However, the practical application of Ir-based catalysts is significantly limited owing to their high cost in addition to the scarcity of the metal. Therefore, it is necessary to improve the efficiency of the utilization of such catalysts. In this study, IrO2-coated Ti felt (IrO2/Ti) electrodes were prepared as high-efficiency catalysts for the OER under acidic conditions. By controlling the surface roughness of the Ti substrate via wet etching, the optimum Ti substrate surface area for application in the IrO2/Ti electrode was determined. Additionally, the IrO2 film that was electrodeposited on the 30 min etched Ti felt had a large surface area and a uniform morphology. Furthermore, there were no micro-cracks and the electrode obtained (IrO2/Ti-30) exhibited superior catalytic performance with respect to the OER, with a mass activity of 362.3 A −2). Therefore, this proposed strategy for the development of IrO2/Ti electrodes with substrate surface control via wet etching has potential for application in the improvement of the efficiency of catalyst utilization with respect to the OER.Under acidic conditions, IrO Hydrogen, which is a pollution-free energy resource with the convenience of long-term storage in small and large quantities without significant loss, has a number of distinct advantages as an alternative to fossil fuels  was dissolved in DI water and stirred for 30 min. Then, 40 mM oxalic acid [(COOH)2·2H2O] and 100 mM hydrogen peroxide (H2O2) were added, and the mixture was homogenized for 10 min. To adjust the pH to 10.5, 340 mM potassium carbonate (34.5% K2CO3) was added, followed by stirring for 3 days to ensure the stability of the prepared electrodeposition solution , which served as the substrate. Prior to the electrodeposition, etching with 5 wt.% oxalic acid was performed to remove any oxide layer or impurities that could be present on the Ti felt. Then, to observe the changes on the surface of the IrO2/Ti electrode with respect to the etching time, etching was performed for 0, 10, 20, 30, and 40 min in the etchant at 95°C.IrO2 electrodeposition was performed using a potentiostat  via the application of a constant current density of 2.5 mA cm−2 to the Ti felt for 10 min in the IrO2 electrodeposition solution at 25°C. The reaction that took place in this anodic electrodeposition is as follows.A three-electrode system was used for the anodic electrodeposition. The titanium mesh and the standard calomel electrode (SCE) were used as a counter and reference electrode, respectively, and IrO2/Ti electrodes. X-ray diffractometry  was performed to observe the crystallinity of the IrO2 layer. For the conditions for the XRD, a Cu target at 40 kV and 200 mA, with a 2θ angle in the range 30–80° and a scanning rate of 1° min−1, was used. Additionally, the crystal structure of the IrO2 film was analyzed via X-ray photoelectron spectroscopy  using an Al Kα (1486.6 eV) light source, and each peak was fitted with carbon (284.6 eV). Inductively coupled plasma-mass spectroscopy  was employed to determine the loading mass of Ir.Field emission scanning electron microscopy  and optical microscopy  were performed to observe the surface morphology of the prepared IrO2/Ti electrode, linear sweep voltammetry (LSV) and stability tests were performed using a potentiostat . IrO2/Ti (1 cm x 1 cm), Ag/AgCl (saturated KCl), and Pt were used as the working, reference, and counter electrodes of the three-electrode system, respectively. A 0.1 M solution of HClO4 was used as the electrolyte, and the LSV was performed within a voltage range of 0.25–2.0 V (vs. RHE). Additionally, the stability tests were conducted via the application of a constant current of 10 mA cm−2 for ~800 min, and electrochemical impedance spectroscopy (EIS) was performed within a frequency range of 100–200 kHz at a potential of 1.34 V.For the electrochemical characterization of the IrO2/Ti- 0, 10, 20, 30, and 40, respectively. After etching, a layer of IrO2 was deposited on the Ti felt via the application of a current density of 2.5 mA cm−2 for 10 min at 25°C in an electrodeposition solution. Following the electrodeposition, the Ti felt was uniformly coated with the IrO2 film (dl) value of the Ti felt with respect to the etching time. Generally, Cdl is proportional to the electrochemical surface area (ECSA), and is related to the roughness factors of the surface  to 1.675 mF (40 min). This indicates that there was an increase in the surface roughness in addition to the surface area of the Ti felt with the etching time  are shown in 2/Ti-30 electrode. Additionally, its Ir 4f XPS spectra showed peaks at 65.1 eV (4f5/2) and 62.1 eV (4f7/2), which corresponded to the Ir3+ state, and others at 66.4 eV (4f5/2) and 63.5 eV (4f7/2), which corresponded to the unscreened component of the Ir4+ state  to 0.234 mg cm−2 (IrO2/Ti-20) (−2 (IrO2/Ti-40) at 40 min. Based on these Ir loading masses, the overpotential was recorded at 20 A 2/Ti-30 electrode also showed the lowest overpotential value at 240 mV, and a comparison of the Ir mass activity at 2.0 V (vs. RHE), revealed the best OER performance  . After 3activity . The IrOformance . When thnditions . The resotential . The Taf0, and A represent the overpotential, current density, exchange current density, and the Tafel slope, respectively , IrO2/Ti-10 (67 mV dec−1), IrO2/Ti-20 (54 mV dec−1), and IrO2/Ti-40 (88 mV dec−1), indicating that the IrO2/Ti-30 electrode surface results in faster reaction rates in the OER. Hence, this demonstrates superior catalytic activity. ECSA enhancement implies an increase in the number of accessible Ir active sites  of the IrO2/Ti-30 electrode, which is proportional to ECSA, was 0.755 mF. This Cdl value was higher than those of IrO2/Ti-0, IrO2/Ti-10, IrO2/Ti-20, and IrO2/Ti-40, which were 0.267, 0.569, 0.580, and 0.639 mF, respectively . These species are susceptible to attack by water molecules or OH species; thus, the potential-determining step and rate-limiting step of the OER, resulting in the formation of O-O bonds, can be accelerated (Jiang et al., 2 layer lower the adsorption energy required to attract H2O molecules. This leads to the accumulation of oxidation equivalents, which are associated with enhanced OER activity, at reaction sites (Bergmann et al., 2 catalyst layers revealed that the IrO2/Ti-30 electrode showed the highest OER activity and was evaluated using 0.1 M HClO4 as electrolyte at 25°C. The variation of its potential with time owing to the application of a constant current density of 10 mA cm−2 is shown in 2/Ti-0 electrode. This superior stability could be attributed to the IrO2 catalyst layer, which serves as a passivation layer that protected the Ti felt from corrosion.The enhanced OER activity of the amorphous IrO2-based catalyst on Ti felt. To increase the surface area of the Ti felt before the electrodeposition, its surface roughness was increased via wet etching. Thus, after etching for 30 min, there was an increase in its surface roughness, and the amorphous IrO2 layer was uniformly deposited. This resulted in superior OER catalytic activity in comparison with the deposition of the crystalline phase, owing to the presence of surface iridium hydroxide. Additionally, the IrO2/Ti-30 electrode showed superior OER catalytic activity and mass activity, which may be attributed to an increase in active sites owing to an increase in surface roughness. Stability tests that lasted ~800 min also confirmed its excellent stability in comparison with the IrO2/Ti-0 electrode. This electrode with increased surface roughness and excellent stability, prepared via electrodeposition and etching, demonstrated superior OER catalytic performance under acidic conditions. Therefore, the results of this study can serve as a promising path for the production of high-performance and low-cost OER electrodes for application in proton exchange membrane water electrolysis.In this study, an electrode for the OER was prepared via the electrodeposition of an amorphous IrOAll datasets generated for this study are included in the article/YJP and JL conceived the idea and designed the experiments. YJP, YSP, and SC synthesized the electrocatalysts and evaluated their electrochemical properties. JY and MJ performed the structure analysis. JL and JJ performed the physical characterizations. JWL, J-DK, and SMC coordinated and supervised the overall project. All authors reviewed the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Continuous circulation and drainage of cerebrospinal fluid (CSF) are essential for the elimination of CSF-borne metabolic products and neuronal function. While multiple CSF drainage pathways have been identified, the significance of each to normal drainage and whether there are differential changes at CSF outflow regions in the aging brain are unclear.Dynamic in vivo imaging of near infrared fluorescently-labeled albumin was used to simultaneously visualize the flow of CSF at outflow regions on the dorsal side  of the central nervous system. This was followed by kinetic analysis, which included the elimination rate constants for these regions. In addition, tracer distribution in ex vivo tissues were assessed, including the nasal/cribriform region, dorsal and ventral surfaces of the brain, spinal cord, cranial dura, skull base, optic and trigeminal nerves and cervical lymph nodes.Based on the in vivo data, there was evidence of CSF elimination, as determined by the rate of clearance, from the nasal route across the cribriform plate and spinal subarachnoid space, but not from the dorsal dural regions. Using ex vivo tissue samples, the presence of tracer was confirmed in the cribriform area and olfactory regions, around pial blood vessels, spinal subarachnoid space, spinal cord and cervical lymph nodes but not for the dorsal dura, skull base or the other cranial nerves. Also, ex vivo tissues showed retention of tracer along brain fissures and regions associated with cisterns on the brain surfaces, but not in the brain parenchyma. Aging reduced CSF elimination across the cribriform plate but not that from the spinal SAS nor retention on the brain surfaces.Collectively, these data show that the main CSF outflow sites were the nasal region across the cribriform plate and from the spinal regions in mice. In young adult mice, the contribution of the nasal and cribriform route to outflow was much higher than from the spinal regions. In older mice, the contribution of the nasal route to CSF outflow was reduced significantly but not for the spinal routes. This kinetic approach may have significance in determining early changes in CSF drainage in neurological disorder, age-related cognitive decline and brain diseases. Most of the CSF then flows around the SAS of the brain and spinal cord towards multiple outflow sites and ultimately enters the systemic vascular system  mice at 2–3 months old , and at 11–13 months  were used. All animal studies were performed in accordance with the National Institute of Health guidelines and using protocols approved by the University Committee on Animal Resources. Mice were housed in the vivarium of the University of Rochester, School of Medicine and Dentistry, on a 12:12 light/dark schedule (6AM: 6 PM) with food and water ad libitum., and placed in their normal prone position on a temperature-controlled stage to maintain body temperature. Anesthesia was maintained with 1–2% isoflurane in oxygen. Once anesthetized, a skin incision was made in the midline from the nose to the end of the spinal cord and the skull and spine column exposed to enhance the NIRF signal intensity. Fat tissues on the back of the neck were dissected to expose the cervical spinal column. The cisterna magna was exposed, cannulated using a 30G needle, connected to a 10 µL Hamilton syringe and to a syringe pump , as reported )at 37 °C , so thisWe did not see any tracer associated with the optic and trigeminal nerves or the dura , but saw tracer in the nasal/cribriform and spinal regions, major pial blood vessels, and brain surfaces of ex vivo samples. We also confirmed these results using Evans blue-albumin. Tracer has been seen previously to be associated with the trigeminal nerve and skull base , 31. Pre and from more recent reports [In fluid balance, the lymphatic system collects excess tissue fluid due to an imbalance in the filtration/reabsorption process at the capillaries, and returns it to blood. The tight junctions between cerebral endothelial cells  and low permeability for polar molecules across the endothelium accounts for the low hydraulic conductivity of the cerebrovasculature , 70, 71. reports , 31, 32.We found no significant CSF flow from the SAS into the brain parenchyma, as reported previously , in contOur data confirm the present of CSF tracers alongside major pial vessels, presumably the perivascular compartment, as described previously e.g., , 67, 72), 7267, 7We show that aging differentially affects the CSF drainage kinetics by reducing delivery to and elimination from the main nasal/cribriform plate outflow site but not significantly changing that for the spinal regions. Using elimination t1/2 to estimate the contribution of these two main routes to the outflow of CSF indicates that the nasal route contributes 1.5 times that of the spinal in young adult mice, and aging reduced the nasal contribution to 0.8 that of the spinal Fig. e. Using In the older mice, the contribution of the two main CSF elimination regions was about the same. Aging has been shown to reduce the rate of CSF secretion and this may lead to a reduction in drainage –41 and CThe primary objective was to determine the elimination rate of tracer in CSF at already-known CSF outflow sites. A major limitation was that our in vivo imaging system cannot assess sites on the ventral brain surface, such as the optic and trigeminal nerves, and the skull base. We used additional ex vivo tissues to look for the association of CSF tracer with those regions previously identified as outflow sites, especially on the ventral brain surface and the skull base. At the end of the 150 min there was some retention of tracer at all the sites imaged in vivo. CSF may be trapped in the various fissures and cisterns in the SAS and remain there until it is eliminated at the CSF outflow sites and a longer imaging time could produce a different result. A further limitation is that the exact location of tracer cannot be distinguished. For example, the longitudinal fissure, which separates the two hemispheres, is under the superior sagittal sinus and dural lymphatics so transcranial recording for the SS also includes signals from this fissure, brain surface blood vessels, subarachnoid CSF. This limitation is also similar for the recordings from the nasal and spinal regions, in that tracer signals come from the subarachnoid CSF, brain, or other surrounding tissues. We used minimal invasive surgery hence our imaging system could not identify the specific spinal drainage sites. Further work is needed to confirm the spinal CSF drainage sites.In mice, using CSF labeled albumin, the nasal regions across the cribriform plate and spinal regions were the main CSF elimination sites using both in vivo and ex vivo data within the conditions of these experiments. There was no significant CSF outflow from the SAS to the cranial dura. Tracer was associated with the nasal/cribriform and spinal regions but not with the optic and trigeminal nerves or the skull base. Also, there was significant retention of NIRF-Albumin on the brain surfaces, including in fissures and around pial vessels. There was no significant inflow from the SAS into the brain parenchyma. In young adult mice, there was a faster CSF elimination rate from the nasal/cribriform plate compared to the spinal regions. For the first time, we show that aging differentially affects the CSF drainage kinetics by reducing delivery to and elimination from the nasal/cribriform plate outflow site but not significantly changing that at the spinal regions. Thus, delayed CSF outflow could be associated with the onset of brain aging, neurodegeneration and age-related decline in cognition.Additional file 1: Figure S1. Analysis of CSF outflow kinetics. Figure S2. Ex vivo images of CSF outflow regions at 150 min. Figure S3. Ex vivo images of CSF outflow regions at 30 mins. Figure S4. Images of the optic nerves and ventral spinal column at 30 min. Figure S5. Albumin distribution at the cervical lymph nodes and Prox1Tom dural expression. Figure S6. Aging alters CSF outflow kinetics. Table S1. Deducible kinetic parameters for the superior sagittal sinus (SS) and transverse sinus (TS).


The aim of this study was to assess cerebrospinal fluid (CSF) concentrations of specific amino acids using a high-performance liquid chromatography system in a sample of patients with functional movement disorders (FMDs) and in a sample of controls. CSF levels of glutamate were significantly lower in patients with FMD than in controls. This finding argues in favor of glutamatergic dysfunction in the pathophysiology of FMD. Growing evidence suggests that abnormalities in glutamatergic neurotransmission, via the N-methyl-d-aspartate receptor (NMDA-R), have a key role in the pathophysiology of several neuropsychiatric conditions, such as schizophrenia, mood disorders, and Alzheimer’s disease4. Recently, a possible role of glutamate + glutamine (Glx) has been suggested in the pathophysiology of FMDs , a neuropsychiatric condition characterized by the presence of motor symptoms that cannot be explained by typical neurological diseases or other medical conditions5. Although they are highly prevalent and have a considerable impact on national health systems, the pathophysiology of FMD remains unclear. In the past decade, new hypotheses based on the integration between psychology and neurobiology have been formulated, gradually shifting the attention of the scientific community from traditional psychoanalytic theories to models considering neurobiological factors6. Functional neuroimaging studies have underlined abnormalities in FMD at the level of brain network activity, connectivity, and specific anatomic areas of altered metabolic demand during tasks (mainly the limbic system)7. With respect to Glx, Demartini et al.5 recently showed, through a brain magnetic resonance spectroscopy (MRS) technique, that patients with FMD presented significantly higher levels of Glx in the limbic system than healthy controls. The authors hypothesized that abnormal increase in Glx in the limbic system might have a central pathophysiological role in FMD onset and maintenance, possibly by altering limbic–motor interactions. However, no studies have been conducted assessing the levels of glutamate and glutamine in the cerebrospinal fluid (CSF) of patients with FMD.Glutamate has an important role as the primary excitatory neurotransmitter in the central nervous systemGiven the evidence of a potential role of Glx in the pathophysiology of FMD, we aimed to retrospectively assess CSF levels of glutamate and glutamine in a sample of patients with FMD and in a sample of controls. Moreover, we also assessed the CSF levels of two other amino acids, alanine and asparagine, serving as controls.p > 0.05).Eight patients with FMD and nine controls were recruited. The Kolmogorov–Smirnov test showed that all the continuous variables respected the assumption of a normal distribution . Multivariate analysis of variance (ANOVA) showed that CSF levels of glutamate were significantly lower in patients with FMD (mean 1.23 ± 0.66) than in controls (mean 2.17 ± 1.05)  = 4.751, p = 0.046). Neither CSF levels of glutamine , asparagine , nor alanine  differed between the two groups  = 0.000, p = 0.994 for glutamine, F  = 0.293, p = 0.596 for asparagine, F  = 0.041, p = 0.841 for alanine)  and date of CSF amino acid measurements Table . No majo4. The discrepancy in CSF glutamate and glutamine levels observed in previous studies might be due to different analytical methods used. Another reason for the inconsistency of results may be a time-dependent in vitro hydrolysis of glutamine to glutamate in the CSF8. In the present study, the mean storage time of patients with FMD and control CSF samples was very similar . In addition, no significant differences in CSF glutamine levels were observed between patients with FMD and control samples. Thus, glutamine hydrolysis, if present, unlikely contributed to differences in glutamate levels observed in the present study. However, previous studies suggested an altered glutamatergic function as a possible key feature in the pathophysiology of different neuropsychiatric disorders, including schizophrenia, depression, and Alzheimer’s disease4. Although an indirect measure of glutamatergic activity, the reduction we found in CSF glutamate of patients with FMD suggests that hypofunction of the glutamatergic system may be present in these patients. It is well known that glutamate plays an important role in brain synaptic plasticity and is involved in several cognitive functions, such as learning and memory, in the hippocampus and neocortex. A proper glutamatergic tone is fundamental for neuronal differentiation and connection. Reduced glutamatergic neurotransmission is known to impair neuronal migration and circuitry maturation, ultimately leading to the development of neuropsychiatric disorders9. Although certain data on FMD are still not available in the literature, we hypothesize that dysregulation of the glutamatergic system might have a specific role in the pathophysiology of FMD. Our hypothesis also fits with the neurophysiological data showing an impairment of cortical excitatory networks, as proven by the well-known paradoxical decrease in motor excitability during motor imagery, which is not restricted to the clinically affected body part11. Moreover, given the absence of any major psychiatric comorbidity in our sample, which could have acted as a confounding factor, we might assume that the abnormal level of glutamate detected is specific for FMD. We did not find any significant correlation between CSF glutamate levels and (i) severity of FMD or (ii) duration of disease. This result, although possibly altered by the small sample size, might suggest that CSF glutamate levels are independent of the severity and duration of symptoms.In this study, which was designed to provide information on CSF glutamate and glutamine and subsequently on pathophysiological mechanisms in FMD, the main finding is that patients with FMD have decreased CSF glutamate compared to a group of controls. To the best of our knowledge, this is the first study exploring the levels of glutamate and glutamine in the CSF of patients with FMD. Previous studies have assessed CSF glutamate and glutamine in different neuropsychiatric conditions, with contradictory results: some studies found glutamate or glutamine to be decreased in patients’ CSF, while others found glutamate or glutamine to be increased5. Nevertheless, first, it is still unclear whether CSF glutamate levels reflect the actual synaptic activity in each corticolimbic region; CSF levels may possibly represent the overall equilibrium of the extraneuronal space12. Thus, it is likely that the difference in glutamate levels between this study and our previous MRS study may be due to the difference between CSF samples and specific corticolimbic areas. Second, here, we specifically measured glutamate separate from glutamine, while in the MRS study, for technical issues, we assessed levels of Glx, namely, the sum of glutamate and glutamine. Third, although it is still not clear whether the low concentration of glutamate in CSF reflects a primary neuronal number or synaptic deficit or whether it represents a secondary compensatory adaptation, we might hypothesize that different areas of the brain, including the limbic system and other areas known to be involved in the pathophysiology of FMD, such as the dorsolateral prefrontal cortex, might abnormally express NMDA-R.Our results might be in contrast with our previous spectroscopic finding according to which Glx in the limbic system of patients with FMD was increased with respect to healthy controls13. Moreover, our data are substantially in line with the results of previous studies assessing levels of CSF glutamate with the same methodology in healthy subjects 8. Concerning this aspect, it is worthwhile to note that although the means of CSF glutamate levels of both patients with FMD and controls fall within this normal range, the range appears to be very wide, and the difference between the two groups, despite the small sample size, is statistically significant.This study has the following limitations: first, the small sample size might limit the generalizability of our results; second, given that previous studies have shown that a good proportion of patients with FMD present psychiatric comorbidities, the absence of psychiatric comorbidities in our sample might limit the generalizability of our results; third, the heterogeneity of controls might also limit our findings. However, to the best of our knowledge, no previous study has ever shown increased CSF glutamate levels in patients with Wernicke encephalopathy, vascular encephalopathy, and Tolosa–Hunt syndrome. With respect to multiple sclerosis, although glutamate seems to be implicated in its pathogenesis, a recent study showed that the levels of CSF glutamate in patients with multiple sclerosis did not differ from those in healthy controlsIn conclusion, our findings argue in favor of glutamatergic dysfunction as a possible biological marker in the pathophysiology of FMD. Moreover, our results might have some therapeutic implications in terms of testing novel pharmacological approaches with drugs acting on NMDA-R in FMD.This was a retrospective study, including all FMD patients whose CSF was stored at −80 °C and registered in the laboratory information system of three Italian university hospitals in Milano  between June 2017 and December 2019. CSF samples were collected in the same way in each center: they were collected into 15 mL polypropylene tubes by LP in the L3/L4 or L4/L5 interspace. The LP was carried out between 8 and 10 a.m. after one-night fasting. Following LP, CSF samples were centrifuged at 2000 r/min for 10 min at 4 °C. The supernatants were aliquoted in polypropylene tubes and stored at −80 °C until use.14, the severity of FMD according to the Psychogenic Movement Disorders Rating Scale (PMDRS) score15, date of symptom onset, date of LP, sample arrival date at the laboratory, and date and result of CSF measurements. Controls were patients who had LP for diagnostic purposes other than FMD. The CSF analysis  in controls led to the following conclusions: the absence of neurological disorders in three subjects, Wernicke encephalopathy in two subjects, multiple sclerosis in two subjects, vascular encephalopathy in one subject, and Tolosa–Hunt syndrome in one subject. Patients with FMD were included if they had “clinically established and documented” FMD according to the Fahn and Williams criteria16. Although participants had CSF collected for diagnostic purposes, they all signed written informed consent for the possible use of their CSF for scientific research purposes. The study was approved by the ethics committee of ASST Santi Paolo e Carlo .Through a review of patient charts, we registered age, gender, major psychiatric comorbidity 17. Briefly, 100 μL of CH3OH was added to 100 μL of thawed sample, vortexed for 20 s, and centrifuged  at 4 °C. Fifty microliters of supernatant was diluted with 200 μL borate buffer , and 50 μL of this solution was transferred to the autosampler mini vial. Samples were derivatized with o-phthalaldehyde-mercaptoethanol reagent according to a previously described procedure17. Amino acid levels were expressed in μmol/LCSF measurements were performed in a single run on March 2020 at the Istituto di Ricerche Farmacologiche Mario Negri IRCCS in Milano. Amino acids were assayed by high-performance liquid chromatography coupled to fluorimetric detection as previously described, with minor modificationst tests. To assess the differences between patients with FMD and controls for the levels of CSF amino acids, a multivariate ANOVA with glutamate, glutamine, alanine, and asparagine as dependent variables and “Group” as a factor was run. Categorical variables were analyzed using the Pearson’s χ2 test. Spearman’s correlation analyses were run between amino acid levels and (i) severity of FMD and (ii) duration of disease.The Kolmogorov–Smirnov test was performed to assess whether the continuous variables respected the assumption of a normal distribution. The differences between patients with FMD and controls for the variable age, PMDRS score, and time elapsed between date of the LP and date of CSF amino acid measurements were calculated using standard NPJPARKD-00462R_nr-reporting-summary


Alzheimer’s disease (AD) is the most common type of dementia and affects millions of adults over the age of 60 years. Accurate diagnosis relies on multiple modalities, including neuropsychological testing, cerebrospinal fluid (CSF) biomarkers, and amyloid positron emission tomography (PET) imaging. Discordant results between CSF biomarkers and amyloid PET can be seen in up to 15% of cases and can lead to exclusion from clinical trials along with anxiety and confusion for the patient and family. We present two cases of discordance  that resulted in the exclusion of both patients from two large anti-amyloid monoclonal antibody trials. Alzheimer’s disease (AD) is the most common type of dementia and affects millions of adults over the age of 60 years. It is initially characterized by a slow progressive decline in declarative episodic memory. Impairment in executive function, as well as procedural memory, may not be apparent until later in the disease. The diagnosis and evaluation of AD involve interviewing and examining the patient, administrating a battery of neuropsychological tests, interviewing a proxy close to the patient, and using biomarkers. In a small subset of patients, Alzheimer’s disease can present with symptoms that overlap with primary tauopathies. Diseases such as frontotemporal dementia (Pick’s disease), progressive supranuclear palsy, and corticobasal degeneration fall into the category of primary tauopathies. In most cases, neuropsychological testing, a thorough history, and neuroimaging can aid in distinguishing between primary tauopathies and AD. Additionally, biomarkers in the cerebrospinal fluid (CSF) can illuminate the underlying pathology.Biomarkers are used for the diagnosis and monitoring of the progression of those with AD. The main biomarkers currently used include structural magnetic resonance imaging (MRI), including volumetric MRI, fluorodeoxyglucose uptake on positron emission tomography (PET) (FDG-PET), PET amyloid imaging, and markers within the CSF. Structural MRI and volumetric studies can be used in the evaluation of patients with symptoms of AD. Patients with early AD often show decreased hippocampal volume and atrophy of the medial temporal lobe. Sequential monitoring of hippocampal volume loss over time with volumetric MRI can be used to predict the progression of AD . FDG-PETAmyloid PET can be used to assess the presence of beta-amyloid plaques and can affect clinicians’ diagnostic confidence and management plans significantly . StudiesAβ42 levels in the CSF reflect the concentrations of soluble amyloid-β, which is hypothesized to correlate with amyloid-β deposits in the brain. A proposed model suggests that the earliest biomarkers of AD are decreased CSF Aβ42 and increased amyloid deposition in amyloid PET imaging, which can often be seen prior to the onset of symptoms . There iIn 2018, the National Institute on Aging and Alzheimer’s Association (NIA-AA) published a research framework to biologically characterize AD by ATN biomarkers  .We present two cases of discordance in amyloid-β PET and CSF biomarkers (PET-/CSF+). These patients were excluded from two major multi-center anti-amyloid monoclonal antibody clinical trials despite showing findings consistent with Alzheimer's on CSF. This case report describes cases of discrepancy given that many clinical trials exclude discordant patients with PET-/CSF+ biomarkers. Additionally, discussion of discordant results is crucial given that discordance could potentially represent up to 15% of those with AD ,6.Case 1A 74-year old male with a history of diabetes mellitus type two, hypertension, and hyperlipidemia presented to the Memory Clinic in September 2013 with a chief complaint of progressive short-term memory loss during the last three to four years. Functionally, he was independent in his activities of daily living with no changes in sleep or appetite. Vital signs and physical exam, including neurologic exam, were normal. His social history was significant for smoking one pack per day for many years and 16 years of education. Family medical history is significant for a maternal aunt diagnosed with Alzheimer’s dementia in her 70s. Initial assessment, including the administration of a 90-minute battery of neuropsychological testing, revealed mild impairments in list learning, delayed recall, and recognition pattern in verbal memory. Therefore, a diagnosis of single-domain amnestic mild cognitive impairment (aMCI) was made. Magnetic resonance imaging (MRI) of the brain was obtained, which showed no significant hippocampal volume loss by visual rating or significant white matter disease . Discordance between visual and quantitative rating can be seen in up to 13% of cases, with quantitative rating categorizing more patients as positive on amyloid PET imaging . The visThe experience of readers appears to be a factor in the interpretation of images, with a more experienced reader having a higher level of agreement between images . TherefoIn our first case, the same scan was read as positive by one nuclear medicine specialist and negative by the second specialist, working with the clinical trial. Reader concordance has been documented in previous literature with a 93.2% concordance rate for positive scans and a 100% concordance rate for negative scans . This hiThe implications of amyloid PET and CSF discordance on the patient and their family cannot be understated. This problem can present two-fold. Anxiety can be experienced by both the patient and caregivers surrounding the diagnosis. Many clinical trials have primary and secondary endpoints measuring CSF and amyloid PET. Excluding individuals from clinical trials due to CSF/amyloid PET discordance can have an undue negative effect on the well-being of the patient and their families. To the best of our knowledge, except for two case reports (both CSF+/PET-), there have been no head-to-head cross-sectional or longitudinal studies to compare in-vivo discordant cases with neuropathological findings -17. GiveAs life expectancy continues to increase, the prevalence of Alzheimer’s will likely increase given that the majority of those diagnosed are over the age of 60. This increased prevalence will further emphasize the need for improved therapies and inclusive clinical trials. These two cases demonstrate that biomarker discordance can obscure an accurate diagnosis of Alzheimer’s disease with implications for clinical trial eligibility and options for treatment.


Cerebrospinal fluid (CSF)-venous fistulas are a recently recognized cause of spontaneous spinal CSF leak and present most commonly with Valsalva (“cough”)-exacerbated or orthostatic headaches. By inducing CSF hypotension, they cause diffuse pachymeningeal enhancement and brain sag on MRI. This unusual case demonstrates the potential for bilateral subdural hygroma development in a patient with an undiagnosed CSF-venous fistula after ventral intermediate nucleus (VIM) deep brain stimulation (DBS) implantation. A 68-year-old gentleman with medically-refractory essential tremor underwent extensive preoperative evaluation by the Mayo Clinic-Rochester DBS Committee. Initial MRI during preoperative evaluation had no evidence of CSF hypotension, but MRI performed the day before surgery demonstrated diffuse pachymeningeal enhancement. He underwent bilateral VIM DBS implantation and presented in the subacute postoperative period with bilateral subdural hygromas. Further testing identified a prominent hyperdense paraspinal vein arising from the T10/T11 nerve root, consistent with CSF-venous fistula. Even when patients undergo rigorous preoperative evaluations for surgical procedures, insidious pathologies can develop and cause unexpected postoperative complications. Cerebrospinal fluid (CSF)-venous fistulas can cause intracranial hypotension, which radiographically manifests as diffuse pachymeningeal enhancement and brain sagging. Diagnosis of CSF-venous fistulas requires CT or digital subtraction myelography as they are not evident on routine MRI or CT scans ,2. TheseMethodsA retrospective chart review was conducted to describe in detail the clinical history and other pertinent details of the reported case. In tandem, we carried out a review of the literature. Three electronic searches were performed using Ovid Embase, PubMed, SCOPUS, and Cochrane databases from each individual database's inception to June 2020. The first literature search used the following string of terms: (CSF venous fistula) OR  AND (DBS) OR (Deep Brain Stimulation). The second literature search used the following string of terms: (CSF venous fistula) OR  AND (hygroma). The third literature search used the following string of terms: (DBS) OR (Deep Brain Stimulation) AND (hygroma). These three literature searches did not identify similar published cases describing postoperative DBS patients who developed bilateral subdural hygromas, which led to the diagnosis of a CSF-venous fistula. ResultsA 68-year-old right-handed gentleman with longstanding, medically-refractory essential tremor (ET), with right upper extremity tremor greater than left, presented for consideration of operative management. His symptoms began in high school and progressed despite maximal pharmaceutical management. He had no history of spine surgery, spinal anesthesia, or lumbar puncture. Neuropsychometric, speech, and psychologic evaluations were normal, without contraindications to surgery. Multichannel surface EMG confirmed an organic asymmetric postural and action tremor at 4.5 Hz. On physical exam, his only positive neurologic findings were bilateral tremor, right worse than left, and bilateral worsening of tremor amplitude with intention and action. As part of his work-up, an MRI brain with gadolinium contrast was obtained, which was unremarkable. His case was discussed at the multidisciplinary DBS Committee meeting for consideration of high-frequency ultrasound versus bilateral VIM DBS. Ultimately, DBS was recommended because of superior system adjustability for tremor treatment over a longer period of time and to treat bilateral symptoms.Six months after the initial MRI, he underwent a repeat preoperative MRI brain with gadolinium contrast the day before surgery, which was co-registered with a non-contrast head CT scan obtained the morning of surgery Figure . During On postoperative day 10, he presented to an outside emergency department for word-finding difficulty and right monocular vision loss, which self-resolved in less than one hour. A non-contrast head CT identified bilateral subdural hygromas Figure . In retrAlthough operative management of the bilateral hygromas was considered, surgical treatment was deferred until the underlying etiology causing CSF hypotension could be made. The patient remained clinically stable, and the hygromas remained stable on repeat CT scan 6 weeks post-operatively. We felt that, even if evacuated, the collections would likely re-develop if the underlying CSF leak was not corrected. Colleagues in our CSF Dynamics Clinic arranged CT myelogram, which revealed a prominent hyperdense paraspinal vein emanating from the left T10/T11 nerve root sleeve, consistent with a CSF-venous fistula Figure .We considered surgical nerve root ligation to treat the CSF-venous fistula. The COVID-19 pandemic delayed elective surgery. During the forced period of observation, he remained clinically stable without further neurological episodes, and the hygromas resolved spontaneously over four months Figure . At the Serious surgical complications from DBS, including intracranial hemorrhage or ischemic events, are rare, with 0.4% 30-day perioperative mortality and 1% permanent neurologic morbidity . One casBetween the completion of the thorough preoperative evaluation for surgical management of our patient’s ET and his bilateral VIM DBS implantation, this patient likely developed a spontaneous CSF venous fistula that led to CSF hypotension and predisposed him to postoperative bilateral subdural hygromas though bilateral postoperative subdural hematomas cannot be completely ruled out. An increased duration of time between initial evaluation and surgical management adds risk of new pathologies developing that may contribute to unexpected postoperative complications. Furthermore, the increased number of indications for and the greater frequency of DBS surgeries may result in increased identification of rare and unusual complications that require prompt diagnosis and disease-specific treatment.


Cerebrospinal fluid (CSF) amyloid-beta (Aβ) 42/40 ratio, threonine-181-phosphorylated-tau (p-tau), and total-tau (t-tau) represent core biomarkers of Alzheimer disease (AD). The recent availability of automated platforms has represented a significant achievement for reducing the pre-analytical variability of these determinations in clinical setting. With respect to classical manual ELISAs, these platforms give us also the possibility to measure any single sample and to get the result within approximately 30 min. So far, reference values have been calculated from measurements obtained in frozen samples. In this work, we wanted to check if the values obtained in fresh CSF samples differ from those obtained in frozen samples, since this issue is mandatory in routine diagnostic work.Fifty-eight consecutive CSF samples have been analyzed immediately after lumbar puncture and after 1-month deep freezing (− 80 °C). As an automated platform, we used Lumipulse G600-II (Fujirebio Inc.). Both the fresh and the frozen aliquots were analyzed in their storage tubes.In fresh samples, a mean increase of Aβ40 (6%), Aβ42 (2%), p-tau (2%), and t-tau (4%) was observed as compared to frozen samples, whereas a slight decrease was observed for Aβ42/Aβ40 ratio (4%), due to the higher deviation of Aβ40 in fresh samples compared to Aβ42. These differences are significant for Aβ40, Aβ42/Aβ40 ratio, p-tau, and t-tau. Nevertheless, the Aβ42/Aβ40 ratio showed a lower variability  with respect to Aβ42. With respect to the AD profile according to the A/T/(N) criteria for AD diagnosis, no significant changes in classification were observed when comparing results obtained in fresh vs frozen samples.Small but significant differences have been found for Aβ40, Aβ42/Aβ40 ratio, p-tau, and t-tau in fresh vs frozen samples. Importantly, these differences did not imply a modification in the A/T/(N) classification system. In order to know if different cutoffs for fresh and frozen samples are required, larger, multi-center investigations are needed. Cerebrospinal fluid (CSF) amyloid-beta 42 (Aβ42), Aβ40, Aβ42/Aβ40 ratio, threonine-181-phosphorylated-tau (p-tau), and total-tau (t-tau) are reliable biomarkers for amyloidosis (A), tauopathy (T), and neurodegeneration (N). These biomarkers are able to identify Alzheimer’s disease (AD) independent of the clinical stage, thus, including the preclinical stages . Also, tThe classical way to measure AD biomarkers is through manual Enzyme-Linked Immunosorbent Assay (ELISA) , which sInterestingly, these platforms offer the possibility to easily and quickly analyze any single, freshly collected CSF sample. This opportunity has two major advantages: (1) it may allow to rule out in real time the diagnosis of subacute encephalitis, namely Creutzfeldt-Jacob disease, and (2) in expert centers collecting every day CSF samples for diagnostic purposes, it might be useful/appropriate to get the result in the same day, as also possible in other medical services/specialties.So far, reference values are represented by measurements obtained in samples aliquoted and frozen. The perspective to analyze fresh CSF samples made us wonder if biomarker measurements can differ between fresh and frozen samples. If yes, then new cutoffs should be defined for fresh samples. Accordingly, in this work, we evaluated the values of Aβ42, Aβ40, Aβ42/Aβ40, t-tau, and p-tau, obtained in fresh and after 1-month freezing CSF samples.g for 10 min, at room temperature, and then aliquoted in 0.5 mL aliquots in sterile polypropylene tubes .CSF samples were obtained from a consecutive series of out-patients referring to Center of Memory Disturbances of the University of Perugia for routine diagnostic work-up and collected according to international guidelines –25. All Fifty-eight CSF samples  were consecutively collected from patients and processed as described above. Patient data  are included in the supporting information in Table Sρ). Bland-Altman plots [blandr [ΔB between freshly acquired (Bfresh) and post-freezing (Bfrozen) values of y Aβ40, Aβ42, Aβ40/Aβ42 ratio, p-tau, and t-tau, from two aliquots originating from the same CSF sample.To evaluate the impact of freezing, biomarkers values measured on fresh samples were compared to the ones obtained from another aliquot of the same sample after 1-month storage at − 80 °C. Due to the non-normality of biomarker data, Passing Bablok regressions  were peran plots  with tre [blandr . To asseΔB values for each tested biomarker, thus one-sample Student’s t test was applied to assess the significance of the relative differences in biomarkers between fresh and frozen CSF samples. A p value below 0.05 was considered significant to reject the null hypothesis. Cutoff values for fresh samples were calculated using both regression analysis [Kolmogorov-Smirnov normality test could not reject the normality of the analysis  and by manalysis . For theIn order to appreciate the variability of fresh vs frozen CSF aliquots, we compared the NIA-AA A/T/(N) classification criteria using standard cutoffs. These standards, developed in our laboratory, are also reported in the biomarker assay cartridge datasheets of the Lumipulse G600-II. Biomarker values were classified as positive (+) or negative (−) by using the cutoff values of 0.069 for Aβ42/Aβ40 ratio, 56.5 pg/mL for p-tau, and 404 pg/mL for t-tau. Samples positive for both Aβ42/Aβ40 ratio and p-tau (A+/T+) have been classified as AD [The levels of Aβ40, Aβ42, p-tau, and t-tau were measured with a fully automated chemiluminescent platform  on two aliquots of the same CSF sample, one analyzed immediately after the sampling (“fresh”) and the other after 30 days of storage at − 80 °C. To investigate the relation between fresh and frozen biomarker levels, Passing Bablok regressions were performed, the results of this analysis are plotted in Fig. t test (see Table ρ = 0.99). Despite of the presence/absence of the freezing step, for each analyte, the measured SD of relative differences between fresh and frozen samples was of the same order of magnitude of the inter-assay variability (CV) of the QC sample  showed higher concentration values for Aβ40, Aβ42, p-tau, and t-tau in fresh samples and a trend of larger differences in magnitude for higher values of biomarkers. However, about 95% of the samples fell within 2 SD from the mean fresh vs frozen difference, confirming the high correlation obtained with Passing-Bablok analysis. As shown in Fig.  showed hFor each aliquot, Aβ42/Aβ40, p-tau, and t-tau have been classified according to the A/T/(N) criteria  by usingConsidering the classification Table , in someTo further investigate the impact of these differences in diagnostics, we recalculated the cutoffs for Aβ42/Aβ40 ratio, p-tau, and t-tau for fresh samples. One way to do that is to transfer frozen-defined cutoffs by applying a linear transformation based on the parameters estimated with the Passing Bablok regression  that did not include the frozen-defined cutoff within its CI. However, considering the variability of biomarkers among measurements, the number of tested samples (N = 58) may have been insufficient to prove or disprove a real discrepancy between frozen-defined and fresh-defined cutoffs.For this purpose, we evaluated if core AD biomarkers, namely Aβ40, Aβ42, Aβ42/Aβ40, p-tau, and t-tau, differ from fresh to frozen CSF samples and if new cutoffs should be defined for freshly measured biomarkers. The effect of freezing on biomarker measurements was previously tested by Sjögren et al. . No signThe wide availability of automated platforms represents an important achievement for reducing the pre-analytical variability of core AD biomarker assays in routine clinical setting . With reAdditional file 1: Table S1. Mean absolute relative differences (MARD) in samples with no cells and 1-2 cells. The measured MARD are higher for samples with 1-2 cells but this difference is significant only for Aβ42. Table S2. Linear regression analysis performed on Mean absolute relative differences (MARD) vs CSF total proteins. No significant correlations were found. Figure S1. Bland-Altman Plot of differences between fresh and frozen CSF aliquots of the same samples vs. the mean of the two measurements  date and raw biomarker measurements in fresh and frozen samples. Two aliquots relative to the same CSF sample were measured with Lumipulse G-600 II, one by fresh and the other one after 30 days of storage at -80°C. AD: Alzheimer’s disease; MCI: mild cognitive impairment; MCI-AD: MCI due to AD; p-AD: preclinical AD; CBS: corticobasal syndrome; V-DEM: vascular dementia; PD: Parkinson’s disease; PD-MCI: PD with MCI; PDD: PD with dementia; DLB: dementia with Lewy bodies; FTD: frontotemporal dementia; SMC: subjective memory complains; PSY: psychiatric disease. Table S4. mean values, SD and coefficient of variation (CV) of biomarker measurements on internal quality control (QC) samples used during the fresh vs frozen measurements with Lumipulse G600-II. Our QC is a pool of 1400 CSF samples belonging to patients affected by neurological and neurodegenerative (mostly AD) diseases (excluding Creutzfeldt-Jacob disease). Table S5. Minimum mean relative difference (MMRD) significantly observable with a t-test power above 0.8. The MMRD was calculated considering the measured SD of relative differences and a sample size of 58.


Neurodegenerative diseases are characterized by the deposition of specific protein aggregates, both intracellularly and/or extracellularly, depending on the type of disease. The extracellular occurrence of tridimensional structures formed by amyloidogenic proteins defines Alzheimer’s disease, in which plaques are composed of amyloid β-protein, while in prionoses, the same term “amyloid” refers to the amyloid prion protein. In this review, we focused on providing a detailed didactic description and differentiation of diffuse, neuritic, and burnt-out plaques found in Alzheimer’s disease and kuru-like, florid, multicentric, and neuritic plaques in human transmissible spongiform encephalopathies, followed by a systematic classification of the morphological similarities and differences between the extracellular amyloid deposits in these disorders. Both conditions are accompanied by the extracellular deposits that share certain signs, including neuritic degeneration, suggesting a particular role for amyloid protein toxicity. Hyperphosphorylated protein tau in Alzheimer’s disease (AD) , tauopatAlpha-synuclein in Lewy bodies in Parkinson disease (PD) and dementia with cortical Lewy bodies (DLB) or in oligodendroglial inclusions in multiple systemic atrophy (MSA);Phosphorylated TDP-43 in frontotemporal lobar degeneration with TDP-43-positive inclusions (FTLD-TDP) [Ubiquitin in frontotemporal lobar degeneration with inclusions positive for ubiquitin-proteasome system markers (FTLD-UPS) ,5;Fused in sarcoma (FUS) inclusions in FTDL-FUS .Deposits of aggregates of particular proteins are specific hallmarks of a wide range of neurodegenerative diseases . AggregaAD ;Prion diseases (Creutzfeldt–Jakob disease (CJD), Gerstmann–Sträussler–Scheinker syndrome (GSS), fatal familial insomnia (FFI), and kuru) .Primary extracellular protein aggregates, in optical microscopy called “plaques,” can be observed in cortical locations in:In all of these diseases, the term cerebral amyloidosis is widely used referring to insoluble fibrillar structures with a predominant beta-sheet conformation detectable by Congo red and thioflavin S binding . These pThe aim of our review is to compare and highlight similarities and differences between the two types of extracellular deposits, i.e., Aβ in AD and amyloid prion protein in prionoses, while simultaneously synthesizing the available information for didactic purposes.Alzheimer’s disease (AD) is a progressive neurodegenerative disease and is the most common form of dementia . The preAmyloid precursor protein (APP), a transmembrane protein existing in several isoforms , is amplThe 42-amino-acid-long Aβ (Aβ42) is the main component of senile plaques, whereas Aβ 40, the more abundant product of APP processing , and whiAn occurrence of familial Alzheimer’s disease (fAD) in patients carrying an autosomal dominant mutation in genes encoding APP.A higher fAD incidence was seen in families carrying the presenilin 1 (PSEN1) and presenilin 2 (PSEN2) mutations, which are the catalytic components of γ-secretase . Most muAPP gene. Many patients suffering from Down syndrome develop AD at an early age. The presence of Aβ plaques in these patients is often described in childhood [Early-onset Alzheimer disease (EOAD) is manifested in patients with Down syndrome. The trisomy of chromosome 21, on which the gene for APP is located, logically leads to a triplicate of the hildhood , and thehildhood . Thence,hildhood .In 1992, Hardy and Higgins  articulaAlthough this theory dominates the field of AD research, it is not universally accepted ,41,42,43There are patients having numerous plaques  but have no clinical signs of cognitive impairment .Conversely, some mouse models of AD show memory deficits before the development of Aβ plaques .While senile plaques appear first in the frontal cortex and then spread beyond the cerebral cortex to the hippocampus and beyond, neurofibrillary tangles initially develop in the limbic system . To thisIt is sometimes questioned for the following reasons:The precise role of Aβ and tau protein in the pathophysiology of AD is still waiting for an explanation.Diffuse/pre-amyloid plaques  that areNon-cored/primitive/immature neuritic plaques see  are ovalCored/classic/dense/mature/focal neuritic plaques  are 20–6Two subtypes of neuritic plaques can be distinguished.Compact/burnt-out plaques  are 5–15Amyloid/senile plaques are extracellular deposits of Aβ that are abundant in the cortex of AD patients , which, It is not entirely clear whether non-cored neuritic plaques progress into cored and then to burnt-out plaques. In addition, it is also not known whether diffuse plaques are a common part of aging or the initial stage of neuritic plaque maturation .Dystrophic neurites in plaques may differ morphologically and immunohistochemically. Type I is described as elongated in shape, whereas type II is dilated, bulbous, or globular . CertainThe results of immunohistochemical examinations showed that diffuse/pre-amyloid plaques contain Aβ42 and other APP fragments lacking the C-terminus , apolipoAccording to Armstrong , non-corCored/classic/dense/mature/focal neuritic plaques consist of an Aβ42 core and a ring of alpha-synuclein. In addition to Aβ42, they contain Aβ40, complement proteins, immunoglobulins, and apolipoproteins D  and E. DApo E genotype does not appear to affect the morphology and distribution of Aβ plaques. Moreover, no differences in plaque density between the sporadic and familial AD variants have been observed [The internal pyramidal layer (layer V) and the external pyramidal layer (layer III) are the most affected . The reaobserved .Prion diseases are transmissible, progressive, and in all cases, fatal neurodegenerative disorders associated with an aggregation of misfolded prion protein . Human tExtracellular deposits and PrPSc plaques are structures visible with hematoxylin-eosin staining, while plaque-like structures can only be visualized by using immunohistochemical methods.  Plaques Cellular prion protein (PrPC) is a glycolipid-anchored cell membrane sialoglycoprotein localized on presynaptic membranes. PrPC appears to have neuroprotective  and pro-Clusterin often co-localizes in PrPSc plaques  and is aKuru was the first human prionosis to be discovered and is defined as a neurodegenerative, non-inflammatory infectious disease ,116. AltPRNP), which accounts for 10–15% of cases [Creutzfeldt–Jakob disease (CJD) is a transmissible and rapidly progressive  degeneraof cases .The other two types can be placed into the category of acquired CJD, i.e., the CJD variant that occurs after consumption of beef from cattle affected by bovine spongiform encephalopathy (BSE). The iatrogenic variant arises during medical or surgical procedures during which pathologically conformed prions are inadvertently transferred  . NeuropaPRNP and the type of proteinase K-resistant prion protein fragments (PrP), using a western blot examination [Different subtypes of sCJD are distinguishable, according to different polymorphisms at codon 129  or methionine and valine (MV) heterozygosity) of the mination .Character and Typical Location of PrP Deposits According to the MV PolymorphismMM1 subtype: synaptic and perivacuolar positivity, although cases with plaques in the white matter are so rarely encountered, we will not mention them in more detail .-Cortical subtype: perivacuolar positivity in all cortical layers;-Thalamic subtype: fewer plaques  MM2MV1 subtype: synaptic and perivacuolar positivity;MV2 subtype: distinctive “kuru-like” plaques in the cerebellum and perineuronal positivity in the cerebral cortex;VV1 subtype: characterized by punctate synaptic positivity in the cerebral cortex;VV2 subtype: perineuronal, with numerous plaque-like areas and some synaptic PrP positivity in the cerebral cortex .As mentioned above, plaques are a neuropathological hallmark, but only for the MV2 subtype, where “kuru-like” plaques are found in the granular and molecular layers of the cerebellum . SometimPRNP mutation. To date, point mutations at codons 102, 105, 117, 131, 145, 187, 198, 202, 212, 217, and 232 have been reported [PRNP gene have been reported. In patients with 4 to 7 multiples, elongated PrP deposits are usually described, while in those having 8 or 9 OPRI, kuru-like or multicentric plaques have been found [Gerstmann–Sträussler–Scheinker syndrome (GSS) is defined as a slowly progressive hereditary autosomal dominant neurodegenerative disease  or encepreported . Some fareported , five [1reported , six [14reported ,150, sevreported , eight [reported , and ninreported  multipleen found . Accordien found .Using silver staining methods, amyloid plaques in prion diseases can mimic burnt-out Aβ42 plaques. Nevertheless, unlike Aβ42 plaques, these PrP plaques can be clearly seen with hematoxylin-eosin staining. After proteinase pre-treatment, the presence of PrPSc can be confirmed by using specific immunohistochemistry. While PrPSc in GSS is partially sensitive to the effects of proteinase .Unicentric/“kuru”/”kuru-like”/stellate plaques  are up tDaisy/florid plaques measure up to 200 µm  and consMulticentric plaques see  are formPure neuritic plaques  are the Both types of plaques are formed by amyloid structures—in AD by Aβ and in TSEs by prion amyloid. We tried to highlight the similarities and differences in their occurrence and behavior.All of these diseases are based on a perturbance of proteins having physiological functions on the neuritic membrane to which they are anchored. Physiologically, they have a neuroprotective function and are able to interact with a number of other agents.They are also similar to each other in the resistance of these extracellular aggregates to degradation by endogenous proteases.In both AD and TSEs, extracellular aggregates may form not only compact structures such as plaques but also diffuse extracellular deposits.For all mentioned diseases, extracellular deposits are mainly found in the cortical areas or in the central grey matter. Their presence in white matter is possible but exceedingly rare in TSEs and absolutely unheard of in Alzheimer’s disease.When forming plaques, they usually contain dystrophic neurites with similar immunohistochemical characteristics in both AD and TSEs. The neuritic morphology can vary from case to case.The most toxic and neuronal death-inducing forms are oligomeric assemblies of both Aβ and PrP.Similarities:While Aβ has thread-like morphology, PrP tends to be more lumpy or globular.In AD, plaques probably mature, i.e., the individual types probably transform from one to the next. Nothing like “plaque maturation” has been recorded in prionoses.Especially in GSS, plaque fusion and the formation of multicentric structures are distinctive. No similar trends are seen in AD.For prionoses, different appearances, locations, and frequencies of extracellular aggregates are reported depending on the form and subtype. In AD, neuropathological differences between early and late-onset or sporadic and familial variants have never been described.In TSEs, PrP deposits may be found intracellularly in some patients, while the occurrence of Aβ is strictly extracellular.In AD and prionoses, there is a different trend relative to the spread of deposits within the brain. In AD, we distinguish five phases, with phase 1 being characterized by the presence of Aβ deposits limited to neocortical areas. During phase 2, the archicortical and paleocortical  regions are affected. This is followed by a spread to the striatum and subcortical nuclei in general during phase 3. Brainstem involvement defines phase 4, and the involvement of the cerebellum defines phase 5 . In prioDissimilarities:To our best knowledge, this is the first systematic classification of the morphological similarities and differences between the extracellular amyloid deposits in AD and CJD. The work also clearly demonstrates the broad spectrum of these specific neuropathological entities. Better clarification of the processes of extracellular aggregate formation of different amyloidogenic proteins may be helpful for understanding the development of individual neurodegenerations and, thus, could be a useful tool for the development of effective and precise biological treatments for these progressive and fatal disorders.


Alligator mississippiensis) has pronounced plasticity in its ventilatory and cardiovascular systems. This study was designed to test the hypothesis that the greater cardiovascular and ventilatory plasticity of A. mississippiensis would result in more variation within the CSF dynamics of this species.Studies of mammalian CSF dynamics have been focused on three things: paravascular flow, pressure and pulsatility, and “bulk” flow; and three (respective) potential motive forces have been identified: vasomotor, cardiac, and ventilatory. There are unresolved questions in each area, and few links between the different areas. The American alligator  duration, and similar duration CSF unidirectional flow pulses were recorded along the spinal cord. Reduction of the duration of the CSF pulsations, as during tachycardia, can lead to a “summation” of the pulsations. There appears to be a minimum duration (~ 1 s) of isolated CSF pulsations. Simultaneous recordings of cranial and spinal CSF pressures reveal a 200 ms delay in the propagation of the pressure pulse from the cranium to the vertebral canal.Alligator may provide unique insights into the evolutionary origins and functional regulation of the human CSF dynamics.Most of the CSF flow dynamics recorded from the alligators, are similar to what has been reported from studies of the human CSF. It is hypothesized that the link between ventilatory mechanics and CSF pulsations in the alligator is mediated by displacement of the spinal dura. The results of the study suggest that understanding the CSF dynamics of  The cerebrospinal fluid (CSF) system is found in all vertebrates , 2. StudThe forces influencing the CSF system are best known in humans. Here, as embodied by the classic Monro-Kellie doctrine , the outAlligator mississippiensis, a species in which the intraventricular and subarachnoid spaces are spatially isolated. We hypothesized that the relatively restricted mobility of the CSF in Alligator, compared to the condition in humans, would alter the cerebral compliance and aspects of the CSF waveform dynamics. In alligators the ventilatory pattern is far more plastic than in humans or other mammals; alligators are capable of sustained apnea [In humans the CSF in the intraventricular and subarachnoid spaces is freely exchanged through the median (Foramen of Magendie) and lateral (Foramen of Lushcka) apertures in the 4th ventricle. These apertures are not present in amphibians and reptiles ; in thesed apnea , 24, anded apnea , 26. Cered apnea , 28. Alled apnea , 30.Alligator would be more variable than in humans, particularly with respect to the cardiac and ventilatory cycles. To explore these hypotheses we surgically implanted pressure transducers into the cranial subarachnoid space of Alligator mississippiensis and analyzed the CSF pressure traces in both the frequency and temporal domains.Thus, when compared to humans, alligators have a different cerebral compliance, and at least three of the known CSF influences  are more variable in alligators. For these reasons, we hypothesized that the CSF dynamics in Alligator mississippiensis) were obtained from the Louisiana Department of Wildlife and Fisheries. The animals were housed communally in a 29 m2 facility that featured three submerging ponds, natural light, and artificial lights on a 12:12 cycle. The facility was maintained at 30–33 ℃; warm water rain showers were provided every 20 min, which helped maintain the facility at > 75% relative humidity. The alligators were maintained on a diet of previously frozen adult rats.Twelve live sub-adult  American alligators , which exceeded the maximum width and length of the alligators used for this study. Six 2.5 cm wide heavy duty straps  were used to secure the alligator to the board; the straps were tight enough to minimize movement of the animal but not tight enough to impede ventilation or circulation.With the alligator’s mouth held open by the bite pad, a laryngoscope was used to depress the gular valve and expose the glottis. A cuffed endotracheal tube was inserted into the larynx and connected to a custom anesthesia system that included a ventilator pump (Harvard), Vaporstick anesthesia machine (Surgivet), isoflurane vaporizer (Surgivet), and Capnomac Ultima respiratory gas monitor (Datex-Engstrom). The alligators were maintained on a steady ventilatory pattern of 6–10 breaths per minute (depending on size) each with a tidal volume of 500 ml. Anesthetic induction was accomplished using 5% isoflurane; once a surgical plane of anesthesia was established the animal was maintained on 2–3% isoflurane depending on the response of the individual alligator.The alligator’s EKGs were recorded using two silver chloride surface cup electrodes , coated with a layer of conducting gel  and placed on the ventral surface of the animal on either side of the heart. The electrodes were connected to a P511 preamplifier (GRASS). A stainless steel surgical burr was used to bore an approximately 4 mm diameter portal through the sagittal midline of the skull just caudal to the orbits. This allowed for direct exposure of the dura mater; a small incision in the dura was used to inset a segment of PE tubing into the subarachnoid space. The PE tubing was connected to a P23AA fluid pressure transducer (Statham), both of which were filled with reptilian Ringer’s solution. The pressure transducer was mounted to the board at a fixed site immediately adjacent to, and level with, the alligator’s head, so that rotation of the alligator did not produce a pressure head between the PE tubing and the transducer. The implantation of the PE tubing was snug enough that no CSF leakage was observed, yet the functionality of the coupling was evident by the (pressure-driven) movement of the CSF along a distance of the PE tubing. The pressure transducer was coupled to a P122 preamplifier (GRASS). In five of the alligators a second surgical exposure was made to the vertebral column between the forelimbs. The exposure was deep enough to enable the implantation of a second PE catheter into the subdural space surrounding the spinal cord; this second implantation site was approximately 12 cm caudal to the implantation site in the skull. The second pressure catheter was connected to a second P23AA pressure transducer and PI22 preamplifier.In seven of the alligators an additional surgical window was used to expose the suboccipital muscles on one side of the vertebral column; this exposure was roughly equidistant  between the two implanted CSF pressure catheters. A hands-free stimulating probe was positioned on the surface of the suboccipital muscles and stimuli provided by a S88 stimulator (GRASS). The CSF pressure, EKG, ventilatory pattern , and synced output from the muscle stimulator were recorded simultaneously (at 5.0 kHz) using the MiDas data acquisition system (Xcitex). The CSF pressure transducers were individually calibrated following each experiment.A. mississippiensis. While the animals were still anesthetized with isoflourane, 0.1 mg/kg of epinephrine was administered (IM) into the triceps.The board holding the alligators was anchored to a rotating spindle machined to have, in addition to a stable horizontal stop, fixed “stops” at 15°, 30°, and 45° above and below the horizon. The animals were subjected to 60 or 90 s rotation trials, with the direction and magnitude of the rotation determined by die roll. The alligators were maintained in the rotated posture for 30 s; to avoid artifacts (vibration in the board) and transitions, only the last 10 s of the postural traces were analyzed. By adjusting the ventilator pump, three different ventilatory patterns were established: (1) resting (6–8 breaths per minute); (2) rapid (3 × the resting rate); and (3) apnea (0 breaths per minute). The alligators were given at least 90 s (at the resting rate of ventilation) between the ventilator trials. Twitch  and train  stimuli were presented to the suboccipital muscles using a hands-free stimulating probe. Visual contractions of the muscles were observed during each trial. Muscle stimulations were only performed on 6 of the While anesthetized with isoflurane the alligators typically had heart rates between 15 and 30 beats per minute (bpm), which roughly corresponded (but see below) to the CSF pulsation rate  microspheres in 5 ml of saline . Doppler ultrasonographic records of CSF flow were quantified using an ultrasonography machine . A linear array probe (Mindary L12-4) was placed contralateral to the exposed suboccipital muscles and pulsed-wave Doppler ultrasonography used to detect the spread of the introduced microspheres in the CSF. The recordings presented were all taken prior to stimulation of the myodural bridge, and thus represent the resting or baseline flow condition. CSF flow velocity was quantified relative to the internally calibrated zero baseline of the ultrasonography machine. Data were collected from five of the alligators.The cranial pressure catheter was disconnected from the transducer and used to introduce into the CSF a suspension of approximately 2.0 × 10Alligator nervous system which may be particularly relevant as the reader puts the experimental data into context. The brain and spinal cord of Alligator are essentially linear, lacking the prominent flexures found in mammals  of 4.6 mm Hg (s.d. = 0.44). The CSF pressure pulses had a mean amplitude (or AMP) of 4.58 mm Hg (s.d. = 0.46); the amplitudes of the pulses were more variable than the durations. Human studies have often described the CSF pulsations by dividing the pulsatile pressure by the duration to the pressure peak ; in thiAlligator; as the heart rate increased above 30 bpm, what appear to be secondary pulses would appear on the diastolic side of the CSF pulsation curve  and head-down  rotations .The subarachnoid space is continuous between the spinal canal and the skull Fig. , so it iThe FFT and power spectral analyses were performed on all 12 alligators and the height (power) of the fundamental frequencies of the ventilatory and cardiac contributions compared. The relative contribution of the cardiac cycle to CSF pressure was on average 3.6 times larger than that of the ventilatory cycle. Interestingly, this mean of 3.6 × was associated with a s.d. of 4.2; the standard deviation was greater than the mean because in some of the records analyzed the relative contribution of the ventilatory cycle was greater than that of the cardiac cycle. The analysis of relative contributions only entailed the fundamental frequencies. A second analysis of absolute contributions was performed; in this analysis the power of each harmonic was added to that of the fundamental frequency. In terms of absolute contribution, the cardiac cycle accounted for 1.7 mm Hg of the CSF pulsation AMP (s.d. = 0.83) while the ventilatory cycle accounted for 1.1 (s.d. = 0.93). The absolute contributions of the cardiac and ventilatory cycles were more similar than their relative contributions because the power spectral analysis more commonly revealed harmonics attributable to the ventilatory than the cardiac cycle.p = 1.11 × 10–16) differences between the three postures. Tukey's Post = hoc analysis revealed no significant difference between horizontal and head-down contributions (p = 0.311); but significant differences between the head-up posture and both the horizontal and head-down postures .The relative and absolute contributions of the cardiac and ventilatory cycles to the CSF pulsations were not static. If ventilatory and heart rates were stable, but the alligator was rotated into a head up or head down posture, shifts were observed in the power spectrum Fig. b. These The CSF flow velocities around the cervical spinal cord were characterized by long-duration pulsatile flow Fig. c. The 10A. mississippiensis, while head-down rotations are associated with a significant increase in carotid blood flow [A. mississippiensis would cause a corresponding change in cardiac output and cranial artery/arteriole pulsation amplitude, and would presumably shift the cranial compliance [The changes in CSF pressure that occur during rotation Figs. d, 5a areood flow . A lack ood flow , 34. Thempliance , 36, whiHeterodontus portusjacksoni) [Pulsations have been reported in the extra brain fluid of the Port Jackson shark (acksoni) , but theacksoni) ; rabbitsacksoni) ; dogs [4acksoni) ; monkeysacksoni) ; horses acksoni) ; mice [4acksoni) ; and humacksoni) . These macksoni) .Alligator the CSF pulsations do not show a consistent temporal relationship to the cardiac cycle, even within an individual and while the animal is horizontal (compare Figs. Alligator than in a mammal, owing in part to the multiple shunts and physiological regulations of cardiac outflow in Alligator [Alligator are driven by arterial pulsation of some form, be it pulsations of the large arteries surrounding the brain [In lligator –50; but he brain , arteriohe brain , or pulshe brain , how canAlligator ventilation is different from that of mammals; alligators are intermittent breathers [Ventilatory movements of the mammalian thorax often produce synchronous CSF pressure pulsations , 55, thoreathers , and canreathers , 59. A rreathers  argued tAlligator is also apparent during frequency analysis. Unlike the situation in mammals, in which the cardiac cycle makes the greatest relative and absolute contribution to CSF pulsations [Alligator there was more variation (Fig. In the experiments reported herein, the alligators were actively ventilated at a frequency and tidal volume similar to what has been reported from non-anesthetized alligators , 61. Altlsations , in AlliA. mississippiensis, there were no corresponding changes in heart rate (Fig. Isoflurane causes respiratory and cardiac depression in reptiles, presumably through increasing vagal tone . Reptileate Fig. c. Previoate Fig. d, 5a butate Fig. c. Frequeate Fig. b.Alligator mississippiensis ventilatory movements result in displacement of the spinal dura adequate to create CSF pulsations. The capacity of the spinal dura to create these pulsations is demonstrated by the activation of the myodural bridge (Fig. Alligator mississippiensis.Accordingly, we follow  and hypodge Fig. b. The myA. mississippiensis had a frequency of approximately 0.3 Hz (Figs. Alligator when anesthetized with isoflurane [Alligator because we see discrete pulsation of slightly greater than 1.0 s (Fig. Alligator the ventilatory contributions create a minimum duration to the CSF pulsations is supported by the observation that only a portion of "summated" CSF curves (Fig. The relationship between between the ventilatory cycle and CSF pulsations in the alligator may also relate to the unusual long duration of the CSF pulsations reported in this study. The CSF pulses in Hz Figs. b, 3a, whoflurane . In mammoflurane  recorded0 s Fig. b, which ves Fig. b increasAlligator. In humans, and other mammals, these CSF flow pulses are of much shorter duration, typically one second or less [Alligator were unidirectional; though the CSF slowed down at the end of the pulse it did not change direction. These Alligator flow velocities were recorded from the subarachnoid space dorsal to the spinal cord; there is no evidence that all of the spinal CSF fluid was flowing unidirectionally. Similar "large scale" unidirectional CSF flow pulses have been reported from zebra fish embryos [Studies of the bulk flow of CSF between the cranial and spinal sub-arachnoid spaces of mammals have demonstrated pulsatile flow, but the pulsations are quite different to those reported herein from  or less , compare or less , though  or less . The flo embryos , 71 and  embryos .Alligator, spectral peaks above a 3rd order harmonic were never recovered. As the spectral analysis of the EKG waveform shows (Fig. Alligator the dura is not fused to the skull [Alligator may be more compliant than those of mammals, which could effectively dampen some of the CSF pulsation.Spectral frequency analysis Figs. c, 6b washe skull , and thePerhaps the most intriguing finding to come out of this study is that the CSF pulsations "differ" between the skull and spinal canal, despite the continuity of the CSF and subarachnoid spaces. When the "same" CSF pulsations are recorded from the cranial and spinal CSF there is a clear synchrony Fig. c. SpectrAlligator mississippiensis CSF pulsations have amplitudes of approximately 5 mmHg and durations around 3 s (Figs. A. mississippiensis, or a shared reptilian characteristic. The current study demonstrated that all of the cardiac, respiratory, vasomotion and orthostatic forces are significant players of CSF dynamics in alligators, and that the link between the pulsatile and bulk CSF flow regimens may be more amenable to experimental exploration and manipulation in this species.All vertebrates have CSF systems involving pulsatile and net flow influenced by cardiac, respiratory, vasomotion and orthostatic forces the relative importance of which are currently debated, particularly in light of the CSF´s role in brain clearance. In  s Figs. a, 3a, an s Figs. c. Furthe s Figs. c, 3b, an


Living donor liver transplantation is a complex surgery, where the donor’s safety is of paramount importance. Despite all precautions, donor morbidity may be inevitable, and long-term follow-up data attest to this fact. However, being a “past donor” all ailments are intuitively attributed to the donation process, which may not always be the case. We present the case of a 47-year-old lady, who developed thrombotic thrombocytopenic purpura secondary to systemic lupus erythematosus 18 months following her liver donation, when she detected to be anti-nuclear antibody (ANA) positive. She developed neurological signs and was managed successfully with therapeutic plasma exchange and steroids. She was discharged home on immunosuppression and remains well on follow-up. We present the medical and social issues that were addressed in the case and highlight the need for a more stringent follow-up protocol in those who are ANA positive. This would also help detect morbidities that may be unrelated to the donation process. Apart from its complex ethical issues, living donor liver transplantation (LDLT) remains a technically demanding procedure. In these surgical procedures, donor safety is of paramount importance, as are recipient outcomes. Donor evaluation is aimed at revealing conditions that could increase the risk of peri-operative complications in a healthy person. This systematic and algorithmic evaluation process excludes unsuitable prospective donors at an early stage, while allowing for suitable candidates to proceed towards donation. This basic concept of minimizing donor risk remains essential to any living donor program. However, despite all precautions, a small risk of complications to the donor remains a reality ,2.There is sizable literature documenting the likelihood of medical and psycho-social decrements after live liver donation. A meta-analysis looking at long-term physical health of live donors (LDs) showed that well over half of all donors (in certain series up to 75%) reported ongoing donation-related health problems, primarily related to gastrointestinal issues, incisional and abdominal pain, and hernia . NotwithWe present one such case of an LD who developed thrombotic thrombocytopenic purpura (TTP) secondary to systemic lupus erythematosus (SLE), and in turn discuss the various issues which were addressed in solving the conundrum.A 47-year-old lady presented with a four-day history of lethargy, vomiting and myalgia. She also gave a history of fever that subsided spontaneously one week prior to her admission. On further elicitation, she complained of alopecia and weight loss over the previous two months. She did not have any history of joint pain, skin rash, altered sensorium or seizures.Eighteen months prior to this episode, she had donated the left-lateral segment of her liver to her grandson who underwent a living donor liver transplant (LDLT) for congenital hepatic fibrosis. Her donor evaluation was unremarkable apart from a positivity of anti-nuclear antibody (ANA) (1:1000 titre). Further evaluation of her autoimmune markers including those for autoimmune hepatitis  was negative. A pre-operative liver biopsy was performed, which was also normal. The donor operation and her post-operative period was uneventful and her routine outpatient donor follow-up  since the operation had been unremarkable.3  along with acute kidney injury with a serum creatinine of 2.5mg/dL (N=0.6-1.2mg/dL). Abdominal imaging showed a well regenerated liver with normal vasculature and her liver function tests were normal apart from mildly elevated indirect bilirubin levels of 1.5mg/dL (N=03-1.2mg/dL). In the ward, she had an episode of transient loss of consciousness without focal neurological deficit. MRI brain revealed an acute left capsuloganglionic infarct. Further blood tests in the form of peripheral smear (abundance of schistocytes), elevated LDH, high reticulocyte count, low haptoglobulin and indirect hyperbilirubinemia were suggestive of microangiopathic haemolytic anemia. Autoantibody screen revealed anti-dsDNA and extractable nuclear antigen panel  antibodies to be positive.Immediately prior to the current admission, she was evaluated at two local health facilities, both of which were wary of her “past-donor” status and referred her on. At admission, her blood tests were suggestive of severe bicytopenia with a haemoglobin of 4g/dL (N=12-15g/dL) and platelet count of 10,000/mmPutting together her constellation of symptoms and signs, a diagnosis of TTP secondary to SLE was made. Her thrombocytopenia remained refractory despite multiple transfusions. Her PLASMIC score of 6  and creatinine) put her in the high-risk category. Following a multi-disciplinary team discussion, as per standard risk-stratified guidelines for the management of TTP, she was started on emergency therapeutic plasma exchange along with high dose steroids . HydroxyThe finite risk of complications and/or death in an otherwise healthy live donor is the single biggest detriment to performing LDLTs. There is also a growing international consensus that the long-term impact of LD demands greater attention in both research and clinical arenas. LD evaluation involves a multi-step protocol that includes exhaustive medical and psychological evaluations of the donor, as well as a precise anatomical study of the liver. LDLT is a directed organ donation and a clear judgment is needed to balance the donor risk versus the recipient benefit with the aim to satisfy the tenet of “double equipoise” . One of To the best of our knowledge, this is the first reported case of SLE with secondary TTP occurring after live liver donation. During the donor evaluation process, our patient was detected to be ANA positive. Despite this, the decision to proceed with her donation was based on the fact that her other autoimmune markers and liver biopsy were normal. Various series have shown 4%-26% of otherwise normal individuals to be ANA positive. On long-term follow-up, 0.3% to 6% of those who are ANA positive go on to develop symptomatic disease . UnfortuTTP, per se whether idiopathic or secondary, has a high mortality of around 90% in untreated individuals and therapeutic plasma exchange along with immunosuppression remain the main modalities of treatment. Inciting factors of TTP include infections, pregnancy, surgery and hematopoietic stem cell transplant . There aAnother interesting consideration is that any physical ailment in a past donor is automatically attributed to the donation process. Many healthcare units are wary of treating these patients. Despite the well-published long-term morbidity to these donors, it is imperative to understand that not all illnesses that occur during the lifetime of a past donor can be attributed to the donor operation. Our patient highlights this tenet, wherein she was referred to our unit from two different regional centers who were unsure of evaluating her medical condition as she was a liver donor. Failure or delay in diagnosis could have been catastrophic to our patient. In cases where health insurance is involved, the “past-donor" epithet could have a major financial impact on the patient’s future medical care.LDLT programs world over have donor follow-up schedules to detect physical and psycho-social issues that may afflict this cohort. Despite the patient’s adherence to our follow-up protocol, we were unable to pre-emptively detect the evolution of SLE in our patient. Reflecting on this case, we feel it might be prudent to have a modified follow-up protocol for a subgroup of donors like those who are ANA positive.We present a case of TTP secondary to SLE in a liver donor who was previously ANA positive. This case posed both medical and social issues that required addressing. Transplant programs often feel responsible for illnesses that arise in donors both in the short and long term even though they may not entirely be related to the donation process. Extensive workup during the donation process often throw up abnormalities that are incidental and do not represent a contraindication for the donor operation. In this instance, some abnormality was indeed detected in the donor, which did not amount to contraindication for the donation process. However, a more stringent follow-up may have identified early signs of SLE in this patient. This case illustrates the need for appropriate referral and focused follow-up in the long term when such abnormalities are detected.


Background: Benign external hydrocephalus (BEH) is defined by rapid increase in head circumference in infancy, with neuroimaging evidence of enlarged cerebrospinal fluid (CSF) spaces. BEH was postulated to predispose to subdural hematoma, neurocognitive impairments, and autism. There is currently no consensus on BEH diagnostic criteria and no biomarkers to predict neurological sequalae.Methods: MRI-based quantitative approach was used for measurement of potential imaging markers related to external hydrocephalus and their association with neurological outcomes. We scanned 23 infants diagnosed with BEH and 11 age-similar controls. Using anatomical measurements from a large sample of healthy infants (n = 150), Z-scores were calculated to classify subject's CSF spaces as enlarged  or normal.Results: Subjects with abnormally enlarged CSF spaces had a significantly wider and longer ON , and a significantly less tortuous ON (p = 0.006). ON deformity demonstrated a high diagnostic accuracy for abnormally enlarged frontal subarachnoid space (AUC = 0.826) and interhemispheric fissure (AUC = 0.833). No significant association found between enlarged CSF spaces and neurological complications . However, cluster analysis identified a distinct subgroup of children  with enlarged CSF spaces and a wider, longer and less tortuous ON, to have an increased risk for neurological complications .Discussion: This is the first report on the association between external hydrocephalus, ON deformity and neurological complications. Our findings challenge the current view of external hydrocephalus as a benign condition. ON deformity is a potential auxiliary marker for risk stratification in patients with enlarged CSF spaces. Benign external hydrocephalus (BEH) is defined by rapid increase in head circumference in infancy, with neuroimaging evidence of enlarged subarachnoid spaces (SAS) and normal or moderately enlarged ventricles . A populThis retrospective cross-sectional study was approved by Soroka Medical Center Institutional Review Board. We searched the term “Benign External Hydrocephalus” in all reports of MRI scans performed between 2012 and 2016 at our institution. We included patients with recorded BEH characteristics , with an adjusted R2 = 0.723. All variables were significant predictors of enlarged frontal SAS width. Multiple linear regression model to predict mean interhemispheric width based on identical combination of variables, showed a significant regression equation , with an adjusted R2 = 0.663. The final regression model, selected by means of backward elimination procedure, included ON width, tortuosity, and age as significant predictors of enlarged interhemispheric width.Linear regression found age-adjusted ON length to have a low positive correlation with increasing frontal SAS and interhemispheric fissure widths   was used to evaluate the diagnostic relevance of ON measurements in identifying patients with enlarged CSF space. For all subjects, Z-scores of the maximal frontal SAS and interhemispheric fissure widths were computed, to determine the deviation from measurements obtained in a large sample of healthy children ( months) . We clas months)  for the p = 0.017 and p = 0.020, respectively, Mann-Whitney U), and a significantly less tortuous ON .We next compared the magnitude of ON anatomical deviation in subjects with enlarged CSF spaces. A total abnormality score for CSF spaces ranging from 0 to 2 was calculated, based on the Z-scores of the frontal SAS and interhemispheric fissure widths. We assigned 1 point for each significantly enlarged CSF space . Subjects scoring 1 or 2 were considered to have abnormally enlarged CSF spaces . The 21 subjects with abnormally enlarged CSF spaces were compared to 13 subjects with CSF spaces under the 95th percentile of normal. Subjects in the “quantified” external hydrocephalus group had a significantly wider and longer ON . Logistic regression to determine association between increased size of CSF spaces and neurological outcomes showed no elevated risk among patients with abnormally enlarged CSF spaces . When controlled for age and ON measurements, this association remained insignificant . Nevertheless, a two-step cluster analysis identified a distinct subgroup of children , to have an increased risk for overall neurological complications . This group was comprised of subjects with enlarged frontal SAS , and enlarged interhemispheric fissure . Furthermore, comparison of the ON anatomical variability between the two subgroups revealed that subjects with an increased risk for neurological complications had a significantly wider , longer , and less tortuous ON .In our cohort, long-term neurological complications  were more prevalent in patients diagnosed with BEH  compared with controls (0/11, 0%, We used a quantitative neuroimaging approach for objective detection of intracranial anatomical variability in a cohort of infants diagnosed with external hydrocephalus and controls. This is the first report on the association between external hydrocephalus, structural ON abnormalities and neurological complications. In our cohort, expanding frontal SAS and interhemispheric fissure were more likely be associated with a wider, longer and less tortuous ON. ON anatomical deviations from the norm were prevalent in subjects with enlarged CSF spaces, had a high diagnostic accuracy for external hydrocephalus and a significant predictive value for neurological complications. These associations challenge the current view of external hydrocephalus as a benign condition. Venous outflow obstruction and decreased CSF absorption have been proposed to underlie BEH and can potentially result in elevated ICP. Reports on elevated ICP in infants with BEH are lacking and measurement of CSF pressure is not part of the initial diagnostic evaluation. Open fontanelles and flexible sutures may enable the ventricles and subarachnoid spaces to expand and can explain why IIH is rarely seen in children younger than 3 years . The litgalben@bgu.ac.il.The datasets presented in this article are not readily available because protection of identifiable imaging data. Requests to access the datasets should be directed to GB-A, The studies involving human participants were reviewed and approved by Soroka Medical Center Institutional Review Board. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.YS and GB-A contributed to literature search, study design, data analysis and interpretation, and wrote the manuscript. SL contributed to data analysis and interpretation. HF, AF, and IS contributed to recruitment of participants, data acquisition, analysis, and interpretation. All authors contributed to the critical revision of the manuscript and approved the final version before 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.