Labyrinthine fistula (LF) is one of the most important complications of cholesteatoma and is defined as an abnormal communication between the inner and the middle ear. This study aims to describe our experience with the partial labyrinthectomy evaluating the post-operative hearing results. Twenty-one patients who presented labyrinthine fistula in the semicircular canals were included in the present study. Hearing impairment was present in 48% of patients (10/21). A pre-operative assessment using the Gardner–Robertson hearing classification showed the following: 52%, Class I; and 48%, Class II. A post-surgical Gardner–Robertson hearing classification evidenced the following: 43%, Class I; and 57%, Class II. The presence of LF is usually considered a negative prognostic factor for hearing preservation. The key point of partial labyrinthectomy surgery is the preservation of structures, keeping them wet with Ringer's solution throughout the procedures, and not performing suction that is close to the opened LF. The bony labyrinth is drilled underwater without suction, removing the entire cholesteatoma matrix and quickly plugging the site before and after the LF. This faster plugging of the labyrinth makes it possible to preserve the peri-lymph and the endo-lymph fluid and the hearing function. This study showed that a partial labyrinthectomy is useful for maintaining serviceable hearing in patients with LF. Labyrinthine fistula (LF) is a pathological condition in which an abnormal communication between the inner ear and the middle/mastoid cavity is present . It is mIn patients with cholesteatoma, CT and MR are the best methods for diagnosing and planning surgical management.Surgical management is, in fact, the treatment of choice in terms of hearing preservation, although which technique should be performed is a source of debate.Some authors retain the cholesteatoma matrix on the side of the LF to avoid damaging the cochlear function. Others claim to manage them by totally removing the cholesteatoma and repairing the labyrinth using bone sealing , 4. In cIn 2008, Magliulo et al. proposed a partial labyrinthectomy as a safe technique for treating LF and for preserving the hearing, using a total removal of the cholesteatoma matrix and the involved bony covering . This prvia MR sequences.This study aims to describe our experience with the partial labyrinthectomy over the last 12 years, evaluating the obtained post-operative hearing results and demonstrating the preservation of the labyrinthine fluids via partial labyrinthectomy and hearing preservation technique. This research was performed following the principle of the Declaration of Helsinki and approved by the local ethics committee of the University “Sapienza,” Rome.The data of 264 patients, who are surgically treated for cholesteatoma, were extracted from a database covering the period from 2007 to 2020. Twenty-one patients, who presented labyrinthine fistula in the semicircular canals, were included in the present study. Patients with fistula of the cochlea or vestibule were excluded. All patients were treated by a senior surgeon (GM) The database was analyzed for presenting the signs and symptoms, surgical details, and post-operative complications.The hearing status was assessed with pure tone audiometry (PTA) and speech discrimination. The degrees of hearing preservation and loss were defined according to the Gardner–Robertson classification system. Only the patients in Classes I–II, with possible hearing preservation, were enrolled.A pre-operative CT showed the extension of the disease and confirmed the presence of LF.Surgical records were cataloged according to the Dornhoffer and Milewski classification, and only the patients in Class II were included .The chosen surgical technique was the subtotal petrosectomy, in view of the extension of the cholesteatoma. All definitive surgical procedures were performed by the senior author (GM).In all cases, a partial labyrinthectomy, as described by Magliulo et al. in 2008, was performed with an exclusive microscopic approach .During a partial labyrinthectomy, the cholesteatoma matrix was radically removed, isolating it from the entire circumference of the LF. Firstly, the membranous labyrinth with the matrix was excised using a sickle knife, and then, the opening in the labyrinth was rapidly closed using a bone wax. While it remained open, the membranous labyrinth was kept wet with a Ringer's solution, and no suction was applied close to the openings of the semicircular canals to protect the anatomy and the function of the membranous labyrinth of the vestibule. Ringer's solution was firstly administered with an irrigation tube, thus, developing a pool. After that, it was kept wet through irrigation when drilling the bone. There is no specific amount of water, but the area should always be wet and viewable through the water.Using a cottonoid, the closure of the fistula was then reinforced with bone patè and temporalis fascia. This allowed the preservation of the vestibule, sealing the openings of the semicircular canals .A post-operative follow-up ranged from 1 to 10 years (average of 4 years) and a MRI was performed to assess the signs of recurrence of cholesteatoma and Labyrinthine Fistula (LF).An evaluation using the Gardner–Robertson classification system was performed at 3, 6, and 12 months follow-up; after the first year of follow-up, they were repeated once a year.For diagnostic purposes for each patient, we performed the MRI study with Fiesta 3D sequences with the Discovery™ MR750 GE 3.0T system Imaging. This equipment is characterized by excellent gradient homogeneity and exceptional stability. The study was performed with a multichannel head and neck coil . Images have been processed with MIP (Maximum Intensity Projection) for the visualization of the membranous cochlea – the Axis of the projected unrolled membranous cochlea was seen superimposed on the volume rendered by the MIP image.Over the study period, 21 patients  presented middle ear cholesteatoma with LF in one of their ears . The lefThe most common symptom was vertigo in 62% of patients (13/21) and 2 of the latter were positive for Hennerbert's sign.Hearing impairment was present in 48% of patients (10/21). A pre-operative assessment using the Gardner–Robertson hearing classification showed: 52%, Class I (11/21); 48%, Class II (10/21).The otomicroscopic exams revealed 7 epitympanic perforations, 5 subtotal or total perforations, 5 normal tympanic membranes associated with swelling of their inferior portion, and 2 cases of canal wall up tympanoplasty: 1 of canal wall down tympanoplasty and 1 radical tympanoplasty.All patients underwent subtotal petrosectomy, and, in 8 patients, a blind closure of the external auditory canal was performed.The LF was located in the lateral semicircular canal (LSC) in 100% of the patients. The lesion was of an isolated nature in 67% (14/21), while 19% presented an association with LF of the superior semicircular canal (SSC) (4/21) and 14% (3/21) with LF of the posterior semicircular canal (PSC). According to Dornhoffer and Milewski's classification, 71% presented IIa LF (15/21) and 29% (6/21) had IIb LF.Post-operatively, the vertigo symptoms reduced, and the vestibular signs (Hennerbert sign) disappeared in 2 months, but the Video Head Impulse Test (vHIT) or caloric tests were not performed.The post-surgical Gardner–Robertson hearing classification evidenced the following: 43% Class I (9/21); 57% Class II (12/21). The two patients, who passed from class I to class II, had an audiometric difference of <10 dB. One of these two cases was a petrous bone cholesteatoma that involved the internal auditory canal.p = 0.7).The Chi-square with Yate's correction did not show any statistical difference between the pre- and post-surgical hearing outcomes  showed a limited recurrence at CT imaging. In all the patients, the T2-weighted MR images demonstrated a post-surgical persistence of intralabyrinthine fluids –4..2% showeLabyrinthine fistula is one of the most important complications of cholesteatoma and is defined as an abnormal communication between the inner and the middle ear.Our data are in line with the current literature that reports an incidence of LF in patients with cholesteatoma ranging from 2.9 to 12.5% with a predominance of LSC, probably owing to its anatomical position . MoreoveThe presence of LF is usually considered a negative prognostic factor for hearing preservation, and surgical management is still a matter of debate. Moreover, previous papers have made a comparison between different types of fistula, different surgical techniques, and pre-operative levels of hearing status. Therefore, we decided to select a homogenous sample belonging to the Class II Dornhoffer and Milewski LF and the Class I-II Gardner–Robertson, treated exclusively by a partial labyrinthectomy performed by the senior author (GM).Traditionally, many authors propose leaving the matrix of the cholesteatoma on the LF, thus, reducing the risk of labyrinth damage and hearing loss. On the contrary, others consider that leaving the matrix in the labyrinth leads to a higher risk of infection/inflammation and advocate a radical removal, despite the risk of a dead ear .A recent review reported a hearing preservation rate similar to the staged and un-staged matrix removal (around 90% of hearing preservation), with no statistical difference between the two procedures , a radical cavity surgery (RCS), and a canal wall up mastoidectomy (CWUM). In all cases, after the complete removal of the cholesteatoma, the matrix is peeled from the LF that is covered with fascia and/or fibrin glue, and bone dust. The required major caution is to avoid accidental suction of the perilymph. The risk of recurrence is relatively high.Thangavelu et al. recently reported their experience with the “underwater technique,” defining it as an evolution of those proposed by Yamauchi and Misale , 10, 11.In 2008, Magliulo et al. reported their experience with partial labyrinthectomy in the treatment of LF . The tecThe hearing outcomes obtained, from 2008 to 2020, are in line with all the techniques described with the preservation of pre-operative hearing levels in 90% of the cases. On the other hand, in our cases, there were no cases of the dead ear, and a serviceable hearing was kept in 100% of cases. Moreover, our data also included a Petrous Bone Cholesteatoma (PBC) fistula that reached the internal auditory canal without any hearing post-surgical deficit. This surgical method, in fact, was previously adopted for Schwannoma surgery by Magliulo et al. who modified the technique proposed by McElveen et al. .Also, the partial labyrinthectomy differs from the other techniques for its reduced risk of recurrence that appeared lower than that described by Thangavelu (9 vs. 20%) and is not comparable with Yamauchi and Misale's papers that did not report any pertinent data.In conclusion, hearing preservation may be possible by employing underwater techniques and by partial labyrinthectomy owing to the ion balance produced by irrigation that allows the preservation of the anatomical structure and the functional activity of the labyrinth. However, to the best of our knowledge, none has radiologically evaluated it. Consequently, we performed a follow-up of the T2 imaging MR to study the labyrinth fluid. The obtained images confirmed the normal anatomical structure and the fluid of the inner ear in all the treated patients.The limitations of this paper are related to the small sample size of patients, resulting from the choice to select only certain patients with LF. Moreover, tests for vestibular function were not performed. Further multicenter studies with larger samples are underway to confirm the described results.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.The studies involving human participants were reviewed and approved by Sapienza University of Rome. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.GM and AP: conceptualization. AMi: methodology. DM: software. GI, SC, and AMa: validation. AP: formal analysis and data curation. VR and CV: investigation. GM: writing—original draft preparation and supervision. CV: writing—review and editing. SC: visualization. All authors have read and agreed to the published version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


We undertook a series of experiments to define the role of cathepsin S in oral cancer pain. We compared cathepsin S activity in human oral cancer tumors versus patient-matched normal tissue; a human oral cancer cell line versus a benign dysplastic oral keratinocyte cell line; and in an orthotopic xenograft tongue cancer mouse model versus normal controls in mice. We localized cathepsin S in macrophages and carcinoma cells in human oral cancers. The injection of cathepsin S caused nociception in a mouse model while the injection of oral cancer cells in which the gene for cathepsin S is deleted generated less nociception. Our findings will lay the foundations for clinical trials of cathepsin S inhibitors for treating oral cancer pain.Oral cancer is often deadly and severely painful. Because this form of cancer pain cannot be adequately treated with current medications including opiates, new treatment approaches are needed. Cathepsin S, a lysosomal cysteine protease may play a role in oral cancer pain through a protease-activated receptor-2 (PAR2) contributes to oral SCC pain. Cathepsin S is a lysosomal cysteine protease released during injury and disease that can activate PAR2. We report here a role for cathepsin S in PAR2-dependent cancer pain. We report that cathepsin S was more active in human oral SCC than matched normal tissue, and in an orthotopic xenograft tongue cancer model than normal tongue. The multiplex immunolocalization of cathepsin S in human oral cancers suggests that carcinoma and macrophages generate cathepsin S in the oral cancer microenvironment. After cheek or paw injection, cathepsin S evoked nociception in wild-type mice but not in mice lacking PAR2 in Nav1.8-positive neurons (Par2Nav1.8), nor in mice treated with LY3000328 or an endogenous cathepsin S inhibitor (cystatin C). The human oral SCC cell line (HSC-3) with homozygous deletion of the gene for cathepsin S (CTSS) with CRISPR/Cas9 provoked significantly less mechanical allodynia and thermal hyperalgesia, as did those treated with LY3000328, compared to the control cancer mice. Our results indicate that cathepsin S is activated in oral SCC, and that cathepsin S contributes to cancer pain through PAR2 on neurons.Oral squamous cell carcinoma (SCC) pain is more prevalent and severe than pain generated by any other form of cancer. We previously showed that protease-activated receptor-2 (PAR Cathepsin S is a lysosomal cysteine protease that degrades proteins along the endocytic pathway, including the invariant chain necessary for MHC class II antigen processing and presentation . Of the Human cancers, including prostate, gastrointestinal , lung, and brain tumors  upregulate cathepsin S ,8,9. Mic2), a G protein-coupled receptor (GPCR) expressed on peripheral nociceptors that mediates neurogenic inflammation and pain ; product size 113 bp). PCR products were analyzed with 1% agarose gel electrophoresis.For reverse-transcription polymerase chain reaction (RT-PCR), total RNA was isolated using TRIzol reagent (Invitrogen). RNA concentration was measured by NanoDrop ND-1000 Spectrophotometer  and cDNA was synthesized from a total of 1 μg RNA using QuantiTect5 cells/well) and were incubated for 24 h. Medium was removed, cells were washed with 3 mL PBS without Ca2+ and Mg2+, and DMEM (500 µL) was added to each well. After 48 h, the medium was collected and centrifuged . The pellet was discarded. Total cellular protein was collected from HSC-3 and OSC-20 cell lines  using standard RIPA lysis buffer (500 µL/well). Cathepsin S concentration was quantified by enzyme-linked immunosorbent assay (ELISA) for human cathepsin S . A standard curve was constructed before each experiment. Protein samples were diluted in dilution buffer and 100 μL was added to each well. The plate was incubated overnight at 4 °C and washed four times with washing solution. The diluted biotinylated anti-human cathepsin S antibody (100 μL) was added to each well and incubated for 1 hour at room temperature. Following the washing steps, 100 μL of 1× HRP–streptavidin solution was added to each well and the plate was kept in a dark environment at room temperature for 45 minutes. After incubation, the plate was washed and TMB substrate reagent  was added to each well. Finally, the reaction was stopped by the addition of 50 μL of stop solution. The absorbance was read at 450 nm in a microtiter plate reader immediately. The absorbance was measured using a 450 nm filter on GloMax®-Multi Microplate Multimode Reader (Promega).HSC-3 or OSC-20 cells were plated in a 12-well plate  [−1, 100 µL) was administered by oral gavage. Mechanical and thermal nociception assays were performed at 1, 3, 6, 12, and 24 h after LY3000328 treatment. At the end of the experiments, the cancer paw was collected and processed for histological hematoxylin and eosin (H&E) staining to confirm viable carcinoma.The paw xenograft cancer model permits the measurement of mechanical and thermal nociception in the paw. Baseline mechanical and thermal withdrawal thresholds were measured prior to tumor cell inoculation. The left hind paw of NU/J el (1:1) ,30. MechTo assess mechanical nociception, mice were placed on a platform with a metal mesh floor and acclimated for 1 h. The paw withdrawal threshold was measured with von Frey filaments  according to the up–down method for rats published by Chaplan et al. in 1994 with modifications for mice ,32. The To assess thermal nociception in the paw, we used a thermal stimulator  . Two weeThe paw xenograft model permits the measurement of cancer paw volume at the time of behavioral assessment. Prior to cancer cell inoculation and at 1, 2, 3 and 4 weeks after inoculation, a plethysmometer  was used to measure the volume of cancer paw in anesthetized mice. Volumes were measured 3 times and reported as the mean.® software. Two independent researchers quantified the area of cancer and the average was recorded.Four weeks after the inoculation of cancer cells into the paw, the cancer paw was collected. The paw was defined as the area from the tip of the digits to the ankle crease. The paw was longitudinally sectioned through the thickest area of the tumor, which generated 2 cut surfaces within the largest area of cancer. The paw was then immersed in 10% neutral buffer formalin (NBF) for 48 h at 4 °C. NBF was then replaced with 10% ethylenediaminetetraacetic acid (EDTA) for paw decalcification. The EDTA solution was changed every 2 days for 14 days and then replaced with 70% Et-OH for 3 days prior to paraffin embedding. One section was made on each cut surface of the cancer paw for H&E histological staining. The H&E image was scanned on a Hamamatsu Nanozoomer. The area of cancer was determined as the average area from the 2 sections as quantified by the NIH ImageJt-test were used for in vivo behavioral experiments. An independent samples Student’s t-test was used to compare values between 2 groups. The relationship between participant pain scores, as measured by the University of California San Francisco Oral Cancer Pain Questionnaire, and cathepsin S activity was analyzed using Spearman correlation.GraphPad Prism 7 and 8  was used for the statistical analysis. Results were expressed as mean ± standard error (SEM). For cell-based assays, triplicate measurements were made; differences were evaluated by one- or two-way repeated measures ANOVA and Dunnett’s or Tukey’s multiple comparisons test. One-way ANOVA and Tukey’s or Sidak’s multiple comparisons and Student’s s = 0.78, p = 0.041)To determine whether cathepsin S is activated in oral SCCs, we collected oral SCC specimens and matched normal oral mucosa from seven patients . Specime+ macrophages in human oral SCC tissue (+) indicating that cathepsin S was expressed by both tumor cells and macrophages within the oral SCC microenvironment   a,b. The e cancer d and come cancer c. PAR2 pl tongue e. Using BMV157, we measured cathepsin S activity in the mouse orthotopic xenograft model in which HSC-3 human oral SCC cells were inoculated into the tongue. Compared to normal mouse tongues, HSC-3 orthotopic xenografts exhibited increased cathepsin S activity and increased total levels of immunoreactive cathepsin S a–c. ThusWe measured cathepsin S activity in HSC-3 cells and dysplastic oral keratinocytes  with BMV157, the cathepsin S-selective probe, and BMV109, a pan cathepsin probe. Cathepsin S activity was higher in HSC-3 than DOK, as measured by both probes a–c. Like2, FLAG-PAR2 plasmids were transiently transfected in HEK cells and PAR2 cleavage was measured with the On-Cell Western assay. These cells expressed PAR2 with an extracellular N-terminal FLAG epitope that is upstream of the site of cathepsin S cleavage  , FLAG immunoreactivity was reduced by 66.02% (1.94 ± 0.17 in vehicle-treated cells versus 0.66 ± 0.14 in Cat S-treated cells). Similarly, the FLAG immunoreactivity was reduced by 42.83% when supernatant from the HSC-3 CTSS+/+) c. LY3 an/+ cells c. Collec2 when injected into tissue innervated by dorsal root ganglia [2. We injected cathepsin S into the cheek of WT C57BL/6J mice and Par2Nav1.8 KO mice which lack the gene for PAR2 in Nav1.8-positive neurons  provoked reduced mechanical allodynia and thermal hyperalgesia relative to xenografts generated from wild-type HSC-3 cells (untreated naïve cells or cells treated with random guide RNA)  c,d,e. Callodynia d and an llodynia e compareCTSS in HSC-3 with CRISPR/Cas9 altered tumor proliferation as measured by tumor volume. The cancer paw volume was measured before (BL) and every week until 4 weeks after HSC-3 inoculation  also exhibit attenuated nociception, indicating a central role for neuronal PAR2 in cancer pain [2-activating proteases  contribute to cancer pain in a PAR2-dependent manner [The primary question addressed by the present study was whether cathepsin S produces cancer pain. We also sought to determine whether the putative nociceptive mechanism involves PARcer pain ,36,37. Sfunction ,36. Micecer pain . Furthert manner ,37 + tumor cells and CD68+ macrophages of oral cancer specimens collected from four patients. Cathepsin S expression in adjacent normal tissue was minimal. We also demonstrated that cathepsin S activity is upregulated in immortalized human oral cancer cell lines compared to dysplastic oral keratinocytes. We demonstrated that cathepsin S is secreted by oral cancer cells, and that it cleaves human PAR2. The orthotopic xenografts of human oral cancer cells also exhibited increased cathepsin S activity compared to naïve mouse tongue tissue.We first characterized cathepsin S expression and activity in human oral squamous cell carcinoma tissues and patient-matched normal oral mucosa using cathepsin S-specific antibodies and BMV157, an activity-based probe that enables the assessment of the proteolytically active fraction of cathepsin S. Total and active cathepsin S were increased in cancer tissues compared to normal tissues. In breast, prostate, colorectal and metastatic brain cancers, cathepsin S is supplied by the tumor cells and associated stromal cells, predominantly macrophages ,38,39. I2-dependent manner, suggesting that cathepsin S would be capable of provoking oral cancer pain.Furthermore, purified cathepsin S was able to invoke facial nociception in vivo, also in an activity-dependent and PARWe next investigated the ability of tumor-supplied cathepsin S to provoke oral cancer pain. The deletion of cathepsin S specifically from HSC-3 tumor cells resulted in a reduction of mechanical allodynia and thermal hyperalgesia  produced by paw xenografts. In the future, complementary studies involving the inoculation of HSC-3 cells into wild-type and cathepsin S-deficient nude mice would shed light on the contribution of stromal (macrophage)-supplied cathepsin S to cancer pain. Ultimately, we showed that the administration of a single dose of a cathepsin S-selective inhibitor LY3000328, which targets both tumor and stromal-supplied cathepsin S, was able to attenuate cancer pain for at least 12 h in two independent xenograft models .2 axis is a strategic target for cancer pain. Cathepsin S is active in the extracellular environment of the cancer and associated neurons. Accordingly, it could be antagonized without cellular uptake, as opposed to other cathepsins, which are active in lysosomes. Furthermore, PAR2 activates several types of TRP channels, including TRPV4 [2 transactivates the epidermal growth factor receptor (EGFR), which also mediates nociception [2 by cathepsin S, we could potentially abrogate the sensitization of the receptors that mediate the nociceptive action of myriad mediators produced and secreted by cancers. A potential concern with the inhibition of cathepsin S is that antigen processing, which is critical to the immune response to cancers, might be affected. This argument is countered by the finding that cathepsin S is upregulated by several cancers. Moreover, to the degree that evolutionary mechanisms underlie tumor progression, the natural selection of tumor cells may be less likely to favor the overexpression of cathepsin S if proliferation were reduced. In fact, the inhibition or deletion of the gene for cathepsin S reverses several hallmarks of cancer [CTSS nor a single dose of the cathepsin S inhibitor, LY3000328, affected the tumor volume; therefore, the mechanism of preventing the expression or inhibiting cathepsin S is antinociceptive. The nociceptive mechanisms that we demonstrated in oral cancer  likely overlap with other cancer pain mechanisms, including bone cancer pain. Nociceptive mediators/mechanisms common to oral cancer and bone cancer include, but are not limited to, endothelin, NGF, and PAR2 [The treatment of oral cancer pain is challenging because cancers are heterogeneous and because oral cancers produce and secrete several nociceptive mediators. Nociceptive mediators produced and secreted by oral cancer include, but are not limited to, nerve growth factor, ATP, endothelin-1 and other proteases ,40,41. Tng TRPV4 , TRPV1 [ng TRPV4 ,44 and Tng TRPV4 , which aiception ,47,48. Tf cancer . Becauseand PAR2 ,50. Cathand PAR2 . Becauseand PAR2 .2) on neurons to contribute to the severe pain that is associated with oral cancer. In humans, cathepsin S activity correlates with reported pain. Genetic deletion of the gene for cathepsin S in a human oral cancer cell line reverses nociception in a mouse cancer model generated with the cancer cell line. We conclude that cathepsin S is responsible for oral cancer pain through PAR2 on neurons.Cathepsin S, a lysosomal cysteine protease, is present and active in the oral cancer microenvironment. The protease cleaves protease-activated receptor-2 (PAR


Damp phlegm and blood stasis pattern (DPBSP) is the main pattern in coronary heart disease (CHD) patients. To quantify and standardize the diagnosis of DPBSP, questionnaires are usually administered. The CHD Damp Phlegm and Blood Stasis Pattern Questionnaire (CHD-DPBSPQ) is the standard metric for measuring CHD-DPBSP signs and symptoms in practice and clinical research. The CHD-DPBSPQ has moderate diagnostic efficiency, as evidenced by its receiver operating characteristic curves. Furthermore, and high reliability and validity have been shown in some studies but not in a multicenter clinical trial. Our purpose was to evaluate the test-retest reliability of a proprietary CHD-DPBSPQ.  The CHD-DPBSPQ uses a standard procedure for measuring symptoms. The (interrater) reliability and validity of this questionnaire have been previously studied. Here, we evaluated the test interval and weighted kappa value of items of test-retest (intrarater) reliability of the CHD-DPBSPQ. The test-retest reliability was evaluated by the intraclass correlation coefficient (ICC) for the total CHD-DPBSPQ score and the phlegm domain and blood stasis domain scores. Weighted kappa statistics were calculated for the individual CHD-DPBSPQ items.  Using the CHD-DPBSPQ, 79 patients with late-stage CHD who were participating in a multicenter clinical trial were assessed twice. The ICCs for the CHD-DPBSPQ score were as follows: 0.827 for the total CHD-DPBSPQ, 0.778 for the phlegm domain score, and 0.828 for the blood stasis domain score. The reliability was slightly better in patients whose test interval was ≤14 days. The weighted kappa values of individual items showed moderate consistency.  The CHD-DPBSPQ was found to have excellent test-retest reliability in this sample of patients. Coronary heart disease (CHD) has been the main cause of mortality and disability in both developed and developing countries in the last 20 years . AccordiEpidemiological investigations of the CHD syndrome have shown that the distributional characteristics are mainly blood stasis and phlegm turbidity. For example, Mao et al. found that the main syndrome characteristics of CHD from 1970 to 2010 were blood stasis, phlegm turbidity, and qi stagnation [The Coronary Heart Disease Damp Phlegm and Blood Stasis Pattern Questionnaire (CHD-DPBSPQ) is the standard metric for measuring CHD-DPBSP signs and symptoms in practice and clinical research –13. The TCM syndromes can be constructed into a set of standards and issued in the form of scales. Several studies have investigated the structure and metric properties of the CHD-DPBSPQ. The CHD-DPBSPQ has been shown to have a moderate diagnostic efficiency based on its receiver operating characteristic curves . Some stThe researchers had to identify patients who could participate in the questionnaire again to ensure that the number of respondents in the two surveys was five times the number of items in the questionnaire. In accordance with the study protocol, the inclusion criteria were as follows: the subjects were aged 18 years or above, provided informed consent for participation, had been diagnosed with CHD according to the guidelines , 18, andThe raters were the main researchers. The raters were all experts in TCM or integrated Chinese and Western medicine in the treatment of cardiovascular diseases, with at least 20 years of clinical experience. All the raters explained the questionnaire to each patient before any evaluations were performed.The questionnaire was validated in a cross-sectional, multicenter, observational, descriptive study that followed a test-retest design. The study was approved by the Research Ethics Committee of Tianjin University of Traditional Chinese Medicine (no. TJUTCMEC2015000). The construction definition, item generation, selection reliability, and validity of the CHD-DPBSPQ were performed according to standard procedures . Next, wFor the statistical analysis, SPSS version 25.0  and SAS software 9.4  were used. The test-retest reliabilities of the different domains of the CHD-DPBSPQ were estimated by the intraclass correlation coefficient (ICC) ; the 95%In our preliminary research, the data of 729 inpatients  were collected and screened from a doctor according to the aforementioned inclusion and exclusion criteria. Ultimately, two experienced CHD experts in TCM or integrated Chinese and Western medicine diagnostic patterns collected 79 inpatient data, which comprised the research sample. The specific data processing flow diagram that was designed is shown in The CHD-DPBSPQ was developed and evaluated according to a set of standard procedures and then issued by the China Association of Chinese Medicine (CACM). To date, there has been no report of a cross-sectional study of TCM syndromes combined with a joint investigation of patients from multiple hospitals.Our results showed that the total CHD-DPBSPQ and blood stasis domain had excellent test-retest reliabilities; the symptom-based subscales also showed considerably good reliability. However, the test-retest reliability of the phlegm domain was somewhat lower than that of the blood stasis domain of the CHD-DPBSPQ. This result may be because of the different characteristics of people in different regions, which reflect the so-called “treating the disease according to the individual condition” and “treating the disease according to the environment” aspects of TCM. However, the test-retest reliability of the phlegm domain was close to 0.75, which is the lower-limit indicator of good diagnostic tests. The ICCs were slightly better for patients with test-retest intervals ≤14 days than for those of patients with intervals >14 days. This finding is consistent with Andrew's report on the time interval . The lowOur study aimed to validate the test-retest reliability of the CHD-DPBSPQ in patients at multiple treatment centers in China. However, the results showed individual differences in the degree of the importance of the different items in the CHD-DPBSP. Even if the individual items of domains show low scores, these results are not necessarily reflected in the total CHD-DPBSPQ score. The CHD-DPBS syndrome is divided into two stages: phlegm in the early stage and blood stasis in the late stage. The average age of our patients was approximately 66 years, at which it is common for late-stage CHD patients to have “blood stasis” as the main stage, and the “phlegm” syndrome score is low. Deng  stated tIn particular, one limitation of our study was the lack of early CHD-DPBSP patients. Thus, future studies should investigate patients from aged 18–66 years. Owing to medication effects, a substantial short-term variability in the CHD-DPBSPQ scores should be expected. Because this investigation comprised the basic work of diagnostic research, the present study lacks the “prescription-syndrome correspondence” to evaluate the reactive evidence of the diagnostic criteria of DPBSP in CHD. That is, we only selected CHD patients with DPBSP for measurement and excluded those DPBSP.Nevertheless, our investigation has some strengths which are as follows. First, we assessed 79 patients to evaluate the test-retest reliability, which met the requirements of a sample size of >20 participants . Second,The CHD-DPBSPQ was found to be reliable and can be recommended for evaluations of CHD. The present study also showed that the CHD-DPBSPQ is a valid instrument for measuring the phlegm domain and blood stasis domain scores. The establishment of DPBSP provides a standard template for determining the test-retest reliability of TCM syndrome types and establishes a foundation for achieving TCM syndrome standardization.


Mycoplasma hyopneumoniae through molecular tools is a growing trend for early diagnosis, highlighting the importance of knowing M. hyopneumoniae dynamics in the respiratory tract upon infection. This study focused on monitoring the infection level and its effects in different anatomic sites of the respiratory tract of experimentally infected swine in four time-points post-infection. To this end, 24 pigs were allocated to either non-inoculated group (n = 8) or inoculated group (n = 16). On day 0 post-infection (dpi), animals of the inoculated group were intratracheally inoculated with M. hyopneumoniae. Nasal swabs were collected weekly for qPCR detection of bacterial shedding. At 14, 28, 42, and 56 dpi, four animals from the inoculated group and two from the control group were necropsied. Bronchoalveolar lavage fluid (BALF) and samples from three different anatomical tracheal sections  were collected for qPCR and histopathology. Bacterial loads (qPCR) in tracheal samples were: 4.47 × 102 copies∕μL (CT), 1.5 × 104- copies∕ μL (MT) and 1.4 × 104 copies∕μL (LT samples). M. hyopneumoniae quantification in BALF showed the highest load at 28 dpi (2.0 × 106 copies∕ μL). Microscopic lesions in LT samples presented the highest scores at 56 dpi and were significantly correlated with the pathogen load on 14 dpi (0.93) and 28 dpi (0.75). The greatest bacterial load of M. hyopneumoniae in CT samples and BALF was registered at 28 dpi, and it remained high in BALF and LT throughout the 56 dpi. The pathogen was able to persist during the whole experimental period, however higher estimated quantification values were registered in the lower parts of the respiratory tract, especially at 56 dpi. These findings are important for improving diagnostics, treatment, and control measures of M. hyopneumoniae infection in swine herds.Direct detection of  Mycoplasma hyopneumoniae is the primary causative agent of enzootic pneumonia (EP) [nia (EP) , a chronnia (EP) , 3. The nia (EP) , disruptnia (EP) .M. hyopneumoniae is known for having a tropism for lower parts of the swine respiratory tract [M. hyopneumoniae was detected in bronchus and bronchioles as early as 7 days post-infection (dpi). Simultaneously, its direct detection in the alveolar macrophage and interstitial macrophages was only seen in the later stages of infection [ry tract –9. The pry tract . By asseM. hyopneumoniae is notorious for its specific membrane proteins that adhere to the tracheal epithelial cilia during infection [M. hyopneumoniae infection in live animals [M. hyopneumoniae [Still, nfection , inducinnfection . Thus, c animals , 13. Coneumoniae . HoweverM. hyopneumoniae infection is essential for decision-making in the sampling methodology [M. hyopneumoniae challenge and the associated effects in different respiratory tract sections of swine during a period of 56 dpi.The knowledge about the dynamics and chronology of hodology . HoweverM. hyopneumoniae DNA (qPCR) and specific M. hyopneumoniae antibodies (ELISA), respectively. While in the mock-inoculated animals, clinical signs compatible with EP were not detected, all inoculated animals presented clinical signs consistent with EP. The dry, non-productive cough was first noted at 10 dpi and thereafter observed in all inoculated animals. At the end of the experiment (56 dpi), at least one animal was still coughing. Beyond that, the animals did not show any clinical signs associated with other diseases.The laryngeal swab and serum samples collected before inoculation were negative for R2 ranged from 0.996 to 1.0, y-intercept ranged from 39 and 40.8, and the slope varied from − 3.36 to − 3.5.All qPCR parameters followed the previously published recommendations described in the Minimum Information for Quantitative Real-Time qPCR Experiments (MIQE) . The effM. hyopneumoniae DNA in nasal swab samples was detected from seven dpi (6∕16) onwards, and all animals, except for pig 2.4, presented at least one positive result across the study. Most animals showed positive results at 14 dpi and 21 dpi, 75% (12∕16) and 91.6% (11∕12), respectively. Bacterial shedding was shown to have an intermittent pattern, as reported in Table Mock-inoculated animals remained negative during the whole study. The presence of en dpi 6∕6 onwardsen dpi 6∕6 onwardsM. hyopneumoniae DNA quantification value in CT, MT, and LT samples at 14, 28, 42, and 56 dpi are shown in Table M. hyopneumoniae burden in CT, MT and LT and dpi.No positive results were obtained from the samples of mock-infected animals. The median of p < 0.05) were only detected between the mean values at 14 dpi and 28 dpi. These results are shown in Table All BALF samples from mock-inoculated animals were negative by qPCR during the whole study, while samples from inoculated animals were positive. Significant differences (p < 0.05). Scar tissue and gray fish-flesh-like areas were noted at 42 dpi and 56 dpi. No gross lesions were observed in the lungs of the mock-inoculated group.Higher GLLS were seen at 14 dpi and 28 dpi than at 42 dpi and 56 dpi. Despite the numeric differences between means, no significant differences were detected using Tukey’s test (p < 0.05).Results of cranial trachea lesion scores (CTLS), medium trachea microscopic lesion score (MTLS), lower trachea lesion score (LTLS), and GLLS are shown in Table M. hyopneumoniae using qPCR. In contrast, lung tissue samples from mock-inoculated animals did not show any positive results in the qPCR test.All lung samples from inoculated animals were positive for M. hyopneumoniae estimated load in BALF samples was significantly correlated with GLLS at 14 (0.93), 42 (0.95), and 56 dpi (0.89). Regarding tracheal samples, M. hyopneumoniae estimated load in CT was only correlated with GLLS at 28 dpi (0.86), while the load in MT correlated with GLLS at 28 (0.78) and 56 dpi (1.0) and with MTLS at 14 dpi (0.89). Finally, the load in LT was correlated with LTLS at 14 dpi (0.93). All coefficient values obtained for significant correlations and the significance values at all four time-points are shown in Table M. hyopneumoniae dynamics is essential for accurate diagnosis of infection, as well as for optimizing treatment and control measures in affected herds. Therefore, this study focused on reporting the infection dynamics upon experimental M. hyopneumoniae infection in specific sites  of the respiratory tract during a period of 56 dpi. To this end, M. hyopneumoniae DNA was quantified by qPCR in BALF and three anatomical sections of the trachea. In addition, gross Mycoplasma-like lung lesions and microscopic lesions of the trachea were scored.Understanding M. hyopneumoniae loads in tracheal sections (assessed by qPCR testing) were detected at 28 dpi (4.6 × 103 copies∕μL) for CT, and at 56 dpi for MT (2.0 × 105 copies∕μL) and LT (1.9 × 105 copies∕μL) samples. Even though no statistical significance was found between M. hyopneumoniae quantification values in the tracheal sections, a numeric increase in M. hyopneumoniae load was observed in MT samples over time. Likewise, the load remained consistently high in LT samples throughout the whole studied period.Throughout the experimental period (0 to 56 dpi), the highest M. hyopneumoniae is more accurately detected in samples collected from the lower respiratory tract; consequently, lower Cq values were obtained from deep tracheal swabs than from nasal and tonsil swab samples in a previous report [This pathogen is known for having tropism for lower parts of the swine’s respiratory tract, being up to 100 times more common in such sites . Previous report . A more s report .M. hyopneumoniae loads were reported in the medium and lower tracheal sections. This fact is mostly related to the better diagnostic sensitivity found in samples obtained from such anatomical regions, like the deep tracheal catheters [The abovementioned findings are in accordance with this study’s results, in which higher atheters , 13.M. hyopneumoniae infection is adhesion to ciliated cells of trachea, bronchi, and bronchioles mucosal epithelial lining, leading to inflammatory cells’ infiltration and their accumulation around airways and blood vessels [The first step of  vessels . The lesM. hyopneumoniae in cultured porcine tracheal cells showed progressive colonization and damage of the mucosal epithelial lining, noted from the 5th dpi onwards and with increasing severity over time [M. hyopneumoniae burden inoculated in swine tracheal cell culture with cilia loss in the same tissue [An in vitro inoculation of ver time , corroboe tissue , patholoe tissue , was repM. hyopneumoniae load in MT (0.89) and at 14 and 28 dpi in LT  samples. Besides, as reported previously, the high load of M. hyopneumoniae found in these samples seems to play a role in the severity of microscopic lesion development in the first weeks of infection [In this research, LT samples showed numerically higher microscopic lesion scores when compared with MT and, especially, with CT. Interestingly, significant correlations were found at 14 dpi between nfection , 19.M. hyopneumoniae quantification value was reported at 28 dpi (1.2 × 106 copies∕μL). Since it did not significantly decrease under the present conditions, the pathogen possibly could have persisted for a longer period and therefore not cleared from the body after 56 dpi. In addition, a previous study showed that all experimentally infected animals were positive up to the 94 dpi, and approximately 60% had positive bronchial swabs results at 214 dpi [Regarding the burden in BALF, the highest  214 dpi .M. hyopneumoniae load in BALF samples was significantly correlated with GLLS at 14, 42, and 56 dpi, highlighting the importance of its burden in lesion severity. Likewise, Vranckx et al. [Interestingly, the M. hyopneumoniae load in CT and GLLS reached the highest values at 28 dpi and were significantly correlated (0.86). Another study showed that M. hyopneumoniae load in nasal swab samples was significantly correlated to the load present in lung lesion homogenate of the same animal, showing that M. hyopneumoniae cells present in cranial regions of the respiratory tract are probably originated from lung lesions [M. hyopneumoniae, considering the chronology of the disease. Nevertheless, further studies are necessary to achieve more accurate results regarding the association between diagnostic samples sensitivity and GLLS at the early stage of infection.In this study, the mean lated 0.8. AnotherM. hyopneumoniae detection in later or chronic stages of the disease [Additionally, on the 56 dpi, GLLS showed its lowest percentage (8.75%) while the LT pathogen load was still high. This result agrees with previous findings, which confirmed that sampling lower respiratory tract sites would be more accurate for  disease , 13, 23 M. hyopneumoniae was first detected in nasal swabs at seven dpi and presented the highest number of positive animals at 21–28 dpi , allowing us to hypothesize that this was the time point when most animals were shedding the agent. Moreover, the detection of the pathogen in swab samples as early as five dpi and 28–30 dpi (peak) was already reported in other studies, as well as the intermittent detection pattern [ pattern , 24, 25.M. hyopneumoniae shedding in nasal swab samples.Unfortunately, the Monte Carlo effect  is an inM. hyopneumoniae (232). The experimental infection model used in this study was successful since all inoculated pigs were confirmed to be positive through qPCR and developed M. hyopneumoniae-like lesions in the lungs, as well as associated clinical signs.The present results originated from an experimental infection using intratracheal inoculation of one particular strain of M. hyopneumoniae burden in tracheal sites may be valid under practical situations. It is also fair to acknowledge that the limited number of animals used in each group could be associated with the wide range of quantification results reported within groups.It is worth mentioning that the experimental infection conditions were different from those on the field, mainly because of the infecting dose, the pathogen inoculation site, and housing conditions . Consequently, the presented results should be extrapolated to field animals with caution, even though some basic principles such as the dynamics of the infection and the M. hyopneumoniae cells but also dead ones. Consequently, the quantification results could be slightly overestimated.Finally, even though qPCR is a sensitive technique for detecting pathogens in biological samples, it is essential to mention that it detects not only live M. hyopneumoniae genome fragments in the lower tracheal samples and BALF were high and remained relatively stable throughout 56 dpi. The CT samples’ load was numerically high at 28 dpi and associated with GLLS. Microscopic tracheal lesion scores seemed to increase with time and were more prominent in LT. To the best of our knowledge, this is the first study to experimentally assess the M. hyopneumoniae burden in different tracheal anatomical sections throughout 56 dpi, demonstrating the association of lung lesion and the pathogen dynamics in the respiratory tract.The amount of M. hyopneumoniae, Actinobacillus pleuropneumoniae, and Porcine Reproductive and Respiratory Syndrome virus (PRRSv) free farm (since Brazil is a PRSS free country), being vaccinated only against swine influenza virus and porcine circovirus 2 (PCV2) with commercial vaccines. Moreover, the pigs remained without any noticeable clinical signs of infectious diseases until the inoculation. The animals were allocated in non-inoculated (n = 8) and inoculated (n = 16) groups. Each group was housed in isolated facilities  with free access to water and fed twice a day, with an acclimatization period of 7 days before the inoculation. Laryngeal swabs and blood samples were collected on the arrival day (dpi − 7) and on the inoculation day (dpi0) to confirm the M. hyopneumoniae -free status by qPCR and ELISA , respectively.A total of 24 male Large White pigs of 28-days of age were selected for this study. The animals originated from an n = 8) were mock-inoculated with 10 mL of sterilized Friis medium. The inoculated animals (n = 16) were given 10 mL of macerated lung inoculum  containing 106 CCU∕mL of M. hyopneumoniae strain 232 (moderate virulence) diluted in sterilized Friis medium. Inoculation was performed with a laryngoscope’s aid to introduce a sterile catheter in the trachea until the trachea bifurcation (approximately 20 cm deep) [M. hyopneumoniae infection.At dpi 0, control animals (cm deep) . After tEvery 14 days, at 14 dpi, 28 dpi, 42, and 56 dpi, two control animals and four infected animals were randomly selected and euthanized according to the guidelines of the Brazilian Federal Veterinary Medicine council: 1 mg∕Kg bodyweight of Xylazine  by intramuscular injection, followed by intracardiac injection of propofol (16 mg∕Kg) . After the euthanasia, a necroscopic evaluation was performed, along with the score of gross lung lesions and the collection of biological samples for further analysis.Nasal swabs were collected weekly using sterilized cotton swabs , which were inserted in both pig’s nostrils, rotated three times clockwise, placed into DNAse and RNAse free microtubes  containing 500 μL of sterilized Phosphate Buffered Saline (PBS) pH 7.4 , and kept at − 80 °C until processing.During necropsy, the respiratory tract from the larynx to the lungs was removed from the carcass and placed on a separate table. BALF samples were collected by injecting 15 mL of sterilized PBS (pH 7.4)  in the bronchial bifurcation with the aid of a sterilized glass pipette and an automatic pipettor. The lungs were gently massaged, and then the PBS was pulled back, placed into 1.5 mL DNAse and RNAse free cryovials , and immediately stored at − 80 °C until use.M. hyopneumoniae infection, were collected to confirm its involvement in the lung lesions seen at the necropsy. Fragments of approximately 1 cm3 were collected in the transition area of affected and healthy tissue using sterilized scalpel and forceps, instantly placed in DNAse and RNAse free cryovials , and stored at − 80 °C until use. Tissue samples of mock-inoculated animals were collected from all lung lobes since no gross lesions were seen during necropsy. For the inoculated animals with lesions in more than one lung region, samples were collected from all lesions and pooled into the same container.Samples of gross cranio-ventral consolidation lesions, considered characteristic of Besides, tracheal samples from three different anatomic sites, namely, cranial trachea (CT) , medium trachea (MT) , and lower trachea (LT) , were collected from each animal. Each tissue sample was divided into two; one was stored in individual DNAse and RNAse free microtubes  and immediately stored at − 80 °C while the other was stored in 10% buffered formalin for 24 h and later transferred to 70% alcohol.g, at 4 °C for 20 min. The sample’s supernatant was discarded, and the pellet resuspended in 250 μL of sterilized DNase and RNase-free PBS . To extract the DNA from tissue samples, 0.5 g of lung tissue and 0.2 g of trachea tissue were used. Regarding DNA extraction from liquid samples, 250 μL of resuspended BALF pellet and 500 μL of PBS from nasal and laryngeal swabs were used in an in-house protocol previously published [gapdh) gene, using a previously published protocol [gapdh gene.BALF samples were initially centrifuged  at 13,000 ublished . The conprotocol . To get M. hyopneumoniae DNA detection was performed with the aid of qPCR in tracheal and lung samples, nasal swabs, and BALF. All samples were tested in duplicates, using M. hyopneumoniae specific primer pair and hydrolysis probe from a previously published protocol [protocol .The reaction was composed of 1X Master mix Go taq® , 0.5 μM of each primer , 0.3 μM of the hydrolysis probe , ultrapure water q.s.p and 1 μL of DNA template, adding up to a final reaction volume of 10 μL. The real-time thermocycler used was a CFX-96 (Biorad - USA) model. The cycling conditions were: one cycle of initial denaturation at 95 °C for 3 min, followed by 39 cycles of 95 °C for 15 s, and annealing∕extension at 55.7 °C for 1 min. Quantification results were only used if the Cq difference was lower than 0.5 cycle . In case7 copies∕μL until 101 copies∕μL, of a synthetic DNA positive control  of the 150 bp amplified fragment. In addition, quantification data was only used if qPCR efficiency was between 90 and 105% [Absolute quantification and detection limits were performed using standard curves. The curve consisted of duplicate 10-fold dilutions, starting at 10and 105% .M. hyopneumoniae infection, like cranio-ventral consolidation lesions in the apical, intermediate, accessory lobes and the cranial part of diaphragmatic lobes. Briefly, each of the lobes was visually assessed, and the percentage of the affected area was estimated and further multiplied by the correction factor, based on each lobe’s proportional weight in the total lung weight, as described previously [M. hyopneumoniae infection were not scored in this study.Lungs of both inoculated and control animals were scored (before tissue sample collection) for characteristic lesions of eviously . Gross lM. hyopneumoniae characteristic microscopic lesions in tracheas [Tracheal samples were submitted to standard histopathology processing. The slides were then stained with Hematoxylin-Eosin and evaluated using a light microscope. The scoring process of the microscopic lesion was based on the description of the tracheas . Lesionsp < 0.05). Significant differences in parametric data were tested through analysis of variance (ANOVA), and multiple pairwise comparisons were made using the Tukey test and Duncan test (p < 0.05). Significant differences in non-parametric data were assessed through Wilcoxon and Kruskal-Wallis tests, and multiple comparisons between data sets were made by using Dunn’s test (p < 0.05). For non-parametric comparison of repeated measurements, the Friedmann test was used (p < 0.05), while for parametric datasets a linear mixed-effects models adjusted for Tukey (p < 0.05) were used. Correlations were tested using Pearson’s correlation (parametric models) test or Spearman’s correlation rank test (non-parametric models).All numeric data was analyzed using the R software . Data normality was assessed through the Shapiro-Wilk (SW) test, and homoscedasticity was tested using the Bartlett test, both with 95% confidence level (


Walking support systems are essential for blind people. In this study, the presentation of phosphene position is focused on as a method to detect obstacles for blind people. When the phosphene is used in a walking support system, it is necessary to accurately present the phosphene in at least three directions of the visual field. Controlling the presentation of phosphene position has been reported in several previous studies. However, methodologies to present phosphene in multiple directions without any electric interference have not as yet been investigated. In this study, therefore, appropriate stimulation factors are clarified by the simulation of electric field on the eyeball surface which is strongly related to the presentation of phosphene position in the visual field. As a result of the simulation, it was revealed that the distance of each electrode does not give a significant effect to the eyeball surface. However, the phase of alternating current significantly changed the electric field on the eyeball surface. From investigation of the simulation results, it was clarified that the transition of the electric field on the eyeball surface can be controlled using anti-phase stimulation. In addition, the methodology to present the phosphene at least in two directions was verified. Phosphene is critical visual information for hands-free walking support systems. Basically, it is a flash which is perceived by giving a magnetic or electrical stimulus to the visual pathway ,2. ThereSeveral previous studies have reported how to control the presentation position of phosphene . HiguchiLaakso et al.  reportedA previous study investigated the current path around the eyeball to explain the presentation position of phosphene without any contradiction. Manami et al.  indicateAs mentioned above, towards realizing a walking support system using phosphene, it is indispensable to present a phosphene in at least three directions. From a previous study , the phoThe rest of the paper is organized as follows: In In this section, related studies on stimulus factors and electrical interference that could be involved in the presentation of phosphenes are mentioned. Moreover, the relation between electrodes’ placement and the position of phosphenes presentation is discussed based on previous studies.Firstly, electric current intensity as a stimulus is focused on. When applying electrical stimulus to a human’s head, it is necessary to consider the magnitude of the current intensity as a stimulus factor. Especially, the side effects such as pain and itch caused by the electrical current stimulus should be taken into account ethically ,5,6,7. HTan et al., simulated how the electric field value on the cortex changes due to the change of the current intensity during brain stimulation by tACS . Using tIt is considered that the electrode placements that present phosphene to an intended direction in the previous study, which was mentioned in Next, the phase of the stimulation current is focused on. In the case of electrical stimulation to the brain, the purpose of the stimulation is to intervene in neurotransmission. Therefore, the selection of the phase of alternating current has been discussed ,18.Ivan et al., varied the phase of the stimulation current from 0 to 360° in 15° steps and reported the magnitude of the electric field in the whole brain with each phase condition . ElectroIn this study, the presentation of phosphene by eye stimulation is focused on, and it is not possible to directly compare this with the response to the electric field in the brain due to the phase change reported by Ivan et al. . HoweverIn this subsection, the relation between electrodes’ placement on the facial surface and the presentation position of phosphene is focused on. As mentioned in Six electrode placements were used in the previous study . The forOn the other hand, when gazing in the center direction, the visual retina is not exposed on the facial surface, hence, the visual retina is not directly stimulated. An indirect stimulation is presumed to explain the presentation of the phosphene in this case. Another related study has reported the presentation position of phosphene when stimulating the cornea with a coil-embedded contact lens . AccordiMoreover, from the report by Mihashi et al., it is clear that the electrical stimulus to the cornea does not flow to the eye orbit and does not stimulate the eyeball from the outer layer of the retina ,21. InjeTherefore, it is considered that when the temporal area of the right eyeball surface is stimulated, the nasal retina of the right eye is stimulated indirectly, and the phosphene is observed on the right side of the visual field. Similarly, when the vicinity of the central cornea of the right eyeball is stimulated, the vicinity of the macula is stimulated, and the phosphene is observed on the central visual field. In the case of the left eyeball, it is considered that the nasal retina is stimulated when the temporal area of the eye is stimulated, and the phosphene is observed on the left side of the visual field. Similarly, when the vicinity of the central cornea of the left eyeball is stimulated, the vicinity of the macula is stimulated, and the phosphene is observed on the central visual field. In other words, the phosphene can possibly present to an intended area in the visual field by stimulating the nasal side, the central side, and the temporal side of the eyeball surface selectively.Therefore, it is considered that controlling the presentation of phosphene in two or more directions of the visual field is possible by clarifying how to stimulate the intended regions on the eyeball surface while avoiding the electrical interference.This subsection describes the simulation tool of the electric field, which was used in the previous study .An open-source software package for simulating non-invasive brain stimulation, namely, SimNIBS ,23,24,25The simulation of tDCS in SimNIBS follows Laplace’s equation in determining the potential .(1)∇·σ∇In Equation (1), σ, φ and Ω indicate the conductivity, the potential caused by an external stimulus, and the region covered by this numerical calculation, respectively. us study ,14, the In this section, based on the background knowledge described in According to , electriVerify how much electrical interference due to the distance between the electrodes affects the transition of the electric field on the eyeball surface. The electrode placements are designed to gradually make the distance between the electrodes larger.Clarify how to simultaneously present the phosphene in two directions of the visual field while avoiding electrical interference. The electrode placements that stimulate intended regions on the eyeball surface reported in the previous study are applied in the simulation.In this study, the change of the electric field value is simulated while changing each factor  is changed, focusing on the transition of the electric field value on the eyeball surface. The simulation of the electric field value on the eyeball surface is performed based on the following two steps.This subsection describes the simulation condition used in the simulation tool, SimNIBS ,23,24,25The head model used in this study is “Ernie”, which is a sample data set of SimNIBS utilized for the electric field simulation. Ernie is a young and healthy man and his written informed consent to publish MR (magnetic resonance) datasets has been obtained. Moreover, Ernie’s structural MR images have been confirmed by a radiologist and are completely anonymized. Ernie’s face was replaced by the average of the datasets of several other subjects obtained in the same MR sequence. The subject’s dataset consists of high-resolution T1 and T2-weighted images and diffusion MR images. The voxel size in high-resolution T1 and T2-weighted images is 1 × 1 × 1 As mentioned above in The electric stimulus value is based on 10 Hz and 1 mA referring to previous studies ,14. BesiThe points on the eyeball surface set for observing the transition of the electric field value are shown in This subsection describes the summarization of the evaluation method in this study. The diagram of the summarizing evaluation method mentioned in this section is shown in Firstly, the electric field change on the eyeball surface during stimulation under the same phase condition shown in Furthermore, the electric field change on the eyeball surface during stimulation under the anti-phase condition shown in Besides, Furthermore, Comparing When two pairs of electrodes’ placements are used under the same phase condition, the effect of electrical interference can be seen. However, the value does not fluctuate significantly within the region of the eyeball surface.When a single pair of electrodes is used under the anti-phase condition, the same result as the norm value of the electric field under the same phase condition is shown.When two pairs of electrodes’ placements are used under the anti-phase conditions, the electric field values near the center point between the electrodes are canceled out due to the electrical interference.The transition waveform and the peak position of the electric field are not changed due to the stimulation current intensity, which is irrespective of the electrode placements.The electric field value with 2 mA is twice that of the one with 1 mA.The coordinates of the peak position are not changed due to the influence of electrical interference, only the norm value of the electric field is changed.From In this subsection, the simulation result of the electric field value on the eyeball surface using the electrode placements that stimulate intended regions on the eyeball surface is described. Firstly, the simulation results under the same phase condition are focused on. In Secondly, the simulation results under anti-phase conditions are focused on. Since In From Focusing on the electrical interference with the electric field on the eyeball surface under anti-phase stimulation, there are significant characteristics of the electric field value shown in Finally, the method of presenting phosphene in two directions is mentioned, which is important in this study. It has been verified that phosphene can be presented in the “right/left direction of the human vision”, “right/center direction of the human vision”, and “center/left direction of the human vision” using the three electrode placements. As a result, it was shown in However, there are still issues to be solved in the presentation of the phosphene toward the center. As mentioned above, the presentation of the phosphene is related to the magnitude of the norm value of the electric field. However, the value of the peak appearing at the 3rd and 4th coordinates from the nasal side of the eye on the eyeball surface, which is currently related to the presentation toward the central vision, is smaller than the value of the peak appearing near the temporal eyeball of both eyes. Moreover, using two pairs of electrodes’ placements simultaneously, the value of a relatively small peak standing at another coordinate becomes large, and a phosphene might be presented at an unintended position. Therefore, when presenting the phosphene in two directions, it is necessary to stimulate only the target region by selecting the stimulation current intensity and the phase to reliably present the phosphene in the central direction.In the verification for presenting the phosphene in two directions under the anti-phase stimulus shown in Stimulate only the right eyeball when presenting the phosphene in the “right/center direction” and stimulate only the left eyeball when presenting the phosphene in the “center/left direction.”Set the stimulation current intensity of the electrode that presents the phosphene toward the center of the visual field higher than the other electrodes.Besides, the following two measures can be taken to improve the presentation of the phosphene in the central direction.Firstly, the first measure will be described. In the electrode placement used in Secondly, the second measure will be described. Electrodes (II) and electrodes (III) shown in In this paper, the electrode placements and stimulation factors that enable the presentation of phosphene in two directions of the visual field were examined. First, focusing on the electric field on the eyeball surface induced by using two pairs of electrode placements, the effect of electrical interference on the norm value was investigated. It was clarified that although the magnitude of the norm value of the electric field changes, the peak of the coordinates does not change. Second, how to present the phosphene only in the intended two directions of the visual field using the specified electrode placements avoiding electrical interference was clarified. As a result, it was shown that the following methods in The relation between the electric field on the eyeball surface and each factor  was clarified. Thereby, a method for presenting phosphenes at the intended position was established. In this paper, the phosphene presentation in three or more directions of the visual field was not focused on. However, even when the phosphene should be presented in a lot of positions in the visual field, it is possible to determine the stimulation method in the same way as mentioned above.In this paper, the phosphene presentation with stimulating eyeball was focused on. However, depending on particular situation, some visually impaired people cannot perceive the phosphene. Since the eyeball is the peripheral part in the visual pathway, the visually impaired people who have damage at the optic nerve or visual cortex cannot process the electric stimulus to the eye. Therefore, how to control the phosphene presentation by stimulating the human brain should be investigated in the future. According to , it was


The group of children born at 35–36 weeks of pregnancy and the control group did not differ significantly. This result suggests that the elevated urinary cathepsin B/Cr. level may be the result of the kidneys’ immaturity in preterm newborns.Increased investment in perinatal health in developing countries has improved the survival of preterm newborns, but their significant multiorgan immaturity is associated with short and long-term adverse consequences. Cathepsin B, as a protease with angiogenic properties, may be related to the process of nephrogenesis. A total of 88 neonates  were included in this prospective study. We collected urine samples on the first or second day of life. In order to determine the concentration of cathepsin B in the urine, the commercially available enzyme immunoassay was used. The urinary concentrations of cathepsin B normalized with the urinary concentrations of creatinine (cathepsin B/Cr.) in newborns born at 30–34, 35–36, and 37–41 (the control group) weeks of pregnancy were  4.00 (2.82–5.12), 3.07 (1.95–3.90), and 2.51 (2.00–3.48) ng/mg Cr, respectively. Statistically significant differences were found between the group of newborns born at 30–34 weeks of pregnancy and the control group ( Preterm birth (before 37 completed weeks of gestation) accounts for 11% of births worldwide . The firCathepsin B is a lysosomal cysteine protease synthesized on the rough endoplasmic reticulum as a pre-proenzyme; cathepsin B in its mature, double-chain form comprises a heavy chain and a light chain ,15,16,17This study assessed whether cathepsin B may be involved in the maturation of the foetus’ kidneys by evaluating the effect of prematurity on the concentration of cathepsin B in the urine of preterm newborns.A total of 88 neonates were included in this prospective study. Sixty of them were born prematurely at 30–36 weeks of pregnancy. The neonates were hospitalized in the Department of Neonatology and Intensive Neonatal Care at the Medical University of Bialystok, Poland between December 2017 and December 2018. The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Local Bioethics Committee of The Medical University of Bialystok . Prior to the study written informed consent has been obtained from the parents of all the neonates. The clinical condition of the neonates was good or average; they were appropriate for gestational age (AGA), with the weight between the 10th and 90th centile of birth weight for their gestational age using normalized growth curves . The groThe inclusion criteria for this study were normal prenatal and postnatal ultrasound examination results of the kidneys, and a good or average clinical condition. The exclusion criteria were: 1-min Apgar scores < 4; congenital abnormalities, including urinary tract defects ; inborn error of metabolism; heart disease; abnormal ultrasound examination of the kidneys and the central nervous system ; abnormal laboratory tests (including elevated levels of inflammatory markers); treatment with catecholamines, antibiotics, diuretics, or mechanical ventilation. Additionally, the children of mothers with a burdened medical history were excluded from the study.Collection of urine samples in the study group was performed using single-use sterile bags . Urine was collected once on the first or second day of life. The urine samples obtained after centrifugation were kept in the refrigerator (4 °C) for no longer than 2 h and then frozen at −80 °C. Repeated freeze–thaw cycles were not used. Collection of the blood samples was conducted during the first or second day of the neonate’s life during routine practice in the Unit. S-Monovette 1.2 mL, Clotting Activator/Serum test tubes  for venous blood sampling were used. A blood cell morphology test and blood biochemistry tests were performed right after taking the blood samples.The levels of urinary cathepsin B were determined using a commercially available ELISA kit , according to manufacturer’s instructions, and were expressed in nanograms per milliliter. The detection range was 0.312–20 ng/mL, according to specifications of the kit. The mean intra-assay and inter-assay coefficients of variation (CV) for cathepsin B were <10% and <12%, respectively.In order to eliminate the potential confounding effect of urinary dilution, we normalized the urinary cathepsin B concentrations for the urinary concentration of creatinine; this was expressed in nanograms per milligram of creatinine (ng/mg Cr). The urinary concentration of creatinine was determined using Jaffé’s method.Calculation of estimated GFR was performed using the Schwartz formula .The determination of urinary cathepsin B was performed in the Department’s Laboratory of Pediatrics and Nephrology, at the Medical University of Bialystok. The morphology and serum biochemistry tests were performed in the Department of Laboratory Diagnostics at the University Clinical Hospital in Bialystok.p < 0.05.The statistical analysis was completed using Statistica 13.3 package . It expressed discrete variables as counts (percentage), continuous variables as median and quartiles (Q1–Q3). In order to determine normal distribution, the Shapiro–Wilk test was used. The Mann–Whitney U test was used for intergroup comparisons of continuous variables because the data were not normally distributed. To establish the direction and power of association between urinary cathepsin B concentrations and other variables, Spearman’s rank correlation coefficients were used. The results were significant at p > 0.05). Birth weight, length, and head and chest circuits were significantly lower in premature neonates than in term children; however, all the children were appropriate for gestational age. Eighty-eight neonates were included in the study. Twenty-eight of them were healthy neonates, and sixty were born prematurely at 30–36 weeks of pregnancy. Both groups were sex-matched (p > 0.05). Birth weight was statistically significantly lower in neonates born at 30–34 weeks of pregnancy than in babies born at 35–36 weeks of pregnancy. All premature babies were appropriate for gestational age, and when we divided both groups of preterm neonates according to the percentile of birth weight (10–50 percentile and 51–90 percentile), they were matched according to their percentile of birth weight. Both 5-min and 10-min Apgar scores were ≥8 in all neonates, and lower 1-min and 3-min Apgar scores characterized younger children.In both examined groups of premature babies (born at 30–34 and at 35–36 weeks of pregnancy), there were no statistically significant differences in the type of delivery . Both groups were sex-matched . Urinary creatinine was significantly lower in premature babies when compared to the reference group (p < 0.01). The lowest concentration of urinary creatinine was found in children born at 30–34 weeks. Similarly, eGFR was significantly lower in premature children compared to healthy controls (p < 0.01); however, we did not notice any difference in eGFR between children born at 30–34 weeks and 35–36 weeks (p > 0.05) . The serum concentration of creatinine did not show a statistically significant difference between the groups. The serum concentration of creatinine was higher in neonates born by cesarean delivery when compared to children born by vaginal delivery. The difference was close to being statistically significant ( > 0.05) .p < 0.05). The highest urinary excretion of cathepsin B/Cr. was found in babies born at 30–34 weeks of pregnancy, and the difference was statistically significant when compared to the reference group (p < 0.01) and when compared to babies born at 35–36 weeks.(p < 0.05). However, no statistically significant difference in the urinary level of cathepsin B/Cr. was observed between children born at 35–36 weeks of pregnancy and the reference group.In the entire group of premature neonates, the urinary level of cathepsin B/Cr. was higher compared to the reference group , Apgar score, prenatal steroid therapy, use of parenteral nutrition, use of nCPAP, or oxygen therapy. No statistically significant differences in the concentrations of urinary cathepsin B/Cr. were found between the groups of boys and girls.Kidney immaturity as a consequence of preterm delivery and perinatal problems connected to prematurity such as respiratory and circulatory failure, asphyxia, and antibiotic therapy are risk factors of renal tubular injury in premature neonates. The aim of this prospective study was to determine the values of urinary cathepsin B in premature neonates. It was hypothesized that cathepsin B may be involved in the processes of kidney maturation in the foetus.p < 0.001) in the urine of diabetic patients as compared to the control group, and they concluded that it may be useful as a non-invasive surrogate marker of incipient nephropathy [Normally, cathepsin B is highly expressed in the proximal tubule. It is the main enzyme involved in the lysosomal digestion of proteins, which are reabsorbed via endocytosis from the glomerular ultrafiltrate. This protease has long been taken under consideration as a biomarker of tubular damage because in response to damages, its levels are lower in tubules and higher in urine . It has hropathy .Knowing that many factors can contribute to tubular damage, we identified a group of sixty children with birth weights appropriate for the gestational age and clinical conditions assessed as good or medium. The children included in this study did not need drugs and mechanical ventilation. Their prenatal and postnatal ultrasound examination of kidneys was normal, and no deviations in laboratory tests and in parameters of renal function were found.The significantly highest urinary level of cathepsin B/Cr. was observed in neonates born at 30–34 weeks of pregnancy. The lack of statistically significant differences between the urinary level of cathepsin B/Cr. in children born at 35–36 weeks of pregnancy and the reference group was caused by the fact that these neonates were born close to the term of delivery. Thus, it may be supposed that the stages of development of the kidneys in both groups were similar. The significantly highest urinary level of cathepsin B/Cr. was found in neonates born at 30–34 weeks. This may have resulted from the fact that cathepsin B may take part in the process of nephrogenesis as a protease with angiogenic properties ,22,23,34Aisa et al. studied cathepsin B activity in the urine of neonates with intrauterine growth-restricted (IUGR)  and preterm neonates , both at 30–40 days of the corrected age. They found that cathepsin B activity in the urine was statistically significantly increased in preterm children, and even more increased in neonates with IUGR, when compared to the control group ): 2.303, (1.7–2.582); 3.633, (2.146–4.848); 1.044, (0.8335–1.372) IU/min mmol, respectively. Taking into consideration significantly lower total renal volume, cortical volume, and observed proteinuria in the IUGR and preterm neonates, they suggested that cathepsin B activity may be useful in the early prediction of renal susceptibility to damage in this group of children [In order to determine if other factors besides immaturity could affect the renal tubules’ functions, the relationship of the concentration of urinary cathepsin B and urinary cathepsin B/Cr. with gender, way of delivery, birth weight, centile of birth weight, Apgar score, prenatal steroid therapy, respiratory disorders , and the use of parenteral nutrition was examined. However, no correlations were found between them. This result suggests that elevated urinary cathepsin B/Cr. level may be the result of the kidneys’ immaturity in preterm newborns.The main limitation for this study was the sample size of recruited children, which was quite small, as it was very difficult to get study consent from some parents.In conclusion, the results showed that preterm neonates born at 30–34 weeks of gestation had elevated urinary cathepsin B/Cr. levels, which may be a result of the immaturity of kidneys. This preliminary observation should be confirmed in a multicenter study, which would consider not only preterm neonates with birth weights appropriate for gestational age and clinical conditions assessed as good or medium, but also those with clinical conditions assessed as bad, with or without AKI. Certainly, extending the study to the following weeks of life of the studied children, performing them both during hospitalization and after discharge from the hospital, would provide valuable information, confirming the role of cathepsin B as a biomarker of kidney maturity.