Maternal and neonatal tetanus (MNT) is still the major public health problem in about 25 countries, mainly in Africa and Asia. However, the utilization of intervention strategies, like tetanus toxoid (TT) immunization remains low in these countries. In Ethiopia, only 49% of the pregnant mothers received TT2+ in 2016. This study was designed to evaluate perceptions and factors affecting the utilization of TT immunization among reproductive-age women in Dukem town, Eastern Ethiopia, 2016.We conducted a community-based cross-sectional study from May to October 2016. A simple random sampling method was employed to select samples of 422 women. Data were collected using a, pretested semi-structured and a face-to-face interviewer-administered questionnaire. We entered data in to Epi Info version 7 and analyzed them by SPSS version 20 software. Odds ratios and a 95% CI at 0 < 0.05 p-value were calculated to ascertain the significance of associations.N = 416). Mean age with standard deviation was 29.25± 5.11 years, and average family size was 4.19. Our study showed the utilization of TT immunization was 39.2% (N = 163). Of the participants, 33.9% (N = 141) had never been vaccinated. ANC follow up service , distance from health facilities , knowing vaccination date , having a TV set in the house , maternal education  were factors significantly associated with the utilization of TT immunization.Response rate was 98.6% (This study indicated the utilization of TT immunization was low. ANC service follow up, distance from health facilities, knowing vaccination date, having a TV in the house, mothers’ educational status, and place of delivery were significant predictors. Our study suggests that policymakers and other stakeholders should consider the need for increasing access to maternal education, like basic adult education, ANC follow up services, providing accessible health facilities, improving varieties of communication media, promoting female occupational status, and providing appropriate vaccination cards. Maternal and neonatal tetanus (MNT) is still the major public health problem, in about 25 countries, mainly in Africa and Asia –3. MaterDespite the initiatives of the World Health Organization (WHO) for tetanus elimination, it still continues to cause a significant maternal and neonatal mortality. Annually, it claims about 180,000 lives worldwide . In the Both maternal and neonatal tetanus are preventable health problems. Proper implementations of maternal immunization programs, like the TT immunization is one of the basic intervention strategies used to alleviate these situations. Tetanus toxoid immunizations are injections given during pregnancy for the prevention of NT. A pregnant woman should receive at least two doses of TT injections during each pregnancy to realize full tetanus protection. Five doses are considered to provide lifetime protection irrespective of the recommended intervals . StudiesIn many parts of low-income countries, however, despite the efforts made to improve maternal and neonatal protection through TT immunization programs, valid doses of TT (two and more) immunization coverage still remains low . For insSimilarly, the majority of the Sub-Saharan African countries could hardly reach the TT immunization target set to be covered. Because of this, they have not yet fully achieved the complete elimination of MNT. Accordingly, a study from Nigeria showed that only 40.8% of TT2 coverage was achieved while 44.4% of mothers did not receive any TT vaccination . EthiopiEvidences show that a range of determinant factors affect the utilization of TT immunization. For instance, women’s education and their wealth index can impose variations in immunization coverage , 19, 20.Surprisingly, we lack evidences demonstrating the number of factors influencing the utilization of TT immunization and mothers’ perceptions of the immunization in Dukem town, East Showa Zone, Ethiopia. Thus, this study intended to investigate the level and ranges of factors affecting the utilization of TT immunization among reproductive age women in Dukem town, Eastern Ethiopia. The finding would probably help us to generate supplementary evidences that strengthen and accelerate current efforts to eliminate MNT.This study was intended to investigate the status and factors determining the utilization of TT immunization among reproductive age women in Dukem town, Eastern Ethiopia.A cross-sectional study design was employed to assess the level and factors affecting the utilization of TT immunization. We collected data based on the number of reproductive -age women currently living in Dukem town from May to October 2016.Dukem town, which is located 35 km to the East of Addis Ababa, the capital of Ethiopia, was our study area. We purposively selected the town and its surroundings. In its four kebeles, the town had a projected total population of 114, 207 according to the 2007 population and housing census of the Central Statistical Agency (CSA) of Ethiopia . Child bThe source population was all child bearing age women who were living in Dukem town at the moment.All women in the reproductive age group who had lived in the study area for at least two years were included.We excluded all reproductive age women who had never given births in the past 2 years.We employed the simple random sampling method to select eligible participants. Participant mothers were proportionally selected based on the number of resident mothers in each of the 4 kebeles. Single population proportion was used to calculate the required sample size. A 50% assumption of prevalence and an absolute precision of 5% were considered. We also assumed of a 95% confidence level to obtain adequate power for analysis. After including an additional grant of 10% for no response, 422 women were included.♣ Child Bearing Age Women (CBAW) - Any women aged 15 to 49 years irrespective of fertility status.♣ Vaccinated by history: Mothers’ self-report of TT vaccination doses received without any documented evidences.♣ Vaccinated by card only: A documented evidence of TT doses on immunization cards only.♣ Vaccinated by card plus history: Both documented and mothers’ self-reported TT doses received.♣ Valid TT doses: Mothers who received at least 2 doses of TT (TT2) in the recommended intervalsWe collected data by using a semi-structured interviewer administered questionnaire. The questionnaire was adopted from the Ethiopian demographic and health survey and other related literature , 24–26. Questions like the accessibility of vaccination site, quality of services provided, behavior of health care providers, time for travel to health facilities, privacy issues during services, and husbands’ encouragement to visit HF were also incorporated to assess mothers’ perceptions toward the utilization of TT vaccinations. We also asked some questions concerning mother’s knowledge of the purpose of TT immunizations.We emphasized quality in the data collection tools. The questionnaire was first designed in English and translated into the local language ‘Afaan Oromoo’ and back to English by language experts to verify its consistency. We also recruited 7 data collectors and 5 supervisors  who had experience and skills in the task. They were trained and oriented for 2 days before the actual survey. The training focused on the purposes of the study, clarity of tools, techniques of interview, confidentiality of information, informed consent, and the roles & responsibilities of data collectors and supervisors. During the data collection process, the principal investigator supervised both groups. Moreover, we conducted a pretest study prior to the actual process to test the validity and consistency of the data collection instrument by using 10% of the sample in a neighboring town, Gelan. Based on the pretest analysis, we modified some misinterpretations, minimized the number of interview questions, and made corrections to some other objections.p-value > 0.05). Odds ratios (OR) with 95% confidence intervals (CI) were applied to ascertain the significance of association.The collected data were checked manually for clarity and completeness. We coded data, labeled, verified, categorized, and entered into EpiInfo version 7. We employed SPSS version 20 to analyze the data. Descriptive statistics, like frequencies, percentages, means and standard deviations were computed. Using a binary logistic regression analysis, we fitted each predictor variable in to a bivariate logistic regression model separately to explore associations with the dependent variable (TT immunization status). Significant predictors at p-value < 0.2 in a bivariate analysis were exported to the multivariable logistic regression model to control possible effects of confounders. Variables were dropped in to the multivariate logistic regression model with a backward variable selection method. We proved goodness of fit model by Hosmer and Lemeshow and the assumption was satisfied  fully responded to the interview questionnaire and included in the analysis. The mean age was 29.25 years and the standard deviation + 5.11. The majority, 414 (99.5%), of the respondents were in the age group of 19–43 years. Average family size was 4.19. Seventy-eight (18.8%) of the respondents never attended any formal school. The majority of the participants, 295 (70.9%) belonged to the Oromo ethnic group (Table Two hundred fifty one (60.3%) of the participants gave birth to 2–4 children in their life. The majority, 218 (52.4%), of the mothers did not have future intention of giving birth. Two hundred seventy-five (66.1%) mothers visited health institutions less than 4 times for ANC services in their last pregnancy. Of the participants, 243 (58.4%), reported that the evidence of TT immunization they utilized was history (Table N = 141) [95% CI: ] reported they were never vaccinated with any doses of TT drug and had no documentation (card). Out of the interviewed mothers, only 39.2% (N = 163) were vaccinated the valid (TT2+) doses, and one hundred twelve (26.9%)  were vaccinated by the TT1 vaccination dose  of the participants indicated that there were accessible vaccination sites closest to their dwellings. One hundred ninety-eight (47.6%) of the mothers ranked the services provided as “good”. More than half, 54.3% (N = 226) of the participant mothers pointed out waiting time for services at facilities was> 1 h  said the purpose was to prevent children alone ]. Participants who used TV in their house showed 1.8 times higher chance for TT immunization usage . The multivariable logistic regression model also showed that mothers utilizing ANC service follow ups were 2.56 times more likely to utilize TT immunization  than those who did not follow ANC services. Moreover, mothers who were far from health facilities by < 30 min were 2.27 times more likely to be immunized compared to those who were far by ≥ 30 min . The model also explained that respondents who knew TT vaccination dates had 1.98 times higher possible utilization of TT immunization  compared to who did not know vaccination dates  utilization was 39.12% [95% CI: ]. The study also revealed that only 41.1% of the participants used TT vaccination by card. In this study, a notable proportion (33.9%) of the participants were never vaccinated any dose of TT.Our finding indicated TT immunization utilization status was relatively lower compared to the national 49% reported by the Ethiopian Demographic and Health Survey (EDHS) of 2016  and a prOur finding was also lower than the TT vaccination status reported by studies in Pakistan  and BangOur finding indicated the level of maternal education, maternal occupational status, ANC service follow up, distance from health facilities, media exposure, knowing vaccination date, and places of delivery considerably predicted the utilization of TT immunization.The result of the multivariate logistic regression model explained that mothers’ educational status was significantly associated with the utilization of TT immunization. Hence, literate mothers were significantly more likely to be immunized than illiterate mothers. This result was consistent with other study reports , 30–32. The 2016 EDHS report  also fouIn this study, maternal ANC service follow up markedly increased the likely hood of the utilization of TT immunization. Women who regularly attended ANC service follow ups had a higher probability of TT vaccination utilization. This was supported by other findings , 34, 35.Our analysis detected distance from health facilities was an important predictor of TT immunization status. In concordance with other studies , 37, womThe odds of TT immunization status were more likely to increase among employed mothers compared to non-employed ones. A number of studies demonstrated that TT immunization utilization depended on mothers’ occupational status , 38, 39.Place of delivery was another determinant factor for the utilization of TT immunization. In this study, we found that mothers who gave birth at health facilities were highly likely to use TT immunization compared to those who gave birth at home. Other studies also verified this association , 35, 38.Similar to the other studies , our anaWomen’s media exposure, like having TV sets in houses significantly determined the status of TT immunization. In line with other studies , 29, 38,Although we found out reliable information that can add to existing literature, some limitations could be distinguished in this study. First, the data collected were based on events that happened within the last two years preceding the study. Therefore, the problem of recall bias and under reporting might be suspected. To decrease such limitations, we focused on the most recent births and utilized vaccination cards and other records of the health facilities. Second, since the data collection technique was a face-to-face interview, there might be a social desirability bias. However, to address this issue, we carefully focused on interview techniques when we trained data collectors and supervisors.This study indicated the utilization of valid TT dose immunization was low in the area. Maternal education, media exposure, ANC follow up services, knowing vaccination date, distance from health facilities, and place of delivery were significant predictors of the utilization of TT immunization. The study suggests that policy makers and other stakeholders should consider the need to ensure access to maternal education, like basic adult education. Moreover, it is advisable to promote ANC follow up services and to provide access to health facilities, varieties of communication media coverage, and appropriate vaccination cards accessible to remind about remaining TT doses.


The Winton Formation is increasingly recognised as an important source of information about the Cretaceous of Australia, and, more broadly, the palaeobiogeographic history of eastern Gondwana. With more precise dating and stratigraphic controls starting to provide temporal context to the geological and palaeontological understanding of this formation, it is timely to reassess the palaeoenvironment in which it was deposited. This new understanding helps to further differentiate the upper, most-studied portion of the formation (Cenomanian–Turonian) from the lower portions (Albian–Cenomanian), allowing a coherent picture of the ecosystem to emerge. Temperatures during the deposition of the Upper Cretaceous portion of the Winton Formation were warm, with high, seasonal rainfall, but not as extreme as the modern monsoon. The landscape was heterogeneous, a freshwater alluvial plain bestrode by low energy, meandering rivers, minor lakes and mires. Infrequent, scouring flood events were part of a multi-year cycle of drier and wetter years. The heavily vegetated flood plains supported abundant large herbivores. This was the final infilling of the great Eromanga Basin. The Winton Formation of central-western Queensland provides a detailed insight into the continental biota of eastern Gondwana during the middle part of the Cretaceous Period, with deposition spanning from the upper Albian to the upper Cenomanian–lower Turonian  portion of the Winton Formation, in which the bulk of the described fossil sites occur. A more refined model of the changing palaeoenvironmental conditions that existed during the time the Winton Formation was deposited will be of use not only for future research on the Winton Formation specifically but also for Cretaceous researchers more broadly. The refined models can also be used as a base from which future finds may be used to clarify, resolve or change our understanding of this increasingly important suite of ‘mid’ Cretaceous terrestrial localities, with the added advantage of considerably simplifying comparisons with other Cretaceous Gondwanan fossil assemblages.Multiple electronic searches were conducted from study conception to February 2018, with no language or period restrictions, in online databases including the ISI/Web of Science, Scopus and Google Scholar. Initial search terms included ‘Winton Formation’, as well as combinations of ‘Cretaceous’, ‘Queensland’, and ‘Australia’. The search included materials in peer-reviewed journal articles, geological reports, conference proceedings and academic books. Non-peer-reviewed sources such as unpublished theses, conference abstract volumes and popular articles were not included. We also searched the reference section of these publications, which aided discovery of older sources, particularly where the age and name of units has been reassigned since publication. Materials were included that were considered to inform the authors on the palaeoenvironment. Articles related to the Winton Formation, but on derived topics , were only included if they also contributed to the broader palaeoenvironmental reconstruction.The Winton Formation covers a large geographic area of western Queensland, northeast South Australia and northwest New South Wales  and has On the basis of detrital zircon chronology, stratigraphy, sedimentology, and architectural analysis, the Winton Formation is now informally divided into Upper and Lower Cretaceous parts . Fossil-It is clear from surface sediment localities in the greater Winton region that the Upper Cretaceous portion of the Winton Formation was derived from terrestrial and freshwater depositional environments . This isNeoceratodus forsteri Krefft, 1870, a living example from the family Neoceratodontidae to which the Cretaceous lungfish found in the Winton Formation belong, live in low-flow to still waters and require a low-flow regime in spring for egg laying on shallow-water plants . Compare this with the Upper Cretaceous portion of the Winton Formation, which is estimated to have received 630 ± 206 mm during the three wettest months and 268 ± 137 mm during the three driest months  similar to modern podocarps, including those that grow on low nutrient soils  de Laub., Prumnopitys taxifolia (Sol. ex D.Don) de Laub., and Podocarpus totara G.Benn. ex D.Don . By comparison, A potential cause of the low diversity is sampling intensity. Taphonomy also must be accounted for, which may bias preservation of parts of a palaeo-landscape over others, and can result in a low apparent diversity of the death assemblage. The Turonian Raritan Formation , for exaProtophyllocladoxylon owensii  portion of the Winton Formation ; Fig. 5 Wintonotitan wattsi is contra this pattern. In their study of sauropod environmental preferences As well as the climatic  and hydrological (see Hydrology) needs of the animals for which we have modern analogues, the environmental preferences of some of the groups that have no modern analogue  are also informative. Some nodosaurid ankylosaurs seem to have a broad range of ecological tolerances  and examThere does not seem to be a clear pattern of distribution of megaraptoran theropods, but that they are almost always found in association with other dinosaurs , from poQM L311 comprises the most well-preserved flora of the Upper Cretaceous portion of the Winton Formation, and although investigation of this flora has been pursued e.g., , undescrThe insect record is also very poor for the Winton Formation, likely due to the rapid destruction of specimens by weathering once exposed . The samUpper portions of the Winton Formation are the most likely to have been exposed to the extensive Cenozoic weathering that hampers understanding of the palaeoecology of this unit and surface exposures of the Winton Formation are very limited. Much of the detailed stratigraphic work to date, identifying Upper and Lower Cretaceous portions of the unit, has been conducted on data core logs or surface concretions. Many of the fossil-bearing localities, particularly those around Winton, do not have site-specific sedimentological and stratigraphic data associated with them, so broad scale stratigraphic correlations that include these sites are tentative and limited to a few key sites . This problem is exacerbated by extensive vertical faulting throughout the area. Continued investigation of the stratigraphy, sedimentology, and geomorphology of the region, along with the application of absolute dating methods to fossil-bearing localities within the Winton exposures, are key steps to guide future prospecting and to inform interpretation of the palaeoecology and palaeoenvironement of this important Australian Cretaceous formation.2 as high as 1000 ppm (When all of the current evidence—sedimentological, taphonomic, floristic, vertebrate, and invertebrate—is combined, a coherent yet complex image of the climate and environment of the Upper Cretaceous portion of the Winton Formation is apparent. The temperatures were warm, mesothermal, and probably similar to those of the sub-tropics today despite the palaeolatitude of ∼50° S. This was in keeping with globally high temperatures that supported forest in the southern polar regions during the Cretaceous  at atmos1000 ppm . Precipi1000 ppm . This reThe landscape that is preserved at the many sites where the Upper Cretaceous portion of the Winton Formation is exposed may be broadly continuous both temporally and stratigraphically, but in terms of their floral communities, they are heterogeneous. In some parts of the palaeolandscape, low diversity appears to be linked to a disturbance regime that may have comprised primarily flooding events. These flooding events were likely infrequent and possibly scouring in places when they occurred, leading to the establishment of long-lived podocarp stands that excluded other plants early in their establishment on the barren sediment. Other parts of the landscape were either less affected by flood events, or were affected at different times, such that their floral community was comprised of a different combination and abundance. These communities appear to have been more diverse, although not as diverse as would be expected of a forest climax community, and not as high or as diverse as at the other Late Cretaceous mid-latitude fossil floras to which it has been compared.Comparing the range of communities recorded within the Upper Cretaceous portion of the Winton Formation to the other well-studied Australian Cretaceous plant localities, the older Albian Otway Basin flora, the Winton communities and their sediments are not as distinct as the six biofacies recorded in the Otway Basin . WhereasThe disturbance events suggested to have shaped this palaeoenvironment may be linked to the influence of a ‘Pacific Decadal Oscillation’-like cycle affecting Queensland during the Late Cretaceous. This may have influenced both precipitation and temperature covariantly, as in modern climate systems. It is likely that precipitation varied between years, possibly on an oscillation of relative drought years and years of higher precipitation similar to the Pacific Decadal Oscillation. Thus, during the earliest part of the Late Cretaceous, when the rocks that now form the Upper Cretaceous portion of the Winton Formation were deposited, this part of eastern Gondwana was a dynamic and heterogeneous environment.


While a novel concept for many, pre-registration is an outlet growing in prevalence that allows researchers to systematically work through future studies from start to finish by laying out research questions, relevant hypotheses, and study design. “Growing up” as a researcher with Open Science, I never felt that pre-registration limited me, my ability to think and ask questions, or any opportunities to explore new ideas with old data. Instead, it strengthened me as a researcher by giving me a methodical perspective to take when considering why I needed to ask a question and how I planned to answer it. In this talk, I will define what pre-registration is and discuss my experiences with it as an early career researcher. Furthermore, I will present different concerns and conceptions people have about it and expound upon the inherent benefits it provides to both critical thinking and study design.


Rat renal transplantation is an essential experimental model for studies of transplantation immunobiology. Harvesting both kidneys from one donor rat for transplantation is widely used to reduce the number of experimental animals. Using the conventional method, both kidneys of the donor rat are harvested simultaneously, which leads to the prolonged warm ischemic times during transplantation of the second donor kidney. Prolonged warm ischemia time is the main risk factor for delayed graft function.Two different approaches are compared. Method 1, conventional method: both kidneys of the donor rat are harvested simultaneously and then transplanted into two recipients. During transplantation, the first and second donor kidneys were regarded as Group 1 and 2, respectively. Method 2, step-by-step method: after left nephrectomy, the donor rat survives, and we perform left renal transplantation (Group 3). Then, the right kidney of the surviving donor rat is incised and transplanted into the left side of the second recipient (Group 4).p < 0.05) but differed non-significantly between groups 3 and 4 (p > 0.05). Serum creatinine levels, blood urea nitrogen and 24-h urine protein level obviously increased after kidney transplantation in group 2 compared with other groups (p < 0.05).The success rates were 86.7, 93.3, 93.3 and 86.7% in groups 1, 2, 3 and 4, respectively. The warm ischemia times increased significantly in group 2 compared with the other 3 groups (We developed an optimized method for reducing warm ischemia time, thereby minimizing delayed graft function. In recent years, bilateral donor nephrectomy in rat kidney transplantation has been commonly used because it can save time and laboratory animals. However, harvesting two kidneys from one donor simultaneously and then transplanting the kidneys cannot guarantee identical warm ischemia times for the two donor kidneys in the case of a single-person operation, and prolonged warm ischemia time is the main risk factor for delayed graft function . Wagner This study were performed at the Laboratory Animal Center of Guangzhou University of Chinese Medicine  in accordance with the Care and Use of Laboratory Animals . Adult male inbred Brown Norway (BN) and Lewis rats (weight 200–250 g) were used as donors and recipients, respectively. The rats were supplied by Vital River Laboratory Animal Technology Co. Ltd. . The animals were fasted 10 h before operation but had free access to water.n = 15) and transplanted into 30 Lewis rats. Both kidneys of the donor rat are harvested simultaneously and then transplanted into two recipients. During transplantation, the first and second donor kidneys were regarded as Groups 1 and 2, respectively. Method 2, step-by-step method: after left nephrectomy, the donor rat (n = 15) survives, and we performed left renal transplantation . Then, the right kidney of the surviving donor rat is incised and transplanted to the left side of the second recipient . In all groups, renal artery (RA) revascularization was performed in an end-in-end fashion via modified sleeve anastomosis [Method 1, conventional method: Both kidneys were harvested from BN rats  via intraperitoneal injections. A long midline abdominal incision was generated, and two retractors were placed to expose the viscera. The bowel was retraced to the right. We can observe the left kidney, abdominal aorta (AO), and inferior vena cava (IVC), and then separated the branches of the vessels, such as the left adrenal artery and vein and testicular vein, ligated, and cut. The left RA and RV were separated at the bifurcation of the AO and IVC, respectively. At this time, the AO and IVC were completely divided. We mobilized the adipose tissue by sharp dissection at about the upper part of the left ureter, preserving adherent ureteral connective tissue and fat to ensure blood supply. The ureter was cut approximately 10 mm to the renal hilum. The distal end of the ureter was ligated. At this moment, the whole left kidney, including the RA, RV, and ureter, was dissected free. Two vascular clips were used to block the AO and IVC together at the lower and upper levels of the left renal vessels. The left RV root proximal to the IVC was tied and transected Fig. . After tThe steps for preparation of the recipient rat are basically the same as the donor. After separation of the left RA and RV, the RA and RV were clamped by two vessel clips as close to the AO and IVC as possible. The surrounding tissues of the ureter were mobilized properly, and then the ureter was cut at about the lower pole of the renal. At last, the left kidney under the capsule was excised. RA revascularization was performed in an end-in-end fashion via modified sleeve anastomosis , 4. RV aAfter the RA and RV anastomoses were complete, the graft was reperfused. As urine passed from the ureterostoma, we began to stitch the ureter. A 10–0 suture line was used to anastomose the ureter end-to-end with 6 interrupted sutures. Finally, in order to hold the graft, two stitches were placed in the upper and lower poles of the donor kidney, respectively. The right renal of the recipient was removed and the first step of left donor orthotopic transplantation was complete.The donor rat subject to left nephrectomy was prepared as described in the first step. After entering abdominal cavity, the bowel was retraced to the left and the right kidney, and AO and IVC were exposed. The AO, IVC, right RA, RV and ureter were separated as described for the left side. After separating the right renal, four vascular clamps were used to block the AO and IVC at both upper and lower ends. The IVC were cut approximately 2 mm below the right RV to anastomose with the recipient RV. Similar to left graft perfusion, the right renal was perfused in situ, and a clear perfusate was obtained from the RV with a segment of the IVC. Thereafter, the right RA was transected close to the AO. The IVC above the right RV was ligated using a 5–0 suture line and cut at the distal end Fig. . The rigThe right donor kidney was rotated 180 degrees around its longitudinal axis. The vessel and ureter anastomosis processes were the same as described for the left renal orthotopic transplantation.Postoperatively, the rats were placed under a heating mat and recovered from the anaesthesia. Blood samples were obtained from the caudal vein. Urine samples were obtained at 1, 5 and 7 days postoperatively for assessment of serum creatinine levels, blood urea nitrogen and 24-h urine protein levels. After the experiment, all animals were euthanized by cervical dislocation.2 test was used to compare qualitative data between the experimental and control groups. p < 0.05 was considered statistically significant.Statistical analysis was performed in SPSS 20.0 . Data are reported as the mean values ± standard deviations. Student’s t-test was used to compare the quantitative parametric data, and the χp < 0.05). In Table p < 0.05) but exhibited no significant difference between groups 3 and 4 (p > 0.05). The warm ischemia times increased significantly in group 2 compared with the other 3 groups (p < 0.05) but exhibited no significant difference between groups 3 and 4 (p > 0.05). The success rates were 86.7, 93.3, 93.3 and 86.7% in groups 1, 2, 3 and 4, respectively. No differences were noted among the four groups (p > 0.05). The main complications after operations were venous thrombosis and ureteral leakage in all animals; however, complications rarely occurred in all groups.In Table In recent years, using both kidneys from one donor rat for renal transplantation has become a commonly applied method to save animals. Yin et al.  employedp > 0.05). Moreover, the extension of the warm ischemia time causes more rapid disease progression in acute renal allograft rejection. In addition, serum creatinine, blood urea nitrogen and 24-h urine protein levels increased significantly in group 2 compared with other groups (p < 0.05). This finding indicates that the warm ischemia time is closely related to the recovery of renal function after transplantation. This finding is explained as follows: the kidney is one of the organs with the largest blood supply in the entire body and is sensitive to ischemia. If the warm ischemia time is longer, the reperfusion injury is more serious. Our study results are consistent with previous results [Rat kidney transplantation requires advanced microsurgery techniques and skills due to vessel and ureter anastomosis, and the techniques are often performed by one person. Our study explored a new technique to harvest two kidneys from one donor in a step-by-step fashion for a single-person operation to ensure that the warm ischemia time of the left donor graft is almost equal to that of the right graft and protect renal function to the maximal extent. The donor rat survives after left nephrectomy. After the left kidney transplantation was completed, we incise the right graft of the surviving rat. Our new step-by-step technique was first described at home and abroad. The results reveal wide variation in the warm ischemia time between groups 1 and 2 when the conventional method is employed. In the step-by-step method, the warm ischemia time of the left and right donor graft did not differ significantly between groups 3 and 4 (Under individual circumstances, our step-by-step methods ensure that the warm ischemia time of the left donor graft is approximately equal to the right graft, thus minimizing delayed graft function and preserving animals. Our technique exhibits a high success rate and low complication rate.


Iatrogenic cerebrospinal fluid (CSF) rhinorrhea in a bilateral frontal decompressive craniectomy patient triggered by strenuous sport is rare. To the best of our knowledge, no similar case has yet been reported.Herein, we report a case of CSF rhinorrhea in a 37-year-old man. He had previously suffered a traumatic brain injury in a traffic accident, and a subsequent bilateral frontal decompressive craniectomy operation was performed. Based on the frontal skull defect peculiarity, strenuous exercise may have caused drastic CSF pressure waves to tear the dura mater of the anterior skull base, resulting in CSF rhinorrhea.The thin-slice computerized tomography (CT) images revealed a frontal skull defect and the open frontal sinus. In addition, in the opened frontal sinus, low-density liquid-filled areas were visible.During surgery, the torn dura was carefully repaired, and the frontal sinus was filled with temporal muscle, fascia, and fibrin glue. A simultaneous cranioplasty was performed.The patient was followed-up postoperatively for 12 months to date without rhinorrhea recurrence. Recovery was uneventful.Patients with skull defects should avoid strenuous sports, and cranioplasty should be performed as early as possible in order to decrease the likelihood of a dural tear and prevent the occurrence of CSF leakage. After cranioplasty, the skull should be restored to a closed state to reduce the damaging effects of CSF waves during movement. It is important to maintain normal intracranial pressure to reduce the recurrence rate of CSF rhinorrhea. Among nontraumatic CSF fistulas, spontaneous fistulas are the most commonly reported at 41% of all fistulas, other types of CSF fistulas are related to iatrogenic trauma after surgery (30%), tumors (5%), or congenital malformation of the skull base (3%). We report the case of a patient who developed bacterial meningitis caused by the leakage of CSF into the frontal sinus. The patient had a bilateral iatrogenic frontal skull defect as the basis of the pathological anatomy, and strenuous exercise was the initiating factor that led to a tear in the dura.Ommaya et al reported that all cases of CSF rhinorrhea, even spontaneous CSF rhinorrhea, had an underlying cause.2A 37-year-old man who was transferred to our hospital presented with a one-week history of CSF rhinorrhea, a four-day history of fever and a one-day history of headache. Six months prior to admission, he had suffered a traumatic brain injury in a traffic accident. Brain computed tomography (CT) revealed bilateral frontal lobe contusions and multiple fractures of the bilateral frontal bones. Under general anesthesia, an emergency contusion cleaning procedure and bilateral frontal decompressive craniectomy were performed.Postoperatively, the patient had a favorable recovery. Head CT showed bilateral frontal bone defects and brain necrosis, and a significantly sunken scalp was noted Fig. A and B. 9/L. A CSF examination revealed pleocytosis , increased total protein (998 mg/L) and a Pandy test result of ++. Streptococcal pneumonia was detected in a bacterial culture of the CSF sample.At admission, he was drowsy upon physical examination. His temperature was 37.6°C. Neck stiffness and meningeal irritation were observed. A laboratory examination revealed an elevated C-reactive protein level of 126 mg/L and a peripheral leukocytosis of 20.1 × 10The patient was therefore treated with high-dose ceftriaxone. Two weeks later, the patient's symptoms returned to normal. Three CSF examinations showed normal results. Two months after the onset of CSF rhinorrhea, the patient still had persistent unilateral clear nasal drainage that worsened when standing or sitting. Axial and sagittal CT revealed an open frontal sinus. In addition, low-density areas indicating liquid were visible in the opened frontal sinus Fig. .A cranioplasty and dural defect repair were planned. A bicoronal scalp incision was made along the primary surgical incision, and a scalp flap was carefully elevated. The frontal sinus was filled with CSF, and the dural tear was identified. After opening the dura, a hyperplastic gliosis scar was visible around the area of the original contusion. We excised the glial scar. Wide apposition of the whole region of the anterior skull base including the ethmoidal roof, orbital roof, and lateral walls extending to the orbital apex was performed.After careful examination of the anterior skull base, no other leaks were found. The dural defect was repaired with artificial dural mater. The frontal sinus was filled with temporal muscle, temporalis fascia, and fibrin glue. Simultaneous cranioplasty was performed using a customized titanium mesh Fig. . The ope3As a case report with written consent, our institution does not require formal ethical approval. Written informed consent was obtained from the patient and his wife for publication of this case report.4 Ommaya et al postulated that there are areas of focal atrophy in the cribriform plate and sella that become filled with a pouch of CSF. Normal CSF pressure waves cause these pouches to enlarge, erode and eventually rupture out of the bone.CSF rhinorrhea after craniofacial trauma mainly occurs in the nasal cavity, and CSF can enter the nasal cavity through defects in both the dura mater and bone in relation to the frontal ethmoid or sphenoid sinuses or along the cribriform plate for multiple reasons.In our case, the patient had an iatrogenic frontal defect, and the roof of the frontal sinus was open and filled with a large pouch of CSF Fig. . This waThe CSF pressure wave was much larger than that caused by normal brain pulsation, and it ultimately lacerated the dura mater at the skull base. Finally, the dura mater overlying the open frontal sinus was torn, and CSF leaked into the frontal sinus and nasal cavities. Therefore, in patients who have skull defects, strenuous sports should be avoided, and cranioplasty should be performed as early as possible to decrease the probability of dural tears and prevent the occurrence of CSF leakage. The mechanism is shown in Figure ,5 In particular, the development of CT thin-section and 3D reconstruction technology enables the identification of smaller cranial defects. In our case, the CT scan allowed clear visualization of the location of the CSF rhinorrhea. The patient was misdiagnosed with a cold, upper respiratory infection or rhinitis due to his major complaints of intermittent serous nasal discharge, fever and headache.,7The use of CT for evaluating skull base defects has become a mainstay in the work-up of CSF fistulas due to its ability to resolve osseous structures. The mortality rate of meningitis caused by CSF rhinorrhea is 14%. The organisms that lead to ascending meningitis or meningoencephalitis typically include flora from the upper respiratory tract. Streptococcus pneumoniae is the most common, and this organism has been implicated in recurrent bacterial meningitis in this patient population.Communication with the intracranial space increases the risk of pneumocephalus, meningitis and brain abscessation, which are potentially lethal. Patients with persistent CSF rhinorrhea require a surgical approach. Endoscopic endonasal fistula repair is safe and has a high success rate. Intracranial access (craniotomy) is utilized where there are skull fractures requiring reconstruction, extensive skull base fractures, intracranial hemorrhages and contusions requiring treatment or sizeable deficits of the dura, for which the probability of relapse is high.,15Because of the attendant risk of meningitis, brain abscessation, and pneumocephalus, all patients with persistent CSF rhinorrhea should undergo surgery if conservative treatment has failed.Iatrogenic CSF leakage always occurs following surgery of the skull base; however, in our patient, CSF rhinorrhea was fundamentally caused by the opening of the frontal sinus during forehead surgery and was triggered by strenuous sport, which is different from other skull defects leading to iatrogenic CSF rhinorrhea. This result suggests to us that sports should be avoided for this population of patients. This case should also remind neurosurgeons that the frontal sinus should be adequately repaired and filled during bilateral frontal decompressive craniectomy.Given the characteristics of the large frontal bone defect in our patient, the craniotomy was used to resolve the CSF rhinorrhea, and simultaneous cranioplasty was performed. During the rhinorrhea repair surgery and because of the defects in the skull and the clearance of the frontal lobe, we could better expose the anterior cranial fossa including the frontal sinus and sieve plate. This allowed direct visualization of the defect for a satisfactory closure.,8,17 During surgery, we used temporalis muscle and fascia to avoid trauma caused by a separate incision.The source of CSF leakage is often repaired with autologous transplantation material, such as cartilage, inner thigh muscle or bone, nasal septum mucosa, turbinate bone, fascia, abdominal fat, ear cartilage, and other autologous tissues.Recurrence of CSF rhinorrhea is not uncommon. In our case, over a follow-up period of 12 months, there was no recurrence of CSF rhinorrhea. This lack of recurrence may be related to the adequate filling and repair of the opened frontal sinus. We also assume that the cranioplasty that closed the cranial cavity may play a role. This maintained the patient's intracranial pressure, reducing the damaging effects of the CSF waves generated during movement.5To the best of our knowledge, no similar case has been reported before. Iatrogenic CSF rhinorrhea should be treated with surgery. Careful and thorough determination of the leakage location and identification of any defects are the keys to a successful surgery. For cranial defect, especially in the frontal region, strenuous exercise may cause strong CSF pressure waves to tear the dura mater and thus should be avoided. Cranioplasty should be performed as early as possible to decrease the probability of a dural tear and to prevent CSF leakage. After cranioplasty, it is important to maintain normal intracranial pressure to reduce the recurrence rate of CSF rhinorrhea.Development of the idea and performance of the surgical operation: Guangming Wang.Development of the idea and editing of the final draft: Jinlu Yu.Data collection and drafting of the original article: Lichao Sun.Drafting of the original article: Wenchen Li.Data curation: Lichao Sun, Wenchen Li.Project administration: Guangming Wang, Jinlu Yu.Writing – original draft: Lichao Sun, Wenchen Li.Writing – review and editing: Jinlu Yu.


Recurrent cerebrospinal fluid (CSF) leak carries significant morbidity and mortality. A 25-year-old Caucasian male developed a symptomatic pseudomeningocele eight days after surgical resection of a cervical schwannoma. With no improvement after lumbar drain placement, the dural sleeve defect over the left C3 nerve root was repaired with suturable DuraGen® . Muscle and fat graft were then laid over the repair site and covered by Tisseel® . Recombinant human bone morphogenetic protein-2 (rhBMP-2) was applied via extra-small sponge laid over the graft. At 15-month clinical and 16-month radiographic follow-up, the patient had complete resolution of symptoms without any evidence of infection, ectopic bone formation, excessive inflammation, neoplasm, or recurrent CSF leak. This case demonstrates the successful use of BMP in the treatment of recurrent symptomatic cervical pseudomeningocele after tumor resection. We believe that the pro-inflammatory effects of rhBMP-2 lead to early scarring of dural defect and resolution of CSF leak. Cerebrospinal fluid (CSF) leak after spinal surgery may lead to significant morbidity including CSF fistulas, meningitis, brain abscess, intracranial hemorrhage, and neurologic deficits ,2. The iWe present a case of recurrent CSF leak and a symptomatic pseudomeningocele formation after resection of a cervical schwannoma in a 25-year-old male who later underwent surgical repair with the use of bone morphogenetic protein (BMP). BMPs have been used successfully to treat recurrent CSF leak after transsphenoidal surgery . In our A 25-year-old male without significant past medical history presented to the neurosurgery clinic with worsening left shoulder and neck pain. Magnetic resonance imaging (MRI) revealed a 2.6 x 1.1 cm intradural extramedullary mass within the left C2-3 neural foramen consistent with schwannoma. The patient underwent left C2-3 hemilaminectomy with complete resection of the tumor. The 2 x 1 cm dural defect was repaired in a water-tight fashion with suturable collagen matrix  and covered by fibrin sealant . There were no complications, and he was sent home four days later. On postoperative day eight, the patient returned to the emergency room with symptoms of nausea, vomiting, headache, and photophobia that worsened when he was upright. MRI showed a large pseudomeningocele at the surgical site  was inserted, and the wound closed in a standard anatomical layer fashion without any complications.The lumbar drain remained in place and was open to drain for five days. The patient was discharged home on postoperative day six. He was followed clinically for 15 months after surgery. The MRI at 16 months post-surgery showed no evidence of recurrent tumor, CSF leak, infection, extraneous bony growth, excessive inflammation, or neoplasm Figure .Spinal intradural tumor resection surgeries carry 5-18% chance of CSF-related complications, including the development of CSF fistula, pseudomeningocele, and meningitis. The complications and their respective treatments of prolonged bed rest, re-operation, and extended use of antibiotics carry significant morbidity and mortality . One of Here we present a case of successful symptomatic CSF leak and pseudomeningocele repair with the use of BMP in a patient who underwent cervical schwannoma resection. BMPs belong to a TGF-β superfamily of signaling molecules discovered in 1965 . Their iIn our study, none of the reported complications associated with BMP use were observed. Although BMPs are scrupulously investigated signaling molecules, more research is required to fully understand their function and use in surgery, wound healing, and tissue repair. Future studies should focus on further demonstrating the safety of BMP in the treatment of CSF leak through stronger study designs, such as a randomized controlled trial.


HIV reservoir, yet little is known about drug exposure in the brain. Our primary objective was to quantify exposure of three common antiretrovirals in brain tissue and compare exposures to plasma and cerebrospinal fluid (CSF). We also sought to identify pockets of brain most vulnerable to inadequate drug exposures and examine the role of meningitis in drug penetration into the CNS. Tenofovir, lamivudine, and efavirenz concentrations were measured using liquid chromatography and tandem mass spectrometry in plasma and CSF from 14 individuals with HIV, 7 with cryptococcal meningitis. In four individuals (three with meningitis) drug concentrations were also measured in 13 distinct brain tissue regions. In subjects with meningitis, geometric mean ratio  of tenofovir CSF to plasma was 66% (7–598%) and 14% (6–31%) in subjects without meningitis. Lamivudine CSF penetration was 100% (25–409%) in subjects with meningitis and 30% (24–37%) in subjects without meningitis. Tenofovir brain tissue concentrations were 36% (14–124%) of plasma and 49% (1–572%) of CSF. Lamivudine brain concentrations were 37% (23–64%) of plasma and 27% (1–104%) of CSF. Efavirenz brain tissue concentrations were 128% (108–179%) of plasma. Tissues collected postmortem provide a unique opportunity to assess drug distribution in tissues difficult to sample in living subjects. CSF is a poor surrogate for drug exposure throughout the CNS. Antiretrovirals differentially penetrate into the CNS and penetration may be enhanced by meningitis.The central nervous system (CNS) is a known  Despite HIV therapy, persistent viral replication occurs in certain anatomic sites, and systemic viral rebound will occur within weeks of treatment interruption.Inflammation at the blood–CSF barrier or the CSF–brain barrier could lead to altered drug distribution via opening of tight junctions, altered pH changing drug ionization, and downregulation of efflux transporters.Postmortem drug quantification has been used in forensics for decades to assist with determining cause of death, in particular when overdose was suspected. Here, we describe the use of brain specimens collected during autopsy to quantify drug exposure of three commonly used antiretrovirals: tenofovir, lamivudine, and efavirenz. We sought to compare exposures to plasma and CSF and to identify pockets of brain most vulnerable to inadequate drug exposures. We also examined the effects of meningitis on drug penetration into CSF.Postmortems were performed in Kampala, Uganda, from 2017–2018 on a cohort of Ugandans with known HIV. Written informed consent was obtained from next of kin under a protocol approved by the Research Ethics Committee of Mulago National Referral Hospital. Time of last dose and time of death were collected by hospital records or caregiver interview. During autopsy, tissue sections (1 g) from discrete brain regions were briefly rinsed with normal saline to remove blood and immediately snap frozen in liquid nitrogen. Whole blood was collected from the femoral vein into EDTA tubes and plasma separated. CSF was collected via cisternal puncture. Samples were stored at −80°C.g. The top supernatant layer was removed for sample extraction. Internal standards  were added to the samples. Sample proteins were precipitated with cold acetonitrile. Detection and quantification of tenofovir and lamivudine was performed in all matrices using an Acquity ultrahigh‐performance liquid chromatograph  coupled with a Quattro Ultima triple state quadrapole mass spectrometer . Efavirenz assay was performed using a high‐performance chromatograph  coupled with a TSQ Quantum triple stage quadrupole mass spectrometer . Calibrator standards and quality control samples were prepared in the matrix and frozen in advance to match the sample tested; bovine brain homogenate was used for brain tissue, a solution of salt and proteins for CSF, and lithium heparin for plasma. Following a 3‐day validation, tenofovir accuracy was 94%, 104%, and 106% in plasma, CSF, and brain, respectively, whereas lamivudine was 95%, 94%, and 95%. Precision was 6.6%, 5.5%, and 5.8% for tenofovir and 2.5%, 7.1%, and 2.8% for lamivudine in plasma, CSF, and brain. Efavirenz accuracy in plasma was 84% with 3% variability; in brain tissue, accuracy was 93% with 4.5% variability. Efavirenz in CSF was not quantified due to low solubility of efavirenz in CSF solution and expected low concentrations of efavirenz in CSF.Prior to drug quantification, ~ 0.3 g of brain tissue was combined with twice the volume (0.6 mL) of 5% bovine serum albumin. Sample was homogenized with Tissue Tearor (BIOSPEC Products) and centrifuged for 15 minutes at 15,000https://www.fairview.org/services/laboratory-services/fairview-diagnostic-laboratories).Plasma creatinine and CSF/plasma albumin ratio (AR)were also quantified by Fairview Diagnostics Laboratory, a CAP‐accredited and CLIA‐accredited clinical laboratory in St. Paul, Minnesota .Data are reported as median (25th and 75th percentile) or geometric mean ratio  assuming a tissue density of 1 g/mL. Mean brain tissue concentrations were calculated for each individual by averaging all brain tissues (except CSF). Spearman rank correlation test was used to test for correlations between variables. Differences between compartments were tested using logarithmic paired Tabler = 0.77, P = 0.005). Lamivudine plasma concentrations in 14 individuals were 1,315  ng/mL. Lamivudine CSF concentrations were 566  ng/mL and highly correlated with plasma . Efavirenz plasma concentrations in 11 individuals were 3,342  ng/mL. The time between death and tissue collection was not correlated with drug concentration in any matrix . Plasma creatinine was significantly correlated with tenofovir and lamivudine concentrations but not efavirenz concentrations . AR was not correlated with drug exposure in CSF  or with CSF to plasma ratios . In addition, there was no relationship between active meningitis and AR (P = 0.9).Seven (50%) individuals had confirmed cryptococcal meningitis by CSF cryptococcal antigen. Plasma and CSF albumin were measured to estimate blood–brain barrier function. CSF to plasma AR was not correlated with postmortem interval . Overall, lamivudine CSF to plasma GMR was 0.54 (0.27–1.11). CSF lamivudine penetration was higher among individuals with cryptococcal meningitis, with GMR 1.00 (0.25–4.09) vs. 0.30  was 0.29 0.11–0.77). CSF penetration was higher among individuals with cryptococcal meningitis, GMR 0.66 (0.07–5.98) vs. 0.14  was greater than intraindividual variability across brain regions (49%). Mean brain tissue to plasma GMR was 0.36 , the National Institute of Neurologic Diseases and Stroke (R01NS086312 and R21NS108344), the National Institute of Allergy and Infectious Diseases (K08AI134262), and the United Kingdom Medical Research Council/Welcome Trust/Department for International Development (MRC MR/M0074131/1).The authors declared no competing interests for this work.M.R.N., K.A.P., J.R., D.A.W., D.B.M., D.R.B., and R.L. wrote the manuscript. M.R.N., K.A.P., J.T., J.R., D.B.M., D.R.B., and R.L. designed the research. M.R.N., K.A.P., J.T., O.C.N., D.A.W., and R.L. performed the research. M.R.N., K.A.P., D.B.M., D.R.B., and R.L. analyzed the data.WHAT IS THE CURRENT KNOWLEDGE ON THE TOPIC?☑ Antiretrovirals have variable penetration into cerebrospinal fluid (CSF). However, methods to assess brain tissue exposure are limited. Inflammation is known to alter the blood–brain barrier but the effects of meningitis on antiretroviral brain penetration are unknown.WHAT QUESTION DID THIS STUDY ADDRESS?☑ We sought to compare antiretroviral exposure among plasma, CSF, and brain tissue to identify pockets of brain most vulnerable to inadequate drug exposures. We also examined the effect of meningitis on drug penetration into CSF.WHAT DOES THIS STUDY ADD TO OUR KNOWLEDGE?☑ Cryptococcal meningitis increased tenofovir and lamivudine penetration into CSF threefold to fivefold. CSF and plasma overpredicted concentrations in brain tissue for tenofovir and lamivudine, whereas efavirenz plasma concentrations were similar to brain concentrations. We provided proof of concept for the use of well‐controlled postmortem tissue collection to quantify tissue exposure.HOW MIGHT THIS CHANGE CLINICAL PHARMACOLOGY OR TRANSLATIONAL SCIENCE?☑ These data lay the groundwork for future studies using postmortem tissues to characterize drug exposure in compartments difficult to sample.Figure S1. Variable antiretroviral tissue to plasma ratios across the CNS.Click here for additional data file.Table S1. Subject characteristics.Click here for additional data file.


Background: The pathophysiology underlying altered blood-cerebrospinal fluid barrier (BCSFB) function in Alzheimer's disease (AD) is unknown but may relate to endothelial cell activation and cytokine mediated inflammation.Methods: Cerebrospinal fluid (CSF) and peripheral blood were concurrently collected from cognitively healthy controls (N = 21) and patients with mild cognitive impairment (MCI) (N = 8) or AD (N = 11). The paired serum and CSF samples were assayed for a panel of cytokines, chemokines, and related trophic factors using multiplex ELISAs. Dominance analysis models were conducted to determine the relative importance of the inflammatory factors in relationship to BCSFB permeability, as measured by CSF/serum ratios for urea, creatinine, and albumin.Results: BCSFB disruption to urea, a small molecule distributed by passive diffusion, had a full model coefficient of determination (r2) = 0.35, and large standardized dominance weights (>0.1) for monocyte chemoattractant protein-1, interleukin (IL)-15, IL-1rα, and IL-2 in serum. BCSFB disruption to creatinine, a larger molecule governed by active transport, had a full model r2 = 0.78, and large standardized dominance weights for monocyte inhibitor protein-1b in CSF and tumor necrosis factor-α in serum. BCSFB disruption to albumin, a much larger molecule, had a full model r2 = 0.62, and large standardized dominance weights for IL-17a, interferon-gamma, IL-2, and VEGF in CSF, as well IL-4 in serum.Conclusions: Inflammatory proteins have been widely documented in the AD brain. The results of the current study suggest that changes in BCSFB function resulting in altered permeability and transport are related to expression of specific inflammatory proteins, and that the shifting distribution of these proteins from serum to CSF in AD and MCI is correlated with more severe perturbations in BCSFB function. Blood-brain barrier (BBB)permeability increases with normal aging and may be an important mechanism in the initiation or worsening of cerebral microvascular disease leading to dementia  integrity as an indicator of BBB disruption. Albumin is a large molecule synthesized predominantly by the liver, so any amounts found in brain or cerebrospinal fluid (CSF) are assumed to be mainly deposited within the extracellular space via the peripheral circulation. Of 10 studies reported in a recent review that examined BBB integrity in AD compared to controls using the “CSF/serum albumin index,” six found evidence of BBB disruption, and four did not  who developed dementia during the follow-up had a higher CSF/serum albumin ratio than those not developing dementia , suggesting that BCSFB disruption may occur before the onset of clinical symptoms  preceded neuritic plaque formation suggesting a central role for early structural changes to the BBB caused by elevated A-beta  and barrier transporter activity (creatinine/creatine), particularly in those with MCI. Urea finely differentiated altered BCSFB permeability from normal barrier function between patients and controls. CSF/serum ratios were consistent with disrupted BBB function that likely occurs even at the prodromal stage of AD, suggesting that measurement of CSF and serum urea and creatinine may be sensitive novel biomarkers of modified fluid biochemistry and loss of BBB function in MCI and AD , but also urea  and creatinine , in order to explore the potential roles of inflammatory markers in earlier AD pathology when BBB disruption may be related to leakage of small molecules or damage to active transport mechanisms.N = 21) and patients with MCI (N = 8) or AD (N = 11). Diagnoses of AD were made according to NINCDS-ADRDA criteria  Alzheimer's Disease and Memory Disorders Center between 2010 and 2016. The biological fluid samples were collected in accordance with the Alzheimer's Disease Neuroimaging Initiative protocol. Paired serum and CSF samples were obtained as part of a neurologic diagnostic evaluation or as add-on donations at the time of a clinical trial or observational research study visit. Following collection, the samples were aliquoted into 2 ml sterile polypropylene screw capped tubes and frozen at −80°C.All subjects signed written informed consent documents approved by the RIH human subjects committee (RIH#210946-17) to allow their serum and CSF samples to be banked for future research.Control patients were evaluated for headache in the Rhode Island Hospital Emergency Department between October 2014 and December 2015, and the biological fluid samples were collected in accordance with standard hospital practice. The control subjects did not carry a diagnosis of active infection and were free of cognitive and primary neurological disorders other than headache based on review of hospital outpatient, inpatient and emergency department records. The main inclusion criteria for the fluid samples were that sufficient volumes of the paired serum and CSF samples be available for analysis, and that routine assays of CSF, including protein and glucose concentrations, cell counts, and Gram stain results be within normal limits. This study (RIH#633481-13) was approved by the Lifespan Hospitals Institutional Review Board.Direct Binding Enzyme-linked Immunosorbent assay (ELISA): Amyloid-beta peptide of the amyloid precursor protein (Aβ1-42) and phospho-tau (pTau-307) immunoreactivity were measured in direct binding ELISAs. Serum samples diluted 1:100 and CSF diluted 1:4 in Tris-buffered saline (TBS) were adsorbed (50 μl each) to the well bottoms of Maxisorp 96-well plates (Nunc) by overnight incubation at 4°C, then blocked for 3 h at room temperature with 1% bovine serum albumin (BSA) in TBS. After washing, the samples were incubated with primary antibody (0.1–0.4 μg/ml) for 1 h at 37°C. Immunoreactivity was detected with horseradish peroxidase-conjugated secondary antibody and Amplex UltraRed soluble fluorophore. Fluorescence intensity was measured (Ex 565 nm/Em 595 nm) in a SpectraMax M5 microplate reader .Multiplex Human Cytokine ELISAs: Bead-based multiplex ELISAs were employed to assess levels of 27 pro-inflammatory cytokines and chemokines and trophic factors in serum and CSF. The list of cytokines, chemokines, and trophic factors, their abbreviations, and functions both systemically and in the central nervous system are summarized in Table d effect sizes (mean difference divided by pooled standard deviation). Conventional interpretation of d is that differences of 0.2, 0.5, and 0.8 demarcate small, medium and large effect size differences. Confidence intervals for Cohen's d were obtained with reference to the non-central t distribution function. The direction of the comparison is such that when d is negative, the mean in the cognitively intact group is lower than in the cognitively impaired group. P-values derived from two-sample t-tests were not corrected for multiple comparisons. With 27 factors, and comparing across serum and CSF, there were 2 × 27 comparisons. The significance level associated with a Bonferroni correction for 54 comparisons is about p < 0.001 (0.05/54 = 0.0009).We compared mean differences in expression levels for cytokines, chemokines, and trophic factors between participants with MCI or AD and those without cognitive impairment, and expressed these differences as Cohen's R2), i.e., an average of all possible combinations of models including (and not including) a given independent variable. We used this to produce a rank ordering of independent variable importance.To evaluate the relative importance of the cytokines, chemokines, and trophic factors to BCSFB ratios for urea, creatinine and albumin, we performed a dominance analysis. Dominance analysis is useful for identifying the relative importance of independent variables and is based on comparisons and combinations of fit statistics from each subset regression Budescu, . We baseR2, reflecting the variable's contribution to the model R2 across all possible models. Because dominance analysis involves fitting all possible subsets of regression equations and this is very computer intensive, we limited our consideration to models with the strongest bivariable association between predictor and outcome. Our criterion for inclusion was that the bivariable association was at least R2 = 0.1, and we allowed a maximum of 12 predictors per model chosen in order of strength of bivariable association.In addition we performed an additive decomposition of the overall model Demographic and clinical characteristics of the subjects are listed in Table The CSF/serum ratios for urea and albumin were higher for patients than controls. The ratio was in the opposite direction for creatinine. This difference in direction is due to the BCSFB to urea and albumin being maintained by passive diffusion mechanism, while creatine and creatinine are governed by active transport between blood, brain and CSF  are proximate proinflammatory mediators of BBB dysfunction in MCI patients as well as in AD  are associated with different degrees of BCSFB disruption. Furthermore, the data show that the apparent evolution of BCSFB changes from increased permeability to smaller probes like urea to more pronounced BCSFB perturbation to a larger molecule, albumin, is correlated with a relative shift of inflammatory factors from serum to CSF. While changes in CSF cytokine levels may reflect transport and distribution of those molecules between blood and CSF, another possibility is that in MCI and AD there is enhanced penetration of leukocytes from blood to CSF, e.g., across the choroid plexus, and that those leukocytes that have accessed CSF are now being stimulated to secrete cytokines into the CSF.Differences in how inflammation patterns in serum and CSF relate to small, then later large BBB permeability changes may shed light on disease pathogenesis and could also be useful as a biomarker to monitor progression. The idea that an inflammatory profile approach is useful to classify AD is supported by previous work which demonstrated that an algorithm comprised of 21 serum inflammatory proteins from 150 AD cases and 150 controls yielded sensitivity and specificity of 0.90 for correctly classifying AD -15, IL-1rα, and IL-2 in serum. Monocyte chemoattractant protein-1 is a chemokine anchored in the plasma membrane that is secreted by monocytes, macrophages and dendritic cells. Induced in astrocytes by PDGF-BB, it attracts monocytes promoting their transmigration through a disrupted blood-brain barrier. Increased levels impair attention; executive function, and psychomotor speed  in CSF. IL-4 is a cytokine that induces differentiation of naïve T cells and regulates humoral and adaptive immune responses. It's anti-inflammatory actions reduce thymus-1 type cytokine, IFN-γ, macrophages, and dendritic cell IL-12. IL-4 is potentially neuroprotective for cortical neurons by modulating excitability , one of which (creatinine) uniquely relates to transporter disruption. Taken together with previous observations, the results of our study suggest that changes in BCSFB function resulting in altered permeability and transport are related to expression of specific inflammatory proteins, and that the complex shifting distribution of these proteins from serum to CSF in AD and MCI is correlated with more severe perturbations in BCSFB function. We did not observe a simple relationship between the physicochemical characteristics of urea, creatinine, and albumin and the cytokines most related to alterations in their BCSFB ratios, arguing somewhat in favor of the cytokines playing a causative role in BCSFB dysfunction, rather than simply being a physical marker of BCSFB sieve characteristics.There are limitations to the current study as well as other valid interpretations of the data. BCSFB permeability and transport perturbation and expression of inflammatory markers may be linked but not causally related. In this regard, in a mouse model of epilepsy, a higher concentration of chemokines and pro-inflammatory cytokines in the serum and higher expression in activated microglia were documented without BBB disruption (Okuneva et al., It is also difficult to assess the effects of peripheral inflammation as distinct and separately arising from inflammatory changes in the brain. A growing literature documents the influence of systemic inflammatory, oxidative and immune processes on biochemical events in the brain (Yu et al., An additional limitation is our small sample size and the relatively high number of comparisons. Small sample size studies are at greater risk of both type-I errors and type-II errors than what would be expected given the nominal significance levels used in forming hypothesis tests (Button et al., Of note, it is possible that the albumin ratio may not accurately reflect the degree of BBB breakdown. Recent studies suggest that albumin levels in the CSF can be affected by many factors which include uptake of extravasated albumin by microglia, astrocytes (Ivens et al., We did not have the ApoE genetic status on most patients. This information was also unavailable in our control subjects. Since ApoE may have a modulating effect on these relationships (Zipser et al., Lastly, while we made efforts to exclude subjects with active inflammatory and infectious disease, the presence of minor inflammation producing illness in some subjects, particularly those drawn from the emergency department, could have influenced our results. We also did not routinely collect details on the subjects regarding various vascular risk factors for dementia like hypertension and diabetes, so future studies in this area should control for such factors, particularly with regard to blood cytokines.In sum, our report should be regarded as preliminary evidence suggesting the possibility that systemic and central neuroinflammatory processes produce BCSFB disruption early in the course of AD. Such damage to the BBB may result in a brain that is highly vulnerable to inward leakage of neurotoxins and weakening of important energy systems dependent on active transport of nutrients such as creatine from the systemic circulation that in turn could lead to or hasten neurodegeneration. Future research should examine whether such processes occur during the preclinical stage of the disease when anti-inflammatory and BBB neuroprotective therapies may have their greatest impact on the prevention of dementia. While our study is essentially exploratory, the unique data on BCFB permeability in relation to cytokines and chemokines should prompt new efforts to experimentally focus on these factors and their mechanisms of barrier impairment, in relation to the changing CSF environment in MCI and AD.BO reports having received research funding from Biogen, Lilly, Avid, Merck, Janssen, AbbVie, and Long Term Care Group, as well as consulting fees from Amgen.BO, RJ, LD, SdlM, ES, CJ, CD, and PG contributed to the conception or design of the work; or the acquisition, analysis, or interpretation of data for the work; and drafting the work or revising it critically for important intellectual content; and final approval of the version to be published; and agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.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.


Plerixafor plus granulocyte-colony stimulating factor (G-CSF) enhances the mobilization of hematopoietic stem cells (HSCs) for collection and subsequent autologous hematopoietic stem cell transplantation (HSCT) in patients with multiple myeloma (MM). This international, multicenter, noninterventional registry study (NCT01362972), evaluated long-term outcomes for MM patients who received plerixafor versus other mobilization regimens. The comparisons were: G-CSF + plerixafor (G-CSF + P) versus G-CSF-; G-CSF + P versus G-CSF + chemotherapy (G-CSF + C); and G-CSF + P + C versus G-CSF + C. Propensity score matching was used to balance groups. Primary outcome measures were progression free survival (PFS), overall survival (OS), and cumulative incidence of relapse (CIR) after transplantation. After propensity matching, 77 versus 41 patients in the G-CSF + P versus G-CSF cohorts, 129 versus 129 in the G-CSF + P versus G-CSF + C cohorts, and 117 versus 117 in the G-CSF + P + C versus G-CSF + C cohorts were matched, respectively. Propensity score matching resulted in a smaller sample size and imbalances were not completely overcome. For both PFS and OS, the upper limits of the hazard ratio 95% confidence intervals exceeded prespecified boundaries; noninferiority was not demonstrated. CIR rates were higher in the plerixafor cohorts. G-CSF + P remains an option for the mobilization of HSCs in poor mobilizers with MM with no substantial differences in PFS, OS, and CIR in comparison with other regimens. Plerixafor (Mozobil®) in combination with granulocyte-colony stimulating factor (G-CSF) is approved by the European Medicines Agency to enhance the mobilization of hematopoietic stem cells (HSCs) to the peripheral blood for collection and subsequent autologous transplantation in patients with lymphoma and multiple myeloma (MM) who are recognized as poor mobilizers of HSCs. Plerixafor is a selective, reversible inhibitor of the chemokine receptor 4 with a (C-X-C) motif (CXCR4) and has a unique mechanism of action compared with other HSC mobilizing agents , 2. The There is a theoretical risk of tumor cell mobilization with any stem cell mobilization method for hematopoietic stem cell transplantation (HSCT). Therefore, the European Union mandated analysis of the European Society for Blood and Marrow Transplantation (EBMT) data registry to evaluate the long-term outcomes of patients with MM who had received plerixafor.The long-term clinical outcomes collected in this postapproval analysis of the EBMT registry included an evaluation of progression free survival (PFS), overall survival (OS), and the cumulative incidence of relapse (CIR) in patients with MM who had undergone mobilization, collection, and transplantation of autologous blood progenitors. The analysis evaluated patients who received plerixafor for stem cell mobilization and HSCT and compared their outcomes with those of patients who had received other mobilization regimens.ClinicalTrials.gov number NCT01362972). The analysis included a prospectively defined cohort of MM patients with data reported retrospectively to the EBMT. Patients from Austria, Belgium, Bulgaria, Czech Republic, Finland, France, Germany, Greece, Hungary, Ireland, Israel, Italy, Netherlands, Poland, Romania, Spain, Sweden, Switzerland, and the United Kingdom were included in the study. Eligibility included all patients’ ≥18 years of age from the EBMT registry with a diagnosis of MM who were to receive an autologous HSCT and were transplanted. This was a noninferiority study. The noninferiority margin was assigned as a 30% increase in PFS and OS corresponding to a hazard ratio (HR) upper limit of 1.3. No lower limit was set. Summary curves for CIR were planned. Due to the observational nature of the study, no formal statistical hypothesis testing was planned with adequate power and the type I error control.This was an international, multicenter, noninterventional registry study with patient follow-up of 3.5–7.5 years to evaluate the long-term outcomes of MM patients who received plerixafor for stem cell mobilization and who completed their first autologous HSCT between 2008 and 2012 . The key safety outcome was transplant complications occurring from the day of transplantation until 100 days post transplant.The primary efficacy outcomes were OS, PFS, and CIR. Key secondary efficacy outcomes were hematological recovery (time to absolute neutrophil counts of ≥0.5 × 10G-CSF plus plerixafor (G-CSF + P) compared with G-CSF alone.G-CSF + P compared with G-CSF plus chemotherapy (G-CSF + C).G-CSF plus plerixafor plus chemotherapy (G-CSF + P + C) compared with G-CSF + C.The following mobilization regimens were compared:9 cells/l) or graft loss .Graft failure was defined as no engraftment  and HR for the effect of treatment were calculated. Survival curves were developed for each treatment group using nonparametric Kaplan–Meier estimates . A compeDue to the observational nature of the study, sample size was not calculated based on power calculations. It has been estimated using the following assumptions: that 85% of transplanted MM patients would receive G-CSF + C and 15% would receive G-CSF alone; that 10% of transplanted patients with each regimen would be treated with plerixafor; and that 70% of plerixafor patients would be matched at a ratio of 1:2 plerixafor to comparator. It was estimated that 4600 patients would be included in the study over a 5-year period and would include: 100 patients in the G-CSF alone group, 540 patients in the G-CSF + C group, 50 patients in the G-CSF + P group, and 270 patients in the G-CSF + P + C group. The predicted number of events for the outcome analysis was 101 for the G-CSF + P compared with G-CSF alone, 101 for the G-CSF + P compared with G-CSF + C, and 546 for the G-CSF + P + C compared with the G-CSF + C group.Overall, 3582 MM patients were screened and, of these, 3566 patients met the study eligibility criteria. These included 141 patients treated with G-CSF + P, 119 patients treated with G-CSF + P + C, 585 patients treated with G-CSF alone, and 2721 patients treated with G-CSF + C , the time to disease progression, PFS, and OS are all significantly shorter compared with successful mobilizers [In line with the licensed therapeutic indication for the use of plerixafor in patients with MM, all patients in the primary analysis had to be poor mobilizers, either as predicted poor mobilizers through exposure to high-dose chemotherapy, or proven poor mobilizers based on their mobilization history. Despite propensity scoring, there were more proven poor mobilizers in the plerixafor cohorts, which may have influenced the outcomes due to an imbalance between comparison groups. A further difference between the plerixafor and comparison cohorts was that the median CD34+ cell counts in the plerixafor group during the current study mobilization were lower compared with comparison cohorts. These differences suggested that the groups may not have been balanced for disease prognosis, which may be important, as it has been reported that in poor mobilizers  was the group with the smallest sample size—a factor that introduced a large variability. The sample size resulting from propensity score matching could be one of the limitations for the demonstration of noninferiority in the current study. Some important variables, such as the amount of prior chemotherapy, cytogenetic risk, or the use of consolidation and maintenance treatments, could not be incorporated in the model due to the lack, or limited availability, of the data. This imbalance in the data collection could have had a substantial impact on outcomes in our study.There are a number of other limitations to our study that mandate caution when interpreting the results. The proportion of patients in the primary analysis who were proven poor mobilizers was numerically greater in the plerixafor cohorts (98.3–100%) compared with the comparator cohorts (72.1–85.4%). There were higher numbers of patients in the plerixafor cohorts who failed to mobilize sufficient CD34+ cells at the predicted peak mobilization times. In line with reimbursement of medicine costs in many European countries, clinicians may have selectively given plerixafor to patients who were the poorest mobilizers at the highest risk of poor outcomes, and this may have contributed to the trend for worse outcomes in the plerixafor cohorts –29. A fuThe findings from this study should be interpreted with caution due to its observational nature, the small sample size of the comparison groups, and the wide 95% CI observed in the HRs. The absence of information on genetic risk and maintenance treatment are further limitations of the study. The cohorts treated with plerixafor had a trend toward numerically shorter PFS and OS times and higher CIR, with similar safety outcomes compared with their respective comparators. Poor mobilization is associated with more aggressive disease and hence poor mobilizers are potentially predisposed to worse outcomes, as may be indicated by the lower baseline CD34+ cell counts at predicted peak of mobilization in the plerixafor cohort . G-CSF +


Background and objectives: In temperate environments, acute orally induced metabolic alkalosis alleviates exercise stress, as reflected in attenuated stress hormone responses to relatively short-duration exercise bouts. However, it is unknown whether the same phenomenon occurs during prolonged exercise in the heat. This study was undertaken with aim to test the hypothesis that ingestion of an alkalizing substance  after dehydrating exercise would decrease blood levels of stress hormones during subsequent 40 km cycling time-trial (TT) in the heat. Materials and Methods: Male non-heat-acclimated athletes (n = 20) lost 4% of body mass by exercising in the heat. Then, during a 16 h recovery period prior to TT in a warm environment (32 °C), participants ate the prescribed food and ingested CIT (600 mg·kg−1) or placebo (PLC) in a double-blind, randomized, crossover manner with 7 days between the two trials. Blood aldosterone, cortisol, prolactin and growth hormone concentrations were measured before and after TT. Results: Total work performed during TT was similar in the two trials (p = 0.716). In CIT compared to PLC trial, lower levels of aldosterone occurred before (72%) and after (39%) TT (p ˂ 0.001), and acute response of aldosterone to TT was blunted . Lower cortisol levels in CIT than in PLC trial occurred before  and after TT , but there were no between-trial differences in the acute responses of cortisol, prolactin or growth hormone to TT, or in concentrations of prolactin and growth hormone before or after TT . Conclusions: Reduced aldosterone and cortisol levels after TT and blunted acute response of aldosterone to TT indicate that CIT ingestion during recovery after dehydrating exercise may alleviate stress during the next hard endurance cycling bout in the heat. McEwen  defined exercise ,18. Datatenuated  or unchatenuated ,21 bloodng bouts ,23. NaHCing bout . CIT bluing bout .3 ingestion alleviates responses of stress hormones to prolonged endurance exercise in warm environments.Thus, the majority of the available data suggest that acute orally induced metabolic alkalosis alleviates exercise stress, as reflected in attenuated stress hormone responses to exercise ,22,23,24Generally, stress induced by exercise is considered to be a stimulus for positive physiological adaptations that, in the course of systematic training lasting several weeks, months or years, leads to improvement of health and performance . Neverth3 [Thus, our purpose was to test the hypothesis that CIT ingestion during 16 h recovery period after DE, in an amount that has been shown to induce metabolic alkalosis and expansion of PV, would attenuate increases in the blood levels of CORT, ALDO, GH and PRL during subsequent 40 km cycling time-trial (TT) in the heat. We experimented with CIT because this substance has been shown to cause less gastrointestinal distress than NaHCO3 .2peak) 57.0 ± 5.9 mL·kg−1·min−1) [A detailed description of the study protocol, participants, research procedures and apparatuses used is given in our previous publication . Briefly1·min−1) .2peak was measured in temperate environmental conditions  50–51%), employing test protocol and equipment described in detail by Suvi et al. [The study was carried out in two stages: preparatory and main phase . In the i et al. . During 2peak until approximately 4% BM loss was achieved due to sweating [Participants were instructed to refrain from strenuous exercise and to record their dietary intake 24 h before the main phase to ensure stable nutritional status. On the first day of the main phase , particisweating .−1) and breakfast , both in the laboratory and ingested gelatine capsules containing CIT (600 mg·kg−1) or PLC (sucrose) in a randomized, double-blind, crossover manner with 7 days between the two trials [−1 initial BM) of CIT or PLC was administered during dinner (approximately 16 h prior to TT), the second dose (200 mg·kg−1) was ingested by participants roughly 1 h before bedtime (approximately 11 h prior to TT) at home, and the third 200 mg·kg−1 dose was consumed the next morning during breakfast at the laboratory at 8:00 AM (2 h prior to TT). The same total dose and identical schedule of CIT ingestion has been demonstrated to expand PV and significantly increase blood pH (from 7.40–7.44), bicarbonate ion concentration (from 25.0–30.45 mmol·L−1) and base excess (from 0.54–5.74 mmol·L−1) [A recovery period lasting for approximately 16 h started immediately after DE. During the recovery period, participants drank bottled water ad libitum, ate prescribed dinner  .c was recorded every 1 min, and they were verbally encouraged to complete the exercise task as fast as possible [Participants’ nude BM was recorded approximately 60 min after the breakfast. After positioning a rectal temperature probe, participants entered the climatic chamber  and spent 20 min in a sitting position prior to donating venous and capillary blood samples. Before starting the TT, participants slightly warmed up by cycling 5 min on a cycle ergometer. During the TT, participants had free access to water, their Tpossible .Venous blood samples were drawn at the antecubital fossa of the arm into Vacutainer tubes containing EDTA, and into sterile serum Vacutainer tubes shortly before and after DE and TT. In blood taken from the EDTA tube, hematocrit and hemoglobin concentration were measured , and on −1 and 0.15 mU·L−1, respectively. The intra- and inter-assay coefficients of variation (CV) were 1.7–8.4% and 5.2–12.8% for ALDO, and 1.8–3.5% and 5.9–10.4% for GH.ALDO and GH concentrations were measured in plasma and serum, respectively, with chemiluminescence immunoassay method and using IDS-iSYS Multi-Discipline Automated Analyser . According to the manufacturer, the minimal detection limits of the assays were 3.7 ng·dL−1 and 1.5 nmol·L−1, respectively. The intra- and inter-assay CVs were 0.8–1.7% and 1.4–2.0% for PRL, and 1.5–2.4% and 1.9–2.8% for CORT.Concentrations of PRL and CORT were measured in serum using electrochemiluminescence immunoassay “ECLIA” on Cobas 6000 e601 analyzer . The lower detection limits of the assays were 1.00 mU·L−1, 0.7–0.8% and 1.2–1.4%, respectively.Glucose concentration in serum was measured using the hexokinase enzymatic method on Roche/Hitachi Cobas 6000 c501 analyser . Minimal detection limit, intra- and inter-assay CVs were 0.11 mmol·LLactate concentration in capillary blood samples taken from fingertip shortly before and after TT was measured using Dr. Lange cuvette test LKM 140 and miniphotometer LP 20 Plus .t-test. A Pearson coefficient of correlation (r) was used to determine the relationship between variables. Significance was set at p < 0.05 level. Data are expressed as mean ± the standard deviation of the mean (SD). The hormone and glucose concentrations measured after the TT are presented as corrected for the individual changes in PV.Data were analyzed with the Statistica 13 software . The normality of data was tested using the Kolmogorov-Smirnov test. Repeated measures analysis of variance (ANOVA) was utilized to assess the differences within and between the trials. Following significant trial by time interactions, Tukeyʼs honest significant difference post hoc analysis was used for multiple comparisons. Between-trial differences in TT time and work done were analyzed using paired samples Studentʼs p ˂ 0.001) and after TT (p ˂ 0.001) (p ˂ 0.001).Plasma ALDO level was lower in CIT compared to PLC trial before (˂ 0.001) A. Duringp = 0.039) and after (p ˂ 0.001) TT (p = 0.257).Serum CORT level was lower in CIT compared to PLC trial before (.001) TT B. Howevep = 0.984 and 0.107, respectively) (p ˂ 0.001) in both trials. The between-trial difference in the extent of an increase in PRL levels did not reach statistical significance (p = 0.064).The levels of serum PRL were similar in CIT and PLC trials before and after TT (ctively) C. Duringp = 0.995 and 0.575, respectively) (p ˂ 0.001) in both trials to a similar extent (p = 0.460).Serum GH concentrations before and after TT were similar in CIT and PLC trials (ctively) D. Duringp = 0.844) and after (p = 0.055) TT, serum glucose concentration did not differ in the two trials (p = 0.020). Therefore, after finishing the TT, serum glucose concentration had fallen below the before exercise level in CIT (p < 0.001) but not in PLC trial (p = 0.643).Prior to . However, during TT, the extent of an increase in blood lactate concentration was greater (p ˂ 0.001) in CIT trial compared to PLC trial. After exercise higher (p ˂ 0.001) blood lactate level occurred in CIT compared to PLC trial .Blood lactate levels before TT were similar in PLC and CIT trials (2.06 ± 0.37 and 2.31 ± 0.42 mmol·Lp = 0.716), and total time also did not differ . There were no between-trial differences in 5 km split times during the TT . Tc before TT was similar in the two trials , but during exercise the magnitude of increase in Tc was smaller in CIT than in PLC trial .Total work performed during TT was similar in PLC and CIT trials  in CIT compared to PLC trial. Compared to pre-DE time point, after DE PV had diminished by 15.7 ± 4.2% in CIT and 15.2 ± 4.6% in PLC trial . However, at the end of the recovery period, i.e., prior to the start of TT, PV exceeded the pre-DE level by 6.9 ± 5.4% in CIT trial and remained 0.9 ± 3.3% below that in PLC trial . After TT, PV had fallen below pre-DE level by 6.2 ± 6.3% in CIT and 12.5 ± 6.9% in PLC trial .Total water intake (4962 ± 739 mL in PLC and 5495 ± 721 mL in CIT) and BM gain (3.67 ± 0.75% in PLC and 4.68 ± 0.71% in CIT) during recovery period were greater (p = 0.009. The only hormone the increases of which significantly correlated with increases in Tc was PRL: r = 0.609 (p = 0.004) in PLC and r = 0.572 (p = 0.008) in CIT trial. Based on pooled data from PLC and CIT trials, plasma ALDO levels before TT correlated with changes in PV during time interval between the beginning of DE and end of the recovery period . Moreover, plasma ALDO levels after TT correlated with changes in PV during time interval between the beginning of DE and end of TT . Finally, based on pooled data from PLC and CIT trials, serum CORT levels before TT correlated with serum glucose concentrations after exercise .During TT, the only hormone the increases of which significantly correlated with increases in blood lactate concentration was CORT and the relationship occurred only in PLC trial: r = 0.566, This study investigates for the first time whether CIT ingestion in the amount that has been shown to induce metabolic alkalosis and an acute increase in PV  influenc+ concentration or renin activity [3 or KHCO3 induced metabolic alkalosis, but neither of these substances suppressed plasma ALDO levels [+ concentrations did not change in NaHCO3 trial, but in KHCO3 trial, parallel increases in ALDO and K+ levels occurred [In goats, metabolic alkalosis suppressed blood ALDO concentration in the absence of detectable changes in plasma Kactivity , which aactivity ,40. Howeactivity . In younO levels . Plasma occurred . Consideoccurred ,40,41,42+ and lower K+ concentrations [+ is considered a strong, and decrease in Na+ a relatively weak, stimulus for ALDO secretion [+ and Na+ concentrations were among the factors that reduced ALDO levels in our participants in resting state and during exercise in the heat.In temperate environmental conditions, CIT compared to PLC ingestion induced higher serum Natrations  and exacecretion ,40,43. Iecretion ,39,40,43Similar BM, urine specific gravity and serum osmolality  suggest 3 compared to PLC ingestion attenuated serum CORT responses to four consecutive 30 s bouts of maximal cycling exercise even though the mean power output was higher in NaHCO3 trial [3 induced metabolic alkalosis was partly responsible for blunted CORT response. Without ingestion of alkalizing substances, parallel increases in blood lactate level and acidity occur during exercise [+ from contracting muscle cells and at the same time more efficiently buffers H+, leading to greater elevations in blood lactate levels, but attenuating increases in blood acidity [3 to their subjects only over a 90 min period preceding the start of exercise.Cortisol is among the most widely studied markers of stress ,46. In aO3 trial . These aexercise . Therefo acidity ,6 and CO acidity , we obse acidity  administc and plasma CORT levels. In our participants, CIT ingestion induced acute PV expansion that may slow down increases in Tc during exercise [c did not differ. Immediately after exercise, Tc was lower in CIT trial, but the between-trial difference in serum CORT level was similar to that observed prior to exercise. Furthermore, increases in Tc did not correlate with increases in serum CORT levels during exercise in either trial. Thus, it is unlikely that Tc induced between-trial differences in serum CORT level in our participants.Cooper et al.  and Rhinexercise . HoweverNutritional status, nutrient timing, intensity and duration of exercise and total amount of work completed are important factors influencing endocrine responses to exercise ,50. Our Several-fold increases in serum PRL concentrations occurred during TT, but the magnitude of this response was similar in the two trials. Exercise stimulates PRL secretion , whereasc and PRL response to exercise [c. Taken together, our data suggest that increases in Tc stimulate PRL secretion during prolonged cycling exercise in the heat and that CIT ingestion does not modulate serum PRL response in this situation.A significant relationship occurs between Texercise ,59,60,61Secretory stimuli are similar for PRL and GH . Similarc. Literature shows, with a few exceptions [c [c and GH levels during prolonged exercise in the cold [c and blood GH levels employed constant-load exercise of fixed duration [c and serum GH in our participants. Nevertheless, our data reveal that in the heat, prolonged self-paced cycling exercise induces immense increases in serum GH levels, and that CIT ingestion does not modulate GH response in these conditions.During the TT serum GH level increased by several times more than that of PRL and, differently from PRL, changes in GH level did not correlate with increases in Tceptions ,62, thattions [c ,55,63, athe cold  and in tthe cold ,49,65. Hduration ,65 or toduration , whereas+ and decrease K+ concentrations [Altogether, our findings of reduced ALDO and CORT levels before and after TT and blunted acute response of ALDO to TT suggest that CIT ingestion after DE alleviates stress during subsequent heavy endurance cycling bout in a warm environment. As suggested by Peart et al. , reducedtrations .CIT compared to PLC ingestion during 16 h recovery period after DE results in decreased blood ALDO and CORT levels prior to and during subsequent cycling TT in a warm environment in male non-heat-acclimated endurance athletes. CIT ingestion also reduces the magnitude of acute increases in blood ALDO, but not CORT level, during TT. CIT ingestion has no influence on blood levels of PRL or GH in these experimental conditions.