We investigated the effect of pre- “race” ingestion of a 1,3-butanediol acetoacetate diester on blood ketone concentration, substrate metabolism and performance of a cycling time trial (TT) in professional cyclists. In a randomized cross-over design, 10 elite male cyclists completed a ~31 km laboratory-based TT on a cycling ergometer programmed to simulate the 2017 World Road Cycling Championships course. Cyclists consumed a standardized meal [2 g/kg body mass (BM) carbohydrate (CHO)] the evening prior to a trial day and a CHO breakfast (2 g/kg BM CHO) with 200 mg caffeine on the morning of a trial day. Cyclists were randomized to consume either the ketone diester (2 × 250 mg/kg) or a placebo drink, followed immediately by 200 mL diet cola, given ~ 30 min before and immediately prior to commencing a 20 min incremental warm-up. Blood samples were collected prior to and during the warm-up, pre- and post- TT and at regular intervals after the TT. Urine samples were collected pre- and post- warm-up, immediately post TT and 60 min post TT. Pre-exercise ingestion of the diester resulted in a 2 ± 1% impairment in TT performance that was associated with gut discomfort and higher perception of effort. Serum β-hydroxybutyrate, serum acetoacetate, and urine ketone concentrations increased from rest following ketone ingestion and were higher than placebo throughout the trial. Ketone ingestion induces hyperketonemia in elite professional cyclists when in a carbohydrate fed state, and impairs performance of a cycling TT lasting ~50 min. Substrate utilization during exercise is influenced by several factors including the relative intensity and duration of exercise, an individual's training status and the effect of the preceding diet on both the substrate pool and the prevailing hormonal milieu , acetone and acetoacetate (AcAc), are produced in the liver mitochondria from acetyl-CoA in response to an increased mobilization of free fatty acids (FFA) from adipose tissue in situations of reduced CHO availability  on performance in trained cyclists. Their ingestion protocol induced a higher blood D-βHB concentration during submaximal cycling (ranging between ~1.5 and 3 mmol/L) and lead to a subsequent improvement in time-trial (TT) performance by ~2% following ketone ester and CHO ingestion compared to the ingestion of only CHO. However, aspects of the study design are inconsistent with conditions of “real world” cycling competition. Accordingly, we examined the effect of pre-“race” ingestion of a ketone diester on blood ketone body concentrations, substrate metabolism and performance under conditions of elite professional cycling; ingestion of a pre-race CHO-rich meal, inclusion of a warm-up, involvement of world-class cyclists and simulation of a real-life course. We hypothesized that this protocol would result in acute nutritional ketosis but that no performance improvement would be observed due to the high intensity nature of a real-life TT event which is dependent on the high rates of energy production from the oxidation of CHO-based fuels.To investigate the potential benefits to metabolism and sports performance, Cox et al.  studied Declaration of Helsinki and was approved (#20161005) by the Ethics Committee of the Australian Institute of Sport (AIS). After comprehensive details of the study protocol were explained to the participants verbally and in writing, all participants provided written informed consent.This study conformed to the standards set by the The study was a randomized crossover, double-blind, placebo-controlled design using elite  cyclists attending a pre-season camp at the AIS, Canberra. On two separate occasions, three days apart, participants completed a 20-min standardized warm-up and rested for 5 min prior to completing a 31 km TT performed on a cycling ergometer  y; body mass (BM), 73.7 ± 7.6 kg; V°O2peak, 71.4 ± 5.6 mL/kg/min, 5.3 ± 0.3 L/min; Maximal Aerobic Power (MAP), 494 ± 20 W] from the ORICA-BikeExchange UCI World Tour (Road Cycling) team participated in this study. Participants included world class elite  and highly trained under 23 riders contracted to the team (n = 3).Eleven internationally competitive male cyclists [age, 25 ± 7  and the experimental exercise protocol .2peak) was calculated as the highest oxygen consumption recorded over a 30-s period. V°O2peak and MAP were used to calculate the PO for the individualized warm up on subsequent trial days, described below.Participants completed a 5-min warm-up at 150 W on the cycle ergometer , thereafter the test protocol started at 180 W and increased 30 W every 60 s until volitional exhaustion. MAP was determined as the power output (PO) of the highest stage completed. If the participant finished partway through a 60 s stage, MAP was calculated in a pro-rata manner. During the maximal test, expired gases were collected into a calibrated and customized Douglas bag gas analysis system, as previously described . The composition and timing of all meals was repeated prior to trial two.t-butylacetoacetate with R,S-1,3-butanediol . The resultant product consisted of a mixture of monoesters and diester, the ratio of which could be adjusted by varying the stoichiometry of reactants. After synthesis the crude product was distilled under reduced pressure to remove all solvents and starting materials and the resultant ketone ester was obtained and assessed for purity using gas chromatography-mass spectrometry (GC-MS).The ketone ester synthesized, 1,3-butanediol acetoacetate diester, is a non-ionized sodium-free and pH-neutral precursor of AcAc , participants were provided their standardized CHO breakfast including 200 mg caffeine , to mimic typical race preparations. Participants were instructed to consume the breakfast within 10 min, with a second blood sample being collected at t = 30 min. At t = 70 min, participants provided a urine sample, were weighed and fitted with a heart rate (HR) monitor. At this time, they ingested the first dose (250 mg/kg BM) of KET or PLAC, followed immediately by 200 mL diet cola. At t = 90 min participants were seated on the Velotron ergometer, blood samples (4 mL) were collected and participants consumed their second dose in the same manner.On each of the trial days, participants reported to the laboratory in a rested and overnight (10 h) fasted state, with the timetable creating a ~30 min time between individuals that was repeated on the subsequent trial day. The trial day protocol commenced with the placement of an indwelling cannula  into a cephalic vein while lying in a supine position to allow for repeat blood sampling. A fingertip sample of capillary blood was collected concomitantly with each cannula sample throughout each trial for immediate measurement of blood ketones . Following a resting blood sample  and continued to rest quietly for a further 40 min. Blood samples were collected at t = TT + 40 min and t = TT + 60 min, with a final urine sample being collected at t = TT + 60 min. Following the removal of the cannula, participants participated in a semi-structured interview with a single researcher using a series of standard questions to probe perceived effort, motivation and comfort rating during the TT. When symptoms  were identified, a standardized Likert scale was used to quantify them into mild, moderate, or severe levels. On completion of the second trial, participants were asked whether they could identify the trial in which they received the ketone ester, and the trial in which they had performed best. The interview technique was used to probe levels of interest in using a ketone ester supplement in real competition.The TT consisted of a simulation of the 2017 Bergen World Championship TT course, based on global positioning system (GPS) mapping data  collected by the Orica cycling team staff . Cyclists completed the 31.17 km TT as fast as possible and during the TT the only feedback provided to the participant was the distance covered (km), cycling gear-ratio (12–27/48–54) and road gradient (%). Participants were only informed of their TT results following the completion of both trials. HR was collected every 5 km and ratings of perceived exertion (RPE) using the Borg 6–20 scale and capillary blood samples were collected at 15.74 km and immediately post TT. Participants ingested 250 mL of commercially available 6% CHO drink (Gatorade) at 15.74 km, as this distance corresponded to the point identified by the cyclists as the most appropriate opportunity to drink on the actual course. Samples of venous and capillary blood, and urine, were collected immediately following the TT and participants were weighed. At Capillary blood samples were analyzed for concentration of ketones and lactate . Venous blood samples were collected into 4 mL SST vacutainers with immediate analysis of a small aliquot for blood glucose concentrations . This venous sample was then centrifuged at 1,500 g for 10 min at 4°C, and aliquots of serum were stored at –80°C for later analysis. Samples were analyzed for FFA concentrations using a non-esterified-fatty acid (NEFA) assay kit , βeta-hydroxybutyrate concentrations using a β-hydroxybutyrate assay kit  and acetoacetate (AcAc) concentrations using an acetoacetate assay kit , as per the manufacturer's instructions. Urine samples were analyzed for urine ketones (namely AcAc) using ketone reagent strips .n = 10) with the exception of respiratory data which includes (n = 9). When analyzing respiratory gases, an RER > 1.0 was not included in analysis as participants were not in steady state . Statistical significance was set at P < 0.05 and data is presented as mean ± standard deviation (SD). TT performance was also analyzed for magnitude-based effect sizes between conditions using a custom spreadsheet . Paired t-tests were used to analyze average PO, cadence, HR and change in BM in the TT. Blood, urine, PO, HR, cadence, RPE, and respiratory data from the two trials were analyzed using a linear mixed model , to moderate nausea (n = 5) or moderate reflux (n = 1), and minor discomfort . No similar symptoms were reported with the PLAC trial. All participants correctly nominated the trial in which they received KET, identifying it via the gastrointestinal side-effects. However, only four of the cyclists correctly identified the trial in which they completed the TT in the fastest time, with one cyclist equivocal. Although each of the riders nominated their gut symptoms as a distraction or interference to performance, six participants identified an “unusual” centrally-derived feeling during the TT in the KET trial that they thought might be associated with better performance. When asked if they would use the current KET supplement in actual competition, prior to the unmasking of performance results, only one participant  nominated being “possibly” interested. The remaining participants identified the need to remove the potential for illness and gut upset as well as to be sure of a robust performance effect before KET would be of value; “racing is hard enough without adding this complication.”Eleven cyclists commenced this study, but one participant experienced severe side effects from KET ingestion during and after the warm-up, including prolonged vomiting and dizziness and was unable to complete the TT. This participant withdrew from further participation in the study. However, data for this participant have been provided in the following analysis in comparison to those of the other riders to investigate a possible explanation for the occurrence of these side effects. All participants reported gastrointestinal discomfort associated with the intake of the ketone diester. Symptoms ranged from major . There was an impairment in cycling performance time in the first segment of the course  and second segment with the climb included  with KET ingestion compared to PLAC. Overall the KET condition was associated with a 3.7% reduction in average PO  for power output and cadence during the TT, as displayed in Figure P = 0.001) and average HR was significantly lower in the KET compared to PLAC condition . There was no difference in RPE between trials despite the lower HR and PO in the KET condition.All cyclists completed the TT in a faster time in PLAC compared with KET, with the crossover allocation of treatments meaning that there was no order effect on performance. Figure P < 0.001) and treatment (P = 0.021) for serum FFA concentrations . FFA concentrations were higher in PLAC vs. KET from pre- to post-TT (P < 0.05). A condition × time interaction was reported for serum βHB concentrations  and βHB remained significantly higher than PLAC trial until t = TT + 60 min. An increase in βHB concentrations from t = 0 was measured in the PLAC trial at the onset of the warm up  and remained higher until t = TT + 60 min . Blood βHB concentrations increased from pre-TT to post-TT in the KET trial (P < 0.001) and this increase was maintained until t = TT + 60 min  and lactate concentrations  in both the KET and PLAC trials but had returned to resting values pre- TT for both trials. Post-TT, blood lactate concentrations were significantly lower in the KET trial compared to the PLAC trial .There was a significant condition × time interaction for blood glucose , where an increase was observed throughout the incremental warm up from stage 1 to stage 3 (Table P < 0.001) where a similar loss was measured in the KET (1.6 ± 0.7 kg) and PLAC (1.4 ± 0.4 kg) trials.There was a main effect of time in the KET and PLAC trials for VO P < 0.00, where aThis is the first study to report the effect of pre-exercise supplementation with a ketone diester on the performance of a cycling TT under conditions simulating real-life competition: laboratory simulation of a World Championship TT course in world-class male road cyclists who undertook nutritional strategies mimicking competition practices with respect to CHO and caffeine supplementation. Although, our protocol achieved hyperketonemia, as evidenced by increases in serum βHB and AcAc concentrations, there was an impairment of TT cycling performance in these elite cyclists. This outcome appears to be linked to the general observation of gut discomfort and intolerance among the study participants, with symptoms ranging from mild to severe. Our investigation adds important information to sports nutrition, by adding a real-world element.The primary aim of our study was to address recent reports of enhanced sports performance associated with an acute increase in blood ketone concentrations following the intake of a ketone ester drink  longer time to completion in the TT following ketone ingestion supports our initial hypothesis that ketone ingestion would not enhance TT performance Figure , our higDue to the lack of a commercial supply, we were unable to obtain the ketone monoester supplement used in the study of Cox et al. . HoweverWe provided the ketone drink in two doses, with the first bolus ingested 70 min prior to the TT. Based on previous research, our aim was to reach peak βHB concentrations at ~1 h following ingestion . Alternatively the high ketone concentrations in the urine suggest that the ketones are not being oxidized at the skeletal muscle. We have not estimated rates of substrate oxidation rates due to βHB and AcAc yielding respiratory exchange quotient values of 0.89 and 1.00, respectively and thus without appropriate correction factors for COn Frayn, . We alsoOf the 10 participants who completed the trials, all reported gastrointestinal discomfort associated with the intake of the ketone ester including dry retching, mild to moderate nausea, reflux and minor discomfort. Furthermore, one participant was unable to start the TT due to prolonged vomiting and dizziness. This participant also experienced the highest concentrations of serum AcAc concentration when compared to the other 10 participants Figure  has provIn conclusion, the results of the current study show that ingestion of a 1,3-butanediol acetoacetate diester under conditions of optimal race nutrition  results in increases in βHB and AcAc concentrations. The diester was associated with gut discomfort and intolerance among the cyclists with symptoms ranging from mild to severe. Despite optimal nutritional support  for performance, ketone ingestion was associated with an increase in perception of effort, leading to an impairment of TT performance in elite professional cyclists.JL, MR, MQ, and JH: Conception and design, Collection and assembly of data, Data analysis and interpretation, Manuscript writing, Final approval of manuscript (required). LB: Conception and design, Financial support, Collection and assembly of data, Data analysis and interpretation, Manuscript writing, Final approval of manuscript (required).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.


Very little is known about the incidence and prevalence of hydrocephalus in patients with mucopolysaccharidoses (MPS). The biggest challenge is to distinguish communicating hydrocephalus from ventricular dilatation secondary to brain atrophy, because both conditions share common clinical and neuroradiological features. The main purpose of this study is to assess the relationship between ventriculomegaly, brain and cerebrospinal fluid (CSF) volumes, aqueductal and cervical CSF flows, and CSF opening pressure in MPS patients, and to provide potential biomarkers for abnormal CSF circulation.Forty-three MPS patients  performed clinical and developmental tests, and T1, T2, FLAIR and phase-contrast magnetic resonance imaging (MRI) followed by a lumbar puncture with the CSF opening pressure assessment. For the analysis of MRI variables, we measured the brain and CSF volumes, white matter (WM) lesion load, Evans’ index, third ventricle width, callosal angle, dilated perivascular spaces (PVS), craniocervical junction stenosis, aqueductal and cervical CSF stroke volumes, and CSF glycosaminoglycans concentration.All the scores used to assess the supratentorial ventricles enlargement and the ventricular CSF volume presented a moderate correlation with the aqueductal CSF stroke volume (ACSV). The CSF opening pressure did not correlate either with the three measures of ventriculomegaly, or the ventricular CSF volume, or with the ACSV. Dilated PVS showed a significant association with the ventriculomegaly, ventricular CSF volume and elevated ACSV.In MPS patients ventriculomegaly is associated with a severe phenotype, increased cognitive decline, WM lesion severity and enlarged PVS. The authors have shown that there are associations between CSF flow measurements and measurements related to CSF volumetrics. There was also an association of volumetric measurements with the degree of dilated PVS. The mucopolysaccharidoses (MPS) are a group of rare genetic disorders of glycosaminoglycan (GAG) catabolism. Each MPS disorder is caused by a deficiency in the activity of a single, specific lysosomal enzyme required for GAG degradation. Lysosomal accumulation of GAGs results in chronic and progressive cellular damage, which can affect multiple organ systems. The neurologic expression of the disease varies among the different MPS types and sometimes also within the same type . VentricBecause brain atrophy and communicating hydrocephalus share common clinical and neuroradiological features in MPS patients, and in addition, they can potentially coexist, these two conditions were previously considered as one . VentricAt present, neuroimaging has only been only used to verify the extent of ventriculomegaly and exclude cases of gross cerebral atrophy and other pathological conditions that might explain the neurological deterioration in MPS patients. Phase-contrast (PC) MRI provides valuable additional information to conventional MRI and could help to distinguish hydrocephalus from brain atrophy in MPS patients by measuring the volume of CSF pulsating back and forth through the aqueduct with each cardiac cycle, the aqueductal CSF stroke volume (ACSV). According to Bradley, hyperdynamic CSF flow through the aqueduct is seen when there is ventricular enlargement without cerebral atrophy . HoweverCurrently, there is a lack of consensus as to which diagnostic test most reliably predicts which MPS patients will benefit from CSF diversion. Moreover, it has proved to be very difficult, using any objective measure, to demonstrate a neurological improvement after CSF drainage that could indicate an eventual positive response to ventriculoperitoneal shunting (VPS) . There iThe purpose of this study was to provide images of both structural and quantitative changes in the brain allowing access to and a better understanding of the brain disease and the consequences of hydrocephalus in patients with MPS. In that sense, we aimed to be able to identify noninvasive potential biomarkers for abnormal CSF circulation. Besides that, we analyzed the relationship between ventricular and cervical CSF flows and ventricular size, brain and CSF volumes, CSF opening pressure, CSF GAGs levels, dilated perivascular spaces (PVS), white matter (WM) lesion load, and craniocervical junction stenosis.l-iduronidase for MPS I, iduronate sulfatase for MPS II, heparan N-sulfatase for MPS III A, α-N-acetyl-glucosaminidase for MPS III B, acetyl-CoA: α-glucosaminide acetyltransferase for MPS III C, galactose 6-sulfatase for MPS IVA, and N-acetylgalactosamine 4-sulfatase for MPS VI). Multiple sulfatase deficiency was excluded by the observation of a normal activity of at least one other sulfatase. Twenty patients were receiving enzyme replacement therapy . Each patient had brain MR imaging immediately followed by lumbar puncture, and neurodevelopmental assessment performed within the same week, except one patient who died before intellectual test could be applied.This was a cross-sectional study. From July 2013 to June 2016, we examined all patients with MPS followed at the Medical Genetics Service of Hospital de Clínicas de Porto Alegre. Of these, 47 patients with confirmed biochemical diagnosis of MPS underwent evaluation for ventriculomegaly. We excluded three subjects who had VPS and one who was unable to undergo the MRI due to respiratory compromise and clinical instability. The remaining 43 patients formed the study group . Of the MPS I patients, 5 had Hurler syndrome, 2 had Hurler-Scheie syndrome and 5 had Scheie syndrome. Of the MPS II patients, 8 had the severe form and 7 had the attenuated form. Of the MPS III patients, 1 had type A, 3 had type B and 1 had type C. Twenty-five patients were male and 18 were female. Each patient presented with typical clinical manifestations of the disorder and had biochemical confirmation of a deficient enzymatic activity  for children younger than 42 months, Wechsler Preschool and Primary Scale of Intelligence—Revised (WPPSI-R) for children between 42 months and 6 years, Wechsler Intelligence Scale for Children Third Edition (WISC-III) for patients between 6 and 16 years, and Wechsler Adult Intelligence Scale Third Edition (WAIS-III) for patients who were 16 years or older. All tests were performed according to their guidelines and to the developmental level of each patient by a psychologist (S.G.P.P.) who was experienced in development neurology. According to the study protocol, full-scale IQ scores were rated and patients presented as having cognitive impairment (CI) or not. CI was considered present when developmental tests (composite score) or intelligence quotient (IQ) <70. Severely affected patients who could not respond to development tests were classified within the CI group.2O  connected to a 20-G spinal needle. In five patients, it was not possible to obtain the opening pressure or the CSF sample due to technical difficulties (3 patients) or refusal to undergo the procedure after performing MRI (2 patients).Each patient underwent a lumbar puncture in a flexed lateral decubitus position whereby 7–10 cc of CSF was removed. The CSF opening pressure was measured with a standard spinal manometer calibrated in mm Hfast spin echo transversal plane FLAIR ; fast spin echo transversal plane T2 ; MPRAGE sagittal plane T1 ; cervical spine included fast spin echo T2 sagittal plane ; fast spin echo T2 axial plane ; fast spin echo T1 sagittal plane ; throughout plane flow was measured with PC gradient echo for CSF at aqueduct and cervical subarachnoid space at C2–C3 spine level with velocity encoding (Venc) equal to 12 cm/s  and 10 cm/s , respectively. For those patients whose CSF flow was very distinct, the Venc was adjusted accordingly to reduce flow void artifacts and achieve higher image contrast.MRI studies were obtained on 1.5T Achieva  software version 2.6.3. For the acquisition of the cerebral images and the cervical region we used a 16-channel neurovascular coil manufactured by Invivo Devices. The research protocol for brain images included Neuroimage post-processing was performed at a workstation by three researchers in agreement . They were blinded to the age, type, and clinical status of the patients.Cerebral segmentation and WM lesion load were measured with FLAIR brain images and CSF with T2-weighted images. The segmentation process and volume quantification were performed as described in detail elsewhere  to obtaiEvans’ index was calculated as the ratio of the greatest width of the frontal horns of the lateral ventricles to the maximal internal diameter of the skull . The thiThe PVS enlargement on T1-weighted images located in periventricular and subcortical WM, corpus callosum, basal ganglia, thalami and brainstem, were graded as follows: 0 = none; 1 = PVS number <10 and PVS size <3 mm; 2 = PVS number ≥10 and PVS size <3 mm; 3 = PVS number ≥10 and PVS size ≥3 mm .For MRI evaluation of the craniocervical junction, sagittal and axial T1- and T2-weighted images were created. The T2-weighted images were also examined for increased signal intensity, suggesting myelomalacia. The presence or absence of a compression of the spinal cord was graded as follows: 0 = no spinal cord compression; 1 = spinal cord compression (absence of CSF in any direction); 2 = signs of myelomalacia .Stroke volumes were defined as the average of craniocaudal and caudocranial volumes displaced through the region of interest (ROI) during the cardiac cycle (CC) . They weTotal CSF GAGs concentration was determined using a thrombin activity assay. The CSF samples were preincubated with human heparin cofactor II (HC II) and then incubated with a fixed amount of thrombin and with 0.5 mmol/l chromogenic substrate S-2238 in assay buffer. The quantification of the GAG concentrations was performed as described elsewhere .s). Due to multiple comparisons, all p values in Table Continuous variables were described using median and range, due to asymmetric distributions. Categorical data were presented as counts and percentages. Comparison of groups was conducted using Mann–Whitney U test or Fisher exact test, accordingly. To evaluate correlations between continuous and ordinal variables we used Spearman’s rank correlation coefficient . Severe forms of the disease (Hurler syndrome and severe form of MPS II) were observed in thirteen patients (30.2%). Macrocephaly (+2SD or 98%) was present in 32.6% of the patients. Based on IQ and development testing, 41.9% of the patients had cognitive impairment.s = −0.61; p < 0.001) and Evans’ index showed the highest correlation with the ventricular CSF volume . In addition, Evans and third ventricle width scores had large correlations  with the total CSF volume. The other factor tested that correlated with the total CSF volume was the dilated PVS. The craniocervical junction stenosis significantly correlated with the cervical CSF flow  and was inversely correlated with the subarachnoid CSF volume.All the scores  used to assess the supratentorial ventricular enlargement in MPS patients presented a moderate correlation with the CSF aqueductal flow. Also, the ventricular CSF volume correlated with the CSF aqueductal flow, which did not occur with the total CSF volume and the subarachnoid CSF volume Table . The this = −0.23, p = 0.177), or the score for craniocervical stenosis . Table The CSF opening pressure did not correlate either with the three measures of ventriculomegaly or with the ventricular CSF volume Table . Also, nTable One of the biggest challenges with MPS patients is to distinguish communicating hydrocephalus from ventricular dilatation secondary to brain atrophy. Although conventional MRI sequences reveal morphological findings, when there is concern that developing hydrocephalus may require surgical management dedicated neuroimaging studies, including CSF flow measurement, may be indicated . VentricEvans’ index presented the highest correlation with ventricular CSF volumetrics and higher brain volume. Despite advances in brain imaging and volumetric analysis, this simple linear measurement continues to be fast, reliable and feasible for neurosurgical practice. The increased third ventricle width, which is a marker of brain atrophy in patients with multiple sclerosis , 34, migAqueductal CSF stroke volume was significantly correlated with ventricular measurements and CSF ventricular volume. Chiang et al. demonstrated that the magnitude of ACSV is linked to the ventricular morphology , which iThe relationship between ventricle enlargement and decreased brain volume, and also the increased total CSF volume, which includes the CSF of subarachnoid spaces, might lead straight to the conclusion that ventriculomegaly is mainly due to cerebral atrophy. However, using the acquired knowledge from the patients with INPH who have also been found to have larger intracranial CSF volumes , it is mCerebrospinal fluid opening pressure did not correlate with ventriculomegaly, CSF volume or CSF flow. In accordance with our results, the relationship between ICP, also considered as CSF opening pressure, and cerebral ventricle indices was shown as unreliable in pediatric and adult patients with communicating hydrocephalus and INPH –14. AlsoIn MPS patients, the WM lesion severity is associated with ventriculomegaly. The loss of myelin, axons and oligodendoglial cells causes the environment to become more hydrophilic . HoweverThis study provides new information for a better understanding of ventriculomegaly in MPS patients, including its relationship with the elevated aqueductal CSF flow. It also reinforces the importance of considering the amount of dilated PVS as a biomarker for the balance between production and CSF reabsorption. Moreover, our study describes a possible temporal correlation of the clinical and neuroimaging findings with some of the histopathological events in the brain of MPS patients. Lee et al. proposed the natural course of cerebral involvement in MPS based on the MRI findings, and also postulated that cribriform changes occurred first, followed by WM changes and, last, atrophy . Our stuHowever, a weakness of the present study is the lack of correlation between ventriculomegaly and intra- and extracranial venous mean flow to test the hypothesis that venous hypertension due to reduced venous blood outflow may also play a role in the genesis of ventricular dilatation. Therefore, further studies are necessary to correlate cerebral venous blood flow with obstruction in cerebral veins, venous drainage anomalies, skull base abnormalities and communicating hydrocephalus in MPS patients. The T2 through-plane used for CSF volumetrics has low resolution. This is a technical limitation of our study instead of using T1-MPRAGE sequence which could increase the sensitivity of volumetric measurements comparison. Moreover, because ventricular size has limited specificity with regard to pathophysiology of hydrocephalus, it is very likely that a stand-alone measurement of ACSV has poor specificity for differential diagnosis, as previously noted in INPH and in predicting the response to shunt surgery . In addiBrain ventricular size and ventricular CSF volume had significant association with ACSV in MPS patients. CSF opening pressure (ICP) had no association with any of the above measurements. It is possible that MPS patients are more heavily reliant on reabsorption via the extracellular space of the brain. Perivascular spaces may represent the initial phase of abnormal CSF circulation, and ventriculomegaly may represent the later stages.Although we have a better understanding of biomarkers associated with ventriculomegaly in MPS patients, these still do not provide a certain diagnosis for hydrocephalus or improve the accuracy of patient selection for surgical treatment. Concomitant analysis of venous and CSF flows using PC-MRI is necessary to search for impaired venous outflow and reduced intracranial compliance due to jugular foramina narrowing and retrograde venous hypertension.


Therefore, transport and capacitance spectroscopy studies have been performed to better understand the origin of unintentional doping in Ga2O3. Previously unobserved unintentional donors in commercially available 2O3 substrates have been electrically characterized via temperature dependent Hall effect measurements up to 1000 K and found to have a donor energy of 110 meV. The existence of the unintentional donor is confirmed by temperature dependent admittance spectroscopy, with an activation energy of 131 meV determined via that technique, in agreement with Hall effect measurements. With the concentration of this donor determined to be in the mid to high 1016 cm−3 range, elimination of this donor from the drift layer of Ga2O3 power electronics devices will be key to pushing the limits of device performance. Indeed, analytical assessment of the specific on-resistance (Ronsp) and breakdown voltage of Schottky diodes containing the 110 meV donor indicates that incomplete ionization increases Ronsp and decreases breakdown voltage as compared to Ga2O3 Schottky diodes containing only the shallow donor. The reduced performance due to incomplete ionization occurs in addition to the usual tradeoff between Ronsp and breakdown voltage.Understanding the origin of unintentional doping in Ga With a bandgap of 4.5–4.9 eV and estimated critical breakdown field of 8MV/cm, Ga2O3 possesses a Baliga figure of merit 10 times greater than SiC and four times greater than GaN2. Indeed, even at this early stage of development, electric fields of at least 3.8 MV/cm4 and 5.1 MV/cm7 have been demonstrated in lateral Ga2O3 FETs and vertical Schottky diodes, respectively, already surpassing bulk critical fields of GaN and SiC. In addition to these promising material properties, melt-growth methods for bulk Ga2O3 substrate growth17 are expected to be more cost-effective than the sublimation techniques used for the growth of SiC substrates, lowering manufacturing costs for Ga2O3 based power electronics.While crystalline Ga2O3 based power electronics devices towards their predicted limits requires further work to understand and eliminate unintentional doping in the material. To illustrate this fact, the dependence of breakdown voltage of p-n single-sided junction and Schottky junction devices, normalized by material parameters, is plotted in Fig. 2O3 devices to those of more mature semiconductor materials gives insight into the future potential of Ga2O3 devices. The plotted experimental data are among the best reported for vertical devices of each material with large breakdown voltage and low doping of the device drift-layer. While edge effects prevent real devices of any material from matching the simplified theoretical limit exactly, the 2O3 to the more mature materials, it is clear that some improvement can be expected from the optimization of device geometries to mitigate edge effects, as shown by the blue arrow. However, much greater improvement in breakdown voltage is possible from reducing drift layer doping in Ga2O3 devices, as indicated by the orange arrow. For instance, if the drift layer doping of a Ga2O3 device can be reduced to about 1014 cm−3 like the 21.7 kV SiC device indicated in Fig. 2O3 device will have a breakdown voltage over 100 kV as indicated by the right axis of Fig. 2O3 is key to increasing the maximum achievable breakdown voltages in Ga2O3 beyond the recently demonstrated 1 kV devices7.While the large breakdown electric field demonstrated in these early experimental studies is certainly promising, pushing the breakdown voltages of Gamentclass2pt{minimmentclass2pt{minim2O3. The transport properties of commercially available unintentionally doped 2O3 substrates from Tamura Corporation grown via the EFG method14 are characterized via temperature dependent Hall effect measurements. High temperature Hall effect measurements up to 1000 K reveal a previously unobserved unintentional donor with an energy level 110 meV below the conduction band edge in addition to previously observed shallow donors attributed to Si17. To confirm the existence of this unintentional donor, temperature dependent admittance spectroscopy measurements were performed on a Ga2O3 Schottky diode structure, with an activation energy of 131 meV observed via this technique. This activation energy agrees well with the donor energy of 110 meV determined via Hall effect measurements. Finally, using the information obtained from the temperature dependent Hall effect measurements, the effects of the 110 meV donor on the specific on-resistance (Ronsp) of Ga2O3 Schottky diodes are assessed via analytical calculations, indicating that incomplete ionization of the 110 meV donor increases the Ronsp and reduces breakdown voltage as compared to Ga2O3 devices with only the shallow donor, beyond the usual tradeoff between on-resistance and breakdown voltage.With these motivations, we have undertaken transport and capacitance spectroscopy studies in order to better understand the origin of unintentional doping in Gam* = 0.3m020 was used to estimate 21, while 22. Carrier activation in previous studies of Ga2O3 was well described by a single shallow donor which they also attributed to unintentional silicon dopants;17 however, the second higher energy donor has not been observed previously.Figure entclass1pt{minima2O3 Schottky diode as a function of frequency for several temperatures. The C vs. ω spectrum exhibits a step transition separating two plateaus, Cd at ω < ωp and Cg at ω > ωp, which indicates the presence of a trap state. The transition frequency, ωp, corresponds to the carrier emission rate from the trap level. To determine the activation energy of the trap state, ωp was obtained from the negative peak in the differential capacitance (ωdC/dω vs ω) spectrum to construct an Arrhenius plot of ln(ωp/T2) vs 1/(kBT), shown in Fig. Ea = 131 ± 5 meV. Furthermore, the trap density, calculated from the height of the capacitance step23, is Nt = 4.4 × 1016 cm−3. A relative dielectric constant of 10 for Ga2O3 was used for this calculation25. These data are well matched to the previously unobserved donor identified via Hall effect measurements, which indicated a donor energy of 110 meV and donor density of 7.5 × 1016 cm−3 as previously discussed. The small differences between the Hall effect measurements and admittance spectroscopy measurements are not surprising and are consistent with sample to sample variation within the two-inch Ga2O3 wafer from which the three samples were cut. Therefore, the trap state observed via admittance spectroscopy confirms the existence of the previously unobserved unintentional donor.Admittance spectroscopy measurements confirm the existence of the previously unobserved donor. Figure 26 and acceptors28, respectively. However, antisites and interstitials could be responsible. Extrinsic impurities, of course, could also be responsible for the donor, and glow discharge mass spectrometry (GDMS) analysis of Tamura substrates indicates the presence of several impurities whose dopant properties in Ga2O3 are unknown22. Last, silicon on the octahedrally coordinated Ga(II) site of Ga2O3 could be responsible for the 110 meV donor. DFT calculations indicate that Si prefers the tetrahedrally coordinated Ga(I) site in which it is a shallow donor;26 however, STM analysis of Si donors near the (100) surface of Ga2O3 indicates that Si occupies both Ga(I) and Ga(II) lattice sites29. This fact suggests that Si on the Ga(II) site should also be considered as a possible origin of the 110 meV donor.While the electrical characterization performed here cannot determine the chemical or crystallographic origin of the 110 meV donor, we nevertheless consider a few hypotheses. It is natural to ask if a native defect is responsible for the observed donor. DFT calculations rule out simple oxygen and gallium vacancies as they are determined to be deep donors2O3 based power devices; namely they reduce the maximum achievable breakdown voltage in a Ga2O3 power device. Because the doping of the drift-layer cannot be reduced below the level of unintentional doping, the density of unintentional donors sets the lower limit on electric field at the metal-semiconductor interface and the upper limit on the depletion width of the Ga2O3 drift-layer for a given applied voltage. Therefore, a limit is set on the tradeoff which allows higher breakdown voltage to be achieved at the expense of larger on-resistance. To mitigate the presence of previously identified silicon shallow donors in the substrate, several methods including LPCVD30, MOCVD32, HVPE34, and MBE36 have been used to grow homoepitaxial Ga2O3 layers on Ga2O3 substrates, achieving lower carrier densities by minimizing unintentional doping. However, the carrier concentration due to unintentional doping still remaining in epitaxial layers is typically 1015 to 1017 cm−3 37. This concentration is similar to the additional carrier concentration due to the 110 meV donor observed in these bulk samples considering incomplete ionization. For example, at a 110 meV donor concentration of 7.5 × 1016 cm−3 as found in this study, with no other donors or acceptors, 3.4 × 1016 cm−3 free electrons are thermally activated into the conduction band at 300 K for an ionization efficiency of 46%. These facts suggest that the 110 meV donor may also play a role in the unintentional doping of epitaxially grown Ga2O3, warranting further study but beyond the scope of this work. The importance of the high temperature Hall effect measurements and admittance spectroscopy measurements should be emphasized. As our calculation indicates, the higher activation energy of the 110 meV donors means that their ionization efficiency These unintentional donors have implications for the performance of Ga2O3 based devices. The 110 meV donor is shallow enough in energy that it becomes fully ionized when the Schottky diode is reverse biased, reducing the depletion width, increasing the electric field at the metal-semiconductor interface, and reducing the breakdown voltage in the same way as the shallow donor. However, the 110 meV donor energy is large enough that incomplete ionization can be significant, meaning that only a fraction of the 110 meV donors are ionized when the Schottky diode is forward biased, reducing the carrier concentration available to conduct the on-current. To quantitatively examine those effects, specific on-resistance (Ronsp) versus breakdown voltage characteristics have been calculated for Ga2O3 Schottky diode devices including the incomplete ionization effect, with the results plotted in Fig. onsp is calculated for a Schottky diode containing both 110 meV donors and shallow donors versus a Schottky diode containing only shallow donors designed for the same breakdown voltage. The results of the calculation are plotted in Fig. 2O3 Schottky diode devices. To limit the increase in Ronsp to one percent, the concentration of 110 meV donors must be less than 5 × 1014 cm−3. A similar analysis of the percent decrease in breakdown voltage due to 110 meV donors is also presented in the supplementary information.While unintentional shallow donors and the 110 meV donor both set a limit on the tradeoff between on-resistance and breakdown voltage, the 110 meV donor poses an additional challenge to maximizing breakdown voltage and minimizing on-resistance due to its four times larger donor energy as compared to the shallow donor. Because of the higher donor energy, incomplete ionization of the 110 meV donor is more severe than for the shallow donor, degrading the specific on-resistance vs. breakdown voltage characteristic for Ga2O3 substrates grown by the EFG method. The existence of these unintentional donors sets a limit on the maximum breakdown voltages that can be achieved in Ga2O3 devices and must be mitigated to achieve the full benefits of Ga2O3 based power electronics. Additionally, incomplete ionization of the 110 meV donor causes increased on-resistance and decreased breakdown voltage in diodes containing the donor, as compared to diodes containing only the shallow donor, beyond the usual tradeoff between on-resistance and breakdown voltage. To achieve 10 kV operation in Ga2O3 Schottky diode devices, analysis indicates that the concentration of 110 meV donors much be reduced below 5 × 1014 cm−3 to limit the increase in Ronsp to one percent.In conclusion, high temperature Hall effect and admittance spectroscopy measurements have revealed a previously unobserved unintentional donor with energy 110 meV below the conduction band edge in commercially available unintentionally doped Ga2O3 Schottky diode. Following device fabrication, temperature dependent van der Pauw and Hall effect measurements were carried out in two separate Hall effect measurement systems. An electromagnet with vacuum cryostat and closed-loop He refrigerator was used for measurements below room temperature, while an electromagnet with a quartz tube furnace with silicon carbide heater was used for measurements above room temperature. The samples were kept under nitrogen gas flow at atmospheric pressure during high temperature measurements. Admittance spectroscopy measurements were carried out under vacuum in a closed-loop He cryostat using a Keysight Agilent 4990 A impedance analyzer.Two van der Pauw test samples were diced from the same two inch wafer into 1 cm × 1 cm square pieces. Following dicing, samples were solvent cleaned and 50 nm/1000 nm Ti/Au contacts were sputtered on the sample corners. To improve contact resistance, the samples were annealed from room temperature up to 450 °C with a 15 min ramp in a tube furnace with argon gas flow. A third sample from the same two inch wafer was prepared for admittance spectroscopy by depositing an indium tin oxide (ITO) transparent contact to form a GaThe data that support the findings of this study are available on request from the corresponding author S.M.Supplementary Information


Our study aimed to investigate whether geometrical features  of the femoral condyle affect the morphology of the trochlear groove.Computed tomography models of 195 femurs  were reconstructed into three-dimensional models and categorised into four types of trochlear groove morphology based on the position of the turning point in relation to the mechanical axis . Only subjects with healthy knees were included, whereas individuals with previous knee trauma or knee pain, soft tissue injury, osteoarthritis, or other chronic diseases of the musculoskeletal system were excluded. The size parameters were: radius of the best-fit cylinder, anteroposterior dimension of the lateral condyles (AP), and distal mediolateral dimension (ML). The shape parameters were: aspect ratio (AP/ML), arc angle, and proximal- and distal- end angles. The alignment parameters were: knee valgus physiologic angle (KVPA), mechanical medial distal femoral angle (mMDFA), and hip-knee-ankle angle (HKA). All variables were measured in the femoral condyle models, and the means for each groove type were compared using one-way analysis of variance.p < 0.05), but not regarding proximal-end angle. There were significant differences when comparing type 90° with types 45°, 60°, and 75° regarding KVPA, mMDFA, and HKA (p < 0.05).No significant difference among groove types was observed regarding size parameters. There were significant differences when comparing type 45° with types 60°, 75°, and 90° regarding aspect ratio and distal-end angle (Among size, shape, and alignment parameters, the latter two exhibited partial influence on the morphology of the trochlear groove. Shape parameters affected the trochlear groove for trochlear type 45°, for which the femoral condyle was relatively flat, whereas alignment parameters affected the trochlear groove for trochlear type 90°, showing that knees in type 90° tend to be valgus. The morphometric analysis based on trochlear groove classification may be helpful for the future design of individualized prostheses. Evaluating the morphology of the trochlear groove represents the theoretical foundation underlying the investigation of the differences between natural and prosthetic knees, or between natural and pathological knees. In a previous analysis of computed tomography (CT) femur models of 100 healthy Chinese subjects (200 knee models), the trochlear groove morphology was defined based on the position of the groove turning point relative to the femoral mechanical axis. Specifically, coaxial cutting planes were defined, rotated about the trochlear groove axis from the proximal to the distal end in 15° increments, and the deepest point of the trochlear groove was marked in each cross section  of the femoral condyle affect the morphology of the trochlear groove.Our study enrolled the same sample population as that involved in the previous study described above (200 knee models from 100 Chinese subjects) [The CT examinations were not performed as part of our study. Instead, our study used the CT images employed in the previous study involving the same cohort . The metBefore measurement, the three dimensional planes were established as follows: the mechanical axis was defined as the line connecting the centre of the femoral head to the apex of the intercondylar notch. The coronal plane was parallel to the mechanical axis and was externally rotated at 3° in relation to the line connecting the most posterior points of both femoral condyles. The sagittal plane was perpendicular to the coronal plane and passed through the mechanical axis at the midpoint of the two condyles. The transverse plane was perpendicular to both the coronal and sagittal planes.A cylinder was established with its axis parallel to both the coronal and transverse planes, and its radius was adjusted to allow the cylindrical surface to closely fit the trochlear groove; its axis represented the trochlear groove axis. Coaxial cutting planes rotating about the trochlear groove axis from the proximal to distal end were created. A 0° cutting plane was represented by the plane parallel to the transversal plane. With 15° increments towards the distal end of the trochlear groove, eight cutting planes were created. The deepest points of the trochlear groove were marked at each cutting plane Figs. . The troThe size parameters consisted of the radius of the best-fit cylinder (R), the anteroposterior dimension of the lateral condyles (AP), and the femoral mediolateral dimension (ML) Figs. , and 3b A test-retest analysis was performed to determine intra-observer reliability, by measuring all parameters three times for 20 randomly selected femurs. The standard deviation of these three measurements was used to represent the accuracy of the measurement for each parameter.p-value of less than 0.05 was considered statistically significant. This sample size was mainly chosen from the data in the pre-experiment of 42 knees and was calculated using a power analysis. The sample size for a power of ~ 0.9 (α = 0.05) indicated a minimum sample size of 48 knees.One-way analysis of variance was performed to determine if the anatomical parameters were significantly different among the groups classified by type of trochlear groove, as well as by age, height, weight, and BMI. The difference in sex ratio between each group pair was analysed using the Chi-square test. A p < 0.05) (Table p < 0.05). The average values of KVPA, mMDFA, and HKA in type 90° were 4.4, 95.0°, and 181.3°, respectively, with significant difference being observed when comparing type 90° with types 45°, 60°, or 90° (p < 0.05). KVPA exhibited a decreasing trend, whereas mMDFA exhibited an increasing trend with the increase in the classified angle, from type 45° to 90°, sequentially  Table . No sign5) Table , with siThe most important finding in our study is that morphological parameters related to shape and alignment influence the morphology of the trochlear groove of type 45° and type 90°. These anatomical indicators may help in characterizing different types of trochlear groove tracking and provide useful information to be used in designing prosthetic components optimally suited for types 45° and 90°.We performed a systematic assessment of the morphological parameters  of the femoral condyle in the context of each type of trochlear groove tracking. In a previous study , the disp < 0.05); moreover, the average value of the arc angle  for type 45°. Furthermore, sexual dimorphism was previously reported in the morphology and shape of the distal femora [p < 0.05). As more than two thirds of the subjects with type 45° groove morphology were men, this may account for the decreased value of the aspect ratio (AP/ML) and arc angle we noted for type 45°.On the other hand, the aspect ratio (AP/ML) and average distal-end angle were smaller in type 45° than in the other types . Furthermore, KVPA decreased, whereas mMDFA increased with the rotation of the coaxial cutting planes relative to the trochlear groove axis. It is possible that, as the turning points lies closer to the sagittal plane, the anatomical axis rotates towards the mechanical axis, and the distal femoral line rotates towards a more pronounced valgus position, away from the mechanical axis, which results in decreased KVPA and increased mMDFA. The HKA was significantly higher in type 90° (p < 0.05). In a study by Wang et al. [We found that KVPA was significantly lower in type 90°, while mMDFA was significantly higher when comparing type 45° with type 90° or type 60° with 90° , 60° (23.5%), 75° (51.5%), and 90° (12.5%) in the previous study [0.0%, 60°The main limitation of our study is that using data obtained via CT measurements, which lies at the core of our approach, implies ignoring the cartilage layer that covers the trochlear surface of the femur. While the geometry of the cartilage surface is known to differ from that of trochlear bone, the difference is relatively small , 17, 18,Our study involving Chinese individuals with healthy knees revealed that, among the size-, shape-, and alignment-related parameters, the latter two showed a partial influence on the morphology of the trochlear groove. Shape-related parameters affected the morphology of trochlear groove type 45°, where the femoral condyle is relatively flat. Alignment-related parameters affected the morphology of trochlear groove type 90°, where the knee tends to be in valgus. Such observations from the morphometric analysis based on the trochlear groove classification may provide useful information for individualized prosthesis design in Chinese populations.


Although quotient of alpha2 macroglobulin (Qα2MG) was previously reported to be useful for the evaluation of blood–brain barrier (BBB) function, it is not commonly used. We therefore evaluated BBB function among the various subsets of neuropsychiatric systemic lupus erythematosus (NPSLE) using quotient Q α2MG. Furthermore, we determined the correlation between Q α2MG and cerebrospinal (CSF) interleukin (IL)-6 level and quotient complement component 3 (Q C3). To determine intrathecal production of C3, the C3 index (Q C3/Q α2MG) was also calculated. Fifty-six patients with SLE were included in this study. Of these, 48 were diagnosed with NPSLE, consisting of 30 diffuse NPSLE patients : n = 14, non-ACS: n = 16) and 18 patients with focal NPSLE. CSF IL-6 concentration, and paired serum and CSF levels of α2MG and C3, were measured by enzyme-linked immuno solvent assay (ELISA). The Q α2MG, Q C3, and C3 index were then calculated. Q α2MG, Q C3, and IL-6 concentrations in the CSF were significantly elevated in NPSLE compared with non-NPSLE. Among the subsets of NPSLE, significant increases in Q α2MG, CSF IL-6, and Q C3 were observed in ACS compared with non-ACS or focal NPSLE. There was a positive correlation between CSF IL-6 level and Q α2MG, as well as between Q C3 and Q α2MG, in diffuse NPSLE. There were no significant differences in C3 index between NPSLE and non-NPSLE, as well as among the subgroups of NPSLE. Our study suggests that BBB disruption is present in ACS, and elevated levels of IL-6 and C3 in CSF in diffuse NPSLE, especially in ACS, might result from their entry to the CSF from the systemic circulation through the damaged BBB, as well as increased intrathecal production. Furthermore, Q α2MG might be useful for the evaluation of BBB integrity. Systemic lupus erythematosus (SLE) is an inflammatory autoimmune disease characterized by the production of autoantibodies and the formation of immune complexes. Deposition of immune complexes triggers the classical complement pathway, resulting in inflammation and tissue injury . NeuropsIn 1999, the American College of Rheumatology (ACR) classified the clinical features of NPSLE in 19 specific neuropsychiatric syndromes, which can be segregated into two main syndromes: central nervous system (CNS) syndromes and peripheral nervous system syndromes. CNS syndromes are further divided into diffuse syndromes , anxiety disorder, cognitive dysfunction, mood disorder, psychosis) and focal syndromes , mostly depending upon the anatomical sites of the pathology in the CNS –6.Multiple mechanisms are involved in the pathogenesis of NPSLE including autoantibody-mediated neurotoxicity, direct action of inflammatory cytokines, microvasculopathy (which may be attributed to complement activation), thrombotic vasculopathy caused by anti-phospholipid antibodies (aPL), intrathecal production of immune complexes, blood–brain barrier (BBB) disruption, and accelerated atherosclerosis –9. GenerThe BBB is a selective semipermeable membrane that separates the circulatory system from the brain. The BBB, formed from polarized brain endothelial cells that are connected by tight intracellular junctions, protects the brain microenvironment. BBB disruption occurs in several pathologies such as stroke, infection, and autoimmune disorders including NPSLE , 11. TheInterleukin-6 (IL-6) is an inflammatory mediator that contributes to the pathogenesis of NPSLE. Recent studies demonstrated that IL-6 is increased in the CSF of patients with NPSLE, especially those with ACS –18, and Complement activation also contributes to the pathogenesis of NPSLE. Reports of brain histopathology showed that microvasculopathy, which may be due to aPL and activation of complement, was the most common finding in patients with NPSLE . EarlierTo assess the role of the complement system, the CSF concentrations of C3 and C4 have been measured in several studies of immune-mediated diseases. However, the measurement of C3 and C4 concentrations in CSF alone is not sufficient to detect the intrathecal production of complement in the CNS. Although complement components are locally produced by resident cells in the brain , the synAlpha2 macroglobulin (α2MG) is a large glycoprotein found in serum with a molecular weight of 718, which inhibits many proteases including trypsin, thrombin, and collagenase. α2MG is mainly synthesized by the liver, and the CSF/serum concentration quotient (Q α2MG) has been reported to be a useful marker to evaluate BBB function –31. AlthKanoh and Ohtani reported that the levels of CSF α2MG as well as Q α2MG in patients with viral meningitis, mycotic meningitis, and bacterial meningitis were significantly elevated compared with healthy controls and that a significant elevation of Q albumin was not observed in patients with viral meningitis, mycotic meningitis, and bacterial meningitis compared with healthy controls , 32.Livrea et al. examined the relationship between the Q of proteins with different hydrodynamic radius (R)  and interpreted it according to pore or vesicular BBB models. The authors suggested that heterogeneous, independent, permeability BBB mechanisms maintain the normal CSF/serum protein concentration gradient, that a set of highly selective pores with a radius less than 120 Å were responsible for the transfer of substantial albumin fraction into the CSF, and that a nonselective permeability mechanism consisting of pores and/or vesicles with a radius greater than 1000–1500 Å were responsible for the transfer of IgG and α2MG. The authors also described that a loss of selectivity was the main feature of BBB in acute meningoencephalitis, and was mediated by pores or vesicles with a radius greater than 1000–1500 Å. These sIn the present study, we evaluated BBB function among various subsets of NPSLE using Q α2MG instead of Q albumin. Then, we determined the levels of CSF IL-6, CSF C3, and Q C3 among the subsets of NPSLE, and evaluated the relationship between these parameters and Q α2MG. Furthermore, we calculated the C3 index using α2MG instead of albumin to determine the intrathecal synthesis of C3.A total of 56 SLE patients who all fulfilled the American College of Rheumatology (ACR) 1982 revised criteria for the classification of SLE  were incAfter obtaining informed consent, CSF samples were obtained from all patients by lumbar puncture and serum samples were obtained at the same time. When CSF samples were collected, routine CSF analysis  were performed in some patients. Serum and CSF samples were kept frozen at −80°C until use.™ Ace, 7.5%; Wako, Osaka, Japan) under non-reducing conditions. For the positive control, 10 ng/lane of α2MG  was used instead of a CSF sample. The separated protein was transferred  to a nitrocellulose membrane , blocked in 1% bovine serum albumin (BSA) overnight, and incubated with goat anti-human α2MG . After overnight incubation at 4°C, the membrane was washed with PBS-T, and then incubated for 60 min with horseradish peroxidase (HRP)-conjugated anti-goat IgG . After washing, the membrane was developed using a SuperSignal West Dura Extended Duration Substrate kit (Thermo Scientific).α2MG in CSF was detected by western blotting. Each CSF sample was diluted 1:10 in sample buffer, and run (1 μl/lane) on 7.5% polyacrylamide gel  at room temperature (RT) for 2 h. Purified human α2MG was used as the standard. After 5 washes with TBS-T, 100 μl of diluted samples  were added to each well, and incubated at 4°C overnight. After 5 washes with TBS-T, the plate was incubated with HRP-conjugated anti-α2MG antibody  for 2 h. After additional washing with TBS-T, TMB substrate (Cappel) was added. To stop the color development, 1 N HCl was added, and the optical density (OD) at 450 nm was determined using a microplate reader . A standard curve was prepared using α2MG  to calculate α2MG concentrations.α2MG levels in CSF and sera were measured in triplicate using an enzyme-linked immunosorbent assay (ELISA). ELISA plates (96-well plates) were coated with 2 μg/ml of goat anti-human α2MG in TBS at 4°C overnight (100 μl/well). After 5 washes with TBS-T (TBS containing 0.05% Tween 20), the plates were blocked with 0.1% BlockAce® kit  and human C3 ELISA kit , respectively, according to the manufacturer’s instructions.CSF IL-6 levels and C3 levels in CSF and sera were measured in triplicate using human IL-6 ELISA Ready-SET-Go!Quotient α2MG was calculated using the following formula: . Quotient C3 was calculated using the following formula: . C3 index was calculated using the following formula: [CSF C3 × serum α2MG/serum C3 × CSF α2MG].U-test for the comparison of median values. Correlation coefficients were determined by Spearman’s rank correlation coefficients. Statistical analyses were performed using SPSS software, version 22 . P values less than 0.05 were considered statistically significant.Statistical analysis was performed using Mann-Whitney’s p = 0.004)  compared with non-NPSLE  (= 0.004) .p = 0.001)  .p = 0.018) and focal NPSLE  (p = 0.002). In contrast, there was no significant difference in CSF α2MG level between non-ACS diffuse NPSLE and focal NPSLE , and focal NPSLE. CSF α2MG levels were significantly increased in ACS diffuse NPSLE  compared with non-ACS diffuse NPSLE  .p = 0.002).We next determined Q α2MG values, which reflect BBB function, in 47 NPSLE and 8 non-NPSLE patients. In 1 patient with ACS diffuse NPSLE, the Q α2MG value was not determined. As shown in p = 0.007) and focal NPSLE  (p = 0.003). In contrast, there was no significant difference in Q α2MG between non-ACS diffuse NPSLE and focal NPSLE  compared with non-ACS diffuse NPSLE  .We determined IL-6 levels in the CSF of 47 NPSLE and 8 non-NPSLE patients. In 1 patient with focal NPSLE, CSF IL-6 was not determined. As shown in p = 0.006) and focal NPSLE  (p = 0.008). In contrast, there was no significant difference in CSF IL-6 concentration between non-ACS diffuse NPSLE and focal NPSLE  compared with non-ACS diffuse NPSLE  . Furthermore, among the subsets of NPSLE, CSF TP level was also significantly correlated with Q α2MG in diffuse NPSLE . In contrast, there was no significant correlation between CSF TP level and Q α2MG in focal NPSLE   .s = 0.517, p < 0.0001)   (CSF IL-6 level was significantly correlated with Q α2MG in NPSLE patients (r 0.0001) . Further= 0.001) . In cont= 0.201) .p = 0.011)  was significantly higher than that in non-NPSLE patients  (= 0.011) . There w= 0.011) .s = 0.383, p = 0.01), although the correlation was weak .We next examined Q C3 values in 45 NPSLE and 8 non-NPSLE patients. In 3 patients with ACS diffuse NPSLE, Q C3 was not determined. As shown in p = 0.043) and focal NPSLE  (p = 0.025). In contrast, there was no significant difference in Q C3 value between non-ACS diffuse NPSLE and focal NPSLE  compared with non-ACS diffuse NPSLE      . Among t= 0.003) . In cont= 0.135) .To examine intrathecal C3 production in NPSLE, we determined the C3 index in 44 NPSLE and 8 non-NPSLE patients. In 4 patients with ACS diffuse NPSLE, C3 index value was not determined. There was no significant difference in C3 index value between NPSLE and non-NPSLE , or betwOur study had three major findings. First, Q α2MG was significantly elevated in NPSLE compared with non-NPSLE. Among the subsets of NPSLE, a significant increase of Q α2MG was also observed in ACS diffuse NPSLE. These results suggest that BBB disruption is present in patients with NPSLE, especially in ACS diffuse NPSLE.Second, CSF IL-6 concentration was significantly elevated in NPSLE compared with non-NPSLE. Among the subsets of NPSLE, CSF IL-6 level was also increased in ACS diffuse NPSLE compared with non-ACS diffuse NPSLE or focal NPSLE. A positive correlation between CSF IL-6 concentration and Q α2MG in NPSLE was determined. Among the subgroups of NPSLE, a positive correlation was also observed in diffuse NPSLE. In contrast, there was no correlation in focal NPSLE. These results suggest that increased IL-6 in the CSF in diffuse NPSLE  is not only a result of intrathecal synthesis but also a result of transfer of IL-6 from the systemic circulation due to a damaged BBB.Third, we observed a significant elevation of CSF C3 concentration and Q C3 value in NPSLE compared with non-NPSLE. Furthermore, CSF C3 concentration and Q C3 were also increased in ACS diffuse NPSLE compared with non-ACS diffuse NPSLE and focal NPSLE. A positive correlation between Q C3 and Q α2MG was determined, especially in diffuse NPSLE. There was no significant difference in C3 index between NPSLE and non-NPSLE, as well as among the subgroups of NPSLE. These results suggest that the source of C3 in the CSF is mainly a result of the transfer of C3 from the systemic circulation due to BBB disruption, especially in diffuse NPSLE.In NPSLE, mild abnormalities in routine CSF analysis commonly include pleocytosis (22–36%), elevated TP level (30–66%), and low glucose level (8–42%) . In thisFurthermore, we observed a strong correlation between CSF TP level and Q α2MG in diffuse NPSLE, especially in ACS diffuse NPSLE. Albumin forms the majority of CSF TP but is not produced in the brain; therefore, our result may support the possibility that BBB disruption is present in diffuse NPSLE, especially in ACS diffuse NPSLE.In NPSLE, two main mechanisms have been implicated in BBB disruption. One is microthrombi in cerebral vessels resulting in ischemia, and the other is immune-mediated endothelial activation resulting in intrathecal cytokine synthesis . PresentSeveral studies have been reported that IL-6 levels are increased in CSF in patients with NPSLE. Hirohata et al. demonstrated that IL-6 activity in CSF in NPSLE patients, including diffuse NPSLE and focal NPSLE, was significantly increased compared with non-NPSLE. They also showed no significant correlation between IL-6 activity and Q albumin. Tryslerg et al. measured IL-6 levels in the CSF in 14 patients with NPSLE (four organic brain syndrome (OBS), three stroke, two transverse myelitis, one meningitis, one movement disorder) and compared them with non-NPSLE patients. They found a 10-fold increase of IL-6 in NPSLE patients, especially in four patients with OBS, consisting mainly of ACS diffuse NPSLE. Katsumata et al. divided NPSLE into ACS and non-ACS , and measured IL-6 levels in the CSF of each group. They demonstrated IL-6 levels were significantly higher in ACS than non-ACS. These findings were supported by a multicenter retrospective study in which CSF IL-6 had high sensitivity and specificity in patients with lupus psychosis .Regarding the source of IL-6 in the CSF of NPSLE, earlier studies suggest that the elevation of CSF IL-6 in NPSLE seemed to be a result of intrathecal synthesis of IL-6, because enhanced expression of IL-6 mRNA is present in the hippocampus of diffuse NPSLE patients , and theStudies previously investigated the relationship between CSF complement levels and NPSLE. Jongon et al.  performeHowever, conflicting results were reported regarding the C3 index in NPSLE from the same authors . Jongon In this study, we found no significant difference in C3 index between NPSLE and non-NPSLE, as well as among the subgroups of NPSLE. However, in SLE patients with hypocomplementemia due to high disease activity, complement index may not reflect intrathecal complement synthesis alone because low serum complement concentration may increase the quotient level, resulting in an increased index. Measuring complement cleavage products  in CSF may be more appropriate for the evaluation of the intrathecal complement activation. Thus, further study is required.In patients with diffuse NPSLE, we observed a significant positive correlation between Q α2MG and CSF TP, Q α2MG and CSF IL-6, and Q α2MG and Q C3. The strong correlation between Q α2MG and CSF TP may be because the majority of CSF TP is albumin, which is not produced in the brain. Compared with the correlation between Q α2MG and CSF TP, the correlation between Q α2MG and CSF IL-6, as well as between Q α2MG and Q C3 were not so strong (moderate). For the correlation between Q α2MG and CSF IL-6, this might be because of the increased synthesis of IL-6 in the brain in ACS diffuse NPSLE , which mOur study suggested that elevated levels of IL-6 and C3 in CSF in diffuse NPSLE, especially in ACS, are the result of entry from the systemic circulation to the CSF through the damaged BBB, as well as the result of increased intrathecal production. Furthermore, it is possible that Q α2MG is a useful measure in addition to Q albumin to evaluate the integrity of the BBB.


The lack of development of local donor tissue acquisition in several regions of the world has resulted in the necessity of performing keratoplasty with imported donor corneas. The greatest concern about the use of donor corneas supplied by foreign eye banks is the effect of the increased donor death-to-operation time which inevitably occurs during the tissue recovery, tissue processing, and tissue transfer between the countries. The purpose of this study was to report the outcomes of descemet stripping automated endothelial keratoplasty (DSAEK) using imported donor corneas.This retrospective, non-comparative case series investigated the outcomes of the 102 consecutive DSAEK procedures using imported donor corneas performed at a single university-based hospital between August 2006–2014. The main outcome measures were postoperative best-corrected visual acuity (BCVA), endothelial cell density (ECD), and complications.2. Fuchs’ endothelial dystrophy was the predominant indication for grafting. The mean follow-up duration was 65.3 months. Ninety-three eyes had improved vision postoperatively (91.18%). BCVA unchanged in 3 eyes due to preexisting macular scar and advanced glaucoma. Primary graft failure occurred in 6 eyes (5.88%). Of the 93 eyes with improved BCVA, 100% had their best corrected vision within the first 1 year. The mean ECD at 6, 12, 24, 36, and 60 months after surgery was 1762 ± 294 cells/mm2, 1681 ± 284 cells/mm2, 1579 ± 209 cells/mm2, 1389 ± 273 cells/mm2, and 1251 ± 264 cells/mm2 respectively. The mean ECD loss at 6 months, 1 year, 2 years, 3 years, and 5 years after surgery was 36.2%, 39.1%, 42.8%, 49.7%, and 54.7% respectively. The most common complication was graft detachment/dislocation (10.78%). There were no cases of any postoperative infection.The mean death-to-operation time was 9.52 ± 1.48 days . The mean preoperative ECD was 2761 ± 285 cells/mmDSAEK with imported donor corneas provides rapid and good visual rehabilitation. The percentages of endothelial cell loss were comparable to those achieved in Western series using domestic corneas in which fresher tissues were available for transplantation. Descemet stripping automated endothelial keratoplasty (DSAEK) has become the first-choice and most popular method for the treatment of corneal endothelial diseases. DSAEK appears to be superior to penetrating keratoplasty (PK) in terms of rapid visual recovery and refractive stability, surgically induced astigmatism, higher order aberrations, structural strength of the eye, postoperative surface keratopathy, wound and suture-related complications, rates of transplant rejection, and intraoperative and late choroidal hemorrhage risk , 2. HoweThe shortage of donor corneas is a major issue worldwide. Many countries, including Thailand, face this problem as cornea donation is not allowed or encouraged in some cultures due to the religious faith and traditional beliefs. To overcome the insufficient donor supply, we have been using both donor corneas from oversea eye banks and domestic donor corneas for keratoplasty. Excellent efficacy and safety of PK utilizing foreign donor corneas have been previously demonstrated –12. AlsoThis was a single-center, retrospective, non-comparative interventional case series, analyzing the outcomes of DSAEK using imported donor corneas in patients with corneal decompensation due to endothelial failure. Clinical data were obtained from a review of the medical records.Declaration of Helsinki. All patients were informed regarding the advantages, disadvantages, and potential complications of this surgery and emphasized the importance of postoperative care and need for regular follow-up and monitoring after surgery. Written informed consent was obtained from each individual prior to enrollment in this study.One hundred and two eyes of 95 patients with endothelial decompensation who underwent DSAEK at Ramathibodi Hospital, Bangkok, Thailand, from August 2006 and August 2014, with a minimum follow-up of 24 months were recruited into this trial. Patients with the accompanying risks of anterior segment abnormalities or further surgeries unrelated to the DSAEK procedure that may influence postoperative endothelial cell survival were excluded from the study. The study was approved by the ethics committee of Mahidol University School of Medicine and adhered to the tenets of the All donor corneas were provided by Eye Bank Association of America (EBAA)-accredited facilities in the United States. Each tissue met EBAA minimum standards of donor age, ECD, and death-to-preservation time. The corneas were transported as corneoscleral buttons housed within corneal viewing chambers filled with Optisol-GS . The viewing chambers were sealed with plastic wrap, packed in a plastic bag, and positioned in a soft foam block to prevent excessive movement. The foam blocks were again sealed in a plastic bag and placed into generic, thick-walled, expanded polystyrene foam shipping containers with wet ice. The packages were sent from the United States to Bangkok Suvarnabhumi International Airport and then transferred to Ramathibodi Hospital via FedEx. The containers were maintained at 2 °C–8 °C in validated shipping containers throughout the entire shipment process. Upon arrival at Ramathibodi Hospital, a technician collected the container and completed a tissue arrival check, including the date and time of arrival, condition of the shipping container, number and status of the ice blocks, number of donor tissues, and status of each viewing chamber. Also, the technician matched and confirmed the documentation accompanying each tissue, and delivered it to the operative room (OR) charge nurse. The tissue and its package must have been received in good condition otherwise it would have been considered not suitable for transplantation. The OR charge nurse placed it in the temperature-controlled refrigerator. On the day of surgery, the tissue was removed from the refrigerator, reevaluated whether it was well suited for transplantation, and allowed to warm to room temperature in the operating theatre for 30–60 min prior to use.Two novice surgeons  performed the DSAEK procedures under either local or general anesthesia. The donor tissue was dissected with a microkeratome in all cases using the Moria ALTK system . Tissue preparation was done by the operating surgeon in 33 eyes and by an US eye bank technician (pre-cut tissue) in 69 eyes. Descemet’s membrane and diseased endothelium were stripped from the planned graft area using the reverse Sinskey hook and a DSAEK stripper. Venting incisions were made to improve graft adherence. A 4.5–5 mm clear corneal incision was created using a 3.0 mm—wide slit knife for graft insertion. The donor cornea was cut to a diameter of 7.5–9.0 mm, depending on the recipient corneal diameter. The trephined donor posterior lenticule was inserted into the recipient anterior chamber using Busin glide . Once the donor graft was properly unfolded and well centered, it was pressed up against the recipient stroma with air. After the anterior chamber was filled completely with air for 10 min, the air bubble was then reduced to prevent pupillary block. Beginning with case 9, the inferior peripheral iridectomy was performed prior to donor insertion to allow leaving nearly a full air fill in the anterior chamber at the end of surgery without producing pupillary block.Postoperatively, each patient received topical corticosteroids (1% prednisolone acetate) 8 times daily and antibiotics 4 times daily for the first 2 weeks. They were gradually tapered at the discretion of the operating surgeons to 1 drop per day dosing, continued indefinitely. Steroid responders received intraocular pressure (IOP)-lowering medications. Also, prednisolone acetate 1% was changed to fluorometholone as needed. Patients were examined daily until the residual air bubble did not cover the pupil when the patient sat up and corneal re-epithelialization was complete and then discharged from the hospital. Follow-up evaluations were performed at 0.5, 1, 3, 6, 9, and 12 months after surgery and then every 6 months thereafter.2 without manual cell border correction. At least 50–100 cells were required to be examined for each image and the postoperative ECD was recorded. Percentage of endothelial cell loss was calculated by subtracting postoperative ECD from preoperative ECD and dividing by preoperative ECD, and then multiplying by 100. Endothelial polymorphism and polymegathism analyses were not performed. Anterior segment OCT was also not performed postoperatively as a result of the unavailability of the equipment at that time. Patients having complications received intensive care in collaboration with other subspecialists.The primary outcome measures included postoperative best-corrected visual acuity (BCVA), endothelial cell density (ECD), and complications. The secondary outcome was the relationships of the postoperative ECD with donor age, death-to-preservation time, death-to-operation time, and baseline donor ECD. Preoperative donor ECD was evaluated by provider eye banks in the United States using specular microscopy. Postoperative ECD was determined by noncontact specular microscopy  in our clinic at 6 months, 1, 2, 3, and 5 years after surgery. The central corneal endothelium was photographed using the auto-alignment and auto-shot functions of the instrument. The best-quality image is automatically selected and analyzed by the preinstalled software of the instrument . The number of analyzed corneal endothelial cells and cell density were automatically calculated using the fixed-frame counting method in an area set to 100 μmP-values less than 0.05 were considered to be statistically significant.Statistical analyses were performed with the statistical software package STATA version 11.1 . Continuous data were expressed as either mean ± standard deviation (SD) or median and range, depending on the normality of distribution. These were compared using independent t test or Mann–Whitney U test respectively. Categorical data described as frequency and percentage, were analyzed with Chi-square or Fisher’s exact tests. Associations between postoperative ECD at 5 years and various donor or recipient characteristics were evaluated using linear regression analysis. Multiple regression analysis was performed for only variables that were statistically significant by univariate analysis. Donor characteristic data including donor age, death-to-preservation time, death-to-surgery time, residual stromal bed thickness after microkeratome cutting, and ECD are summarized in Table P < 0.01). Preoperative BCVA was ≤20/200 in 80 eyes (78.43%) and ≤20/40 in 101 eyes (99.02%). Ninety-three eyes had improved vision postoperatively (91.18%). BCVA unchanged in 3 eyes (2.94%) due to preexisting macular scar (2) and advanced glaucoma (1). Six eyes (5.88%) experienced a decline in vision secondary to primary graft failure. Of the 93 eyes with improved BCVA, 100% had their best corrected vision within the first 1 year. Postoperatively, 9.80% achieved a BCVA of ≥20/25, 50.98% achieved ≥20/40, and 79.41% achieved ≥20/100 by the last follow-up , representing an average gain of 5 Snellen lines from preoperative vision .The mean preoperative ECD was 2761 ± 285 cells/mm2 range, 2361–3015). Among eyes examined with clear grafts, the mean ECD at 6, 12, 24, 36, and 60 months after surgery was 1762 ± 294 cells/mm61–3015. Postoperative complications after DSAEK surgery are shown in Table Primary graft failure occurred in 6 eyes and one of them was the eye with eccentric mis-cut. Two eyes required reinsertion of the donor lenticule into the anterior chamber. Two eyes had a thin, precut donor lenticule, causing more tissue manipulation during transfer to the glide. Another eye underwent uneventful DSAEK using an eye bank-prepared precut cornea. Of the 6 eyes with primary graft failure, the donor grafts were surgeon-prepared in 4 eyes and technician-prepared in 2 eyes. Five patients developed episodes of endothelial graft rejection and all were treated with systemic and topical corticosteroids. Graft rejection was observed within 1 year post-surgery in 2 eyes and after 1 year in 3 eyes. Six eyes experienced late graft failure; 2 was post-rejection, 1 was post-additional IOL surgery, and 3 had no obvious precipitating factor. Pupillary block occurred in 6 eyes without surgical peripheral iridectomy at the time of DSAEK. It was relieved by partial removal of the air bubble or peripheral iridectomy. There was no pupillary block occurred in any cases after inferior peripheral iridectomy was adopted as a routine policy. One eye having a diagnosis of PBK secondary to complicated phacoemulsification with posterior capsular tear experienced posterior IOL dislocation after DSAEK. It possibly resulted from unrecognized small zonular dialysis or undetected placement of a haptic in the capsular bag. The IOL was successfully repositioned by a retinal specialist; however, the graft eventually failed 13 months following IOL surgery.Corneoscleral rim cultures were obtained in all cases and none had a positive donor rim culture. There were no cases of infectious keratitis or endophthalmitis.The lack of development of local donor tissue acquisition in several regions of the world has resulted in the necessity of performing DSAEK with internationally shipped donor corneas. The greatest concern about the use of donor corneas supplied by foreign eye banks is the effect of the increased donor death-to-operation time which inevitably occurs during the tissue recovery, tissue processing, and tissue transfer between the countries. In addition, the shipping-related vibration effects and the changes in the air pressure caused by air transport might affect the quality of the donor corneas , 14. Thu2 after the 10th post-harvest day [Our study demonstrated that despite having longer transportation time, there was a satisfactory BCVA improvement in 91.18% of patients after DSAEK using imported donor corneas. Additionally, although the postoperative mean ECD at 6 month, 1, 2, 3, and 5 years of this study was lower than those of the Japanese report and previous studies of domestically shipped corneas, the percentage of endothelial cell loss at each time point in our study was similar to those of such studies . Since tvest day . In this2 and were available for DSAEK [With respect to the relationship between donor tissue storage time and the probability of endothelial survival, there has still been conflicting evidence in the literature. A significant correlation between prolonged storage time (> 7 days) and increased risk of graft failure has been reported in PK with imported donor corneas . Neverthor DSAEK , 14. Theor DSAEK . No assoor DSAEK . Furtheror DSAEK . Also, nor DSAEK . These for DSAEK  and the or DSAEK , 18, usiIt is interesting that the endothelial cell density was slightly changed between 36 and 60 months. This corresponds to the earlier finding that long-term endothelial cell loss in DSAEK patients plateaus more quickly than in those who undergo PK , 8. The Considering the occurrence of other postoperative complications, graft rejection was found in only 4.9%, secondary angle closure glaucoma in 2.94%, and no infectious keratitis or endophthalmitis was seen in this series. This observation suggests that DSAEK using foreign donor corneas with prolonged storage time have a good safety profile, resembling those using domestic donor corneas .The limitations of this study included the small and heterogeneous patient populations, 2 different surgical techniques used (precut and surgeon-cut), short follow-up time, no comparison of endothelial cell loss between the early and later cases, and inherent shortcomings in any retrospective study.In summary, the outcomes of DSAEK performed with internationally shipped donor corneas were acceptable and the percentages of endothelial cell loss were comparable to those achieved in Western series using domestic corneas in which fresher tissues were available for transplantation. This study also supports the efficacy and safety of imported donor corneal tissues stored greater than 1 week and up to 13 days. Cornea donation from overseas eye banks may be of substantial benefit to patients in countries with inadequate donor cornea supply.