articles. Articles available in English language and articles focusing on the lymphatic system were included. Different combinations of following MESH terms were used when formulating the search: “lymphatic senescence” and “lymphatic aging”.The initial search provided 1060 articles entries on “lymphatic senescence” (402) and “lymphatic aging” (658). After application of inclusion/exclusion criteria, a total number of 114 studies (35 on lymphatic senescence and 79 on lymphatic aging) were further considered. Included were human and animal studies as well as articles focusing on lymphatics and all related structures or pathologies. Excluded were studies not focusing on the lymphatic vessels and duplicates. Finally, the most appropriate 57 studies that specifically addressed lymphatic senescence have been selected for the preparation of this review. The research articles were categorized and discussed in specific sections as follows:Different types of animals and different breeds have been used in the selected articles. About 42% of these animals were rats of which 37.5% of Fisher-344 rats , 12.5% C57Bl/6 mice (in 25% they studied mesenteric lymphatic vessels), 9.4% Wistar rats (in 100% they studied mesenteric lymph duct), 6.2% Sprague-Dawley rats  and 3.1% BALB/c mice.About 3% of the studies were carried out on humans, in the following tissues: femoral regions, vocal folds and drainage larynx, cervical, auxiliary, inguinal, popliteal, mesenteric, capillaries, nasal and cerebral lymphatic vessels, lymphatic intact skin, lymph supply of the knee menisci, interstitial lymphatics fluid traffic and endothelial lymphatic vessels.When discussing aspects related to aging, it is important to distinguish between the effects of biological aging and that due to environmental factors . Dermal As aging progresses, a decrease in lymphatic vessel function and lymphatic flow is observed, arising due to a significant reduction in pumping rates, including pump frequency, amplitude, and fractional lymph flow . In the Furthermore, it has been shown that aging alters the glycocalyx (and consequently the permeability of the lymphatic collectors), lymphatic contractility and reduces junctional proteins . It is iA decrease in the function of the lymphatic pump associated with aging in the mesenteric lymphatic vessels (MLV) has also been observed. This suggests that aging can significantly affect the lymphatic muscles . For insIn aged lymph nodes, decreases in the total number of double-negative (podoplanin-CD31) cells as well as a disorganization in their distribution has been reported. Podoplanin and CD31 are expressed in the lymphatic vessels and are used for immunohistochemical staining of the endothelial subtype . The strThe senile involution seen in lymph nodes of humans affects the structure of the lymph node. Moreover, this lymph node degeneration process is progressive . In the The musculature of the thoracic duct reaches its maximum size around 30 years of age . In elde2+ type-T and type-L channels (characteristic of smooth muscle cells and involved in both cardiac and lymphatic tissues) [The lymphatic muscle is composed by a mixed type of myosin, resembling both smooth and striated muscle myosin. Recent studies have shown that it is actually a new class of muscle, which is quite distinct from cardiac and smooth muscles. Lymphatic muscles are also modulated by mechanical, neuromodulatory and vasoactive factors. The peculiarity of the lymphatic muscle lies in the fact that there are Catissues) . Up to 9tissues) . With re2O) [2O), which may lead to an insufficient or total absence of fluid transport [Functionally, aging-associated changes in lymphatic muscle cells can lead to periods of increased functional demands required to contract the lymphatic vessels . In summary, decrease in the amplitude of lymphatic contraction, along with the lowering of lymphatic tone in the segments of the thoracic duct, are seen in the aged thoracic lymphatic vessels, even at low pressure. Additionally, it has been observed that as the pressure increases, magnitude of the lymphatic contraction decreases to a greater extent in older lymphatic vessels. This negative inotropy is also accompanied by a decrease in the frequency of contraction, especially at high transmural pressure levels (5 cm H2O) . Similar2O) . It has ransport .Another consequent cause of the loss of muscle efficiency is a lower efficacy of the lymphatic valve closure causing a consequent reflux of lymph into the elderly lymphatic collecting vessels . It has Aging is associated with a reduction in the levels of proteins, which regulate muscle contraction . The conAdministration of 100 µM L-NAME into aged mesenteric lymphatics in rats showed that active pumping was severely decreased, mainly due to the associated decrease in contractile speed in aged lymphatics. These changes are believed to be related to the enlargement of the MLVs with the aging process. At the same time, pumping of aged MLVs can be restored to pumping levels of adult vessels by increasing the rate of contraction induced by NO elimination . NO moduInflammation can increase lymphatic flow and is also accompanied by increases the volume of interstitial fluid, which arises due to increased microvascular permeability. The increase in volume induces a stretch on the lymphatic vessel wall, increasing the frequency and/or the contractile force. On the other hand, decreases in lymphatic drainage occurs due to synthesis of certain neuromediators linked to inflammatory and immune responses. These include substance P, the peptide related to the calcitonin gene (CGRP), neuropeptide Y, prostaglandins and vasoactive intestinal polypeptide. It is believed that all of these substances affect the contractile function of the lymphatic vessels.Aging introduces significant changes in the modulation of contractility of the lymphatic wall related to the functional increase of histamine as an endothelium-derived relaxation factor in MLVs and the simultaneous elimination of NO . IncreasThe lymphatic system exhibits a rich innervation with many different neuropeptides and neurotransmitters. The presence of sympathetic and parasympathetic nerve systems on lymph vessels has been reported. These innervations play important roles in the maintenance—and regulation—of the hydrodynamic forces and humoral factors that are important regulators of lymphatic functions. Furthermore, the innervation of the lymphatic vessels is not the same throughout the body. In the mesenteric and femoral lymphatic vessels, there is a greater distribution of nerve fibers than in the cervical region, thus allowing better lymphatic flow from the lower regions of the body . In addiA change in the functions of these nerve fibers and neurotransmitters can negatively affect the physiological homeostasis of the organism. Moreover in this context, aging-related changes play a key role. Aging is associated with a reduction of all specific nerve fibers, thus influencing the lymphatic function and flow regulation, which can lead to the development of lymphatic pathologies and/or aggravate existing pathologies in the lymphatic system .The glycocalyx acts as a barrier between lymphatic fluid and the endothelium thus decreasing the permeability of the lymphatic collectors and prevents pathogens and immune cells from adhering to the endothelium . In agedLymphatic vessels can display a coordinated contraction that can propagate in the opposite direction to the flow . After tLymphatic vessels have been shown to play an important role in skin inflammation and in the skin response to ultraviolet B (UVB) ray exposure . ChronicApelin, a bioactive peptide, has been shown to be important in the maintenance of the lymphatic vascular system as it plays a key role in containing UVB-induced skin inflammation . OverexpReactive oxygen species (ROS) in moderate quantities act as molecular signals and play a critical role in maintenance of vascular cell functions . Damage Aging also produces alterations in the activity—and expression—of superoxide dismutase (SOD) in different tissues, including blood and lymphatic vessels . SOD actAging-induced changes in mast cell function influence the responses of MLVs to acute inflammation . The relHistamine is known to play a role in the functioning of immune cells, including multidirectional crosstalk between mast cells and macrophages and the chemoattraction of several immune cells . FurtherAlterations in the lymphatic system and function, as e.g., seen during aging, can accompany metabolic syndrome, obesity, cancer or infections lead to modulation of the immune response. This can occur via alterations in the transport of antigens, trafficking of antigen-presenting cells, regulation of T-cells differentiation and modulation of immunosuppressive responses. These changes can sometime evolve into a vicious cycle of events . It has A notable finding from isolated MLVs in 9-month old mice was that bacteria tended to accumulate near the valves of lymphatic vessels . These vLipopolysaccharide (LPS), an endotoxin, applied at the MLVs of cattle and rats leads to relaxation and inhibition of spontaneous phasic contractions of the MLVs . Other sIn the elderly, there is up to 70% reduction of circulating lymphocytes as compared to in young adults . The decThe main risk factor for many neurological disorders, such as Alzheimer’s disease, is aging . In addiRecent work has shown that glymphatics  is modulated by meningeal lymphatic function suggesting that there is a link between the lymphatic and glymphatic vascular systems. Aging also leads to a progressive deterioration of the cerebral vascular function, which in Alzheimer’s (AD) is aggravated by amyloid angiopathy. The malfunction of the meningeal lymphatic vessels and the flow alone, also caused by aging, is related to the reduced inflow of paravascular cerebrospinal fluid and reduced outflow of interstitial fluid. Therefore, this leads to a reduction of macromolecules solutes in the brain via the glymphatic pathway, manifesting cognitive impairment in amyloid pathology. Some data suggest that aging-induced alterations in immunity and meningeal lymphatic drainage could lead to cognitive decline . It remaA possible limitation could be that only “aging” instead of “ageing” was used to search for literature. From our previous experiences and publications, and to prevent confusion, we have always used one spelling. We do not believe this is a major limitation, as “aging” in American and Canadian English is the preferred spelling and moreover, because it is in major use.Although in recent decades, important advances have been made on the principles of physiological regulation of the transport of lymphatic fluids, we are still a long way from having a complete understanding. One of the factors that determines lymphatic function is determined by complex factors of lymphatic hydrodynamics, for example, passive and active lymphatic pumps, pressure and lengthening of the lymphatics, the consequent combination of extrinsic and intrinsic flows. Not to mention the important changes in the transport capacities and sensitivities of the flow of the lymphatic vessels largely influenced by perturbations. Finally, some normal and pathological conditions are capable of impairing the various lymphatic transport functions causing important biological and clinical problems. An example is the effect of aging on lymphatic transport. Therefore, it is essential to understand the systems that guide and regulate lymphatic transport functions, all to improve the knowledge of lymphatic dysfunction on the different pathological processes, with the aim of promoting the development of therapies to treat these processes more effectively and to counteract these dysfunctions.


The Covid-19 pandemic caused lockdown of economies, which in turn led to the worsening of the economic situation of many households. During the first wave of the Covid-19 pandemic governments undertook various measures to support economies and societies, including jobs protection along with financial support provision to people who suffered financial loss during the economic crisis. We analyse the economic situation of older Europeans, depending on their socio-economic status as well as country of residence characteristics, including economic and labour market changes during the first phase of the pandemic, the strictness of government policies but also the country development level using the Human Development Index. We use the results of the Survey of Health, Ageing, and Retirement in Europe (SHARE), including the SHARE Corona Telephone Survey, which was conducted during the first wave of the Covid-19 pandemic. Our results indicate that individual characteristics have a higher impact on individual economic stress, compared to country characteristics. However, country’s response to the consequences of the Covid-19 pandemic, but also the overall level of development influences the economic situation and ability to cope with the economic risks people aged 50 and over face. People in more developed countries have smaller difficulties in making ends meet, while the economic crisis and more stringent policies reduce chances to receive financial support and increase economic risks. In response to the Covid-19 pandemic outbreak, governments introduced lockdown measures that affected the labour market and economic situation of many Europeans. Many industries were forced to reduce employment due to the closures. Many households, especially those who had already faced difficulties with making ends meet, experienced a further increase of economic stress due to the loss of employment income, and an increased dependence on financial support provided by employers, from public sources or private transfers.The European countries were affected differently by the Covid-19 pandemic; they also introduced different policies that influenced their economic growth and labour market. These policies and their outcomes, in turn, affected the economic situation of European populations. Our main contribution is to provide evidence on the role of government intermediate policies, changes in the economic situation in the country as well as the overall country development on the economic stress of people aged 50 years or over in Europe. This gives a further insight on if and how people in the second half of their life course are protected against economic stress in the crisis situation caused by the Covid-19 pandemic.Our results are based on the responses from SHARE Corona Telephone Survey that was conducted during the first wave of pandemic, from the beginning of June 2020. The use of harmonised SHARE Corona Telephone Survey data as well as ability to use longitudinal information from earlier SHARE Wave 7, gives a unique opportunity to measure and interpret the observed differences in a cross-country dimension, including 25 European countries. The obtained data reveal that there are large differences between the countries with regard to different aspects of economic situation of older people. These differences concern the entire analysed population, also people who were working before the Covid-19 outbreak and faced the risk of job loss. Finally, it is also important to see, how people who are most vulnerable economically, particularly those who had problems before the pandemic, adapt to their worsening situation in order to meet their current consumption needs.In our analysis, we focus on the economic situation of the above-mentioned groups in the European countries and how it relates to individual and country characteristics. Our contribution to the literature is that we provide cross-country comparisons based on internationally harmonised data, but also that we analyse different symptoms of economic stress of the older Europeans during the first wave of the Covid-19 pandemic, which is not jointly discussed in the literature.Our hypothesis is that the overall country’s development prior to the crisis, short-term economic outcomes, the severity of the Covid-19 pandemic and undertaken direct policy measures, have a significant impact on the symptoms of economic stress, including the risk of job loss, economic situation of households of people in age group 50 or over, including the most vulnerable as well as their access to financial support.The article is structured as follows. First, we present the findings on the impact of crisis on the situation of people 50 + , including the Great Recession and first evidence from the Covid-19 crisis. We also present main statistical indicators showing differences in the economic and labour market outcomes in 2020 that we use as the independent variables in the analysis. Then, we present our analytical approach and findings related to the economic situation of the analysed population during the first wave of the pandemic. We discuss the role of individual and country characteristics in explaining the analysed aspects of the economic situation of people aged 50 or over. Finally, we present the discussion of the results and conclusions, including policy implications.While the evidence on the impact of the Covid-19 pandemic on the economic situation of the population aged 50 and over is still limited, there is a growing body of literature that indicates risks that are emerging for this group. Miller With respect to the labour market situation, Johnson  providesThere are several reasons why the impact of the Covid-19 crisis on the employment of older workers can be more acute. As pointed out by Ghilarducci and Farmand , older wOlder workers also face higher risk of job loss due to employers’ preferences. Evidence from previous research shows that employment reductions during a crisis affect workers who have early retirement packages, which increases intergenerational tensions . It is a composite measure of nine metrics including: school closures; workplace closures; cancellation of public events; restrictions on public gatherings; closures of public transport; stay-at-home requirements; public information campaigns; restrictions on internal movements; and international travel controls, mean number of confirmed deaths, and duration of stay-at-home requirements  in Croatia, Cyprus, and Italy and above 80 in France, Malta, Romania, Slovenia, Greece, Poland, Spain, Portugal, Belgium, and Lithuania. It fell between June and August and increased again in October  from 2020 , we analyse the strategies of coping with current consumption that may have a longer-term impact on their financial situation in the future:Finally, for respondents who were employed before the Covid-19 outbreak (4666 respondents) we additionally look at the occurrence of job loss, since the outbreak of the Covid-19 pandemic, due to unemployment, lay off, or business closure.In the analysis, we use the following individual socio-economic characteristics as explanatory variables: age group, sex, household size, educational attainment, labour market status as well as the economic situation of the household before the pandemic, measured by the ability of the household to make ends meet in 2017 . The description of the sample used in the analysis is presented in Table The sub-sample for postponed bills payment Table b compristhe overall level of development before the Covid-19 outbreak, measured by the Human Development Index (HDI) from 2020  between first and second quarter of 2020;Covid-19 policy responses measured by the Stringency Index   and increased difficulties to make ends meet for the entire population. In the case of the economically vulnerable group (that is people who face difficulties in making ends meet) the risk of postponing of the payment of bills and using savings to finance current expenditure varies between countries. Finally, the risk of job loss among those who were employed prior to the pandemic also depends on the country of residence characteristics Table .Table 4SFor illustration of the distribution of the analysed symptoms of economic stress, we also present them on maps Fig. .Fig. 1AnDuring the first wave of the pandemic, relatively few respondents in general reported receiving some kind of financial support (around 8%). In this case, there is one outlier–Slovenia, where almost 57% of respondents received financial support. In the rest of the countries such help was received by more than 10% of respondents in Italy, France, Cyprus, Belgium, and Greece, while in Denmark, Sweden, Luxembourg, and Finland (that is mainly Northern countries) only less than 5% of respondents declared receiving financial support Table , Fig. 1aMore than a third of the surveyed population declares that their ability to make ends meet worsened after the outbreak of the pandemic. Again, people living in the Southern Europe as well as most of the “new” EU countries, faced such difficulties more often, with more than 90% of people indicating worsening economic situation in Greece and more than 70% in Hungary, Romania, and Bulgaria. In Denmark, Czechia, Luxembourg, and Sweden the economic situation of the households of people 50 and over was better; less than 15% of respondents have reported problems with making ends meet Table , Fig. 1bAmong those who face economic difficulties, arrears in the payment of bills are also an indicator of economic difficulties, but it also signals the household strategies to deal with the financing of the current consumption. Around 12% of respondents that face economic difficulties indicated that they postponed the regular payment of bills. SHARE results again show large differences between countries, which are like the ones observed in case of job loss. The largest share of people postponing bills payment was noted in Greece, Italy, Cyprus, Finland, Belgium, and Latvia, while in Slovenia, Lithuania, Estonia, Slovakia, and Czechia Table , Fig. 1cFinally, when we focus on the group that was economically active before the outbreak of the pandemic, one in five workers aged 50 and over in the analysed countries lost their job after the outbreak of the pandemic. The incidence of job loss was lowest in Czechia, Poland, Latvia, and Denmark and the highest in France, Greece, Slovenia, Cyprus, and Italy. Overall, the risk of job loss was higher in the Southern European countries, but also many conservative ones . The risk of job loss was lower in Northern Europe, but also in many Central and Eastern European countries that joined the EU in 2004 and later Fig. e. ResultComparison of the economic difficulties experienced by older people since the outbreak of the Covid-19 pandemic by countries reveals some similarities and differences. Older people living in Southern European countries, both in the “old” and “new” European countries, face the largest difficulties in making ends meet during the pandemic. At the same time, despite a steep drop in the GDP, respondents in France and Denmark face less difficulties to make ends meet. Yet, the risk of job loss in France was the highest, but to some extent it was offset by the provision of financial support. In general, we also observe that people living in the “old” European Member States faced higher risk of job loss and used their savings more often, compared to the “new” countries. In those countries, where the incidence of job loss was higher, people also postponed payment of bills and more frequently received financial support. It is also worth to notice that despite a smaller exposure to economic risks, in some countries, people declared using their savings more frequently . Finally, while people in the “new” Member States seem to have a lower risk of job loss, they more frequently indicated increased difficulties in making ends meet, which shows that in general they are more exposed to the economic hardships caused by unexpected shocks. We also observe that the share of people receiving financial support was lower in the “new” countries, with notable exception of Slovenia, which can be also linked to a lower share of people that lost their jobs during the first months of the pandemic.tet)tet coefficients are the largest between job loss and receiving financial support (0.52), which is intuitive, as well as between job loss and postponing of the bills payment (0.48), which may indicate that in case of job loss people first tend to finance their current consumption needs at the expense of rising indebtedness. The rtet coefficient between postponing bills payment and dipping into savings (0.43) indicates that those that are most economically vulnerable tend to combine these two strategies to finance the current consumption. The rtet coefficients between discussed variables justify the modelling of all five symptoms of economic stress separately.The tetrachoric correlations , people in age group 55–59 received financial support more frequently . At the same time, those living in larger households also have a higher risk of such difficulties (by 4.4%). Those living in larger households (3 or more people) and struggling to make ends meet were also more likely to dip into their savings (by 6.1%).The experienced symptoms of economic stress also depend on the educational attainment. Compared to those with secondary education (ISCED 3–4), people with lower educational attainment have 7.6% more chances of facing difficulties in making ends meet, while those with higher education have 9.1% less chances of such situation.Compared to the retired people, those still employed were more likely to receive financial support (by 6.3%); they also had smaller difficulties in making ends meet (by 6.6%). Among those more vulnerable, employees were more likely to postpone the bills payment (by 8.3%). Those who were neither working nor retired also more frequently postponed the bills payment (by 3.8%).Finally, earlier exposure to economic difficulties understood as the difficulty in making ends meet observed in 2017 also increased chances to face economic risks during the Covid-19 pandemic. They have much bigger difficulties in making ends meet after the pandemic outbreak (by 37.0%), as well as higher chances to receive financial support (by 6.2%). Those more vulnerable earlier also more frequently postponed their bills payment (by 5.6%).Our results also reveal the interactions between age group and sex, which are shown in regression results in Table Results of the models also confirm that the country characteristics are related to the observed symptoms of economic stress. The drop in the GDP observed in the Q2 of 2020 led to the increased share of people receiving financial support , but also to a large extent reduced the risk of difficulties in making ends meet (by 28%). This means that countries with a higher level of social and economic growth are more resilient to economic stress and at the same time have a potential to provide financial support to a larger share of the population. At the same time, those most vulnerable were more likely to dip into savings or postpone bills payment in countries with a higher HDI. We can hypothesise that they treat their economic difficulties as a transitory situation and believe that with the return to the growth path their situation will improve again.We observe a direct impact of introduced policies captured by the Stringency Index. Higher government policy stringency is associated with lower chances of receiving financial support, while higher ones in facing difficulties to make ends meet. At the same time, those with economic difficulties less frequently decided to postpone their bills payments in countries that introduced more strict stringency policies.As the sub-population of people at risk of job loss differs from the entire analysed population, we discuss the results of the logistic regressions for this symptom separately. Results of the logistic regression for the job loss are also included in Tables The risk of job loss was less frequent among those in age group 70–74 (compared to those in age group 65–69); women in this age group were more likely to lose a job compared to men, which is shown in interaction effects. Job loss was also less frequent among people with higher educational attainment. People with this level of education had 9.9% less probability of losing a job, compared to those with secondary education. The job loss among those who were employed prior to the Covid-19 outbreak and already faced economic risk was higher by 6.4% compared to those that did not have such difficulties earlier.The immediate response of the economy, measured by the GDP decline, was also associated with a higher risk of job loss (by 1.3%). Overall change in employment rate (20–64 years) affected the risk of job loss among those 50 and over (by 4%). Interestingly, the share of those that lost a job was higher in countries with higher HDI. The short-term labour market adjustment was therefore combined with the higher ability of these countries to provide financial support and also higher resilience of the population to difficulties with making ends meet.To assess the explanatory power of individual and country characteristics, separate models with only individual and only country characteristics were estimated. The values of pseudo-R-squared for the models are presented at the end of Table The results of the SHARE Corona Telephone Survey reveal differences in the economic risks faced by individuals both with respect to their individual characteristics, but also countries which they live in. This indicates that immediate country policies, their impact on the economic growth and labour market, but also country development affect economic situation of the older population in Europe.Stringent lockdown policies, including workplace closures, led to increased risk of making ends meet, but also lower chances of receiving financial support. While in general the governments introduced policies to replace the lost income of people affected by workplace closures, it seems that in the case of more stringent policies, the financial support was less available.The income support was mainly addressed to the working population. Others (including pensioners) received their social insurance transfers, and therefore, their incomes did not suffer as much. Increased vulnerability among people aged 50 and over, who are not receiving pensions, triggered adjustments such as postponing the bills payments or dipping into savings. We might hypothesise that those that are still economically active treat the income loss as temporary, and they assume that they will regain their income, so they will be able to make up for the arrears against their future income. If such strategies are continued, the economic situation of the older population might worsen. Some of the decisions, such as using savings to finance current consumption, particularly for those who will soon reach retirement age, might also have long-term consequences on their future income after retirement, as they will not be able to rebuild their savings before retirement.The SHARE Corona Telephone Survey results also show that the economic vulnerability of people living in countries with lower Human Development Index is higher. This can relate to the relatively weaker welfare state provisions . At the same time, people living in these countries might not have sufficient savings to use for their consumption in the case of reduced household income.Contrary to our expectations, we see that the employed people in general assessed their economic situation slightly better than pensioners, but they more frequently resorted to strategies such as postponement of the payment of the bills as well as they received financial support to cover their wages, which could contribute to the relatively weaker influence on the ability to make end meet.However, it should be noted that these findings capture only the first reaction to the policy measures introduced at the outbreak of the Covid-19 pandemic and there is a need to monitor the long-term effects of the crisis on the labour market and employment, as longer periods of lockdown and workplace closures, as well as worsening public finance situation can further affect the economic risks faced by the older workers and pensioners.