Background. The aim of this investigation was to compare the sedative effects of oral midazolam/chloral hydrate and midazolam/promethazine combinations on fearful children needing dental treatment.Methods. This crossover double-blind clinical trial was conducted on 30 children aged 2‒6 years, who had at least two similar teeth needing pulp treatment. Standard vital signs were recorded before and after premedication. Wilson sedation scale was used to judge the level of sedation. Cases were divided into two groups based on the sequence of medication received. This was to overcome the sequence effect. Group I received oral midazolam  at the first visit while they received midazolam (0.4 mg/kg)/promethazine (5 mg/kg) in their second visit. Group II received the premedication in the opposite sequence. The operator and child were blinded to the medication administered. Sedative efficacy of the two combinations were assessed and judged by two independent pediatric dentists based on the Wilson scale. Data were analyzed with ANOVA and paired t-test.Results. Only 10% of children who received chloral hydrate with midazolam exhibited high improvement in their behaviorwhile 53% showed reasonable positive changes and 12% had no change or even deterioration of behavior. The differencebetween the effect of the two combination drugs was statistically significant (P<0.05) in favor of the chloral hydrate group.Conclusion. The results showed a significant difference in the sedation level induced between the two groups. Midazolam/chloral hydrate combination more effectively improved the co-operation for dental treatment. Late-onset effect is considered as a disadvantage to oral sedation along with unpredictable absorption rate and titration limitations. Appropriate case selection is the key to having the best and reasonably effective oral sedation results. In most instances, the initial sedative signs are seen at and around half hour of administration wken the pick plasma level of the drug is reached to obtain its anxiolytic effects.4Dental visits have long been a concern and for many as a major challenge at young age. Fear and anxiety could lead to serious avoidance of treatment when it is in its critical stages. This will, in turn, lead to a more complicated status, which sometimes compromises the chance for saving the teeth. Many dental professionals seldom see children due to the pretext of being unable to cope with their treatment, usually ending in an uncompleted dental procedure.10 The use of inhalation sedation is routine when associated with other routes, including oral sedation.12 Oral midazolam has been used as a short- and fast-acting benzodiazepine prior to GA and several other medical diagnostic approaches for many years.14 Promethazine is a known antihistaminic drug with degrees of sedative effect, particularly when associated with other agents. It is well absorbed in the gastrointestinal system with its effect starting within the first 20 minutes of oral administration. This effect will diminish after a period of 4‒6 hours. Promethazine is also used to control postoperative nausea, vomiting and apprehension.7 Most sedative drugs reach their highest level of effect after 30 minutes of their administration.8 As most of the sedative agents have the potential to take the patient to deep states, it is always desired to look for a more moderate sedative agent to enable a lighter sedation mode with closer monitoring to better control the patient while sedated. This is thought to be provided with the use of antihistamines along with low doses of benzodiazepines. Titration problem in oral sedation is a pitfall and obstacle to obtaining an efficient clinical state while parenteral routes of the same drugs provide lasting effects of sedation (3‒4 hours) for efficient treatment; however, the risks of airway depression increases, too. Besides, deeper sedation would trigger late discharge due to late sedative effects, highlighting the benefits of lighter oral sedation.14Oral sedative drugs usually include anxiolytics, sedatives, barbiturates, narcotics and antihistamines.8 As an example, ketamine and midazolam have already been used successfully with lower doses compared to their single use for dental treatment of young fearful children.12 Potential side effects of ketamine and chloral hydrate when used orally include nystagmus, hallucination and hypersalivation.15 A few different cocktails of sedative agents have been tested successfully for children, among which less attention has been paid to the use of promethazine or chloral hydrate along with midazolam, for dental procedures. The use of promethazine/midazolam allows lower doses to be used with almost the same effect comparable to two sedatives of chloral hydrate/midazolam. This can be administered in a safe way in clinics.On the other hand, several earlier studies have looked at the combined sedative effects of various drugs used alongside midazolam. This is believed to have a synergic effect on sedation and therefore a lower dose is needed, which would provide efficient sedation level suitable for delivering dental treatment.This study was designed to evaluate and compare the sedative effects of two combinations of oral drug regimens, including chloral hydrate/midazolam and promethazine/midazolam in a group of uncooperative child dental patients aged 2‒6 years.This crossover double-blind clinical trial was conducted on 30 children aged 2‒6 years, who had at least two similar teeth needing pulp treatment. Case selection was performed based on their attendance and in a sequential manner (simple sampling). Cases that were judged definitely negative in Frankl behavioral scale were included, who were also classified as ASA I. Cases were selected from referrals to outpatient clinic in a Children’s Hospital, Tehran, for arrangement to receive dental treatments under general anesthesia or sedation. The combination of drugs was administered at the start of each dental session by an anesthesiologist with dental operator and patient blinded to the drugs combination during the visit.16Each patient was assigned to receive two dental visits with two similar teeth needing relatively similar dental treatment in one jaw. Each case would therefore attend two appointments for two different combination drugs as the test and control. To avoid the sequence effect, the patients were randomly allocated to two separate groups in order to receive the test or control combination drugs in a specific order. Inclusion criteria were: cases that were confirmed as having no signs of any systemic problem, cold or other nasal or respiratory infections, no enlarged tonsils, no problem with neck movements and no oversized tongue. All the required information and instructions were given to parents in writing preoperatively, including the consent form for the child’s participation in this trial. Ethical approval was obtained from the Ethics Committee of Shahid Beheshti University of Medical Sciences prior to the commencement of the investigation. A 6-hour period of fasting (NPO) was instructed, preoperatively. The drugs were administered in the first or second visits of each patient in a random manner while each patient served as self control. A combination of 0.4 mg/kg of midazolam  mixed with sweetened water was administered along with 5 mg/kg of chloral hydrate elixir  in one session while 5 mg/kg of midazolam was administered along with 5 mg/kg of promethazine orally in the next visit. The dental procedure was started after a 30-minute waiting period in order to achieve the best possible oral effects of sedation.14 Blood pressure (BP) and oxygen saturation (OS) were measured and recorded as the two main components of the child’s vital status.13 In case of refusal due to lack of sufficient sedation the case was excluded from the study and treatment was completed using deeper sedation. The patients were discharged after full recovery from sedative signs as judged by the anesthesiologist in charge. The parents were encouraged to report all the potential postoperative complications. Data were statistically analyzed using ANOVA for vital sign differences, Friedman's test for sedation differences and paired t-test for differences before and after sedation in each group. Wilcoxon’s signed rank test was also used for inter-group comparisons.14The second combination consisted of 0.4 mg/kg of midazolam with 5 mg/kg of promethazine . All the vital signs were constantly monitored, including blood pressure, oxygen saturation and pulse rate, starting at baseline followed by recording every 15 minutes up to the discharge point, using a vital sign monitor . A pediatric dentist performed all the treatment procedures while child’s behavior rating was conducted by another independent pediatric dentist. Wilson’s Sedation scoring system was used to record child’s behavior pre-, intra- and post-operatively.The mean age of the subjects was 43.5 months (±10.9) with 48.4% of them being male. There was a greater improvement in cooperation level of those receiving midazolam/chloral hydrate combination when compared to those receiving midazolam/promethazine combination (P<0.05). The least value for oxygen saturation level was 94% among the entire population of the study with no statistically significant difference between 6 readings of the groups (P<0.001). Pulse rate changes clearly changed from pre-operation to the time of treatment and after sedation when chloral hydrate was administered compared to the promethazine group with a significant difference between groups using paired t-test (P<0.002). Wilson behavioral evaluation scale showed a considerable rise in cooperation level of children treated with chloral hydrate (48.4%) . However4 (2008) reported midazolam as an agent capable of inducing sufficient level of sedation when prescribed as the only medication.There are still many children who suffer from severe dental problems due to their extreme anxiety and fear. Many pediatric dentists and even dental schools still use routine office behavioral control techniques, including physical restraints as their only available resource. More recent intensive research indicates that premedication is a useful tool to shape the child’s behavior in the dental clinic. Oral route of sedation has been widely agreed on as the most convenient yet effective way to overcome fear and anxiety in young fearful children. The taste of medication remains a challenge for the operator, which can be resolved when it is added to sweetened water or a juice. Damle et al17 The results of this study showed the benefits of the use of cocktail drugs in favor of reducing the sedative dose required to achieve the optimum result. The use of an antihistaminic drug such as promethazine reduces the risk of postoperative nausea and vomiting as an expected consequence of sedation. No significant differences were seen between the level of blood oxygen saturation before and after sedation was induced in both groups (P=0.119). Similar findings were reported by Vahid et al18 (2012) with ketamine/midazolam. Chawdhury and Vargas19 also reported that chloral hydrate and meperidine did not show any significant differences in their sedative effect. Sheroan et al15 (2006) reported a slight change in blood oxygen saturation level when meperidine/chloral hydrate combination was administered. Any drop in blood oxygen saturation was treated seriously when a patient was sedated unless it was within the normal range of 95‒100%.20 Clearly the drug type and its dose have a direct impact on oxygen saturation rate and its reduction in certain cases can lead to airway obstruction.21 Dallman et al22 (2001) did not report any major oxygen saturation change following the use of midazolam with and without chloral hydrate and promethazine.22 Heart rate changes were noticeable to some extent in both groups of this study at different stages, indicating the direct effect of the drugs used.Chloral hydrate has been the drug of choice for many years based on its highly desired efficacy; however, it has potential risks which make its daily dental use compromised.Since young children are highly vulnerable to the medication used for various purposes, including sedation, it is important to make every attempt to bring the risks to the minimum level possible. This is valuable if the effectiveness of the drugs used could provide the optimum level of cooperation for the desired dental procedure to be performed in a safe mode. The combination drug use consists of a strategy in which studies, including the current one, confirm that lower doses of the known sedative agents can be administered safely in children while achieving maximum required sedation level to satisfactorily carry out the procedure. Evaluation of the potential effect of promethazine along with midazolam revealed no significant differences when stronger chloral hydrate/midazolam combinations were used, which was expected to act more effectively (P>0.05).23 Oral sedation combinations clearly produce various levels of sedation and it is the direct responsibility of the operating clinician to use the best method and regimen in a carefully selected manner.24It appears that with the sedative effect of midazolam used in both groups there seems to be a good capacity for the use of a more conservative promethazine agent over more controversial chloral hydrate in pediatric dental patients in order to avoid untoward complications when cocktail sedation agents are being used. Interestingly, chloral hydrate use has declined in recent years based on its side effects, supporting the outcome of this investigation.There was no significant difference between the sedation levels induced by chloral hydrate/midazolam and promethazine/midazolam.Child’s postoperative problems were much less in the promethazine group.There were no significant differences between the two groups in their vital signs.No sequence effect was seen following the use of one combination over the other.Authors would like to express their appreciation for support and help from Mr Moatamedi, Ms Oghbaei, Patients and their parents and Dental School Research Institute at Shahid Beheshti Medical University for their financial and scientific support.Authors would like to express their appreciation for support and help from Mr Moatamedi, Ms Oghbaei, Patients and their parents and Dental School Research Institute at Shahid Beheshti Medical University for their financial and scientific support.MM, GhA, ShSh and MVG were involved in designing the study and collection of data. The rest of the authors were involved in drafting and final approval of the manuscript.The study was financially supported by the Research Center of Shahid Beheshti Medical University.The authors declare no competing interests with regards to the authorship and/or publication of this article.The ethical clearance was obtained from the institutional ethics committee. All the patients’ parents involved in this study gave written consent for participation and publication of the research data.


We followed 72 northern leopard frogs (Lithobates pipiens) in two agricultural wetlands for insight into where and when individuals are at high risk of pesticide exposure. Novel passive sampling devices (PSDs) were deployed at sites where telemetered frogs were located, then moved to subsequent locations as frogs were radio-tracked. Pesticide concentration in PSDs varied by habitat and was greatest in agricultural fields where frogs were rarely found. Pesticide concentrations in frogs were greatest in spring when frogs were occupying wetlands compared to late summer when frogs occupied terrestrial habitats. Our results indicate that habitat and time of year influence exposure and accumulation of pesticides in amphibians. Our study illustrates the feasibility of quantifying the amphibian exposome to interpret the role of habitat use in pesticide accumulation in frogs to better manage amphibians in agricultural landscapes.This is the first field study of its kind to combine radio telemetry, passive samplers, and pesticide accumulation in tissues to characterize the amphibian exposome as it relates to pesticides. Understanding how habitat drives exposure in individuals ( Our knowledge of the impact of pesticides on amphibians is limited in part because route, timing, and rate of exposure in the field is unknown3. Pesticides occur frequently in amphibian habitats and accumulate in their tissues7; however, pesticide exposure relative to habitat use at the population or individual level has not been investigated. Sensitivity to pesticides varies by amphibian species and by the type of chemical or chemical mixtures to which an animal is exposed8. Timing of exposure is also important, and metabolism and excretion play a role in pesticide accumulation and residence time within tissues9. Compounding this complexity is the aquatic/terrestrial duality of many amphibian species. Due to their unique physiology and life history, amphibians are more vulnerable to pesticides than other wildlife species11. In addition, amphibians use both aquatic and terrestrial habitats, which are prone to change not only between years but also within a single season, contributing to fluctuations in pesticide presence and concentration12. Furthermore, amphibians have highly permeable skin which allows for dermal uptake of pesticides14. Contact with pesticides can cause death or a range of sub-lethal effects2. Health effects vary from immune suppression to reproductive and behavioral changes that may lead to population declines18. This is especially true in the agricultural landscape where pesticides are frequently applied but vary in type and amount20.Pesticides are one of multiple stressors that contribute to amphibian declines, but their population-level effects on health are still poorly understood21. For example, some species  spend much of their adult life in terrestrial environments, and may be exposed to greater amounts of pesticides outside of wetland habitats22; however, this has not been tested in the field. Several field studies have noted that pesticides detected in frog tissues could not be explained by the aquatic habitats in which they were captured7, leaving questions about the role of habitat use in pesticide exposure and accumulation. To our knowledge, pesticide exposure based on habitat use has never been studied explicitly in any amphibian species under field conditions.A better understanding of exposure potential will provide insight into threats that pesticides pose to amphibian health. Researchers have proposed that the terrestrial environment has the potential to significantly contribute to an amphibian’s pesticide accumulation and is a topic that warrants further investigationUnderstanding the complex relationships among amphibians, pesticides, and their environments is vital for conservation, and investigating pesticide exposure in the field is a central piece of this understanding. Specifically, to design mitigation strategies, we need to know where and when individuals are at the highest risk of pesticide exposure. Our goal was to understand the role of habitat use in determining pesticide exposure in frogs in an agricultural landscape. This information will facilitate an assessment of the potential health risks of agrochemicals under field conditions. We hypothesized that habitat use, measured as time spent in a particular habitat, would influence an individual frog’s exposure to types and amounts of pesticides, which would correlate with the types of pesticides accumulating in their tissue.23 to assess pesticide presence near individual animals occupying different habitat types, and to facilitate comparisons of pesticides in habitats to pesticides in tissues of northern leopard frogs We radio tracked 72 northern leopard frogs during the summers of 2015 and 2016 . Females had a larger home range than males, but total movement and average daily movement for male frogs were greater than for female frogs  as they moved across the landscape for over 100 pesticides and pesticide degradates Fig. . The thrSeventeen pesticides  and included 2 fungicides (fenbuconazole and tebuconazole), 1 insecticide (bifenthrin), and 1 breakdown product of DDT . Thus, for this short-term study, it is likely that differences in survival relative to habitat are indicative of predation risk rather than exposure to pesticides. However, in terms of management, frogs did spend a small but measurable amount of time in agricultural fields (on average frogs spent 6% of their time in a corn or soybean field) and because the effects of pesticides are accumulative27, even short periods of time spent in habitats where pesticide exposure is high can have negative consequences over their lifetime, especially when use of agricultural fields coincides with pesticide application34.Frogs in our study spent most of their time (58%) in grasslands, the habitat where pesticide exposure was the lowest, but where predation risk to amphibians is typically higher than in wetlands2. Understanding potential exposure during hibernation is another piece of the exposome that is understudied. Amphibian skin is highly permeable, a characteristic that is magnified during hibernation, when frogs undergo physiological changes at low temperatures (<5 °C). These changes lower metabolism which reduces the metabolism of pesticides37. Liver condition and function is also lowest at the end of hibernation (early spring), which makes it more difficult for an individual to efficiently metabolize contaminants38. In our study, four pesticides  were identified in overwintered (aquatic) PSDs, with atrazine and metolachlor at the highest concentrations. These results indicate that exposure during winter hibernation is significant for northern leopard frogs.Northern leopard frogs, like many ranid species, hibernate at the bottom of lakes and ponds, resting on the substrate and covered with a thin layer of silt that allows for cutaneous uptake of Oi.e., chlorpyrifos peaked in August in all habitat types). Frogs used agricultural fields infrequently indicating that the pesticide application rates (exposure concentrations) tested in laboratory studies21 may not represent the actual environmental concentrations to which frogs are exposed in their preferred grassland habitats.In the spring, northern leopard frogs selected for aquatic habitats coincident with the application of herbicides  and then moved into terrestrial habitats  when insecticides are commonly applied  the physical-chemical properties of the pesticides (may not be accumulated), (2) metabolism/elimination by the frog (accumulated but quickly broken down), or (3) limited time spent in each habitat (slow assimilation into tissues). In contrast, two pesticides  were only identified in frog tissues. Discrepancies between pesticides identified in tissues versus the environment could be related to low environmental concentrations, limitations in the ability of PSDs to sequester more hydrophobic compounds – although this is unlikely40, or effect of maternal transfer rather than dermal uptake.A number of pesticides were identified in PSDs  that were not detected in frog tissues. Other studies have reported differences in pesticides identified in the environment (water and sediment) compared to those accumulating in tissuee.g., grass buffers). We assessed pesticide exposure in the environment and differences in the use of habitat by frogs, relative to pesticides found in their tissues, to understand how these factors interact to affect the persistence of frogs on the landscape. These data illustrate that understanding the extent and magnitude of pesticide exposure is foundational in assessing risk and developing mitigation strategies for the conservation of frogs; and our study was unique in its innovative use of silicone PSDs to facilitate these assessments. More broadly, the distances frogs moved, patterns of habitat use, and home range sizes are similar to previous telemetry studies on northern leopard frogs in the Midwestern United States42, such that our findings may be applicable to other altered  landscapes. This study provides initial steps towards quantifying the amphibian exposome and interpreting the role of habitat use in pesticide accumulation as it contributes to population declines.Our results corroborate the importance of multiple habitat types to anurans, highlighting the need to protect wetlands and surrounding habitats  appropriately sized for radio telemetry, (2) relatively abundant in our study area, and (3) similar to other anuran species in their use of both aquatic and terrestrial habitat. Northern leopard frogs are distributed widely across the United States and parts of Canada50. Frogs were located daily, from May release, until August. We recorded the habitat type where frogs were observed and marked their location with GPS (Garmin eTrex® 30). Frogs were recorded in 1 of 5 habitats: grassland ; wetland ; agriculture (corn and soybean fields); forest (large groups of deciduous trees); or developed (man-made structures such as roads). If individuals died or could not be located for >2 wks they were replaced with newly captured and telemetered individuals  and cleaned23. Three PSDs were set aside as “cleaning blanks”, frozen, and extracted with the environmental samples. Additionally, during each PSD field deployment a “field blank” was also deployed at the site to assess potential contamination. Clean latex gloves were worn when handling PSDs. In 2016, we assigned each telemetered frog  a PSD , in July–August in 2015, and June–August in 2016  in habitats where we most frequently observed our telemetered animals Fig. . We depl52. PSDs were not collected and replaced during the winter because sites were inaccessible due to ice cover.Finally, we assessed the potential pesticide exposure in overwintering habitat by placing PSDs at four aquatic locations in each wetland from November 2015 to April 2016. PSDs were zip-tied to a metal O-ring that was attached to a metal T-post driven into the substrate in 1 m of water  mode or an Agilent 1260 bio-inert LC coupled to an Agilent 6430 MS/MS. Data was collected in multiple reaction monitoring (MRM) mode with each compound having 1 quantifier MRM and at least 1 qualifier MRM. Limits of detection (LODs) for all compounds ranged from 2–5 ng/PSD.All PSDs were sent to the USGS California Water Science Center for analysis. PSDs were extracted and analyzed for over 100 pesticides and pesticide degradates. In the laboratory, PSDs were rinsed gently with deionized water to remove particulates and allowed to dry in the fume hood. Prior to extraction PSDs were weighed and placed in a pre-cleaned flask. Samples were spiked with d53. Individual liver and gonads were dissected in the field, frozen, and sent to the USGS California Water Science Center for analysis6.In 2016 we captured and euthanized individuals in mid-May prior to tracking while they were breeding and residing at the wetlands (n = 10). In August we captured and euthanized all remaining radio telemetered frogs (n = 10). Frogs were euthanized with an overdose of Tricaine Methanesulfonate (0.5 g/1 L water)5 (see Supplementary Methods).All tissues were extracted using pressurized fluid extraction and analyzed for 98 pesticides and pesticide degradates using an Agilent 7890 GC coupled to an Agilent 7000 MS/MS operating in EI mode. Method detection limits (MDLs) for all compounds ranged from 0.5 to 4.2 μg/kg wet weight54 to estimate survival probabilities of the radio tracked frogs (n = 72). We included daily habitat use  as an individual covariate to evaluate its influence on frog survival. Each frog was assigned to 1 of 8 groups that reflected site, year, and sex (see Supplementary Table c)55.We performed a known fate analysis in Program MARK (V6.1)56. We used the “kde” and “isopleth” functions in Geospatial Modeling Environment 57 using kernel density estimates.We estimated core home range (50% quantile) and total home range (95% quantile) of 41 frogs  each frog moved using the “pointdistances” function in GME, which assumes a straight-line moved between successive locations. We summed the daily distances each frog moved and divided the total by the number of days the frog was tracked to get average daily movement.58. Available habitat was calculated within a 1 km buffer42. GSRs reflect the proportion of time an animal uses each habitat relative to the proportion of that available habitat. For example, a habitat with a GSR > 1 indicates that the animal shows positive selection for that habitat.We determined the proportional use of habitat types, relative to their availability by calculating GSRs using the Manly Resource Selection function in R (V3.3.3)We combined PSD data from 2015 and 2016 and used Analysis of Variances (ANOVAs) in R to test for a difference in number of pesticides and total pesticide concentration in the PSDs among the habitat types . We also performed ANOVAs to test for a difference in number of pesticides and pesticide concentrations within habitat types over time . Data were log transformed to meet assumptions of constant variance and normality. All non-detect values received ½ MDL for statistical analyses.59 and an independent t-test to test for differences in concentration of pesticides found in individuals collected in May vs August 2016. Data were log transformed to meet assumptions of constant variance and normality. All non-detect values received ½ MDL for statistical analyses.We assessed differences in pesticide presence in frog gonad and liver tissue using a Kruskal-Wallis testData generated in this study are available at the U.S. Geological Survey data release, 10.5066/F7D799PV.Supplementary Information


The rate of CSF/ICV changes, however, was not significantly different between groups over time. CSF volume increased linearly over bilateral sensorimotor cortices  and in the supracerebellar space (p < 0.001) within two years. An acceleration of the enlargement within the third (p = 0.017) and the fourth (p = 0.006) ventricles was observed in patients over time. Sample size estimation for six-month trials with CSF volume requires 25 patients per treatment arm to detect a hypothetical treatment effect in terms of slowing of atrophy rate of 30%. This study shows that SCI-induced changes in CSF/ICV ratio and ventricular expansion rate provide additional information on the neurodegenerative processes after injury. The sensitivity to scoring treatment effects speaks to its potential to serve as a sensitive biomarker in addition to local atrophy measures.Next to gray and white matter atrophy, cerebrospinal fluid (CSF) volume and ventricular dilation may be surrogate biomarkers for brain atrophy in spinal cord injury (SCI). We therefore aimed to track brain atrophy by means of CSF volume changes and ventricular enlargements over two years after SCI. Fifteen patients with SCI and 18 healthy controls underwent a series of T1-weighted scans during five time points over two years. Changes of CSF/intracranial volume (CSF/ICV) ratio, CSF volume, and ventricular enlargement rate over time were determined. Sample sizes with 80% power and 5% significance were calculated to detect a range of treatment effects for a two-armed trial. There was a significant cross-sectional increased CSF/ICV ratio in patients compared with controls at each time point ( The neuronal mechanisms underlying neurological and functional recovery, however, are still not well understood because of the complex relationship between neurodegeneration and plasticity. The ability to track trauma-induced structural changes across the neuraxis provides the opportunity to quantify neurodegeneration in vivo3 and recovery-related plasticity,4 which may identify new treatment targets.T5 and sensory systems6 above the level of injury. Recently, a follow-up study in the same patient cohort revealed that these changes continued for at least two years post-SCI.7 Crucially, the magnitude of neurodegeneration at the level of the spinal cord, brainstem, and cortex over the first six months predicted clinical outcome at two years, independent of early clinical changes.7After SCI, serial magnetic resonance imaging (MRI) studies revealed progressive neurodegeneration, both in the gray matter (GM) and white matter (WM), along the entire trajectory of the motor8 Parkinson disease,9 Huntington disease,10 and traumatic brain injury (TBI).11 For instance, early ventricular dilatation was observed in the course of significant cognitive decline in patients with Parkinson disease.9 In addition, it has been shown that the CSF/ICV ratio, besides its potential to quantitate general brain atrophy,12 does not depend on sex and therefore may be used in mixed sex studies as well.13Nevertheless, potential neuroimaging biomarkers for global brain atrophy such as intracranial volume (ICV), cerebrospinal fluid (CSF) volume changes, and ventricular enlargement were not investigated. ICV, CSF volume, and ventricle enlargement measurements are reliable morphometric features to determine atrophy patterns in patients with mild cognitive impairment, Alzheimer disease,14–17 Chronic inflammation is associated with neurodegeneration and hence could lead to volumetric fluctuations in brain tissue.19 Interestingly, measures of global brain atrophy as well as longitudinal MRI findings are now used as surrogate end-points in clinical trials19 next to measures of focal GM and WM neurodegeneration to complement clinical assessments for disease-modifying trials. It remains to be established, however, whether MRI-derived measurement of global brain atrophy and ventricle expansions (both are reflected by increases in CSF volume) are sensitive and accurate in identifying disease-related changes in patients with SCI.The rationale for the use of these markers of global brain atrophy measures in SCI arises as the trauma to the spinal cord triggers a cascade of inflammatory processes that spreads across the central nervous system (CNS).7 To track brain atrophy, we applied longitudinal Voxel Based Morphometry (VBM)21 on serially acquired high resolution T1-weighted MR images. We estimated the sample sizes for a six-month trial of CSF volume that might inform the design of future clinical trials to detect a range of treatment effects with 80% statistical power.The aim of this study was to investigate the trajectory of progressive global brain atrophy  and local brain atrophy  over two years in the same SCI patient cohort that previously showed enduring neurodegenerative changes in the cortical GM and WM after SCI.22 at baseline and at two months, six months, 12 months, and 24 months follow-up.The longitudinal study was approved by the local ethics committee of Zurich (EK-2010-0271), and written informed consent was obtained from each subject before the examination. Fifteen patients with SCI  was acquired at 49.67 (± 22) days post-injury, the second scan at two, the third scan at six, the fourth scan at 12, and the fifth scan at 24 months after injury. The images on the first four time points were acquired using a 3T Magnetom Verio , and for measurements on the fifth time point, the scanner was upgraded to a 3T Magnetom Skyrafit. We used a 16-channel radiofrequency receive head and neck coil in combination with a spine matrix coil.The 3D-MPRAGE sequence comprises the following parameters: field of view = 224 × 256 mm21Trained radiographers positioned all participants in the same supine position for each scan. Image acquisition over five time points was completed successfully in 14 patients and in 18 healthy controls. One patient died of causes unrelated to SCI after the second time point. A total of 156 MRI datasets were analyzed from 33 participants. All T1-weighted 3D-MPARGE images acquired from subjects were included in the VBM analysis.23 and the total ICV was expressed as the sum of volumes of all tissue classes. A previously established global measure of CSF volume is the CSF volume-to-ICV (CSF/ICV) ratio, calculated as the CSF volume divided by total brain volume  to adjust for intersubject differences in brain size.24 CSF/ICV ratio is used as a global atrophy marker associated with CSF volume.Global tissue volumes for GM, WM, and CSF at each time point were calculated using the segmented T1-weighted images with applying unified segmentation,20 Non-linear template generation and image normalization were performed using a geodesic shooting procedure.25 The template was affine registered to the standard brain template from Montreal Neurological Institute for all subsequent modeling steps. Consecutively, normalized CSF tissue segments from all subjects and time points were modulated by the Jacobian determinants encoding individual volume changes over time. Morphometric images were smoothed using Gaussian kernels of 6 mm full width at half maximum. Subsequent modeling and analysis were performed for smoothed, normalized CSF segments within specific brain areas.To assess local change of CSF volume over time, longitudinal VBM was applied within SPM12 (Wellcome Trust Centre for Neuroimaging). Diffeomorphic registration was applied for longitudinal MRI, and resulting midpoint images were segmented,To statistically assess cross-sectional and longitudinal changes of the CSF/ICV ratio, we used pairwise comparisons for each time point and linear mixed effects models with a group indicator and group by time interaction to assess changes over time using STATA 14 .0 + β1 t + β2 t2 and obtained intercepts (β0), rate of change (β1), quadratic effects (β2), and time since injury (t) for all subjects in the sample independently. In a second stage, we used two-sample parametric t tests , comparing the parameters across clinical groups, while adjusting for age and sex as covariates of no interest. Group differences of linear  and quadratic  effects were assessed using random field theory for correction of multiple comparisons within each considered ROI.To assess group differences in trajectories of local CSF volume and ventricular enlargements, we followed a conservative two-stage summary statistics approach commonly used in fMRI and longitudinal image analysis. In a first stage, we estimated individual quadratic trajectory models y (t) = βp values were corrected for multiple comparisons using family-wise error correction, and cluster significance was tested (after applying a cluster-forming–threshold of 0.001), using Gaussian random field theory. Regression models were applied to determine associations between CSF volume and ventricular expansion and clinical outcomes over two years of follow-up. The mean age difference between patients and controls was not found to be statistically significant . However, age was included as a covariate of no interest in all statistical tests.The associated 26 The required Pearson correlation coefficient between baseline data and six-month CSF volume was estimated using the available data. The associations between MRI readouts  and clinical outcomes were investigated using regression model in SPM 12 and Stata 13.For CSF volume, we applied the six-month effect size to calculate estimates of the sample sizes necessary to detect a 100% treatment effect with 80% statistical power and 5% significant differences between healthy controls and patient group, by use of the standard formula based on two-group trials, assuming a baseline adjusted comparison of mean .7 The mean scan intervals between time of injury and imaging baseline, two, six, 12, and 24 months were 49.67 (standard error of the mean [SE] 5.91), 103.5 (SE 12.40), 220.36 (SE 18.69), 389.93 (SE 29.60), and 881.14 (SE 43.07) days, respectively.The local WM and GM changes over two years were estimated previously from these 33 participants, and the results have been presented before.p < 0.01). The ratio remained constantly increased , but the linear slope of rate of change was not significantly different between patients and controls over time  in patients relative to controls while accounting for age and sex. Over 24 months, local CSF volume increased linearly  over bilateral sensorimotor cortices  and within the left supracerebellar space  and fourth  ventricle enlargements accelerated (positive quadratic effect) in patients compared with the controls  and clinical outcomes showed no significant correlations between MRI parameters and clinical outcome measures.7 Sample size calculations demonstrated the sensitivity of acute CSF neuroimaging biomarkers to render them viable candidates for scoring the effects of treatment, including rehabilitation.This longitudinal study shows that progressive global brain atrophy is evident—next to GM and WM atrophy as reported in the same cohort—early after SCI. At baseline, the CSF/ICV ratio had increased already and remained so over time without substantial fluctuations while changes of CSF volume over the sensorimotor cortex and within the supracerebellar space showed sustained increases. Interestingly, the rate of expansions of the third and fourth ventricle showed linear and non-linear changes  over time. These dynamic volumetric expansions may be indicative of enduring neurodegenerative processes within the cortical GM and WM as reported in this cohort before over two years post-SCI.5–7 focused previously on progressive GM and WM atrophy after SCI but did not address global measures of brain atrophy such as the CSF/ICV ratio, local CSF volume, and ventricular expansion. Although non-specific to any single disease process, global measures of brain atrophy have proven sensitive in tracking disease processes and in picking up treatment-induced changes in neurodegenerative diseases,8–10 thus rendering them viable tools in clinical trials.10 Moreover, a previous MRI study on 123 patients with TBI showed that TBI results in the expansion of CSF spaces, particularly in the temporal horns and third ventricle, which preceded subsequent reduction in total brain volume.11 A gradual process of diminishing arborisation of surviving neurons as a result of disruption of neuronal circuitry might be one possible explanation for this observation.11Several studies7 The reported focal GM changes within the sensorimotor cortices (restricted to the leg area) and thus at the border to the CSF27 might be because of increases in CSF volume, however, which had been assigned falsely to GM during segmentation.In the current SCI cohort, next to baseline differences of the CSF/ICV ratio and enlarged third and fourth ventricle, linear increases of CSF volume were detectable in the supracerebellar space and bilaterally over the sensorimotor cortices. The increase of CSF volume over the sensorimotor cortices might reflect active neurodegeneration within the GM in the output deprived leg representing area of primary motor cortex.28 Crucially, patients with depression showed a higher CSF volume in other neurodegenerative diseases,29 which may also be the case in SCI.16 Because these symptoms have been associated with brain atrophy in other diseases,29 it seems likely that global brain atrophy in our cohort could be associated with signs of cognitive impairment. In particular, we have shown previously that the limbic system in this cohort shows signs of neurodegeneration.6Cognitive decline, anxiety, and depression are reported to be elevated in patients with SCI when compared with the normal population.10 Changes in imaging outcomes in response to treatment might not always be accompanied by functional improvements, and clinical function might sometimes improve in the absence of changes in imaging measures.10We did not anticipate that the global measures of the CSF changes were related to specific functional measures of recovery. This finding is in line with a previous study in Huntington disease in which no significant correlations were detected between ventricular volume changes and clinical measures.Thus, although anticipated, our results provide no evidence that the severity of trauma is related to the rate of change of CSF measures. This is interesting because it also points to the fact that a focal CNS injury, such as SCI, might induce a cascade of secondary neurodegenerative events that progress with a distinct time profile. The conjoint analysis of CSF volume has the potential to elucidate whether the chronic inflammatory process within the CSF or the WM and GM is the main factor driving the observed changes in our study cohort.30 Hypothetical treatment effects, defined by slower longitudinal structural changes in these imaging measures, could be detectable over a realistic time scale with significantly lower sample sizes than required for traditional clinical readouts.31 Thus, these objective outcome measures hold considerable promise for quantifying the effects of treatment. In short, quantitative MRI biomarkers of neurodegeneration therefore represent promising instruments for the stratification of patient cohorts and the improvement of trial efficiency.30Based on the estimated longitudinal effect sizes of CSF volume, we recommend CSF volume as an outcome measures to power clinical trials in SCI. These MRI-based measures may afford the opportunity to assess site-specific effects of intervention, essential for the translation of trial efficacy into clinical effectiveness.A limitation of the study is the relatively small number of participants that were recruited. In total, however, 156 data points from patients and controls were included in the analysis and the summary statistics used maximize the efficiency because all data points were included. Hypothetical treatment effects defined by slower longitudinal structural changes in these measures would be detectable over a realistic time scale with practical sample sizes and be useful in monitoring trauma-induced changes before, during, and after treatment. Our sample size calculations are made under several assumptions. We do consider full compliance, no dropout, any possible effects of treatment response heterogeneity, all of which might inflate within group variance. The impact of these confounds, however, is an order of magnitude smaller than the sample sizes that we present.33 and global brain atrophy should be addressed next.We found that CSF/ICV ratio, CSF volume, and ventricular enlargement rate are sensitive to neurodegenerative changes in SCI by way of group differences between patients and healthy controls. The sensitivity to scoring treatment effects speaks to its potential to serve as a sensitive biomarker in addition to local GM and WM atrophy measures. The link between inflammatory effects detectable within the CSF


Spermatogenesis, which is the complex and highly regulated process of producing haploid spermatozoa, involves testis-specific transcripts. Recent studies have discovered that long noncoding RNAs (lncRNAs) are novel regulatory molecules that play important roles in various biological processes. However, there has been no report on the comprehensive identification of testis-specific lncRNAs in mice.We performed microarray analysis of transcripts from mouse brain, heart, kidney, liver and testis. We found that testis harbored the highest proportion of tissue-specific lncRNAs . Testis also harbored the largest number of tissue-specific mRNAs among the examined tissues, but the proportion was lower than that of lncRNAs . We categorized the testis-specific lncRNAs and found that a large portion corresponded to long intergenic ncRNAs (lincRNAs). Genomic analysis identified 250 protein-coding genes located near (≤ 10 kb) 194 of the loci encoding testis-specific lincRNAs. Gene ontology (GO) analysis showed that these protein-coding genes were enriched for transcriptional regulation-related terms. Analysis of male germ cell-related cell lines  revealed that some of the testis-specific lncRNAs were expressed in each of these cell lines. Finally, we arbitrarily selected 26 testis-specific lncRNAs and performed in vitro expression analysis. Our results revealed that all of them were expressed exclusively in the testis, and 23 of the 26 showed germ cell-specific expression.This study provides a catalog of testis-specific lncRNAs and a basis for future investigation of the lncRNAs involved in spermatogenesis and testicular functions.The online version of this article (10.1186/s12864-018-4931-3) contains supplementary material, which is available to authorized users. Spermatogenesis is the complex and tightly regulated differentiation process through which haploid spermatozoa are produced in the seminiferous tubules of the testis. This process can be divided into three successive phases: the mitotic division of spermatogonia, the meiosis of spermatocytes and the morphological change of spermatids during spermiogenesis . It is eLncRNAs are arbitrarily defined as noncoding RNAs longer than 200 nucleotides. Compared with protein-coding transcripts, they tend to be shorter and have lower expression levels, fewer exons, less sequence conservation and more tissue and cell type-specific expression patterns –11. StudRecent studies have identified lncRNAs in the mouse testis and examined their biological roles during male germ cell development –27. HoweHere, we used microarray analysis to profile the previously annotated lncRNAs and mRNAs expressed in various mouse tissues, including testis. By comparing the lncRNA and mRNA expression profiles among the tissues, we identified lncRNAs and mRNAs that showed differential expression in each tissue. We found that testis has the largest number of tissue-specific lncRNAs. We also profiled lncRNA and mRNA expression in three mouse male germ cell-related cell lines . To the best of our knowledge, this is the first study to identify mouse testis-specific lncRNAs and analyze their characteristics. Our data provide a valuable resource for future investigation of the lncRNAs that are involved in male germ cell development and testicular functions.To identify lncRNAs and mRNAs expressed in major tissues  in ICR mice and germ cell-related cell lines F9, GC-1 and GC-2) in mice, we performed a microarray analysis using the Arraystar mouse lncRNA microarray V3.0, which contains probes for 35,923 lncRNAs and 24,881 mRNAs  and mRNAs (1090) among the analyzed tissues  and mRNAs  were observed in testis compared to the other tissues, suggesting that testis is characterized by a high level of transcriptomic diversity and complexity. To perform tissue-specific expression profiling, we compared the expression levels of transcripts among the five tissues across three microarray experiments and classified the transcripts as present (P), marginal (M) or absent (A) of which expression level was lower than the background level. We regarded a transcript as a tissue-specific lncRNA or mRNA with the expression showing a fold-change ≥5 and 4,256 andes Table . We specTnp1) gene, which encodes a testis-specific mRNA [To validate our microarray-based selection of tissue-specific lncRNAs, we performed quantitative real-time PCR (qRT-PCR) on several arbitrarily selected tissue-specific lncRNAs Fig. . As contfic mRNA . The tiscis-regulation between the two groups of testis-specific transcripts.To characterize the genomic nature of the identified testicular lncRNAs and mRNAs, we examined the chromosomal localization of the loci that expressed these transcripts. Our analysis revealed that they were widely distributed throughout the mouse chromosomes. Chromosome 5 and the X chromosome contain the highest numbers of loci for testis-specific lncRNAs and mRNAs, respectively Fig. . To furtWe categorized all of the identified lncRNAs based on the position and direction of their genomic regions relative to nearby mRNAs. The lncRNAs were classified into six subtypes: exonic sense, exonic antisense, intronic sense, intronic antisense, intergenic and bidirectional Fig. . If an lcis-regulatory manner [cis-regulatory targets of 829 testis-specific lincRNAs by searching for protein-coding genes within 10 kb up- and downstream of their encoding loci. We found that 23.4% (194 of 829) of the lincRNAs co-localized with 250 protein-coding genes , suggesting the expression regulation of these genes by the lincRNAs through transcriptional or post-transcriptional mechanisms. Gene Ontology (GO) [P-value ≤0.05), including regulation of transcription DNA-templated (GO:0006355), transcription DNA-templated (GO:0006351), positive regulation of transcription from RNA polymerase II promoter (GO:0045944), negative regulation of transcription from RNA polymerase II promoter (GO:0000122) and cell differentiation (GO:0030154)  of the mouse testis-specific lncRNAs were partially conserved in the human genome  to examine their distribution in 10 mouse tissues. Our results showed that all 26 of the testis-specific lncRNAs were transcribed only in the mouse testis  mutant mice [W/Wv mutant mice  [Eif2s3y) [cis-regulatory mechanism, and thus identified and examined 250 protein-coding genes within the 10-kb regions up- and downstream of 194 testis-specific lincRNAs. Our GO analyses revealed that these genes were mainly involved in regulating transcription. It seems possible that the testis-specific lincRNAs could contribute to spermatogenesis through such function. Interestingly, some of the identified cis-acting target protein-coding genes are known to be involved in spermatogenesis , raising the possibility that the testis-specific lincRNAs could play direct regulatory roles in male germ cell development. Alternatively, they may act in trans by targeting protein-coding genes located on the other mouse chromosomes. To answer whether testis-specific lincRNAs indeed regulates the expression of these protein-coding genes requires further investigation.We observed that the testis-specific lncRNAs and mRNAs were enriched on the Y chromosome relative to the other mouse chromosomes. Genes of the mammalian Y chromosome are known to play important roles in sex determination   and sperEif2s3y) , suggestEif2s3y) . We hereEif2s3y) . We hypoAlthough it was previously reported that lncRNAs have a very low sequence conservation among species , we usedWe used microarray analysis to examine the expression profiles of the selected lncRNAs and mRNAs in the cell lines, F9, GC-1 and GC-2 , which exhibit germ cell characteristics. A total of 128, 143 and 126 testis-specific lncRNAs and 187, 167 and 175 testis-specific mRNAs were found to be expressed in the F9, GC-1 and GC-2 cell lines, respectively. Fifty five lncRNAs and 105 mRNAs were expressed in an overlapping manner in all of the cell lines. It is possible that these transcripts are expressed during the whole period of spermatogenic stages from which the cell lines were derived. It should be noted that these cell lines do not possess all the characteristics and phenotypes of male germ cells, such as meiosis and spermiogenesis, suggesting weak and/or no expression of some of the testis- or germ cell-specific transcripts in the cell lines. Nonetheless, these cell lines could prove useful for studying the expression and functions of certain testis-specific lncRNAs.We arbitrarily selected 26 of the novel testis-specific lncRNAs and examined their tissue distributions in 10 mouse tissues. All of the examined lncRNAs were, indeed, specifically expressed in the mouse testis. Additionally, both developmental expression analysis during the first round of spermatogenesis and expression analysis in germ cell-less mutant mice confirmed the germ cell-specificity of these lncRNAs. Interestingly, 16 of the 26 selected lncRNAs were expressed between 16 and 21 days after birth in mouse, suggesting that these lncRNAs undergo a transcriptional change between the late spermatocyte and haploid round spermatid stages. During this period, epigenetic regulation  occurs actively in testis . As lncRWe herein used microarray analysis to identify and characterize lncRNAs in mice, and then focused on comprehensively cataloging the lncRNAs specifically found in the testis, which showed the highest proportion of tissue-specific expression. Our findings provide a basis for further investigating the functions of testis-specific lncRNAs, which should improve our understanding of the lncRNA-mediated regulatory mechanisms that may be associated with mouse germ cell development.2. Three biological replicates were performed in all cases, and samples were stored at − 80 °C until use.All animal experiments were performed in accordance with Korean Food and Drug Administration (KFDA) guidelines. Protocols were reviewed and approved by the Institutional Animal Care and Use Committees (IACUC) of Gwangju Institute of Science and Technology (GIST) (permit number: GIST-2017-013). We used 8-week-old ICR male mice  for our microarray analysis. Mouse brain, heart, kidney, liver and testis tissues were obtained and immediately frozen in liquid N2 in Dulbecco’s modified Eagle’s medium (DMEM)  supplemented with 10% fetal bovine serum . The culture dishes for the F9 cells were pre-coated with 0.1% gelatin. Because retinoic acid (RA) is known to induce F9 cell differentiation [F9 (CRL-1720), GC-1 (CRL-2053) and GC-2 (CRL-2196) cells of mouse origin were obtained from the American Type Culture Collection  and maintained at 37 °C and 5% COntiation , it was Total RNA from five major mouse tissues and three mouse germ cell-related cell lines was isolated with an RNeasy Plus Mini kit  according to the manufacturer’s instructions. Total RNA was quantified and checked for quality using an ND-1000 spectrophotometer . RNA degradation and DNA contamination were examined by 1% denaturing agarose gel electrophoresis. RNA samples with RNA Integrity Number (RIN) scores > 8 were used for microarray analysis. Each 1 μg of total RNA was reverse-transcribed into cDNA using a MMLV-RT kit (Life Technologies). The cDNA was transcribed and labeled with Cyanine 3-CTP (Cy3) using a Quick-Amp labeling kit  according to the manufacturer’s protocol. The labeled cRNAs were quantified using an ND-1000 spectrophotometer.P-value ≤0.05. The raw and processed microarray data have been deposited in the Gene Expression Omnibus (GEO) database at the National Center for Biotechnology Information (NCBI), under GEO accession number GSE105024.Total cRNA was hybridized to a Mouse LncRNAs Microarray  using a hybridization oven (Agilent). The hybridized microarray chip was washed according to the manufacturer’s protocol, and the hybridized images were scanned using a DNA microarray scanner and quantified with the Feature Extraction Software (both from Agilent). Data normalization and selection of fold-changed transcripts were performed using GeneSpring GX 7.3 (Agilent). Total lncRNAs and mRNAs were detected in three independent sets of the samples in each tissue; those above background were flagged as present or marginal . For each tissue, specifically expressed lncRNAs and mRNAs were chosen using the following criteria: 1) in three independent samples, they were flagged as P or M in a single given tissue but absent (A) in the other tissues; 2) fold-change ≥5; and 3) 2O to a final volume of 20 μl. The reactions were performed on a StepOnePlus Real-Time PCR System (Thermo Fisher Scientific) as follows: 95 °C for 12 min 30 s, followed by 40 cycles of 95 °C for 10 s, 60 °C for 15 s and 72 °C for 23 s. The melting curve analysis was performed from 65 °C to 95 °C with increments of 0.3 °C. The primer sets used to amplify the selected lncRNAs and control genes are listed in Additional file -ΔΔCt method [Gapdh mRNA.Total RNA was extracted from five mouse tissues and three germ cell-related cell lines, and reverse-transcribed using RNeasy Plus Mini and Omniscript RT kits (Qiagen). qRT-PCR was carried out using the TOPreal™ qPCR 2X premix . The reaction volume contained 10 μl of TOPreal™ qPCR 2X PreMix, 1 μl of 10 μM forward and reverse primers, 50–100 ng of template cDNA and dHt method , and norcis-regulatory mechanism [P-value ≤0.05 were considered significantly enriched.As several lincRNAs act as through a echanism , 30, we echanism . GO termW/Wv mutant mice to investigate whether these lncRNAs were expressed in somatic cells of testis [Gapdh) were used as a loading control.To determine the tissue distribution patterns of 26 testis-specific lncRNAs arbitrarily selected from our microarray analysis, we performed RT-PCR using cDNA from 10 mouse tissues . We also used cDNA from germ cell-lacking testes of f testis . To invet-test, and P ≤ 0.05 was taken as indicating a significant difference. Data are presented as the mean ± standard error of the mean (mean ± SEM).All experiments were performed in triplicate for each sample. Statistical analysis was performed using the Student’s Additional file 1:Table S1. LncRNAs and mRNAs in the mouse brain, heart, kidney, liver and testis. (XLSX 18613 kb)Additional file 2:Table S2. Tissue-specific lncRNAs and mRNAs in the mouse brain, heart, kidney, liver and testis. (XLSX 4020 kb)Additional file 3:Table S3. The numbers and ratios of testicular lncRNAs and mRNAs on each mouse chromosome. (XLSX 11 kb)Additional file 4:Table S4. The numbers and ratios of six types of testicular lncRNA. (XLSX 10 kb)Additional file 5:Table S5. Nearby protein-coding genes located within genomic regions 10 kb up- and downstream of the testis-specific lincRNAs. (XLSX 24 kb)Additional file 6:Table S6. GO terms of the nearby protein-coding genes. (XLSX 19 kb)Additional file 7:Table S7. Mouse testis-specific lncRNAs that show conservation in human. (XLSX 26 kb)Additional file 8:Table S8. LncRNAs and mRNAs in three mouse germ cell-related cell lines . (XLSX 16402 kb)Additional file 9:Table S9. Testis-specific lncRNAs and mRNAs in three mouse germ cell-related cell lines . (XLSX 712 kb)Additional file 10:Table S10. Primers used for qRT-PCR. (XLSX 10 kb)Additional file 11:Table S11. Primers used for RT-PCR. (XLSX 11 kb)


After publication of this supplement , it was Introduction We have previously demonstrated that intracranial pressure (ICP) is elevated ~ 24 h after small-moderate ischaemic strokes in rats. Oedema or cerebral blood volume increase was not the primary cause of this ICP rise, suggesting a role for cerebrospinal fluid (CSF) volume increase.Objective To determine whether resistance to CSF outflow is responsible for ICP elevation post ischaemic stroke in rats.Methods Outbred male Wistar rats (aged > 12 weeks) were subjected to photothrombotic stroke or sham procedure, and ICP was measured from 18 h post-stroke using a fibre optic pressure sensor probe  inserted through a burr hole to access the epidural space. Another burr hole was made, and a catheter was inserted into the left lateral ventricle. Artificial CSF was then infused at rates that were increased step-wise. Resistance to CSF outflow (Rout) was determined by modifying the original constant rate infusion technique [Davson et al. 1970] to establish a continuous, low infusion rate (up to 30 μl/min) method.Results Photothrombotic stroke technique resulted in reproducible, small-moderate sized infarcts with well-defined boundaries. ICP was significantly higher (p = 0.0002) in rats subjected to stroke compared to sham animals (9.8 ± 1.1 mmHg (n = 11), and 3.3 ± 0.4 mmHg (n = 10), respectively) 18 h after intervention. CSF Rout was significantly increased (p = 0.0004) in rats subjected to stroke compared to the sham group . Values are mean ± SD.Conclusion CSF volume is very hard to measure accurately, but is dependant on CSF production and outflow. Our previous preliminary data showed that CSF production rates were not significantly different between stroke and control groups. The results from the current study strongly suggest that resistance to CSF outflow is increased in rats subjected to stroke compared to sham animals. These data support our hypothesis that ICP elevation post-stroke is most likely due to CSF volume increase caused by reduced CSF outflow and not because of increased CSF production.Grant support Supported by a NSW Health EMC Fellowship to AP and the NHMRC (NS).


To the EditorWe read with great interest the recent article “Is the absence of Right Hepatic Vein opening into Inferior Vena Cava a contraindication for right lobe liver donation in Living Donor LiverTransplantation? Common hepatic venous trunk—A rare hepatic vein anomaly: A case report and review” published by Ray et al. . The autFirst, the authors present the presence of inferor right hepatic veins as a vascular anomaly. We strongly disagree with this opinion of the authors. According to my experience from a liver transplant center, more than half of both recipients and donors have one or more inferior right hepatic veins, which is supported by the literature ,3. ThereSecondly, the authors no provided any information about back-table venous drainage reconstruction model, graft implantation techniques, and whether the recipient experienced any complications related to postoperative venous drainage.Thirdly, the presence of variations or anomalies in the vascular structures of the liver and their incidental detection during radiological examinations mean nothing in clinical terms. Because these patients are often asymptomatic. In our opinion, the most important issue is how to proceed if vascular anomaly or variation is detected in living liver donor and whether these donor candidates will be refused. The answer to these questions depends on whether the recipient has a chance to find another living liver donor and the transplant center's experience on vascular reconstruction.When the title of the study is examined, it is seen that the article is prepared as case report and literature review. Moreover, when the text of the article is examined, it is understood that such an anomaly has not been identified in the living liver donor before. However, this is not true. The first case of congenital right hepatic vein absence in the living liver donor was reported by our team .No conflict of interest about this letter to the editor.No received funding.No required ethical approval.This work was written in the form of ”letters to the editor”. Therefore, no patient was presented.This letter was designed and written by Akbulut.No.No.Editorially reviewed, not externally peer-reviewed.


Beta-cell replacement is the best therapeutic option for patients with type 1 diabetes. Because of donor scarcity, more extended criteria donors are used for transplantation. Donation after circulatory death donors (DCD) are not commonly used for pancreas transplantation, because of the supposed higher risk of complications. This review gives an overview on the pathophysiology, risk factors, and outcome in DCD transplantation and discusses different preservation methods.Studies on outcomes of DCD pancreata show similar results compared with those of donation after brain death (DBD), when accumulation of other risk factors is avoided. Hypothermic machine perfusion is shown to be a safe method to improve graft viability in experimental settings.DCD should not be the sole reason to decline a pancreas for transplantation. Adequate donor selection and improved preservation techniques can lead to enhanced pancreas utilization and outcome. In a select group of patients with type 1 diabetes mellitus (DM) with severe complications, beta-cell replacement by either pancreas or islets of Langerhans transplantation is the treatment of choice, leading to restoration of normoglycemia, reduction of long-term diabetes complications, and improved quality of life . Resultsp = 0.001) for pancreas acceptance: pancreata from donors with P-PASS > 17 were three times more likely to be discarded = 0.003)  and evenNowadays, protocols for DCD pancreas preservation are quite similar to DBD protocols and are based on the principle of reducing cellular metabolism by lowering the temperature of the organ by SCS. With every 10 °C drop in temperature, cellular metabolism decreases two- to threefold, thereby leading to reduced oxygen and ATP use and reduction of ischemic injury . HoweverPreservation solutions have been developed to counteract ischemic injury . These aIn 1988, the two-layer method (TLM) was developed by a Japanese group with the intention to improve pancreas viability during preservation for islet isolation. This technique involves the addition of perfluorocarbon (PFC) to the preservation solution, in order to combine the characteristics of the preservation solution and PFC together to prevent injury and supply oxygen during preservation. PFC is an inert solution with a high capacity for dissolving oxygen . By addiIn organ persufflation, humidified and filtered oxygen is bubbled directly to an organ via its vasculature. Oxygen persufflation can either be performed anterograde (through the arteries) or retrograde . The technique was first discovered coincidentally in 1902 when compressed oxygen instead of blood was perfused through a feline heart, leading to continuation of heart contractility . In the Hypothermic machine perfusion (HMP) is a technique which enables continuous circulation of the microvasculature with cold preservation solution, removing toxic metabolites and supporting ATP synthesis by delivery of oxygen and adenosine . HMP hasEarly studies on HMP of canine pancreata concluded that pancreas storage time could be extended up to 24 h, while retaining viability  and funcIn the first study on human pancreata, four human donor pancreata were perfused for 4 h by HMP after 13 h of SCS, resulting in an adequate amount of islets that could be isolated with excellent in vitro viability . In a reNormothermic machine perfusion (NMP) can be used to improve organ quality during preservation and facilitate the administration of drugs, and it allows viability testing in a physiological environment prior to transplantation . In expeDespite a growing incidence in type 1 DM, pancreas transplantation rates are declining worldwide. As beta-cell replacement is the best therapeutic option for these patients, better assessment of possible pancreas donors is needed to expand the donor pool without compromising the outcome. This overview shows that excellent results can be achieved with DCD pancreas transplantation, when other detrimental donor characteristics are kept to a minimum. Donor selection tools are developed in order to assess potential pancreas donors. PDRI did show to have a predictive value in outcome; however, conflicting cutoff points have been found in different studies. PDRI can be used as a tool to estimate the risk of a certain donor pancreas; nevertheless, other transplant and recipient factors should be kept in consideration. DCD should not be the sole reason to decline a pancreas for transplantation, though accumulation of risk factors in these donors should be avoided. Hypothermic oxygenated machine perfusion seems a promising preservation method for these organs, as it safely replenishes ATP concentrations.


A cerebrospinal fluid (CSF)-mask algorithm has been developed to reduce the adverse influence of CSF-low-counts on the diagnostic utility of the specific binding ratio (SBR) index calculated with Southampton method. We assessed the effect of the CSF-mask algorithm on the diagnostic performance of the SBR index for parkinsonian syndromes (PS), including Parkinson’s disease, and the influence of cerebral ventricle dilatation on the CSF-mask algorithm.123I-Ioflupane single-photon emission computed tomography (SPECT) images of these patients. We compared the diagnostic performance of the corresponding indices and evaluated whether the effect of the CSF-mask algorithm varied according to the extent of ventricle dilatation, as assessed with the Evans index (EI). A receiver-operating characteristics (ROC) analysis was used for statistical analyses.We enrolled 163 and 158 patients with and without PS, respectively. Both the conventional SBR (non-CSF-mask) and SBR corrected with the CSF-mask algorithm (CSF-mask) were calculated from p < 0.001; attenuation correction, AUC = 0.930 [95% CI 0.902–0.957] vs. 0.903 [95% CI 0.870–0.936], p < 0.001). When not corrected for attenuation, no significant difference in the AUC was observed in the low EI group between the non-CSF-mask and CSF-mask algorithms ; in the middle and high EI groups, the CSF-mask algorithm performed better than the non-CSF-mask algorithm . When corrected for attenuation, significant differences in the AUC were observed in all three EI groups .ROC analyses demonstrated that the CSF-mask algorithm performed better than the non-CSF-mask  0.887–0.947] vs. 0.895 [95% CI 0.861–0.929], The CSF-mask algorithm improved the performance of the SBR index in informing the diagnosis of PS, especially in cases with ventricle dilatation. Dopamine transporter (DAT) single-photon emission computed tomography (SPECT) is an imaging modality that can effectively differentiate neurodegenerative parkinsonian syndromes (PS), including Parkinson’s disease (PD) and dementia with Lewy bodies (DLB) from other neurological disorders not characterized by dopaminergic degeneration, such as Alzheimer disease, drug-induced Parkinsonism, and essential tremor , 2. HoweTossici-Bolt et al. developed the Southampton method, a semi-quantitative method based on the volume of interest (VOI), that has gained widespread use. Specifically, the method applies a large pentagonal prism-shaped VOI setting that encompasses a wide area around the striatum , therebyRecently, the CSF-mask algorithm has been developed to reduce the aforementioned influence of CSF-low-counts . Camicion = 14 and 177, respectively) or insufficient image quality (n = 17), which included cases with cerebral hemorrhage or brain infarction. Concerning the 177 patients who were clinically undiagnosed cases, they did not meet the diagnostic criteria for any disease finally. In addition, some patients who were difficult to follow due to hospital change were also excluded. Of the remaining 321 patients , 163 with PS and 158 without PS (NPS) were included in the assessment of the accuracy of the SBR index for diagnosing PS . Of the 163 patients with PS, 114 were diagnosed with PD based on the clinical diagnostic criteria of the UK Parkinson’s disease society brain bank [n = 22), multiple system atrophy (MSA) (n = 11), and progressive supranuclear palsy (n = 16), on the basis of established diagnostic criteria [n = 92, 92, and 91, respectively). A flow diagram presents the number of participants at each stage of the study .123I-Ioflupane (185 MBq). The imaging parameters were as follows: matrix size, 128 × 128; pixel size, 4.4 mm; slice thickness, 4.4 mm; and energy window, 159 keV ± 10%. The projection data acquired for 30 min were reconstructed on a Xeleris workstation . The ordered-subset expectation maximization method  and a Butterworth filter  were applied to the SPECT images. Both data with and without attenuation correction were generated . Scatter correction was not used.Using the Discovery NM/CT 670 or Discovery NM 630  mounted with a FAN beam collimator, SPECT images were acquired 3 h after the injection of We used a commercially available software package for VOI based analysis: DaTView . This software enables the semi-automatic calculation of the SBR index based on the Southampton method and mounts a function of the CSF-mask algorithm.σ are the median and standard deviation, respectively, of the background histogram with a Gaussian fit normal distribution and k is a coefficient. Previous reports informed our use of 1.0 as the k value [The CSF-mask algorithm is a threshold process that eliminates low counts caused by brain atrophy or ventricular dilatation. In the CSF-mask algorithm, the threshold is defined as follows: k value , 10.The SBR with the CSF-mask algorithm is calculated according to Southampton method as follows:Vs is the striatal volume (assumed to be 11.2 ml); CtcVOI represents the total counts in the striatal VOI, excluding the threshold value; Ccr is the count concentration in the reference background, excluding the threshold value; and VcVOI is the volume of the striatal VOI, excluding the threshold volume.Where We calculated the SBR indices with and without CSF-mask correction according to the method described above. Figure n = 119) or MR (n = 156) images obtained within 6 months of the DAT SPECT examination. The calculated EIs informed the delimitation of the enrolled cases into three approximately equally sized groups: low EI, middle EI, and high EI.We assessed ventricular size using the EI, which is usually used to diagnose normal pressure hydrocephalus . The EI t test was used for comparisons of age and sex distribution. The Mann-Whitney U test was used to compare the SBR indices between the PS and NPS groups. A receiver-operating characteristics (ROC) analysis was performed to evaluate the area under the curve (AUC). The DeLong method was used to examine the difference between the two AUCs [p values of < 0.05 (two-sided) were considered to be statistically significant.The Fisher’s exact test or two AUCs . The sent test, and the Mann-Whitney U test were performed using SPSS software . The ROC analysis was performed using the statistical package R .The Fisher’s exact test, the p < 0.001, Fig. p < 0.001, Fig. p < 0.001, Fig. p < 0.001, Fig. Table p < 0.001, Fig. p < 0.001, Fig. Figure Table n = 92), 0.279 , and 0.325 , respectively. Figure p = 0.11; Fig. p < 0.05, Fig. p < 0.01, Fig. p < 0.05, Fig. p < 0.005, Fig. p < 0.005, Fig. Table The CSF-mask algorithm has been developed to reduce the influence of CSF-low-counts . In thisOur overall analysis demonstrated that the diagnostic accuracy of the SBR with the CSF-mask algorithm was higher than that of the SBR without it, revealing the value of routinely using the CSF-mask algorithm when calculating the SBR index with the Southampton method in the diagnosis of PS. The ROC analyses indicate that cut-off values of 3.80 in NC and 3.97 in AC may differentiate PS from NPS when using the SBR with the CSF-mask algorithm; without the CSF-mask algorithm, the recommended cut-off values for the SBR are 4.58 in NC and 4.78 in AC, which are almost the same values as those previously proposed by Tossici-Bolt et al. [Our study also demonstrated that the effect of the CSF-mask algorithm tends to improve the diagnostic performance in cases with higher EI values. This result confirmed our hypothesis: the stronger the extent of brain atrophy, the more effective the CSF-mask algorithm. Because brain atrophy or ventricular dilatation may occur even early in the course of cognitive decline in patients with PD , our resThe present study is subject to some limitations. First, the diagnoses of PS and NPS were based on clinical diagnoses; thus, we did not use a pathological diagnosis. Although this may have influenced our results, it is difficult to perform DAT SPECT and pathological examination at the same time, and the pathological diagnosis can change with disease progression . It shouFor PS, the diagnostic performance of the SBR index was enhanced by the CSF-mask algorithm, especially in cases with ventricular dilatation.