This term emphasizes the borderline conditions for neurons placed within such a critical region. Area penumbra separates the ischemic core, where frank cell loss occurs, from the surrounding healthy brain tissue. Within such a brain region, nervous matter, and mostly neurons are impaired concerning metabolic conditions. The classic biochemical marker, which reliably marks area penumbra is the over-expression of the heat shock protein 70 (HSP70). However, other proteins related to cell clearing pathways are modified within area penumbra. Among these, autophagy proteins like LC3 increase in a way, which recapitulates Hsp70. In contrast, components, such as P20S, markedly decrease. Despite apparent discrepancies, the present study indicates remarkable overlapping between LC3 and P20S redistribution within area penumbra. In fact, the amount of both proteins is markedly reduced within vacuoles. Specifically, a massive loss of LC3 + P20S immuno-positive vacuoles (autophagoproteasomes) is reported here. This represents the most relevant sub-cellular alteration here described in cell clearing pathways within area penumbra. The functional significance of these findings remains to be determined and it will take a novel experimental stream to decipher the fine-tuning of such a phenomenon.The peri-infarct region, which surrounds the irreversible ischemic stroke area is named ischemic  This term provides an operational definition, which emphasizes the borderline conditions for neurons placed within such a critical region. Area penumbra is shaped like a belt, which separates the ischemic core, where frank cell loss occurs, from the surrounding healthy brain tissue. Within such a brain region, nervous matter, and mostly neurons are impaired concerning metabolic conditions. In this peri-infarct area, maturation phenomena take place early, which may lead either to delayed cell death or, conversely, to reversed metabolic impairment and restored neuronal function. For such a reason, the fine analysis of sub-cellular organelles and biochemical pathways, which operate within area penumbra is key to understanding the biology of ischemia-induced brain damage. In turn, this is key to planning beneficial therapeutic interventions to obtain a morpho-functional rescue of such a brain area. Depending on various concomitant phenomena, opposite outcomes may occur in this area, consisting either in a reduction or magnification of the infarct volume [penumbra are analyzed in an effort to plan beneficial post-ischemic treatments to improve the amount of rescued brain tissue and the clinical outcome of ischemic events.The peri-infarct region, which surrounds the irreversible neuropathology following an ischemic stroke is named ischemic t volume . Thus, sArea penumbra is originally identified by using magnetic resonance imaging (MRI) [penumbra) region, being the ischemic core frankly dark, while the surrounding healthy tissue appearing as intensely white. This corresponds to the etymological definition of the word “penumbra”  as it appears in the peri-infarct region surrounding the infarct (shaded) area. The optimal planning of a beneficial modulation of area penumbra requires an in-depth knowledge of those biochemical pathways, which operate at this level. The classic biochemical marker, which reliably overlaps with MRI-defined “area penumbra” is the over-expression of a chaperone, heat shock protein 70 (HSP70) [area penumbra. Among these, proteins belonging to the autophagy machinery were probed [area penumbra although their significance remains controversial [ng (MRI)  using T2 HSP70) . Howevere probed ,5. In faoversial ,5,6,7,8.0 . Howevarea penumbra also features an altered expression of proteasome-related proteins [area penumbra and no evidence is available concerning the concomitant expression of LC3 and P20S within area penumbra when referring to intracellular compartmentalization within neurons. This is key especially when considering the role played by these proteins in neuronal survival, and considering recent data showing co-localization of LC3 and P20S within the same vacuoles [area penumbra is carried out. This allows us to measure the co-localization of autophagy- and proteasome-related markers, as well as the occurrence of the autophagoproteasomes within area penumbra, compared with control tissue.In keeping with cell clearing systems, proteins . In factproteins  or vice-proteins . This isproteins . For insvacuoles ,14,15. Tvacuoles ,17,18. Ivacuoles , which svacuoles . This suWhen permanently occluding the distal, superficial aspect of MCA , the outThis is carried out to show the model of permanent focal ischemia; the site of middle cerebral artery (MCA) occlusion corresponds to the centre of the red circle. This corresponds to the superficial course of the cortical extent of MCA after all perforant branches were sent off.area penumbra, as shown in × 1.3 mm = 1.3 mm2 area placed at the dorsal and ventral border of the ischemic region. In this way, the further staining of each semithin section was already placed with area penumbra and the focus provided by the visualization of the semithin section allowed to further dissect an ultrathin area from area penumbra to be counted at electron microscopy. This is further evident in area penumbra as shown in area penumbra were compared with homologous regions from sham mice and homologous regions in the contralateral (non-ischemic) hemisphere. These multiple comparisons were carried out to avoid bias potentially produced by paracrine or systemic alterations triggered by the ischemic side. In fact, the expression of LC3 and P20S may depend on both focal and non-focal, even systemic, determinants. This may be triggered by a number of mechanisms, which would be relevant on the hemisphere contralateral to the ischemic region.The counts within area penumbra from the right ischemic  hemisphere. Homologous regions are isolated from the left non-ischemic  hemisphere. Slices were stained with thionin. When applying electron microscopy, additional control tissue was obtained from homologous regions from both hemispheres from sham-operated mice.The term Dorsal-peri and Ventral-peri refer to the dorsal and ventral peri-infarct regions (respectively), both corresponding to area penumbra, HSP70 marks the area to be stained for LC3 and P20S .As shown in representative pictures of and P20S . In detaand P20S . The staand P20S , along warea penumbra, while the amount of P20S is dramatically reduced compared with the homologous region from a sham mouse  is concomitant with an increase in the other (LC3). Thus, a further decrease in LC3*P20S immunostaining cannot be the consequence of their stochastic combination. Therefore, within the ischemic HSP70-positive area penumbra something else occurs, which leads to a specific loss of autophagoproteasome (merging of LC3 with P20S). This represents a major issue of the manuscript, and it is specifically investigated in the second part of the present study by ultrastructural morphometry and quantitative stoichiometry. Data obtained at light microscopy already indicate that the suppression of merging between LC3 and P20S cannot be considered to depend solely on a decrease of P20S, since the concomitant increase in LC3 should have mitigated the loss of merging rather than worsening the merging of LC3 and P20S. The outcome of this first piece of investigation leads to some ad interim conclusions: (i) within cells from area penumbra there is an increase in LC3 which is concomitant with a decrease in P20S; (ii) within cells from area penumbra the increase in LC3 is reminiscent of the strip-like area stained by HSP70 and the same is true for the decrease in P20S, which suggests two additional markers to stain area penumbra; (iii) within area penumbra there is a suppression in the merging between P20S and LC3, which surpasses the decreases in P20S and it remains unexpected when considering the concomitant increase in LC3.The amount of immunofluorescence for LC3 and P20S is reported in representative am mouse . When coam mouse , LC3-poss region . The incnificant B. In str ventral C compare ventral D. In the ventral E and dor ventral F regionsarea penumbra, there is a marked increase in LC3 (area penumbra). These findings were further confirmed by using ultrastructural stoichiometry.The amount of LC3 and P20S reported following immunofluorescence was confirmed by Western Blotting . This pre in LC3 A with a e in LC3 B. This iarea penumbra and within the ventral sham region or within the contralateral homologous area. These three figures reporting representative images indicate the placement of both LC3 and P20S particles within multi-membrane gold standard autophagy vacuoles owing a cytosol-like electron-density as per Autophagy Guidelines [The occurrence of LC3 , P20S F, and theidelines . The quaarea penumbra from sham-operated mice. In this way, systemic effects on these proteins induced by focal ischemia could be disclosed.When performing electron microscopy, we added as a control the contralateral side, in order to provide a quantification of those potential changes occurring within the non-ischemic side of mice undergoing the mono-lateral (right) irreversible occlusion of MCA. In this way, graphs for quantitative stoichiometry report the ischemic penumbra (Ipsi/Ischemia) along with the homologous region from the contralateral (left) side (Contra/Ischemia). Additionally, homologous regions in the right hemisphere of sham mice (Sham) were included. Such an add on was provided to assess potential changes induced in the whole brain by ischemia. In fact, the fine quantitative assessment provided by ultrastructural stoichiometry allows us to detect changes that are slighter to be assessed by light microscopy. The amount of immunofluorescence indicates a rough variation in the amount of a protein, but it does not tell the real number of the antigen molecules, which instead are detected by immuno-gold-based stoichiometry. Thus, by profiting off single-molecule detection provided by immuno-gold, we compared the contralateral side of ischemic mice with regions homologous to The combination of quantitative stoichiometry with plain ultrastructure also allows us to detect the number of vacuoles within various brain regions and the number of proteins within vacuoles compared with cytosol.area penumbra, respectively. The increase is similar in both regions when measured as the amount of whole cytosol LC3 immuno-gold particles  compared with what was measured at light microscopy, where the number of P20S-positive cells was reduced three-fold compared with controls. The stoichiometric counts of P20S were relevant. In fact, when the number of P20S-positive vacuoles  or slightly decreased (P20S). The striking effect concerns the vacuole compartment where a similar massive decrease was measured for both antigens (LC3 and P20S) within area penumbra compared with control tissue.Thus, by profiting off the stoichiometric count of LC3 and P20S within specific compartments, it was possible to demonstrate that indeed, the discrepancy between LC3 and P20S as measured at light microscopy within area penumbra, which is the concomitant decrease in the amount of both LC3 and P20S from vacuoles. This is independent of the net amount of either LC3 (moderately increased) or P20S (slightly decreased) in the cytosol since the massive decrease in the amount of both antigens from vacuoles prevails at large over the effects measured in the cytosol. It is likely that concomitant measurement of both molecules which are decreased from vacuoles within the peri-infarct region (area penumbra) may explain why in this case we measured a difference between homologous areas contralateral to ischemia compared with sham-operated mice. The compartmentalization of both autophagy and proteasome markers within area penumbra was confirmed by quantitative ultrastructural morphometry in mice sacrificed at 6 h or 72 h following ischemia  at light microscopy showing redistribution of LC3 within area penumbra neurons [area penumbra by studying LC3 compartmentalization by quantitative ultrastructural morphometry. In fact, here we measure a decrease in the amount of LC3 within autophagy vacuoles which is redistributed outside the vacuoles towards cytosolic compartments.In the present study, we provide light and electron microscopy evidence showing that LC3 increases in the whole cells of  neurons . This geThis may be due either to a reduction in the entry of LC3 within the vacuoles or it may be caused by a loss of LC3 from previously filled vacuoles. The additional time intervals  indicate that even a few hours after the ischemia, the same decrease in the amount of LC3 is measured and this persists at 3 days. Again, such a decreased amount may be the consequence of a potentiated degradation of LC3 within vacuoles. Such a hypothesis remains to be investigated. Nonetheless, one should consider that a similar loss within autophagosomes is measured for P20S, which markedly reduces the amount of autophagoproteasomes and rules out a proteasomal degradation of LC3. Again, when another autophagy marker such as Beclin1 was measured, its amount was decreased as well in the vacuoles despite an increase in the cytosol just like LC3. Similarly, a decrease in the combined concentration of Beclin1 + P20S within vacuoles was measured in the present study.area penumbra, and redistribution of LC3 in the cytosol compared with vacuoles does occur.Apart from these hypotheses, it remains solid evidence of the present study that, the ratio between the amount of LC3 within vacuoles and cytosol decreases in area penumbra is also characterized by altered expression of proteasome-related proteins [In keeping with cell clearing systems, proteins . In factproteins  or vice-proteins . This isproteins .area penumbra and no evidence is available concerning the concomitant expression of LC3 and P20S within area penumbra when referring to intracellular compartmentalization within neurons. This is key, especially when considering the role played by these proteins in neuron survival and in the light of recent data showing co-localization of LC3 and P20S within the same vacuoles [area penumbra, which mimics what is described following the staining with HSP70. Concomitantly, when the occurrence of P20S is analyzed, area penumbra is characterized by a loss of this proteasome antigen. To our knowledge, this is the first evidence showing the sub-cellular compartmentalization of LC3 within area penumbra and the first study, which report the occurrence of P20S within area penumbra so far. Compared with HSP70 and LC3, the trend of P20S expression which marks area penumbra is opposite, i.e., P20S is suppressed within this peri-infarct area. Since LC3 and P20S were recently described to co-localize within the same vacuoles named autophagoproteasome [area penumbra.No study so far had investigated the compartmentalization of autophagy and proteasome markers within vacuoles ,14,15. Toteasome ,14,15, warea penumbra, neurons autophagy like vacuoles are similar to control tissue, however, LC3 is decreased within vacuoles. Such a cytosolic redistribution is likely to produce fluorescent puncta, which are due to cytosolic LC3 aggregates. This could also be due to a loss of antigenicity of LC3 within vacuoles. In any case, the autophagy vacuoles are supposed to become defective despite the net amount of LC3 in the cell is increased since they do not store LC3 anymore or LC3 is altered. Similar to LC3, the proteasome component P20S was shown to decrease markedly within vacuoles from area penumbra neurons. In contrast to LC3, P20S also decreases in the whole cell from area penumbra. Indeed, the decrease of P20S within the whole cell is slight (20%) when compared to its massive decrease from vacuoles (300%). Thus, within area penumbra cells, the amount of P20S is reduced from vacuoles similarly to LC3. Similarly, the amount of LC3 is greatly reduced from autophagy vacuoles to an amount, which is in excess compared with its increase within the cytosol. In this way, both LC3 and P20S may no longer be committed to produce their specific cell clearance and protect neurons from ischemia-induced damage.Within area penumbra is also slightly detected within the contralateral (left) homologous area of mice undergoing mono-lateral (right) MCA occlusion. This might depend on the powerful stressful conditions, which are produced in the course of a stroke, which is supposed to alter the activity of reticular nuclei, which project directly to the cortex. Recent studies indicate that a marked LC3 redistribution is produced by the neurotransmitter norepinephrine [The loss of autophagoproteasomes, which is severe within nephrine , which iarea penumbra. In keeping with this, when the ultrastructural morphometry of the autophagoproteasome is measured, the suppression of such an organelle appears to be the most impressive within area penumbra. In these neurons, the amount of total vacuoles stay steady, while the decrease in total proteasome is slight, and the increase in LC3 is evident. All these changes are slighter compared with the massive decrease in autophagoproteasome, which is documented in the present manuscript. The functional significance of these findings deserves an appropriate extensive and dedicated experimental design. Nonetheless, the functional significance of an increased expression of autophagoproteasome is evident following mTOR inhibition exerted by rapamycin [area penumbra, a deficiency of autophagoproteasome may be rescued by mTOR inhibitors to improve the outcomes of neurons within area penumbra by restoring an effective cell clearance.The evidence recently published  about enapamycin . In thesapamycin ,14,15. Iarea penumbra showing a decreased amount of these proteins from their natural (LC3) or putative (P20S) sites (vacuoles), where they are needed to properly exert cell clearing function. The functional significance of these ultrastructural changes remains to be determined and it will take a novel experimental stream to decipher the fine-tuning of this phenomenon.The present study is the first report to describe the distribution of LC3 and P20S within n = 38 Adult C57BL/6 male mice  weighing 25 g, were housed under controlled conditions  on a 12 h light-dark cycle with food and water ad libitum. The experimental protocol was approved by the Ethical Committee of Neuromed Institute  further supervised by The Italian Ministry of Health (Authorization number 1194/2020-PR). The animals were anaesthetized with chloral hydrate . With the aid of an operating stereomicroscope, an incision was made between the outer canthus of the eye and the external auditory meatus. The temporal muscle was bisected and retracted to expose the temporal  surface of the skull. The MCA was exposed in its lateral superficial course through a small drill-operated craniotomy (0.5 mm2). When drilling, the deepest bone layer was preserved to avoid drill penetration through the dura mater and cortical mechanical and thermal damage. In fact, such a layer was carefully removed manually to reach out to the distal middle cerebral artery (MCA), which was occluded by using electrocoagulation. The arrest of blood flow in the distal segment of MCA was visualized by a stereo-microscope. The site of occlusion corresponds to the centre of the red circle pointed by the red arrow of A total of Brains were dissected out and immediately placed into a Carnoy fixing solution composed of ethyl alcohol (60%), acetic acid (10%), and chloroform (30%) for histochemistry and immuno-histochemistry. Twenty-four hours later, brains were placed into 70% ethanol to be wax-embedded (paraffin). The brains were cut at microtome  into 10 μm-thick coronal slices, which were regularly spaced 550 µm through the whole rostro-caudal extent of the ischemic region. Sections were used for histological and immuno-histochemical analysis.Sections were de-waxed and processed for staining with thionin (a kind of Nissl staining) to assess the localization of stroke-induced brain damage. Further morphological analysis was carried out on 10 μm thick slices, which were regularly spaced every 550 µm, through the extension of the ischemic region. This space interval corresponds to a standardized procedure, which allows us to measure ischemic brain volume based on 10 slices. The infarct area in each stained section appears as reported in Brain slices were used for LC3, P20S, and gold standard HSP70 immune-staining. Slices were treated with normal sera for 1 h (10% in TBS).Double immunofluorescence for LC3 and P20S was performed by incubating mouse brain sections overnight with polyclonal rabbit anti LC3B , polyclonal mouse anti-P20S , and then for 1 h with secondary CY3-coupled anti-rabbit or Fluorescein anti-mouse IgG  . ControlHSP70 or Caspase 9 active (cleaved) immunoperoxidase was performed by incubating mouse brain sections with normal serum for 1 h (10% TBS). Sections were incubated overnight with mouse anti-HSP70 antibody  or rabbit anti-Caspase 9 antibody , and then for 1 h with a secondary biotinylated-anti mouse. Control staining was performed without a primary antibody.Brain tissues were homogenized at 4 °C in ice-cold lysis buffer with phosphatase and protease inhibitor. One microliter of homogenates was used for protein determinations (Bradford procedure). Proteins (20 µg) were separated on sodium dodecyl sulphate gels (polyacrylamide PVDF precast gel 4–20% gradients) and transferred on transblot-turbo  for 7 min mixed molecular weight. Filters were blocked 2 h in Tween-20 Tris-buffered saline (TTBS)  containing 5% non-fat dry milk. Blots were incubated overnight at 4 °C with the following primary antibodies: rabbit polyclonal anti HSP70 , mouse monoclonal anti LC3B , mouse monoclonal anti P20S . For the housekeeping, we have used beta actin. Blots were incubated with primary mouse monoclonal anti beta actin antibody  for 1 h at 22 °C. The filter was washed 3 times with TTBS buffer and then incubated for 1 h with secondary peroxidase-coupled antibodies . Immunostaining was revealed by enhanced chemiluminescence luminosity . Densitometric analysis was performed with IMAGEJ software. Data were expressed as the mean ± S.E.M.n = 6) and from the contralateral side (n = 6) as schematically marked within representative area penumbra as a dorsal and ventral area penumbra of the side ipsilaterally to ischemia and a similar number of regions in the contralateral non-ischemic side. Therefore, the number of area penumbra corresponds to 2  ipsilateral for each mouse (n = 6) undergoing ischemia for a total of 6 × 2 = 12 area penumbra  and the peri-infarct area (area penumbra). This allows a qualitative ultrastructural comparison between area penumbra and the core of the ischemic region. Twenty-four hours following ischemia, blocks from the cerebral cortex (1 mm × 1.3 mm × 1 mm) were dissected in the ipsilateral hemi-encephalon of ischemic mice  are reported as red-lined squares in 4) for 1 h at 4 °C. Then, the blocks were dehydrated in a serial gradient of ethanol solutions , and finally, they were embedded in epoxy resin. The concentration of the fixing and post-fixing solutions, along with the use of the epoxy embedding resin, were validated in previous studies performing immuno-gold-based ultrastructural morphometry [4, and epoxy resin allows a minimal epitope covering while preserving sub-cellular architecture [In addition, two other time intervals, namely 6 h and 72 h following ischemia, were added. In detail, for the analysis at 6 h, phometry . In factitecture ,32,33. Fitecture  and obseUltrathin sections were used both for plain, and post-embedding immuno-gold electron microscopy. Ultrathin slices were stained with uranyl acetate and lead citrate , and the4) for 30 min, at 22 °C to remove OsO4. This step is recommended for antigen unmasking, while keeping optimal preservation of cell ultrastructure [Ultrathin sections, collected on nickel grids were processed for proteins detection using primary antibodies detailed in tructure . After wtructure ,37. To ctructure . We counn = 6) leading to 120 cells from each group.Grids were analyzed in order to count immuno-gold particles within 20 cortical neurons for each mouse (2) were calculated in brain slices stained with LC3 and/or P20S. Slices spaced 550 microns apart were calculated within each region . Four slices were counted for each animal. Values for neuronal density (number of neurons/mm2) were calculated within a squared area on a computer monitor, ranging in size around 82,500 μm2 (330 × 250 μm) Results are given as cell density per mm2. Number of positive neurons are given as the mean ± S.E.M. Inferential statistics to compare groups was carried out by using Student’s t-test analysis .Neuronal density values  was used to compare the experimental conditions and Bonferroni test was used to compare the mean values for all groups. The null hypothesis was rejected for For electron microscopy, assessment of vacuoles and measurement of immunogold particles were carried out according to Lenzi et al. (2016) . For ult0 was rejected when p < 0.05.For TEM analysis, the counts were carried out by two different observers, who were blind to treatment for each specific tissue blocks. Data are given as the mean ± S.E.M. Data between groups were compared by using one-way ANOVA with Fisher’s Test. Null Hypothesis H


Recently, researchers have focused on the biocompatibility and mechanical properties of highly porous structures of biomaterials products. Porous composites are a new category of bioengineering that possess excellent functional and structural properties. In this study, the physical and mechanical properties of prepared doped silver (Ag)-hydroxyapatite (HA) by the mechanochemical and spark plasma sintering (SPS) methods were investigated. The influence of dopant on phase formation, structural properties, mechanical properties and morphological characteristics was investigated. Furthermore, in this case, as a new approach to produce a porous scaffold with an average size of >100 µm, the hair band was used as a mold. According to the Monshi–Scherrer method, the crystal size of scaffold was calculated 38 ± 2 nm and this value was in the good agreement with average value from transmission electron microscopy (TEM) analysis. In addition, the stress–strain compression test of scaffold was considered, and the maximum value of compressive strength was recorded ~15.71 MPa. Taking into account the XRD, TEM, Fourier-transform infrared (FTIR), scanning electron microscope (SEM) and energy dispersive X-Ray analysis (EDAX) analysis, the prepared scaffold was bioactive and the effects of doped Ag-HA and the use of polyvinyltrimethoxysilane (PVTMS) as an additive were desirable. The results showed that the effect of thermal treatment on composed of Ag and HA were impressive while no change in transformation was observed at 850 °C. In addition, PVTMS plays an important role as an additive for preventing the decomposition and creating open-microporous in the scaffold that these porosities can be helpful for increasing bioactivity. One of the most desirable and well-known CaPs groups is associated with hydroxyapatite (HA) ,2. The ce tissue . Accordi10PO6(OH)2 ane tissue . On the e tissue ,8. Moreoe tissue ,10. Howee tissue . In addituration . Polyvinturation . PVTMS ituration . Recentlturation ,16. Overturation ,18, the turation , the grituration ,19 and tturation . Anotherturation . The genturation . As mentturation . In thisturation . There aturation . In addituration .Simon et al. fabricated composites of bioactive glass/polymer and used poly-96L/4D lactide for strength . In anot3)2·4H2O), phosphorus pentoxide (P2O5), silver nitrate (AgNO3) and vinyltrimethoxysilane (VTMS) (merck) were utilized as the precursors. Powders X-Ray, phase series were confirmed by X-ray diffraction (XRD) and performed on a Philips XRD diffractometer, Cukα radiation was used at 40 KV, 30 mA, step size of 0.05° (2ϴ) nd scan rate of 1°/min . Furthermore, X’Pert software was used for qualitative analysis and report of width diffraction peaks  at full width half maximum (FWHM) in different 2θ values according to the situation of peaks (Version 4.9.0). In addition, differential scanning calorimetry (DSC) were carried out by STA-BAHR . Fourier-transform infrared spectroscopy (FTIR) spectra of the compounds was attached in the potassium bromide (KBr) powders and the instrument that was used was a Perkin-Elmer Spectrum BX FT-IR spectrometer . Moreover, transmission electron microscopy (TEM) Tecnai G2 F20 X–TWIN with acceleration voltage from 50 to 80 KV was utilized . For chemical elements of components, an energy dispersive X-Ray (EDX) spectrometer Phillips/FEI 149 Quanta 200 was utilized . In addition, scanning electron microscope analysis (SEM) Phillips/ FEI Quanta 200 was used to study the morphology of the compounds and scaffold . In addition, simulated body fluid (SBF) was prepared according to the method of Kokubo et al. [2OH)3CNH2 and HCl (merck) were added (dropwise) to achieve a pH of 7.40 , then the temperature was decreased (20 °C) and distilled water was added [In this study, calcium nitrate tetrahydrate 2·4H2O) and phosphorus pentoxide (P2O5) were used in the molar ratio of 10:3. The following steps were performed: (1) Ca(NO3)2·4H2O) and P2O5 were dissolved in 10 mL of ethyl alcohol (C2H5OH) and distillated water. (2) The product was stirred at 350 rpm for 2 h. (3) The gel was prepared at the bottom of the dish. (4) The gel was then dried at 110 °C in air for 20 h. (5) Heat treatment at 850 °C for 15 h was considered for sintering. This is similar to the method is presented in [According to ented in ,31. The Vinyltrimethoxysilane (VTMS) was polymerized to 20-mers. In addition, PVTMS was caused to enhance the thermal stability and physical properties of scaffold. PVTMS was prepared with tertiary butyl peroxide as an initiator under reflux for 2 h at 150 °C in flowing nitrogen ,33. In a3) with 99% purity was used as the dopant. The amount of replacement of Ca2+ ions by Ag+ corresponds to the formula 4)63 were ground by using a high-energy planetary ball mill via a mechanochemical process at 4 h. In mechanochemical process, hard chromium steel shells and balls with a diameter of 20 mm were used. This process was carried out at ambient atmosphere and rate of 500 rpm with a ball to powder ratio of 15:1. In addition, to prevent agglomeration of the particles, the machine was stopped per 45 min. (2) After the end of mechanochemical process, calcination was carried out at 900 °C, at a heating rate of 10 °C per minute for degassing (90 min). (3) Taking into account the fact that HA is very sensitive to temperature, transformation and decomposition, the SPS process was considered. In this process, the powder was placed in the graphite mold (diameter = 20 mm), and pressure and temperature values were set 50 MPa and 600 °C for 10 min, respectively. The route of the fabricated Ag-doped HA composite is shown in In this study, silver nitrate (AgNO2O (1 wt%)/CH3COOH (1 wt%) were prepared. (5) In addition, a hair band  was chosen as a new approach to obtain open porosities, (6) which was immersed in the slurry for 5 days at room temperature. According to 3COOH) has played an important role as a dispersant for particles, especially for Ag, to prevent the formation of colloidal particles and to prepare small particles. PVTMS is bioactive polymer, as well as PVTMS is an organo-silane molecule and provides a hydrophobic environment in the composite. Since water can easily diffuse into the polymer structure, and it can lead to the breakdown of intermolecular forces and create the voids in the polymer [2, sp3 are more stable and uniform, as well as, PVTMS is an intermediate for bonding and it will lead to improvement in mechanical properties and adhesion. Therefore, PVTMS prevents a hydrophilic ambient and the collapse of the scaffold [(4) Component of a slurry consisted of Ag-doped HA (93 wt%)/PVTMS (5 wt%)/H polymer . The effscaffold . PVTMS played an important role as a drying control chemical additive (DCCA) to prevent cracking and shrinkage of scaffold. As shown in 3COOH +H2O is shown in 4 and crystallization of HA and decarburization-phase transformation into HA such as beta-tricalcium phosphate (β-TCP) [The DSC of the rolled hair band consisted of Ag-doped+HA+PVTMS+CH (β-TCP) . Accordi (β-TCP) ,41. Acco3)2·4H2O), P2O5 and AgNO3 disappear, and single phase of Ag-doped HA+PVTMS is produced. In addition, the absence of specific peaks of components such as AgNO3 and Ag3PO4 and the agreement of the XRD pattern  at 2θ = 25.86°, (211) at 31.86°, (300) at 32.89°, (130) 39.75°, (222) at 46.72°, (213) at 49.45° and (004) at 53.11° are investigated, showing that the cationic substitution not only prevents the formation of Ag-doped HA+PVTMS but also stabilizes at room temperature, as confirmed in [X-ray diffraction of the Ag-doped HA+PVTMS scaffold is shown in  pattern  with theirmed in . Moreoveirmed in . Similarirmed in . (−5.6194) = 0.0036, therefore TEM) is ~ less than 50 nm  vs. Ln (1/(Cos ϴ)) (degree) can be a linear plot for all chosen peaks a. In equtal size ,48. The an 50 nm b. For scarticles ,50,51.(−1 and the broad spectrum is due to the bonding between O and H. The evidence of the existence of carbonate groups in HA corresponds to the wave numbers at 1442 and 1486 cm−1 (belonging to the –CH3 group) and all –CH3 groups have implied the existence of PVTMS. Moreover, the wave number values of 927 and 985 cm−1 are due to the effect of PVTMS [3 content in the fabrication process of scaffold, there is no impressive tangible difference between the FTIR of pure HA and the FTIR of Ag-doped HA+PVTMS, except the intensity and broadening of the spectrum in some regions, which can be attributed to the substitutions of cations and anions in the HA structure. Furthermore the existence of main bonds are corresponded with [The FTIR spectrum of the Ag-doped HA+PVTMS scaffold is shown in of PVTMS . Based oof PVTMS . Therefoded with .3PO4 [3PO4 into metallic Ag through sintering process.The SEM image of Ag-doped HA powder is shown in 3PO4 . The choSEM images of the Ag-doped HA+PVTMS scaffold are shown with different magnification in 3 content. The role of Ag is mainly related to the antibacterial properties, and this amount can support this feature. In this case, the substitution of Ag instead of Ca is very important, because it affects the chemical properties of hydroxyapatite, decreases the crystallinity and increases the solubility [The results of elemental analysis using EDAX stoichiometry of SEM are shown in lubility . The EDA2O (1 wt%)/CH3COOH (1 wt%) without using metals with high concentration. (2) In this case, the bioactivity characteristics and creating the large size of porosities were purpose and, considering the initial bioactivity of ingredients, use of high concentration metals such as Mg, Zn and Pt can improve the value of compressive strength and mechanical properties.The stress–strain compression curve of the Ag-doped HA+PVTMS scaffold is shown in Scaffold was immersed in SBF  and keptThe X-ray diffraction and FTIR spectra, as well as the SEM images and EDAX data analysis of the scaffold after immersion in SBF, are shown in −1 can be associated with the Si-O-Si groups originating from PVTMS or with the overlap of spectra attributed to the carbonate and silanol groups [−1 can be attributed to the potential occurrence of hydrogen bonding between the carbonyl, hydroxyl and phosphate groups. This evidence indicates the presence of a bioactive scaffold in the structure. The transmittance values increased over the days, suggesting more precipitation of HA on the surface of the porous scaffold. In addition, a broad peak in the range from 2800 to 3760 cm−1 is associated with the bonding between O and H, which occurs as a bending mode, and the reason for the presence of these peaks is related to the hydroxyl groups of the precursor and water. Additionally, the researchers reported that the peak at 1480 cm−1 belongs to C–O [In the FTIR spectrum , the weal groups . Moreoves to C–O ,70. Thuss to C–O .According to the SEM images  of the pThe synthesis of the composite consisted of Ag-doped HA through the utilizing mechanochemical process was successful. SPS process prevented temperature deviation and phase decomposition of composite, in addition, SPS was caused to fabricate uniformity of Ag-doped HA and as a result, the distribution of Ag in HA was desirable through using the SPS process.The effect of Ag and PVTMS loading on the HA, was assessed. Ag provided antibacterial environment and PVTMS preven ted from cracking and shrinkage of scaffold and the free radicals of PVTMS structure provided flexibility of the scaffold through the bonding between C-C and siloxane (Si-O-Si), as well as PVTMS prevented to collapse via carbon chains during the heat treatment, because it is hydrophobic polymer due to the silane group.The crystal size of Ag-doped HA was calculated 38 ± 2 nm and this value was corresponded with value extracted by TEM analysis (~less than 50). In addition, the a and c parameters  of the Ag-doped HA+PVTMS structure increased, which is due to the larger ionic radius of Ag compared to Ca and it is related to the proper bonding between Ag ions and the HA structure.A new approach to fabricate porous scaffold through the utilizing hair band was carried out and as a result, the average porosity value was obtained at >200 µm, so this value was suitable for sitting blood cells in these porosities and the coefficient of bioactivity was enhanced.For investigating in vitro bioactivity test, the SBF was successfully synthesized, and the scaffold was placed in the SBF for 3, 5, 10 and 20 days. As the result, according to the FTIR spectrum of immersed scaffold in SBF, the resulting bands were attributed to HA, with the most prominent bands related to the phosphate groups of the HA structure. In addition, the XRD, SEM and EDAX analysis of the immersed scaffold were studied and HA was nucleated and grown on the surface of the scaffold; as a result, the scaffold was bioactive.The maximum value of compressive strength reached 15.71 MPa, and this value could be suitable according to the content of Ag and not using high amount of metals.


Multivariable logistic regression analysis revealed that Hunt and Hess grade ≥ IV (p = 0.023), DAPK1 (p = 0.003), and BNIP3L (p = 0.039) were related to DCI. Increased mitochondrial dysfunction associated with autophagy and mitophagy could play an important role in DCI pathogenesis.Decreased mitochondrial membrane potential in cerebrospinal fluid (CSF) was observed in patients with subarachnoid hemorrhage (SAH) accompanied by delayed cerebral ischemia (DCI). However, whether abnormal mechanisms of mitochondria are associated with the development of DCI has not been reported yet. Under cerebral ischemia, mitochondria can transfer into the extracellular space. Mitochondrial dysfunction can aggravate neurologic complications. The objective of this study was to evaluate whether mitochondrial dysfunction might be associated with autophagy and mitophagy in CSF cells to provide possible insight into DCI pathogenesis. CSF samples were collected from 56 SAH patients . We analyzed CSF cells using autophagy and mitophagy markers  via qRT-PCR and western blotting of proteins . Confocal microscopy and immunogold staining were performed to demonstrate the differentially expression of markers within dysfunctional mitochondria. Significant induction of autophagic flux with accumulation of autophagic vacuoles, increased expression of BECN1, LC3-II, and p62 degradation were observed during DCI. Compared to non-DCI patients, DCI patients showed significantly increased mRNA expression levels (2 Medical complications following SAH are still a challenge in neurocritical care, even if ruptured aneurysm is treated successfully. Among complications, delayed cerebral ischemia (DCI) is still a major contributor to poor neurologic outcomes of patients with SAH. The clinical syndrome of DCI is characterized by newly developed neurologic deficits with symptom fluctuation, and usually occurs between 4 and 14 days after SAH ictus3. DCI pathogenesis is known to be associated with cerebral vasospasm, cortical spreading depolarization, and microthrombosis or early brain injury (EBI). However, the mechanisms underlying DCI have yet to be elucidated. Unlike other complications including EBI, DCI is considered to be a treatable and preventable cause. Therefore, most studies on DCI are needed to provide a viewpoint different from existing ones.Subarachnoid hemorrhage (SAH) is defined as acute bleeding in the subarachnoid space due to ruptured intracranial aneurysm. Despite advances in minimally invasive surgery, the mortality rates in patients presenting with poor-grade SAH remains high: 24% for Hunt and Hess (H–H) grade 4 and 71% for grade 54. In particular, when healthy mitochondria are maintained though the selective removal of damaged or dysfunctional mitochondria via autophagy, this is referred to as mitophagy6. Since the early 2010s, reports on mitochondrial dysfunction involved in stroke have increased7. Mitophagy controls the quality of the mitochondria by eliminating dysfunctional mitochondria known to contribute to the production of reactive oxygen species (ROS)8. Lee et al.5 reported a higher expression of beclin-1 (BECN1), microtubule-associated protein light chain-3 (LC3) conversion, and cathepsin-D in the frontobasal cortex, suggesting increased autophagy in neurons following SAH. Zhang et al.9 also showed that mitophagy was associated with oxidative stress-related neuronal death in rat SAH models induced by endovascular perforation. However, most of these studies were mainly concerned about the association of autophagy and mitophagy with EBI. EBI refers to secondary complications of SAH that usually occur within the first 72 h after ictus10. Accordingly, it is thought that studies on autophagy and mitophagy in DCI have hardly been done.Autophagy is a reparative process in which cellular homeostasis is maintained through the lysosomal degradation of dysfunctional cytoplasmic components11. Hayakawa et al.7 reported that cerebral ischemia can result in the entry of astrocytic mitochondria into adjacent neurons in mice while suppression of CD38 signaling can reduce extracellular mitochondria transfer and neurological deterioration. Under cerebral ischemic conditions, mitochondrial depolarization can lead to increased synthesis of ROS, PTEN-induced kinase 1 (PINK1), and elevated unfolded protein response13. Compared to cerebral ischemia caused by a decrease in intravascular cerebral blood, DCI has a more complex mechanism. SAH occurs in subarachnoid space which surrounds cerebral arteries. Thus, inflammation or immunological changes in cerebrospinal fluid (CSF) can affect vascular responses11. In addition, mitochondria can transfer into the extracellular space14. Therefore, analysis of CSF mitochondria may be valuable to assess the relationship between mitochondrial dysfunction and DCI.DCI literally means secondary brain ischemia that occurs over time rather than immediately after SAH15. Chou et al.14 reported that higher mitochondrial membrane potential in the extracellular mitochondria in CSF was associated with favorable neurologic outcomes of SAH patients. Recently, Youn et al.15 showed that the membrane potential was significantly decreased in mitochondria isolated from of SAH patients with DCI, but not in those without DCI. In their study, the decrease in mitochondrial membrane potential was remarkable at 5 days after ictus15. Moreover, mitochondria of endothelial origin shown as von Willebrand factor (vWF)-positive were increasingly detected in the CSF of DCI patient concomitant with a decreased mitochondria membrane potential. Considering the facts mentioned, we hypothesized that mitochondrial dysfunction associated with autophagy and mitophagy in CSF cells may be associated with DCI pathogenesis following SAH. However, in the case of craniotomy and clipping, there is a possibility that the analysis of mitochondria in the CSF might be affected by retraction of the brain and draining of the CSF to expose the aneurysm during surgery. Therefore, in this study, we only included SAH patients who underwent endovascular coil embolization.Mitochondria in CSF show a dynamic nature following SAH. Mitochondrial membrane potential as a parameter of mitochondrial function is increased up to 3 days after ictus. It decreased thereafterA total of 56 SAH patients treated with endovascular coil embolization were included in the analysis Table . Twenty-Transmission electron microscopy (TEM) was used to investigate the changes in subcellular ultrastructure of CSF cells obtained from SAH patients with DCI Fig. . Numerou−ΔCt) than non-DCI patients: death-associated protein kinase (DAPK)-1, 0.279 (0.220–0.297) in DCI vs. 0.043 (0.021–0.086) in non-DCI; BCL2 interacting protein 3 like (BNIP3L), 0.134 (0.060–0.202) in DCI versus 0.045 (0.020–0.101) in non-DCI, and PINK1, 0.064 (0.044–0.810) in DCI versus 0.045 (0.012–0.063) in non-DCI. Other mRNAs expressed such as Bcl-1 antagonist X (BAX), 0.204 (0.167–0.294) in DCI versus 0.166 (0.095–0.223) in non-DCI; Unc-51 like autophagy activating kinase 1 (ULK1), 0.013 (0.003–0.027) in DCI versus 0.004 (0.001–0.008) in non-DCI; and nuclear dot protein 52 (NDP52), 0.258 (0.045–0.323) in DCI versus 0.155 (0.030–0.229) in non-DCI  analysis was performed to evaluate mRNA levels of autophagy and mitophagy markers in CSF cells of SAH patients with DCI compared to those in CSF cells of SAH patients without DCI. DCI patients exhibited significantly higher expression 2−ΔCt thanCI Table . In addiTo investigate whether mitochondrial dysfunction with increased autophagy and mitophagy occurred in vWF-positive CSF cells in SAH patients with DCI, multi-colored immunofluorescence staining was conducted with antibodies specific for vWF and MTDR as a marker of dysfunctional mitochondria Fig. . The fluWe further performed immunogold labeling with anti-DAPK1 to confirm the subcellular localization of enhanced DPAK1 in CSF cells obtained from SAH patients with DCI Fig. . Interes12. Autophagy and mitophagy are induced to remove damaged mitochondria after an ischemic insult. However, results did not always show only neuroprotection12. Baek et al.16 reported that attenuated autophagic signaling by carnosine, an endogenous pleiotropic peptide, was associated with improvement of mitochondrial function against ischemic brain injury. On the other hand, BNIP3L causes excessive mitophagy leading to delayed neuronal loss17. Chen et al.18 reported that -(-) Epigallocatechin-3-Gallate (EGCG) administration was beneficial for maintain autophagy flux by regulating Beclin-1 and LC3B. EGCG decreased oxyhemoglobin-induced mitochondrial dysfunction with decreased ROS production following SAH. Cao et al.19 also reported that melatonin lessened mitophagy-associated NLRP3 inflammasome for EBI after SAH. Nonetheless, the therapeutic role of mitophagy in the DCI is still unknown. Compared with EBI, which occurs in the brain parenchyma in the first 72 h after SAH onset, DCI usually develops 3 days thereafter and peaks from days 7 to 14. SAH literally refers to acute hemorrhage within the subarachnoid space surrounding the cerebral arteries. Free hemoglobin within the subarachnoid space triggers oxidative stress and inflammation, resulting in DCI20. Chou et al.14 initially investigated the functional relevance of mitochondria in human CSF samples following SAH. In their study, higher mitochondrial membrane potential reflected favorable functional outcomes during the 3-month follow-up. In particular, the higher percentage of mitochondria potentially originating in astrocytes was associated with a favorable outcome. In this study, we further evaluated autophagy and mitophagy markers in the CSF cells to reveal the underlying DCI pathogenesis based on mitochondrial dysfunction. Our study showed that increased mitochondrial dysfunction associated with autophagy and mitophagy in CSF cells may drive DCI pathogenesis. Among the markers, the DAPK1 expression varied most significantly between the DCI and non-DCI patients.The clinical significance of mitochondrial dysfunction has not been well studied in the DCI following SAH. In the event of cerebral ischemia, mitochondria are damaged through a series of following steps: (1) depolarization of mitochondrial membrane potential; (2) PINK2 accumulation with decreased adenosine triphosphate (ATP) reduction; (3) abnormal mitochondrial fission and fusion; and (4) broken cellular homeostasis due to increased ROS and matric calcium and subsequent cell death21. Mitochondrial toxins, such as rotenone, and carbonyl cyanide m-chlorophenylhydrazone (CCCP), which is an uncoupler of mitochondrial oxidative phosphorylation induce loss of membrane potential and mitochondrial swelling leading to cell death via DAPK1 activation in neuroblastoma cells. In damaged mitochondria, the accumulation of PINK1 and LC3 on the outer membrane is accompanied by loss of membrane potential22. Stroke-induced neuronal cell death is related to DAPK1 signaling mediated via DAPK1-N-methyl-D-aspartate receptors (NMDARs), DAPK1-p53 and DAPK1-Tau in cerebral ischemia23. Tu et al.24 reported that DAPK1 directly binds with NR2B subunit of NMDARs in the cortex of mice, leading to aggravation of calcium reflux by enhancing the NR1/NR2B channel conductance. In their study, the genetic deletion of DPAPK1 or administration of NR2BCT, defined as carboxyl tail region consisting of amino acids 1292–1304, inhibited calcium reflux and protected neuronal injury against ischemic insults24. DAPK-1 was also involved in the regulation of inflammation. Activation of DAPK led to decreased T cell activation and IL-2 production26. Chuang et al.26 reported that DAPK was a regulator of T cell receptor (TCR)-activated NF-kappa B and T lymphocyte activation. Therefore, a further study elucidating the role of DAPK1 and T cell immunity in DCI pathogenesis in the CSF space is required.DAPK1 acts as a sensor of mitochondrial membrane potential in mitochondrial toxin-induced cell death14. The findings demonstrated colocalization with DAPK1, BNIP3L, PINK1, and BECN1-positive autophagosomes in mitochondria, suggesting mitochondrial dysfunction with autophagy and mitophagy in DCI pathogenesis. Endothelial dysfunction is related to cerebrovascular disease. Nitric oxide and endothelin released from endothelium regulates vascular function27. Compared with healthy controls, SAH patients exhibit elevated synthesis of endothelial microparticles28. In addition, increased levels of CSF and plasma endothelin were observed in SAH patients with vasospasm27. In DCI patients, a higher percentage of vWF-positive mitochondria was observed compared with non-DCI patients15. Accordingly, future studies should focus on the alleviation of the mitochondrial dysfunction in CSF cells of endothelial origin in DCI patients.In this study, we further evaluated the role of confocal microscopy to confirm the differential expression of autophagy and mitophagy markers in vWF positive-CSF cells of SAH patients with DCI29. Mitochondrial dysfunction is likely to vary depending on the initial SAH severity and thus may be associated with the patient’s outcome. Although there was no statistical significance of autophagy and mitophagy markers according to the SAH severity and patient’s outcome  adult patients > 18 years old; (2) SAH due to ruptured aneurysm; (3) dense localized clot and/or vertical layer of blood greater than 1 mm in thickness on computed tomography (CT); and (4) SAH patients who were treated with endovascular coil embolization. The exclusion criteria were: (1) non-aneurysmal SAH such as trauma, infection or perimesencephalic SAH, (2) patients treated with surgical clipping and (3) previous history of central nervous system disorder or mitochondrial diseases32. Confocal microscopy was used to identify colocalization of differentially expressed genes in vWF-positive CSF cells, which represented endothelial cell origin, and were increased in SAH patients with DCI15.TEM was used to detect the autophagic vacuoles and morphological changes of mitochondria in CSF cells derived from SAH patients with DCI. We evaluated and compared the autophagy and mitophagy biomarkers in CSF cells of SAH patients with and without DCI using qRT-PCR. The markers included DAPK-1, BNIP3L, BAX, PINK1, ULK1 and NDP52. We further evaluated the expression of autophagy executor gene of BECN1 and autophagy adaptor protein of p6233. When DCI was suspected, treatment to increase blood pressure was given first. Blood pressure was targeted with the mean systolic blood pressure 20 mm Hg above the baseline, up to 200 mm Hg while looking at the clinical response. Intra-arterial chemical angioplasty using nimodipine was performed when DCI-related clinical symptoms worsened or severe vasospasm was suspected based on a TCD greater than 200 cm/s in the middle cerebral artery or 85 cm/s in the basilar34, despite treatment to induce hypertension. The procedure was carried out once or twice a day from 1 to 7 days after identification of DCI. Poor outcome was defined when a modified Rankin scale (mRS) score was three or more at 3-month follow-up.In our study, the diagnosis of DCI wad performed through the following criteria: (1) new developed neurological changes such as motor weakness, dysphasia, and sensory change; (2) decrease consciousness by more than 2 points via the Glasgow Coma Scale score or National Institutes of Health stroke scale; (3) fluctuation of symptoms lasing more than 1 h; (4) cerebral infarction identified on CT or MRI, but not complications related to the endovascular coil embolization; (5) concomitant severe cerebral vasospasm with narrowing more than 50% compared to the initial radiological tests; and (6) excluding other causes that may neurological changes such as re-bleeding, hydrocephalus, seizures or electrolyte imbalances15, we analyzed the CSF samples obtained from days 5 to 7.After coil embolization, continuous lumbar drainage of CSF was maintained in the neurointensive care unit in every SAH patient for 1 week after ictus in our institution. Because difference in mitochondrial membrane potential of the CSF cells was most prominent on day 5 post ictus in SAH patients with and without DCIClinical, laboratory, and radiological information was reviewed by the two investigator independently. Disagreements were resolved by the third investigator. The protocol of DCI diagnosis among reviewers are presented in detail in the 15. In the present study, TEM was used to investigate the changes in subcellular ultrastructure of CSF cells in SAH patients with DCI. CSF samples were centrifuged at 4000 rpm for 10 min, and the pellets were analyzed by electron microscopy15. The pellets were fixed overnight in 2% glutaraldehyde in cacodylate buffer  at 4 ℃. After washing three times with cacodylate buffer at 4 ℃, the samples were post-fixed in 2% osmium tetroxide for 1 h at 4 ℃. The samples were rinsed with deionized water and dehydrated through a gradient series of ethanol, ranging from 50 to 100% ethanol, 20 min each step. The samples were incubated with progressively concentrated propylene oxide dissolved in ethanol followed by infiltration with increasing concentration of Eponate 812 resin. Samples were baked in a 65 °C oven overnight and sectioned using an Ultra microtome. Sections were viewed with a Field Emission TEM unit  at the Korean Basic Science Institute, Chuncheon, South Korea15.Previously, SAH patients with DCI showed depolarization of mitochondrial membrane potential, which triggers alteration in mitochondrial function and morphologiesCSF cells obtained from SAH patients were lysed with RIPA buffer . Protein lysates of the supernatant were quantified using the BCA protein assay kit . Equal amounts of protein were separated on 10% SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes . After blocking the membranes with 2% BSA in TBS-T  for 1 h, the membranes were incubated with the primary antibodies overnight at 4 ℃. After extensive washing, the membranes were incubated with HRP-conjugated secondary antibodies, and developed using an enhanced chemiluminescence (ECL) kit . Antibodies used in this study were: BECN1 , p62 , LC3 , pBECN1Ser15 , DAPK , PINK1 , BNIP3L/NIX , and actin .14. CD41  was used to differentiate endothelial and platelet, and it was confirmed that vWF was an endothelial specific marker in our study. To assess the dysfunctional mitochondria undergoing autophagy in vWF-positive CSF cells, autophagy- or mitophagy-related markers were subjected to co-immunofluorescence stained with MTDR , which is widely used as a mitochondria-selective dye to determine mitophagy35. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole dihydrochloride . Fluorescence images were obtained via confocal microscopy at 400 × magnification.To evaluate the colocalization of autophagy and mitophagy markers with dysfunctional mitochondria in vWF-positive CSF cells, multi-color immunofluorescence staining was performed via confocal microscopy. CSF cells were fixed with paraformaldehyde (4% w/v) and then washed with PBS. After blocking with 2.5% blocking solution  for 1 h, the cells were incubated with antibodies specific to DAPK1 , PINK1 , BECN1  and BNIP3L/NIX  overnight at 4℃. After incubation with anti-rabbit Alexa Fluor 750-conjugated secondary antibody , the coverslips were mounted in Fluoroshield™ with DABCO (ImmunoBioScience Corp. USA). vWF-FITC  was used as an endothelial marker to confirm the endothelial origin of CSF cells22. Total RNAs were isolated from the CSF cells using TriZOL  according to the manufacturer’s instructions. The cDNA was synthesized from 5 μg of RNA using Maxime RT PreMix Kit . The expression of mitophagy-related genes was measured by qRT-PCR using the 2 × Rotor-Gene SYBR Green PCR Master Mix  in the Rotor-Gene Q . Primer sequences are presented in the Supplemental Table The loss of mitochondrial membrane potential leading to mitochondrial dysfunction results in activation of mitochondrial autophagy. Therefore, autophagy and mitophagy markers such as DAPK1, PINK1, BAX, BNIP3L, and NDP52 were examined in CSF cellsWe further performed immunogold staining to confirm the location of the most significant differentially expressed markers within the mitochondria of CSF cells in SAH patients with DCI. CSF cells were fixed with 0.1% glutaraldehyde and 2% paraformaldehyde in phosphate buffer at pH 7.4 for 1 h at 4 °C, followed by incubation with post-fixed osmium tetroxide for 30 min at 4 °C. The samples were dehydrated in a graded series of ethanol. The samples were treated with graded ethanol series and embedded in LR white resin (EMS). The sections were then sliced into ultra-thin sections of 80 nm each, and placed on a copper grid. The sections were treated with 0.02 M glycine for 10 min to ensure quench-free aldehyde groups. Sections were then washed with deionized water, blocked in 1% BSA and incubated with rabbit anti-DAPK1 antibodies  for 1 h. The grid was washed five times with 0.1% BSA in PBS, and incubated with a secondary antibody conjugated with 10 nm gold  in 0.1% BSA in PBS. The final samples were stained with uranyl acetate and lead citrate, and then visualized under a transmission electron microscope  at 200 kV.t test was carried out to identify meaningful differences between DCI and non-DCI patients. Comparative analysis via qRT-PCR was performed using the Mann–Whitney U test. The results were presented as the median and 25th–75th percentiles. Quantification of western blots using the relative optical densities with actin protein as the reference and presented as the mean ± standard error of the mean (SEM). Univariate analysis was performed to find relevant factors for the development of DCI. A multivariable logistic regression analysis was then performed to identify independent variables associated with the development of DCI using variables showing p value less than 0.20 in the univariate analysis. Statistical analysis was performed with SPSS V.21  and GraphPad Prism software  with a statistical significance indicated at p < 0.05.Continuous variables are expressed as the mean and ± standard deviation (SD). A chi-square or Student’s Sample collection and study design were performed according to the principles of the Declaration of Helsinki and were approved by Coordinating Ethnics Committee of the Chuncheon Sacred Heart Hospital.Supplementary Information.


Central nervous system (CNS) infections are serious and debilitating diseases with significant mortality, and high prevalence in the context of human immunodeficiency virus (HIV) pandemic in Africa. However, their diagnosis remains challenging due to outdated technical platform. We aimed to determine the frequency of CNS infection and to describe the epidemiological, clinical and outcome of this at the Douala General Hospital (DGH), Cameroon. To carry out this study, we collected the medical records of patients hospitalized for CNS infections in the internal medicine department of DGH from January 2015 to December 2019.n = 56) of them on regular follow-up. The most common clinical signs were fever (84.8%), headache (68.6%), meningeal syndrome (38.7%), and seizures (36.3%). Cerebral toxoplasmosis (24.5%), cryptococcal meningitis (21.1%), and acute bacterial meningitis (8.3%) were leading aetiologies. Of the 143 CSF samples, 70.6% (n = 101) were sterile. The in-hospital mortality rate was 23.5% with CNS infection of unknown cause (22.1%) be independently associated to this .Among 8430 files reviewed, 336 cases of CNS infection were identified giving a frequency of CNS infection of 3.99% among which 204 files were included in the study . HIV infection was found in 147 patients (72.1%) with 38.1% (Clinical presentations of CNS infections are same with classical data. HIV-related opportunistic infections are the main aetiologies. About one over four patients with CNS died. Two thirds of CSF are sterile using basic laboratory assessment giving a need to identify simple tests to increase sensibility and specificity of diagnostic tools in our setting. Central nervous system (CNS) infections are potentially life-threatening conditions that constitute a diagnostic and therapeutic emergency –5. It isStreptococcus pneumonia (46%) and Neisseria meningitis (30%) were the main aetiologies among 135 cases of meningitis reported in Cameroon [The global incidence of CNS infections between 1990 and 2016 is estimated at 389/100,000 . In AfriCameroon .Morbidity and mortality associated with CNS infections is higher compared to infections of other systems . DespiteA retrospective cohort study was conducted in the internal medicine department of the Douala General Hospital (DGH). Medical files were reviewed for patients aged 16 years and above, hospitalized between January 1, 2015 and December 31, 2019 for CNS infection diagnosed via neuroimaging (brain CT scan and MRI) with and without cerebrospinal fluid (CSF) analysis. All patients performed at least brain CT scan/MRI and/or CSF analysis. Each file stored in the archives was recorded in a register, as well as in a computer which contained the hospital reports of each patient. Incomplete files, especially without neuroimaging and/or CSF analysis results were excluded. Ethical approval was obtained from institutional ethics committee of the University of Douala (No. 2162CEI-UDo/01/2020/T).The following data were collected from selected medical files: (1) sociodemographic data: age, gender, profession, marital status; (2) comorbidities: HIV, diabetes, hypertension, chronic kidney disease (CKD); (3) clinical data: neurological and extra-neurological signs; (4) paraclinical workups: neuroimaging (brain CT scan or MRI), CSF analysis when done, and other biological testing, such as CBC, CRP, blood culture; and (5) outcome: mean length of hospitalization and mortality.Mycobacterium tuberculosis), Sabouraud dextrose agar (fungi), soluble bacterial antigens, cryptococcal antigens, and Veneral disease research laboratory (VDRL)/Treponemal Pallidum Hemagglutination assay (TPHA). When requested, a real-time polymerase chain reaction (PCR) for Mycobacterium tuberculosis also called Xpert MTB/RIF Molecular Beacon Assay was performed at the laboratory of the Regional Delegation of Public Health of the Littoral. Hypoglycorachia was defined as a CSF glucose level less than half of the glycaemia; hyperproteinorachia was defined as a CSF protein level in the CSF greater than 0.45 g/L; and pleocytosis was considered as a white cell count > 5 cells/mm3.The CSF analysis carried out by the laboratory of the DGH are classically: biochemistry (protein and glucose level), cell count (leucocytes and red blood cells), direct detection , culture on blood or chocolate agar (bacteria), Lowenstein–Jensen medium . χOut of the 8430 files reviewed, 336 cases of CNS infection were identified, of which 204 met the inclusion criteria. From 2015 to 2019, the hospital-based frequency of CNS infections ranged from 2.2% to 2.6% . The most common age groups were 30–40 years (29.9%) and 40–50 years (28.4%). Male patients accounted for 54.4% of cases. HIV infection was found in 147 cases (72.1%) with 61.9% of them not on regular follow-up with highly active antiretroviral therapy (HAART) , impaired consciousness (44.1%), signs of meningeal irritation (38.7%), and epileptic seizures (36.3%), while altered general state (93.1%), fever (84.8%) and vomiting (35.8%) were main extra-neurological signs Table .Table 2Cn = 67; 46.6%), hyperproteinorachia , and pleiocytosis . CSF microbiological analysis was positive for Cryptococcus neoformans (n = 38), Neisseria meningitidis (n = 1), Streptococcus pneumoniae (n = 1) and Pseudomonas aeruginosa (n = 1). No acid-fast bacilli were detected through the Ziehl–Neelsen stain. PCR was positive for Mycobacterium tuberculosis in three cases. Among the patients with HIV, the mean CD4 count (in 94 patients) was less than 350 cells/mm3 in 92.3% of cases. The most frequent aetiologies were cerebral toxoplasmosis (24.5%) and cryptococcal meningitis (21.1%), while unknown aetiology was founded in 34.8% of cases . Results were abnormal in 117 cases with brain mass representing the commonest lesion reported. These abnormalities were found in 65.6% and 67.4% of brain CT scan and brain MRI, respectively. Lumbar puncture with CSF analysis was performed in 143 patients. Results showed hypoglycorachia , 40–50 (p = 0.011) and CNS of unknown aetiology (p = 0.006). After multivariate analysis, only CNS infection of unknown aetiology was independently associated to the in-hospital mortality , p = 0.039) , CNS infections are frequent causes of admission in neurology . The oveYoung adults represented the main age group. Several studies in Africa reported similar data , 25–27. 3). This could explain the lower inflammatory response, hence the low frequency of patients with meningeal syndrome [Clinical manifestations are often similar to those of classical data . In thissyndrome .The leading aetiologies were cerebral toxoplasmosis (24.5%), cryptococcal meningitis (21.1%), and acute bacterial meningitis (5.9%). These findings are consistent with a study done by Luma et al. in 2013, which found cerebral toxoplasmosis (32.3%) and cryptococcal meningitis (25%) as the commonest aetiologies . Given tp = 0.039). This could be explained by the fact that without knowing of the causes, specific treatment cannot be instituted, thereby increasing the risk of death [In 2013, Luma et al. reported a mortality of 49% in PLWH which is more than two time higher than our finding . SeveralThis study places the foundations for epidemiological data on CNS infections in Cameroon. However, the retrospective design of this study leads to data collection challenges with up to 132 incomplete medical files excluded. This could have impacted the reliability of results. In addition, this study was conducted in one hospital, and therefore, data cannot be generalized in the entire community.Clinical presentations of CNS infections are same with classical data. HIV-related opportunistic infections are the main aetiologies. Mortality remains high in our context. Two over third CSF are sterile using basic laboratory assessment giving a need to implement simple tests to increase sensibility and specificity of diagnostic tools in our setting.


Basic Documentation for Psycho-Oncology (PO-Bado) is a hetero-assessment and psychosocial burden documentation tool for cancer patient caregivers . Recently, the psychometric properties of the standard 12-item version of PO-Bado were published. However, the standard version is relatively time-consuming for the caregivers. Here, we developed and examined psychometric properties of a French short-form of PO-Bado (PO-Bado-FSF) with seven items derived from the validated standard version.Mage = 58.4 years, SD = 13.9 years; 68.6% were women) participated in this study during a supportive care following the first diagnosis of cancer or a relapse. All patients completed the Hospital Anxiety and Depression Scale (HADS) and General Health Questionnaire (GHQ), in addition to the PO-Bado-FSF.One hundred and twenty-one cancer patients ; all items loaded significantly on the single CFA factor and yielded coefficients 0.40 or higher.The results of this study highlight the value of using PO-Bado-FSF to identify psychological distress in cancer patients in research and practice. PO-Bado-FSF presents good psychometric properties and is less time-consuming than the standard version. The scientific literature emphasizes the importance of integrating the assessment of patients’ psychological distress at each stage of cancer .This hetero-assessment tool can be used in cancer patients across all types and stages. It was developed between 2001 and 2006 in Germany . One of the six selected items is not present in the German short version , but is heavily saturated in our analysis. In the French version, the item ‘tiredness/fatigue’ was not retained, contrary to the German version, and was replaced by a more general question ‘physical distress’  which not only encompasses fatigue but also explores loss of appetite and pain. Furthermore, we chose to retain a 7th item ‘Other problems e.g. social or family problems’, also present in the German short version despite a moderate factor loading in the results of the French version, because it of an indisputable clinical interest for professionals. This makes it possible to tackle family or socio-professional problems and to set up early support mechanisms (the Po-Bado-FSF is presented in the Appendix).The PO-Bado-FSF was devised similar to the standard version and pre-tested with four caregivers. All topics were considered to be clearly expressed, easily understandable and no changes to the text were required.Overall, 121 cancer patients participated in this study, out which 83 were women (68.6%). The average age was 58.4 years (SD = 13.9 years). Sociodemographic and cancer characteristics of these patients are presented in The interviews were conducted by caregivers  trained to administer the Po-Bado. These interviews took place after a ‘breaking bad news’ consultation with the doctor. The interviewers carried out the supportive care consultation and PO-Bado interview with the patient. One of the purposes of this interview was to detect psychological issues early and point to supportive care and psychological consultations. For test-retest assessment purpose, 40 randomly selected patients, agreed to have their interviews recorded and to complete a second interview. These interviews were carried out during their consultation or treatment visits, in a quiet room. They also agreed to fill out a new questionnaire, one month later. Full ethics approval was obtained from the national authorities . In addition, 15 PO-Bado-FSF protocols were recorded and rated by the caregiver conducting the interviews, and by three additional caregivers to assess inter-rater reliability.In addition to the PO-Bado-FSF interview, all patients completed two questionnaires:The Hospital Anxiety and Depression Scale (HADS)  . H coefficients between .40 and .50 are indicative of medium scales, and H coefficient >.50 suggest strong scales ; where ‘SE’ means the standard error of H-value. As suggested by Gillespie et al. . Consequently, we opted for the Maximum likelihood estimation with robust standard errors  available in lavaan package  statistic, the comparative fit index (CFI), the standardized root mean square residual (SRMR), and the root mean square error of approximation (RMSEA) and its 90% confidence interval (90% CI) were used to evaluate the goodness-of-fit of the measurement models. As recommended by Hu and Bentler  coefficient  coefficient as well as Raykov’s omega )  was assessed using a two-way random, consistency, average-measures ICC , and the depression scale of the HADS . There were no gender differences on the PO-Bado-FSF scores and on the anxiety subscale of the HADS scores.The main characteristics of the patients are presented in iH values of the PO-Bado-FSF items were found to be above the suggested a lower bound cutoff value of .30, and ranged from iH = .38 for item 7, which was the least scalable item, to iH = .58 for item 5, which was the most scalable one. All these coefficients were significantly different from zero at p < .000. The H coefficient for the whole set of the 7 items of the PO-Bado-FSF was .49 (SE = .046). The 95% confidence interval for this estimate  ranges from a moderate Mokken scale to a strong Mokken scale.All χ² (14) = 34.81, p = .002, CFI = 0.911, RMSEA = 0.126, 90% CI , SRMR = 0.065). Modification of indices showed a misspecification associated with the pairing of error terms associated with item 1 and 2 (see Appendix). Thus, freeing the correlation of these two measurement errors had significantly improved the model fit (χ² (13) = 7.73, p = .86, CFI = 1.00, RMSEA = 0.000, 90% CI , SRMR = 0.027). As shown in A single-factor measurement model for the PO-Bado-SF data was tested. The results showed that this solution did not fit the sample data (r = 0.46 (item 7) to r = 0.73 (item 3). Deletion of any item was not likely to improve the reliability of the scores.The alpha coefficient value was 0.84, and the omega coefficient Raykov,  was 0.80We performed a 4-week test-retest reliability measure among 40 patients with an intra-class correlation coefficient ). The results revealed the average ICC was 0.81 . The mean total scores from the first and the second administrations were 6.71 (SD = 3.95) and 6.43 (SD = 3.70), respectively. This difference was not statistically significant.To estimate the inter-rater reliability (IRR), 15 PO-Bado-FSF interviews recorded and quoted by 4 nurses were randomly selected (2 interviews were subsequently deleted due to missing data). The resulting mean ICC (i.e. an average of the 13 ICCs) was in the excellent range, ICC = 0.71 (range 0.43–0.95), indicating that raters had a good degree of agreement and suggesting that PO-Bado-FSF was rated similarly across coders. The high ICC suggests that a minimal amount of measurement error was introduced by the independent coders, and therefore statistical power for subsequent analyses using the PO-Bado-FSF is not substantially reduced .H-value) indicating that this whole set of items form a moderate Mokken scale. Except item 7 which displayed weak scalability, the 6 other items displayed moderate  or strong scalability . Recall here that item scalability (iH) measures the homogeneity of a particular item with respect to all other items. As noted by Gillespie et al.  was 0.66, indicating that the PO-Bado-FSF scores capture more psychological distress than simple depressive mood or anxious mood .Since the psychosocial burden of cancer diseases could not be reduced to negative mood  or any psychological morbidity, the PO-Bado-FSF has all the required psychometric properties to capture specific psychosocial challenges and burden among cancer patients.Time-consuming tests are often tiring to the patient and perceived as invasive; even more so when professionals have to evaluate other needs in care of support. PO-Bado-FSF allows, in ten minutes, exploring the intensity of the psychological distress of the patient. As pointed out in the French National Cancer Institute's Recommendations (Institut National du Cancer [INCa], One of the limitations of this work is the difference between the German and the French versions. A collaborative effort between the German and French research teams is possible to make a cross-cultural comparison of the two PO-Bado-SFs. The differences between culturally based items and clinical practices are pointed out in the Appendix. We note that for cost control reasons, this study is monocentric. The study needs to be replicated in other populations to test the robustness of the 7-item structure of PO-Bado-FSF.The PO-Bado-FSF scores exhibit sound psychometric qualities. This version takes into account organizational constraints and limited time. In a clinical setting, it is a useful tool to help provide suitable treatment and improve the quality of life of cancer patients. We have shown that this tool significantly helps caregivers to better manage their interview and better identify patients’ distress. We hope to continue the work to refine our analyses notably, at the clinical level and to extend the PO-Bado to other chronic progressive diseases.


N-methyl-D-Aspartate receptor (NMDAR) and Leucine-rich Glioma-Inactivated 1 (LGI1) encephalitis, have been known for more than a decade. Nevertheless, no well-established biomarkers to guide treatment or estimate prognosis exist. Neurofilament light chain (NfL) has become an unspecific screening marker of axonal damage in CNS diseases, and has proven useful as a diagnostic and disease activity marker in neuroinflammatory diseases. Only limited reports on NfL in AE exist. We investigated NfL levels at diagnosis and follow-up in NMDAR and LGI1-AE patients, and evaluated the utility of CSF-NfL as a biomarker in AE.The two most common autoimmune encephalitides (AE), Patients were included from the National Danish AE cohort (2009-present) and diagnosed based upon autoantibody positivity and diagnostic consensus criteria. CSF-NfL was analyzed by single molecule array technology. Clinical and diagnostic information was retrospectively evaluated and related to NfL levels at baseline and follow-up. NMDAR-AE patients were subdivided into: idiopathic/teratoma associated or secondary NMDAR-AE .A total of 74 CSF samples from 53 AE patients (37 NMDAR and 16 LGI1 positive) were included in the study. Longitudinal CSF-NfL levels was measured in 21 patients. Median follow-up time was 23.8 and 43.9 months for NMDAR and LGI1-AE respectively. Major findings of this study are: i) CSF-NfL levels were higher in LGI1-AE than in idiopathic/teratoma associated NMDAR-AE at diagnosis; ii) CSF-NfL levels in NMDAR-AE patients distinguished idiopathic/teratoma cases from cases with other underlying etiologies  and iii) Elevated CSF-NfL at diagnosis seems to be associated with worse long-term disease outcomes in both NMDAR and LGI1-AE.CSF-NfL measurement may be beneficial as a prognostic biomarker in NMDAR and LGI1-AE, and high CSF-NfL could foster search for underlying etiologies in NMDAR-AE. Further studies on larger cohorts, using standardized methods, are warranted. N-methyl-D-aspartate receptor encephalitis (NMDAR-AE) and Leucine-rich Glioma-Inactivated 1 encephalitis (LGI1-AE)  are LGI1-AE) . LGI1-AELGI1-AE) –7. WhileLGI1-AE) –10. NMDALGI1-AE) , 11–13.AE’s can be challenging to treat, and good biomarkers to assist physician’s decision-making are scarce. Naturally, immunological biomarkers have been in focus in AE patients. Thus, Chemokine (C-X-C motif) ligand 13 (CXCL-13) has been reported as a potential biomarker of treatment response and relapse rate in a bigger cohort of NMDAR-AE patients, but long-term outcomes in relation to CXCL-13 remains to be further elucidated . SimilarNeurofilament light chain (NfL) are scaffolding proteins solely expressed in the neuronal cytoskeleton and a reliable indicator of axonal damage. They have been studied as emerging biomarkers of neurodegeneration, but also increasingly as a biomarker in neuroinflammation-related axonal damage. NfL has been shown to correlate with disease severity and progression/outcome in diseases like optic neuritis, multiple sclerosis, neurosarcoidosis and myelin oligodendrocyte glycoprotein associated diseases –27.Here we investigate CSF-NfL levels in the acute phase of NMDAR- and LGI1-AE patients and evaluate its utility as a diagnostic and prognostic biomarker in the two commonest types of AE.From 2009-2018, 74 CSF samples (49 NMDAR and 25 LGI1 IgG positive) from 53 patients (37 NMDAR-AE and 16 LGI1-AE) were obtained from the two only test centers in Denmark . Medical charts and diagnostic work-up were retrospectively evaluated using the consensus criteria for NMDAR-AE and autoimmune limbic encephalitis (LE) . Thus, aAntibody positivity was reported qualitatively as weakly (+), moderately (++) and strongly positive (+++) and based on a commercial cell-based assay . Abnormal CSF was defined as pleocytosis (>5 cells/μL), elevated protein levels and/or presence of CSF specific oligoclonal bands (OCB). An abnormal MRI was defined as T2/FLAIR hyperintensities in NMDAR-AE and temporal lobe T2/FLAIR hyperintensities in LGI1-AE. The modified Rankin Scale (mRS) was used to determine neurological disability at last follow-up. A mRS score > 2 was considered a poor outcome and last follow-up was the last obtainable clinical neurological assessment. The CSF sample NfL was measured on, was obtained within one month of diagnosis, and prior to any treatment in all patients.CSF-NfL was measured blinded to clinical data by single molecule array technology ), using a commercial NfL assay . CSF samples used were routine samples from patient’s initial hospital stay, stored at -80°C. Quality control was performed using two commercial controls provided by the manufacturer. In addition, an in-house CSF pool, from routine CSF samples, was used as internal control. Lower limit of detection and quantification was 0.038 pg/mL and 0.174 pg/mL, respectively. Age adjusted CSF-NfL values were determined according to a previous study and applied when performing univariable analysis . Thus, eEthical approval was obtained by the Danish Medical Authorities (3-3013-3124/1) and the Danish Data Protection Agency, with waived requirement for participant consent.Stata Statistical Software: Release 17. College Station, TX: StataCorp LLC). NfL data were skewed and thus natural logarithmic transformed. Post-HSE and secondary NMDAR-AE forms were excluded from all stastistical analyses  and stataCorp. 2021  were included in this study. Twenty-three (62%) NMDAR-AE patients were female with a median age of 27 years Table 1.All NMDAR-AE patients had a classic phenotype with a variety of changed behavior, memory impairment, psychiatric symptoms, insomnia, reduced verbal output, decreased consciousness and dysautonomia. Five patients had underlying tumors . Three patients had concomitant demyelinating diseases; two multiple sclerosis and one acute disseminated encephalomyelitis. The three patients with teratomas showed a similar phenotype and disease course to idiopathic NMDARE patients, while patients with malignancies and demyelinating diseases showed a more extensive clinical syndrome than regular AE. NMDAR-AE patients were therefor subdivided into three groups: idiopathic/teratoma associated, secondary post-HSE and secondary other  Table 1.The median CSF-NfL concentration at diagnosis in LGI1-AE patients (n=16) was higher than in idiopathic/teratoma associated NMDAR-AE (n=27)    of the LGI1-AE patients had autoantibodies present in the CSF at diagnosis   similarly revealed that higher CSF-NfL at diagnosis was associated with a poorer outcome in idiopathic/teratoma associated NMDAR-AE and LGI1-AE patients combined  Table 2.We examined the relation between CSF-NfL levels and neuronal surface autoantibody titers, clinical presentation, ancillary findings and long-term outcomes in a national AE cohort, including 74 CSF samples from 53 patients. We found differences in CSF-NfL levels not only between NMDAR- and LGI-AE patients, but also between different NMDAR-AE etiologies. NfL levels at diagnosis appeared to be associated with long-term outcomes in both our univariable and multivariable analysis. This is in line with not only previous findings in other inflammatory diseases, but also a smaller study in AE , 24, 31.We evaluated NMDAR-AE patients based on underlying etiology and found that gender distribution, median age and outcomes were widely different between idiopathic NMDAR-AE or NMDAR-AE secondary to HSE or concomitant with malignancies or demyelinating disease. Teratoma patients were grouped with idiopathic NMDAR-AE, as they turned out to present a similar clinical phenotype and disease course, although a paraneoplastic phenomenon. In addition, our study revealed that CSF-NfL levels were lower at diagnosis in idiopathic NMDAR-AE (teratomas included) when compared to secondary NMDAR-AE forms.Higher CSF-NfL values in NMDAR-AE patients reported by previous studies may reflect combined evaluation of all NMDAR-AE etiologies , 32, 34.A recent study reported lower CSF-NfL values in patients with ovarian teratoma . In lineOur study suggests that idiopathic and teratoma induced NMDAR-AE patients have lower CSF-NfL levels than patients with LGI1-AE at disease onset. This is in accordance with two previous studies and could be expected due to age differences between the diseases , 33. NevIt has been shown that intrathecal LGI1-IgG synthesis correlates with worse outcome, suggestive of greater neuronal injury in these patients . LGI1-AEFollow-up measurements in our AE cohort indicated steadily decreasing levels in NMDAR-AE and a more heterogenous pattern in LGI1-AE patients. This might reflect a subtle, but ongoing, neurodegeneration in LGI1-AE patients, even during recovery. A recent study reported persisting long-term cognitive impairment in LGI1-AE, but whether these are temporarily progressing is unknown . The oneLGI1-AE patients with abnormal CSF findings and with hyponatremia had the highest CSF-NfL concentrations. This may reflect, that patients with CSF abnormalities  are more severely affected by inflammation. The mechanisms behind the hyponatremia in LGI1-AE is speculated to be caused by hypothalamic neuronal impairment or LGI1 channel dysregulation in the renal epithelium. Higher CSF-NfL levels in patients with hyponatremia may reflect a more widespread disease, affecting the brain more globally and with a greater systemic affection compared to patients without hyponatremia.We found no connection between elevated CSF-NfL levels and seizure activity or ICU admission. To our knowledge, scarce publications on seizures/epilepsy and serum NfL levels exist, that show none or sparse temporal elevation of NfL levels after seizure activity .When comparing studies on NfL, the method used for evaluation is of essence. We used Simoa technology which has a higher sensitivity than ELISA and Electrochemiluminescence assays . Thus, SReference values and clinical validated cut-off values (decision limits) for NfL levels are still lacking. Further studies on paired CSF and serum samples, including healthy controls, would be beneficial to enlighten NFL’s utility as a disease monitoring entity.The strength of our study is the national NMDAR- and LGI1-AE cohort database used to provide accurate clinical and paraclinical information, coupled with a long follow-up time and sensitive CSF-NfL testing on Simoa. This allowed us to test a substantial number of patients.Limitations include the small sample size and the retrospective approach, which constrained statistical analysis. However, our main aim was to evaluate CSF-NfL levels at time of diagnosis and relate to long-term outcomes. Performing exploratory univariable analysis, as we do in this study, composes a risk of overinterpretation and overlooking potential confounders. To accommodate this, we performed multivariable testing. However, due to the sample size, the model could only fit three predictors, and unfortunately did not allow to adjust for antibody subtype (NMDAR or LGI1) or treatment regimen (secondline), which are two important variables , 43, 44.Using the mRS to evaluate long-term outcome of AE patients comprises a restriction, as recent studies have shown that AE patients often suffer from cognitive impairment even years after treatment , 46. ConIn conclusion, CSF-NfL levels at diagnosis were higher in LGI1-AE patients than in idiopathic and teratoma associated NMDAR-AE and our data indicate that high CSF-NfL may relate to worse long-term outcome. Furthermore, NfL levels might help distinguish idiopathic/teratoma associated NMDAR-AE from secondary NMDAR-AE forms. Additional studies on larger cohorts, with standardized methods and cut-off values are warranted to further validate CSF-NfL as a potential biomarker in AE.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Danish Medical Authorities (3-3013-3214/1), the Ethical Committee of the Region of Southern Denmark (nr. S-20180026) and the Danish Data Protection Agency. Written informed consent from the participants’ legal guardian/next of kin was not required to participate in this study in accordance with the national legislation and the institutional requirements.MN and MB, drafting/revision of the manuscript for content, including medical writing for content, major role in the acquisition of data, study concept or design, and analysis or interpretation of data. MR, JM, and DO, drafting/revision of the manuscript for content, including medical writing for content, and analysis or interpretation of data. UH, major role in analysis and interpretation of data. AN and ML, drafting/revision of the manuscript for content, including medical writing for content, and major role in the acquisition of data. ZI, drafting/revision of the manuscript for content, including medical writing for content, study concept or design, and analysis or interpretation of data. All authors contributed to the article and approved the submitted version.MN received funding from Odense University Hospital and the University of Southern Denmark.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.