App knock-in mouse models, AppNL-F/NL-F and AppNL-G-F/NL-G-F, exhibiting AD-like Aβ pathology to analyze how the brain pathologies translate to CSF proteomes by label-free mass spectrometry (MS). This identified several extracellular matrix (ECM) proteins as significantly altered in App knock-in mice. Next, we compared mouse CSF proteomes with previously reported human CSF MS results acquired from patients across the AD spectrum. Intriguingly, the ECM protein decorin was similarly and significantly increased in both AppNL-F/NL-F and AppNL-G-F/NL-G-F mice, strikingly already at three months of age in the AppNL-F/NL-F mice and preclinical AD subjects having abnormal CSF-Aβ42 but normal cognition. Notably, in this group of subjects, CSF-decorin levels positively correlated with CSF-Aβ42 levels indicating that the change in CSF-decorin is associated with early Aβ amyloidosis. Importantly, receiver operating characteristic analysis revealed that CSF-decorin can predict a specific AD subtype having innate immune activation and potential choroid plexus dysfunction in the brain. Consistently, in AppNL-F/NL-F mice, increased CSF-decorin correlated with both Aβ plaque load and with decorin levels in choroid plexus. In addition, a low concentration of human Aβ42 induces decorin secretion from mouse primary neurons. Interestingly, we finally identify decorin to activate neuronal autophagy through enhancing lysosomal function. Altogether, the increased CSF-decorin levels occurring at an early stage of Aβ amyloidosis in the brain may reflect pathological changes in choroid plexus, present in a subtype of AD subjects.Cerebrospinal fluid (CSF) biomarkers play an important role in diagnosing Alzheimer’s disease (AD) which is characterized by amyloid-β (Aβ) amyloidosis. Here, we used two The online version contains supplementary material available at 10.1186/s40478-022-01398-5. Alzheimer’s disease (AD), as the most common cause of dementia, has two pathological hallmarks: extracellular amyloid-β peptide (Aβ) plaques and intracellular tau aggregation. Aβ could also accumulate in the vasculature and lead to changes in the blood–brain barrier (BBB) and in choroid plexus (ChP), which is part of blood-cerebrospinal fluid barrier (BCSFB), and potentially worsen disease progression –3. The BApp knock-in mice exhibit AD-like phenotypes including robust Aβ pathology whereas App expression is at endogenous levels. AppNL-F/NL-F mouse model harbors two familial AD (FAD) mutations, the Swedish (NL) and the Beyreuther/Iberian (F), in the humanized mouse App gene which increases the human Aβ42 levels in the mouse brains [AppNL-G-F/NL-G-F mouse model has an additional Arctic mutation (G) leading to a much earlier and severer Aβ pathology which eventually cause more aggressive neuroinflammation, synaptic alteration and memory impairment [e brains . The Apppairment .App knock-in mice by label-free mass spectrometry (MS) and compared their proteomes with protein changes in CSF of human subjects across the AD spectrum from a previously published large AD cohort [In a translational approach to understand how pathological changes in the brain are mirrored in the CSF, we analyzed CSF of D cohort . We idenApp knock-in mouse models, AppNL-F/NL-F and AppNL-G-F/NL-G-F, have been described previously [App knock-in models were performed under ethical permit ID 12570-2021 approved by Stockholm animal ethical board and ID 407 approved by Linköping animal ethical board except the CSF collection from 13 months old AppNL-F/NL-F mice approved by RIKEN Center for Brain Science (W2021-2-020(2)). Two APP transgenic (Tg) mouse models were used, APP23 mice harboring only the Swedish mutation [The two eviously . Most ofmutation . The CSFThe mice were anesthetized by isoflurane and placed on the heating pad with fixed heads (the head angle was around 135° from the body) on the stereotaxic instrument. The skin was incised sagittally after removal of the fur. Under the dissection microscope, subcutaneous tissues and muscles were separated to expose the dura mater of cisterna magna. To reduce blood contamination, the exposed dura mater was cleaned by PBS-soaked cotton swabs and punctured with a 27-gauge needle avoiding blood vessels. CSF was sampled with a glass capillary and collected in low affinity Eppendorf tubes and directly frozen. The collected CSF was discarded if there was any blood contamination under microscope inspection. Thereafter, the mice were perfused with PBS through cardiac perfusion, and hippocampus and cortex were dissected.Appwt/wt mice E16-E18 and the heads were kept in HBSS  on ice for brain dissections. Brains were dissected under the dissection microscope to separate the cortex/hippocampus. Cortex/hippocampus were chopped and transferred to the falcon tube together with HBSS. HBSS was removed until only tissues were left at the bottom. Neurobasal medium  97% + B-27  2% + Glutamax  1% were added and tissues were separated by pipetting up and down 20–30 times. Cells were counted by a hematocytometer with trypan blue staining. 1.5 × 105 cells for 24 well plates, 3 × 105 cells for 12 well plates and 5 × 106 cells for 10 cm petri dishes were seeded. After one week, 50% of the medium was replaced by a fresh medium.Twenty-four well plates were coated with Poly-D-lysine  for one hour at room temperature and washed with Milli-Q water followed by drying overnight. Embryos were separated from For decorin treatment study, after 18 DIV, cells were treated with 200 nM recombinant mouse decorin  and/or 100 nM bafilomycin A1  for six hours and thereafter the cells were collected in PBS. Cell solutions were centrifuged at 1500 rpm for five min at 4 °C to pellet the cells, which were lysed in RIPA buffer . The lysate was sonicated for one min and centrifuged at 15,000 rpm for 20 min at 4 °C. The supernatant was transferred to new tubes for western blot. Each condition was triplicated and the whole set of experiments repeated twice.For human Aβ42 treatment study, after 12 DIV, cells were treated for 24 h with 50 nM recombinant Aβ42 (Met-Aβ42) which was prepared as described previously . Then thThe paraffin-embedded mouse brains (12 months old) were sectioned into four µm thick sections for most of immunofluorescence staining except for vessel staining for which 10 µm thick sections was used. For decorin staining, slides were treated with 0.5 units of chondroitinase ABC  in 0.1 M Tris-acetate (pH: 7.3) for four h in 37 °C before blocking . For othThe cell lysates were boiled at 95 °C for 3 min. 10–20 μg proteins were loaded onto 4–20% SDS-PAGE for separation and transferred to nitrocellulose membranes. The nitrocellulose membranes were blocked by 5% skim milk and were probed by primary antibodies, anti-p62  (1: 500), anti-LC3  (1: 1000), anti-decorin  (1: 100), anti-LAMP1  (1:500), anti-p-TFEB S142  (1:200), anti-TFEB  (1:200), anti-NBR1  (1:200), anti-β-actin   overnight at 4 °C. Next day, the nitrocellulose membranes were incubated with fluorescently labeled secondary antibodies (LI-COR Biosciences), Donkey anti-rabbit (Cat. 926-32213)  or Goat anti-mouse (Cat. 926-68070)  for one hour at room temperature. Images were acquired by a fluorescence imaging system  and were analyzed by Image Studio Lite (LI-COR Biosciences) software.The phosphorylation profiling  following the manufacturer’s protocol.One μL of mouse CSF samples were applied to Mouse Exploratory Panel, Olink according to manufacturer’s protocol.6. The 10 most intense ions were selected for higher-energy collisional dissociation fragmentation  and MS/MS spectra were generated with an automatic gain control target of 5 × 105 at a resolution of 17,500. The mass spectrometer was operated in data-dependent mode. Acquired raw files were processed by MaxQuant (version 1.5.1.2) (the software is available at http://www.maxquant.org). Tandem mass spectra were searched with Andromeda against the UniProt Mus musculus database (release January 2017). The search settings were set as: maximum 10 ppm and 0.02 Da error tolerance for the survey scan and MS/MS analysis respectively; enzyme specificity was trypsin/Lys-C; maximum two missed cleavage sites were allowed; cysteine carbamidomethylation was set as static modification, and Oxidation (M) was set as dynamic modification.An aliquot of eight μL of each CSF sample was used for in-solution digestion. Briefly, the proteins were re-dissolved in 50 μL of digestion buffer . A volume of 10 μL of 45 mM aqueous dithiothreitol was added to all samples and the mixtures were incubated at 50 °C for 15 min to reduce cysteine disulfides. The samples were cooled to room temperature and 10 μL of 100 mM aqueous iodoacetamide was added before incubating the mixtures for an additional 15 min at room temperature in darkness to carbamidomethylate the cysteines. Finally, 10 μL of 0.1 μg/μL trypsin/Lys-C mixture dissolved in 50 mM ammonium bicarbonate was added to the samples. The tryptic digestion was performed at 37 °C overnight. Prior to MS analysis, the peptides were purified and desalted using SPE Pierce C18 Spin Columns (Thermo Fisher Scientific). The columns were activated by 2 × 200 μL of 50% acetonitrile and equilibrated with 2 × 200 μL of 0.5% trifluoroacetic acid. The tryptic peptides were adsorbed to the media using two repeated cycles of 40 μL sample loading and the column was washed using 3 × 200 μL of 0.5% trifluoroacetic acid. Finally, the peptides were eluted in 3 × 50 μL of 70% acetonitrile and dried. Dried peptides were resolved in 21 μL of 0.1% formic acid prior to nano-LC–MS/MS. The nano-LC–MS/MS experiments were performed using a Q Exactive Orbitrap mass spectrometer  equipped with a nano electrospray ion source. The peptides were separated by C18 reversed phase liquid chromatography using an EASY-nLC 1000 system (Thermo Fisher Scientific). A set-up of pre-column and analytical column was used. The precolumn was a 2 cm PepMap Acclaim  (Thermo Fisher Scientific) while the analytical column was a 10 cm EASY-Spray column . Peptides were eluted with a 150 min linear gradient from 4 to 100% acetonitrile at 250 nL min-1. The mass spectrometer was operated in positive ion mode, acquiring a survey mass spectrum with resolving power 70,000 , m/z 400–1750 using an automatic gain control target of 3 × 10The search criteria for protein identification were set to at least two matching peptides. No proteins were identified and quantified using only one peptide. A maximum false discovery rate of 1% for peptides and proteins was selected. Both razor and unique peptides were used for quantification. For decorin, at least five peptides per sample were used for the quantification  were compared.The same protein identifiers were used to compare mouse and human CSF proteomes. We performed two types of comparisons. In the first exploratory comparison, proteins with fold change > 1.0 was considered to be upregulated whereas < 1.0 was considered downregulated in the mouse and human CSF proteomes. In the second comparison only signifcantly changed proteins in the mouse and human CSF samples  and CSF Aβ42, t-tau and p-tau levels (predictors) were determined using linear regression across the total group, and differences between subgroups  in NC group were tested with the ‘emtrends’ function from the emmeans R package (v1.5.2). ROC curve analyses were performed for CSF-decorin levels and AUC with 95% CI were calculated with R package pROC (v1.18.0).Appwt/wt and AppNL-F/NL-F mice and Appwt/wt and AppNL-G-F/NL-G-F mice, respectively.The correlation analysis between parameters of Aβ pathology and decorin expression in the mice were made by Spearman’s rank correlation coefficient since some of the parameters were not normally distributed. The data were grouped from App knock-in mice which exhibit robust Aβ amyloidosis. To stratify the data, the mouse CSF proteomes were next compared with the proteomes of human CSF from a previously published large AD cohort [To investigate how AD pathologies driven by Aβ translate to CSF, we used label-free MS to analyze the CSF of D cohort . This reAppNL-F/NL-F and AppNL-G-F/NL-G-F mice which exhibit different degree of Aβ pathology  resulted in a tighter clustering  chain, basement membrane-specific heparan sulfate proteoglycan core protein, fibronectin, SPARC-like protein 1, fibulin-1, vitronectin and ecm1 protein  changes in additional ECM related proteins  using a mouse proximity extension assay (PEA) panel. Indeed, two ECM proteins, matrilin-2 and CCN family member 4, were significantly (p < 0.05) altered  cohort (n = 310) [App knock-in mice and a + t − human subjects were higher as compared to a + t + subjects, indicating that the alterations in the CSF proteomes of App knock-in mice are, at least to some extent, more similar to the CSF alterations observed in a + t − human subjects (Table App knock-in mice have a strong Aβ pathology whereas the tau pathology is less pronounced [In an attempt to further understand how changes in the CSF proteomes of n = 310) . In thisn = 310) . The groonounced . FurtherAppNL-F/NL-F mice showed that 33 proteins exhibited relative expression levels above one  was significantly upregulated in both AppNL-F/NL-F mice and a + t − NC subjects (p < 0.05)  and fibronectin (FN1)  . In contrast, within a + t − NC subjects CSF-decorin levels significantly correlated with CSF-Aβ42 levels (beta(se) = 0.61(0.25), p = 0.01), a correlation that was significantly (p = 0.016) different from that of a-t- healthy subjects       was 0.61  = 0.50, 0.73) and not significant within this rather limited cohort (p = 0.058)   as the App knock-in mice, by enzyme-linked immunosorbent assay (ELISA). APP23 mice contain only the Swedish mutation and form Aβ plaques from around 12 months of age, while tg-ArcSwe mice having both the Swedish and the Arctic mutation start to form Aβ plaques at around six months of age [AppNL-F/NL-F mice -II and LC3-II/LC3-I ratio . However, decorin could indeed classify with high sensitivity and specificity a subtype of CSF-amyloid positive subjects including NC, MCI, AD and whose CSF proteome was characterized by innate immune activation, increased ECM proteins and increased transthyretin indicating a potential ChP dysfunction . Further analysis of ChP pathology in AD brains of this AD subtype and its correlation with CSF-decorin is warranted. Moreover, the CSF-decorin levels significantly negatively correlated with both CSF-t-tau and CSF-p-tau in a + t − NC subjects as well as over the whole AD spectrum. This additionally supports the link of decorin and the tau pathology in AD brain which needs to be addressed in mouse models of tau pathology.In this study we aimed at determining how AD brain pathologies are reflected in CSF and their translational potential toward clinical use, using data from both AD mouse models, and human subjects. Here, we used two se brain . The perse brain . Most imia stage . The ROCApp knock-in mouse models and a + t − MCI and AD human subjects showing that such changes are translational between mice and human. Considering that ECM is an important constituent of BBB/BCSFB, which are most likely affected in AD [App knock-in mice could also have changes in the BBB/BCSFB.A growing number of studies have shown that BBB breakdown occurs in early AD and is closely related to cognitive dysfunction –37. Henced in AD , 4, our e.g., glia cells, induced by Aβ amyloidosis may eventually lead to CSF-decorin changes. This needs to be further investigated. Furthermore, we showed that decorin expression in ChP was increased in AppNL-F/NL-F mice which correlated both with Aβ pathology and elevated CSF-decorin level. Taking these data together indicates that early Aβ amyloidosis, potentially involving soluble Aβ species, in the brain may increase the decorin expression in ChP which may also lead to elevated decorin levels in the CSF since CSF is produced in ChP. A time course analysis of CSF-decorin in AppNL-F/NL-F mice revealed an increase of CSF-decorin levels at three months of age even before the establishment of Aβ plaque deposition followed by a continuous decrease upon Aβ pathology development, which is similar to the CSF-decorin alterations in a + t − human subjects. An increase in CSF-decorin was also found in the AppNL-G-F/NL-G-F mice by ELISA. The discrepancy between MS and decorin ELISA data of the 12–13 months old AppNL-G-F/NL-G-F mice may be due to differences in detection of different proteolytic fragments of decorin by the two methods  altered proteins in AppNL-F/NL-F mice as compared to AppNL-G-F/NL-G-F mice. Proteins were sorted into upregulated and downregulated proteins in the AppNL-G-F/NL-G-F mice and by significance from highest to lowest (top to bottom).Additional file 5: Table S4. Significantly altered ECM associated proteins in mouse CSF determined by PEA.Additional file 6: Table S5. Description of the participants in EMIF-AD MBD cohort.Additional file 7: Table S6. Proteome comparison of AppNL-F/NL-F and AppNL-G-F/NL-G-F mice vs NC/MCI/AD .Additional file 8: Table S7. Proteome comparison of AppNL-F/NL-F and AppNL-G-F/NL-G-F mice vs NC/MCI/AD .Additional file 9: Fig. S2. CSF-decorin negatively correlates with CSF-t-tau and CSF-p-tau in the whole EMIF-AD MBD cohort. a Correlations of CSF-DCN and CSF-Aβ42, b CSF-DCN and CSF-t-tau, c CSF-DCN and CSF-p-tau in the whole EMIF-AD MBD cohort (n = 310), including NC (n = 139), MCI (n = 92) and AD (n = 79) were analyzed. The regression coefficients (Beta) and p-values are displayed. DCN decorin.Additional file 10: Fig. S3. Decorin is expressed in vessels on the brain surface but not in vessels of brain parenchyma. a Double immunostaining of decorin with αSMA (vascular smooth muscle cell marker) in 12 months old Appwt/wt mouse brain showing decorin expression in the arteries and b veins of the brain surface, but not c in the vessels of brain parenchyma. Scale bars, 200 µm , 100 µm (c). d Immunostaining of decorin in 12 months old Appwt/wt, AppNL-F/NL-F and AppNL-G-F/NL-G-F mouse brains showing decorin expression in the vessels under the hypothalamus. Scale bars, 200 µm. e The intensities were quantified. (n = 4). Data in (e) were analyzed by one-way ANOVA followed by Dunnett’s multiple comparisons test. Data are represented as mean ± SEM. DCN decorin, a artery, v vein, ns not significant.Additional file 11: Fig. S4. CSF-decorin levels correlates with Aβ pathology in the brains of AppNL-F/NL-F mice. a Spearman’s correlation analysis of parameters related to Aβ plaque load and decorin levels between 12 months old AppNL-F/NL-F and Appwt/wt mice, bAppNL-G-F/NL-G-F and Appwt/wt mice. The corresponding figures for each parameter are denoted. The correlation coefficients are displayed . The red color represents positive correlation, and the green color represents negative correlation according to the scale bars in the right column.Additional file 12: Fig. S5. Autophagy main upstream pathways are not affected by decorin in the neurons. Phosphorylation profiling of proteins involved in autophagy upstream pathways in non-treated and decorin-treated mouse primary neurons.


Improper utilization of antibiotics harms the patient, the public, and the economy. The overuse of injections is one of the key factors in the irrational use of medicines. However, little is known about intravenous (IV) to peroral (PO) conversion practice in the Ethiopian healthcare setting, specifically in the Northwest part of Ethiopia.  To assess antibiotics IV to PO conversion practice and its associated factors at the internal medicine ward of the University of Gondar Comprehensive and Specialized Hospital (UOGCSH). P value≤ 0.05 was considered statistical significance.  A prospective observational study was conducted on 324 study participants who were admitted to the University of Gondar Specialized Hospital from October 3 to November 14, 2021. A systematic random sampling technique was employed to select the study participants. Stata version 14.2 was used for the analysis. Descriptive statistics result was presented using mean and standard deviation. Logistic regression analysis was done to determine the association between independent variables and dependent variables. The association between independent variables and dependent variables was tested at 95% CI and  A total of 324 study participants were included in the study, and the mean age of the patients was 41.4 ± 18.6. Of the 324 study participants, 63.3% were male. The most frequently prescribed antibiotics used for empiric treatment were ceftriaxone (45.4%), followed by metronidazole (33.2%), and cloxacillin (11.4%). A total of 34.5.57% of patients who took antibiotics were converted to PO antibiotics. The most frequently converted type of conversion practice was sequential (23.1%), followed by the switch type of conversion (7.4%). Tachypnea, unavailability of medication, higher temperature, hospital stay greater than 10.78-days, and the presence of comorbidity were predictors of IV medications not being converted to PO medications.  Intravenous to peroral conversion practice was infrequent. The most frequently applied conversion practice was sequential type conversion practice, followed by switch type of conversion practice. IV to PO conversion practice was significantly associated with tachypnea, unavailability of medication, higher temperature, hospital stay greater than 10.78-days, and comorbidity. Awareness of IV to PO conversion practice and short-term training for healthcare teams is vital for better antibiotic conversion practice. Antibiotic therapy has become vital for the effective management of infectious diseases . A prevaThe ideal route of administration for any medication is to attain the ideal serum concentration without producing any adverse effects . Hence, The route of administration of antibiotics, either PO or IV, depends on the patient's condition and site of infection. The PO route of administration has many advantages over the IV route. Among these, it is the most convenient, cheapest available route, easy to use, safe, easy to prepare, and less expensive than the IV route , 11. AppAn institutional-based prospective observational study was conducted from October 3 to November 14, 2021, in the medical ward of UOGCSH. The hospital is located in Gondar town, North Gondar administrative zone, Amhara regional state. This is located 750 km away from of Addis Ababa, the capital city of Ethiopia. The University of Gondar Hospital has more than 400 beds with different units and clinics, which include medical wards, pediatric ward, psychiatry ward, surgery, emergency ward, chemotherapy center, gynecology and obstetrics ward, and HIV clinic. In addition, the hospital provides outpatient medical services to 250,000 patients. Besides, the hospital serves as a teaching hospital, research center, and referral center for more than five million people.The study was conducted using a prospective hospital-based observational study.All patients admitted to the medical ward taking antibiotics during the study period and who volunteered to take part in the study were the study population.Patients whose age was above 18 years old who had received an IV antibiotic for more than 48 hours, were able to eat or tolerate enteral feeding, patients with a gastrointestinal tract, absence of bowel abnormalities, and who adequately absorbed oral medications via the oral, gastric, or nasogastric tube route were included in the study.Staphylococcus aureus bacteremia, and endocarditis. Patients unable to respond to oral medication, patients with grade three and above mucositis, patients with unstable conditions, patients refusing oral medications, and immunocompromized patients (febrile neutropenia on cancer chemotherapy) were excluded from the study.For those patients who received IV prophylactic antibiotics, a prolonged course of IV antibiotics was required in the case of osteomyelitis, meningitis, Zα/2 and the proportion (P) of IV to PO antimicrobial conversion in these groups with a 95% confidence interval (CI) and marginal error (d) of 5%.X = 307.5 population plus 5% (m) contingency = 16, N = 1530 population, and n = 384 population.Because no research had been published on this topic in Ethiopia before October 2021, we took a 50% prevalence of IV to PO conversion. The sample size was calculated based on the single population proportion formula using the following assumption: (1.96) 2 were used n = the number of calculated patients from the aforementioned formula. N = the number of patients expected to be admitted within three months in the ward. Based on the above formula, assumptions, correction formula, and 5% of contingency, the sample size (n) was calculated to be 323.5. Finally, we selected 324 patients using a systematic random sampling technique. Then, every third patient who arrived at the clinic was selected for the study. The following figure shows the procedure of how to exclude and include the study participant during the study period  in the data abstraction format before the main data collection was done. The pretested data were not included in the study, and the appropriate adjustment was done to the data abstraction format. In addition to this, the authors supervised the data collector during the data collection process. The collected data were checked for completeness and consistency on a daily basis.(1). Ethical Approval and Consent to Participate. Ethical clearance with ethical approval code SOPs/117/2021 was obtained from the ethical clearance committee of the department of clinical pharmacy, school of pharmacy, University of Gondar. Permission to access the medical records of patients was then obtained from the UOGCSH clinical directorate. This study was conducted following the Declaration of Helsinki, in which it is stated that in medical research using identifiable human material or data, physicians must normally seek consent for the collection, analysis, storage, and/or reuse. Confidentiality of the information regarding patients was ensured in such a way that the data was only used for the study purpose. To ensure privacy and confidentiality, information collected was not directly linked to the respective participants; names of patients were not used. Codes were used as an identifier.P value of less than 0.2 were included in multivariate logistic regression analysis to identify independent predictors of IV to PO conversion. Those variables having a P value of <0.05 were considered statistically significant. The Hosmer–Lemeshow goodness of fit test for logistic regression was done, and the model was well fitted for IV to PO conversion (P=0.95). A contingency coefficient test was done to see whether categorical variables have multicollinearity. A variance inflection factor was used to test whether continuous variables had multicollinearity and had no correlation between independent variables.The data was entered into a computer database using Epi-data version 4.6 and exported to STATA version 14.2 for analysis. Categorical variables were summarised as frequencies and proportions. Continuous variables were first to be tested for normal distribution using a histogram. All continuous variables were normally distributed. Simple descriptive statistics such as mean ± SD, frequency, and percentages were determined. A bivariable logistic regression analysis was done to see the association between the independent variable and the outcome variable. Then, independent variables having a Sequential therapy: the act of replacing a parenteral version of medication with its oral counterpart of the same compound.Switch therapy: the conversion of an IV medication to a PO equivalent that is in the same class and has the same level of potency but is a different compound.Stepdown therapy: the conversion of injectable medications to oral medications in another class or to different medications within the same class where frequency, dose, and spectrum of activity may not be the same. It converts IV to PO agents with reduced potency.There are mainly three types of IV to PO conversions .SequentiOut of 324 study participants, 63.3% were males. The majority of them were in the age group of 18–29 (35%). The most frequent site of infection was soft tissue infection (SSI) 106 (32.7%), followed by respiratory tract infection 101 (31.2%). Among enrolled patients, 127 (39.2%) had one or more comorbidities. The major comorbidities encountered were tuberculosis (TB) 18(12.3%), hypertension 16 (10.9%), and other 47 (32.2%). The mean duration of antibiotics was 8.6 days and the mean length of hospital stay (LOHS) was 10.8 days .The most frequently prescribed antibiotics used for empiric treatment were ceftriaxone 226 (45.4%), followed by metronidazole165 (33.2%), and cloxacillin 57 (11.4%) . A totalP value less than 0.2 and were candidates for multivariate logistic regression analysis. Tachypnea, unavailability of the medication, longer length of hospital stays, presence of comorbidity, and higher temperature significantly affected IV to PO conversion in multivariate logistic regression. Accordingly, the presence of tachypnea increased the risk of IV medication not being converted to PO medications by an odds of 1.89 as compared to patients without tachypnea . The presence of higher body temperature increased the risk of IV medication not being converted to PO medications by an odds of 1.43 as compared to afebrile patients . The unavailability of medication increased the risk of IV medication not being converted to PO medications by an odds of 3.27 as compared to the availability of the medication . Patients with a hospital stay greater than 10.78 days increased the risk of IV medication not being converted to PO medications by an odds of 1.82 as compared to patients with a hospital stay ≤10.78days . The presence of comorbidity increased the risk of IV medication not being converted to PO medications by odds of 2.74 as compared to patients without comorbidity   .This study aimed to evaluate the practice of conversion from IV to PO antibiotic conversion and its associated factors. The prevalence of IV to PO conversion practice in this study was 34.5%, of which 23% was the sequential type (23%), followed by switch types of conversion (7.4%).The prevalence of IV to PO antibiotic conversion practice in this study was higher than in the study conducted in India. This difference could be due to the fact that the India study included a sampler sample. In addition to the above factors, the availability of oral formulation antibiotics, the knowledge and practice of prescribers, and healthcare policy are the determinant factors of conversion practice of IV to oral PO of administration. On the other hand, a lower practice of IV to PO conversion practice was reported in Lebanon at 26.1% . This diw, including spasms, pain, hematoma, infiltration, air embolism, overhydration, and an increased risk of cannula-associated and catheter-related adverse events [Although patients were eligible for conversion of antibiotics from IV to PO formulations, 63.43% of antibiotics were not converted to oral formulations. This implies that the practice of IV to PO conversion practice in the University of Gondar Comprehensive Specialized Hospital was poor. In this regard, patients were exposed to several complications e events .For those patients who were not converted to PO medication, the cost of the IV route is comparably higher than the cost of PO counterparts. The cost of IV medication was increased unless the antibiotics were converted to PO medications , 22. ThiIn this study, the most frequent type of IV to PO conversion practice was sequential type 75 23%) followed by the switch type of conversion 24 (7.4%). This study was different from a study conducted in India that reported the most frequent type of conversion practice was a step-down (45.3%) and a sequential type (18.7%) [% followeIn this study, the mean length of hospital stays was 10.78 ± 6.6 days. This was a longer hospital stay as compared to a study conducted in Lebanon which reported a shorter mean length of hospital stay (6.67 ± 2.74) days . This diRegarding the associated factor of IV to PO conversion practice, the presence of tachypnea is significantly associated with IV medication not being converted to PO medication. This study was in line with a study conducted in the Netherlands . This miThe unavailability of suitable oral medication was significantly associated with IV medication not being converted to PO medications as compared to the availability of the medication. This may be due to the fact that the number of administered antibiotic courses of treatment that were switched to a suitable oral dosage form was very small and the involved antibiotics were mostly macrolides, fluoroquinolones, metronidazole, ceftriaxone, vancomycin, and ceftazidime classes. These antibiotics were converted in a sequential type to oral formulations .Higher temperatures are significantly associated with IV medication not being converted to PO medications compared to patients with normal temperatures. This factor was in line with a study conducted in the Netherlands . PatientThe prospective nature of the method increases the quality of data as it enables us to follow the patients till discharged.It is a single-center study and cannot be generalized to other hospitals. In addition, the cost of medications was not assessed as it may have affected conversion practice.The findings of this study showed that IV to PO conversion practice was frequent. The most frequent conversion practice was sequentially followed by switch conversion. Tachypnea, unavailability of medication, higher temperature, hospital stay of more than than 10.78-days, and comorbidity were all significantly associated with IV to PO conversion practice. Awareness of IV to PO conversion practice and short-term training of healthcare teams is vital for better antibiotic conversion practice.Based on the findings of our study we forwarded the following recommendations:Create awareness about IV to PO conversion practice by preparing for hospital-specific IV to PO conversion.To PO conversion practice guidelines to be included in the curriculum and short-term training and create awareness for healthcare teams at the national level.


Although these two β-amyloid biomarkers may be considered interchangeable, they sometimes show discordance, particularly in early stage of Alzheimer's disease. Individuals with cerebrospinal fluid β-amyloid positive only or β-amyloid positron emission tomography positive only may be at early amyloidosis stage compared to those who are cerebrospinal fluid β-amyloid negative and β-amyloid positron emission tomography negative orcerebrospinal fluid β-amyloid positive and β-amyloid positron emission tomography positive. Besides, β-amyloid pathology may play an initiating role in Alzheimer's disease onset, leading to subsequent tau increases. However, it is still unclear whether individuals with different β-amyloid pathways have distinct spatial patterns of cortical tau tangles in early amyloidosis stage. In this study, we analyzed 238 cognitively unimpaired and 77 mild cognitive impairment individuals with concurrent  18F-flortaucipir tau positron emission tomography, β-amyloid (18F-florbetapir or 18F-florbetaben) positron emission tomography and cerebrospinal fluid β-amyloid42 and β-amyloid40 and cerebrospinal fluid p-Tau181 and divided them into four different cerebrospinal fluid/positron emission tomography groups based on the abnormal status of cerebrospinal fluid β-amyloid42/β-amyloid40  and β-amyloid positron emission tomography (±). We determined the cortical regions with significant tau elevations of different cerebrospinal fluid/positron emission tomography groups and investigated the region-wise and voxel-wise associations of tau positron emission tomography images with cerebrospinal fluid β-amyloid42/β-amyloid40, β-amyloid positron emission tomography and cerebrospinal fluid p-Tau/β-amyloid40 in early  and late  amyloidosis stages. By compared to the cerebrospinal fluid negative/positron emission tomography negative individuals (Ref) without evidence of tau increase measured by cerebrospinal fluid or positron emission tomography, cerebrospinal fluid positive/positron emission tomography negative individuals showed higher tau in entorhinal but not in BraakIII/IV and BraakV/VI, whereas cerebrospinal fluid negative/positron emission tomography positive individuals had significant tau elevations in BraakV/VI but not in entorhinal and BraakIII/IV. In contrast, cerebrospinal fluid positive/positron emission tomography positive individuals showed significant tau increases in all the cortical regions than the Ref group. The voxel-wise analyses provided further evidence that lower cerebrospinal fluid β-amyloid42/β-amyloid40 was associated with higher tau in entorhinal, whilst higher β-amyloid positron emission tomography was related to higher tau in BraakV/VI regions in early amyloidosis stage. Both lower cerebrospinal fluid β-amyloid42/β-amyloid40 and higher β-amyloid positron emission tomography were correlated with tau aggregation in all the Braak stages regions in late amyloidosis stage. These findings provide novel insights into the spatial patterns of cortical tau tangles in different amyloidosis stages of Alzheimer's disease, suggesting cerebrospinal fluid β-amyloid and β-amyloid positron emission tomography discordant groups may have distinct characteristics of cortical tau tangles in early amyloidosis stage.Extracellular β-amyloid plaques and intracellular neurofibrillary tau tangles are the primary hallmarks of Alzheimer's disease. β-Amyloid pathology can be directly quantified by positron emission tomography imaging or indirectly by measuring the decrease of cerebrospinal fluid β-amyloid et al. found that cerebrospinal fluid and positron emission tomography β-amyloid discordant individuals have distinct cortical tau deposition patterns in non-demented elderly adults, indicating different cerebrospinal fluid β-amyloid positive-first and positron emission tomography β-amyloid positive-first individuals may have initial tau tangles in distinct cortical regions in early amyloidosis stage of Alzheimer's disease.Jiang   Nevertheless, either CSF Aβ  or Aβ PET may become abnormal first.22–31 Together our laboratory7 and other group's findings,32 the individuals with CSF Aβ positive only (CSF+/PET−) or Aβ PET positive only (CSF−/PET+) may be at early amyloidosis stage compared to the individuals who are negative (CSF−/PET−) or positive (CSF+/PET+) at both CSF Aβ and Aβ PET. This may represent two different Aβ pathology progressing pathways . Notably, the ‘Aβ pathway’ described in this study represents the different Aβ pathology progressing sequences rather than the biological mechanism of Aβ pathology.Extracellular β-amyloid (Aβ) plaques and intracellular neurofibrillary tau tangles are the primary hallmarks of Alzheimer's disease,34 or longitudinal tau changes.35–37 The BraakI–VI stages38 have been proposed to characterize the spatial patterns of cortical neurofibrillary tau tangles based on the autopsy data. PET imaging studies39–43 provide further evidence that the cortical tau tangles may initially present in entorhinal cortex, following by spreading to the BraakIII/IV and BraakV/VI cortical regions in the presence of substantial Aβ burden. However, it is still unclear whether individuals who are on the Alzheimer's continuum but with different Aβ pathways have distinct spatial patterns of cortical tau aggregation. Exploring the spatial distribution of cortical tau tangles is important for understanding the characteristics of Alzheimer's disease pathophysiology in different stages and may provide novel reference for designing anti-tau clinical trials of Alzheimer's disease.Furthermore, Aβ pathology may play an initiating role in Alzheimer's disease onset, leading to subsequent tau increases42/Aβ40, phosphorylated tau (p-Tau), Aβ PET and tau PET data and divided them into four different CSF/PET amyloidosis stages based on the abnormal status of CSF Aβ42/Aβ40 and Aβ PET. In order to explore the spatial patterns of cortical tau tangles in different amyloidosis stages, we determined the cortical regions with significant tau elevation of different CSF/PET Aβ stages and investigated the region-wise and voxel-wise associations of tau PET images with CSF Aβ42/Aβ40, Aβ PET and CSF p-Tau/Aβ40 in early (CSF+/PET− and CSF−/PET+) and late (CSF+/PET+) amyloidosis stages.In this study, we analyzed non-demented Alzheimer's Disease Neuroimaging Initiative (ADNI) participants who had concurrent (within 1 year) CSF Aβ18F-flortaucipir (FTP) tau PET, amyloid [18F-florbetapir (FBP) or 18F-florbetaben (FBB)] PET, CSF Aβ42 and Aβ40 and CSF p-Tau. We divided these 315 participants into four CSF/PET groups according to Aβ positivity defined by the thresholds of CSF Aβ42/Aβ40 and Aβ PET as described below: CSF−/PET− (concordant Aβ negative), CSF+/PET− (discordant CSF Aβ positive), CSF−/PET+ (discordant Aβ PET positive) and CSF+/PET+ (concordant Aβ positive). In order to control for the influence of non-Alzheimer's related tauopathy, 43 CSF−/PET− participants with either abnormal CSF p-Tau/Aβ4036 or abnormal FTP tau PET  were excluded from the CSF−/PET− group, and the rest of the CSF−/PET− group were defined as the reference (Ref) group.The data were obtained from the ADNI database (ida.loni.usc.edu). The ADNI study was approved by institutional review boards of all participating centres, and written informed consent was obtained from all participants or their authorized representatives. In this study, we identified 238 cognitively unimpaired (CU) and 77 mild cognitive impairment (MCI) ADNI participants with concurrent  http://adni-info.org). As described previously,3 FBP or FBB scans were coregistered to their corresponding baseline structural MRI scans. FreeSurfer  was used to extract cortical Aβ tracer retention in 68 regions of interest (ROIs) defined by Desikan–Killiany atlas,45 and FBP or FBB standardized uptake value ratios (SUVRs) were calculated by referring regional florbetapir or florbetaben to that found in the whole cerebellum. A cortical summary COMPOSITE SUVR was created from a COMPOSITE cortical area, including frontal, cingulate, parietal and temporal regions.46PET data were acquired in 5-min frames from 50 to 70 min (FBP), 90–110 min (FBB) and 75–105 min (FTP) post-injection, and more details are given elsewhere (FBP) – 196.03 and Centiloid = (159.08 × SUVRFBB) – 151.65.The FBB Aβ positivity of the COMPOSITE region was defined as SUVR  ≥  1.08, and FBP Aβ positivity of the COMPOSITE region was defined as SUVR  ≥  1.11. Finally, FBP and FBB SUVRs were converted to Centiloids using the equations Centiloid =  FTP SUVR was calculated by referring to a mean inferior cerebellar grey matter uptake.47 In order to evaluate tau deposition in different Braak neurofibrillary tau stages,38 we also calculated mean FTP SUVRs in BraakIII/IV and BraakIV/VI that correspond to anatomical definitions of Braak stage III/IV  and V/VI .39 The thresholds of entorhinal FTP SUVR and Temporal-meta ROI FTP SUVR were set as ≥1.21 and ≥1.25 respectively according to an ROC analysis using the Youden index classifying 280 Aβ– ADNI CU participants and 183 Aβ+ ADNI MCI and Alzheimer's disease patients as the endpoint as described previously36 and also in FTP tau PET scans of 5 minutes × 4 frames were realigned, averaged and registered to the baseline MRI scan that was closest in time to the baseline FTP scan. FTP uptakes in 68 cortical ROIs defined by Desikan–Killiany atlas47 and smoothed using a Gaussian kernal of 8 mm in SPM12 .For the voxel-wise analyses, FTP PET images were spatially normalized to the MNI space, intensity normalized at the voxel-wise level by a mean inferior cerebellar grey matter uptake40, Aβ42 and p-Tau181 data were analyzed by the University of Pennsylvania ADNI Biomarker core laboratory using the fully automated Roche Elecsys and cobas e 601 immuno-assay analyzer system. CSF data (UPENN-BIOMK10_07_29_19.csv) were downloaded from the ADNI website. Considering that using a CSF p-Tau/Aβ40 ratio may reduce measurement error likely related to individual differences in CSF production rather than pathology, and improve associations with AD biomarkers compared to using CSF p-Tau alone,36 we decided to use CSF p-Tau/Aβ40 ratio to represent CSF tau in this study. The CSF Aβ42/Aβ40 ratio and CSF p-Tau/Aβ40 ratio36 were calculated, and their thresholds were defined as ≤0.054 and ≥0.0012 respectively according to the ROC analysis using the Youden index classifying 181 Aβ- CU participants and 163 Aβ+ cognitively impaired participants (MCI and Alzheimer's disease patients) as the endpoint as described in CSF AβNormality of distributions was tested using the Shapiro–Wilk test and visual inspection of data histograms. Data are presented as median (interquartile range [IQR]) or number and percentage. Different CSF/PET groups were compared using a Mann–Whitney U test for continuous characteristics unless otherwise noted. We assessed categorical differences using Fisher's exact test. A false discovery rate (FDR) of 0.05 using Benjamini–Hochberg approach was employed for multi comparisons correction.III/IV FTP SUVR, BraakV/VI FTP SUVR of different CSF/PET groups with the Ref group.In order to investigate tau elevations of different CSF/PET groups, we used generalized linear model (GLM) to compare FTP SUVRs in 68 FreeSurfer-defined ROIs of CSF+/PET−, CSF−/PET+ and CSF+/PET+ groups with the Ref group (CSF−/PET− without evidence of elevated tau measured by either CSF or PET), controlling for age and sex. We also compared entorhinal FTP SUVR, Braakt-test in SPM12, controlling for age and sex. The voxel-wise comparison between the CSF+/PET− group and the Ref group was presented using an uncorrected voxel threshold of P < 0.001, whilst the other comparisons were presented using an uncorrected voxel threshold of P < 0.001 and with family-wise error (FWE) corrected P < 0.05 at the cluster level.In addition, the voxel-wise FTP PET images of the CSF+/PET−, CSF−/PET+ and CSF+/PET+ groups were compared with the Ref group using two-sample 7 both CSF+/PET− group and CSF−/PET+ group were defined as early amyloidosis stage whilst CSF+/PET+ individuals as late amyloidosis stage of Alzheimer's disease. In order to determine the associations of different Aβ biomarkers as well as CSF p-Tau/Aβ40 with cortical tau deposition of different Braak stages  cortical regions in different amyloidosis stages, we used GLM model to investigate the association of FTP SUVRs in entorhinal, BraakIII/IV and BraakV/VI with CSF Aβ42/Aβ40, Aβ PET and CSF p-Tau/Aβ40 in early (CSF+/PET− and CSF−/PET+ groups and including the CSF−/PET− group as the reference) and late (CSF+/PET+ group and including the CSF−/PET− group as the reference) amyloidosis stages separately, controlling for age and sex. We also investigated the voxel-wise association of FTP PET images with CSF Aβ42/Aβ40, Aβ PET and CSF p-Tau/Aβ40 in early and late amyloidosis stages separately, controlling for age and sex. The voxel-wise association between FTP SUVR images and CSF Aβ42/Aβ40 in early amyloidosis stage was presented using an uncorrected voxel threshold of P < 0.005, whilst the other comparisons were presented using an uncorrected voxel threshold of P < 0.001 and with FWE corrected P < 0.05 at the cluster level.As we described previously,42/Aβ40 and Aβ PET SUVR, we also repeated all the analyses after excluding the borderline participants who were within ±5% of the CSF Aβ42/Aβ40 and Aβ PET (SUVR) thresholds.We noticed that one CSF+/PET− individual and one CSF+/PET+ individual had extremely high entorhinal FTP SUVRs; thus, we repeated all the analyses after removing them from the dataset. In addition, in order to control for the influence of those individuals around the thresholds of CSF AβStatistical analyses were performed in the statistical programme R  unless otherwise noted.https://adni.loni.usc.edu).All data used in the current study were obtained from the ADNI database  was limited. In contrast, the CSF−/PET+ group showed significant tau increases in a few ROIs in BraakIV stage  and most of the ROIs in BraakV/VI stage  than the Ref group. The CSF+/PET+ group had significant tau elevation in all the cortical regions than the Ref group.As shown in P = 0.012) but not in BraakIII/IV and BraakV/VI than the Ref group. In contrast, the CSF−/PET+ group showed higher FTP SUVR in BraakV/VI FTP SUVR  but not in entorhinal and BraakIII/IV than the Ref group. The CSF+/PET+ group had higher FTP SUVR in entorhinal , BraakIII/IV  and BraakV/VI FTP SUVR  than the Ref group.As illustrated in P < 0.001 without cluster correction) than the Ref group, whereas the CSF−/PET+ group showed significant increases of FTP SUVRs in BraakIV stage ROIs  and most of the BraakV/VI stage ROIs  (P < 0.001 with FWE P < 0.05 cluster correction). In addition, the CSF+/PET+ group showed significant (P < 0.001 with FWE cluster correction) elevated tau in most of the cortical regions except for pericalcarine and cuneus.In the voxel-wise analysis, the CSF+/PET− group showed higher tau PET in the left entorhinal cortex and parahippocampal     −0.22 , P = 0.004) but not in BraakIII/IV and BraakV/VI  and BraakV/VI  but not in entorhinal cortex. However, we found positive association between CSF p-Tau/Aβ40 and tau PET in entorhinal, BraakIII/IV and BraakV/VI , BraakIII/IV  and BraakV/VI . Higher Aβ PET was positively related to higher FTP SUVR in entorhinal , BraakIII/IV  and BraakV/VI FTP SUVR . Higher CSF p-Tau/Aβ40 was positively associated with higher FTP SUVR in entorhinal , BraakIII/IV  and BraakV/VI .In late amyloidosis stage, lower CSF Aβ40 in early amyloidosis stage disappeared, whilst the other associations retained  in late amyloidosis stage.In early amyloidosis stage, lower CSF Aβl cortex , higher l cortex , and CSFBANKSSTS . In contis stage , D and F40 in early amyloidosis stage disappeared, whilst the other associations retained  individuals without tau increase in CSF or cortex, we found individuals with abnormal CSF Aβ42/Aβ40 only (CSF+/PET−) showed higher tau in entorhinal but not in BraakIII/IV and BraakV/VI, whereas individuals with abnormal Aβ PET only (CSF−/PET+) had significant tau elevations in BraakV/VI but not in entorhinal and BraakIII/IV. The voxel-wise analyses provided further evidence that lower CSF Aβ42/Aβ40 was associated with higher tau in entorhinal, whilst higher Aβ PET was related to higher tau in BraakV/VI ROIs in early amyloidosis stage (CSF+/PET− and CSF−/PET+). In contrast, individuals with abnormal CSF Aβ42/Aβ40 and abnormal Aβ PET (CSF+/PET+) had significant tau elevations in all the Brank ROIs, and both lower CSF Aβ42/Aβ40 and higher Aβ PET were correlated with higher tau in entorhinal, BraakIII/ IV and BraakV/VI in late amyloidosis stage. These findings provide novel insights into understanding the cortical tau aggregation in different amyloidosis stages of Alzheimer's disease, suggesting CSF Aβ and Aβ PET discordant individuals may have initial tau tangles in distinct cortical regions in early amyloidosis stage of Alzheimer's disease.In this study, we investigated the cortical tau deposition measured by tau PET imaging of different amyloidosis stages defined by CSF Aβ16 we found CSF+/PET− individuals (CSF-first Aβ pathway) and CSF−/PET+ individuals (PET-first Aβ pathway) had similar proportion, and both of them had more Aβ pathology measured by either CSF or PET imaging than the CSF−/PET− individuals, supporting that CSF Aβ and Aβ PET may measure different features of Aβ pathology48 and two distinct Aβ pathways22 exist in early amyloidosis stage of Alzheimer's disease. Furthermore, both CSF+/PET− and CSF−/PET+ individuals had higher CSF p-Tau/Aβ40 ratios than the CSF−/PET− individuals, implying that the early abnormal tau increases in CSF50 are detectable in early amyloidosis stage. Notably, the CSF+/PET− individuals also showed higher CSF p-Tau/Aβ40 ratio than the CSF−/PET+ individuals. Together, these findings suggest that the CSF-first Aβ pathway (CSF−/PET− → CSF+/PET− → CSF+/PET+) may have more soluble Alzheimer's disease pathophysiology, which typically present in early stage of Alzheimer's disease,50 than the PET-first Aβ pathway (CSF−/PET− → CSF−/PET+ → CSF+/PET+).In line with previous studies,50 PET imaging offers spatial information on where tau deposits. Notably, our group36 and other laboratories50 very recently observed evidence that CSF p-Tau may detect early tau increase than the tau PET imaging, which was also supported by postmortem studies.51–53 Importantly, both dichotomous and continuous analyses showed that the CSF-first Aβ pathway had Aβ-related tau increase in entorhinal cortex, which has been regarded as the earliest cortical region of tau aggregation.38–43 In contrast, the PET-first Aβ pathway showed significant Aβ-related tau increase in BraakV/VI but not in entorhinal and BraakIII/IV. In concordance with our findings, one recent important ADNI study32 also found that the CSF+/PET− and CSF−/PET+ individuals have numerically (not significant) higher and lower entorhinal tau measured around 5 year post-baseline CSF Aβ and Aβ PET than the CSF−/PET− individuals respectively, although they used CSF Aβ42 to define CSF Aβ status, which may be less reliable than CSF Aβ42/Aβ40 used in this study. They also found CSF−/PET+ but not CSF+/PET− individuals had significant tau increase in BraakV/VI but not in entorhinal and BraakIII/IV, which was consistent with our findings. Together with our findings and previous study,32 it is probably that individuals with PET-first Aβ pathway may not have tau increases in entorhinal and BraakIII/IV cortical regions due to their lower CSF p-Tau, which plays an important role in tau spreading in cortical regions of early Braak stages .50 The voxel-wise results provide further evidence to support the notion that CSF Aβ42/Aβ40 may be related to tau aggregation in entorhinal cortex whereas cortical Aβ burden correlates with elevated tau in cortical regions of BraakV/VI stage in early amyloidosis stages. Notably, the CSF−/PET+ individuals had smaller CSF p-Tau/Aβ40 ratio than the CSF+/PET− individuals, suggesting we may not be able to use CSF p-Tau biomarker to represent complementary BraakV/VI tau increase in CSF−/PET+ individuals.Whilst CSF measurement of p-Tau provides complementary early tau increase,7 and other laboratory54 previously observed that CSF+/PET− individuals were accumulating cortical Aβ burden with a similar rate to the CSF+/PET+ individuals, and will become CSF+/PET+ in future. Besides, the CSF-first Aβ pathway has been observed more frequent than the PET-first Aβ pathway according to the previous reports.17 In this study, we found similar proportion of CSF and PET Aβ discordant groups, but our previous longitudinal analyses7 also support that CSF Aβ may become abnormal earlier than Aβ PET. Consequently, it is probably that the CSF-first Aβ pathway may represent the typical evolution of Alzheimer's disease which shows Alzheimer's typical tau spreading pattern,38–43 whilst the PET-first Aβ pathway may have cortical Aβ-burden related hippocampal-sparing elevated tau in early amyloidosis stage of Alzheimer's disease. The Temporal-metaROI 44 composite regions have been commonly used to detect Alzheimer's-related tau deposition in human brain.55–60 However, our findings suggested that we may not be able to use Temporal-metaROI regions to capture the cortical tau increase in early amyloidosis stage among these individuals who have a PET-first Aβ pathway, implying different cortical regions should be selected to detect early tau increase in early Alzheimer's disease.Our group44 composite regions. Furthermore, the voxel-wise analyses revealed that lower CSF Aβ42/Aβ40, higher Aβ PET, and larger CSF p-Tau/Aβ40 ratio were related to significant tau elevations in all the cortical regions and with Temporal-metaROI44 composite regions showing the strongest association. These findings suggest that it is reasonable to use Temporal-metaROI regions to detect cortical tau increase in CSF and PET Aβ concordant individuals.In contrast, we found CSF Aβ positive and Aβ PET positive  individuals showed significant tau increases in all the cortical regions than the control group, and the highest tau elevations were found in the Temporal-metaROI42/Aβ40 was defined on the basis of the ADNI database, which requires validation with other databases.This study has several limitations. First, as CSF Aβ and Aβ PET have a very high agreement, the sample sizes of the discordant CSF/PET Aβ groups with concurrent tau PET imaging were relatively small and the results need to be replicated in a larger cohort. To the best of our knowledge, there is currently no larger cohort available with the measurements needed for this analysis. Second, the ADNI participants overall are a highly selected sample, recruited to reflect the exclusionary criteria and types of individuals likely to participate in clinical trials; thus, it would be extremely useful to validate these findings in other aging cohorts. Third, our analyses were limited to cross-sectional PET measured with FTP, which may need to be replicated using longitudinal data and other PET ligands. Forth, the threshold of CSF Aβ61–64 suggest that Alzheimer's disease may have distinct biological features of tau spreading patterns, which are important for explaining the heterogeneity of tau-related neurodegeneration and cognitive decline and the design of anti-tau clinical trials. Our findings are useful for understanding the subtypes of tau spreading patterns in Alzheimer's disease and provide novel reference for cortical tau detection in individuals who are at early amyloidosis stage.In conclusions, we found that CSF and PET Aβ discordant individuals have distinct cortical tau deposition patterns in non-demented elderly adults. Recent studiesfcac084_Supplementary_DataClick here for additional data file.


Biomarkers used for predicting longitudinal cognitive change in Alzheimer’s disease (AD) continuum are still elusive. Tau pathology, neuroinflammation, and neurodegeneration are the leading candidate predictors. We aimed to determine these three aspects of biomarkers in cerebrospinal fluid (CSF) and plasma to predict longitudinal cognition status using Alzheimer’s Disease Neuroimaging Initiative (ADNI) cohort.A total of 430 subjects including, 96 cognitive normal (CN) with amyloid β (Aβ)-negative, 54 CN with Aβ-positive, 195 mild cognitive impairment (MCI) with Aβ-positive, and 85 AD with amyloid-positive (Aβ-positive are identified by CSF Aβ42/Aβ40 < 0.138). Aβ burden was evaluated by CSF and plasma Aβ42/Aβ40 ratio; tau pathology was evaluated by CSF and plasma phosphorylated-tau (p-tau181); microglial activation was measured by CSF soluble TREM2 (sTREM2) and progranulin (PGRN); neurodegeneration was measured by CSF and plasma t-tau and structural magnetic resonance imaging (MRI); cognition was examined annually over the subsequent 8 years using the Alzheimer’s Disease Assessment Scale Cognition 13-item scale (ADAS13) and Mini-Mental State Exam (MMSE). Linear mixed-effects models (LME) were applied to assess the correlation between biomarkers and longitudinal cognition decline, as well as their effect size on the prediction of longitudinal cognitive decline.Baseline CSF Aβ42/Aβ40 ratio was decreased in MCI and AD compared to CN, while CSF p-tau181 and t-tau increased. Baseline CSF sTREM2 and PGRN did not show any differences in MCI and AD compared to CN. Baseline brain volumes  decreased in MCI and AD groups. For the longitudinal study, there were significant interaction effects of CSF p-tau181 × time, plasma p-tau181 × time, CSF sTREM2 × time, and brain volumes × time, indicating CSF, and plasma p-tau181, CSF sTREM2, and brain volumes could predict longitudinal cognition deterioration rate. CSF sTREM2, CSF, and plasma p-tau181 had similar medium prediction effects, while brain volumes showed stronger effects in predicting cognition decline.Our study reported that baseline CSF sTREM2, CSF, and plasma p-tau181, as well as structural MRI, could predict longitudinal cognitive decline in subjects with positive AD pathology. Plasma p-tau181 can be used as a relatively noninvasive reliable biomarker for AD longitudinal cognition decline prediction. Alzheimer’s disease (AD) is the most common neurodegenerative disease characterized by progressive cognitive decline. Amyloid beta (Aβ) cascade and hyperphosphorylated tau (p-tau) hypothesis are considered as the major pathogenic mechanisms of AD .11C-PK11195-PET is extensively used for the neuroinflammation imaging study.Except for Aβ and p-tau aggregation, neuroinflammation and microglial activation have been considered as the third pathological hallmark of AD . NeuroinTREM2 is an AD susceptibility gene that is related to microglial activation. It is determined in genome-wide association studies analysis and confirmed by several subsequent studies . The conHowever, biomarkers used for predicting longitudinal cognitive change in the AD continuum are still elusive. Among all the biomarkers, tau pathology, neuroinflammation , and neurodegeneration are the leading candidate predictors of longitudinal cognitive impairment according to the previously reported studies but still need to be ascertained. Besides, their effect sizes on the prediction of longitudinal cognitive decline are unknown.A better understanding of the biomarkers predicting a decline in AD will help to monitor disease progress and develop reliable prognostic and outcome measures in clinical practice. Here, we evaluated the role of microglial activation, tau pathology, and neurodegeneration in predicting the longitudinal cognitive deterioration in the AD continuum using the baseline assessments of CSF sTREM2, PGRN, p-tau181, t-tau, plasma p-tau, t-tau, and brain volumes.All participants were enrolled in the Alzheimer’s Disease Neuroimaging Initiative (ADNI). In the ADNI database, the classifications of subjects  were based on subjective memory complaints, Mini-Mental State Exam (MMSE) score, Clinical Dementia Rating score, and Wechsler Memory Scale–Revised score. In addition, subjects with MCI should be largely intact in general cognition and functional performance except for memory area and could not meet the diagnosis of dementia, while subjects with AD should meet the NINCDS-ADRDA diagnostic criteria for probable AD . In ADNIUsing these criteria, we finally selected 430 participants in this database, including 96 CN with Aβ-negative, 54 CN with Aβ-positive, 68 EMCI, and 127 LMCI with Aβ-positive, and 85 AD with Aβ-positive. These subjects were divided into four groups, namely, CN group with Aβ-negative (CN A-), CN group with Aβ-positive (CN A+), MCI group (EMCI+LMCI) with Aβ-positive (MCI A+), and AD group with Aβ-positive (AD A+).Cognition was examined annually in the baseline and the following 8 years using MMSE and Alzheimer’s disease Assessment Scale Cognition 13-item scale (ADAS13). As MMSE was a rudimentary assessment of cognition, we also used ADAS13 to evaluate the global cognition of each subject more precisely. Take note that not every subject had a complete record of an 8-year evaluation. The average number of visits and average follow-up years in each group are shown in T1-weighted MP RAGE was acquired for each subject at baseline view (1.5 T scanning for ADNI1 subjects and 3.0 T scanning for ADNI2/GO subjects). A 1.5 T data was run in FreeSurfer version 4.3, and 3.0 T data was run in FreeSurfer version 5.1. Each image was processed through several complicated steps for cortical reconstruction and volumetric segmentation (see “UCSF FreeSurfer Methods”). Then, hippocampal, entorhinal, and middle temporal lobe volume for each subject were calculated and adjusted for intracranial volume (ICV). These data are derived from UCSFFSX51_11_08_19.csv and UCSFFSX_11_02_15.csv.CSF sTREM2 and PGRN concentrations were measured by the ELISA protocol established by the Haass’ group in MSD platform . The datp-values using Holm adjustment. To test the correlation between baseline cognition score and baseline CSF biomarkers or brain volumes in cognitive impairments subjects, we conducted a multivariate linear regression model. For CSF sTREM2, PGRN, and brain volumes, diagnosis , age, gender, years of education, APOE genotype, CSF p-tau181, and CSF Aβ42/Aβ40 ratio were used as covariates. Moreover, for CSF p-tau181 (or Aβ42/Aβ40 ratio), only age, gender, years of education, APOE genotype, and CSF Aβ42/Aβ40 ratio (or CSF p-tau) were used as covariates. Then, we applied an LME regression model to identify whether baseline CSF sTREM2, PGRN, Aβ42/Aβ40 ratio, p-tau181, and brain volumes could predict cognitive decline. Diagnosis , age, gender, education, APOE genotype, CSF p-tau181 × time, and Aβ42/Aβ40 × time were used as covariates with subject-specific intercepts and slopes . All the statistical analyses were performed using R statistical software version 4.0.4. The p-values in LME models were acquired using the R package “lmerTes1”; the regression coefficients β in LME models were acquired using function “HLM_summary” in R package “bruceR”; the d scores in LME models were done using the R package “EMAtools.” A significance criterion was set to be p < 0.05.CSF markers did not comply with normal distribution. Regional brain volume complying with normal distribution but did not show homogeneity of variance. As a result, Kruskal–Wallis test was used to evaluate differences in baseline biomarkers  among CN, MCI, and AD groups. For pairwise comparison, we used the Wilcoxon rank-sum test with adjusted d slopes . The p-vp < 0.001, respectively). We also observed an increased level of p-tau181 in the AD group compared to the MCI group . However, there was no difference in CSF Aβ42/Aβ40 ratio between MCI A+ and AD A+ groups , and CN A+ showed no difference with CN A-, and CSF Aβ42/Aβ40 ratio was not associated with ADAS13 score as well . Subjects with higher CSF p-tau181 did not show higher ADAS13 score  or in the MCI A+ group , but not in the AD A+ group  in the MCI+AD A+ population , whereas in the AD A+ group, we did not reach similar results  and MCI A+ combined AD A+ group . For plasma p-tau181, we confirmed higher plasma p-tau181 concentration could predict faster ADAS13 score change  showed maximal prediction effect size while CSF p-tau181 and sTREM2 exhibited similar effect size. For plasma and CSF comparison, plasma p-tau181 showed a smaller effect size than CSF p-tau181 but still a medium effect. These findings extended the clinical use of CSF sTREM2 and plasma p-tau181.Several studies have confirmed Aβ42/40 ratio was better in AD diagnosis and better concordance with Aβ PET than CSF Aβ42 , 2017, wMicroglial activation is believed to be one of the core pathologies of AD. Some studies demonstrated that CSF sTREM2 exhibited a dynamic change during the AD progression . In thisNext, we assessed the role of neurodegeneration biomarkers using CSF t-tau and brain volumes in predicting cognition decline. Our result showed that brain atrophy could predict cognitive change with a large effect size while CSF t-tau could not. These results coincided with several previous studies. A recent study showed that the cortical thickness and sulcus depth indices were significantly changed during AD progression, suggesting that gray matter characteristics can track AD progression . SimilarFinally, we evaluated the differences between CSF and plasma biomarkers in predicting cognition decline. We discovered plasma Aβ42/Aβ40 ratio and t-tau were not correlated with longitudinal cognitive decline while plasma p-tau181 predicted longitudinal cognitive decline. Recent studies confirmed that plasma Aβ42/Aβ40 ratio was correlated with PET measured brain amyloid deposition. However, due to different environment, plasma Aβ42/Aβ40 ratio was less prominent in predicting amyloidosis compared to CSF Aβ42/Aβ40 ratio and shows a larger overlap between subjects with PET measured amyloid-positive and amyloid-negative . SimilarRare studies have compared the role of CSF and plasma p-tau181 in predicting longitudinal cognitive decline. In this study, we found that plasma p-tau181 had a smaller effect than CSF p-tau181. As plasma p-tau181 was derived from CSF p-tau181, and there is a different environment between peripheral circulation and central nervous system, it is not surprising that plasma and CSF p-tau showed a bit different effect size. Nevertheless, our study supported that plasma p-tau181 can be used as a reliable relatively noninvasive biomarker for longitudinal cognition decline prediction as higher baseline plasma p-tau181 predicted faster longitudinal cognitive decline.There were several limitations in this study. First, most of the AD subjects only had a follow-up visit for 2 years and did not have enough longitudinal ADAS13 and MMSE scores, which might affect the results of the LME model computed in the AD group . Hence, Collectively, our study reported that baseline CSF sTREM2, CSF, and plasma p-tau181, as well as structural MRI, could predict longitudinal cognitive decline in subjects with positive AD pathology. CSF sTREM2, CSF, and plasma p-tau181 had similar medium prediction effect, while brain volumes showed stronger effects in predicting cognition decline. Plasma p-tau181 can be used as a reliable relatively noninvasive biomarker for longitudinal cognition decline prediction.The original contributions presented in this study are included in the article/The studies involving human participants were reviewed and approved by regional ethics committees of all institutions joined in ADNI. The patients/participants provided their written informed consent to participate in this study.Y-HC: analysis of the data and drafting the original manuscript. R-RL, H-FH, and Y-YX: data acquisition and analysis. Q-QT: funding, study design, and critical revision of the manuscript. All authors have contributed to the article, revision of the manuscript, and approved 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Esketamine showed more potency, more rapid recovery from anesthesia, and less psychotomimetic side effects when compared with ketamine. However, the patients still experience psychotomimetic side effects of esketamine. In order to investigate whether midazolam can attenuate the esketamine-induced overactive behaviors and neuronal hyperactivities, midazolam 0, 40, 80, and 120 mg/kg combined with esketamine 50 mg/kg were administrated on Kunming mice to assess the behaviors changes during anesthesia. The indicators, including action time, duration of agitation before the sedation, duration of sedation, duration of loss of pedal withdrawal reaction (PWR), duration of loss of righting reaction (RR), duration of agitation during the recovery, and recovery time, were monitored for up to 3–4 h after intraperitoneal administration. The results demonstrated that midazolam 40, 80, and 120 mg/kg efficiently decreased the esketamine-induced overactive behaviors including ataxia, excitation, and catalepsy before sedation. Midazolam and esketamine synergically improved the anesthesia quality assessed by PWR and RR. However, even high doses of midazolam were not able to suppress the esketamine-induced psychotomimetic effects during the recovery. S)-ketamine enantiomer (esketamine) and (R)-ketamine enantiomer (R)-ketamine and ketamine (Ketamine is an anesthetic of N-methyl-D-aspartic acid receptor (NMDA receptor) antagonist, which can produce unique dissociative anesthesia –3. Ketamantiomer . Esketamketamine , 5. Now,ketamine . Both keketamine . Howeverketamine . Esketamketamine . The neuketamine , 9. The ketamine . Midazolketamine , 11. Eskketamine , 13. HowThis experiment was approved by the ethics committee of the Zhengzhou Central Hospital. All the processes were strictly adhered to the Chinese Animal Welfare Act.The male Kunming mice weighing around 25 g were purchased from the Animal Center of the Medical College of Zhengzhou University. Every 2–3 mice were allocated to a cage, where the ambient temperature was 22 ± 2°C, humidity was maintained at about 40 ± 5%, and sufficient food and water were ensured with wood bedding in accordance with the Chinese guidelines. Before the experiment, each mouse was acclimated to the testing room for 24 h. The mice were randomly divided into the different groups. Adequate oxygen supply was ensured during medication treatment.The doses of esketamine  were 50 mg/kg , and theThe experimental mice were randomly divided into the following groups in which the treatment details were descripted in During the experiment, each mouse was placed separately in a cage and given 100% pure oxygen to observe and record any changes in behaviors before and after the administration of the drug. The behaviors registered were agitation, sedation, pedal withdrawal reaction (PWR), and righting reaction (RR). Agitation was characteristic by involuntary myoclonic twitching, running aimlessly, stiffness, loss of coordination and balance in walking, and inability to keep normal posture. Sedation was characterized by a stage of immobilization, sleep-like posture, but still responsive to stimulation. PWR was recorded when mice attempt to withdraw the limb to toe pinch . The pinAction time was defined as the time from the injection drug to emergence of any signs of ataxia, agitation, or sedation , 17. DurAfter the full recovery from anesthesia, the mice were euthanized by decapitation under anesthetized by chloral hydrate. The mice brains were retrieved for western blot and immunohistochemistry (IHC).2O2 solution for 10 min at room temperature. The primary antibody c-Fos  was incubated with the sections over night at 4°C. After washing, the biotinylated secondary antibody and streptavidin-HRP were sequentially incubated with the sections. After washing, the sections were stained using DAB substrate solution . The sections were counterstained by hematoxylin. Then, the sections were dehydrated and cleared before mounting coverslips. Finally, sections were visualized under a microscope at × 400 or × 100 .Mice brains were put into 4% paraformaldehyde for at least 24 h at 4°C. The brains were embedded in paraffin after dehydration and clearance. The paraffin-embedded tissue blocks were cut into sections at 4 μm thickness on a microtome. Sections on glass slides were deparaffinized and rehydrated before immunohistochemistry. Sections were boiled in citrate buffer (pH 6.0) for 20 min for antigen retrieval. The endogenous peroxidase was blocked by incubating in 3% HThe retrieved mouse brains were stored at −80°C and the proteins were extracted using the RIPA buffer . The concentration of the extracted protein was determined using the BCA protein assay kit . The same amounts of protein were run on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were incubated in 5% non-fat milk for 1 h at room temperature. The membranes and the primary antibodies c-Fos  were incubated together and overnight at 4°C, and then incubated with an HRR-conjugated secondary antibody . The GADPH protein  serves as the internal reference protein. The proteins expression was analyzed using an ESL scanner with Omni-ECL Enhanced Pico Light Chemiluminescence kit , and signal intensities of the protein expression were quantified using Chemidoc EQ system .∧2 (Z∧(α/2) + Z∧β)∧2/d∧2  procedure of holm, and then by the false discovery rate (FDR) procedure of Benjamini-Hochberg. Western blot results were presented as mean ± SD. ANOVA was used to analyze the c-Fos expression differences. P < 0.05 was considered as significant difference.The sample size was calculated according to duration of agitation before the sedation which was attained from our previous paper . The samβ)∧2/d∧2 . Some grAfter injection of esketamine, the mice gradually appeared ataxia including loss of coordination in walking, imbalance, inability to keep posture. Subsequently, the mice displayed agitation, some of them catalepsy, which demonstrated immobilization associated with intensive muscle tone all over body, like arched-back rigidity. After excitation and catalepsy, the mice returned to ataxia again and regained coordination and balance gradually.The median of action time was about 1.5 min for esketamine. There is a significant difference between Mida120 and Esk groups. Esk+Mida80 and Esk+Mida120 groups had a shorter action time than Esk groups. However, these differences were not clinically significant because all action times were within 2 min , Table 2The agitation before sedation was found in esketamine and Esk+Mida groups. The durations of agitation before sedation in all Esk+Mida groups were shorter than that of Esk group. There were no differences in the duration of agitation before the sedation among Esk+Mida groups. The duration of agitation before the sedation was transient and lasted <3 min in all Esk+Mida groups , Table 2There was almost no sedation in esketamine group. Midazolam dose-dependently increased duration of sedation when used alone or with esketamine , Table 2There were no differences in duration of loss of RR between esketamine and midazolam groups. However, the durations of loss of RR were extended significantly when combination of esketamine and midazolam were administrated. Esk+Mida40 group showed a shorter duration of loss of RR than Esk+Mida80 or Esk+Mida120 groups. There were no differences in duration of loss of RR between Esk+Mida80 and Esk+Mida120 groups , Table 2Esk, Mida40, and Mida80 did not have significant effects on PWR. Only Mida120 could extend PWR a little longer than Mida40 or Mida80 groups. However, the durations of loss of PWR were extended significantly when combination of esketamine and midazolam were administrated. Esk+Mida40 group showed a shorter duration of loss of PWR than Esk+Mida80 or Esk+Mida120 groups. Esk+Mida80 group had a shorter duration of loss of RR than Esk+Mida120 group , Table 2The agitation during the recovery was found only in combination of esketamine and midazolam. There is no agitation during recovery in Esk group because esketamine did not induce any sedation , Table 2The median of recovery time of esketamine was 30 min which was less than all midazolam and Esk+Mida groups. The recovery time of Mida120 group was longer than that of Mida40 and Mida80 groups. There were no differences in recovery time between Mida40 and Mida80 groups. The Esk+Mida groups had a longer recovery time than the corresponding Mida groups. Midazolam dose-dependently increased recovery times in Esk+Mida groups , Table 2Because anesthesia stage was defined as the duration of loss of both PWR and RR in this study, anesthesia stage could be identified as the lower duration from the duration of loss of PWR and RR. Therefore, the results regarding anesthesia stage are available in duration of loss PWR and RR.Esketamine group had more c-Fos-positive neurons in a high-power field under microscope than saline or midazolam groups in the mice brain, including cerebral cortex, hippocampus, thalamus, amygdala, and hypothalamus. However, All Mida +Esk groups demonstrated the similar numbers of c-Fos-positive neurons as the esketamine group .Midazolam at 40, 80, and 120 mg/kg had no effects on esketamine-induced c-Fos expression , 7.In this study, we found that midazolam decreased esketamine-induced agitation before the sedation. However, midazolam had no effects on the agitation during the recovery. Midazolam dose-dependently extended sedation and recovery time not only alone but also combined with esketamine. Combination of esketamine and midazolam synergically enhanced each other by increasing duration of loss of RR and PWR to get an anesthesia state which were not reached by either esketamine or midazolam alone.Psychoactive effects were the main concern for esketamine or ketamine in anesthesia . BecauseThe aim of this study was to determine if midazolam could inhibit the central nervous excitation of esketamine through behaviors observation. One of these behaviors was agitation, characterized by ataxia, aimless running, myoclonic twitching, catalepsy, and overactive behaviors. Because these behaviors changed too fast to record, agitation was used to include all these overactive behaviors. We found that the agitations before sedation were attenuated efficiently by midazolam in mice. It is consistent with our hypothesis that midazolam can suppress esketamine-induced overactive behaviors as dexmedetomidine .To our surprise, midazolam could not decrease the agitation during the recovery. At first, we expected that the agitation can disappear when the dose of midazolam was increased. However, high doses of midazolam still had no effects on the duration of agitation during the recovery. There were no significant differences in the duration of recovery agitation among 40, 8, and 120 mg/kg midazolam with esketamine. The duration of recovery agitation among these groups ranged from 20 to 75 min. Comparing to the esketamine-induced agitation in Esk group, the duration of the recovery agitation was longer (10–20 vs. 20–75 min) in Esk+Mida groups, but the intensity of agitation was milder according to the subjective observation. It was reported that mice would experience ataxia during the recovery from anesthesia of s-ketamine and medetomidine . This longer elimination time may account for the recovery agitation after a long sedation of high dose of midazolam in this study (Why did midazolam have no effects on the recovery agitation in combination with esketamine? The obvious reason may be that midazolam has a shorter effect than esketamine. However, midazolam 120 mg/kg extended the duration of sedation by 3 times that of 40 mg/kg midazolam, the recovery agitation still started after 2–3 h delay and lasted for the similar range of duration. The possible reason may be that the recovery agitations come from the metabolite of esketamine –29. Ketais study , 23, 33.There are several limitations in this study including small sample size, lots of subjective data, and non-specific indicator for neuronal activity. First, the sample size was just set to detect the difference in duration of agitation before the sedation. So, this study may have no enough power to find any other difference. Second, all the behaviors data were attained through observation, which is easy to be affected by observer's subjectivity. Third, c-Fos was used to reflect neuronal activity in this study. However, c-Fos expression is very sensitive to any stimulation and is not specific to reflect neuronal activity induced by esketamine. Last, only male mice were chosen in this study. The findings will not be generalized to both male and female when combination of midazolam and esketamine was applied in anesthesia.Midazolam 40, 80, and 120 mg/kg efficiently decreased the esketamine-induced overactive behaviors including ataxia, excitation, and catalepsy before sedation. Midazolam and esketamine synergically improved the anesthesia quality assessed by PWR and RR. However, even high doses of midazolam were not able to suppress the excitatory effects of esketamine during the recovery. It suggested that it is not an ideal strategy to administrate one dose of midazolam to minimize esketamine-induced psychoactive effect in anesthesia. A further study was warranted to set up a continuous or multiple administration method to combine midazolam with esketamine in animal anesthesia.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by the Ethics Committee of Zhengzhou Central Hospital. All processes were strictly adhered to the Chinese Animal Welfare Act.QC prepared and wrote the manuscript, performed data analysis, and contributed to manuscript review and preparation. MM collected data, prepared tables and figures, and contributed to manuscript review and preparation. YB aided in the study design, collected and analyzed data, and contributed to manuscript writing and review. LS prepared and performed IHC experiment. DZ contributed to the experiment of western blot. PZ contributed to article writing and data analysis. XJ designed the study, performed data analysis, contributed to manuscript writing and review, and supervised all aspects of the study. All authors read and approved 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Heart transplant surgery is considered the destination therapy for end-stage heart disease. Unfortunately, many patients in the United States of America who are eligible candidates for transplants cannot undergo surgery due to donor shortage. In addition, some donors' hearts are being labeled as unacceptable for transplant surgery because of the rigorous and restricted rules placed on the approval process of using a donor's heart. Over the last few decades, the rising discrepancy between the scarcity of donor hearts and the demand for such organs has led to the discussion of expanding the donor heart selection criteria. A softer view on using marginal hearts for transplants would help those on the waitlist to receive a heart transplant. Marginal hearts that contain the hepatitis c virus (HCV), COVID-19, older age, or repairable heart defects have become viable options to use for a heart transplant. Also, the prioritization based on the new heart allocation system would help efficiently decide which recipients would be the first to get a donor's heart. Recently there has been a consensus to broaden the eligibility of donor's hearts by accepting valvular abnormalities, coronary artery disease, and congenital abnormalities. This review highlights some of those expansions in selection criteria in particular using repairable hearts, which could be fixed in the operating room on the back table before transplantation. In the United States of America, less than 50% of the potential organ donors do, in fact, become actual donors. The field of medicine has evolved as better medicine and technology have become available . With thThe process of getting a transplant candidate approved for receiving a heart donation is exhaustive. The transplant centers begin the process by placing the patient on the transplant waitlist. Once placed on the waitlist, the potential heart donor matching process starts by identifying and surveying different candidates. The survey lists information about the donor, such as confirmation of brain death, consent for donation, blood type, etc. When a donor's heart has been matched, a transplant surgeon is sent to the location of the donor's heart for final inspection and approval to be used for transplant. Finally, once the donor's heart has been accepted, it is brought to the recipient facility to be implanted .Since the list of the waitlist has been growing at a rapid rate, there has been greater pressure of expanding the selection criteria. With the scarcity of donor hearts available, there has been an urgency to use marginal hearts. Marginal hearts have problems that would not generally be acceptable under current United Network for Organ Sharing (UNOS) guidelines . With grRepairable heartsFor a long time, a donor heart that had the valvular disease or needs valve surgery was disqualified for a heart transplant. Any donor heart with echocardiographic evidence of valvular disease abnormality is normally considered a contraindication to donation in the United States . RecentlHeart failure is a major global health issue, affecting approximately 5.8 million people in the United States and over 23 million people worldwide. Heart failure is a leading cause of hospitalizations among individuals aged 65 and older, with a mortality rate of over 10% every year. As a result, heart failure consumes a significant amount of healthcare resources, putting immense pressure on the healthcare system -13. HaviA repairable heart transplant case by Sultan et al. reports on a 53-year-old male who had ischemic cardiomyopathy. The recipient received a heart of a 37-year-old donor who had a bicuspid valve with severe aortic insufficiency. A concomitant aortic valve replacement was performed on the donor's heart. The recipient is doing well at the six-month and one-year follow-ups, and CT scans showed no aortic dilation . As the In our institution, we have used hearts with congenital anomalies, such as persistent total left superior vena cava (LSVC) The heart was repaired on the back table and transplanted successfully Figures , 2B. BefEven though using a repairable can be a great option for expanding the donor selection pool, there are a few limitations. When using a repairable heart for a heart transplant, the repairable heart must first be operated on before placing inside the recipient. Repairing the donor's heart will lead to a longer ischemic time of the donor's heart and an increase in the primary graft dysfunction, which will increase the overall operative time -23. AlsoHepatitis C virus Two decades back, hepatitis C virus (HCV) infection was considered highly fatal due to its association with hepatocellular carcinoma and lack of any effective treatment . The troAgeAnother way to broaden the donor pool is by accepting the hearts of older patients. Even though there is no age limit to be a candidate for a heart organ donation, it is generally less desirable to receive a donor heart above the age of 50 years . Donor hPrioritizationRecently, a new heart allocation policy was enacted with the purpose of giving new hearts to patients who are the sickest in order to decrease the amount of time on the waitlist . The preOpioid overdose deathDue to the worsening of the opioid epidemic, there has been a rise in opioid overdose death donors (OODDs). Sadly, most of the patients who contribute to the OODDs are younger than the age of 45 years and greatly contribute to the donor pool . It is wCOVID-19Heart organ donation and transplantation were significantly affected in the United States due to the COVID-19 pandemic. Recipients on the waitlist had an increased risk of acquiring COVID‐19 . From MaDue to the relative lack of supply of donor hearts and the growing number of patients on the waiting list, cardiologists and surgeons have become more enlightened in their donor heart selection. There is substantial evidence that using marginal donors produces satisfactory results, making it justifiable to address the growing donor organ shortage. With the increasing need for heart transplant surgery, especially for those who are older and have severe medical conditions, recipients may benefit from donors with marginal hearts who have been properly examined. The purpose of expanding the selection criteria is to broaden the range of patients' hearts that can be used successfully and increase the number of hearts in the donor pool. With the continuous advancement in medicine, ways to combat the need for a heart transplant and the shortage of heart donors will continue to be created. Further studies are needed to discuss the possible risk factors using the methods discussed to expand the selection criteria.