BackgroundCSF3 gene encoding the G-CSF protein to delineate the mechanism of G-CSF production by the cancer cells.Granulocyte colony-stimulating factor (G-CSF) is a member of the CSF family of glycoproteins that regulate the proliferation, differentiation, and mobilization of neutrophils. G-CSF-producing malignant cancers have been reported to occur in various organs and are mostly associated with poor clinical prognosis. Here, we analyzed the structure of the MethodologyTwo cases of G-CSF-producing urothelial cancers and three cases of G-CSF-nonproducing bladder cancers were enrolled for genetic analysis.ResultsCSF3 gene. No somatic mutations in the CSF3 gene were detected in another case of G-CSF-producing renal pelvic cancer and G-CSF-nonproducing bladder cancers. Copy numbers of the CSF3 gene were not increased in G-CSF-producing urothelial cancers.In one case of G-CSF-producing bladder cancer, six somatic mutations were detected in the 5’- upstream region of the ConclusionsCSF3 gene may cause G-CSF protein overproduction.Somatic mutations in the 5’- upstream region of the  CSF3 genes encoding G-CSF protein are located on the human q21-22 region of chromosome 17 and murine chromosome 11, respectively, and both genes consist of ﬁve exons and four introns [CSF3 gene codes for two different mRNAs that are generated by the alternative use of two 5’ splice donor sequences in intron 2 [Granulocyte colony-stimulating factor (G-CSF) is a member of the colony-stimulating factor (CSF) family of glycoproteins that regulate the proliferation, differentiation, and mobilization of neutrophils ,2. The hTumor-related leukocytosis is a paraneoplastic syndrome that is rarely observed in patients with nonhematologic malignant tumors  and can G‑CSF produced by cancer cells also expressing G-CSF receptor can stimulate cancer growth, induce angiogenesis, and promote metastasis by the autocrine signaling pathway . G-CSF-pEscherichia coli infection in the urinary tract [Innate immune response and subsequent granulopoiesis can be activated by pattern recognition receptors, such as Toll-like receptors (TLRs), expressed on nonhematopoietic cells . For exary tract . TLR sigry tract . The L26ry tract . The frery tract .CSF3 gene encoding the G-CSF protein and intracytoplasmic domains of TLRs to delineate the mechanism of G-CSF production by those cancer cells. Specifically for the former, the CSF3 gene and its upstream sequences in G-CSF-producing urothelial cancer were analyzed by Sanger sequencing, and the copy numbers of the CSF3 gene were also determined.Here, we observed two cases of G-CSF-producing urothelial cancers  and analyzed the structures of the Cases of G-CSF-producing urothelial cancersCase 1A male in his 60s presented with gross hematuria. The diagnosis was invasive bladder cancer. The white blood cell (WBC) count was 17,300 at diagnosis. Transurethral resection of the bladder tumor (TURBT) was performed, and the pathology was urothelial cancer with muscle invasion. In addition, immunohistochemistry showed G-CSF expression in the cytoplasm of tumor cells. Radical cystoprostatectomy with ileal conduit was performed, and the pathology revealed no remaining tumor, with pathological stage pT0N0M0. Nevertheless, multiple lung metastases occurred after one month. Gemcitabine plus cisplatin chemotherapy was not effective, and the patient died due to cancer 3.4 months following the diagnosis of metastases. At death, the WBC count was 139,500.Case 2A male in his 60s presented with gross hematuria. The diagnosis was a left renal pelvic tumor with multiple lung metastases. The WBC count was 20,000. Administration of two courses of gemcitabine plus cisplatin chemotherapy appeared to stabilize the original and metastatic tumors. Then, a left nephroureterectomy was performed. Before the operation, the WBC was 25,700, and the serum level of G-CSF was 172 pg/mL . The pathology of the surgical specimen was urothelial cancer of the left renal pelvis, with pathological stage pT2N0M1. Immunohistochemistry showed G-CSF expression in the cytoplasm of tumor cells. Six weeks after the nephroureterectomy, lung metastases increased. Pembrolizumab, an anti-programmed death receptor-1 (PD-1) antibody, was administered, but it was not effective. At cancer death 6.5 months following left nephroureterectomy, the WBC count was 94,400.Case 3A female in her 60s presented with gross hematuria. The diagnosis was large bladder cancer. The WBC count was 25,100 and the serum level of G-CSF was 409 pg/mL at diagnosis. TURBT was performed, and the pathology was urothelial cancer with submucosal invasion. Immunohistochemistry showed G-CSF expression in the cytoplasm of tumor cells. After two months, a bladder tumor measuring 7 cm in diameter rapidly relapsed and transurethral tumor resection was incomplete. Then, an anti-PD-1 antibody therapy using pembrolizumab was introduced. A temporary increase in the bladder tumor and worsening of the bilateral hydronephrosis were observed, but at the end of four courses, the WBC was normalized to 7,800, and the disappearance of the bladder tumor was confirmed by a CT scan and cystoscopy. Eight courses of pembrolizumab were administered in total, and complete remission was achieved with no bladder tumor recurrence up to 20 months after starting the therapy. In Case 3, the bladder tumor specimen for genetic analysis could not be collected.Control 1A male in his 80s underwent TURBT for a bladder tumor. TURBT pathology report showed urothelial carcinoma, pT1, G3>G2. Preoperative WBC was 5,800. Bacille Calmette-Guerin bladder instillation therapy was performed after TURBT. One and two years later, the patient developed a recurrence in the bladder and underwent TURBTs again. There has been no recurrence since then.Control 2A male in his 60s underwent TURBT for a bladder tumor. TURBT pathology report showed urothelial carcinoma, pTa, G2>G3. Preoperative WBC was 6,400. One and two years later, the patient developed a recurrence in the bladder and underwent TURBTs again. Four years after the initial treatment, the patient died of colorectal cancer.Control 3A male in his 80s underwent TURBT for a bladder tumor. TURBT pathology report showed urothelial carcinoma, pT1, G3>G2. Preoperative WBC was 5,300. One year after TURBT, the patient died of pneumonia.Two cases of G-CSF-producing urothelial cancers (Cases 1 and 2) and three cases of G-CSF-nonproducing bladder cancers (Controls 1-3) were enrolled for genetic analysis. Genomic DNAs were extracted and purified from surgical specimens kept at −70°C until use, as well as whole blood samples collected in ethylenediaminetetraacetic acid-containing tubes using kits , following the manufacturer’s instructions. Bladder and renal pelvis cancers share a similarity in that both are included in the category of urothelial carcinoma.Immunohistochemistry of G-CSFCat.no. sc-53292), following the manufacturer’s instruction.Paraffin-embedded sections of surgical specimens were immunohistochemically stained with anti-human mouse monoclonal G-CSF antibody  encompassing the whole gene using sense/antisense pairs of primers. Polymerase chain reactions (PCRs) were performed using a thermal cycler  in volumes of 50 μL with 0.3 μM of sense and antisense primers, one unit of KOD Plus ver.2 polymerase , 4% dimethyl sulfoxide (DMSO), and the buffer supplied with the enzyme as follows: 94°C for two minutes, 36 cycles  of two-step PCR. Amplified PCR products (10 μL) were resolved by electrophoresis in 1% agarose gel. The primers used for amplification were 5’-tcgagaccagcctgaccaccaacatgg-3’/5’-ctgggccaagacactcacccatcagct-3’ for Fragment 1 , 5’-gggcaaggcgacgtcaaaggaggatca-3’/5’-cccgaggccacccagaaaaacaggaga-3’ for Fragment 2 , and 5’-ccaggcctctgtgtccttccctgcatt-3’/5’-ggaaagcagcttcccttccttggagcc-3’ for Fragment 3 . PCR products were directly Sanger sequenced with PCR primers and additional sequencing primers; 5’-atcacgaggtcaggagatcgtgac-3’ and 5’-aactctccggaggctgcctgtctg-3’ for Fragment 1; 5’-gcttcctgctcaagtgcttagagc-3’ for Fragment 2; 5’-cttgagtccagctggtgcctggcc-3’ and 5’-gtcacattgtaactgaacttcagg-3’ for Fragment 3. The analyzed sequences were compared with a reference sequence obtained from NCBI Reference Sequence: NC_000017.11.For genomic DNAs, the CSF3 geneCopy number analysis of the Assays were designed according to the guidelines from Bio-Rad Laboratories. Copy Number Determination Assay kits for CSF3 and AP3B1 were purchased from Bio-Rad Laboratories .2O was incubated using a T100 Thermal Cycler for restriction enzyme treatment, followed by droplet generation using Automated Droplet Generator Oil for Probes (Bio-Rad).A 22-μL mixture containing 1.1 μL of Copy Number Determination Assay: CSF3, 1.1 μL of Copy Number Determination Assay: AP3B1, 11 μL of ddPCRTM Supermix for Probes (no dUTP) (Bio-Rad), 0.5 μL of HaeIII enzyme (New England Biolabs), and 20 ng of DNA resuspended in dHCSF3 gene were expressed assuming that the copy number of the AP3B1 gene in the corresponding genome is two. Copy number analysis of the CSF3 gene was performed by Riken Genesis Co., Ltd. .PCR with the droplets was performed using a Veriti® Thermal Cycler (Thermo Fisher Scientific) with the following cycling parameters: 10 minutes at 95°C (one cycle), 30 seconds of denaturation at 94°C and one minute of annealing and extension at 60°C (40 cycles), 10 minutes at 98°C, and hold at 4°C. All steps had a ramp rate of 2°C/second. Fluorescence signals of the droplets were measured using a QX200TM Droplet Reader (Bio-Rad). Fluorescent data from each well were analyzed using QuantaSoftTM Analysis Pro Software, calculating copy numbers based on the Poisson distribution. Copy numbers of the MyD88 geneSequencing of TLR2 TIR, TLR4 TIR, and CSF3 gene, except the annealing and extension time was 30 seconds. Primers for amplification were 5’-ccgtttccatggcctgtggtatatgaa-3’/5’-ctaggactttatcgcagctctcagat-3’ for TLR2 TIR (526 bps), 5’-acctgatgcttcttgctggctgcataaag-3’/5’-tcagatagatgttgcttcctgccaattgca-3’ for TLR4 TIR (551 bps), and 5’-gggatatgctgaactaagttgccac-3’/5’-gacgtgtctgtgaagttggcatctc-3’ for MyD88 (726 bps). The PCR products were directly sequenced with PCR primers.The PCR and gel electrophoresis conditions were similar to those in the Immunohistochemistry of G-CSFBy immunohistochemistry, G-CSF-producing urothelial cancers showed cytoplasmic G-CSF protein expression, whereas G-CSF-nonproducing bladder cancers did not Figure .CSF3 geneSequencing of CSF3 gene  or three cases of G-CSF-nonproducing bladder cancer.In the case of G-CSF-producing bladder cancer (Case 1), six somatic mutations were detected in the 5’-upstream region of the  Figures , 3, but CSF3 geneCopy number analysis of the CSF3 gene were not increased in G-CSF-producing urothelial cancers.As shown in Table MyD88 geneSequencing of the TLR2 TIR, TLR4 TIR, and MyD88 gene or the TIRs of TLR2 and TLR4 compared with reference sequences in all patients.There were no variations in the DNA sequences of the CSF3 gene in G-CSF-producing bladder cancer. These mutations may affect the promoter activity of the CSF3 gene and lead to G-CSF protein overproduction -1, GPE-2, and GPE-3, respectively. Mutations introduced further upstream of GPE-1 (CSF box) did not affect transcription [CSF3 gene cannot be excluded.Nishizawa et al. analyzed the promoter structure and function of the mouse cription . The sixCSF3 gene was not increased in the two G-CSF-producing urothelial cancers, so the increased copy number of the CSF3 gene was not considered a cause of G-CSF overproduction in the cases examined herein.In general, an increased gene copy number may be responsible for increased protein production. However, the copy number of the Granulopoiesis can also be triggered by signaling from TLRs. In this study, we examined the sequences of the TLR2 and TLR4 TIR domains, as well as MyD88, the major adaptor to TIR, in G-CFS-producing urothelial cancer tissues, but found no somatic mutations. Thus, TLRs may not be involved in the overproduction of G-CSF by tumor cells.CSF3 gene caused the overproduction of G-CSF protein in the G-CSF-producing bladder cancer examined in this study. However, in the case of G-CSF-producing renal pelvic cancer (Case 2), neither the somatic mutation in the CSF3 gene, the increased CSF3 gene copy number, nor the abnormal signaling from TLRs can explain the overproduction of G-CSF protein. In this case, genomic structures other than the CSF3 gene, such as those adjacent to the CSF3 gene, aberrated methylation of the CSF3 gene, or factors other than TLR signaling that can induce granulopoiesis, may have had an effect. For example, GeneHancer [CSF3 gene.It is undeniable that somatic mutations in the 5’-upstream region of the neHancer  has showAs seen in Cases 1 and 2, the prognosis of G-CSF-producing urothelial cancer is poor. While cisplatin-based chemotherapy has been widely used for advanced and metastatic urothelial cancer, effective treatment for chemotherapy-resistant urothelial cancer is almost nonexistent. Recently, immunotherapy using anti-PD-1 and anti-programmed cell death ligand 1 (PD-L1) antibodies has been introduced and shown some efficacy -17. As sA limitation of this study is that it presents only one possible cause for the formation of G-CSF-producing tumors. Various other causes of G-CSF-producing tumors should be investigated in multiple cases, with the hope of elucidating therapies specific to G-CSF-producing tumors.CSF3 gene, suggesting that they cause G-CSF protein overproduction. An increased copy number of the CSF3 gene was not observed in the two cases of G-CSF-producing tumors studied.In one case of G-CSF-producing bladder cancer, six somatic mutations were detected in the 5’- upstream region of the


DAZL knockout (KO) causes defects in GC commitment and differentiation. We characterized DAZL-KO pigs as SSC transplantation recipients. While there were GCs in 1-week-old (wko) KO, complete GC depletion was observed by 10 wko. Donor GCs were transplanted into 18 DAZL-KO recipients at 10–13 wko. At sexual maturity, semen and testes were evaluated for transplantation efficiency and spermatogenesis. Approximately 22% of recipient seminiferous tubules contained GCs, including elongated spermatids and proliferating spermatogonia. The ejaculate of 89% of recipients contained sperm, exclusively from donor origin. However, sperm concentration was lower than the wild-type range. Testicular protein expression and serum hormonal levels were comparable between DAZL-KO and wild-type. Intratesticular testosterone and Leydig cell volume were increased, and Leydig cell number decreased in transplanted DAZL-KO testis compared to wild-type. In summary, DAZL-KO pigs support donor-derived spermatogenesis following SSC transplantation, but low spermatogenic efficiency currently limits their use for the production of offspring.Spermatogonial stem cell (SSC) transplantation into the testis of a germ cell (GC)-depleted surrogate allows transmission of donor genotype via donor-derived sperm produced by the recipient. Transplantation of gene-edited SSCs provides an approach to propagate gene-edited large animal models. DAZL is a conserved RNA-binding protein important for GC development, and  Spermatogonial stem cell (SSC) transplantation is a technique that holds great potential for addressing male infertility, as well as for endangered species conservation, genetic preservation, and generation of genetically modified animal models ,5,6,7,8.The transplantation of gene-edited germ cells to recipient males is an effective strategy for engineering targeted mutagenesis and offers advantages over traditional methods of generating transgenic models, especially in large animals, such as shortened time and cost efficiency in comparison to cloning or embryo-based technologies, or even by replacing techniques that may be inefficient or unavailable in some species ,22,23,24To optimize SSC transplantation efficiency, the recipient testis is ideally devoid of germ cells, as the lack of endogenous spermatogenesis creates available niches for transplanted cells to colonize and differentiate ,29,30. TNANOS2 knockout were previously generated and used as recipients for SSC transplantation with limited success [Gene-edited pigs with germline ablation via  success ,42. AlthDAZL gene have been associated with male infertility and conditions such as azoospermia, where no sperm are present in the ejaculate [DAZL-deficient animals present a germ-cell-depleted phenotype, with defects in germ cell lineage commitment, spermatogonial differentiation, and meiosis, impairing their ability to progress through spermatogenesis and produce sperm [DAZL, also known as “Deleted in Azoospermia-like”, is a highly conserved RNA-binding protein ,44,45 exjaculate ,56,57,58ce sperm ,59,60,61DAZL-KO male pigs [Therefore, herein we took advantage of previously generated ale pigs ,54 and hDAZL knockout (DAZL-KO) males were obtained using transcription activator-like effector nuclease (TALEN)-mediated gene editing in pig fibroblasts as described by Tan and colleagues [+/− clones with confirmed mutation were used to generate a breeding herd of DAZL+/− pigs, which was then used when colony expansion and sample collection were necessary. The experimental pigs obtained were a Landrace/Yorkshire crossbreed.lleagues . BrieflyDAZL-KO male recipients when they were 10–13 weeks old, with one testis being injected with donor GCs while the contralateral testis was kept intact. Transplantation was performed as previously described, with aseptic surgical conditions and under general anesthesia [We performed SSC transplantation into 18 esthesia . Germ ceesthesia . The resesthesia , germ ceEjaculates were evaluated according to sperm presence, concentration, and motility by light microscopy. Sperm genomic DNA was differentially extracted to reduce the recipient’s non-sperm cells within the seminal plasma and concentrate the sperm heads. Single-nucleotide polymorphisms (SNP) identified for the recipient tail and donor genomic DNA were PCR-amplified and Sanger-sequenced to confirm the origin (donor/recipient) of the sperm collected . DAZL-KO boars were castrated only at sexual maturity, when blood was also collected for hormonal analyses. Blood was allowed to clot for approximately 30 min at room temperature before being centrifuged at 1000× g for 10 min at 4 °C. Blood serum was collected, aliquoted, and frozen immediately. For intratesticular testosterone measurements, fragments of the transplanted and non-transplanted testes were homogenized in PBS containing protease inhibitors and detergent according to the service provider guidelines, centrifuged at 10,000× g for 10 min at 4 °C, and supernatant was frozen immediately. Samples were submitted to the service provider  and analyzed using the “Steroid-Thyroid Hormone 6-Plex Discovery Assay”.Non-transplanted control, heterozygotes, and knockout pigs were castrated at 1 week and 10 weeks of age and at sexual maturity for evaluation of the testicular parenchyma, while transplanted Collected testes were fixed in 2% paraformaldehyde or Bouin’s solution and routinely processed for histomorphometry and immunohistochemistry analyses through dehydration via graded alcohol series and embedding in paraffin. Slides were prepared with sections of 5 μm thickness, stained with hematoxylin and eosin or periodic acid-Schiff, and imaged under light microscopy for histological evaluation and testicular morphometry.The volume densities of tubular and intertubular components of the testicular parenchyma were determined at 400× magnification using Image J software (version 1.53n) with a 713-point grid over digital images. Ten randomly chosen images  were counted for each animal, and points were classified as one of the following: seminiferous tubules, comprising tunica propria, seminiferous epithelium, germ cells, and tubular lumen; intertubular compartment, including Leydig cells, connective tissue, and blood vessels. The volume of each component was calculated as the product of its volume density and testis volume, considering 1.0 for the specific density of the testis and excluding the testis capsule from the testis weight .3) according to the formula v = 4/3πr3, where r = nuclear diameter/2. The total number of Sertoli cells per testis was determined as follows: total number of Sertoli cells per testis = total volume of Sertoli cells in the testicular parenchyma (mL)/Sertoli cell nuclear volume (µm3). The volume of individual Leydig cells was obtained from the measurement of their nuclear and cytoplasmic volume. To calculate the proportion between nucleus and cytoplasm and relative nuclear and cytoplasmic volumes, a 713-point square lattice was projected over digital images at 630× magnification, and 1000 points over Leydig cells were counted for each animal. The total number of Leydig cells per testis was calculated from their individual volume and the volume density occupied by Leydig cells in the testis parenchyma.The mean Sertoli and Leydig cell nuclear volumes were determined by measuring the nuclear diameter of 30 nuclei showing an evident nucleolus per animal and the calculation of nuclear volume  were evaluated and categorized into having or not having germ cells in the seminiferous epithelium.Testis sections were deparaffinized with xylene and rehydrated. Antigen retrieval was performed by boiling for 10 min in citrate buffer . Slides were allowed to cool, washed in PBS, and permeabilized in cold methanol for 10 min, followed by 10 min in 0.2% Triton-X in PBS. Slides were then washed in PBS twice and unspecific binding was blocked by incubating with CAS-Block for 30 min . Sections were then incubated with the primary antibodies  diluted t-test. The limit of statistical significance considered was p < 0.05 .Statistical analysis was performed using GraphPad Prism v6.0 . Data were tested for normality (D’Agostino and Pearson) and analyzed accordingly using either one-way ANOVA with Tukey’s multiple comparison tests or Student’s DAZL-KO (KO) pigs at 1 week old (1 wko) and in adulthood. We first characterized the testes of heterozygote (Het) and homozygote DAZL-KO pig testes, complete germ cell depletion was observed at 10 weeks of age (10 wko) as well as in adult testes  of transplanted males. Peak sperm concentration ranged from 5 × 10analysis c confirmDAZL-KO transplanted testis at the time of castration. Morphometric quantification revealed that 22.2%  of the seminiferous tubule cross sections contained germ cells at different stages of development, and most germ-cell-containing tubules were observed at the region close to the transplantation site (mediastinum/rete testis). Immunostaining of the recipient testes that received donor germ cells confirmed the presence of elongating spermatids expressing TNP1 (nuclear transition protein 1) (otein 1) a as wellotein 1) b.DAZL-KO pigs. Given the low sperm production efficiency and sperm quality observed, we further evaluated the recipient testes for markers of somatic cell function at 1 wko and in adulthood, as well as the hormonal profile in adult p < 0.05, p < 0.05). In the adult pigs, the differences in testis component occupancies were more pronounced. The absence  of germ cells led to a smaller proportion of the testis being occupied by seminiferous epithelium and the tubular compartment itself in both Het and KO groups when compared to WT (p < 0.05), while the tunica propria occupancy was higher in KO in comparison to WT (p < 0.05). A consequence of reduced tubular compartment occupancy is the observed increase in the intertubular compartment proportion in Het and KO pigs (p < 0.05), which was accompanied by higher occupancy of both the Leydig cells and connective tissue present in this compartment (p < 0.05).At 1 wko, the only significant difference observed was the proportion of the testis occupied by connective tissue that was increased in both Het and KO pigs when compared to control  in Het when compared to WT, and in KO when compared to Het and WT at 1 wko (p < 0.05), but no difference was observed in mature Sertoli cell nuclear volume in adulthood , an intermediate filament protein present in the cytoskeleton of testicular somatic cells; the Anti-Müllerian Hormone (AMH), produced by immature Sertoli cells; occludin (OCLN), a protein present in tight junctions among adjacent mature Sertoli cells and some interstitial cells; alpha-smooth muscle actin (SMA), present in peritubular myoid cells and endothelial cells; and aromatase (CYP19), a steroidogenic enzyme present in Leydig cells. The expression of these markers in 1 wko  and adulp < 0.05) (p < 0.05)  a. No dif < 0.05) b.DAZL-KO pigs as hosts for SSC transplantation.The successful application of the SSC transplantation technique will allow the development of novel strategies in assisted reproduction, preservation of genetic diversity in livestock species, rescue and propagation of difficult-to-breed genotypes, and generation of genetically modified animal models. We previously reported targeted gene editing in porcine germ cells that can serve as donor cells for SSC transplantation and enable the successful generation of gene-edited pig models . In the DAZL-KO pigs [DAZL-KO seminiferous cords still harbor gonocytes, but these GCs are no longer present in the testis of knockout pigs at 10 weeks of age and onwards. The exact age and the mechanism by which the postnatal GC development is disrupted in this model were not within the scope of this study, but these aspects certainly deserve further investigation [We confirmed the impaired spermatogenesis previously observed in -KO pigs , which i-KO pigs ,59,60,61tigation ,48,59. Ttigation ,31. Thertigation ,42,66.DAZL-KO recipient pigs were able to produce sperm, and all sperm evaluated were exclusively from donor origin. This confirmed that testes of DAZL-KO pigs can be hosts for transplanted SSCs and can regenerate donor-cell-derived spermatogenesis. However, the peak sperm concentration was low when compared to controls, and motile sperm were rarely observed except for one recipient which showed better sperm motility. This probably means that this individual had better colonization of the testis parenchyma by donor cells, therefore showing improved sperm parameters. However, at this point, there is no definite explanation for this observation. Based on histological analyses, the degree of colonization obtained was not very high, which could be one reason why fewer sperm were produced. Similar findings have been reported, including when NANOS2-KO pigs were used as recipients for SSC transplantation [The majority ~89%) of antation ,42. The 9% of antantation ,66,67,68DAZL-KO testes may not be optimally supportive for GC colonization, maintenance, and differentiation. To investigate this, we evaluated somatic cell function and hormonal homeostasis in these pigs. Most changes observed in the occupancy of the testicular parenchyma components were related to the lack of germ cells inside the seminiferous tubules, which led to a proportional reduction of tubular compartment occupancy and a higher proportion of intertubular compartment. No major change was observed in Sertoli cell number in either age group, while Leydig cells in the adult DAZL-KO testes were larger in size but reduced in numbers. We suspect that the changes observed in these steroidogenic cells may be related to the increase in intratesticular testosterone levels in the KO-transplanted testis in comparison to WT, and the increased serum E2 levels in DAZL-KO pigs, considering that high testosterone levels can lead to increased aromatization into E2. These results show that the steroidogenic potential of Leydig cells is maintained in DAZL-KO pigs and can be activated in the presence of spermatogenic cells, as testosterone is required for the regulation of spermatogenesis at several steps, such as meiosis and spermiogenesis [DAZL-KO pigs.Other factors of the microenvironment within ogenesis ,70,71. Togenesis ,72. The DAZL-KO pig testes support donor-derived spermatogenesis and produce sperm following SSC transplantation, but low spermatogenic efficiency limits its use for the production of donor-derived offspring. Therefore, the technique needs to be further improved, possibly focusing on increasing the level of GC colonization of the testis parenchyma, by investigating the potential mechanisms involved or stimulating this process via hormonal modulation for example [In summary, in the present study, we showed that  example . Current


Extracellular vesicles and particles (EVPs) are released from virtually all cell types, and may package many inflammatory factors and, in the case of infection, viral components. As such, EVPs can play not only a direct role in the development and progression of disease but can also be used as biomarkers. Here, we characterized immune signatures of EVPs from the cerebrospinal fluid (CSF) of individuals with HTLV-1-associated myelopathy (HAM), other chronic neurologic diseases, and healthy volunteers (HVs) to determine potential indicators of viral involvement and mechanisms of disease.We analyzed the EVPs from the CSF of HVs, individuals with HAM, HTLV-1-infected asymptomatic carriers (ACs), and from patients with a variety of chronic neurologic diseases of both known viral and non-viral etiologies to investigate the surface repertoires of CSF EVPs during disease.p = 0.0002 and p = 0.0003 compared to HVs, respectively, and p = 0.001 and p = 0.0228 compared to MS, respectively), consistent with the immunopathologically-mediated disease associated with CD8+ T-cells in the central nervous system (CNS) of HAM patients. Furthermore, CD8+ (p < 0.0001), CD2+ (p < 0.0001), CD44+ (p = 0.0176), and CD40+ (p = 0.0413) EVP signals were significantly increased in the CSF from individuals with viral infections compared to those without.Significant increases in CD8+ and CD2+ EVPs were found in HAM patient CSF samples compared to other clinical groups (These data suggest that CD8+ and CD2+ CSF EVPs may be important as: 1) potential biomarkers and indicators of disease pathways for viral-mediated neurological diseases, particularly HAM, and 2) as possible meditators of the disease process in infected individuals. Human T-lymphotropic virus type 1 (HTLV-1), the first human retrovirus to be discovered, results in a chronic, lifelong infection –4. DurinChronic neurologic disease encompasses a wide spectrum of etiologies including genetic, autoimmune, protein disorders, and infection-driven pathologies . DiffereCSF samples used in this study were collected from subjects under the National Institute of Neurologic Disorders and Stroke protocols #98-N-0047, 89-N-0045, 13-N-0149, 13-N-0017, and 15-N-0125. Prior to study inclusion, written informed consent was obtained from the subject in accordance with the Declaration of Helsinki.g for 10 minutes. The cell-free supernatants were collected in cryotubes in 1 mL aliquots and immediately frozen at -80°C until use. The collected CSF cells were counted using a Muse cell analyzer (Millipore) and freshly used for immunophenotyping analysis. For this study, the cell-free CSF material was used for our assays. We selected 10 HV, 10 MS, 10 HAM, and 14 OND CSF samples to provide sufficient numbers for this exploratory study , as previously described . All flo9/mL. Acquired output files were imported into MATLAB  where the spike-in population was used to calibrate the sample size and concentration across acquisitions. Statistical analysis of summary data was performed using GraphPad Prism (v9.3.1).CSF EVP size and concentration was measured using MPRS . Measurements were accomplished using TS-400 microfluidic cartridges. Neat, cell-free CSF samples were measured immediately after first-time freeze-thaw with the addition of a 240 nm NIST bead spike-in population at a final concentration of 1 x 10Cell-free CSF samples were titrated  for incubation with 10 µL of MACSPlex Exosome Kit  antibody-capture bead mixture which contains 39 separate fluorescently barcoded bead populations, each specific to a different cell surface marker, in 1.5 mL low protein binding tubes overnight at room temperature, rotating and protected from light. A 0.2 µm PES filter plate was washed using 150 µL of MACSPlex buffer and cleared using a vacuum manifold. Fifty microliters of MACSPlex buffer were added to each well before incubated EVP-capture bead samples were aliquoted into each of the wells. The EVP-capture bead mixtures were washed using vacuum manifold and immediately resuspended in 200 µL MACSPlex detection antibody solutions . Wells were reverse pipetted to mix, and plates were left on a shaker for two hours at room temperature and protected from light. Samples were washed three times using the vacuum manifold and immediately resuspended in 200 µL MACSPlex buffer. Each well was then transferred to 96-well U-bottom polypropylene plates. Sandwiched EVPs were then analyzed via flow cytometry and signals were detected for EVPs that co-expressed the capture bead marker as well as one or multiple of the EV-enriched tetraspanins. All data acquired by Multiplex assay (MPA) required the sample to be bound by both a capture bead and an EVP tetraspanin-specific detection antibody, and thus we refer to these markers as EVP-associated.-1) until 5,000 single bead events had been acquired. Post-acquisition .fcs file arbitrary unit scales were calibrated to APC molecules of equivalent soluble fluorochrome (MESF) units using reference standards .Beads were triggered using a forward light scatter trigger threshold and optimal gains for each detector  found by performing voltration on 8-peak beads . Each plate well was acquired using a medium flow rate   where a database was built. A detailed protocol can be found at http://dx.doi.org/10.17504/protocols.io.bm3gk8jw  , 21. Conftware/) , 22. Mulbm3gk8jw . A normaPreviously, we have studied EVPs from both peripheral blood mononuclear cells (PBMCs) and the CSF of HAM and MS patients and found that EVs from HTLV-1-infected cells can contain Tax protein and were able to sensitize an HTLV-1-specific CTL response in targets . Because9 particles/mL) that pre-isolation of EVPs prior to incubation with multiplex beads did not improve EVP attachment or detection with multiplex capture  and ONDs , but not to HVs and MS patients. Additionally, ACs and ONDs commonly had increased signals of tetraspanins compared to HVs . EVP-associated T-cell markers CD8 and CD2 were increased in HAM patients compared to HVs  and MS patients , whereas CD4+ EVPs had overall lower signals and were not statistically different between groups (p = 0.0085) and CD44+ EVPs which had elevated signals in ACs and ONDs compared to HVs , no other groups differed substantially from each other. Finally, CD133/1+  and MS . Additionally, CD133+ EVP signals were reproducibly decreased in HAM compared to MS patient CSFs (p = 0.0406). The patient demographics for the second cohort used are shown in Selected MPA results were converted to MESF units, background subtracted, and compared by clinical group. While all groups had tetraspanin signals > 1 x 10d tumors , 25) EVPp = 0.0202, r = 0.7333) and the CD8+ T-cell concentration  in the CSF of HAM patients  with CD133/1+ EVPs in HAM CSF , whereas we were able to detect levels of CD8+ EVP signals between 1 x 103 to 5 x 104 MESF units using 250 µL of CSF  and tumor necrosis factor-α (TNF-α), which is proposed to result in substantial bystander destruction of resident CNS tissue and neural cells associated with demyelination , 30. MorWe found that EVP CD44 and CD40 markers were also increased in CSF samples from individuals with viral neurological diseases in our cohort and in CSF samples from asymptomatic carriers of HTLV-1 . CD44 isFinally, we found that CD133/1+ EVPs in the CSF were significantly decreased in HAM patients compared to MS patients , 25. IncHere, we have optimized and performed MPAs of various neurological disease group CSF samples to determine EVP surface marker differences between pathologies. We have demonstrated higher levels of CD8 and CD2 on CSF EVPs from patients with virus-associated neurologic diseases and infections in this cohort. There are some potential limitations we acknowledge with this work. It is possible that containing our analysis to 39 surface markers that we are excluding several important and biologically relevant other signatures on CSF EVPs. Neural-specific markers such as L1CAM, MOG, MBP, GLAST, and neurofilament, which were not commercially available in a comparable kit at the time of this study, will be of particular interest to investigate going forward. Several studies have shown compelling evidence that viruses, particularly Epstein-Barr virus (EBV), may act as a trigger for MS through interaction with genetic and other factors , 46; howSince many neurological diseases caused by viruses, including HAM, involve the infiltration of CD8+ CTLs into the CNS, it is possible that EVPs originating from activated CD8+ T-cells may play important functional roles in the neural proinflammatory response, both through antigen presentation and activation of other immune cell types, and/or by inducing inflammation, cytokine production, and cell death in neural lineage cells. These data collectively suggest that CD8+ and CD2+ EVPs may be significant in two respects: 1) CSF EVPs possessing CD8 and/or CD2 may be used as a potential biomarker for viral-mediated neurological diseases such as HAM, and 2) CD8+ and/or CD2+ EVPs may themselves mediate areas of the disease process in infected individuals. Ultimately, as methods for evaluating these EVP subsets and their cargo improve in sensitivity and analytical depth, these CD8 and CD2 EVP populations may be useful for monitoring treatment responses or disease activity in patients with chronic viral CNS conditions.The original contributions presented in the study are included in the article/The studies involving humans were approved by National Institutes of Health Institutional Review Board. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.MP and JW performed the experiments, data analysis, and wrote the manuscript. ES, SC, D-AJ, BK, and TT helped with portions of experiments and optimization of experimental pipelines. AC, RH, MM, NN, JO, YE-A, AN, IC, and DR were responsible for patient sample acquisition, storage, and research-based and clinical data procurement from patients at the NIH clinical center. JJ and SJ oversaw all experimental designs, sample usage, and overall direction of the manuscript. All authors contributed to the article and approved the submitted version.


To enrich pork with valuable n-3 PUFA, it is common practice to include flaxseeds in the swine diet. However, due to the high susceptibility of these acids to oxidation, this treatment requires an additional supply of antioxidants. Thyme herb can be used for this purpose, which in addition to high antioxidant activity is characterized by numerous health-promoting properties. The present study aimed at evaluating the impact of the inclusion of 1% and 3% of thyme herb in mixtures with 4% of extruded flaxseeds as a source of n-3 PUFA on the performance results and health status of fatteners. The experiment was carried out on 120 weaners with an initial body weight of about 30 kg and kept until the end of fattening. They were divided into three experimental groups of 40 animals each (5 pens with 8 pigs in each). The control group (C) consisted of pigs receiving a base mixture with 4% of extruded flaxseeds. In the experimental groups, an additional 1% (T1) or 3% (T3) of thyme herb was added to the mixture. By supplementing the diet with 3% of thyme, an increase in average daily weight gain (P = 0.001) and a better feed conversion ratio (P < 0.001) were obtained. This could be the result of an improvement in the small intestine histology (greater villus height–P < 0.001) and better digestibility of basic nutrients  found in experimental animals. In addition to these effects, thyme herb supplementation contributed to the stimulation of immune mechanisms . The obtained results indicate the advisability of the use of thyme as a feed additive beneficially influencing the health and performance results in pigs. The obtained results indicate the advisability of the use of thyme as a feed additive beneficially influencing the health and performance results in pigs. It is extremely important to include adequate levels of n-3 polyunsaturated fatty acids (PUFAs) in a well-balanced diet for both younger and older pigs. For this reason there is a clear trend of adding various significant sources of these fatty acids to feed. Rich sources of n-3 PUFAs include vegetable oils, e.g. hemp or rapeseed oil, animal oils, such as fish oil, and flaxseed or flaxseed oil .Our previous research has shown that the inclusion of extruded flaxseed as a source of n-3 PUFAs in the diet of pigs improves the digestibility of basic nutrients (EE and NfD) and performance parameters, as well as meat quality (an increased concentration of n-3 in the tissues) and animal health . HoweverThymus vulgaris) is an aromatic plant of the family Labiatae. According to Dauqan and Abdullah [There are few reports, however, on the use of thyme herb in the diet of pigs. Thyme  will have a positive effect on the intestinal histology, which will result in an increase in the digestibility of nutrients and, as a consequence, an improvement in the performance parameters of fattening pigs.Therefore the aim of the study was to assess the effect of the inclusion of 1% or 3% thyme herb in diets with 4% extruded flaxseed as a source of n-3 PUFAs on the growth performance and health of fattening pigs.The experiment was conducted on 120 piglets (Polish Large White x Neckar) weighing approximately 30 kg (about 84 days of age), which were kept until the end of the fattening period . The animals were divided into three experimental groups matched for sex and body weight. Each group consisted of 40 animals, were housed in 5 pens with 8 pigs in each (4 gilts and 4 barrows). In order to minimize the influence of factors that could affect the obtained result, variation in body weight within and between pen replicates and groups was minimized to the extent practically possible. The pigs were kept in pens on straw.All methods were carried out in accordance with relevant guidelines and regulations regarding to life animal studies and the procedures complied with the Directive 2007/526/EC of the European Parliament and of the ouncil on the protection of animals used for scientifc purposes. All tests on animals were performed with the approval of the Local Ethics Committee on Animal Experimentation of the University of Life Sciences in Lublin, Poland .ad libitum. The ingredient composition of the diets is presented in •+  radical) - 2.11mmol Trolox/g; DPPH•  - 1.76mmol Trolox/g.In the presented experiment, the control group (group C) comprised animals receiving a diet with 4% extruded flaxseed but without thyme herb. In groups T1 and T3, 1% or 3% of thyme was added to the basal diet. The animals were fed complete Grower and Finisher diets The choice of thyme herb was influenced by a pilot experiment in which the dietary preferences of piglets were observed. In that experiment piglets received diets with a 3% share of herbs with antioxidant properties: thyme, caraway, coriander, or purple coneflower. The diet most preferred by the piglets was observed to be the one with thyme herb.The thyme herb used in the experiment was obtained from a single farm on which thyme was cultivated. Dried thyme herb was added to the diets when they were being prepared in the feed mixer.All animals were weighed prior to the start of the experiment, and again when the diet was changed from grower to finisher (at about 70 kg BW) and at the end of the experiment (about 110 kg BW). Then average daily weight gain (ADG) was calculated. Feed intake (FI) was recorded for each pen throughout the experiment. The FI values were calculated per pig (by dividing by the number of pigs per pen). The FI and feed conversion ratio (FCR) are expressed as the pen average for each period. At the end of the experiment (about 110 kg BW) the animals were slaughtered with the use of the electric shock method.Before the start of each stage of the experiment, the thyme herb and the experimental diets were sampled–from three different places in the batch of feed and from three different places in the batch of thyme herb–for analysis of proximate chemical composition, i.e. the content of dry matter (103˚C for 20h), crude protein  determined by the Kjeldahl method), ether extract (procedure 935.38), and crude fibre (procedure 962.09), according to AOAC . The dieContents of amino acids were determined on a Sykam Amino Acid Analyzer . Prior to analysis, the samples were hydrolysed with 6 N HCl at 110˚C for 24 h. Methionine and cysteine were analysed as methionine sulfone and cysteic acid after cold performic acid oxidation overnight prior to hydrolysis.Prior to analysis, the samples were homogenized using a BUCHI mixer B-400 with ceramic blades. All analyses were performed in triplicate.2O3) per kg of diet was added to the feed of all groups as a digestibility marker, prior to pelleting [At the end of the growing period (grower: 65–75 kg BW) and fattening period (finisher: 100–110 kg BW), 3 g of chromium oxide . It was used to determine the CrThe content of chromium oxide in the diets and faeces samples was determined according to Suzuki and Early .The apparent total digestibility (ATTD) of the nutrients were calculated according to the following formula:g/kg DW) .Blood was collected twice from the same pigs that were used for digestibility analysis (6 pigs from each group), at a body weight of about 70 and 100 kg. The animals had no access to feed for 12 h before blood sampling. Blood was collected by a veterinarian from the jugular vein into 10 ml heparinized tubes.The blood samples were centrifuged at 3500 g for 15 min to obtain plasma, which was frozen (–20°C) until analysis.White blood cell parameters, i.e. white blood cell count (WBC) and differential leukocyte count–the percentages of granulocytes (GRA), lymphocytes (LYM) and MID (sum of eosinophils + basophils + monocytes), were analysed in whole blood using an ABACUS-Vet analyser.The titre of IgG (QY-E40124), IgA (QY-E40199), IgM (QY-E40123) immunoglobulins and the content of lysozyme (QY-E40200) and IL-6 (QY-E40023) in the plasma were determined by an immunoenzymatic ELISA assay using kits from AssayGenie 2); lean meat content; average backfat thickness from 5 measurement points (BT 5); and the weight of the kidney, liver and heart. The intestines were also collected for morphometric analysis.Carcass analysis was performed after slaughter (∼110 kg) on 6 pigs from each group–the same animals used for the blood and digestibility analyses. Carcass analysis involved determination of dressing percentage (%); meat percentage in the ham (%); loin area , and embedded in paraffin. Sections 3 μm thick were prepared from the paraffin blocks and stained by standard H&E (haematoxylin and eosin) staining. Morphometric analysis of the slides was then performed using the LUCIA NIS Elements BR 2.20 image processing and analysis system , according to methods described by Hedemann et al. . MeasureAll data are expressed as means and SEM (standard error of the mean).Data were analysed by ANOVA. The performance parameters were measured based on replication groups . For slaughter parameters single animal (randomly chosen from each replication group) was treated as a scientific unit, it allowed to have 6 values of each parameters per group. It is minimal number of samples for statistical purposes and also according to the Local Ethical Committee, approval. Resolution No. 64/2011.To determine the effect of the addition of thyme and the level of thyme, the data were analyzed using pre-planned contrasts according to the following model:T = difference between control group and supplementation of thyme (C vs T1+T3)D = difference between treatment with supplementation level of thyme (T1 vs T3).Treatment means were compared using Tukey’s multiple comparison test in Statistica 13 .The analysis showed that the thyme herb used in the experiment contained 58.7 g/kg crude protein, 39.2 g/kg ether extract, and 212.2 g/kg crude fibre .P = 0.005 and P = 0.001, respectively), as well as their average daily gains (ADG), , compared to the control group and group T1 (P = 0.038), and compared to groups C and T1 during the finisher period (P = 0.014). Animals from the group receiving 3% thyme had a significantly lower feed conversion ratio (FCR) than in the control group and group T1 in the finisher period (P < 0.001). The use of thyme significantly influenced mean ADG (P = 0.007) and FCR (P = 0.003) in comparison with the control group (C vs. T), , mean ADG (P = 0.026) and mean FCR (P < 0.001).The inclusion of 3% thyme in diets for fatteners significantly increased their body weight in both the grower and finisher period (group T1 . FI in g vs. T), . The dosP = 0.030 grower and P = 0.027 finisher), (P = 0.050) and of Cfibre compared to group C (P = 0.023). The dose of thyme significantly influenced CP (finisher P = 0.023), EE .Faecal digestibility of crude protein was significantly higher in pigs receiving a diet with 3% thyme than in groups C and T1 during the entire experiment (nisher), . The incP = 0.042).The inclusion of thyme in diets for fatteners did not significantly influence carcass analysis parameters, i.e. dressing percentage, meat % in ham, lean meat content, BT5, kidney weight, liver weight, and heart weight . It onlyHistological analysis of the sections of the pigs’ intestines showed that the inclusion of 3% thyme significantly increased the length and width of the villi and the thickness of the muscularis externa in the ileum relative to the control group , . The useP ≤ 0.05), (P = 0.02 –grower and P = 0.048 finisher). Grower pigs from group T3 also had a significantly lower number of granulocytes than pigs from groups C and T1 (P = 0.048).In each stage of the experiment, the blood of pigs receiving a diet with 3% thyme had significantly higher WBC and LYM counts compared to the control group (≤ 0.05), . The lymP < 0.05), , .In the first part of the research, published by Klimiuk et al.  and Czecin vitro) against haemolysing strains of E. coli isolated from them, but showed no effect on growth performance. Experiments on the effectiveness of the use of components of essential oils in diets for piglets weaned from the sow, such as thymol or carvacrol, do not yield clear conclusions. Some studies have shown no effect of these substances on growth performance [There are numerous reports indicating that diets containing thyme herb or thyme oil improve the growth performance of animals raised for meat, mainly poultry . Howeverformance , while oformance  found thformance , substanformance , used asformance .The improved growth performance in the group of pigs receiving a diet with 3% thyme herb was likely due to the improvement in the digestibility of crude protein as well as ether extract and Cfibre, which was particularly evident in the first stage of the fattening period. The study by Li et al. , cited aThyme herb did not affect most of the parameters of the carcass analysis in the present study or in a study by Rhouma et al.  in rabbiImproved nutrient digestibility and fattening efficiency may also be linked to the beneficial effect of the active substances in thyme on the histomorphology of the intestine . The morMany studies indicate that the presence of polyphenolic compounds in thyme is responsible for improvement of the immune response . The preIn this study the inclusion of 3% thyme herb in the diet enriched with PUFA (extruded flaxseed) of fattening pigs improved the histology of the intestinal over its entire length, causing a significant increase in villus length which translated to improved nutrient digestibility and production efficiency. It also stimulated immune processes, resulting in increased levels of class G and A immunoglobulins and lysozyme in the blood plasma.Improved feed intake corresponding to better weight gains in pigs receiving diets with 3% thyme herb, as well as stimulation of immune processes, suggests that this is a promising phytobiotic that can be used throughout the fattening period.S1 File(XLS)Click here for additional data file.


Kidney transplantation is often regarded as the preferred therapy forend-stage renal disease. Several surgical procedures have been developed toreduce postoperative donor complications, while maintaining kidneyquality.This study aimed to compare the preoperative and postoperative outcomes ofliving kidney donors who underwent either transperitoneal laparoscopicnephrectomy or open nephrectomy.Retrospective study conducted in Istanbul, Turkey.Fifty-five living-related kidney donors underwent nephrectomy and wereretrospectively divided into two groups: 21 donors who underwent opennephrectomy (Group 1) and 34 donors who underwent transperitoneallaparoscopic nephrectomy (Group 2).In comparison to the donors who underwent open nephrectomy, those whounderwent transperitoneal laparoscopic nephrectomy had significantly shorterpostoperative hospital stays ,duration of urinary catheterization , operating times , and less blood loss . There was nostatistically significant difference between the two groups with regard tothe estimated blood transfusion and warm ischemia time. The preoperativeweek, first postoperative week, and 1-month postoperative serum creatininelevels were comparable between the groups.Laparoscopic donor nephrectomy can be safely performed at centers withexpertise in laparoscopic surgery. Laparoscopic donor nephrectomy has betteroutcomes than open donor nephrectomy in terms of length of hospital stay,duration of urinary catheterization, operating time, and blood loss. Ratner introduced this technique in 1995; it is now performed morefrequently, with higher success rates. Donor nephrectomy is distinguished from other surgical procedures in that thesurgery is performed on a healthy individual to improve the health of another. Thisplaces a strong emphasis on reducing donor morbidity and implementing minimallyinvasive approaches. There is an increasing gap between organ supply and demand,which has also played a role in the recent trend toward living-donor kidneytransplantation.Improvements in technique have resulted in better outcomes for laparoscopic donor nephrectomy. Some of the advantages of transperitoneal laparoscopic nephrectomy over openmethods are reduced intraoperative blood loss, improved aesthetics, shorter hospitalstay, and faster overall postoperative recovery, which allows the recipient toreturn to normal activity in a shorter period. As a result of these advantages, thenumber of living-donor kidney transplants has increased. Currently, most transplantation centers harvest living-donor kidneys using aconventional laparoscopic surgical approach. Transplantation teams accept living kidney donations under conditions thatsuggest a safe long-term outcome for the donor.Transplant recipients receive numerous benefits from living donations, and theoperation can be planned. However, donors do not receive the same benefits.We aimed to evaluate and compare early complications and renal function followingdonor nephrectomy performed by an experienced surgeon using either an open orlaparoscopic approach.This study included 55 living-related kidney donors who underwent nephrectomybetween March 2010 and March 2014. Twenty-one of these patients underwent opennephrectomy (Group 1), and 34 underwent laparoscopic nephrectomy (Group 2).Donors were interviewed regarding their surgical preferences, which includedboth open and laparoscopic donor nephrectomies. Patients aged between 18 and 75years with end-stage renal disease, ,who received an organ from a live donor from their family were included in thestudy. The exclusion criteria were nephrectomy of cadaver origin, a follow-upduration less than 1 month, and pre- or post-operative contrast-enhancedimaging. Patient data were collected from the hospital's medical recordsdatabase and through patient interviews.The donations were voluntary and in accordance with the Human Organ Transplantpolicies and regulations in Turkey. This study was approved by the EthicsCommittee of the Marmara University School of Medicine . The ability of the MDRD formula to predict GFRs was analyzed by comparingthe results obtained from other prediction equations of healthy participantswithout any known kidney disease. Age, sex, and serum creatinine levels were recorded for the studyparticipants to estimate GFR using the abbreviated version of the MDRD formula:A detailed assessment of the donors is routinely performed to ensure long-termsafety. According to the United Network for Organ Sharing (UNOS), follow-up andmonitoring of serum creatinine levels are required after a post-donationduration of at least 2 years. The functional performance of the kidney is mainlyevaluated using the best overall measure: the glomerular filtration rate (GFR).The Modification of Diet in Renal Disease (MDRD) formula was used to calculateand perform a detailed evaluation of pre- and post-operative kidney functionusing estimated glomerular filtration rates (eGFRs). This formula was developedby the MDRD study group.Before the procedures, the donors and recipients underwent a comprehensivemedical assessment, and light bowel preparation was performed before surgery.The renal vessel anatomy of all donors was evaluated using abdominal computedtomography (CT) imaging.The best use of renal vein length was achieved by left donor nephrectomy, whichwas routinely performed. A retroperitoneal flank incision was used for classicopen nephrectomy. Transperitoneal laparoscopic nephrectomy was performed on theleft side with the patient in the decubitus position. The procedure wasperformed using a video laparoscope and dissecting instruments. The procedurestarted with the inflation of the abdomen using a Verres needle. The abdominalcavity was inspected to ensure that there was no damage after inserting a 12-mmtrocar. Two additional trocars were then inserted, the first superolateral tothe umbilicus and the second at the midline of the rib cage. We often preferredusing 10-mm trocars because they were easy to interchange with laparoscopicinstruments. Dissection started with a Toldt line incision and reflection of thedescending colon and continued until Gerota's fascia was seen. The medialgonadal vein was observed, and the dissection was traced up to the renal hilum.The renal artery and gonadal, renal, and adrenal veins were then carefullydissected and transected. The progression of the level of the iliac vessels wasmade by ureteral dissection, and the ureter was transected distally. Each of therenal arteries, veins, and ureters were stapled across before the kidney couldbe removed from the bag. In the final step, the completely freed kidney wasremoved from the Gibson incision.The data were analyzed for frequencies, and the chi-square test was used tocompare categorical variables. The mean values of the numerical variablesbetween the groups were compared using the Mann–Whitney U test. SPSS for Windows was used for thestatistical analysis of all data. Statistical significance was set at P <0.05.Donor demographics, estimated blood loss, operative characteristics, mean hospitalstay, mean operative time, warm ischemia time of the graft, number of vessels,reduction rate of donor serum creatinine levels in the first seven days and onemonth after renal transplantation, and donor complications were compared between thetwo surgical approaches.Living-donor nephrectomies were performed on all 55 donors . The donor demographics andindications for surgery were similar in both groups. A comparison of donorcharacteristics is shown in The creatinine levels in the preoperative week and first postoperative week and monthwere comparable between the two groups . The mea2, respectively (P = 0.28). After postoperative week 1, theMDRD values decreased to 34 and 34.3 ml/min/m2 in Group 1 and Group 2,respectively    and 3. AA within-group assessment of the surgical learning curve was also performed for Group2 (laparoscopic nephrectomy), which showed a significant reduction in surgery timefollowing the first 10 cases of living donors (P = 0.0001). From an average of morethan 200 min for the first 10 cases, surgery time was reduced to less than 200 minafter the initial 10 procedures (249 ± 19 versus 197 ± 13 minutes). No statisticallysignificant differences were found based on the learning curve between the twogroups  in terms oflaboratory parameters, perioperative blood loss, analgesic requirement, andpostoperative clinical parameters. No deaths occurred among the 55 donors includedin this study.Limitations in the organ donor supply continue to pose a significant challenge toimproving the outcomes of patients with end-stage renal failure. It has becomeimperative to expand the potential living-donor pool; this has been successfullyachieved with the advent of laparoscopic donation because of the rapid recovery andreturn to normal activities. Donor nephrectomy is a unique operation in that it exposes a person incomplete health to the potential complications of major surgery for the benefit ofthe recipient. Therefore, donor safety should be the priority in kidney transplantation.Living-donor nephrectomy is considered the most stressful intervention in urologybecause, by definition, it involves an altruistic organ donation by healthy individuals. There is an association between the introduction of transperitoneallaparoscopic nephrectomy and the expansion of the living kidney donor pool inparticular, and renal transplantation in general; evidence of this has beenpreviously reported.The rate of live kidney donation in the United States, Europe, and Turkey hasincreased directly with the increased use of living transperitoneal laparoscopic nephrectomy. Additionally, four of the five conversions occurred in the first 40 cases. The learning curve for laparoscopic nephrectomy flattened after 10 cases,even in the hands of an experienced laparoscopic surgeon. Based on thetransperitoneal laparoscopic nephrectomy experience gained during this study, weadopted a point of view that seemed promising in terms of minimizing the morbidityassociated with the learning curve. Moreover, from the recipients’ standpoint, thetransperitoneal laparoscopic nephrectomy results were comparable to those obtainedusing the well-established open approach. From the donors’ standpoint, thetransperitoneal laparoscopic nephrectomy results were superior to those of the openapproach.However, morbidity and complication rates have been found to increase among surgeonsjust beginning to learn the technique for laparoscopic donor nephrectomy. Tominimize the warm ischemia time, careful handling of the vessels and kidney, rapidspecimen extraction, and extensive vascular dissection are required, and advancedlaparoscopic skills are necessary. The operative time and complication rates wereused to measure the surgical learning curve. After significant gains in experience,the incidence of delayed graft function and operative time decreased considerably.Leventhal et al. reported that the majority of complications occurred during thefirst 30 cases. In a study by Berber et al., the postoperative serum creatinine levels werewithin normal limits. Therefore, the development of kidney dysfunction or failure in a donor ishighly unlikely. Despite the limited follow-up and number of patients, severalstudies have examined changes in serum creatinine levels. Hartmann et al. reported 1,800 cases of living donors, of which only sevendeveloped end-stage renal disease. Another study reported 402 cases of living donors in Sweden, and only onerequired hemodialysis due to postoperative renal failure. In our study, the donors did not develop end-stage renal disease in thelong-term follow-up, a result consistent with the findings of previous studies.The increasing number of living donors has resulted in the need for more informationabout the potential risks of living with one kidney. Our findings indicate that mostkidney donors have a favorable renal course. However, additional donors should beevaluated to confirm these findings. After donation, numerous donors developedincreased serum creatinine levels, which may be associated with increasedcardiovascular mortality. In a study of 500 cases of laparoscopic donor nephrectomy reported byLeventhal et al., the average warm ischemia time was 2.6 min. Previous studies have reported shorter warm ischemia times fortransperitoneal laparoscopic nephrectomy in comparison to open donor nephrectomy. Inour study, the mean duration of warm ischemia was 2.7 min. Ideally, the warmischemia time should not exceed 3 min in transplant surgery. In general, the warm ischemia time is expected to be shorter in minimallyinvasive donor nephrectomy than in open donor nephrectomy. However, in our study, weobserved a longer warm ischemia time in the open donor nephrectomy group than thatreported in the literature. Specific factors may have contributed to the longer warmischemia time such as the complexity of the procedure, surgical team experience, orvariations in the technique used. It is also possible that there are issues relatedto the preservation and handling of the kidney after removal that may affect thewarm ischemia time. Another reason could be that open donor nephrectomy involves alarger surgical incision and more extensive dissection of the kidney and its bloodvessels, which increases the risk of bleeding and prolongs the warm ischemia time.The longer operative time for open donor nephrectomy differed from that reported inthe literature. This discrepancy may be because the duration of open donornephrectomy varies depending on the surgeon's experience, the patient's anatomy, andthe type of surgical technique used.During transperitoneal laparoscopic nephrectomy, minimization of the warm ischemiatime is crucial to avoid renal injury. In one study, the reported warm ischemia timeranged between 2.6 and 6 min.We found that the results from the donors’ standpoint corresponded with thosereported in other studies that compared transperitoneal laparoscopic and opennephrectomy. These include shorter hospital stay, less blood loss, and similar ratesof complications.Our study had some limitations. First, although the collection of laparoscopic datawas prospective, this study was retrospective, and the majority of open nephrectomydata were historical. Consequently, a significantly longer follow-up period wasobserved in the open nephrectomy group (Group 1). Second, the higher AmericanSociety of Anesthesiologists status of the open nephrectomy group constituted adiscrepancy between the two study groups that was unlikely to account for the longerhospital stay. Another limitation was the relatively small sample size of eachgroup. An additional limitation was the inadequacy of our findings, which indicateda difference in the length of the donors’ hospital stay between the two groups.Additional outcomes, such as functional status and patients’ quality of life, couldhave been more detailed and informative.Transperitoneal laparoscopic living-donor nephrectomy is a less invasive approachthan open nephrectomy. This has a significant influence on kidney donor operations.Consequently, donor morbidity decreased while a higher-quality allograft for therecipient was maintained. Transperitoneal laparoscopic donor nephrectomy can besafely performed in centers with expertise in laparoscopic surgery. From the donor'sperspective, transperitoneal laparoscopic donor nephrectomy has better outcomes thanopen donor nephrectomy in terms of the length of hospital stay, duration of urinarycatheterization, operating time, and blood loss.


Injured pericytes in the neurovascular unit release platelet-derived growth factor β (PDGFRβ) into the CSF. However, it is not clear how pericyte injury contributes to Alzheimer disease (AD)–related changes and blood-brain barrier (BBB) damage. We aimed to test whether CSF PDGFRβ was associated with different AD-associated and age-associated pathologic changes leading to dementia.APOE ε4 genotype and MRI measurements of cortical thickness, white matter lesions (WMLs), and cerebral blood flow. We also analyzed the role of CSF PDGFRβ in the relationship between aging, BBB dysfunction , and neuroinflammation .PDGFRβ was measured in the CSF of 771 participants with cognitively unimpaired , mild cognitive impairment , and dementia (n = 188) from the Swedish BioFINDER-2 cohort. We then checked association with β-amyloid (Aβ)-PET and tau-PET standardized uptake value ratio, b = 19.1, β = 0.5, 95% CI 16–22.2, p < 0.001), increased CSF neuroinflammatory markers of glial activation YKL-40 , GFAP , and worse BBB integrity measured by QAlb . Age was also associated with worse BBB integrity, and this was partly mediated by PDGFRβ and neuroinflammatory markers . However, PDGFRβ showed no associations with APOE ε4 genotype, PET imaging of Aβ and tau pathology, or MRI measures of brain atrophy and WMLs (p > 0.05).The cohort had a mean age of 67 years , and 50.1% were male . Higher CSF PDGFRβ concentrations were related to higher age (In summary, pericyte damage, reflected by CSF PDGFRβ, may be involved in age-related BBB disruption together with neuroinflammation, but is not related to Alzheimer-related pathologic changes. Furthermore, the relationship of CSF PDGFRβ to MRI measurements of cortical thickness, white matter lesions (WML), and CBF were studied. Finally, we analyzed the role of CSF PDGFRβ in the relationship between aging, BBB dysfunction  and neuroinflammation .The aim of this article was to determine whether CSF PDGFRβ is indeed associated with aging and key AD pathologic changes (measured with Aβ-PET and tau-PET) and All participants gave written informed consent. Ethical approval was given by the Regional Ethical Committee in Lund, Sweden.12 were to be 40 years and older, being fluent in Swedish, having Mini-Mental State Examination (MMSE) scores between 27 and 30 for cognitively unimpaired (CU) participants, between 24 and 30 for MCI, and equal to or above 12 for patients with AD dementia. MCI diagnosis was established if participants performed below 1.5 SD from norms on at least 1 cognitive domain from an extensive neuropsychological battery examining verbal fluency, episodic memory, visuospatial ability, and attention/executive domains. Patients with AD dementia, vascular dementia (VaD), behavioral variant of frontotemporal dementia (bvFTD), and dementia with Lewy bodies (DLB) fulfilled the respective criteria of the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition.13 Semantic and nonfluent variants of primary progressive aphasia  were defined according to the Gorno-Tempini criteria.14 All patients were genotyped for APOE. Exclusion criteria included severe somatic disease and current alcohol/substance misuse. CSF sampling and imaging investigations were performed at the time of enrollment, in conjunction with the clinical examination and cognitive tests. The study was approved by the Regional Ethics Committee in Lund, Sweden. All participants gave written informed consent to participate.The cohort included participants from the Swedish BioFINDER-2 study (NCT03174938). All participants were recruited at Skåne University Hospital and the Hospital of Ängelholm, Sweden. The cohort covers the full spectrum of AD, ranging from adults with intact cognition or subjective cognitive decline, mild cognitive impairment (MCI), to dementia. The main inclusion criteria, as described previously,15 PDGFRβ was measured with the Human Total PDGFRβ DuoSet IC ELISA  with few adaptations. In brief, the standard curve followed a 1:3 dilution, starting from 12,000 pg/mL. Capture antibody was diluted in phosphate-buffered saline (PBS). One percent MSD Blocker A buffer  in PBS was used to dilute standards and as blocking buffer. Detection antibody and streptavidin were diluted in 20 mM Tris, 137 mM sodium chloride, Tween 0.05%, and 0.1% bovine serum albumin, pH 7.2–7.4. Interassay variability (coefficient of variation %) measured over 14 runs was 7.3%. For a detailed description of the protocol, see supplementary material . Aβ42, Aβ40, p-tau181, YKL-40, and GFAP were measured with NeuroToolKit . Cutoff for an Aβ-positive (Aβ+) status was calculated with the Youden index in the cohort, based on CSF Aβ42/40 (cutoff = 0.08).12CSF was collected by lumbar puncture and stored at −80°C in polypropylene tubes following the Alzheimer's Association flow chart for lumbar puncture and CSF sample processing.12 A neocortical meta-region of interest (ROI) for Aβ-PET  was calculated, as previously described.16 According to the enrollment protocol, Aβ-PET was not performed in the dementia group.Aβ-PET images were acquired on digital GE Discovery MI scanners 90–110 minutes after the injection of ∼185 megabecquerel (MBq) [18F]flutemetamol. Standardized uptake value ratio (SUVR) was calculated with pons as reference region. For the analysis, Aβ PET measures were considered both as continuous SUVR and as binarized data using a cutoff derived from mixture modeling in the BioFINDER-2 cohort (0.53 SUVR).12 A temporal meta-ROI for tau-PET  was created, as previously described.17Tau-PET images were acquired on digital GE Discovery MI scanners 70–90 minutes postinjection of ∼370 MBq [18F]RO948. Tau-PET SUVR was created using the inferior cerebellar cortex as the reference region.18 The AD-specific cortical thickness meta-ROI (AD signature) was measured in regions with known susceptibility to atrophy in AD , adjusted for cortical surface area. Automated segmentation of WML using the LST toolbox implemented in SPM8 generated a total lesion volume (in milliliters), which was then normalized for intracranial volume, as previously described.19 Total gray matter CBF was measured in a smaller cohort of participants in the AD continuum  with arterial spin labeling, see Structural MRI was performed using a Siemens 3 T MAGNETOM Prisma scanner , with high-resolution T1-weighted anatomic magnetization-prepared rapid gradient echo images (1-mm isotropic voxels). T1 images underwent volumetric segmentation and parcellation using FreeSurfer (version 6.0). Cortical thickness was measured as the distance from the gray matter-white matter boundary to the perpendicular pial surface, as previously described.p values <0.05 were considered significant. Group differences were assessed in univariate general linear models, with post hoc least significant difference tests for pairwise group comparisons. Biomarker values were log10 transformed before this analysis. Linear regression models were used to determine the associations between aging, biomarkers, and imaging measures to PDGFRβ and to test for interaction between variables. For each linear model, participants were excluded if they had 1 or more missing data in the variables included in the individual model. Mediation analysis was performed in SPSS with the PROCESS version 3.5 extension with a bootstrap method for the CIs of the mediated effect . Mediation effect was considered significant if the 95% CI did not include 0. Unless described otherwise, analyses were adjusted for age, sex, diagnosis, and ventricular volume. Numbers after the decimal point were rounded to the first significant figure.Statistical analysis and data visualization were performed with SPSS version 26  and R software version 4.2.3. Anonymized data will be shared by request from any qualified investigator for the sole purpose of replicating procedures and results presented in the article and as long as data transfer is in agreement with EU legislation on the general data protection regulation.APOE status, MMSE score, Aβ status, and Aβ-PET and tau-PET SUVR between the CU, MCI, and dementia groups (p < 0.001) . There were no differences in CSF concentrations of PDGFRβ between APOE ε4 carriers  and noncarriers .The study cohort consisted of 771 participants diagnosed as CU patients (n = 408), patients with MCI (n = 175), or patients with dementia (n = 188) . Disorde< 0.001) . Men hadb = 19.1, β = 0.5, 95% CI 16–22.2, p < 0.001; b = 5.3, β = 0.6, 95% CI 0.6–10, p = 0.03), but significant associations between age and CSF PDGFRβ survived in the diagnostic subgroups .CSF PDGFRβ was overall significantly associated with age , small vessel disease expressed as WMLs, neuroinflammation, and BBB dysfunction.p > 0.05 for all pairwise comparisons; eFigure 3B, links.lww.com/WNL/C795). Furthermore, no associations were observed between CSF PDGFRβ and Aβ-PET SUVR . Interaction between diagnosis and Aβ-PET or tau-PET had no significant effect on CSF PDGFRβ (p > 0.05).CSF levels of PDGFRβ did not differ within diagnostic groups divided according to Aβ status or according to type of dementia . The group sizes of the smaller cohort that underwent CBF analysis were consistent with those of the whole cohort . Interaction between diagnosis and measures of cortical thickness, WML volume, or CBF had no significant effect on CSF PDGFRβ (p > 0.05).There were no associations between CSF PDGFRβ and cortical thickness in the temporal AD signature regions . Association with QAlb was not significant in the dementia subgroup (p > 0.05). CSF PDGFRβ levels also showed overall strong associations to the neuroinflammatory markers YKL-40 .CSF PDGFRβ was overall associated to the CSF/plasma albumin ratio (QAlb)  and R2 for the models with combined effects of predictors was higher than that for individual effects (eTable 2), we performed a sequential statistical mediation analysis to determine whether neuroinflammation and pericyte damage affect the relationship between age and QAlb. We observed that CSF PDGFRβ fully mediated the effect of YKL-40 on QAlb , accounting for 33%–50% of the total effect .Because age, CSF PDGFRβ, and CSF markers reflecting neuroinflammation  were associated with QAlb  and glial activation/neuroinflammation (CSF YKL-40 and GFAP). We also found that both age and the glial biomarkers are associated with QAlb. Interestingly, the effects of age on the BBB integrity were partially mediated by pericyte damage and neuroinflammation. CSF PDGFRβ was not related to other age-related pathologies such as AD pathologic changes, as reflected by the lack of association with 22 For instance, some  studies have shown correlations of PDGFRβ with age as well as with QAlb.23 Here we report that in a large cohort of well-characterized participants, older age was consistently associated with higher CSF levels of PDGFRβ and that the association was unaffected by clinical diagnosis and possible concomitant AD pathology. QAlb was also consistently associated with PDGFRβ in CU and MCI and at whole cohort level, with the exception of the dementia subgroup. Taken together, these findings provide support that age-related pericyte injury is associated with BBB dysfunction and not with AD pathology.Aging is associated with morphological and functional changes in BBB, and preclinical evidence indicates that age-related pericyte degeneration and reduced pericyte coverage could cause BBB breakdown, impairment of protein transcytosis, vascular damage, and alterations in blood flow  and pericyte damage mediate the effects of age on the BBB. We also propose a model where age triggers increase in neuroinflammation and pericyte damage, which are both involved in the disruption of the BBB. Furthermore, we suggest that, based on their individual and combined effects, neuroinflammation and pericyte damage interact in the disruption of the NVU.Aging also increases the neuroinflammatory activity in astrocytes, and astrocytic processes directly connect to the BBB in the NVU  or within cohorts defined by A/T/N that only included preclinical AD.22 Most importantly, this is one of the few and the largest study using PET imaging and not only CSF biomarkers. PET imaging accurately defines the load of the core AD pathologic changes, that is, the amount and spread of insoluble Aβ and tau aggregates, which is not influenced by possible CSF dynamics that can affect biomarker concentration.31 Method-wise, some of these studies used a western blot method for detection of PDGFRβ in CSF instead of ELISA, which might have led to lower accuracy in the measurements.29 The studies where ELISA was used had a smaller sample size than ours.23 In the only study that compared the Western blot and ELISA methods in parallel, the authors suggest that the 2 techniques measure different species of PDGFRβ, which might have led to discrepancies in the results between different studies.9 Another possible limitation of the study is the use of QAlb to measure integrity of the BBB, which raised questions on whether this is the best method.32 QAlb has been shown to perform satisfactorily in this sense, especially in dementia studies33; however, more sensitive methods for detecting BBB dysfunction using MRI neuroimaging have been used in other studies,34 showing that BBB permeability is affected differently by AD pathology and cardiovascular risk factors. This warrants adjustment for cardiovascular risk scores in future studies.In contrast to our findings, increases in CSF PDGFRβ concentrations have been observed in AD defined clinically or by A/T/N classification.Despite convincing evidence of the interplay between age, pericyte injury, neuroinflammation, and BBB damage, the actual extent of their role in aging and disease remains unclear. Targeted longitudinal studies in clinical cohorts and in vivo models are needed to confirm these observations and investigate the relationship between microglia, pericytes, and BBB in the aging brain.In conclusion, we observed that the levels of CSF PDGFRβ increase with age and are associated with neuroinflammation and BBB dysfunction, but not with other age-related pathologies such as AD pathologic changes or WMLs. We also propose that pericyte damage partially mediates the disruptive effects of age on the BBB, together with neuroinflammation. Further studies are however needed to clarify the role of pericyte injury in aging, BBB dysfunction, and neurodegenerative diseases.


Spontaneous cerebrospinal fluid (CSF) leakage at the clivus is rare. In previous reports, reconstructive materials used to treat such leakage were typically autografts. Considering the pathology, rigid reconstruction is preferred. We here describe a case of spontaneous CSF leakage at the clivus with multiple bony defects. In this case, in addition to using artificial material instead of autografts, such as fat or fascia, that require additional extranasal invasive harvesting site, a rigid material layer of septal cartilage and bone was also used, enabling more stable multilayer reconstruction. One month postoperatively, computed tomography revealed that the bony defect at the clivus had been well reconstructed. All nasal structures were preserved, and the nasoseptal flap was well engrafted. At eight months post-surgery, the patient remained in good condition. This method allows minimally invasive repair of the leaking clivus, according to the underlying pathophysiology. Spontaneous cerebrospinal fluid (CSF) leakage is relatively rare, accounting for approximately 5-10% of all types of CSF leaks . These aMost fistulas related to CSF leakage at the clivus are only a few millimeters in diameter ,3,13, anHere, we report a case of CSF leakage at the clivus, in which we utilized artificial materials, nasal septal cartilage, and bone for reconstruction, in a method that is less invasive than those previously reported. Additionally, two small bony defects were resected and reshaped to create one large defect. This approach allowed for precise delineation of the bone and dura layers, resulting in successful rigid reconstruction. This case highlights the utility of such minimally invasive reconstruction with precise layer identification.2, who had been experiencing serous rhinorrhea for the past month presented to our Neurology Department with a headache and high fever. A lumbar puncture revealed bacterial meningitis, and the patient was subsequently referred to the Otorhinolaryngology Department for identification of the source. In the absence of a rhinologist, another otolaryngologist attended to the patient using an endoscope and evaluated her computed tomography (CT) images but could not determine the cause. The patient was discharged after the symptoms of bacterial meningitis improved with conservative treatment.A 72-year-old woman with a body mass index of 18.6 kg/mFour months after discharge, she developed bacterial meningitis again. She was readmitted to the Neurology Department and subsequently referred to the Otorhinolaryngology Department. The patient also had a persistent serous nasal discharge. When this discharge was collected and analyzed, the glucose level in the fluid was 93 mg/dl, strongly suggesting CSF leakage. A review of thin-slice CT images revealed multiple bony defects in the clivus Figure . MagnetiTM Dural Graft Matrix ), a type of artificial material, was implanted over the dural defect, with its central part carefully shaped to conform to the defect, serving as a “bath plug” in the first layer (Figure Surgery was performed after the improvement of meningitis. Using a transseptal approach, the sphenoid sinus was opened, the sphenoid crest was extensively resected, the septum of the sphenoid sinus was removed Figures -3C, and r Figure . The sizr Figure . The colr Figure . The borr Figure , 4D. Subr Figure -4H. FinaNo findings suggestive of postoperative spinal fluid leakage were encountered; thus, the lumbar drain was removed on the third postoperative day. Owing to the persistent low-grade fever, around 37°C, after surgery, intravenous antimicrobial infusion was continued, but was terminated after fever resolution on the ninth postoperative day. No significant complications were noted, and the patient was discharged on the 14th postoperative day. One month postoperatively, CT images showed that the bony defect in the clivus had been well reconstructed Figure . Two monIn this case, we propose the feasibility of minimally invasive reconstruction surgery for CSF leakage at the clivus, without the need for harvesting autografts at the other operative site, and highlight a novel and effective technique for merging multiple bony defects into a single large defect, facilitating dura defect repair.For skull base reconstructions due to spontaneous CSF leakage at the clivus, previous studies have reported harvesting autografts and conducting a multilayered reconstruction Table  1-13]. -13. 1-13By merging multiple bony defects into a single large defect, we were able to identify the defect in the dura mater clearly. Additionally, it allowed for the detachment and insertion of reconstructive materials between the bone and dura mater layers. While it led to the drawback of making a large bony defect, it also presented the advantage of allowing for rigid reconstruction. Considering the possible influence of increased intracranial pressure and a pulsating basilar artery on the development of perforation, rigid reconstruction can be considered a suitable method. This method is applicable wherever endonasal endoscopic skull base surgery is possible. Rigid reconstruction has been reported in three patients, including the present case, with bony defects of 7 mm , 8 mm, aRegarding the diagnosis of this condition, half of these patients have a history of meningitis before the diagnosis of CSF leakage -6,8-12, This case report illustrates that spontaneous CSF leakage from the clivus can be reconstructed in a minimally invasive, without the extranasal autograft harvesting. Additionally, rigid reconstruction can be performed for multiple bony defects, while considering the pathophysiology of the condition. This is considered to be a generalizable surgical procedure because it uses a readily available artificial material. Further similar cases should be accumulated to verify the utility of this approach.