CircZBTB46 exerts a critical role in promoting the expression of SCD to protect AML cells from ferroptotic cell death. Importantly, our findings may offer a potential therapeutic target, circZBTB46, to broaden treatment options for human AML, especially concerning the use of combined treatment with ferroptosis inducers.It is urgent to identify new biomarkers for diagnosis, prognostication, and therapeutic targets of acute myeloid leukemia (AML) so as to develop more effective surveillance and treatment programs. Ferroptosis is a crucial, iron-dependent regulated cell death driven by excessive accumulation of lipid hydroperoxides. Accumulating evidence has proven that dysregulated ferroptosis is implicated in tumor progression and is becoming an emerging therapeutic target for the treatment of AML. However, the effect of circRNAs on ferroptosis and the development of AML remain unclear. In this study, we provide the first line of evidence that circZBTB46 was significantly upregulated in AML patients and AML cells. Moreover, the expression of circZBTB46 was associated with the stages of AML patients and showed high sensitivity and specificity for diagnosing AML. Silencing of circZBTB46 inhibited AML cell proliferation and induced cell cycle arrest. Importantly, the depletion of circZBTB46 notably increased ferroptosis and enhanced RSL3-induced ferroptosis in AML cells. Mechanistically, circZBTB46 upregulated the expression of stearoyl-CoA desaturase 1 (SCD) possibly by acting as a miRNA sponge. Finally, the circZBTB46 knockdown repressed the tumor growth of AML in vivo. In conclusion, circZBTB46 protects AML cells from ferroptosis and promotes the proliferation by upregulating SCD, thus suggesting that circZBTB46 may be a potential therapeutic target for AML.Circular RNAs (circRNAs) have been shown to be closely linked to the tumorigenesis and treatment response of hematological malignancies. However, the biological functions and clinical implications of circRNAs in acute myeloid leukemia (AML) remain largely unknown. CircRNA microarray datasets were analyzed to screen differentially expressed circRNAs in AML patients. It was found that  Acute myeloid leukemia (AML) is a clonal hematopoietic stem cell malignancy characterized by an accumulation of immature progenitor cells with arrested differentiation, thus resulting in suppression of hematopoiesis . As the Circular RNAs (circRNAs) are a novel type of ubiquitous, stable, and conserved noncoding RNA (ncRNA), which, unlike linear RNAs, have covalently closed structures and lack the 3′- and 5′-ends . CircRNAZBTB46) gene (termed circZBTB46 hereafter), was significantly upregulated in AML and was associated with stages of AML. Moreover, circZBTB46 exhibited high sensitivity, specificity, and diagnostic simplicity by evaluation not only in bone marrow (BM) but also in peripheral blood (PB) samples. Importantly, circZBTB46 knockdown significantly impeded the proliferation of AML cells and induced ferroptosis. Furthermore, we found that circZBTB46 executed its regulatory functions by upregulating SCD. Therefore, inhibiting circZBTB46 may provide a therapeutic opportunity and a unique synergistic antitumor effect with ferroptosis inducers for AML.In this study, we sought to explore the roles played by circRNAs in AML. Our screening strategy recognized that hsa_circ_0002805, a circRNA derived from the zinc finger and BTB domain containing 46 (p < 0.05) between AML patients and healthy subjects were identified through bioinformatic analysis. A cutoff fold change (FC) of 4 was applied to select the markedly upregulated circRNAs in AML.CircRNA microarray datasets GSE94591  and GSE163386  were downloaded from the Gene Expression Omnibus (GEO) database. Both of the microarray datasets used the Arraystar human circRNA microarray to analyze the circRNAs’ profiles in bone marrow samples as based on the GPL19978 platform. Differentially expressed circRNAs  samples were collected from 25 AML patients and 25 age- and gender-matched (1:1) healthy subjects at the Third Xiangya Hospital of Central South University. Bone marrow (BM) samples were obtained from 18 out of the 25 AML patients and 9 out of the 25 healthy volunteers. After standard treatment, 12 out of the 25 AML patients achieved complete remission (CR), and 7 out of the 12 CR patients relapsed from remission during the follow-up, whose PB samples were also collected. All clinical samples were obtained with the informed consent of the patients, and the study was approved by the Ethics Committee of the Third Xiangya Hospital of Central South University (No. 22186) according to the principles of the Declaration of Helsinki.2.Human AML cell lines , and the human BM stromal cell line (HS-5) were purchased from the American Type Culture Collection  and were cultured in RPMI 1640  with 10% fetal bovine serum . All cells were cultured at 37 °C in a humidified atmosphere containing 5% CO® Reagent  according to the manufacturer’s instructions. Nuclear and cytoplasmic RNA were isolated from AML cells using a Cytoplasmic & Nuclear RNA Purification Kit . Reverse transcription was performed using ReverTra Ace qPCR RT Master Mix with gDNA Remover , and qRT–PCR was performed using a KOD SYBR® qPCR Mix Kit  according to the manufacturer’s protocol. The relative expressions of circRNAs and mRNAs were calculated using the 2−ΔCt method (ΔCt = CtcircZBTB46 − CtGAPDH), whereas the fold change (FC) was calculated using the 2−ΔΔCt method (ΔΔCt = ΔCttest − ΔCtcontrol), and GAPDH was utilized as an internal control. All primer pairs were designed and synthesized by Tsingke Biotechnology Co., Ltd. , and the sequences of the primers are listed in Total RNA was extracted using TRIzolcircZBTB46 and ZBTB46 mRNA. For the actinomycin D assay, AML cells were exposed to 3 µg/mL actinomycin D  to block gene transcription for 4, 8, 12, and 24 h. Then, the cells were harvested, the total RNA was extracted, and the half-lives of the circZBTB46 and ZBTB46 mRNA were analyzed using qRT–PCR.For the RNase treatment assay, 2 μg of total RNA was incubated with 3 U/μg RNase R  at 37 °C for 30 min, and RNase-free water was used as a control (Mock). The digested RNA was subsequently purified with an RNeasy MinElute Cleaning Kit , and then qRT–PCR was performed to detect the abundance of circZBTB46 were designed and synthesized by GenePharma . Lentivirus containing shRNA targeting circZBTB46 were purchased from GeneChem . The full-length sequence of circZBTB46 was amplified and cloned into a circRNA-specific overexpression lentiviral vector, GV689 . SCD overexpression plasmids (GV367) were also purchased from GeneChem . Lipofectamine 3000  was used for the cell transfection according to the manufacturer’s instructions. Forty-eight hours post-transfection, the knockdown or overexpression efficiencies of the corresponding genes were evaluated using qRT–PCR. The siRNA sequences are: si-circZBTB46#1, GCTGTCCCAGTCTGTAGAAGA-dTdT; si-circZBTB46#2, CACTCGCTGTCCCAGTCTGTA-dTdT.Two siRNAs targeting the head-to-head splicing junction of 2. Then, 10 μL of the CCK-8 solution was supplemented into each well at the indicated time points , and the cells were incubated at 37 °C for 2 h. Finally, the absorbance of the cells in each well was measured using a BioTek microplate reader  at a wavelength of 450 nm.The proliferation of AML cells was assessed with the CCK-8 . Treated cells were seeded into 96-well plates containing 100 µL of medium and cultured at 37 °C with 5% COTreated AML cells were fixed using 70% cold absolute ethyl alcohol and stained with propidium iodide  containing RNase A for 30 min at 37 °C. Cells were analyzed for DNA content using flow cytometry , and ModFit software was used for data analysis.2. The next day, cells were incubated with the indicated treatments. After that, the cells were stained with propidium iodide (PI) for 30 min at 37 °C. Cells were also stained with DAPI to visualize the nuclei. A fluorescence microscope  was used to capture images at 100× magnification.Cells were seeded into 6-well plates and cultured at 37 °C with 5% COA BODIPY 581/591 C11 probe  was used to detect lipid ROS according to the manufacturer’s protocol. Treated cells were incubated with BODIPY 581/591 C11 at a final concentration of 3 µM at 37 °C for 30 min. Then, cells were washed three times with PBS and were measured using flow cytometry . The MDA levels of the treated cells were detected using an MDA colorimetric assay kit  according to the manufacturer’s instructions.The EZ-Magna RNA-Binding Protein Immunoprecipitation Kit  was utilized to validate the interaction between RNA and RNA-binding protein (RBP) according to the manufacturer’s instructions. In brief, cells were lysed in RIP lysis buffer, and the cell lysates were then incubated with RIP buffer containing magnetic beads conjugated with antibodies at 4 °C overnight. Finally, RNA was extracted from the RNA-protein-bead complexes and subjected to qRT–PCR to calculate enrichment. The following antibodies were used in this assay: anti-AGO2  and IgG .7 AML cells were harvested and lysed. The biotin-labeled circZBTB46-specific probe, or NC probe, was incubated with streptavidin-coupled dynabeads at room temperature for more than 30 min to generate probe-bound dynabeads. After the treated beads were washed, the RNA complexes bound to the beads were isolated and further analyzed using qRT–PCR.The RNA pulldown assay was performed using a Pierce™ Magnetic RNA-Protein Pull-Down Kit  according to the manufacturer’s protocol. In brief, a total of 1 × 10A total protein extraction kit  was used to extract total protein from cells, and the protein was quantified using a BCA kit  and following the manufacturer’s protocol. Protein samples were then separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes . After blocking with 5% defatted milk at room temperature for 2 h, the membranes were washed with Tris buffered saline Tween, and then incubated with primary antibodies at 4 °C overnight. The next day, the membranes were incubated with a secondary antibody at room temperature for 1 h, followed by the visualization of the signals on the membranes using an Odyssey CLx Infrared Imaging System . The following antibodies were used in this assay: anti-SCD  and anti-GAPDH . Uncropped Western blot images can be found in 6sh-NC or sh-circZBTB46 HL-60 cells in 150 μL of Matrigel solution into the left armpit of each mouse (n = 5 mice/group). Four weeks after inoculation, xenografted mice were euthanized for analysis. The tumor volume was calculated as 0.5 × length × width2.Five-week-old male NOD-SCID mice were maintained under specific pathogen-free (SPF) conditions in the Department of Laboratory Animals of Central South University , and procedures were performed according to the institutional ethical guidelines for animal experiments. NOD-SCID mice were randomly split into 2 groups, and the xenografted AML in these mice was generated by injecting 5 × 10http://www.circbank.cn/index.html (accessed on 22 July 2022)), circInteractome (https://circinteractome.nia.nih.gov/index.html (accessed on 22 July 2022)), and ENCORI (https://starbase.sysu.edu.cn/ (accessed on 22 July 2022)), were used to predict candidate miRNAs interacting with circZBTB46. The target genes of miRNAs were collected from miRTarBase (https://mirtarbase.cuhk.edu.cn/ (accessed on 1 August 2022)), ENCORI (https://starbase.sysu.edu.cn/ (accessed on 1 August 2022)), and TargetScan (https://www.targetscan.org/vert_80/ (accessed on 1 August 2022)). A circRNA-miRNA-mRNA crosstalk network was built using Cytoscape software (Version 3.8.2). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses for target genes were performed using the clusterProfile R package (Version 4.2.1), and the results were presented as graphs using the ggplot R package.Three open-access databases, including circBank  as indicated in the corresponding figure legends. Receiver operating characteristic curve (ROC) was used to assess the value of circZBTB46 as a diagnostic indicator in AML. In addition, the areas under the curves (AUC) were calculated. All calculations were performed using GraphPad Prism version 8.0.1 , and a two-tailed p < 0.05 was considered to indicate a statistically significant difference.Experiments were repeated at least three times, and one representative experiment was shown in the results. Error bars represented the mean plus or minus standard deviation of the mean. Statistical analysis was performed using either Student’s p < 0.05, Log2 FC > 2.0) (hsa_circRNA_103104 and hsa_circRNA_100199) were identified  had been verified to be specifically accumulated in AML and to play a pathogenic role in AML [hsa_circRNA_103104, which has not been studied. By searching the human reference genome (GRCh37/hg19), we found that hsa_circRNA_103104 was generated from the exons 2 and 3 regions within the ZBTB46 gene locus; thus, we termed it as circZBTB46. In GSE94591 and GSE163386 microarray datasets, circZBTB46 was remarkably increased in AML patients when compared with healthy controls   screenedC > 2.0) A,B, respentified C. Intrige in AML . Therefocontrols .circZBTB46 expression was consistently and significantly increased in the bone marrow and peripheral blood of patients with AML as compared with healthy controls  after treatment  compared with a bone marrow stromal cell line (HS-5) (circZBTB46 expression was relatively higher in AML cells compared to hematopoietic stem and progenitor cells (HSPCs), whereas ZBTB46 mRNA was expressed at a relatively lower level in AML cells  J. AdditiML cells K. In sumcircZBTB46 in the circBase database (circBase ID: hsa_circ_0002805) and circBank database (circBank ID: hsa_circZBTB46_002), and we found that circZBTB46 was an exonic circRNA generated from chr20: 62407030–62422143 with a splice length of 1255 nucleotides (nt). According to the UCSC Genome Browser (http://genome.ucsc.edu/ (accessed on 7 August 2022)), four other circRNA isoforms, namely hsa_circ_0061176, hsa_circ_0061177, hsa_circ_0061178, and hsa_circ_0061179, were identified in the ZBTB46 gene locus 18 primers (circZBTB46 does not contain a poly(A) tail.Then, we verified the circularization of hodology . Diverge primers B, suggescircZBTB46 in AML cell lines using RNase R or actinomycin D (an inhibitor of DNA transcription) treatment assay. Resistance to digestion with RNase R exonuclease confirmed that circZBTB46 harbored a circular RNA structure  region of circZBTB46 were designed, and they specifically inhibited circZBTB46 expression  but not by the pancaspase inhibitor (ZVAD)   . Thus, tinducer) B, supporcircZBTB46-knockdown cells. The depletion of circZBTB46 increased the levels of lipid ROS in HL-60 cells  in HL-60 cells  to estimate the level of ferroptosis-associated lipid peroxidation in wild-type and 60 cells C and exa60 cells D. Accord60 cells E and enh60 cells F, which xidation G–I despi62 cells . Furtherstatin-1 B,D,F–I. circZBTB46 regulates the proliferation and ferroptosis of AML cells. Given that ACSL4 [GPX4 [SLC7A11 [ALDH3A2 [HMGB1 [circZBTB46 knockdown and overexpression. As shown in circZBTB46 did not significantly change the expression levels of these genes in AML cell lines, indicating that circZBTB46 may have targeted other genes to inhibit ferroptosis of AML cells. Given that cytoplasm-localized circRNAs often participate in translational regulation by acting as competing endogenous RNAs (ceRNAs), we then conducted anti-AGO2 RIP assays to determine whether circZBTB46 functions as a miRNA sponge in AML cells. The results showed that ciRS-7 (a circRNA binding with AGO2) [circZBTB46 were significantly enriched by the anti-AGO2 antibody, but circMYBL2   and circind AGO2 ) was notR-671-5p B. Furthel probes C. These circZBTB46. Based on the miRTarBase, ENCORI, and TargetScan databases, a total of 581 genes were predicted as target genes of the three miRNAs (circZBTB46 shared miRNA response elements (MREs) of miR-326 and miR-671-5p with HEXA, HTT, SCD, TMEM184B, MTHFR, LGALS3BP, HDLBP, and PTBP1, and shared MREs of miR-339-3p and miR-671-5p with TOMM40L, BTBD3, ANKRD52, ELK1, SH3PXD2A, CBX6, and PTBP1  or without (sh-NC) circZBTB46 knockdown. The results showed that tumors derived from circZBTB46-knockdown cells were smaller and weighed less  have been identified and proven to play a vital role in AML [circZBTB46 knockdown significantly induced ferroptosis and enhanced the lethality of ferroptosis inducer, supporting the hypothesis that circZBTB46 acts as an endogenous inhibitor of ferroptosis in AML cells. Ferroptosis, a recently described cell death process, is a crucial, iron-dependent, regulated cell death driven by excessive accumulation of lipid hydroperoxides . During e in AML ,19. Moree in AML ,27,28,29e in AML . In the circZBTB46 may act as a miRNA sponge for miR-326, miR-339-3p, and miR-671-5p in AML cells, a finding based on bioinformatic analysis and validation experiments. Previous studies showed that miR-326 and miR-339-3p are significantly decreased in AML-derived samples [circZBTB46 may mediate the upregulation of some pluripotency genes and play a regulatory role in AML by sponging these miRNAs. The competitive endogenous RNA (ceRNA) hypothesis has attracted notable attention as a unifying function of ncRNAs as well as an alternative function of mRNAs . Most cy samples ,38 and tCircZBTB46 shared miRNA binding sites of miR-671-5p with SCD; we then confirmed the regulatory role of circZBTB46 on SCD in AML cells. Stearoyl-CoA desaturase (SCD), an enzyme that catalyzes the rate-limiting step in monounsaturated fatty acid synthesis, has recently been identified as a ferroptosis-related gene in various cancers, including bladder cancer [circZBTB46 knockdown. Importantly, we substantiated this concept in a mouse xenograft model. Similarly, Dong et al. found that circKDM4C induces ferroptosis by upregulating P53 via sponging hsa-let-7b-5p in AML [circZBTB46 and SCD may play a key role in ferroptotic cell death and identifies circZBTB46 or SCD inhibition as a therapeutic strategy for targeting ferroptosis in AML.r cancer ,41, cervr cancer , ovarianr cancer , and lunr cancer . Emerginr cancer ,44. In ar cancer . In thisp in AML . Our worcircZBTB46 is a critical oncogene as well as a diagnostic and prognostic biomarker for AML. CircZBTB46 exerts important functions in upregulating the expression of SCD to protect AML cells from ferroptotic cell death. Moreover, our findings offer a potential therapeutic target, circZBTB46, which may broaden the treatment options for patients with AML, especially for the application of combined treatments with ferroptosis inducers.In conclusion, we first demonstrated that


The integration of structure and function for tissue engineering scaffolds is of great importance in mimicking native bone tissue. However, in contrast to the increasing demands, how to rapidly prepare various sizes of such Haversian system mimicking scaffolds in batch becomes a major challenge. In this study, we designed three types of platforms with different sizes in combination with the freeze-drying approach. Chitosan/type I collagen composite materials were used to study the structure, morphology, and performance of the production, and the effects of the controlled architecture on osteogenesis. Results showed that the physicochemical effects of the mass fabricated scaffolds of various sizes met the requirements of bone repair materials. In addition, the scaffolds had good cytocompatibility and excellent  Z-direction, the distribution of bone tubules in the bone plate is centered on the Haversian canal in the XY plane , rigidity close area polyurethane , and extruded polystyrene sheets . Chitosan , PBS , type I collagen , and vacuum freeze drier .The platform components models were created using AutoCAD 2019, and three types of the platforms were designed. Based on the cranial defect dimensions used in most Sprague Dawley (SD) rats models, platform B was with the size of Φ5 mm × 1.2 mm. Platform A and platform C were designed with a transverse diameter of c. a. 20 mm for the inner condyles and a transverse diameter of c. a. 30 mm for the outer condyles, for the large animal models, as shown in The difference in thermal conductivity of the materials results in the rate of thermal radiation transfer from the preform being transformed from a uniform undirected value to a vector value with direction and magnitude. The thermal radiation of the preform is transferred faster in one or more directions, resulting in the ice crystals in that direction being the first to form and occurring of phase separation; the ice crystals will always grow in that direction as they conduct heat faster than the water phase which has not yet phase separated and crystallized. The specific parameters of the preliminary modeling are as follows.Platform A design data: Thermally conductive component a: overall size 100 mm × 100 mm × 3 mm, six evenly spaced holes, 20 mm diameter, 1.2 mm length, and bottom surface taken out 80 mm × 80 mm × 1.8 mm. Thermally conductive component b: overall volume 200 mm × 200 mm × 19 mm. Thermally insulation component a: overall volume 80 mm × 80 mm × 10 mm and two pieces. Thermally conductive placeholder component a: 10 mm diameter and 50 mm height.Platform B design data: Thermally conductive component c: overall volume size 50 mm × 50 mm × 3 mm, evenly distributed six holes, hole diameter 5 mm, hole length 1.2 mm, and bottom surface taken out 30 mm × 30 mm × 1.8 mm. Thermally insulation component b: overall volume 30 mm × 30 mm × 10 mm and two pieces. Thermally conductive placeholder component b: overall size 2 mm diameter and 50 mm height.Platform C design data: Thermally conductive component d: overall volume 200 mm × 200 mm × 20 mm, six perforated holes, 30 mm diameter, and 5 mm hole length. Thermally insulation component c: overall volume 200 mm × 200 mm × 30 mm.4Col50 and CS4Col25 pre-solutions; 50 ml 2% CS solution was added 0.5 and 0.25 g type I collagen, respectively, to obtain CS2Col50 and CS2Col25 pre-solutions.A volume of 50 ml 4% chitosan (CS) solution was added with 1 and 0.5 g type I collagen, respectively, to obtain CS2Col25 pre-solution was added to a 30-ml screw-top vial. The solution was mixed with a cross-linking agent and kept stirring. The solution was left to stand for 12 h, the pH was adjusted to 7, placed in a −20°C environment for 12 h, and then freeze-dried for 24 h to obtain CS2Col25 samples. CS2Col50, CS4Col50, and CS4Col25 were used as the CSCol experimental groups and CS2Col25 as the CSCol control group and placed in a 4°C environment until use. The scaffolds made by different types of the work platforms were named ACSCol, BCSCol, and CCSCol, respectively.The experimental solution was fed into the work platforms, and the CSPlatform A and B operation as follows. The thermally conductive component a and the thermal insulation component a are assembled into the sample tank component a. The pre-solutions and cross-linking agent are added to the sample tank component a. The sample tank component a and thermally conductive placeholder component a are assembled into the molding component a. The overall ambient temperature of the bio-incubator and thermally conductive component b is adjusted to −50°C using liquid nitrogen. Overall ambient temperature is maintained at this temperature until ready for use. The molding component a is put with the bio-incubator. Then the bio-incubator is closed. After 30 min, the molding component a is removed and freeze-dried for 24 h. The thermal insulation component a and thermally conductive placeholder component a are removed from the molding component a; the thermally conductive component a is kept upside down on the thermally conductive component b, and this forms the molding component b. The molding component b is kept in the bio-thermal adjusted overall ambient temperature to 4°C. Pre-solutions and cross-linking are added to the remaining space. The overall ambient temperature is lowered to −50°C. After 30 min, thermally conductive component a is freeze-dried for 24 h.Platform C operation as follows. The pre-solutions and cross-linking agent are kept in the hole of thermally conductive component d. The overall ambient temperature of the bio-incubator is adjusted to −50°C using liquid nitrogen. Thermally conductive component d is put with the bio-incubator. Then the bio-incubator is closed. After 30 min, the thermally conductive component d is freeze-dried for 24 h.The scaffold morphology was observed and analyzed by using a scanning electron microscope , Fourier transform infrared spectrometer (FT-IR), and GT; the porosity of the gel scaffold was tabulated using the liquid displacement method . SpecifiThe macroscopic structure of the ACSCol and CCSCol scaffolds was observed by stereomicroscopy. The macroscopic structure of the BCSCol and the microscopic structure of the scaffolds were observed by SEM. The cross-sectional morphology and pores were analyzed.The mechanical properties of each group of gel scaffolds were analyzed by a stress and strain analysis using electromechanical universal testing machines . In total, three sets of parallel tests were made for each sample in both directions and averaged. The compression rate was 1 mm/min and the strain was set at 70%/60%.The porosity of the scaffolds was tabulated using the liquid displacement method. The volume of the gel scaffold was recorded as V, the container full of ethanol was obtained as M1, and the remaining ethanol in the container after the scaffold was immersed was recorded as M2.Cell activity was assessed using acridine orange/ethidium bromide (AO/EB) staining, and at the appropriate time points, AO/EB working solution was added to each well and cell activity was recorded using an inverted fluorescence microscope  observations. The effect of CSCol on cell proliferation was probed using a Cell Counting Kit-8 (CCK-8). After culturing human umbilical vein endothelial cells (HUVEC) for 1, 3, and 5 days, cell proliferation was detected using the CCK-8 kit, and the optical density (OD) of each well was recorded at 450 nm using a multifunctional microplate reader  .SD rats were anesthetized with sodium pentobarbital intraperitoneally; the right leg was shaved at the knee joint to expose the surgical site, and the skin was disinfected with iodine. A 1-cm linear skin incision was made at the distal femoral epiphysis of both hind limbs and the muscle was bluntly dissected to expose the femoral condyles. A 3-mm diameter to 3-mm depth osteotomy perpendicular to the bone axis is drilled at a slow pace using a dental ring drill. After rearing to week 1 (1W)/2W/4W, the rats were anesthetized and the material was taken. The material was processed for hematoxylin-eosin (H&E) staining, Masson’s staining, TRAP staining, immunofluorescence staining, immunohistochemical staining, and micro computed tomography (micro-CT) imaging for observation and evaluation .The platform is based on bi-directional freezing, which is a step up from directional freezing. The essence of directional freezing technology is to transform the temperature conduction from a three-dimensional non-vector value to an approximate two-dimensional vector value, which requires human control of the temperature conduction speed and direction to form a differential thermal conductivity; bi-directional freezing is to transform the temperature conduction into two or several approximate two-dimensional vector values. The platform design utilizes materials with different rates of thermal conductivity to absorb heat the precast fluid differently at different locations, resulting in bi-directional freezing of the precast fluid and ultimately the preparation of mimicking Haversian system gel scaffolds. The role of the bio-thermal chamber component is to stabilize the low temperature environment around the molding components a, b, c, and d and the thermally conductive component d during platform operation. The thermal conductivity of pure copper is known to be better among common materials, slightly less than precious metals such as pure silver and graphene, which are expensive, and the processing of graphene or graphite is difficult. Pure copper is divided into T1, T2, and T3 licenses according to Chinese national standards, of which T1 copper has the least number of impurities, and fewer impurities can make the thermal conductivity of the material closer to isotropic, which is conducive to the preparation of gels.2(OH)2CO3 and Cu(HCO3)2 because the product is relatively single; the later platform maintenance is easy and efficient and conducive to extending the platform life.After research, high purity copper and most biocompatible materials and their cross-linking agent short contact reaction is not violent after long contact with water, and carbon dioxide and oxygen are easy to form CuThere is a dimensional difference between the thermally conductive components of the three types of platforms, but none of them are welded. The welding process is likely to lead to changes in the density, strength, thermal conductivity, and specific heat capacity of the components and raw materials; the prefabricated liquid is easy to react with the raw materials and lead to corrosion of the components; and the liquid nitrogen quenching process has a great chance of weakening the strength of the welded joints, leading to shortening the service life of the platform and increasing the maintenance cost of the platform.The platform is a modular solution to the need for rapid fabrication of different parts and different defect repairs. The design of the modules is based on relative thermal conductivity volumes. Module designs are based on relative thermal conductivity volumes and temperature transfer vectors.The use of materials with low thermal conductivity to control heat transfer can lead to an increase in the vector value of differential thermal conductivity. By the common materials thermal conductivity and national standards in the selection, rigid closed-cell polyurethane and XPS thermal conductivity is low and closed-cell rate is high so that when it receives extrusion it will not immediately expel gas in the material, to protect the thermal insulation performance and its mechanical properties, high closed-cell rate so that the thermal insulation components a will not be infiltrated by the prefabricated liquid, conducive to quantitative prefabricated liquid and waterproof performance. In summary, rigid closed-cell polyurethane and XPS meet the requirements for platform erection due to their processing properties, water resistance, and mechanical properties. Compared to rigid closed-cell polyurethane, XPS is simpler to prepare on the basis of sacrificing certain performance and meets the design purpose of the C-shaped platform, so it was replaced.The platform component test results compared to Chinese national standards have been placed in support of the information .This chapter only discusses the relevance of the choice of materials for building platform preparation to the principle of oriented freezing, and the principle of gel scaffold preparation will be discussed in conjunction with the analysis of gel scaffold morphology.2Col25 samples had no significant differences in the morphology of the bi-directional cut profiles in the three types of platforms, which are homogeneous, disordered and unoriented structures. Other CSCol scaffolds show arranged bi-directional alignment morphologies, wherein the outer oriented channels radiate from the center core, which forms a longitudinal, highly oriented honeycomb canal. The size of the pores was in the range of 80–300 μm, similar to that of the Haversian Canals in the human Haversian system. Compared with the CS2Col50, the structures of the CS4Col50 scaffolds are denser, due to the larger feeding concentration, ice crystal formation and the bi-directional compression force of the sample tank compartment. As can be seen from It was observed that the CSThe small holes in the walls of the aligned channels are in fact the “defects” that occurred when the other two phases were unable to resist the two-way compressive stress of the ice crystals and the bulk at low temperatures, and it is these defects that allows the ice on either side to be joined together. These “defects” cause the collapse of the oriented pores purely in terms of the mechanical stability of the materials but have the advantages that the size is both the minimum size of the endothelial cells and the effective transport of nutrients between the oriented pores, so that these “defects” can effectively increase biocompatibility. The small pores, mimic the Volkmann canal of Haversian system, can provide a constant supply of nutrients to the cells in the oriented pores.4Col25 showed a similar situation, while CS2Col50 of the three types of platform types were the most stable with the least sample variation. No significant interfacial effects were found at any of the interfaces at the junctions of the bi-directional pores, and the spatial connection between the radially oriented pores and the axially oriented pores was good. It can be concluded that an increase in parameters proportional to the bi-directional compressive stress  will lead to the growth of oriented pores.The pore lengths were affected by the bi-directional compression forces failing to extend to the top of the material, in fact a very few samples of CSThe forming mechanism of the three types of platforms was analyzed in conjunction with SEM. After the molding component a is placed in the bio-thermal incubator at a constant temperature, the already cold thermally conductive component b is brought into contact with the thermally conductive component a via a connector, and only 5 min later the temperature of the thermally conductive component a has dropped below 5°C when the liquid nitrogen is added for the first time under the infrared temperature measurement gun, while the thermal insulation component a remains above 10°C. When the incubator is opened for the second time after 10 min and the liquid nitrogen is added, the temperature of the thermally conductive component has dropped to about 1°C, while the thermal insulation component a was still maintained at about 3°C. At this point the low temperature of the thermally conductive component a had started to induce a phase transition in the water, which then caused the ice crystals in contact with the thermally conductive component a to start to grow radially centripetally along the circular hole, and by 15 min the temperature of the thermally conductive component a had dropped to below 0°C, while the thermal insulation component a had also dropped to around 1°C. The temperature of the upper thermal insulation cover was tested at around 5°C, and Thermally conductive placeholder component a was around 1°C. As the ss316 used in the occupancy device was also very fast in conducting heat at 25 min the overall temperature of all components of the molding component a had fallen below 0°C, indicating that the preform had completely completed its phase transition. The freeze-dried ice crystals sublimate leaving radially oriented pores.Unlike the molding component a, the molding component b has the thermally conductive component b. Due to the relatively large area of the direct contact the thermally conductive component b, the thermal conductivity of the thermally conductive component b is superior by virtue of its larger cross-sectional area and direct contact with the liquid nitrogen, and the effect of differential thermal conductivity can be achieved without pre-cooling. The ice crystals start to grow from the bottom and eventually form axially oriented pores throughout the central part after freeze-drying. The overall temperature change was also slightly different, as the thermally conductive component b was in direct contact with the liquid nitrogen, which resulted in the temperature of the thermally conductive component b dropping to around 3°C by 5 min. The temperature of the gel in the centripetal aperture was then affected by the thermally conductive component a. The temperature of the thermally conductive component a also dropped to around 5°C. At 10 min the temperature of both the thermally conductive component a and the thermally conductive component b reached around 0°C. As the temperature of the sample could not be measured directly, the whole platform was kept in operation for 30 min to ensure that all the water in the sample had completed its phase transition. The gel in the radially centripetal oriented pore section was converted to a hydrogel due to direct contact with a small portion of the prefabricated liquid hydrogel that had not yet been lyophilized, which resulted in the overall gel not showing a significant interface after freeze-dried.It is worth noting that the temperature of both the molding component a and the molding component b decreases more and more slowly with time, the rate at which the temperature decreases imply the rate at which the aqueous phase separates and freezes, as will be explained in the next paragraph in relation to the phase transition and separation and the generation of oriented pores.When the two-way compressive stress is applied to the water molecules that have not yet formed ice crystals, the water molecules that have not yet formed ice crystals undergo a relative enthalpy change, and the enthalpy change in thermodynamics is not discussed in depth here. The process of generating ice crystals is an exothermic process, and it is this recurring cycle that makes the temperature drop more and more slowly, but of course this “slow” is a very inaccurate description because the platform environment is around −50°C this “slow” is a relative process, the actual situation is in the it is only at the beginning of the platform’s operation and near −1°C that temperature statistics can be found, and only at the stage near the end of all ice crystal growth is the pressure sufficient to affect the aqueous phase transition temperature, and many experiments have found that at temperatures below −5°C, the temperature of the molding component drops rapidly until it approaches −50°C.In fact, after several attempts, the 20 mm aperture is already the limit size of the thermally conductive component a. For this platform, exceeding the 20 mm aperture will lead to a radially oriented pores that not only cannot be fully extended to the center as the ice crystal extends further and further away from the cold source and the pressure increases under the influence of the lowering of the freezing point the growth rate of the ice crystal slows down making the volume of the ice crystal larger, making it difficult to ensure the size of the aperture This is one of the limitations of this platform.In order to achieve faster thermal conductivity, the platform B was reduced from a 20 mm aperture to a 5 mm aperture and maintained a 1.2 mm aperture length compared to the platform A, resulting in a 93.75% reduction in sample volume and a 75% reduction in the thermally conductive component, which is 125% of the relative volume of the thermally conductive component of the platform A. The reason for maintaining the 1.2 mm aperture is that the efficiency of heat radiation is not only related to the specific heat capacity and volume of the heat transfer material but also to the area of contact. A larger volume of material at the same temperature and specific heat capacity will provide or receive more heat radiation, while a larger contact cross-sectional area at the same temperature, specific heat capacity and volume of material will provide faster heat radiation. The reduction of the radial area is due to the conclusion reached during the preparation of the platform A sample that increase in the parameter that makes the pressure rise in both directions  leads to the growth and elongation of the oriented pores. As a result of this improvement, the length of the radially oriented pores has increased significantly, the number of penetrated axially oriented pores has increased significantly.The overall temperature variation is slightly different for the platform B compared to the platform A. The platform B was not found to find the phenomenon that the temperature of the molding component c and the molding component d decreases more and more slowly with the passage of time, which is due to the small volume of the molding component c and the molding component d itself with small heat travel, large relative area, and strong ability to receive thermal radiation from the prefabricated liquid, and the freezing point reduction brought about by enthalpy change has minimal impact.Compared to the platform A and B the thermally conductive components, the thermally conductive component of the platform C is larger and more difficult to prepare, but still cannot be prepared by the welding process of choice because not only does the density strength change after welding, but welding also has an effect on the thermal conductivity and specific heat capacity, and the welded joints are very susceptible to corrosion. The most fatal problem is that the sudden cooling of liquid nitrogen can significantly weaken the strength of the welded joints and thus shorten the life of the platform and increase maintenance costs. Therefore, the thermally conductive components s a, b, c and d are machined in one piece.The radial dimensions, axial dimensions, overall dimensional uniformity and overall structure of the gel scaffold orientation orifices prepared for the three types of platforms have proven to be satisfactory. For mixed feedstocks with solvents with freezing points below the temperature of liquid nitrogen, the three types of platforms were able to prepare Haversian system mimicking scaffolds, initially achieving the construction of a platform from an individual case to a universal platform. The length of the access pore is one of the limitations of this set of platforms.For a qualitative comparison of the “elasticity”, according to the stress and strain curves and the mechanical properties of the material, there are three stages in the compression process: elastic deformation, inhomogeneous plastic deformation, homogeneous plastic deformation. This is defined by looking at the ratio of the deformation to the total deformation when the upper yield point was reached: the larger the value, the more elastic the material.2Col25 scaffolds. The values in the longitudinal direction are all higher than those in the transverse direction, in some degree.As can be seen from 4Col50 with higher Col content is lower than that of the CS4Col25. In terms of the elasticity of the gel scaffold, CS4Col25 is significantly the stiffest, although it possesses the same collagen concentration with the CS2Col25, and the same chitosan concentration with the CS4Col50. Scaffolds fabricated by the three types of platforms show obvious differences of the strength. BCSCols possess the highest strength and modulus, and CCSCols have the lowest, due to the radial sacrifice of a certain length of oriented pores in platform C.In terms of 70% compressive strength in combination with Young’s modulus data, the scaffolds fabricated with 4%  chitosan solution are better than those with 2% (wt) chitosan solution, mainly due to the increased content of chitosan as the main load-bearing materials. With the loss of the central oriented central core structure support, the chitosan content fraction is less susceptible to intrinsic structural defects with lower porosity. A comparison between the two 4% chitosan groups shows that the compressive strength of the CSThe SEM characterization showed that almost all the pores in the void structure of the bionanogel scaffold were larger than 500 nm, in which case the nitrogen uptake and desorption method was not suitable. Therefore, the liquid displacement method was chosen to characterize the porosity of nanofiber membranes in this topic, calculated as follows:2Col50 has the highest porosity and the lowest in the experimental group is CS4Col50. CS2Col25 should exhibit a high degree of isotropy, but as a direct freeze-dried sample its porosity is affected by the freezing speed and freezing temperature, and the overall stability and structure of the material is difficult to achieve isotropy. CS2Col50 performed very well in this porosity test, with the advantage of a lower feeding volume compared to the other two in the test group, which allowed the ice crystals to grow with less spatial resistance after phase separation. However, the consequence of this porosity is that the CS2Col50 has thinner oriented pore walls, which not only means that the walls themselves are less able to support pressure but also means that adjacent ice crystals are more likely to be connected and create more ‘defects’. As the feed volume increases, the ice crystals grow with greater spatial resistance and smaller pore size. The magnitude of the spatial resistance is not only related to the feed rate but also to the physicochemical properties of the other two phases of the feed material outside of the aqueous phase.As can be seen from The Haversian system is mostly found in the cortical bone, and low bone density or excessive defect size necessarily does not provide space for the Haversian system to exist. This results in the inability to generate tissues such as blood vessels or generating too little of them. It can be concluded from this that the self-healing ability of the organism will be dramatically increased after the generation of tissues such as blood vessels in the presence of mechanically supportive fixation devices, which is the reason why the Haversian system is present in the initial stages of bone defect repair.in vivo through in vitro cell studies, then HUVEC cells are an excellent choice.In summary, if the intention is to examine whether scaffolds can function similarly to the Haversian system in vitro cytocompatibility assay of mimicking Haversian system gel scaffolds. After co-culture of HUVEC cells on the scaffolds for 1, 3 and 5 days, respectively, as shown in 4Col50, BCS4Col50 and CCS4Col50 were all better than other samples in the same group.In order to investigate the cytocompatibility of the scaffolds constructed by different platforms, the growth of HUVEC cell lines after co-culture with the materials was analyzed using cell live-dead staining (AO/EB) as well as cell proliferation assays (CCK-8). The Haversian system is primarily responsible for nutrient transport and angiogenic conduits in the early stages of bone healing, and with this property in mind, umbilical vein vascular endothelial cells were used as experimental cells in the Since all parts of the Haversian system  play an important role in the bone repair process, and these parts cooperate with each other.4Col50 performed better in the cell study, BCS4Col50 was finally chosen as the scaffolds for the animal study.Since the prepared scaffolds mimic a complete Haversian system, the scaffolds used in the animal study should be complete in order to truly take advantage of the significant benefits of the scaffolds. Since the size of the bone defect in the model for the animal study in this study could only accommodate the scaffolds prepared by platform B, it was chosen as the scaffolds for the animal study model, and since BCS4Col50 promoted more new bones. At week 4, the blank control group had fluffy new bone structures, but both experimental groups had significant new bone generation. This may be due to the better mechanical properties of the BCS4Col50 scaffold materials, which can maintain the stability of the scaffolds more effectively and mimic the biomechanics of bone tissues to stimulate new bone regeneration through stress stimulation and postcondition. In addition, the BCS4Col50 scaffold has a rough internal surface and a large number of microchannels, which facilitates the exchange of nutrients and metabolic products within the scaffold, thus facilitating the growth of surrounding tissues and blood vessels.From 4Col50 group was the best in the first 2 weeks. Both material groups showed a lower separation of bone trabeculae compared to the blank control group and exhibited better pro-bone repair. Bone trabeculae thickness failed to show significant changes throughout the repair process, indicating that the bone tissue did not develop severe acute inflammation leading to osteonecrosis or infected osteonecrosis as a result of the defective zone.The macroscopic bone repair capacity of mimicking Haversian system gel scaffolds was further assessed by statistical analysis. Bone density and relative bone volume are relatively important indicators of bone repair and their level can indicate whether the bone discontinuity has occurred. The results show that the both of the values increased with time, wherein the BCSThe reason why Haversian system mimicking scaffolds can enhance osteogenesis is that in the early stages of defect repair, the use of the bionic cavities provided by scaffolds and The good biocompatibility of scaffolds provides space for the growth of tissues such as blood vessels and uses these bionic cavities to transport nutrients and metabolic wastes, thus serving to guide the body to produce new bone and scabs.4Col50 caused moderate inflammation throughout the first week, indicating the initiation of the wound healing process, which is consistent with the statistical analysis of the imaging. At week 2, the BCS4Col50 group showed significantly better bone defect healing than the other groups, indicating that mimicking Haversian system gel scaffolds facilitated bone regeneration. At week 4, new bone tissue such as bone trabeculae and bone units were formed at the bone defect in the BCS4Col50 group. On the other hand, the blank group at week 1, 2 and 4 showed no significant bone crust formation and slow osteogenesis.H&E staining allows clear visualization of the nucleus and ribosomes (blue) as well as the cytoplasm and extracellular matrix (red), as shown in 4Col50 group showed varying degrees of myofibrils, collagen fibers, new blue bone trabeculae and pink striated osteoid, with a large proliferation of fibroblasts, indicating a significantly greater degree of healing than the other two groups, suggesting that mimicking Haversian system gel scaffold facilitated collagen repair at the injury site.Masson’s staining  revealed4Col50 group, osteoclast was significantly less than the blank group and the CS2Col25 group at week 4, indicating that the scaffolds were beneficial to bone reconstruction.Activation of osteoclasts is another important aspect of bone reconstruction. Upon activation, they degrade bone by secreting acid and protein hydrolases such as tissue proteinase K (CTSK), and then dissolve collagen and other matrix proteins produced during bone resorption to absorb aged and damaged bone. However, excessive osteoclasts can lead to bone resorption, so the process of bone reconstruction is a dynamic balance between osteogenesis and osteolysis. The amount of osteoclasts and their status were observed by TRAP staining at the site of bone damage. In the BCS4Col50 group was the highest, higher than that in the CS2Col25 group, indicating that the constructed mimicking Haversian system structure had a significant bone-enabling effect.Immunofluorescence staining of osteocalcin (OCN), osteopontin (OPN), type I collagen (Col-1), osteogenic transcription factor (Runx 2) was utilized to analyze the production of new bone at the site of bone injury, as shown in 4Col50 experimental group was significantly lower than that in the other two groups, indicating that mimicking Haversian system gel scaffolds maybe reduce the occurrence of inflammation through regulation of the microenvironment or new tissue regeneration.Interleukin-6 (IL-6), tumor necrosis factor (TNF-α) and Interleukin-1β (IL-1β) are typical pro-inflammation related factors. From the immunohistochemical results , it was The main innovation of this study is, first, to mimic the structure of the Haversian system formed in the early stage of bone tissue healing and to construct a universal preparation platform based on the directional freezing technology, which can be used to prepare scaffolds with cavities roughly arranged at right angles to each other; second, to use the directional freezing technology to prepare chitosan/collagen mimicking Haversian system gel scaffolds. Physiochemistry, cells, and animal experiments showed that Haversian system mimicking scaffolds possess an anisotropic structure and can effectively accelerate bone regeneration to heal of bone defects.


This phenotype was partially recapitulated by application of an amyloid-β peptide to healthy arteries. Our results will direct further research to restore cerebrovascular function, which is damaged in Alzheimer’s disease, leading to potentially new treatment options.Patients with Alzheimer’s disease show hypoperfusion of the brain and this may contribute to disease progression. To elucidate underlying mechanisms, we studied pial arteries from 18-mo-old mice with Alzheimer’s disease due to overexpression of amyloid precursor protein. We found enhanced pressure-induced constriction of arteries because of reduction in ryanodine receptor-mediated, local calcium-release events (“Ca 2+ sparks”) in arterial smooth muscle cells and a consequent decrease in the activity of large-conductance Ca2+-activated K+ (BK) channels. The ability of the endothelial cell inward rectifier K+ (Kir2.1) channel to cause dilation was also compromised. Acute application of amyloid-β 1-40 peptide to cerebral arteries from wild-type mice partially recapitulated the BK dysfunction seen in APP23 mice but had no effect on Kir2.1 function. If mirrored in human Alzheimer’s disease, these tandem defects in K+ channel-mediated vasodilation could account for the clinical cerebrovascular presentation seen in patients: reduced blood flow and crippled functional hyperemia. These data direct future research toward approaches that reverse this dual vascular channel dysfunction, with the ultimate aim of restoring healthy cerebral blood flow and improving clinical outcomes.The brain microcirculation is increasingly viewed as a potential target for disease-modifying drugs in the treatment of Alzheimer’s disease patients, reflecting a growing appreciation of evidence that cerebral blood flow is compromised in such patients. However, the pathogenic mechanisms in brain resistance arteries underlying blood flow defects have not yet been elucidated. Here we probed the roles of principal vasodilatory pathways in cerebral arteries using the APP23 mouse model of Alzheimer’s disease, in which amyloid precursor protein is increased approximately sevenfold, leading to neuritic plaques and cerebrovascular accumulation of amyloid-β similar to those in patients with Alzheimer’s disease. Pial arteries from APP23 mice (18 mo old) exhibited enhanced pressure-induced (myogenic) constriction because of a profound reduction in ryanodine receptor-mediated, local calcium-release events (“Ca Small vessel diseases of the brain are increasingly recognized as important contributors to the functional and cognitive decline of patients with Alzheimer’s disease (AD) , 2. ThisAD and cerebral amyloid angiopathy are also linked from a pathophysiological perspective, with both conditions being characterized by deposition and accumulation of amyloid β (Aβ) peptide. Two isoforms predominate, with the Aβ(1-40) isoform localizing to the walls of small arteries, whereas the Aβ(1-42) isoform is found in neuritic plaques . PreviouBroadly speaking, the microcirculation of the brain fulfills two key functions: maintenance of blood flow in the face of moment-to-moment changes in blood pressure  and neuronal activity-dependent increases in local blood flow .2+ signals, known as Ca2+ sparks, that reflect release of intracellular Ca2+ through the opening of a small cluster of ryanodine receptors in the sarcoplasmic reticulum membrane  channels in the plasma membrane of vascular smooth muscle cells (VSMCs), resulting in K+ efflux, membrane potential hyperpolarization, deactivation of voltage-dependent L-type Ca2+ channel-mediated Ca2+ influx, and diminished Ca2+-dependent contraction.In the first of these two functions, cerebral arteries  respond to increases in blood pressure with a contractile response that serves to maintain constant flow. One major contributor to cerebral autoregulation is contraction of arterial smooth muscle cells in response to an elevation of pressure, a depolarization-dependent response intrinsic to smooth muscle myocytes termed “myogenic constriction” . In addi+ (Kir2.1) channel  increase in extracellular [K+] induces a retrograde (against the flow of blood) membrane potential hyperpolarization that propagates via ECs of the microvasculature (capillary to arteriole to artery) to induce upstream dilation. The resultant increase in local blood flow toward the site of signal initiation thus supports ongoing neural activity  inwardly rectifying K channel , which aactivity . Interesactivity , whereasactivity . AD appeactivity  and a siactivity .2+ sparks-to-BK coupling, which is responsible for setting basal arterial tone and therefore blood flow to the brain, and endothelial Kir2.1 activity, which in capillaries is crucial to a major neurovascular coupling mechanism underpinning functional hyperemia. We also show that exposure of cerebral arteries from young, healthy wild-type (WT) mice to Aβ(1-40) peptides disrupts the Ca2+ spark–BK channel vasoregulatory mechanism in VSMCs, partially recapitulating the resistance artery dysfunction phenotype seen in the APP23 mouse. From a clinical perspective, both blood flow to the brain and neurovascular coupling are disrupted in patients with AD . Cerebrovascular amyloid staining patterns in APP23 mice were consistent with previous reports .Amyloid was observed throughout the vasculature, including leptomeningeal vessels, in the cortex of 18-mo-old APP23 mice  . BK currents were subsequently isolated using paxilline (1 μM) (i). Paxilline-sensitive currents did not differ between WT and APP23 pial artery VSMCs  . Becausee (1 μM) D, i. Paxry VSMCs D, sugges2+ sparks to activate BK channels. We investigated this by imaging Ca2+ sparks in intact pressurized arteries loaded with Fluo-4-AM using high-speed spinning-disk confocal microscopy (ii). Using our recently developed imaging analysis techniques (iiii) in which each active site within the imaged region is a column, and the Ca2+ events at that site are represented by their spatial spread (width), duration (length), and relative Ca2+ release (intensity). From here, each Ca2+ event was converted into pixels and quantified by calculating an individual Z-score (Zscr) based on the average quiescence intensity and SD of the event (Methods and iiiiii). Since each cell has a different background fluorescence depending on the loading of Fluo-4-AM, pixel conversion removes background noise and allows individual events to be categorized into Ca2+ sparks or waves based on their duration and spatial spread. Ca2+ sparks can be visualized as the repetitive, small dot-like events in ST map columns, whereas Ca2+ waves are larger, usually occupying the width of the column (larger spread) and exhibiting a longer duration. Ca2+ sparks and interspersed Ca2+ waves were clearly detected in cerebral arteries from 18-mo-old WT mice . There were no alterations in Ca2+ wave frequency or amplitude in the APP23 mice compared to WT controls  channels in VSMCs also regulate the membrane potential, and hence diameter, of resistance arteries. However, in experiments designed to study changes in these channels associated with AD, we found that the Kv1 channel blocker, 4-aminopyridine (1 mM) produced statistically indistinguishable effects on the diameter of pressure-constricted pial arteries from WT and APP23 mice , indicating that these channels likely play no role in the increased basal tone in APP23 mice. We did not examine the role of VSMC Kv2.1 channels  and small-conductance (SK) K+ channels. Activation of SK and IK channels results in a membrane hyperpolarization that is transmitted to VSMCs through gap junctions, causing vasodilation. Thus, SK and IK channels can be regarded as important adjuncts in endothelial-dependent hyperpolarization. Using the SK/IK activator NS309 to assess SK/IK-dependent dilation of pressurized arteries, we observed a reduced dilatory effect in arteries from APP23 mice compared with that in WT mice . Focusing on the BK channel, we examined the effect of Aβ(1-40) on STOCs in VSMCs using patch-clamp electrophysiology. Consistent with the observed increase in pressure-induced constriction of the arteries and the reduction in the response of arteries to paxilline, Aβ(1-40) significantly reduced STOC frequency  and pixel ST maps (iiiiii), obtained using the same analysis technique as used for APP23 Ca2+ imaging, showed a clear increase in wave activity in vessels exposed to Aβ(1-40) compared with those exposed to the scrambled(1-40) peptide (i and Movie S3). The waves initiated by Aβ(1-40) exhibited a relatively short duration and occurred in a regular and repetitive manner throughout the recording (Movies S3 and S4), an unusual phenotype not previously described in VSMC. There was no difference in the amplitude (Zscr) of waves in Aβ(1-40)–treated arteries compared with scrambled peptide-treated arteries (ii), and no significant differences in spark frequency or amplitude (It has been established that Aβ(1-40) has acute effects on cerebral vessels, constricting cerebral resistance arteries , 7 and nrequency C. There arteries D, ii, anmplitude C were fo2+ spark frequency in VSMCs and the consequent loss of Ca2+ spark-induced BK channel activity is responsible for the enhanced basal constriction of arteries. Additionally, dysfunction of the Kir2.1 channel in the vascular endothelium was associated with compromised vasodilation to external K+. Incubation of healthy arteries with the (1-40) isoform of Aβ blunted BK channel function, partially recapitulating the phenotype of arteries from the APP23 mouse.AD affects one in eight people over 65 y of age, and as the population of the world ages, the lack of effective treatment strategies becomes ever more acute. However, in the last decade—and over 100 y since the disease was first identified—there is growing recognition that a significant degree of the clinical presentation seen in patients with AD reflects progressive cerebrovascular dysfunction due to small vessel disease of the brain , 5, 30. 2+ channels and Substance P have been identified in cortical vessels and pericytes associated with amyloid plaques in AD mouse models , and the tempering influence of a pressure-sensitive vasodilatory pathway comprising Ca2+ spark activation of BK channels  increases Ca2+ spark frequency approximately threefold, an increase sufficient to engage juxtaposed plasma membrane BK channels to oppose membrane depolarization  and Aβ(1-42), both of which have been implicated in the development of cerebrovascular damage . Both Aβ+ channel vasodilatory pathways, which could account for our observations in the APP23 mouse. Incubation with Aβ(1-40) did indeed affect the BK channel, as evidenced by an increase in pressure-induced constriction, reduction in paxilline-induced constriction, as well as a decrease in the frequency of BK channel-mediated currents . However, in contrast to our findings from APP23 mice, Aβ(1-40) had no effect on Ca2+ sparks in VSMCs. Instead, the principal observation was a striking initiation of rhythmic, medium-sized Ca2+ waves. Compromised BK channel activity in the presence of normal Ca2+ spark activity could be attributable to several mechanisms. BK channels can only be activated by Ca2+ sparks in direct proximity to the channel  could be due to effects on K channel . This na channel  and STIM channel , which timpaired , 37. Wer2+, the latter of which is dependent on functional association of the BK channel β1-subunit (2+ sparks would be unable to generate STOCs. A third possibility is that the generation of rhythmic medium-sized waves seen after incubation with Aβ(1-40) represents a failure of sarcoplasmic reticulum function such that, although Ca2+ sparks are still generated, their coupling to the BK channel is interrupted. Although generation of rhythmic Ca2+ waves in VSMCs is not well described, generation of pathological Ca2+ waves within cardiac myocytes is a well-recognized phenomenon that leads to contractile dysfunction and arrhythmias. For example, digitalis increases sarcoplasmic reticulum Ca2+ load, leading to Ca2+ wave development  on cerebral arteries could be an effect on the BK channel itself. BK channels are regulated both by voltage and Ca-subunit . If Aβ translates into a reduction in BK channel availability, despite the absence of an effect on Ca2+ spark frequency. Furthermore, the phenotype of vascular dysfunction observed as a result of acute administration of Aβ(1-40) differed significantly from that seen in APP23 mice where Ca2+ spark frequency was reduced. The reduction in Ca2+ spark frequency in cerebral VSMC of the APP23 mice could be accounted for by lower sarcoplasmic reticulum Ca2+ stores or defective Ca2+ release, potentially implicating the sarcoplasmic reticulum Ca2+ ATPase pump or ryanodine receptor function, respectively. Precisely how acute effects of the peptide in triggering pathogenic Ca2+ waves in healthy cerebral arteries are converted into the reduction in Ca2+ sparks seen in the cerebral arteries from 18-mo-old APP mice remain unclear. Further work is now warranted, potentially involving longer exposure of cerebral arteries to Aβ(1-40), using mice of different ages, or even chronic administration of Aβ(1-40) to induce sarcoplasmic reticulum failure.Within the arteries studied here, how Aβ(1-40) triggered the rhythmic, medium-sized VSMC Ca+ currents through SK/IK channels activate endothelial Kir2.1 channels within a functional microdomain, thereby potentiating SK/IK-induced hyperpolarization and boosting vasodilation. Thus, vasodilatory responses to activation of SK/IK reflect the combined action of SK/IK and Kir2.1 channels, whereas electrophysiological recordings of SK/IK currents measure SK/IK activity only. The observed reduction in the dilation of APP23 arteries in response to NS309 can therefore be seen as further evidence of the far-reaching impact that damage to Kir2.1 channel activity has on endothelial—and thus microcirculatory—function.Vasodilation in response to direct pharmacological activation of endothelial SK/IK channels was also reduced in APP23 mice compared with WT mice, an observation seemingly at odds with the results of a patch-clamp electrophysiological assessment, which showed that endothelial SK/IK channel current density was equivalent between the two genotypes. This apparent discrepancy between the two approaches for assessing SK/IK channel function can be accounted for by the fact that SK/IK channels and Kir channels are functionally linked, as previously reported . Specifi2+ spark-induced BK channel activity and dysfunction of the Kir2.1 channel in the vascular endothelium—may be amenable to pharmacological rescue. On the endothelial side, we recently reported that retro-orbital application of a water-soluble analog of the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2), which is an essential cofactor for Kir2.1 channels   . Notablese (PKG)  that shoarteries . Thus, m+ channel deficits in resistance arteries of the brain: an increase in arterial contractility due to a failure of VSMC Ca2+ spark–BK channel vasoregulation, and a reduction in external K+-induced vasodilation resulting from EC Kir2.1 dysfunction. Although numerous studies have demonstrated small vessel disease of the brain in AD in vivo, ours reports a specific signature of vascular ion channel dysfunction ion within the native resistance arteries in AD. Aβ(1-40) alone also compromises BK channel function, although this appears to have more to do with global dysregulation of Ca2+ signaling than an immediate effect on Ca2+ sparks. However, pharmacological approaches for restoring Ca2+ spark–BK channel vasoregulation, together with restoration of Kir2.1-driven neurovascular coupling using PIP2-boosting approaches, may represent a strategy for targeting the two principal cerebrovascular deficits in AD: cerebral hypoperfusion and neurovascular uncoupling. Given that both cerebral hypoperfusion and neurovascular uncoupling are also seen in mild cognitive impairment associated with the prodromal stage of AD  and hemizygous APP23  mice were allowed to age to 17 to 19 mo prior to experimentation. Homozygous mice die at a young age (3 to 4 mo), and thus cannot be used in an aged protocol. Following killing of the mice, cerebral pial resistance arteries were dissected from the brain and studied using pressure (isobaric) myography and high-speed spinning-disk confocal microscopy. Arteries were digested enzymatically and both VSMCs and ECs were studied using patch-clamp electrophysiology. Brain tissue was also embedded in paraffin prior to immunohistochemistical study of Aβ deposition. Full details of all materials and methods can be found in SI Appendix.Procedures involving animals were performed in accordance with UK Home Office Guidance on the implementation of the Animals (Scientific Procedures) Act of 1986 under appropriate project license authority and with the approval of the University of Manchester Animal Welfare Ethical Review Board. The APP23 mice used in this study express an isoform of the human Supplementary FileSupplementary FileSupplementary FileSupplementary FileSupplementary File


Function’s most eminent Executive Editors, it seems particularly poignant. In the spirit of Mahler’s song cycle, this editorial is partly a short “In Memoriam” for this great scientist (Function will publish a more detailed obituary in due course) and partly a brief account of plans for our journal as we move on, into 2023.Gustav Mahler’s famous valedictory song cycle “Das Lied von der Erde” (The Song of the Earth), dealing with the eternal cycle of death and (re)birth, has always been very close to my heart. Just now, only a couple of weeks after the death of Tullio Pozzan, one of Function, he immediately accepted. When discussing the plans for the journal in more detail—at the symposium celebrating Tullio’s 70th birthday , the US National Academy of Sciences (2006), and the Royal Society (2018). For almost the whole of his professional life, he worked at the University of Padua in Italy, except for a period (1978–1981) as a Long Term EMBO Fellow at the University of Cambridge.2+ in living cells with fluorescent indicators that could be trapped intracellularly.2 This new technique had a colossal influence on the Ca2+ signaling field and rapidly became the dominant technique for assessing changes in the cytosolic Ca2+ concentration, a key parameter in the control of almost all cellular activities.During his Cambridge period, he was a part of the trio [with Roger Tsien (Chemistry Nobel Prize 2008) and Tim Rink] that developed the revolutionary technique for measurements of Ca2+ measurements in selected subcellular localizations. With the help of this new technique, they published a landmark paper in Science, demonstrating for the first time that microdomains with a high Ca2+ concentration close to IP3-sensitive channels in the endoplasmic reticulum (ER) membrane are sensed by neighboring mitochondria.3 This paper initiated a revolution in our understanding of the role of mitochondria in cellular Ca2+ homeostasis. It also solved a key problem in cellular physiology, namely, how increased cell activity—elicited by cytosolic Ca2+ signals—was linked to the required increase in mitochondrial ATP production.Tullio’s most important work was carried out at the University of Padua, in close collaboration with his younger co-worker Rosario Rizzuto. They developed the first genetically encoded probes for CaScience paper3 started a new era of cellular signaling work with a focus on subcellular microdomains, also reflected in other work published that same year.4 In 1998, a further Science paper by Tullio and his collaborators5 provided impressive high-resolution evidence for linkage between local Ca2+ signals, generated by release from the ER, and mitochondrial Ca2+ uptake. This Ca2+ uptake stimulates the Krebs cycle, thereby generating ATP that is needed to power cellular activity.The 1993 6Tullio continued to generate useful and important tools for cellular physiologists, also focussing on another critical intracellular messenger molecule, namely, cyclic adenosine monophosphate (cyclic AMP). He generated the first genetically encoded fluorescent probe for this messenger and established the concept of cyclic AMP microdomains.Function, describing a new transgenic mouse line for imaging mitochondrial Ca2+ signals.7 Anant Parekh, in his accompanying Perspective, wrote: “The erudite, innovative and timely advance takes us to the next level, the ability to study mitochondrial Ca2+ signaling in vivo. This landmark contribution will no doubt lead to new vistas in Ca2+ signaling in health and disease.” 8More recently, Tullio published an important paper in Function. As we mourn Tullio, we nevertheless have to continue our work and we need to plan for the future of Function. In this context, I am very pleased to report that Annette Dolphin FRS, Professor of Pharmacology at University College London, has accepted the invitation to join the group of Executive Editors from January 1, 2023. She has been a member of the editorial board of Function from the beginning and also been a consistent and important contributor. Annette’s innovative work on voltage-gated Ca2+ channels has led the field for many years.9Tullio’s death is a heavy loss for the cellular signaling community and for Function has already for some time been included in PubMed Central and Web of Knowledge (Clarivate). More recently, Scopus has also started to list our content and this is now visible.One of the most important problems facing every new journal is the long time it takes before its content is listed in the most important databases, even when the journal has a distinguished editorial board and is published by major well-known organizations. Happily, this period has now come to an end for our journal. Function, it was planned to combine publication with in-person scientific conferences. However, the COVID pandemic prevented this from happening. Now that the world is opening up, we are planning a Function symposium early in 2023. Following the remarkable interest in the editorial published in Function’s previous issue (“Are scientists sufficiently ambitious?”),10 the symposium will be ambitious, aiming to capture, assess, and discuss the most important progress in the broad areas of physiology and pathophysiology. We do not want to drown in data, but rather aim to gain real and useful knowledge.10 We have a stellar list of speakers and much time will be devoted to interactive discussions. The symposium will take place in the heart of Europe, at the German National Academy of Sciences Leopoldina in Halle on March 7 and 8, 2023. The conference is co-sponsored by the American Physiological Society and the Leopoldina Academy and organized in co-operation with Academia Europaea’s Cardiff University Knowledge Hub. Registration will soon open and all details will be available at https://aecardiffknowledgehub.wales/2022/10/21/physiology-pathophysiology-2023-symposium/From the very start of The symposium will be a unique opportunity to gain a high-level overview of where Physiology is right now and to learn from world leaders in the field where we are heading.None declared.


Urticaria is one of the most frequent dermatological diseases and it usually occurs in paroxysmal, recurrent form. Urticarial wheal is a visible edema of the skin, which is caused by the increased permeability of blood vessels. Urticaria manifests as a sudden appearance of wheals, angioedema or both. Urticarial wheals usually disappear within 24 h, and angioedema in 72 h . The mosCurrently, two endotypes of CSU can be distinguished, which vary in terms of pathogenesis and markers of the disease, as well as clinical course and sensitivity to treatment applied: type I and IIb. Type I CSU is related to the occurrence of IgE antibodies to autoantigens such as TPO (thyroid peroxidase), TG (thyroglobulin), ds-DNA (double-stranded DNA) and Il-24. In type I, it is observed that the occurrence of allergic diseases and the total concentration of IgE are normal or even increased. Antihistamines and omalizumab used in this endotype therapy are usually effective [The diagnosis of urticaria is based on the skin lesions’ appearance, and its causes are potentially determined through carefully collected medical history and diagnostic tests. The following diagnostic procedures are recommended for urticaria: basic laboratory tests , as well as additional tests in more complex cases, such as ANA, cryoglobulins, components of the complement system, number of eosinophils in peripheral blood, skin prick tests (SPT), autologous serum skin tests, patch tests, provocation tests with food and medications, skin biopsy  and physical tests—exercise, warm bath provocation, temperature and pressure .In the treatment of urticaria, the elimination of the triggering factors and avoiding factors that intensify the symptoms play a major role. The basics of first-line symptomatic treatment are second generation antihistamines (AH). In case of continuous disease with a lack of control after applying the standard dosage of AH, it is suggested to increase the dosage four-fold. The third line of therapy is adding the AH human monoclonal antibody—omalizumab. Nowadays, biological treatment with omalizumab is a recommended therapy for patients over the age of twelve. The medication is administered subcutaneously in 2–4-week intervals. ,7. The fAngioedema (AE) is an edema of the subcutaneous or submucosal tissue, which develops as a result of an extension and increase in vascular permeability. Most frequently, this symptom is well demarcated, asymmetrical, located around the eyelids, lips, genital areas and distal parts of the limbs, as well as the mucous membrane of the upper respiratory tract. It usually occurs in the form of a single focus rather than several changes of different sizes. Skin lesions may be painful. Recurrent edema might appear in the same location. Laryngeal and/or throat edema with dyspnea is life-threatening and potentially related to acute respiratory failure caused by the obstruction of the upper respiratory tract . AE freqA significant increase in the interest in diseases affecting patients’ quality of life has been observed in recent years. It does not only refer to urticaria and its angioedema form, but also the deterioration in quality of life caused by the coexistence of these symptoms. Undoubtedly, the symptoms of CSU are a cause of distress for patients, e.g., itching intensity in CSU has been proven to be related to stress ,5. One oUrticaria is a multiform disorder with partially unknown pathophysiology, and therapy effectiveness is still limited, in spite of several options. Recurrent angioedema is a form of chronic urticaria. The symptoms of angioedema, with or without coexisting wheals, constitute a significant cause of the deterioration in urticaria patients’ quality of life. A lower sense of coherence in this group of patients leads to an increased manifestation of anxiety. Identifying patients with angioedema symptoms and a lowered sense of coherence may potentially create wider interdisciplinary therapeutic possibilities for this group.


Encephalitozoon cuniculi is a eukaryote, unicellular, spore-forming, obligate intracellular microorganism of the phylum Microsporidia, with domestic rabbits as its main host. Another important species in which this pathogen has been identified are humans, the infection being therefore called a ”zoonosis”. The transmission takes place via the horizontal route or the vertical route, and cell-mediated immunity plays the biggest role in the infected hosts’ protection. Encephalitozoonosis can manifest itself as an acute infection, with neurological signs, renal signs, and ocular lesions, or as a chronic or subclinical infection, which is usually the case for asymptomatic carriers. The diagnostic techniques usually carried out are histological examination, serological tests, and molecular genetic techniques. The treatment of encephalitozoonosis is usually symptomatic, with unrewarding results, and prevention methods include periodical serological screening, prophylactic administration of fenbendazole, and maintenance of a clean environment. The purpose of this article is to review the current data regarding the pathogenesis, host immunity, clinical signs, diagnostic methods, treatment, and prevention methods of encephalitozoonosis in the domestic rabbit, as well as to analyze the prevalence of this disease in different countries of the world. Nosema, Vittaforma, Brachiola, Pleistophora, Enterocytozoon, and Encephalitozoon . (EC11C5) . E. cuni.  is another option, where all of the proliferative stages  of ’s study , all of E. cuniculi as the causative agent of the disease [Even if severe histological lesions can be identified in the brain and kidneys, it has been proven that they are not necessarily associated with the manifestation of clinical signs. Therefore, the severity of the histological lesions cannot determine  disease .E. cuniculi infection is one of the most widely utilized ante-mortem diagnostic methods, as the options are quite limited. Through this method, the specific antibodies IgM and IgG are titrated; however, a positive result only indicates whether there was a previous exposure of the animal to E. cuniculi, with no exact information as to when this occurred. It is recommended that more than one titer be obtained from an individual in order to establish the exposure to the microorganism. Clinical signs are not necessarily correlated with a high antibody titer, and only a negative result can rule out encephalitozoonosis [Serological testing in the case of a suspected zoonosis . The teczoonosis .E. cuniculi in rabbits reported a significant correlation between the qualitative results of ELISA and IFAT methods, as well as between ELISA and CIA and between IFAT and CIA [Studies conducted on the serological diagnosis of  and CIA ,21. Quan and CIA . Western and CIA .E. cuniculi is the causative agent of the disease [Determining the IgM and IgG antibody titers in a suspected case of encephalitozoonosis is an indicator of a humoral response of the host organism, but these do not show anything more than the fact that an exposure to the parasite took place ,8. Altho disease ,19. The  disease .E. cuniculi exposure show high IgM titers between 0 and 35 days post-infection that can persist for up to 18 weeks—a time frame in which the animal can eliminate the spores via urine, which makes it very important that they are isolated from the rest of the population. Even so, after 5–18 weeks, the IgM titers will start to decrease and will no longer be helpful in establishing whether the animal is suffering from an acute infection [During the course of infection, serum levels of IgM decrease, while those of IgG increase . A studynfection .E. cuniculi has not taken place. A recently exposed immunocompetent rabbit can show negative titers for up to 2 weeks post-infection; therefore, only a second negative IgG titer in the clinically healthy animal 3 weeks later can confirm non-exposure to the pathogen. If a clinically healthy individual presents a single positive IgG titer, this can mean either that it is an early infection, a chronic infection, and/or previous infection and recovery from encephalitozoonosis. In the presence of neurological clinical signs and two negative IgG titers 3 weeks apart, E. cuniculi can be ruled out as the causative agent of the disease [It is important to highlight that a negative result does not automatically mean that exposure to  disease .The significance of age at the time of testing is also essential to take into account. Young rabbits have maternal antibodies passed from the mother up until 4 weeks of age, during which time they will test seropositive, followed by the disappearance of the antibodies from 4 to 8 weeks of age, where they will be seronegative . This coE. cuniculi in rabbits of multiple origins and with different clinical statuses, whether they are clinically healthy, or with specific or non-specific encephalitozoonosis signs , and organs collected through necropsies, such as brains, kidneys, eye lenses, livers, lungs, hearts, and spleens [Molecular genetic techniques of diagnosing  spleens ,31. Spec spleens .The first step of this diagnosis is the extraction of DNA from the collected materials, with the organ samples being deposited beforehand at −20 °C. This is usually performed according to the instructions of the kit manufacturer, with small modifications depending on the author. Because of the thick wall of microsporidians, a pretreatment is usually required in order to destroy the wall so that the DNA is released, which is performed with either liquid nitrogen, mechanical disruption with glass beads, and/or digestion with proteinase K. The only exception from this are the eyes with phacoclastic uveitis, where the spore load is sufficiently high that DNA can be extracted and amplified easily ,19.E. cuniculi detection using nested PCR [E. cuniculi in urine, but no concrete information on its sensitivity has been offered [E. cuniculi DNA from these materials [PCR amplification follows after obtaining the DNA isolates, the type of PCR reaction being different depending on the sample tested and the author. In the case of conventional PCR, the primers ECUNF (5′-ATGA-GAAGTGATGTGTGTGCG-3′) and ECUNR (5′-TGCCATGCACTCACAGGCATC-3′) have been used to target the 549 bp fragment of the 16S small subunit ribosomal RNA gene . The consted PCR . RT-PCR  offered . Diagnosaterials . DiagnosE. cuniculi isolates, semi-nested PCR can be used, targeting the polar tube protein (PTP) gene with the first pair of primers PTPF (5′-GCAGTTCCAGGCTACTAC-3′) and MPTPR (5′-CATGACAGGGTTTGGAATCT-3′) to amplify a 423 bp fragment for genotypes I and II and a 345 bp fragment for genotype III. For the second PCR, the pair of primers PTPF and PTPR (5′-AGGAACTCCGGATGTTCC-3′) can be utilized to target a 363 bp fragment for genotypes I and II and a 285 bp fragment for genotype III [For the genotyping of the type III .Different studies report the prevalence of encephalitozoonosis in rabbits using molecular genetic techniques, with the highest being detected in eye lens and brain tissues .E. cuniculi diagnosis in rabbits. Some options include hematology for evaluating infection markers, serum protein electrophoresis for evaluating the specific proteins and biochemistry, and kidney ultrasound for assessing renal function [There are several tests that can help assess the prognosis of an acute infection or guide us in the direction of a differential diagnosis, but these certainly cannot give us a definitive function ,9. In adfunction .E. cuniculi that showed signs of renal disease revealed changes such as increased absolute heterophil counts and a low hematocrit (<33%)—changes that can, however, go unnoticed [Hematology from rabbits infected with nnoticed . Biochemnnoticed . Anothernnoticed , but decnnoticed .E. cuniculi. A comparison between infected and clinically healthy rabbits revealed that the affected ones usually had a lower albumin-to-globulin ratio, a lower β-globulin fraction, and a higher γ-globulin fraction [Serum protein electrophoresis has been used for the measurement of the albumin-to-globulin ratio in rabbits infected with fraction . Anotherfraction . This cafraction .Pasteurella multocida, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Listeria monocytogenes, while among the less frequently diagnosed pathogens are human herpesvirus 1, Toxoplasma gondii, and rabies. Non-infectious causes of CNS signs in rabbits include lymphosarcoma or metastatic neoplasia, lead toxicity, hydrocephalus, cerebral infarcts, hepatic encephalopathy, enterotoxemia, and sepsis. Focal spinal disease or disease anywhere along the cerebral–brainstem–spinal cord axis can also be etiologies of paresis, plegia, or ataxia [Keeping in mind that the clinical signs of encephalitozoonosis in rabbits are not really specific to the disease and that establishing a diagnosis can be quite hard and expensive for the owners, looking into other similar pathologies is essential in making a correct diagnosis. The causes of neurological signs affecting the CNS can be infectious and non-infectious. The infectious pathogens that cause central vestibular syndrome are of bacterial, viral, protozoal, and parasitic origins. The bacterial agents are the most frequently encountered, such as r ataxia .In cases of peripheral vestibular syndrome, a few differential diagnoses include bacterial otitis interna/externa, traumatic rupture of the tympanic bulla, aminoglycoside-mediated ototoxicity, and idiopathic vestibular disease .Although renal impairment is rarely manifested clinically in rabbits with the disease, weight loss and emaciation despite a normal food intake can be associated with chronic azotemia. Ultrasonographic changes of the kidneys in acute onset have been observed in rabbits with chronic renal failure .Pasteurella multocida infection [The differential causes of ocular disease are lens-induced uveitis secondary to geriatric cataract formation, and bacterial uveitis—a lesion that is usually bilateral and can be encountered in nfection ,20.Therapy can be just as challenging as establishing a diagnosis of encephalitozoonosis in rabbits, since this disease has no specific cure and acute cases in immunocompromised animals usually have a deadly outcome . The mosThe parasitic spores cause the inflammation of the ruptured host cells that they have invaded, which is why treatment goals consist of reducing spore proliferation and migration, reducing spore-mediated inflammation, and management of severe neurological signs and concurrent disease .E. cuniculi is part parasite and part fungus, first on the medication list are antiparasitic and antimycotic drugs. The study of Wei et al. [E. cuniculi infection in humans [Since i et al.  regardinn humans . On the n humans  outlinedn humans .E. cuniculi in rabbits showed seronegativity after 21 days post-infection and the absence of spores in brain tissue upon postmortem analysis. Most authors use the off-label treatment with fenbendazole orally dosed at 20 mg/kg body weight daily for 28 days, with the success rate in acute or chronic cases of encephalitozoonosis being lower than in prophylactic administration [A better option for antiparasitic medication with fewer adverse effects in rabbits is fenbendazole—also from the benzimidazoles class—recommended both for treatment of the disease and as prophylactic administration. The preventive use of fenbendazole prior to experimental infection with stration ,9.E. cuniculi [In the treatment of severe systemic inflammation, steroids have shown promising results, although their use is controversial due to the anti-inflammatory versus immunosuppressive effects. The administration of dexamethasone at 2 mg/kg body weight intramuscularly for three doses every 6 h in experimentally infected rabbits caused a significant decrease in inflammatory activity of the organism, but at the same time led to acute immunosuppression that exacerbated and/or caused clinical signs of encephalitozoonosis. In cases of cerebral trauma, the use of dexamethasone is also not recommended, because this can worsen the clinical signs, and a therapeutic effect has not been achieved in chronic infection . Anothercuniculi .Rabbits with signs of neurological disease  should receive small doses of diazepam at 0.5 mg/kg body weight subcutaneously/intramuscularly/intravenously or midazolam at 0.07–0.22 mg/kg body weight intramuscularly/intravenously administered as a mild sedative. Even though rabbits are not capable of vomiting and lack the vomiting center in the medulla oblongata, antiemetic drugs such as metoclopramide , prochlorperazine , or meclizine  have been used in cases of severe torticollis .In acute manifestations of encephalitozoonosis in rabbits, in addition to fenbendazole administration, broad-spectrum systemic antibiotherapy is recommended for any concurrent or secondary bacterial infection. The most frequently used substances are trimethoprim–sulfamethoxazole  or enrofloxacin  for 7–10 days .E. cuniculi in rabbits, it was reported that a combination of fenbendazole, oxytetracycline, enrofloxacin, and dexamethasone or prednisone showed a 54.2% clinical recovery rate. Another study [In the study of Künzel et al.  on treater study , where ter study .Ocular lesions of encephalitozoonosis in rabbits—more specifically, uveitis—have been treated conservatively with systemic administration of dexamethasone and oxytetracycline together with topical ophthalmic dexamethasone and tetracycline. In cases of focal granuloma and persisting cataracts, surgical removal of the lens by phacoemulsification is another option to consider. It is important to recall that dexamethasone, with an immunosuppressive effect, can be absorbed systemically through ocular administration; therefore, the use of non-steroidal anti-inflammatory drugs is preferred when the renal biochemical values are in their reference ranges . Other tThe in vitro efficacy of various other drugs—such as fumagillin, sparfloxacin, polyoxin D, and nikkomycin Z—has been demonstrated, but their clinical use in rabbits has not yet been documented .Other aspects to be taken into consideration in the acute disease of rabbits are fluid therapy, assisted feeding if needed, prokinetic drugs, and a stress-free environment with protection against self-inflicted lesions that can occur due to neurological manifestations ,9.E. cuniculi infection in both rabbits and other susceptible species—especially in humans—is of the utmost importance. Because of the wide spread of the organism and all of its characteristics detailed in this study, this is not the simplest thing to accomplish. The first and most essential step would be setting up E. cuniculi-free rabbit breeding colonies, which unfortunately for breeders can require a lot of time and expense. This should be achieved through periodic serological testing of rabbit populations, with the isolation of healthy seronegative individuals from the others in separate cages. Especially in breeding facilities, the testing of antibody levels should be performed on young rabbits every 2 weeks for 2 months, with the removal of all seropositive animals. Only rabbits that show negative results for an entire month should be used for future breeding, followed by monthly serological testing to confirm their disease-free status [The prevention of e status . It is pe status .E. cuniculi-free colonies, the best way of reducing the spread of infection would be prophylactic administration of fenbendazole orally or in the food, using the same 28-day protocol mentioned earlier. This should also be applied for newly acquired animals before putting them in contact with other rabbits [For rabbits that are not assured to come from  rabbits . What ne rabbits .Supplementation of the diet with probiotic bacteria is also thought to help by increasing the immune response and by forming a defense against pathogen infection ; howeverE. cuniculi in the environment is high, but the spores may be destroyed using certain disinfection protocols, such as 0.1% bleach with a 10 min contact time or ethanol (70%) with a 30 s contact time. Sodium hydroxide (1%), formaldehyde (0.3%), and hydrogen peroxide (1%) are other options that can kill spores with a contact time of 30 min [The resistance of f 30 min .Other prophylactic measures that should be applied are reducing of rabbits’ contact with other rabbits or even wildlife, thorough disinfection of the environment using substances such as those mentioned above, appropriate hygiene through hand-washing and disinfection after handling rabbits, and the use of raised food dishes and water bottles that reduce the risk of urine contamination ,40.E. cuniculi is a eukaryotic, unicellular, obligate intracellular and ubiquitous parasitic organism from the phylum Microsporidia, with the domestic rabbit (Oryctolagus cuniculus) being its main host, but also transmissible in numerous other species—most importantly, in humans. The transmission in rabbits takes place either via the horizontal route, through contaminated water and food or, rarely, through spore inhalation, or via the vertical route, from doe to kit, through the ocular structures. The hosts’ superior protection mechanism is cell-mediated immunity provided by the CD4+ and CD8+ T lymphocytes, followed by humoral immunity, where specific IgM and IgG antibodies are produced. The clinical signs in infected rabbits are mainly of a neurological, renal, and ocular nature, but the disease can also be subclinical, with a significant number of asymptomatic carriers. The diagnosis of encephalitozoonosis through histopathology reveals lesions of granulomatous meningoencephalitis and chronic interstitial nephritis together with spores in the targeted organs. Serological testing is the most widely used ante-mortem diagnosis tool, where specific IgM and IgG titers are detected, but with no value of certainty. Diagnosis through molecular genetic techniques is becoming more popular in recent years, but with a questionable sensitivity of the method. Conventional PCR, nested PCR, and RT-PCR are the methods through which specific DNA has been identified previously—especially from urine and postmortem organs. The treatment of infection usually includes fenbendazole, systemic antibiotherapy, supportive therapy, and the treatment of eye lesions with anti-inflammatory drugs or even surgical intervention, but with a variable rate of success. Prophylactic administration of fenbendazole in rabbits, periodic serological testing of rabbit populations, and maintaining a clean environment are the best ways to reduce the transmission of this widespread pathogen, not only among rabbit populations but, more importantly, in humans, in whom the zoonosis can cause severe outcomes in immunocompromised individuals.