To select a non-rodent species for regulatory toxicology studies, we conducted PK and PD evaluation of BPN-14136 in dogs and non-human primates (NHP). PK properties were determined following oral and intravenous administration of BPN-14136 in beagle dogs and cynomolgus monkeys. Dynamics of plasma RBP4 reduction in response to compound administration was used as a PD marker. BPN-14136 exhibited favorable PK profile in both species. Dose-normalized exposure was significantly higher in NHP than in dog. Baseline concentrations of RBP4 were considerably lower in dog than in NHP, reflecting the atypical reliance of canids on non-RBP4 mechanisms of retinoid trafficking. Oral administration of BPN-14136 to NHP induced a strong 99% serum RBP4 reduction. Dynamics of RBP4 lowering in both species correlated with compound exposure. Despite adequate PK and PD characteristics of BPN-14136 in dog, reliance of canids on non-RBP4 mechanisms of retinoid trafficking precludes evaluation of on-target toxicities for RBP4 antagonists in this species. Strong RBP4 lowering combined with good PK attributes and high BPN-14136 exposure achieved in NHP, along with the biology of retinoid trafficking that is similar to that of humans, support the choice of NHP as a non-rodent safety species.Accumulation of lipofuscin bisretinoids in the retina contributes to pathogenesis of macular degeneration. Retinol-Binding Protein 4 (RBP4) antagonists reduce serum retinol concentrations thus partially reducing retinol delivery to the retina which decreases bisretinoid synthesis. BPN-14136 is a novel RBP4 antagonist with good  ABCA4 gene [trans- and 11-cis-retinaldehyde [trans-retinol from serum to the RPE, it was suggested that pharmacological reduction of serum retinol may represent a target area in a search for macular degeneration treatment. Serum retinol is delivered to the RPE by the specific serum carrier protein, Retinol-Binding Protein 4 (RBP4) [in vitro RBP4 binding potency as well as a strong in vitro ability to antagonize retinol-dependent RBP4 interaction with TTR [in vivo efficacy (plasma RBP4 lowering) in both rodent species [Abca4-/- mouse model of Stargardt disease significantly inhibited bisretinoid synthesis and normalized dysregulation of the complement system in the retina [in vitro ADME  properties. The important objective of the PK-PD and in vitro ADME studies was the selection of the appropriate non-rodent species suitable for a formal evaluation of BPN-14136 safety in GLP studies as well as confirmation that canine retinal degeneration models, such as the cmr model of BVMD, can be used in accessing pre-clinical efficacy of BPN-14136 and similar compounds.Dry (atrophic) form of age-related macular degeneration (AMD) represents a slowly progressing neurodegenerative disorder in which specialized neurons (rod and cone photoreceptors) die in the central part of the retina called macula . PhotoreCA4 gene . Best ViCA4 gene . There aCA4 gene –15. Bisraldehyde –18. Upta4 (RBP4) , 20. Mos4 (RBP4) . RBP4-TT4 (RBP4) , 24. Ret4 (RBP4) , 25. We 4 (RBP4) –30. One with TTR . The com species , 28 whicIn vitro ADME tests were conducted at AMRI, Albany, NY. Plasma protein binding for BPN-14136 was determined (in triplicates) by equilibrium dialysis of plasma against phosphate buffered saline (pH 7.4). Plasma spiked with BPN-14136 at a concentration of 1 μM was loaded to one side (donor) of the dialysis device insert, and phosphate buffered saline was loaded to the other side (receiver). After four hours, the concentration of BPN-14136 was assessed in both the donor and receiver sides.BPN-14136 was synthesized as described previously , 28. In Metabolic stability determinations for BPN-14136 and testosterone (positive control) were conducted in the presence of human, dog, and cynomolgus monkey liver microsomes. All measurements were done in duplicate. Pooled mixed gender human donor microsomes, male beagle dog microsomes and male cynomolgus monkey were obtained from BioIVT . BPN-14136 was prepared as a 10 mM stock solution in DMSO. A mixture containing 50 mM potassium phosphate buffer pH 7.4 and 1 mg/mL liver microsomes was pre-warmed for 10 min at 37°C in a shaking water bath, followed by the addition of test compound. Final DMSO concentration in the reaction mix was 0.1%. Reactions with cofactor were initiated by adding a NADPH-regenerating system to the incubation mixtures . The final volume of the reaction mixture was 800 μL, containing 1 mg/mL liver microsomes, and 1 μM test compound. Aliquots (100 μL) of reaction mixtures were removed from the incubation plate at pre-defined timepoints and mixed with 150 μL of ice-cold acetonitrile, incubated on ice for 15 min, and samples were centrifuged  to precipitate protein. The supernatants were diluted 1:1 (v/v) with water containing the internal standard and subjected to the LC/MS analysis. The low limit of quantitation for BPN-14136 was0.01 μg/ml.6 cells. Final DMSO concentration in the reaction mix was 0.1%. Aliquots (100 μL) of reaction mixtures were removed from the incubation plate at different timepoints and mixed with 150 μL of ice-cold acetonitrile. After incubation on ice for 15 min, the samples were centrifuged  to precipitate protein. The supernatants were diluted 1:1 (v/v) with water containing internal standards and subjected to the LC/MS analysis. Hepatic clearance was calculated using the well-stirred liver model [Metabolic clearance in hepatocytes experiments were conducted to assess BPN-14136 stability in the presence of human, dog, and cynomolgus monkey hepatocytes. All measurements were done in duplicate. Testosterone and 7-hydroxycoumarin were used as positive controls. Pooled cryopreserved human hepatocytes, cryopreserved male beagle dog hepatocytes, and cryopreserved male cynomolgus monkey hepatocytes were obtained from BioIVT . Compound at 1 μM was incubated with 1 x 10er model ad libitum. Dogs were maintained on a 12:12 hour light:dark cycle at the 72–73°F temperature and 40–71% humidity. Every effort was made to minimize, if not eliminate, pain and suffering in all animals in this study. No restraining devices were used in blood collecting procedures. Dogs were gentle hand-held to restrain by trained technicians with the dogs foreleg extended for sample collection of blood from cephalic vein. Animals were awake during blood sample collection with no anesthesia/analgesic agents used, and positive reinforcement by the technician was provided following collection. No animals were required to be euthanized during this study due to their health status. All dogs were released back to the colony after the study was completed. Male beagle dogs and male cynomolgus monkey received a single i.v. dose of a test compound at 0.5 mg kg−1 (dog) and 1 mg kg−1 (monkey) or a single p.o. dose of a test compound of at 2 mg kg−1 (dog) and 5 mg kg−1 (monkey). The number of animals that was used in PK experiments is 3 per study group. Intravenous administration vehicle was 3% DMA/45% PEG300/12% ethanol/40% sterile water; p.o. vehicle was 2% Tween 80 in 0.9% saline. Blood was collected from dogs and monkeys at pre-dose and at 5, 15 and 30 min, and 1, 2, 4, 6, 8, 12, 24, 36 and 48 h post-dose in the i.v. arms and at 15 and 30 min, and 1, 2, 4, 6, 8, 12, 24, 36 and 48 h post-dose in the p.o. arm. Blood samples were processed to plasma. Following protein extraction with acetonitrile, compound concentrations in plasma were measured by LC-MS/MS. The plasma drug level data were analyzed using WinNonlin software package; Model 201 (for i.v. bolus administration);was used for the analysis of i.v. data, and Model 200 (for extravascular administration) was used for the analysis of the p.o. data. Aliquots of pre-dose plasma samples, as well as aliquots of plasma PK samples, were used for the analysis of the plasmabiomarker, RBP4.Pharmacokinetic studies were conducted in male beagle dogs and male cynomolgus monkeys at SRI International, Menlo Park, CA. General procedures for animal care and housing were in accordance with the National Research Council (NRC) Guide for the Care and Use of Laboratory Animals (1996) and the Animal Welfare Standards incorporated in 9 CFR Part 3, 1991 and complied with guidelines set forth by the ARVO Animal Statement for the Use of Animals in Ophthalmic and Vision Research. During the course of the PK experiments, cynomolgus monkeys were individually housed in stainless steel primary enclosures in an AAALAC-accredited facility. Animals were fed a commercial monkey chow, and supplemental food was provided . Reverse osmosis-purified drinking water was available at all times. Cynomolgus monkeys were maintained on a 12:12 hour light:dark cycle at the 64–84°F temperature and 30–70% humidity. Monkeys had access to enrichment opportunities  and were provided auditory stimuli and human interaction enrichment throughout the study. Blood samples were collected by venipuncture of a peripheral vein. Animals were acclimated to the squeeze back mechanism in their home cage by repeated, short periods of restraint during general husbandry procedures for at least 2 weeks prior to dosing. Animal were not removed from their cages for collection, and no anesthesia/analgesic agents were used for blood collection. Monkeys were observed at each study blood collection time point and twice daily at least 6 hours apart (a.m. and p.m.). None of the cynomolgus monkeys became ill during the course of the study and all primates returned to the home colony after the completion of the study. For PK experiments, beagle dogs were individually housed in enclosed run (≥ 4 ft x 6 ft). Dogs were exposed to their daily ration of food, Harlan Teklad 2025C Certified Global 25% Protein Dog Diet. Water was provided from the public water supply  Concentrations of RBP4 in cynomolgus monkey plasma samples (diluted 1:5000) were measured using the RBP4 (Human) ELISA kit  following the manufacturer's instructions. RBP4 concentrations in dog plasma samples at the 1:3000 dilution were measured using the Canine RBP4 ELISA kit  following the manufacturer's instructions.For Western blotting, serum and plasma proteins were separated by NuPAGE Bis-Tris gels (4%–12%)  and transferred to PVDF . After blocking, membranes were incubated with rabbit polyclonal anti-human RBP4 antibodies  overnight at 4°C. Membranes were washed 3 times for 10 min in Tris-buffered saline (pH 7.5) containing 0.2% Tween (TBST), and then incubated in goat anti-rabbit IgG-HRP secondary antibody  for 1 hour at room temperature. After washing, the protein bands were visualized with enhanced chemiluminescence labeling using the ECL immunoblotting detection system . The developed films were scanned and the pixel volumes of the bands were determined by using NIH’s ImageJ software, with the values in ratios of intensity. Transferrin loading control was detected using rabbit anti-human transferrin polyclonal antibodies . HEK293-expressed purified dog RBP4  and HEK293-expressed purified human RBP4  were used as control of antibody specificity in Western blot experiments.in vitro RBP4 binding potency (IC50 = 12.8 nM in SPA binding assay) as well as the superb in vitro ability to antagonize retinol-dependent RBP4 interaction with transthyretin (IC50 = 43.6 nM in HTRF RBP4-TTR interaction assay) [50 values > 34 μM), displayed very good stability in human and rat liver microsomes (>90% remaining at 30 min.), no PXR activation, and no significant off-target activity at the hERG channel or within a standard screening panel of fifty-five GPCRs, enzymes, ion channels, and transporters [in vitro ADME characterization of this compound.As previously reported, BPN-14136 exhibits very good n assay) . The comsporters . The comsporters  and moussporters  and normsporters . Encourain vitro ADME characteristics in rodents [in vitro ADME studies in dog and non-human primate. The pharmacological profile of BPN-14136 is presented in In light of good  rodents  we extenint values in hepatocytes suggest very low predicted hepatic clearance. The extent of plasma protein binding was in the high range for all three species with low fraction unbound. Satisfactory in vitro ADME characteristics justified BPN-14146 characterization in dog and non-human primate PK studies. Single dose PK studies were conducted with cynomolgus monkeys at 2 mg kg−1 i.v. and 5 mg kg−1 p.o. and with beagle dogs at 0.5 mg kg−1 i.v. and 2 mg kg−1 p.o. BPN-14136 possessed favorable PK profiles in cynomolgus monkey and dog  and the estimated bioavailability was 84.3%. Comparably to cynomolgus monkey, BPN-14136 was rapidly absorbed in dog reaching the peak concentration of 7.5 μg/ml at 0.33 hr after post oral dose. CL and Vss values were higher in dog than in cynomolgus monkey (inf) in dog ranged between 10.3 hr•μg/ml for i.v. dosing and 35.4 hr•μg/ml for oral administration. The oral bioavailabilities (%F) for BPN14136 for both species was around 84%. BPN-14136 exhibited overall good PK profile in both species . The overall good PK profile of the compound in dog and NHP is consistent with good PK characteristics that BPN-14136 exhibited in our previous rat [−1) was lower than in PK experiments conducted in three other species  we compared exposure in four species by analyzing dose-normalized AUCinf and Cmax values  were highest in cynomolgus monkey and rat while lower exposures were seen in mouse and dog PK studies , and an observed Cmax of 31.4 μg/ml after oral administration and corresponding Tmax at 1.67 h (max) of 7.5 μg/ml achieved at Tmax of 0.33 h post oral dose (−1) was lower than in PK experiments conducted in cynomolgus monkey as well as in two rodent species that we studied earlier  we compared exposure in four species by analyzing dose-normalized AUCinf and Cmax values Click here for additional data file.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file.


Shortly, the subarachnoid space was accessed in the anesthetized minipig by puncture with a Tuohy needle; when CSF was flowing through the needle a catheter was advanced and thereafter tunneled and fixed on the back. The PK of peptide A administered subcutaneously was performed and CSF could be sampled in the conscious animals for up to 48 h. When compared to the plasma kinetic data, there was a clear difference in the elimination phase of Pept. A from CSF, with an apparent longer average terminal half-life in CSF. The 3Rs are addressed by reducing the number of animals needed for a pharmacokinetic profile in central nervous system and by improving the validity of the model avoiding biases due to anesthesia, blood contamination, and inter-individual variability.Drug concentrations in cerebrospinal fluid (CSF) are typically used as a as a surrogate measure of their availability in the CNS, and CSF penetration in animal studies are used for assessment of CNS drug delivery in early preclinical drug development. The minipig is a valid alternative to dogs and non-human primates as non-rodent species in preclinical research, but this species presents anatomical peculiarities that make the serial collection of CSF technically challenging. A minimally-invasive serial cerebrospinal fluid collection model  As first, rodent models are used and subsequently translated to larger animals. CSF can be sampled at the cerebral ventricles (CV), cisterna magna (CM) or lumbar thecal sites in conscious or anesthetized animals [via lumbar puncture under anesthesia or after euthanasia, with all disadvantages listed above. Spinal catheters for CSF collection could also be placed surgically by laminectomy [via catheterization of the subarachnoid space allowing for serial CSF collection in conscious minipigs. The aim for the study presented was both to describe a minimally invasive method for CSF sampling through catheterization of the subarachnoid space and to validate this method within a pharmacokinetic study for a compound under investigation as therapeutic agent in CNS related disorders.In central nervous system (CNS) drug research, drug concentrations in cerebrospinal fluid (CSF) are typically considered a good surrogate of concentrations at pharmacological target site l phases . For dru animals . To perf animals . The min animals ,9. Pigs  animals , the CV  animals  or via linectomy . Still tn = 3, female, aged 25–37 months, BW 43.7–52.2 kg). Animals were individually housed in an Association for Assessment and Accreditation of Laboratory Animal Care International (AAALACi) accredited facility with olfactory, visual, and auditory contact to their conspecifics in pens sized 8.6 m2, under a 12 h light, 12 h dark cycle. The environment was temperature-controlled (18°C ± 2°C) with relative humidity ranging from 40%–80%. Animals were offered commercially available laboratory swine diet  twice a day and tap water ad libitum. Apart from a straw box for each animal, enrichment devices  were offered on a rotational basis. All animals underwent an acclimatization phase of at least three weeks before study start.All animals were Göttingen Minipigs purchased at Ellegaard Göttingen Minipigs A/S, Dalmose, Denmark  and all experiments performed according to the Swiss regulations and as described in the permission.®, AbbVie AG, Baar, Switzerland) with a flow of 2 L/min (1:1 air/O2) administered via a leak proof face mask, using a Matrx VMS® anesthesia machine with a circle breathing system and a Tec7 Sevoflurane vaporizer. As soon as consciousness was lost, sevoflurane was reduced to 2%–4% and the carrier gas flow was set to 0.5–1 L/min (air/O2 = 50%/50%) for the duration of catheterization. A local anesthetic was injected subcutaneously  at the insertion site to guarantee perioperative analgesia. Animals were covered with the warming blanket of a forced air warming device  to ensure temperature close to the physiologic body temperature of 37°C–38°C in conscious Gottingen minipigs [®, Bayer AG, Zürich, Switzerland) was applied over the cornea to avoid desiccation. Heart rate (HR) and oxygen saturation of blood (SpO2) were continuously monitored via a pulse oximeter  in addition to rectal body temperature.For catheterization, anesthesia was induced by using 8% sevoflurane . The IVS cranial to the last lumbar vertebra was chosen as insertion point and was identified with anatomical landmarks . Briefly, the two tubera coxae were palpated and marked with a waterproof marker. The lumbosacral intervertebral space was identified just caudal to an imaginary line drawn between both tubera coxae, indicated by a depression in the spinous processes. All spinous processes and IVS cranial to the last lumbar vertebra were marked. These landmarks are valuable for identifying the targeted IVS without imaging techniques or when using pigs with a higher amount of fatty tissue. The lumbar area was clipped and thoroughly washed and disinfected with iodine soap . The skin was sprayed with alcoholic iodine solution  and covered in sterile self-adhesive surgical drapes  .Animals were placed in either sternal or lateral recumbency, with the hind limbs stretched cranially in order to widen the IVS . An epidural needle   was advanced with a ventro-cranial orientation forming an angle of 45° with the spinal cord through the skin, subcutaneous tissue, and interspinous ligament towards the subarachnoid space . Entry into the epidural space was determined using either the loss-of-resistance (LOR) technique or the hanging drop (HD) technique. With the HD technique, the needle was filled with sterile saline and advanced until an inward movement of the saline was observed, as sign of the negative pressure present in the epidural space. With the LOR technique, a syringe filled with air was attached to the epidural needle, and loss of resistance to injection of air was examined intermittently while slowly advancing the needle, with clear loss of resistance identifying the epidural space. The needle was then carefully advanced further 5–10 mm into the subarachnoid space until CSF backflow was seen, indicating correct intrathecal positioning . Thereafter the stylet was removed and the epidural catheter  was fed through the epidural needle and gently advanced cranially in the subarachnoid space. Segmental markings on the catheter allowed determining that its tip was advanced approximately 8 cm into the subarachnoid space . This length was chosen to place the catheter at the level of the 1st lumbar vertebra for study related sampling. Patency of the catheter was assessed by viewing the free CSF flow. The external part of the catheter was tunneled subcutaneously to add stability by using a small trocar to exit around 6 cm cranial to the insertion point . The external tip of the catheter was connected to Snap catheter connector (part of the described Perifix® set). The skin incisions made prior to entry of the needle and for tunneling were adapted using non-absorbable monofilament suture material . Thereafter the catheter was covered with gauze compresses then both, catheter and compresses were sutured to the skin .Catheterization was performed under aseptic conditions. An incision of about 5 mm was made in the skin above the intervertebral space cranial to the last lumbar vertebra with a disposable scalpel  . Extrapolating from man the expected intensity of pain for this intervention, no post interventional analgesia was given. The minipigs were visually checked twice daily for integrity of the protective dressing and catheter and wellbeing. Action and endpoints were defined based on the recognition of clinical signs indicative of spinal cord impairment, neurologic deficit, pain or infection, and animals were monitored for 48 h post-catheterization using the clinical scoring system (Table 1). Depending on the severity, intervention consisted either in close monitoring for up to 48 h if signs did not get worse or cutoff with euthanasia.Post-catheterization, sevoflurane administration was discontinued and the animals were visually supervised until recovery  under clean conditions by plunging the Snap connector with a sterile syringe and then applying gentle suction. The first sample withdrawn of CSF remaining in the catheter dead space was discarded. Sampling occurred in the home pen, restraining the minipig against one wall.n = 3) was performed on the day before the longitudinal CSF PK study  as described above. Pept. A, a highly hydrophilic cyclic small peptide with a molecular weight (MW) of about 1 kDa, was administered twice at 4.2 mg/kg subcutaneously on study day 1 and again on study day 2, with a dosing interval corresponding to 24 h. Based on results of previous pharmacokinetic studies in rats, Pept. A was known to be able to reach the CSF after peripheral administration. Blood (100 µl) and CSF (100 µl) aliquots for drug concentration assessment were collected pre-dose, on study day 1 at 0.5, 1.5, 3, 6, and 24 h post first dose, and on study day 2 at 1.5 h post the second dose (corresponding to 25.5 h from study start) . All blood samples were collected via microsampling  from the lateral ear vein because blood sampling from the jugular vein would have required placing the animals on their backs and thereby potentially compromising the patency of the CSF catheters. CSF was sampled as described above. On Study day 2, last samples collection was performed 1.5 h after the second administration of Pept. A . Thereafter, animals were euthanized under anesthesia [8% sevoflurane  by intravenous administration of an overdose of pentobarbital , routinely embedded in paraffin , and 5 µm slides sections were cut  and deparaffinised, and routinely stained with hematoxylin eosin stain . EDTA-containing plasma was prepared from the collected blood samples by centrifugation; both CSF and plasma were stored at −20°C until bioanalysis. Bioanalytical assessment of Pept. A concentration in plasma and CSF matrices was performed by Liquid chromatography–mass spectrometry (LC-MS/MS).Catheterization  containing Bosentan (50 ng/ml) as internal standard, mixed and centrifuged . Thereafter, 50 µl of the supernatant was diluted 50:50 on-line with [90% HDue to the small animal number, no statistical analysis was performed, but data were presented as mean values with standard deviation (SD) and coefficient of variation (CV%) calculated using Microsoft Excel 2010.n = 3) was performed on the day before the longitudinal CSF PK study : Anesthesia was uneventful with HR between 52 and 88 beats per minute (BPM) for subject #220613 and 102 to 110 BPM for subject #222445, SpO2 was stable between 95 and 100% for both animals, and sevoflurane was kept at 4 Vol% for both animals. The anesthesia record of subject #219514 was missing.Catheterization . Serial CSF could be sampled in the conscious animals with minimal or no restraint; the samples were clear and transparent, thus not visibly contaminated with blood.Recovery from anesthesia was smooth and animals returned to their normal behavior and activities within 1h after recovery , subject #220613 showed mild monolateral ataxia of the right hind limb, without reaching cutoff criteria. Details of the pathological changes in the symptomatic minipig are shown in Figure 4 and in Table 2 and Table S1. Pept. A was rapidly distributed in the circulation after SC injection, with an average Tmax of 0.5 h and short (3.5 h) derived average terminal half-life. In CSF, Pept. A concentration was above the lowest detection limit (> 10 ng/ml) until 24 h post first dose. When compared to the plasma kinetic data, there was a clear difference in the elimination phase of Pept. A from CSF, with an apparent longer average terminal half-life in CSF in agreement with previous observations in rats and estimated to be driven by the CSF flow rate. Regarding the uptake of Pept. A in the brain (CSF) from the circulation, an average AUC0-24CSF/plasma value of ca. 2% was calculated, explained by the limited passive permeability of the peptide and its hydrophilic nature. Despite of similar PK profiles obtained in plasma between animals, subject #220613 showed a remarkably higher Pept. A exposure in CSF. This observation could not be further explored in a follow-up work, but was considered to be related to the intactness of the Blood-Brain-Barrier or Blood-CSF-Barrier, likely lower in this animal, and/or to a more efficient active brain uptake of Pept. A. Finally, the exposure of Pept. A in the CSF at 1.5 h after the second dosing obtained from lumbar catheterization and choroid plexus (CP) sampling was comparable, indicating a homogenous distribution of the test compound in the CSF (Table 2).The compound tested in the PK validation study was a small cyclic peptide with a MW of ca. 1 kDa, here named “Pept. A”, under investigation as therapeutic agent against a CNS-related disorder. Due to company policy and restrictions on disclosure of confidential information, its molecular structure and target cannot be reported here. The plasma and CSF concentrations and PK data are reported in To the best of our knowledge this is the first description of minimally-invasive serial CSF sampling in conscious minipigs. The IVS cranial to the last lumbar vertebra was chosen for needle insertion, as it is the largest facilitating access . When deFig. 3); therefore, the smallest size of epidural catheter allowing free flow of CSF should be selected.The rationale for selecting an off-label epidural catheter instead of a spinal catheter was based on the larger lumen facilitating retrieval of CSF; spinal catheters of the necessary size are not commercially available. Another important technical feature of the chosen epidural catheter is its 3 lateral openings avoiding adhesion of its tip to the arachnoid membranes and consequent occlusion while sampling. The use of a large catheter in the narrow spines can lead to compression of the spinal cord clinically resulting in neurological deficits as observed in one animal , it was known that Pept. A displayed a very low membrane passive permeability (below quantification) as well as no binding to plasma proteins. In rodents, systemic clearance of Pept. A was very high as expected due to the nature of the molecule, resulting in a very short terminal half-life in plasma < 0.5 h. The compound quickly reached the CSF after subcutaneous dosing and the kinetic in CSF was clearly different from those observed in plasma, suggesting that the plasma kinetic data of Pept. A could not be used to predict its exposure in the brain, and that measurements of longitudinal CSF PK profile in large animals were needed to progress the project into clinical trials. In minipigs, Pept. A was rapidly distributed in the circulation after SC injection, in line with the expectations for a small peptide showing no binding to plasma proteins, and aligned with previous PK data acquired in rodents. Regarding the uptake of Pept. A in the brain (CSF) from the circulation, the very low AUC0-24CSF/plasma value was aligned with data from previous rodent PK studies (AUC0-24CSF/plasma 1%–1.5%) where a CSF sample was collected at animal termination from CP site, increasing the confidence of human CSF PK translations.From previous In conclusion, we have described a new method refining serial CSF sampling in conscious minipigs. This technique is highly valuable for measuring the longitudinal CNS PK profile of drug candidates targeting the brain as it allows minimally-invasive determination of drug concentration at target site (CSF) in a time-dependent manner from the same animal. The availability of compound concentration kinetic at target site is essential for understanding the pharmacodynamic effect, for enabling drug candidate optimization and selection, and for allowing extrapolation of preclinical PK/PD data to human during early drug research phases. The described methodology reduces the number of animals, enhances PK data reliability by excluding inter-individual differences compared to a composite PK profile, and avoids technical biases of anesthesia or blood contamination. Although the approach is technically challenging, it is minimally invasive and well tolerated by the minipigs without disturbing their normal behavior. Overall it can be concluded that the 3Rs concept was successfully addressed and animal welfare improved.Supplementary informationTable S1. Individual and average PK raw data obtained in minipig plasma and CSF.Supplementary information of this article can be found online athttp://www.jbmethods.org/jbm/rt/suppFiles/265.


In particular, whether macroscopic connectivity supports this given the disproportionate volumetric differences between species in the brain regions that project to the ventral striatum, including the prefrontal and limbic areas, has not been established In this study, the human and macaque striatum was first tractographically parcellated to define the ventral striatum and its two subregions, the nucleus accumbens (Acb)-like and the neurochemically unique domains of the Acb and putamen (NUDAPs)-like divisions. Our results revealed a similar topographical distribution of the connectivity-based ventral striatal components in the two primate brains. Successively, a set of targets was extracted to construct a connectivity fingerprint to characterize these parcellation results, enabling cross-species comparisons. Our results indicated that the connectivity fingerprints of the ventral striatum-like divisions were dissimilar in the two species. We localized this difference to specific targets to analyze possible interspecies functional modifications. Our results also revealed interspecies-convergent connectivity ratio fingerprints of the target group to these two ventral striatum-like subregions. This convergence may suggest synchronous connectional changes of these ventral striatal components during primate evolution.Although the evolutionarily conserved functions of the ventral striatal components have been used as  The primate ventral striatum is comprised of the nucleus accumbens (Acb), the broad continuity between the caudate nucleus and the putamen ventral to the rostral internal capsule, the olfactory tubercle , and theGiven that the ventral striatum  extends slightly into the caudate nucleus and putamen, reaching the medial caudate nucleus border , the dora priori knowledge for further studies  comparative analyses and is one of the few techniques that can truly bridge the gap between species by using identical non-invasive data acquisition schemes and data handling . DiffusiThe goal of this study was to compare the macroscopic connectivity of the ventral striatum and its subregions between humans and macaques. To address this issue, we first recursively parcellated the striatum, based on probabilistic diffusion tractography, to define the connection units of the ventral striatal components for each species. Then, we selected a set of target areas to construct the fingerprint framework for cross-species comparisons. Using these target areas, we characterized the anatomical connectivity patterns of the ventral striatum and its subregions. Finally, we evaluated the similarity of the corresponding connectivity fingerprints between the two species and detected the interspecies single connectivity differences to analyze possible interspecies functional modifications.A human MRI dataset  was extracted from the Human Connectome Project HCP;  because ex vivo; age: 4–23 years; two males; for details, see b-value = 1000 s/mm2 and four non-diffusion weighted images. The data quality controls used on these macaque diffusion images are described in A macaque MRI dataset consisting of eight adult rhesus macaque brain specimens  was used as a reference to decide which subregions should be chosen as the connection units of the ventral striatum and its major components.Unlike previous parcellation procedures , the conThe connection units that had corresponding locations in the two species were treated as seeds for cross-species comparisons. The connectivity fingerprint was chosen as a comparative characteristic  to detecEarlier studies calculated relative connectivity strength (RCS) by normalizing the data to the maximum regional connectivity strength in the brain to build connectivity fingerprints for cross-species comparison . Howeveri) is one of the n ventral striatal subregions; RCS) is the RCS between the given target and seed(i); CR) is the connectivity ratio of seed(i).Where, seed(i)) has values in the interval , and high values indicate that the target tends to connect with the seed(i) when compared with the other ventral striatal subregions. Therefore, the connectivity ratio fingerprint reflects the contrast in the general connectivity trend across the ventral striatal subregions. We used the CR fingerprint to detect possible interspecies differences in the connectivity trends for the ventral striatal components.The CR to reduce false positive connections as was done in earlier studies , temporal pole (TP), caudate nucleus (Ca), putamen (Pu), pallidum (Pa), hippocampus (Hipp), amygdala (Amyg), mediodorsal (MD) part of the thalamus, and midbrain (MidB). The areas located in the lateral orbitofrontal cortex were eliminated from the target group considering the substantial interspecies differences reported in previous studies .The anatomical RCS fingerprints of the human and macaque ventral striatum-like division were calculated and compared . Given tTwo ventral striatum-like subregions, namely the Acb-like and NUDAPs-like divisions, were characterized using the CR fingerprint in both species . When coThe corresponding CR fingerprints of the Acb-like and NUDAPs-like divisions between the two species were extracted and compared to detect their cosine similarity. The permutation tests indicated that the observed cosine similarity between the two ratio fingerprints was not a rare value in the permutation distribution, as can be seen in the histogram. The observed cosine similarity was lesser than the calculated test criterion at the 5% significance level in the right tail . Thus, wBoth the human striatum and macaque striatum were recursively parcellated based on tractography. The ventral striatum-like, Acb-like, and NUDAPs-like connection units were defined in both species and treated as seeds for cross-species comparisons. A set of areas meeting specific criteria was extracted as targets to construct connectivity fingerprints, which were used as the comparative characteristics to characterize and compare the seeds. Our results revealed that the RCS fingerprint of the ventral striatum-like division in humans differed considerably from that of macaques. However, the general connectivity trend of these targets to the ventral striatum-like subregions, the Acb-like and NUDAPs-like divisions, was conserved, based on their convergent CR fingerprints.The gross correspondence between connectivity architecture and microstructural features  has yet to be clarified; in fact, both similar  and discex vivo macaque MRI data enabled the accurate identification of both the ventral striatum-like and the Acb-like connection units in both species. The lateral ventral striatum  was excluded from the Acb-like division given its large localization error with the histological Acb, and was called the NUDAPs-like division only because the histologically-defined patch-like NUDAPs defined in histology is mostly distributed in this region  and the decision-making process via its projection to the ventral striatum . A signiAlthough dissimilar RCS fingerprints of the ventral striatum-like division and some significantly different single connections were revealed between humans and macaques, conservative connectivity trends of these targets to the Acb-like and NUDAPs-like divisions were present. The synchronous changed (strengthen or weaken) connectivity observed in the two subregions in both types of primate brains indicated that the labor division and the cooperation of the two ventral striatum subregions may have not been noticeably altered across species.The same tractography-based recursive parcellations of the striatum were performed in both humans and macaques to delineate their ventral striatum-like, Acb-like, and NUDAPs-like connection units. A set of targets was chosen to enable the construction of fingerprints to characterize and compare such components across species. A dissimilar RCS fingerprint of the ventral striatum-like division was revealed between the species. Furthermore, this connectivity difference was localized to specific targets and such single connectivity differences were used to infer possible interspecies functional modifications. Finally, our results revealed distinct connectivity fingerprints of the Acb-like and NUDAPs-like divisions and interspecies convergent CR fingerprints of this target group to the two ventral striatum-like subregions.Eight adult rhesus macaque brain specimens were used in this study. These monkeys were judged by the veterinarian as appropriate subjects for euthanasia due to the presence of serious physical diseases. All the brain specimens were obtained at necropsy immediately following euthanasia due to reasons not related to the study and acquired following protocols approved by the National Animal Research Authority of China. All experimental procedures were performed in strict accordance with the recommendations in the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All of the animals were handled according to the protocol (#IA-2016-05) approved by the Animal Care and Use Committee of the Institute of Automation, Chinese Academy of Sciences.TJ and HL conceived and designed the experiments. XX, LF, CC, RY, HD, and DZ carried out the research and analyzed the data. XX, LF, HL, and TJ wrote the manuscript. All authors reviewed and approved the manuscript for submission.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The decision to evacuate or shelter in place during a natural disaster such as a hurricane is complicated and poses risks to long-term care residents. While research has documented the difficulty of the evacuation decision for nursing home administrators, little is known about how assisted living residence (ALR) administrators make this decision. This is a concern given the physical and cognitive impairment level of many ALR residents, the increasing number of ALRs in the U.S., and the frequency of natural disasters. The purpose of this paper was to explore the factors that influenced whether assisted living administrators evacuated their ALRs for Hurricane Irma, a large hurricane that made landfall on Florida’s Southwest coast in September, 2017. This qualitative study used semi-structured interviews and focus groups with ALR owners or administrative staff (N=60) with questions including how they prepared for Hurricane Irma, their experiences during the hurricane, including whether they evacuated or sheltered in place, and lessons learned. The sample includes small (< 25 beds) and large ALRs in the multiple Florida counties affected by the hurricane. A content analysis approach was used. Atlas.ti version 7 was used for initial and axial coding. Prevalent themes included “emergency management planning”, “logistics”, “pressure”, “storm characteristics,” and “staffing”. The results of this study have implications for long-term care policy and training, potentially leading to changes in how ALR leaders prepare for and respond to disasters to improve the safety of residents.


The outflow of the cerebrospinal fluid (CSF) in animals was over the years the subject of detailed analysis. For a long time it was stated that arachnoid granulations of the venous sinuses play a key role in CSF circulation. However, recent studies on this subject have shown that a considerable part of the CSF is drained to the lymphatic vessels. Moreover, disorders in the CSF passage may result in severe central nervous system diseases such as e.g. hydrocephalus. In this paper, we summarize the current knowledge concerning the lymphatic drainage of the CSF in mammals. We present in detail comparative anatomy of different species taking into account cranial and spinal compartment. In addition, we clarified role of the lymphatic vessels in the CSF outflow and the relationship between impairment in this transport and central nervous system diseases. In the author’s opinion knowledge on CSF circulation is still poorly examined and therefore required comment. Until recently, arachnoid granulations protruding into dural venous sinuses were seen as the main outflow tract of cerebrospinal fluid (CSF) from the subarachnoid space (SAS). However, some authors reported other CSF outflow tracts as far back as in nineteenth century Schwalbe . For a lIt is widely accepted that nerve tissue has no lymphatic vessels. At the same time, there is ample evidence that dyes and markers introduced (both in vivo and postmortem) intraventriculary or directly into the SAS enter the lumen of lymphatic vessels  a rich network of lymphatic vessels that drained CSF to the nearby lymph nodes originated in the meninges of the spinal cord, at the level of the cervical and the first thoracic segments. This network was mainly located in the epidural connective tissue surrounding area of the spinal nerve roots and discharged its contents into the deep cervical and prevertebral lymph nodes. In the remaining part of the thoracic region and the lumbar and sacral regions, in the area of spinal nerve roots there were only a few lymphatic vessels observed. The existence of the connection between the SAS of the spinal cord, and the lymphatic system were also found in mice. In this species, 125I-Human Serum Albumin administered intraventricularily reached the lumbar lymph nodes in large quantities (Boulton et al. The cranial cavity is not the only area from where CSF is drained by the lymphatic vessels. Lymphatic vessels are also involved in the absorption of CSF from the SAS area of the spinal cord. Miura et al.  proved tThe information mentioned above describes a wide network of connections between the SAS and the lumen of lymphatic vessels. On this basis it might be concluded that the lymphatic system plays an important role in CSF absorption.According to the literature there are considerable differences between species in the lymphatic vessels role in the CSF drainage from the cranial SAS. Various studies with isotopically labeled albumin administered intraventriculary found that at least 50% of CSF in rat, 30% in rabbit, 10–15% in cat and 40–48% in sheep was transported to the lymphatic vessels (Bradbury and Cole The discrepancies in the results described above, may be due to differences between species, as well as to the use of different research methodologies. Mollanji et al.  observed131HSA injected into the cerebellomedullary cistern. The authors compared radioactivity of blood samples collected from the cranial venous system and the peripheral blood under different intracranial pressure states. The authors found that under normal intracranial pressure conditions, the main tract of CSF outflow was through the lymphatic vessels. The transportation to the cranial venous system, with the participation of arachnoid granulations, was triggered only when this pressure raised. In conditions of high intracranial pressure, the flow of lymph through the lymphatic cervical vessels also increased, which clearly indicated an increase in CSF transportation (Hasuo et al. The quoted studies included a detailed description of lymphatic system involvement in CSF outflow and contested the dominant role of arachnoid granulations in some species. However, it did not specify what percentage of the CSF drained through arachnoid granulations and when this route gained importance. Zakharov et al.  continue14C] sucrose administered intraventriculary did not reach the SAS located over the cerebral cortex, thus it did not reach arachnoid granulations protruding into the sagittal dorsal sinus (Ghersi-Egea et al. Studies on rat confirmed that the lymphatic system plays the main role in CSF outflow from the cranial cavity. In this species, [The cranial cavity is not the only compartment where CSF is absorbed from the SAS. A significant role in this respect is also played by the spinal compartment. In meninges of the spinal cord, in the vicinity of the roots of the spinal nerves, the presence of both the arachnoid granulations and the lymphatic vessels was identified (Kido et al. CSF transportation from the cerebellomedullary cistern to the lymphatic vessels occurs rather quickly. In studies carried out intravitally on rat, Brinker et al.  demonstrSeveral research teams reported that in rat CSF transportation to the cribriform plate took place in SAS in proximity and along to the base of skull (Kida et al. Similar conclusions might be drawn from other studies on rat, in which kaolin was deposited in basal cisterns. In those animals, a significant impairment of CSF outflow through the cribriform plate into the nasal cavity was obtained. It is worth noting that kaolin did not exceed rostrally the level of the optic chiasm and it did not contact with the cribiform plate in the examined specimens. Moreover, the studied animals developed communicating hydrocephalus, the degree of which was related to the degree of impairment of CSF outflow into the nasal cavity (Nagra et al. Pdn/Pdn (Naruse and Ueta Other research that are worth considering are those carried out on homozygous mice Duong et al. , analyzeImpaired lymph drainage from the area of head may also be responsible for the brain edema. Casley-Smith et al.  caused cThe effects of abnormal CSF outflow through lymphatic vessels do not appear only in the chronic cases. Mollanji et al.  demonstrDespite differences between species, the information presented in this article clearly prove that in mammals, lymphatic vessels, and mainly lymphatic vessels of the nasal cavity, play a very important role in CSF absorption from the area of SAS. It seems that it is necessary to harmonize the methodology of research in this field, which would allow for an objective assessment of interspecies differences in terms of the percentage of the lymphatic system in the CSF drainage. Further studies would provide a better understanding of CSF absorption and, perhaps, would allow to determine the etiology and effective treatment of certain CNS diseases, for instance hydrocephalus.


Patient-reported outcome measures (PROMs) are widely available for use in musculoskeletal care. However, there is little research exploring the implementation of PROMs in clinical practice. This qualitative study explored chiropractors’ views on PROMs to identify any barriers and facilitators to implementing PROMs in chiropractic care and the training needs of chiropractors regarding the use of PROMs.. Semi-structured interviews were conducted with eight chiropractors, either face-to-face at their place of work or over the telephone. The interviews were transcribed verbatim and analysed using thematic analysis. The data were coded inductively by two authors.A qualitative study of chiropractors’ views on PROMs was undertaken as part of a larger project to address the feasibility of conducting a randomised controlled trial of PROM use in chiropractic clinics for patients with low back pain. Contact was made with chiropractors working in chiropractic companies with multiple clinic sitesChiropractors discussed their knowledge and engagement with PROMs in clinical practice, identifying reasons for their use, such as understanding clinic performance, clinical practice, and research. They also discussed how they used PROMs within their clinical practice and the benefits of using them with individual patients, for example during the consultation, identifying yellow flags, and tracking patient progress. Chiropractors voiced concerns about patient engagement with PROMs, questioning if patients find them burdensome, and the appropriate PROMs to use with patients with pain. Finally, chiropractors acknowledged the organisational barriers and facilitators to using PROMs within their practice, such as busy practices, electronic systems, and use of reception staff.Using participating chiropractors’ views of PROMs, the study identified barriers and facilitators to implementing PROMs in chiropractic care, such as clinician knowledge, engagement, and organisational concerns and identified the potential training needs of chiropractors regarding PROMs. The results from the study suggested chiropractors use PROMs with their individual patients, but PROMs should be meaningful to patients and chiropractors to improve engagement. Patient-reported outcome measures (PROMs) are instruments and questionnaires which can be used to collect patients’ perceptions and views on their own health –4. Data Since their development, research has been conducted to identify the impact of PROMs when used in clinical practice. Literature indicates that PROMs may impact clinically and psychologically on patients when used in clinical practice. One review of PROM research suggested that using PROMs may influence detection of psychological problems and facilitate communication between healthcare professionals and patients . Three rAlthough PROMs are increasingly being used to collect patient outcomes, with a NHS report in 2008 highlighting the importance of using PROMs to measure patients’ perspective on their care , there iDespite the availability of musculoskeletal PROMs, there has been very little published research in the context of musculoskeletal care and PROMs, with just three studies exploring how PROMs can be used in clinical practice –17. MuchChiropractors are increasingly encouraged to use PROMs within their practice , howeverIt is necessary to further explore the barriers and facilitators of using PROMs within the specific context of chiropractic care to ensure successful implementation. It is important to understand patients’ and clinicians’ views on using PROMs and the values of implementation. Greenhalgh et al.,  identifin = 40). Chiropractors received an email invitation to the study, explaining the purpose of the study and the procedures involved in participating. Recruitment continued until data saturation was reached (no new themes were generated from the data), this was assessed by two authors. This study received ethical approval and research governance approval from the University of Southampton (ID: 20715). All participants provided informed consent to participate in the interviews. Additionally, the data collected fit with the Data Protection Act of 1988 and the Data Policy of the University of Southampton  and getting them to grade things on 1 - 10 is fairly subjective anyway. When you say “how do you feel, are you very much improved or just much improved?” You know you can go to a smiley face system can’t you.” - James.Some chiropractors lacked clarity on the details of their clinic’s procedure relating to PROMs, especially when done via an electronic system. For example, there was uncertainty on the timing of follow-up PROMs: “I do think that many practices can benefit” – Clare. They suggested highlighting the benefits of using PROMs and the simplicity of the process. “So assuming everybody knows absolutely nothing about them and saying ‘this is what they are, this is why they are useful, this is how it can help you’ erm.. is probably the way to go.” – James.Participants believed that, in general, chiropractors might not have any previous knowledge on PROMs. In the interviews, chiropractors were asked about training on PROMs. Training on PROMs delivered via the internet, with e-resources and a physical guide was seen as acceptable by chiropractors: “I do think.. the longer you’re out in practice, either the more you pick up on things and use it, or the more settled to your routines you become and maybe forget the importance about the psychological aspect of the care, because you get so.. so sort of swaddled in your own routine, that you forget about renewing yourself. I think this sort of feedback is useful for renewing us, if we choose to use it actively.” - Clare.Chiropractors discussed their use of PROMs throughout the treatment process with patients. PROMs were used from the first consultation to look at the patients’ story. They identified that sometimes the information they received from PROMs differed from patients reporting during the initial visit. One chiropractor stated PROMs enabled chiropractors to see things from the patient’s perspective.“For me it’s about identifying those that are at risk of not.. not responding as you would expect them to and then being able to intervene a lot earlier” - James.Chiropractors also talked about being able to identify ‘yellow flags’ by using PROMs. This triggered chiropractors to reassure the patient, educate them about recovery, and discuss self-management. One chiropractor stated that PROMs affected the management plans of his patients: “I think they’d put that it hadn’t improved. So I phoned and err.. yeah we had a discussion about it, and they just started coming back” - Charlotte. Chiropractors also mentioned discussing PROMs with patients and visually showing them their progress.“I did do a guy this morning, literally like that.. cos he was chronic and erm.. he’d had pain for three years, and I started seeing him about six months ago, and he came in for a sort of final check-up, cos I think there’s an argument for doing check-ups on people with chronic problems, and he hadn’t been for three months, and he’s pretty good, he’s almost fine really. And I pinged up his little graph and said ‘look, there you are’ and he went ‘oh, yeah, that’s loads better isn’t it’ so for him, it was really neat.” – Cameron.Chiropractors also used PROMs at follow up to track patients and identify any improvements. They were also used to identify patients who were not progressing. One chiropractor discussed how they used the PROMs to follow up a patient: cos they can physically.. go.. ‘oh last time I remember the last time I filled that in I was 8 and now it’s only a 4’” – James. Chiropractors also believed that PROMs improved patient adherence for treatment and self-care exercises “for patient compliance.. if a patient is.. that is the advantage if the patient sees or remembers what they’ve done, then they themselves can see improvement” - James. Chiropractors also thought that PROMs may improve the patient-clinician relationship.“So for the majority of patients I feel like it makes them, feel like we are really interested, like we want to know everything. Especially if they can fill it out at home, and then when they come in for the initial like consultation, I’ve already looked at it, and have some insight into their pain. I think it’s really good, and I think they feel.. kind of reassured by it.” - Gemma.This visual depiction, and showing patients their improvement after a change in behaviour, was seen as positive reinforcement ““I know I’ve ended care for patients because it hasn’t seemed to be helping, but I’ve continued care but in a different way. Erm.. because maybe it was maybe more appropriate to.. to refer to someone else” - Clare.Chiropractors also identified using PROMs to change treatment for their patients: “I suppose it gives me a handle on the psychosocial stuff and the barriers, and the chronic ones. And therefore I might tweak them more psychosocially, or more in terms of exercise and something different, from just treating.” – Cameron. One chiropractor also said discussion was down to their personal rapport with the patient:“So for example, if I’ve got a builder in, who’s talking about football, or rugby, I’m not going to ask him.. I’m not going to start talk about emotions and stuff like that” - Neil.Despite chiropractors identifying how they might discuss PROMs, many chiropractors reported not discussing PROMs with their patients. This was often in cases where patients were improving. Practitioners would also not talk to patients about anxiety and depression unless they scored highly. Several chiropractors thought discussing PROMs may only be beneficial for certain patients, such as with chronic patients. This is just a pain, literally”; she along with other chiropractors expressed that PROMs were bothersome for patients, stating that patients do not enjoy completing a large volume of paperwork. Some chiropractors reported comments from patients, asking them to stop sending the questionnaires, although one chiropractor did state this was a small minority of patients. Chiropractors also had concerns that it might put off patients coming back to the practice, due to the paperwork involved “I mean the more questionnaires you give people, the more annoyed they are going to be with questionnaires” – Cameron.Chiropractors voiced views on patient engagement with PROMs. Charlotte felt that for patients: ““there’s a small minority that kind of just can’t be bothered to do it, and are just not interested” - Gemma. However, many participants had ideas to improve patient engagement. Chiropractors explained that most patients come in for treatment and are not necessarily expecting to fill out a form, and they do not understand why they are filling in the questions. One method recommended to improve engagement, was to explain to patients that completing the questionnaires is a component of their care and explain its inherent value. For example: “this a positive thing as part of your management plan, which gives us information about how you are improving” – James. Chiropractors also suggested reception staff may have to explain this to patients, as they often explain and provide the questionnaires to patients in advance of their appointment with the chiropractor.“I think it totally hinges on how you explain it to the patient and the way that they.. that initial conversation and how it’s explained initially will probably determine whether that patient stays with that or not. The better it’s explained and the more they understand it, the better their compliance will be later down the line.” - Rachel.Participants did acknowledge some barriers to patients completing PROMs, such as: email and computer access, IT skills, literacy, age, and time. Chiropractors stated that some patients might be too busy to fill them in. Some chiropractors believed that you cannot change patient engagement with PROMs “I always think ‘we keep going back to the pain’ and really we don’t want them to focus on the pain.” - Gemma. Several chiropractors commented that these pain scores might have a negative effect for the patient.“By always focusing on how much things hurt, and how difficult it is to do things, and how bad it’s been, for that kind of patient.. you’re perhaps, kind of, maintaining them in that, sort of, slightly negative spiral.” - Rachel.Chiropractors discussed some of the PROMs currently used in clinical practice for low back pain. Chiropractors also expressed wanting to focus more on a functional scale rather than a pain or quality-of-life scale. Although chiropractors acknowledged the need to know how much pain patients are in, several expressed concerns about getting patients to routinely quantify their pain. One chiropractor expressed that this might remind patients of their pain. “I work both here and in a private practice, tried different things in both places, and really ended up with.. the same thing overall.. going with measures of what they feel they can achieve and do. In my opinion I feel that’s what matters in the end.” – Clare.Chiropractors often chose to focus on functional outcomes, changing the focus to a patient’s abilities rather than their pain.“It says something about.. yeah.. ‘how depressed have you been feeling?’ and she was like ‘I didn’t think I was supposed to be depressed with back pain’. And it hadn’t even crossed her mind” - Rachel.Some chiropractors also had concerns about questionnaires that used the word ‘depressed’. Noting that although this was pertinent for some patients, this may not always be appropriate.I do think time pressure, is a big limitation, forgetting and then sometimes you think.. ‘oh I forgot on that one patient, or no I’m just going to leave that one patient out, I need to get on to the next one’ - that’s the challenge.” - Clare.“Chiropractors discussed the organisational barriers and facilitators to implementing PROMs within routine clinical practice. Chiropractors also voiced concerns about practicalities and human error. Chiropractors often spoke about forgetting to look at the data and that PROMs did not come to mind easily. It was an active task to remember to look at PROMs during follow-up treatments. They also discussed their busy practices and that time was an important factor. Chiropractors expressed that in practice they are trying to be efficient, on time, so they do not keep the patients waiting, and this can restrict their use of PROMs within treatment sessions.“You can sit there for ages trying to figure out” - Rachel. One chiropractor had issues with the electronic system not functioning correctly. However, using a paper version often required more administrative time for both the reception staff and the chiropractor. One key message was that all data should be on the same medium. One practice used paper clinic notes and an electronic system for PROMs, which was deemed to be inconvenient. Chiropractors within this clinic spoke about the difficulties switching between the two systems when the clinic is busy, and PROMs often got forgotten.One debate was the best medium to use for PROM collection, to use paper or via an electronic system. One chiropractor highlighted that results were easier to view and use when on the computer. The computer system they used generated patients’ results into a graph, which would be time consuming to do if PROMS were paper-based. However, some chiropractors had difficulties with using the electronic system I just leave it to the receptionist to do all that” - Cameron.One key facilitator in the collection of PROMs was reception staff. All chiropractors spoke very highly of their reception teams. Some chiropractors noted it was a joint effort, with reception staff explaining the importance of PROMs to patients and chasing patients for follow-ups. One chiropractor, who did not have the support of a reception team, noted that this would improve data collection. However, other chiropractors saw PROMs as mainly an administrative task that they did not get involved with: “This study aimed to explore chiropractors’ views on the use of PROMs in clinical practice. In order to address the knowledge-to-practice gap of using PROMs, it is essential to understand the implementation process . Five thWithin the interviews, chiropractors stated a preference for functional scales, which focus on a patient’s functioning rather than pain levels, with chiropractors finding them meaningful for their clinical practice. These preferences will need to be addressed, both in selection and practitioner education on appropriate measures, for PROMs to be successful in clinical practice. One systematic review has similarly identified that PROMs must be appropriate for successful implementation and use in clinical practice , suggestAlthough two chiropractors suggested issues with an electronic PROM system this was preferred over a paper-based system. In a study to assess the feasibility and acceptability of PROMs with patients with rheumatoid arthritis, patients’ perceived a web-based PROM as easy, and reported willingness to fill in the questionnaires at home . During A commentary by Chang  also ideInterviews also explored how to improve patient engagement. Two reviews previously identified that co-operative colleagues and staff encouragement facilitated patient engagement and the use of PROMs in clinical practice , 37. ChiThe qualitative interviews identified that chiropractors often had a lack of knowledge and engagement with PROMs, despite using them in clinical practice. It is also acknowledged in the findings that chiropractors’ attitudes and beliefs around the consultation process may influence their use of PROMs, with some chiropractors explicitly rejecting the use of PROMs, despite clinical procedures. Antunes et al.  and DuncChiropractors indicated that training would be acceptable and would improve knowledge and engagement with PROMs in clinical practice. It is important that training should also include the use of PROMs for clinical audit, feedback, and research purposes. Understanding the multitude of ways PROMs can be used within clinical practice may improve uptake and engagement.Generally, the results suggest that clinician training, selection of PROMs, and organisational barriers should be considered when implementing PROMs in clinical practice. However, the sampling of participants may limit the transferability of the results. Socioeconomical region of the clinics and details on the experience and training of the sample were not collected. It is therefore not possible to compare the chiropractors to the wider profession. The findings also only represent the views of chiropractors who have volunteered to take part in research and therefore may not be representative of the population. These findings also do not include the patient perspective of PROMs. While the sampling may limit the transferability, this study provides an insight into PROMs use in chiropractic practice.Further research in this area should consider the views of patients completing PROMs in chiropractic practice. Patients’ views will provide necessary insight into the process and benefits of implementing PROMs into clinical practice. Additionally, as interviewees spoke about how they use PROMs with individual patients, research should also explore how PROMs may potentially affect the process and outcomes of patient care when used clinical practice. A recent review of PROMs examining their use with individual patients found PROMs could influence the clinician-patient relationship, consultation discussions, and decision-making around treatment . Not allChiropractors are increasingly using PROMs in their clinical practice. The aim of this qualitative study was to examine the views of chiropractors on using PROMs. Exploring chiropractors’ experience of using PROMs, this study identified how clinician knowledge and engagement and organisational barriers and facilitators affect implementing PROMs in chiropractic care, such as choosing the appropriate PROMs and systems to use in their practice. Chiropractors also identified possible training needs of chiropractors regarding PROMs, with training including the process and benefits of using PROMs in clinical practice. The results from the study also demonstrated the necessity of ensuring PROMs are meaningful to patients and chiropractors. It is clear there are differing views and engagement with PROMs within clinical practice; in addition, future research must consider patients’ views on completing PROMs and how it affects the process of clinical practice and outcomes.


Streptococcus pneumoniae in recurrent meningitis may be a clue to the presence of a skull base abnormality. Craniopharyngeal canals have been classified depending on their qualitative and quantitative imaging features. Such imaging based classification is important for identification of patients with associated potential pituitary involvement and also for appropriate surgical planning. Controversy exists as to the approach to surgical treatment of craniopahryngeal duct. The persistent craniopahryngeal duct in our patient was successfully treated by an endoscopic transsphenoidal approach.Identification of the cause of recurrent meningitis may pose a diagnostic challenge. Evaluation of a patient with recurrent meningitis calls for meticulous review of skull base structures by cross sectional imaging to exclude any underlying anatomical abnormality. Our case highlights the importance of excluding persistent craniopharyngeal duct, a rare but treatable cause of recurrent meningitis. The isolation of  We describe a patient who presented with recurrent meningitis and was subsequently investigated to identify any underlying cause.Cranial anatomical defects may predispose to recurrent meningitis.Streptococcus pneumoniae serotype 23B from the blood and cerebrospinal fluid (CSF) cultures.A 5-year-old girl presented to the emergency department with headache, vomiting, increasing drowsiness and fever. A clinical diagnosis of meningitis was made. She was treated with intravenous antibiotics  for a period of 14 days and made a full recovery from this episode. A diagnosis of pneumococcal meningitis was made following the identification of 6 l−1 (50% neutrophils)]. Blood culture grew S. pneumoniae serotype 21 and polymerase chain reaction analysis of CSF was positive for S. pneumoniae.15 months later, the patient presented to the emergency department of our hospital again with symptoms suggestive of meningitis. A CT scan of the head was performed to exclude a suspected intracranial abscess and was reported as normal. The patient was treated for meningitis with intravenous antibiotics and made a full recovery. CSF showed an increased white cell count [7160 × 10The patient did not have history of concurrent infective illnesses, including middle ear or mastoid infections on either admission. There was no history of other recurrent infections, head injury, CSF rhinorrhoea or otorrhoea. The patient’s perinatal history was uneventful and she had normal developmental milestones. On examination, there were no stigmata of spinal dysraphism.Following treatment for the second episode of pneumococcal meningitis, the patient was reviewed by the infectious disease team and was commenced on long-term prophylactic antibiotics (amoxicillin) pending further investigations to ascertain an explanation for her recurrent episodes of pneumococcal meningitis.To investigate the cause of her recurrent meningitis, further immunological tests were performed. The immunoglobulin levels, complement activity, and T and B lymphocyte counts were normal. She continued to have protective levels against the 13 pneumococcal serotypes that she had been vaccinated against (Prevenar 13), although these levels had decreased since they were last measured following her booster dose.S. pneumoniae  access to the CSF. The review of the initial CT scan of the head confirmed the presence of a bony canal, measuring about 6 mm in diameter, extending from the sella, through the sphenoid and opening into the nasopharynx. A diagnosis of a persistent craniopharyngeal duct was made  and fibrin glue were used to repair the defect in the pituitary fossa. The distal end of the craniopharyngeal tract was seen ending in a blind sac in the nasopharynx. This was punctured and obliterated with electrocautery.A postoperative MRI  showed o1 Anatomical defects may be acquired or congenital. Acquired causes may be as a result of head injury causing CSF fistulae or secondary to neoplasms causing erosion of the skull base. Congenital abnormalities may include a wide spectrum of defects in the skull base. These defects may range from craniopharyngeal duct to large cephalocoeles with craniofacial defects.2Several conditions may predispose to the development of recurrent meningitis. These include anatomical abnormalities, immunodeficiency disorders and chronic parameningeal infections.3 It can persist as a corticated defect of the midline sphenoid body. Craniopharyngeal duct is believed to arise owing to a defect in the normal development of the pituitary. The adenohypophyseal (Rathke’s) pouch is formed at about the fourth week of gestation from the primitive mouth cavity (stomatodeum) and extends towards the brain.2,4 Around the fifth to sixth week, the pouch elongates further, forming an elongated stalk between the pouch and the stomatodeum The cartilaginous skull base forms at around the sixth to seventh week of gestation, obliterating the adenohypophyseal stalk.4 Incomplete fusion of the cartilaginous elements results in incomplete obliteration of the adenohypohyseal stalk, which persists as the craniopharyngeal duct or canal, extending from the sella turcica to the nasopharynx.2,4,5 Some authors have proposed that the craniopharyngeal duct represents a vascular channel.3 However, the histological demonstration of normal as well as adenomatous pituitary tissue in craniopharyngeal ducts argue strongly in favour of the craniopahryngeal duct being of pituitary origin.4,5Craniopharyngeal duct is a bony channel that connects the floor of the sella turcica, along the midline, to the nasopharynx.6 Currarino et al7 classified the abnormality on the basis of size: the hypophyseal channel or the small craniopharyngeal canal, which have a maximum width of 15 mm, and the large craniopharyngeal canal or transspehnoidal channel, which are more commonly associated with other craniofacial abnormalities such as encephalocoeles, cleft palate and lips and such. More recently, a classification of the craniopharyngeal duct has been proposed by Abele et al4 based on the size and spectrum of other associated pathologies . In the case series forming the basis of the classification system, the quantitative evaluation showed a significant difference (p < 0.0001) in the anteroposterior diameters of types 1, 2 and 3 canals, thus described as small, medium and large canals, respectively. The classification is summarized in Small craniopharyngeal canals have been reported to occur in up to 0.42% of the asymptomatic population.8 In cases of meningitis with predisposing factors, such as skull base fracture, middle ear and mastoid infection, imaging is necessary to identify the underlying cause. Our case illustrates the importance of reviewing the skull base in patients with recurrent meningitis, as early identification and treatment of an abnormal communication can potentially prevent further intracranial infections.Brain imaging is indicated only when the diagnosis of meningitis is clinically uncertain and when there is unusual presentation or suspected complication .T2 weighted sequence acquired volumetrically may also be helpful in further characterizing the abnormality, as in our case.Standard CT imaging of the head as well as MR sequences may at times be suboptimal for demonstrating a narrow craniopharyngeal duct. A high-resolution CT scan of the skull base postprocessed with bone algorithm and viewed with wide (bone) window may be necessary to demonstrate a narrow craniopharyngeal duct. MRI is necessary to localize the pituitary and exclude the presence of associated neoplasms. A sagittal heavily 9 There is a predisposition of S. pneumoniae in patients with intracranial encephalocoeles. The isolation of S. pneumoniae from the CSF and blood on two occasions in our patient prompted the review of the CT scan, which led to the diagnosis of the persistent craniopharyngeal duct.Correlation of the type of organism isolated from the CSF and/or blood in recurrent meningitis and the site of anatomical defect has been described.5 Our patient was treated with transsphenoidal endoscopic approach with good outcome.There are different approaches to the repair of a craniopharyngeal duct that include transcranial, transoral–transpalatal and transsphenoidal endoscopic, with some controversy existing regarding the choice of approach.The skull base is a “review area” in imaging of patient with intracranial infection, particularly in cases of recurrent meningitis, in order to exclude underlying defects such as a persistent craniopharyngeal duct.S. pneumoniae may point towards the presence of such a defect.In recurrent meningitis, the isolation of High-resolution CT scan of the head with reconstruction in bone algorithm can identify a persistent craniopahryngeal duct, and an MRI is necessary to gather important information such as position of the pituitary and presence/absence of associated neoplasms.A useful classification system of craniopahryngeal canals based on qualitative and quantitative criteria has been described.Although controversy exists regarding the surgical approach, our case describes successful treatment using an endoscopic transsphenoidal approach.


A, and GABAB receptors as well as DPPX, CASPR2, LGI1, IgLON5, or glutamate decarboxylase (GAD), with respect to the reported basic CSF findings comprising CSF leukocyte count, total protein, and the presence of oligoclonal bands (OCB) restricted to the CSF as a sensitive measure for intrathecal IgG synthesis. Our results indicate that these basic CSF findings are profoundly different among the 10 different AIE subtypes. Whereas, AIEs with antibodies against NMDA, GABAB, and AMPA receptors as well as DPPX show rather frequent inflammatory CSF changes, in AIEs with either CASPR2, LGI1, GABAA, or glycine receptor antibodies CSF findings were mostly normal. Two subtypes, AIEs defined by either GAD, or IgLON5 antibodies, did not fit into this general pattern. In AIE with GAD antibodies, positive OCBs in the absence of other changes were typical, while the CSF in IgLON5 antibody-positive AIE was characterized by elevated protein.Autoimmune encephalitides (AIE) comprise a group of inflammatory diseases of the central nervous system (CNS), which can be further characterized by the presence of different antineuronal antibodies. Recently, a clinical approach for diagnostic criteria for the suspected diagnosis of AIE as well as definitive AIE were proposed. These are intended to guide physicians when to order the antineuronal antibody testing and/or facilitate early diagnosis even prior to the availability of the specific disease-confirming test results to facilitate prompt treatment. These diagnostic criteria also include the results of basic cerebrospinal fluid (CSF) analysis. However, the different antibody-defined AIE subtypes might be highly distinct with regard to their immune pathophysiology, e.g., the pre-dominance of specific IgG subclasses, IgG1, or IgG4, or frequency of paraneoplastic compared to idiopathic origin. Thus, it is conceivable that the results of basic CSF analysis might also be very different. However, this has not been explored systematically. Here, we systematically reviewed the literature about the 10 most important AIE subtypes, AIE with antibodies against NMDA, AMPA, glycine, GABA Autoimmune encephalitides (AIE) are inflammatory diseases of the central nervous system (CNS) . As a diRecently, diagnostic criteria for AIEs were proposed, which also incorporate CSF findings . Of noteIn this analysis, we systematically reviewed the literature regarding 10 AIE subtypes with well-defined antibodies. We extracted the reported CSF findings both on the basis of group findings as well as of data reported for individual patients whenever possible. In these different data sets, we analyzed the cumulative reported frequencies and levels of CSF pleocytosis, elevated total protein as well as the frequency of positive OCB with respect to the antibody-defined specific AIE subtype. In addition, we analyzed the typical combination of the three basic values when reported for individual patients to characterize the typical CSF result pattern in the 10 AIE subtypes.https://www.ncbi.nlm.nih.gov/pubmed) of the National Center for Biotechnology Information for publications published until December 31st, 2018 with regard to AIE with AMPA receptor (AMPAR), CASPR2, DPPX, glutamate decarboxylase (GAD), glycine receptor (GlyR), IgLON5, GABAB receptor (GABABR), GABAA receptor (GABAAR), LGI1, and NMDA receptor (NMDAR) antibodies using the following search terms: “NMDA,” “AMPA,” “CASPR2,” “DPPX,” “GAD,” “GlyR,” “IgLON5,” “GABABR,” “GABAAR,” or “LGI1” in combination with “encephalitis,” “IgLON5” combined with “case report,” “CASPR2” in combination with “seizure.” The hits were critically reviewed and all publications that reported CSF findings for two or more patients for one of the 10 specific antibodies were selected for further analysis. However, in AIE subtypes for which this strategy identified very few patients (<15), namely AIEs with CASPR2, DPPX, GABABR, and GABAAR antibodies, for individual CSF data comprising all three parameters, we also incorporated data from reports of two or one patients. It was carefully checked whether some patients might be reported in more than one publication. These patients were only included once. As characterizing the CSF abnormalities in children was beyond the scope of this paper, patients younger than 13 years were excluded.We reviewed the published literature using the PubMed data base  with GABABR antibodies and in 2 of 30 patients (7%) with AMPAR antibodies, 2 of 15 patients with DPPX antibodies (13%) and 18 of 52 patients (35%) with NMDAR antibodies but not in the other AIE subtypes. Maximal cell counts well above 500 cell/μl were reported for patients with GABABR (950 cell/μl) and NMDAR antibodies (730 cells/μl) only. However, for GABABR antibody-positive AIE this cell counts can be judged as exceptionally high as the next highest cell count was considerably lower (159 cells/μl).The cumulative reported frequencies of CSF pleocytosis were also highly divergent among the 10 different antibody-defined AIE subtypes: while present in 50% or more of the patients with NMDAR, AMPAR, GABAectively . Patientanalysis . Median frequent . HoweverAR as well as GlyR  among cases with individual exact values reported were substantially higher when these were compared to group data  were available. We thus omitted their graphical depiction as these two values were judged as not representative  with low anti-Caspr2 antibodies in serum only, and finally cerebellar ataxia , 27. HowAR antibodies have been reported in a variety of neurological diseases, even in patients finally diagnosed to suffer from a hereditary disease  associated with NMDAR antibodies is the most frequent AIE subtype , prefereBR, NDMAR, and AMPAR antibodies these were classified as pre-dominantly IgG1  and member of the executive board of the German Society for Cerebrospinal Fluid Diagnostics and Clinical Neurochemistry. He received funding from the BMBF for a project have covers the topic of autoimmune encephalitides. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The ears of many mammals have a set of uniformly spaced horizontal ridges that form groove arrays. Contact of coherent waves (e.g. acoustic waves) with a series of slits or grooves causes diffraction, which produces constructive and destructive interference patterns. Increases in signal strength will occur but will depend on the frequencies involved, the groove number and their separations. Diffraction effects can happen for a wide range of frequencies and wavelengths, but no array can diffract wavelengths greater than twice the groove separation, and it is for those wavelengths comparable in size with the groove separation that the effects are greatest. For example, when ridges in bat ears are 1 mm apart, the strongest influence will occur for a 1 mm wavelength which corresponds to a frequency of 343 kHz. If bats could use these wavelengths, it would help them to resolve objects or surface textures of about 0.5 mm. Given how critical acoustics are for bat function, we asked whether bats may be taking advantage of diffraction effects generated by the grooves. We hypothesize that groove number varies with bat foraging strategy. Examining 120 species, we found that groove number is related to both guild and ear length. Bats in guilds that glean prey items from foliage or ground have on average more grooves than bats in other guilds. Harmonics generated by echolocation calls are the most likely source for the wavelengths that would correspond to the groove separations. We apply the physical principles of wave reflection, diffraction, and superposition to support the hypothesis that acoustic responses generated from grooves may be useful to bats. We offer an explanation why some bat species do not have grooves. We also discuss the presence of groove arrays in non-echolocating Chiropterans, and five additional mammalian orders. Many nocturnal mammals rely on auditory cues for orientation, resource detection, and predator avoidance –4. The pI is the peak intensity, I0 is the incident intensity, a is the slit or groove width, λ is the wavelength, and θ is the outgoing angle, p is a parameter to count grooves, n is the number of slits or grooves, and d is the distance between the ridges . The. The13]. = 0) Eq I=4I0, a 1 mm wavelength (λ) will correspond to a frequency of 343 kHz. A 1mm diffraction grating will begin to cause diffraction effects at ~172 kHz and above with the peak response occurring at 343 kHz.Therefore, an array with 12 grooves produces a very narrow central peak with the intensity 144 times greater than that from a single groove. Note also that diffraction effects are more pronounced the more monochromatic (single frequency) the source. While for a given grating diffraction effects can happen for a wide range of frequencies and wavelengths, no grating can diffract wavelengths greater than twice the groove separation, therefore it is for those wavelengths comparable in size with slit separation that the effects are greatest. According to the relationship It is important to appreciate that lobes produced by plane waves impacting flat diffracting gratings are described as lying in a plane orthogonal to the grating surface and parallel to the grooves. Diffraction effects redirect wave energy transversely from the grooves in the arrays. Because the array is oriented horizontal to the ear’s long axis and centered above the ear canal, the diffraction lobes are directed toward the ear drum. Note that when an array is present in an ear, the grooves are always oriented in this direction. If the grooves in an array were vertical, the grooves would direct most of the energy away from the ear canal and would be unable to provide benefit.Given how critical acoustics are to bats, it is possible that bats are taking advantage of the effects generated by the grooves. While the overall shape of the ear focuses all sound frequencies in a conventional way, we postulate that the grooves are concentrating diffracted frequencies into primary diffraction lobes aligned with the reflection angle, which is controlled by groove dimensions and number.To take advantage of the diffraction effects created by 1mm grooves in some way, bats would need to be able to make use of frequencies greater than 172 kHz with a peak response occurring at 343 kHz. Although it would be surprising to learn that bat species could hear frequencies as high as 343 kHz, there is evidence that the hearing of some species goes as high as 250 kHz. For example, Myotis oxygnathus responded to sounds as high as 250 kHz, which was the highest frequency the equipment was able to test . Some baGlossophaga soricina can discriminate structural differences as small as 380 μ m using calls with three overlapping harmonics that starts with a fundamental FM sweep of ~55–95 kHz. The authors conclude that the most likely source of that ability comes from wave interference patterns generated from suitable sized wavelengths of ~190 kHz found in the third harmonic [G. soricina to discriminate that level of detail, then it means the intensities within harmonics will be greatly diminished and that the bat is using those frequencies somehow. The diffraction effects attributable to the number of grooves (G. soricina has 6 grooves) within the arrays may be making it possible to increase the intensities to usable levels. But that still would not explain how many species would be able to use frequencies generated as harmonics that could be well above their hearing range. We could not find information on the hearing range for G. soricina. Using the principles of wave behavior, we offer a possible explanation of how the grooves may be assisting the bats to access information from frequencies that may be above their hearing range.Target resolution is related to wavelength . Wavelenharmonic . If harmEcholocating bats have evolved highly specialized auditory structures including variability in ears along with a diversity of feeding strategies. In this study, we evaluate if the groove number varies with bat foraging strategies. We hypothesize that the grooves reflect incoming acoustic waves in patterns that make them useful to bats and discuss the wave behaviors that may play a role. Throughout this manuscript we consider a 1mm separation array for the ease of maintaining a consistent discussion, but it must be kept in mind that groove separation in bats can be above and below this value. We also discuss the presence of the groove arrays in the ears of non-echolocating bats, and four additional orders of mammals.To determine the number of grooves in the ears of bats, we took photos of bats that we captured in Arizona, United States, and Nicaragua. Animals were captured and handled following guidelines of the American Society of Mammalogists  and withWe measured groove separation in bat ears by laying a ruler next to the grooves when taking photographs. From these photos, we counted the grooves and measured groove separation as the distance from ridge peak to peak . We counWe computed Pearson's correlation coefficients to determine the relationship between ear length and groove number within the pooled data set and within each guild separately. We analyzed the effect of guild and ear length on groove number using generalized linear models in R . We onlyThe number of grooves in bat ears varied between 0 and 20 among the 120 bat species that we assigned to guilds . Groove The Pearson’s correlation coefficient describing the relationship between ear length and groove number was 0.6 in the pooled data set. Within the guilds the coefficient ranged from -0.18 to 0.68 .The number of grooves modeled as guild and ear length is the best model, followed by the number of grooves modeled as ear length and number of grooves modeled as guild alone . Log-likOur cursory review of mammals other than bats found that at least 12 other species of mammals in 4 orders also have recognizable groove fields associated with their ears .Molossus sinaloa) have relatively short 13 mm-pinnae with 9 grooves, whereas some serotine bats (Eptesicus serotinus) have long 20 mm-pinnae with only 5 grooves. If these differences in ear size and groove number are related to acoustic needs, it may lend support to the hypothesis that the groove number may have a stronger link to foraging strategy than ear length.Our data show that groove number is related to both guild and ear length. Bats in the narrow space, passive gleaning guild (guild 5) have on average more grooves and some of the longest ears compared to bats in the other guilds , Table 3in and θout are the incident and outgoing angles, d is the groove separation, and m  is the diffraction order. For normally incident waves, and first order diffraction, θin is 0 and sin(θout) = θ /d. Because outgoing waves with θout greater than 90 degrees are not possible, sin must be less than 1, and the maximum diffracted wavelength for normal incidence has to be less than the groove separation d. By allowing non-normal incident waves, the constraint can be relaxed to λ< 2d. Therefore, under normal incidence, waves longer than d will simply be reflected, but not diffracted. For extreme incidence, waves up to 2d can be diffracted. This implies that a 1mm based groove array should cause diffraction effects to begin for frequencies ~>172 kHz with the peak response occurring at 343 kHz. In addition, because different wavelengths emerge from the array at slightly different angles, bats would have the ability to make micro-adjustments to the array angle to monitor different frequencies being diffracted from the array.Diffraction gratings can work with a range of wavelengths, but no grating can diffract wavelengths greater than twice the groove separation. The general diffraction equation for incident waves of arbitrary angle is d. The ability to retract the ears during roosting may help to reduce injury, conserve energy, and reduce water loss [Nycteris have no grooves; conversely, other species such as Artibeus gnomus have many grooves but small ears that may not need to be folded. Therefore the grooves might have multiple purposes; assist the ear to flex during folding, and to maneuver the array in a manner that directs sound energy into the ear and to offer diffraction effects.When measuring the groove separations, we noticed that the bats can change the shape of their ears, including the groove arrays, sometimes extending them or contracting them which altered the groove separation measurements. Schneider  found thter loss . HoweverKerivoula, Murina, Ceolops, Natalus, Fruripterus, Thyroptera and Nycteris. Bats without grooves have been shown to produce some of the highest fundamental frequencies documented in the vocalizations of all echolocating bats. For example, echolocation calls of bats in the genus Kerivoula have been recorded as high as 292 kHz [Glossophaga soricina attributed the ability to discern minute structural details to the use of ~190 kHz found in the third harmonic [Some echolocating bat species have indistinct or no grooves in their ears, including species in the genera  292 kHz  with ban 292 kHz . A studyharmonic . PresumaNycteris grandis of the grooveless gleaning bats does not appear to use the distinct vocalization differences when compared to grooved species like those we just described for aerial foraging strategies  appear to have no grooves. Information on the vocalizations for most of the grooveless species are not readily available but it appears that at least rategies . In ordeIn addition to echolocating bats, other bat species that do not echolocate (i.e. Pteropodidae) and species in other mammalian orders, have grooves in their ears , often wThere are still many unanswered questions about how a groove array in the ear of a mammal influences acoustic signals and how that information may be of use, if at all. Basic wave theory leads us to believe the ear grooves are offering additional auditory capabilities over simple reflections, such as enhanced directionality and wavelength separation/selectivity. The different groove separation measurements may provide insight into the frequencies that are most useful to the species. Changing the angle of the array may help to monitor specific frequencies being diffracted. The highest groove numbers are found in echolocating bats known to glean prey from the ground or foliage. Based on our review of echolocating bats, the groove number appears to have a relationship to foraging strategy and therefore could help to clarify either guild definitions (in the case of bats) or provide greater insight into niche partitioning. Future studies evaluating the use of grooves in echolocating bats should try to document the presence of the frequencies that correspond to groove separations by ensuring the microphones are properly placed to overcome attenuation and capable of processing the information associated with those frequencies.S1 Table(XLSX)Click here for additional data file.