For an increasing trend in treatment duration, a decreasing trend in marker score was observed, suggesting an inverse correlation. The expression of the markers was different depending on the trigger factor, on MRONJ localization (maxilla/mandible), and the healing status, being more intense in patients cured per primam compared to those who had relapses. The patient’s immune response was negatively influenced by the duration of the treatment, the trigger factor, the location of the lesion in the mandible, and the recurrence of MRONJ.Impairment of the immune response in MRONJ (medication-related osteonecrosis of the jaws) is one of the still unclear etiopathogenic mechanisms of this condition encountered in cancer patients treated with bisphosphonates, with negative effects on the patient’s quality of life. The aim of the present study was to correlate the immune response with etiopathogenic factors via immunohistochemical evaluation of the maxillary tissues in zoledronic acid osteonecrosis. The retrospective study included a group of 51 patients with various types of cancers, diagnosed with stage 2 or 3 MRONJ at zoledronic acid and treated surgically. Immunohistochemical expressions of αSMA, CD3, CD4, CD8, CD20, CD79α, CD68, CD204, and tryptase were evaluated. Immunohistochemical markers expressions were statistically analyzed according to the duration of the treatment, the trigger factor, the location of the MRONJ, and the healing status. Analysis of the immune response included T lymphocytes, B lymphocytes, plasma cells, macrophages, and mast cells. The duration of treatment significantly influenced the immunohistochemical expression of most markers ( Osteonecrosis of the jaw occurring as an adverse effect of antiresorptive and antiangiogenic medication, is a multifactorial condition that greatly reduces the quality of life of cancer patients ,2,3,4,5.The objective of this retrospective study was to correlate the immune response defined by the expression of immunohistochemical markers  with etiopathogenic factors of MRONJ by using the information obtained from the immunohistochemical analysis of MRONJ tissues from cancer patients treated with zoledronic acid.The study group included 51 patients, 26 from Craiova and 25 from Constanta, with ages between 46 and 85 years old, mean value of 69.94 ± 8.50 years old, comprising 32 females (representing 62.7% of the entire study lot) and 19 males (37.3%).All patients were diagnosed with cancer and bone metastasis and developed MRONJ . PatientThe most frequent location of the primary tumor was the breast (49.02%), followed by the prostate (29.41%) and colon (7.84%). Most of the patients in the study lot suffered from comorbidities such as hypertension  or obesity .All patients were treated with zoledronic acid (ZA), administered intravenously at 4 mg once a month, and osteonecrosis appeared after a period varying between 24 and 35 months , and between 36 and 48 months . BP treatment duration varied between 12 and 48 months, with a mean value of 29.51 ± 9.50 months (approx. 2.5 years).The trigger factor for MRONJ development was extraction , periapical infection , and periodontal disease , and in most cases, the local pain appeared spontaneously. MRONJ location was mostly at the lower jaw for 34 patients (66.7%), compared to only 17 patients with MRONJ at the upper jaw (33.3%).Most patients diagnosed and treated for osteonecrosis presented stage 2 MRONJ , and the rest of the 12 patients (22.4%) had stage 3 MRONJ. Almost all patients had a poor health status, with ulcerations and exposure of the necrotic bone in 64.7% of cases, fistulae (41.17%), or even abscesses and purulent secretions (29.41%). Two patients with stage 3 upper jaw MRONJ presented maxillary sinusitis. All patients were surgically treated, and samples of tissues were analyzed histopathologically and immunohistochemically.2(1) = 11.754, p = 0.002 (Fisher’s Exact test). The association was strong with Cramer’s V = 0.490, as more than half of the patients with MRONJ stage 3 suffered a relapse after the first surgical intervention (63.6%), compared to patients with MRONJ stage 2, where only 13.2% experienced relapse (Relapse occurred for 13 patients (25.5%), while 38 patients (74.5%) were considered healed after the first surgical intervention. There was a statistically significant association between relapse status and MRONJ stage, χ relapse .p > 0.05), and variances were homogeneous. There was a statistically significant correlation between relapse status and age, rpb(49) = 0.314, p = 0.025, with relapse occurring more in older patients than in younger patients (74.461 ± 8.79 versus 68.394 ± 7.93) (pb(49) = 0.227, p = 0.110, even if relapse occurred more for patients with an increased treatment duration rather than a smaller treatment duration (33.153 ± 10.78 versus 28.263 ± 8.82)  . The sam ± 8.82) .Bacterial colonies, inflammatory infiltrate, and lymphoplasmacytic infiltrate were present in more than half of all patients, as indicated in Various histopathological aspects of the bone tissue were highlighted in the examined samples. These ranged from areas of bone necrosis that alternated with areas of bone tissue in different stages of degradation, intact bone tissue, and bone tissue with early osteonecrosis to bone tissue that was completely necrotic, resulting in a mosaic appearance. Areas of advanced osteonecrosis showed an inhomogeneous, vacuolar appearance with irregular borders and a “moth-eaten” appearance, empty osteocytic lacunae with no surrounding defense reaction, no osteoblasts, and no osteoclasts, and Haversian canals with central bone necrosis a,b. In tImmunohistochemical images of oral mucosa fragments showed damage to the oral epithelium and the chorion. In epithelium, a positive AE1/AE3 immunohistochemical reaction was revealed, which varied from moderately positive in areas with epithelial necrosis and disruption of the superficial layer to an intensely positive reaction in areas with thickened or spongy epithelium and the presence of intraepithelial inflammatory cells. The underlying chorion was characterized by the presence of moderate infiltrate with immune cells in fragments with epithelial necrosis and by the presence of abundant diffuse inflammatory infiltrate in fragments with thickened or spongy epithelium a–c.Markers’ expressions were evaluated as Negative (N), Low (L), Moderate (M), and Intense (I). p > 0.05, for 7 out of 9 markers. For two markers, the expressions were statistically significantly different between the two medical centers: for αSMA: U = 213, z = −2.379, p = 0.017, and for MASTOCYTES, with U = 220, z = −2.102, p = 0.036.A Mann–Whitney U test was run to determine if there were differences in markers’ expression between patients enrolled in the two medical centers. Distributions of the expression levels for the two groups of patients were not similar, as assessed by visual inspection. The expressions for the patients in Craiova were overall higher for all markers compared to patients from Constanta, and the differences were not statistically significant p < 0.05 , there was a moderate or strong negative association between treatment duration and markers’ expression, which was statistically significant, p < 0.05 .T lymphocytes were present in low numbers in tissues affected by osteonecrosis with weakly positive immunolabeling. Immunoexpression of CD3, CD4, and CD8 markers was moderate intense for images from the periodontium .Macrophages were observed in the periodontium in large numbers in the areas with rich inflammatory infiltrate, showing intensely positive CD68 expression. They were rare in the muco-osseous fragments with necrosis, showing moderate positive immunolabeling .Mast cells were observed in both bone and periodontal tissue fragments. In the bone tissue, mast cells immunolabeled for tryptase were identified in large numbers at the periphery of the osteonecrosis zone and the periodontal tissue, both intraepithelial and in the underlying chorion .Each possible value of markers was numerically quantified, to obtain a general score per patient . This score was the sum of all markers’ values, thus obtaining one single numerical value for every patient included in the study lot. 2(2) = 6.061, p = 0.048. Subsequently, pairwise comparisons were performed using Dunn’s (1964) procedure with a Bonferroni correction for multiple comparisons. This post hoc analysis revealed statistically significant differences in the markers’ expression between periodontal disease (35.86) and periapical infection (19.90) (p = 0.042), but not between other group combinations , “extraction” (n = 34), and “periapical infection” (n = 10) trigger factors. Distributions of markers’ expressions were not similar for all groups, as assessed by visual inspection of a boxplot. Overall, the markers’ expression for MRONJ triggered by periodontal disease was higher (Moderate–Intense) for all markers, followed by lower values for MRONJ triggered by extractions (Low–Moderate), and Low values for MRONJ triggered by periapical infection. Only for αSMA, the expressions were statistically significantly different between the different trigger factors, χinations .The intensity of the αSMA immunohistochemical reaction varied in appearance from low intensity with the significance of reduced vascularization in the areas of inflammation to high intensity with the significance of rich vascularization with numerous vessels of angiogenesis, in the case of patients with an abundant inflammatory reaction and high regeneration capacity .For patients with extraction as a trigger factor, the markers’ score had the highest values at the beginning of the BP treatment  and maintained this characteristic even for high treatment durations, remaining around 10, similarly to the average score of the group a.For patients with periodontal disease, the markers’ score at the beginning of treatment was a little higher than the average markers’ value for the entire study group and maintained this trend until the end of the study period b.For patients with periapical disease, the markers’ score at the beginning was below the average for this period c.p > 0.05 for all markers.Markers’ expression in the upper jaw was predominated by Intense values . Only T/B lymphocytes immunolabeled with CD20 were agglutinated in lymphoid follicles or disseminated forming a field of B lymphocytes with an inhomogeneous arrangement in the inflammatory periodontium a,b. Plasp > 0.05 for all markers. For the subgroup of Healed patients, the immunohistochemical analysis revealed that markers were mostly expressed as Moderate or Intense. The following markers were mostly expressed as Moderate: αSMA, T/CD8, B/CD20, PLASM/CD79, and MASTOCYTES, while Intense expression was predominant for T/CD3, T/CD4, CD68, and CD204. In contrast, the Relapse group emphasized more Low expressions. For the subgroup of Relapse patients, only αSMA and B/CD20 were mostly expressed as Moderate, while all others presented mostly Low intensities  compared to Relapse patients (mostly Low expression) and the differences were not statistically significant ensities .p > 0.05 for all markers.A Mann–Whitney U test was also carried out to determine if there were differences in markers’ expression between gender, MRONJ stage, HTA presence, obesity, and localization. Distributions of the expression levels for each of the two groups of patients were not similar, as assessed by visual inspection. The expressions for the healed patients were overall higher for all markers (mostly moderate expression) compared to relapse patients (mostly low expression) and the differences were not statistically significant, The present study showed that zoledronic acid has negative effects on bone, periosteum, and oral epithelium, as well as on blood vessels and immune cells in oral tissues. If, at the bone level, major effects were observed that led to the death of bone cells , and at the epithelial level the effects on keratinocytes showed destruction and ulceration of the epithelium, the expression of immunohistochemical markers varied greatly depending on the etiopathogenic factors and the prognosis of osteonecrosis healing. Patients with osteonecrosis presented with poor oral health status, the trigger factors of osteonecrosis being represented by recent extractions, periapical infections, or periodontal disease. Patients who underwent surgery as treatment for MRONJ had advanced stages of osteonecrosis, 2 and 3. More than 25% of them experienced a recurrence of the disease after the first surgery, the majority being in stage 3 osteonecrosis. Recurrence of the disease occurred especially in elderly patients. Although the longer the duration of treatment, the number of patients with relapse increased, this correlation was not statistically significant for the study group.Despite the increasing amount of literature generated over the years (1400 articles only in the last 6 months), the pathogenesis of MRONJ is still not fully elucidated. Several theories have been proposed for the pathogenesis of MRONJ ,36,37, iFrom other research on MRONJ ,34,35,38MRONJ occurs exclusively in the jawbones, associated with a very active oral tissue barrier from an immune perspective . The uniThe data from our study corroborated the data extracted from the literature, showing us that treatment with BPs, in this case, zoledronic acid, has effects on all tissues of the oral cavity: bone, periosteum, oral mucosa, and immune cells.The histopathological analysis in the present study showed that bone remodeling was affected, as all bone cell lines  were influenced by MRONJ. The histopathological appearance of the bone showed the presence of multiple foci of necrosis, without inflammatory infiltration, with sharp, scalloped bone edges. Thus, the lack of osteocytes in the osteocyte lacunae, and the absence of osteoclasts and osteoblasts in histopathological preparations were observed . On the N-BPs (nitrogen bisphosphonates) interact with soft tissue cells such as fibroblasts and keratinocytes, producing gingival ulceration followed by bone exposure, the main clinical sign seen in BRONJ. Impaired soft tissue biological activity may result in delayed mucosal healing after tooth extraction or dentoalveolar surgery in patients treated with BPs . It is aInflammation, a manifestation of the body against infection, is characterized by persistent infiltration of immune cells and increased levels of multiple pro-inflammatory cytokines and chemokines . InflammThe present study showed that the expression of immunohistochemical markers varied according to etiopathogenic and prognostic factors. The duration of drug administration is correlated with the intensity of immunohistochemical expression of all studied markers. The markers’ expression for patients from Craiova was generally higher for all markers compared to patients from Constanta, and the differences were statistically significant for two markers, αSMA, and mast cells. For 8 of the 9 markers , there was a moderate or strong negative association between treatment duration and marker expression that was statistically significant. For an increasing trend of the duration of treatment, we identified in opposition a decreasing trend of markers’ scores. The markers’ expression according to the trigger factors varied from Low to Moderate-Strong. Overall, marker expression for periodontal disease-triggered MRONJ was higher (Moderate-High) for all markers, followed by lower values for extraction-triggered MRONJ (Low-Moderate) and low values for periapical infection-triggered MRONJ. Only for αSMA, expressions were statistically significantly different between different triggers. There are statistically significant differences in markers’ expression between periodontal disease and periapical infection. In a histopathological and immunohistochemical study of specimens from patients with periodontal disease, it was shown that periodontal disease is the result of bacterial aggression that triggers inflammation and mobilizes the immune system as a defense system. The inflammatory process observed as a local immune response, arising as a reaction to bacterial invasion, is characterized by the presence of defense cells, especially immune cells. Lymphocytes are present both in the superficial area of the chorion, immediately subepithelial, and in the rest of the chorion, having the appearance of a diffuse infiltrate or, in some cases, they were identified as a grouped, nodular, especially perivascular formation, indicating an increase in vascular permeability. Vascularization was increased, being located both subepithelial and in the rest of the chorion, due to angiogenic factors associated with mediators of the inflammatory process. The angiogenesis process was exclusively capillary, starting from pre-existing vessels . Bănică In our study, markers’ expression for maxillary MRONJ patients was generally higher for all markers (mostly intense expression) compared to mandibular MRONJ patients (mostly low and moderate expression) and the differences were not statistically significant. Markers’ expression by lesion healing showed that expressions for healed patients were generally higher for all markers (mostly moderate expression) compared to relapsed patients (mostly low expression), and the differences were not statistically significant. For the subgroup of cured patients, the following markers were mostly moderately expressed: αSMA, T/CD8, B/CD20, PLASM/CD79α, and MASTOCYTES, while intense expression was predominant for T/CD3, T/CD4, C D68 and CD204. In contrast, the Relapse group emphasized more low expressions. For the subgroup of relapsed patients, only αSMA and B/CD20 were mostly expressed as moderate, while all others showed low intensities. An environment in which the immune system is suppressed favors the occurrence of MRONJ. N-BPs cause immune system dysfunction in MRONJ patients , affectiRecently, the important role of immune responses and inflammation in the development and progression of BP-induced osteonecrosis of the jaw (MRONJ) was emphasized as a result of massive infiltration of lymphocytes mixed with inflammatory cells in the tissue affected by MRONJ . BPs stiThe Immune system is closely related to bone loss and bone regeneration. More recent data investigate the effects of antiresorptive drugs on components of the immune system and introduce potential changes in the immune response as novel elements in the pathogenesis of MRONJ . The altGamma delta T cells representing innate lymphocytes are important in bone regeneration. Such T cells are significantly reduced in osteoporotic patients who are treated with BPs, indicating a link between MRONJ and gamma delta T cell deficiency . NeutropMacrophages are other immune cells sensitive to BPs, which have an inhibitory effect and reduce the viability and differentiation capacity of macrophages. Macrophage function is disrupted by increased matrix metalloproteinases (MMP) expression, leading to reduced wound healing in areas affected by MRONJ ,89. In vInhibition of angiogenesis is another central hypothesis believed to be a possible cause of pathogenesis of MRONJ. Inhibition of angiogenesis is hypothesized to adversely affect bone regeneration capacity after bone injury, delay bone remodeling or healing, and may increase susceptibility to superinfection. Zoledronate has direct inhibitory effects on angiogenesis, with MRONJ occurring due to reduced angiogenesis affecting healing after surgery ,90. BispAnother studied marker was αSMA. Actin alpha 2 (ACTA2), also known as alpha-smooth muscle actin (αSMA), is one of six actin isoforms that form the cytoskeleton and is found predominantly in smooth muscle cells. ACTA2 plays a crucial role in cells, promoting focal adhesion, migration, transcriptional and shape regulation, and contractile activity. In the oral cavity, ACTA2 is expressed in periodontal tissues and supports cytoskeletal dynamics, cell motility, and contractility . VasculaThe commensal microbiome may play a protective rather than a pathological role in the early stages of MRONJ development . InvasivA limitation of this study is the absence of data from cancer patients without MRONJ but under treatment with zoledronic acid—to compare the effects of zoledronic acid on MRONJ patients with non-MRONJ patients . Follow-up work includes a new study lot characterized by other inclusion criteria.The retrospective study used databases from Dolj County Emergency Clinical Hospital and Constanța County Emergency Clinical Hospital, as well as from the Center for Microscopic Morphology and Immunology in Craiova, Romania. The database contains information on patient demographics , cancer diagnosis, comorbidities, MRONJ, surgical treatment of MRONJ, and histological and immunohistochemical data. The study data were collected between March 2019 and December 2022. The Ethics Committee of the University of Medicine and Pharmacy in Craiova approved the study with no. 59/22.03.2019. The study meets the STROBE criteria .The primary outcome was to correlate the immune response defined by the expression of immunohistochemical markers  with etiopathogenic factors of MRONJ, by using the information obtained from the immunohistochemical analysis of MRONJ tissues from cancer patients treated with zoledronic acid. Secondary outcome referred to establishing an overall marker score for each patient, and its relationship with the trigger factors.The retrospective study included a group of 51 patients with cancer under bisphosphonate treatment for bone metastasis who suffered complications of osteonecrosis of the jaw and presented themselves for treatment at the Department of Oral and Maxillo-Facial Surgery of the Craiova County Clinical Hospital or the Oral and Maxillo-Facial Surgery department of the Constanta County Clinical Hospital in the period March 2019–December 2022. All participating patients gave their consent for the treatment and the histological and immunohistochemical processing of the obtained specimens.All patients included in the study were patients with various types of cancer, treated for bone metastases with bisphosphonates, who developed osteonecrosis of the jaw bones during treatment, such as exposed necrotic bone or intra- or extraoral fistulas with persistence greater than 8 weeks, without previous radiation therapy in the jawbones area, and without cervicofacial cancers or metastases.Patients with malignant tumors in the maxillary area and patients who received radiation therapy in the facial area in the past were excluded from the study.From the data extracted from the medical charts, each patient received the indication of local decontamination of the oral cavity with 0.2% chlorhexidine mouthwash twice a day and topical application on the lesion with 1% chlorhexidine gel three times a day, as well as empiric oral antibiotic therapy with amoxicillin and clavulanic acid 875/125 mg twice daily for non-beta-lactam allergic patients and clindamycin 600 mg twice daily for allergic patients. Before the institution of empiric antibiotic therapy, the antibiogram was collected from the lesion. Inpatients were subsequently switched to targeted IV antibiotic therapy according to the antibiogram result.The surgical interventions were performed with general or local anesthesia, with an intraoral approach, and consisted of curettage and sequestrectomy in most cases, with beveling of the sharp edges of the bone and closing the wound with a mucoperiosteal flap. In cases of recurrence, a second surgical intervention included bone resection associated with osteosynthesis (when appropriate) and reconstruction with proximity flaps. In all cases, the removal of bone sequestrations and curettage into the bone tissue was performed until clear bleeding from the underlying bone occurred. A-PRF has been used in very few cases, especially when the resulting bone defect could not be covered with a mucoperiosteal flap or when it was difficult to protect the remaining bone. Due to the long half-life of zoledronic acid, the surgeon did not recommend discontinuation of zoledronic acid. Treatment with zoledronic acid was stopped only in patients who received this recommendation from their primary care oncologists.After surgery, antibiotic therapy continued for 10 to 14 days, intravenously, until suture removal (in severe cases), followed by oral antibiotic therapy for 7 days. In less severe cases, after discharge, the patient continued oral antibiotic therapy until 2–3 weeks after the surgical procedure.Age, sex, type of cancer, type of bisphosphonate, location of osteonecrosis, and stage of osteonecrosis were recorded for each patient.v/v) and sent to the Department of Histopathology where they were processed for histopathological analysis and immunohistochemistry. The histological and immunohistochemical analysis was carried out at the Research Center for Microscopic Morphology and Immunology Studies, University of Medicine and Pharmacy of Craiova. A total of 78 samples were collected.In the treatment of osteonecrosis, patients underwent sequestrectomy or partial resection of the necrotic bone. The bone and marginal periodontal specimens taken at the Oral and Maxillofacial Surgery Clinics  were included in 10% formalin  and the trichrome technique, according to the Goldner–Szekely (GS) method. A qualitative histopathological analysis was performed, taking into account changes in the epithelial, periosteal, deep periodontal, and alveolar bone levels. The analysis was performed on samples collected from osteonecrosis foci located in the anterior and posterior areas of the upper and lower jaws. These samples were used to analyze the following components: inflammatory infiltration, blood vessels, osteocytes and empty lacunae, and viable bone. Analysis of histological parameters was performed by a single calibrated investigator using a Nikon Eclipse 55i binocular microscope equipped with planar-fluorine objectives, DS-Fi1(5Mp) digital camera as well as Nikon NIS-Elements image acquisition and analysis software . Images were captured using 100× and 200× objectives. The clinical diagnosis of osteonecrosis of the jaw bones was confirmed by the results of the histopathological examination, the general histopathological evaluation revealing different degrees of osteonecrosis.The immunohistochemical study of the oral tissues  from the area of osteonecrosis to zoledronic acid sought to highlight the main types of cells involved in the immune response and their role in the pathogenetic mechanisms of osteonecrosis. The studied cells were T lymphocytes, B lymphocytes, plasma cells, macrophages, mast cells, keratinocytes, and endothelial cells; and the immune response of patients with cancer and MRONJ to zoledronic acid was highlighted depending on the duration of treatment with bisphosphonates, the location of MRONJ, the trigger factor , and wound healing after surgery. The following antibodies were used: AE1/AE3, monoclonal antibodies used to detect cytokeratins, which are the predominant structural proteins in epithelial cells; alpha smooth muscle Actin or αSMA (ACTA2) Mouse Mono-clonal Antibody, an antibody for identification of myofibroblasts and smooth muscle cells in vascular walls; CD3  for enhancing T lymphocytes; CD4, for highlighting CD4 positive T lymphocytes, clone MT310, M0716, Dako, dilution 1:50; CD8, for highlighting CD8 positive T lymphocytes, clone C8/144B; CD20  for B-lymphocyte study; CD79alpha  for plasma cells; CD68  for macrophages; tryptase  for mast cell study.The qualitative assessment of the immunohistochemical reaction to the studied markers was achieved by assessing the intensity of the staining as follows: the intensely positive reaction (+++) was defined as the reaction present in more than 80% of the cells, diffusely distributed, well visible under microscopic examination with low magnification objectives; the reaction of moderate intensity (++) was defined as the reaction present in 30–80% of the cells, with a focal disposition, well visible during microscopic examination with objectives with medium power of magnification; the reaction of weak intensity (+) was defined as the reaction present in 5–30% of the cells, visible during microscopic examination with high-power objectives; and the reaction was absent (−) when no cell with positive immunomarking can be identified.Immunohistochemical analysis was performed by a single calibrated investigator using a Nikon Eclipse 90i binocular microscope  equipped with planar-fluorine objectives, DS-Fi1(5Mp) digital camera, and Nikon NIS image acquisition and analysis software -Elements . The images were captured using 100× and 200× objectives.2) were employed for categorical parameters. The following p values were accepted: p < 0.05 significant based on a confidence interval (CI) of 95%, as well as p < 0.001 highly significant (CI of 99.9%).All primary data acquired in this study were recorded using Microsoft Excel 365 , and included clinical and non-clinical data related to demographic aspects , primary tumors’ location and comorbidities, BP treatment (acid type and duration), MRONJ data , and immunohistochemical markers’ expression. Continuous variables were expressed as mean ± standard deviation (SD) to complete the descriptive analysis, while categorical data were expressed as absolute and relative frequencies (%). All statistical analyses were performed using IBM SPSS Statistics 20.0 software . Potential associations involving the acquired parameters were analyzed using the following tests: Shapiro–Wilk’s for normality distribution, Levene’s test for homogeneity of variances, point-biserial or Kendall’s tau-b for correlations, Mann–Whitney U, and Kruskal–Wallis H (completed with pairwise comparisons performed using Dunn’s procedure with a Bonferroni correction for multiple comparisons) for group distributions. Chi-square and Fisher Exact tests (χThe immunohistochemical expression of the markers is statistically significantly lower with increasing duration of bisphosphonate treatment. The patient’s immune response was negatively influenced by the duration of the treatment, the trigger factor, the location of the lesion in the mandible, and the recurrence.


The counting of surgical instruments is an important task to ensure surgical safety and patient health. However, due to the uncertainty of manual operations, there is a risk of missing or miscounting instruments. Applying computer vision technology to the instrument counting process can not only improve efficiency, but also reduce medical disputes and promote the development of medical informatization. However, during the counting process, surgical instruments may be densely arranged or obstruct each other, and they may be affected by different lighting environments, all of which can affect the accuracy of instrument recognition. In addition, similar instruments may have only minor differences in appearance and shape, which increases the difficulty of identification. To address these issues, this paper improves the YOLOv7x object detection algorithm and applies it to the surgical instrument detection task. First, the RepLK Block module is introduced into the YOLOv7x backbone network, which can increase the effective receptive field and guide the network to learn more shape features. Second, the ODConv structure is introduced into the neck module of the network, which can significantly enhance the feature extraction ability of the basic convolution operation of the CNN and capture more rich contextual information. At the same time, we created the OSI26 data set, which contains 452 images and 26 surgical instruments, for model training and evaluation. The experimental results show that our improved algorithm exhibits higher accuracy and robustness in surgical instrument detection tasks, with F1, AP, AP50, and AP75 reaching 94.7%, 91.5%, 99.1%, and 98.2%, respectively, which are 4.6%, 3.1%, 3.6%, and 3.9% higher than the baseline. Compared to other mainstream object detection algorithms, our method has significant advantages. These results demonstrate that our method can more accurately identify surgical instruments, thereby improving surgical safety and patient health. In recent years, with the development of computer vision (CV), it has been widely used in the medical field, covering multiple aspects, such as medical image analysis ,2, pathoSurgical instruments play a vital role in surgeries, assisting doctors in performing surgical procedures, controlling bleeding, and reducing the duration of surgeries. They are essential tools for doctors to complete surgeries. The proper selection and use of surgical instruments directly affect the success rate of surgery and the patient’s recovery. Each type of surgical instrument has a specific function, and any omission or confusion of surgical instruments during surgery may result in surgical failure and unnecessary harm to the patient. Moreover, if surgical instruments are inadvertently left in the patient’s body after surgery, it may lead to consequences, such as pain, infection, tissue inflammation, organ damage, and even death, resulting in serious medical accidents. A study  in 2019 The traditional method for counting surgical instruments typically involves medical staff checking the name, quantity, and model of each instrument on a list to ensure consistency with the surgical plan, as well as inspecting them for damage. However, due to the wide variety of surgical instruments and the potential for confusion between instruments with similar appearances, as well as factors, such as patient cases, individuals, and environments, the efficiency and accuracy of manual instrument counting are limited . This meAlthough there has been some progress in visual recognition for surgical instrument counting, there are still some problems. In real scenarios, surgical instruments may be densely arranged or obstructed by each other, and they may be affected by different lighting environments, all of which can affect recognition accuracy. In addition, some surgical instruments have very similar appearances, making it difficult to distinguish them through visual recognition, so achieving accurate recognition in different surgical scenes remains a challenge. To address these issues, this paper improves the YOLOv7x  and applA surgical instrument detection model was proposed to help hospitals achieve automated surgical instrument counting.The model can accurately identify 26 commonly used surgical instruments and has good recognition performance for some instruments with similar shapes and dense occlusions.https://aistudio.baidu.com/aistudio/datasetdetail/198164, accessed on 4 April 2023). It contains 452 images, covering 26 types of surgical instruments, each appearing 60 times. To enhance the model’s recognition accuracy and generalization ability, the surgical instruments were placed, with varying levels of dense occlusion, and data were collected under multiple lighting conditions. In addition, various data augmentation techniques were used to expand the data set.The OSI26 surgical instrument data set was created and made public , model scaling, reparameterization, auxiliary training heads, and multi-label assignment . The YOLThe entire model consists of four parts: the input end (Input), the backbone network (Backbone), the neck (Neck), and the prediction head (Head) . The imaThe model’s neck uses the Path Aggregation Feature Pyramid Network (PAFPN)  to extraRepLKNet  uses larThe network architecture of RepLKNet mainly consists of the RepLK Block, ConvFFN, and Transition Block. The RepLK Block contains deep convolution and shortcut connections. The use of large convolutional kernels may result in difficulty in capturing local features. The shortcut connections explicitly make the model a combination of models with different receptive field sizes, allowing for improvement in larger receptive fields without losing the ability to capture small-scale features. RepLKNet employs multiple repeated convolutional layers, each of which includes a large-sized convolutional kernel, a batch normalization layer, and a ReLU activation function. This repeated layer structure can make the network deeper, while the large-sized convolutional kernel can extract more abundant features, thereby improving the performance of image classification.ODConv  utilizesIt can be described in the following form:n DyConv  and Condn DyConv .The improved structure of the YOLOv7x algorithm, as shown in The experimental environment is built on the Windows operating system, Python 3.8.0, PyTorch 1.11.0 deep learning framework, and Cuda 11.3. The GPU model is a RTX 3090 (with 24 GB of memory), and the CPU model is an Intel (R) Xeon (R) Platinum 8255C CPU @ 2.50 GHz. The model is trained with an initial learning rate (lr0) of 0.01, using a cosine annealing learning rate decay strategy with a cosine annealing hyperparameter (lrf) of 0.1. The loss function is optimized using the stochastic gradient descent (SGD) method, with a weight decay coefficient of 0.0005 and a learning rate momentum parameter of 0.937. Warm-up learning is performed in the first three epochs, with a warm-up learning rate of 0.8 and a warm-up learning rate momentum of 0.8. The input image size is set to 640 × 640, and the batch size for training is 16. A total of 150 epochs are trained, using transfer learning  to pre-tThe experiment uses precision P, recall R, F1 (F-score), and mean Average Precision mAP as indicators to evaluate the performance of surgical instrument detection. Precision reflects the proportion of true positive samples in the predicted positive samples, while Recall reflects the proportion of correctly predicted positive samples in the total positive samples . TP (TrumAP (mean average precision) is an important indicator for measuring detection accuracy in object detection, which represents the average AP (average precision) of each category. The AP value of each category can be calculated by integrating the PR  curve, and the calculation formula is as follows.In the above formula, n represents the number of categories. We utilize common object detection evaluation metrics from the COCO evaluation, including AP, AP50, AP75, APS, APM, and APL. AP represents the mean average precision (mAP) across all categories. AP50 and AP75 represent the AP values at IoU thresholds of 0.5 and 0.75, respectively. APS represents the AP value for object bounding boxes with a pixel area smaller than To validate the effectiveness of the proposed improvement methods for surgical instrument category detection, we conducted ablation experiments. In these experiments, we tested each improvement method individually. “√” indicates that the corresponding improvement method was added, while “-” indicates that the method was not used. The experiments were conducted on the OSI2 surgical instrument data set, with input images uniformly resized to 640 × 640 and a batch size of 16. We utilized the pre-trained model YOLOv7x and trained the models for a total of 150 epochs. The performance comparison of different improvement methods on the validation set is presented in Based on the data in As shown in According to the data in To compare the improvement effects of the model more intuitively, we selected densely unoccluded, sparsely occluded, and densely occluded surgical instrument images from the test set for testing. The confidence threshold of the model was set to 0.25, and the IOU threshold was set to 0.45. The results are shown in In In order to comprehensively demonstrate the advantages of the improved algorithm, comparative experiments were conducted with classical object detection algorithms, such as YOLOv5l, YOLOv7, YOLOX-tiny, YOLOv6n, Faster RCNN, DETR, and Dynamic RCNN. Transfer learning was performed on the official pre-trained models, using the same data set, training epochs, and input image sizes. The performance of different models on the validation set is shown in The results in From the data in In hospitals, the commonly used method for surgical instrument counting is the double-checking method, which requires two surgical personnel to simultaneously count and cross-check the instruments to ensure accuracy. Typically, it takes 1–2 s for one person to manually count a surgical instrument. In contrast, the improved model proposed in this study can effectively identify six to seven images per second with a high recognition accuracy. Each image contains three to ten surgical instruments. Therefore, it can significantly assist in the manual counting of surgical instruments, greatly improving work efficiency, as well as reducing counting errors, thereby ensuring surgical safety.We also evaluated the classification performance of the improved model using the confusion matrix for surgical instrument classification, as shown in From This paper proposes an improved model based on Yolov7x for detecting surgical instruments during the instrument counting phase, aiming to improve counting efficiency and reduce error probability. Although the current improved model has achieved good results, it still has certain limitations.Firstly, the improved model exhibits high accuracy in recognizing the 26 surgical instruments studied in this research. However, there may still be recognition errors for some instruments that have very similar appearances, especially for the (q) and (s) instruments depicted in Secondly, the proposed method in this paper is only applicable to common surgical instruments used in operating rooms. For special types of surgical instruments, such as those used in neurosurgery or microsurgery, dedicated counting methods need to be developed. Future research can focus on these specific types of instruments to devise counting approaches that are tailored to their characteristics, thus enhancing surgical safety and accuracy.Lastly, the method presented in this paper is implemented based on computer vision technology. Although efforts have been made to consider factors, such as lighting, angles, and camera resolution during image data collection, there may still be counting errors caused by lighting variations, viewing angles, or camera limitations. Therefore, future research can explore new image processing techniques and deep learning models to improve the accuracy and reliability of surgical instrument counting.Based on the real-time and low false positive requirements for surgical instrument detection in actual application scenarios, this paper proposes an improved YOLOv7x algorithm based on RepLK Block and ODConv to address issues such as dense arrangement of surgical instruments, mutual occlusion, difficulty in distinguishing similar instruments, and varying lighting conditions. The improved algorithm introduces the RepLK Block module into the original backbone network, which utilizes the advantages of large convolutional kernel structure to reduce the loss and blurring of image feature information, enhance the effective receptive field of the model, and thus improve the detection performance of surgical instruments under dense occlusion and the recognition accuracy of similar instruments. At the same time, the neck network introduces the ODConv structure, a full-dimensional dynamic convolution, to obtain more abundant feature information and enhance the robustness of the algorithm. The ODConv structure adjusts the convolutional kernels dynamically by obtaining multi-dimensional feature information, reducing the limitations of feature extraction ability in traditional convolutional neural networks, and improving the generalization ability of the model’s detection, thus further improving the accuracy of surgical instrument detection. After testing on the OSI26 data set, the improved algorithm achieved excellent performance in terms of precision, recall, F1 score, AP, AP50, AP75, and APL. The respective values are 92.6%, 97.0%, 94.7%, 91.5%, 99.1%, 98.2%, and 91.5%. These results represent improvements of 4.2%, 5.1%, 4.6%, 3.1%, 3.6%, 3.9%, and 3.1% compared to the baseline model. Compared with other object detection algorithms, such as YOLOv5l, YOLOv7, YOLOX-tiny, YOLOv6n, Faster RCNN, DETR, and Dynamic RCNN, the improved algorithm proposed in this paper also has significant advantages. The number of parameters in the improved model is 84.0MB, and it can detect six to seven densely arranged surgical instrument images per second, helping medical staff to quickly and accurately identify surgical instruments, improving the efficiency and accuracy of counting, and reducing the occurrence of medical accidents.


In this study, we used a combination of bovine proteoglycan and dimethyldioctadecylammonium (DDA) to establish a mouse model of proteoglycan-induced spondylitis (PGISp). To evaluate the therapeutic effects of UCMSCs, we treated PGISp mice with different doses of hUCMSCs 7 cells/kg, iv, QOW×2, n=10). To assess the therapeutic effects of hUCMSCs, we evaluated Th cell subsets in the spleen, spleen and thymus coefficients, peripheral blood inflammatory factors, and pathological and imaging observations of the spines and lumbar spines in the PGISp mice.At week 13, the PGISp mice exhibited thickened, erythematous paws, erythema in the extremities, and lameness. CT scans revealed necrotic lysis of chondrocytes, formation of fissures, visible hemorrhage, connective tissue hyperplasia, and focal infiltration of lymphocytes in the intervertebral discs. At week 14, the PGISp mice were randomly divided into three groups and administered different doses of hUCMSCs  in the serum decreased after hUCMSC injection. CT imaging and pathological analysis indicated that hUCMSC treatment inhibited ectopic osteogenesis and maintained clear small joint gaps, which slowed down the progression of structural lesions in the disc, nucleus pulposus, fibrous ring, and cartilage in PGISp mice.Administering hUCMSCs at the 14th week after modeling proved to be an effective treatment for PGISp mice. This experiment offers a valuable reference for the pre-clinical use of hUCMSCs in the treatment of AS. Ankylosing spondylitis (AS), a form of spondyloarthritis, is an autoimmune disease that primarily involves the spinal joints, sacroiliac joints, and their adjacent soft tissues, such as tendons and ligaments. Retrospective epidemiological studies have shown that the prevalence of AS in the United States ranges from 0.2% to 0.55% . The aveCurrently, effective treatments for AS are limited and are divided into traditional, pharmacological, and surgical treatments , 8. The In summary, AS requires more aggressive and effective modalities or means of treatment which can effectively suppress the systemic inflammatory response during all stages of AS disease progression, promote spinal repair during all stages of the disease, improve and maintain spinal flexibility and normal posture, relieve symptoms, reduce functional limitations, and reduce other complications such as peripheral joint development . NumerouTo further elucidate the underlying pathological mechanisms for AS, researchers established a series of tonic animal models including transgenic animal models, inflammation-induced animal models, and other animal models, according to different pathogenic mechanisms . Based oEighty SPF-grade female BALB/c mice, weighing approximately 17-19 g, were purchased from Zhejiang Viton Lever Laboratory Animal Technology Co. The mice were housed and used with the approval of the ethics committee of the Shanghai Institute of Pharmaceutical Industry and in compliance with all applicable institutional and governmental regulations regarding the ethical use of animals. After acclimatization, mice were injected intraperitoneally with a mixture of 1 mg/ml proteoglycan solution and 10 mg/ml DDA micelles at 0.2 ml per mouse at 3-week intervals according to 1:1 (v:v), i.e., the second and third sensitization reactions were performed at weeks 3 and 6, respectively .6, 5×106, 10×106 cells/kg, respectively, and the same dose of PBS was used as placebo in the control group.After the third immunization, the extent of lesions on the extremities of mice was observed twice a week, and the mice in each group were selected for CT photography in week 13. In week 14, all mice were stratified and randomly grouped to ensure the number of animals in each group was 10, and thereafter therapeutic administration was started, intravenously, at one-week intervals, twice. The low, middle and high doses of treatment groups were designed as 2.5×10At the 20th week, the blood of the mice was collected to detect the content of inflammatory factors and chemokines, the spleen and thymus were collected to weigh and calculate the organ coefficient, and the proportion of T cell subsets in the spleen was detected, and the spinal lesion status of the mice was detected by CT and HE Figure 18 cells/10 ml and lyophilized by programmed cooling instrument. The preparation of hUCMSC is carried out in a sterile and clean GMP workshop.hUCMSC was provided by Shanghai Quan Sheng Biotechnology Co., Ltd. The human umbilical cord was obtained from Dongguan Chang’an Hospital and passed ethical filing. The hUCMSCs were prepared by enzymatic digestion  from the umbilical cord of newborns and cultured in α-MEM medium containing 10% FBS, and after culturing to P6 generation, cell lyophilization solution was added according to the ratio of 1×106 hUCMSCs were tested for surface markers including CD14, CD19, CD31, CD34, CD45, CD73, CD90, CD105, and HLA-DR. Surface markers of hUCMSCs were detected using flow cytometry .Pre-life-storage hUCMSCs were taken for washing and counting. 3×10To validate the immunomodulatory function of hUCMSCs, co-culture experiments were performed by incubating hUCMSCs with PBMCs extracted from different donors’ peripheral blood. The PBMCs were stimulated with PHA, and the inhibitory effect of hUCMSCs on PBMC proliferation, differentiation of Th cell subsets , and secretion of inflammatory cytokines  were examined.MesenCult™ Lipogenic Differentiation Kit was used to detect the lipogenic differentiation ability of hUCMSCs. After 3 weeks of culture in lipogenic differentiation medium, cells were stained using Oil Red O stain and observed for lipid droplet formation under high magnification. The osteogenic differentiation ability of hUCMSCs was detected using MesenCult™Osteogenic Differentiation Kit. After 3 weeks of culture in osteogenic differentiation medium, cells were stained using Alizarin Red staining solution and observed under low magnification for the presence of visible red staining dots, and altered red staining dots for calcium nodules. The chondrogenic differentiation ability of hUCMSCs was measured using StemPro™ Chondrogenic Differentiation Kit. After 1 week of culture in chondrogenic differentiation medium, cells were stained using Alizarin Blue staining solution and observed for the presence of blue cartilage tissue.6 cells/ml. The next day, 1.5 µl of stimulation inhibitor was added to each sample, and cells were collected after 4-6 h of culture for flow cytometry . Th1, Th2, Th17 and T-reg detection indexes were CD3+CD4+IFN-γ+, CD3+CD4+IL4+, CD3+CD4+IL17A+ and CD3+CD4+CD25+FOXp3+, respectively. Among them, CD3, CD4, and CD25 are surface markers, IFN-γ and IL17A are intracellular factors, and FOXp3 is an intranuclear factor. The detection of intracellular factors and nuclear factors requires cells to be fixed and stained after membrane rupture.At the 20th week, the spleens of PGISp mice in each group were ground and prepared into single cell suspensions. After counting the cells, the cells were resuspended in 1640 medium (10% FBS) and seeded into cell culture dishes at a density of 5×10The spleen and thymus of PGISp mice were collected at the 20th week of the experiment, and the organ weights were weighed using an analytical balance and divided by the corresponding body weight of each group of mice to calculate the spleen coefficient and thymus coefficient obtained.Peripheral blood was collected from PGISp mice by eye blood sampling at week 20, and the serum was centrifuged at 10,000g for 15 minutes. The pro-inflammatory cytokines  and anti-inflammatory cytokines  levels in peripheral blood were measured by the LEGENDplex Multifactor Assay Kit (Biolegend).The PGISp mice were anesthetized and examined by CT scan  at the 13th and 20th weeks of the experiment, respectively, while the reference The lumbar vertebrae of mice were decalcified, paraffin-embedded, sectioned, HE stained, and sealed at week 20 after CT scan. The sections were browsed under the microscope or viewed digitally, and the tissue sections were carefully observed under 20x and 200x conditions, and the basic pathological changes in the sections such as congestion, bruising, hemorrhage, edema, degeneration, necrosis, hyperplasia, fibrosis, mechanization, granulation tissue, and inflammatory changes were described in text and reflected the differences between the pathological findings, while the typical lesion sites were arrowed with different colors to markings.Statistical analysis was performed with SPSS 22. The ROUT (Q = 1%) method was used to remove Outliers. Multigroup comparisons were analyzed using one-way ANOVA. A value of p < 0.05 was considered statistically significant. A value of p < 0.01 was considered statistically significant.60% of the mice induced by bovine proteoglycan combined with DDA started to develop peripheral joint disease and relieve symptoms at the 7th and 19th weeks, respectively, and about 40% of the mice showed no or very mild disease characteristics. At the 13th week, the limbs of the mice were observed, and the mice with no disease or mild disease were shaved. The limbs and spines of normal mice did not have any lesions  has the function of promoting immune cell activation, by co-culturing hUCMSCs with PBMCs and stimulating with PHA, hUCMSCs demonstrated a proliferative inhibition effect on PBMCs, CD3+ T cells, and CD4+ T cells  of PGISp mice showed an increasing trend. The thymus weight and thymus coefficient of the low-dose, middle-dose, and high-dose groups  also increased significantly , IL-6 and IL-22 in serum , macrophages, natural killer (NK) cells, and adaptive immune cells. These immune cells produce various innate cytokines that play a crucial role in the development of AS , 40. RecIn vitro experiments showed that co-culturing hUCMSCs with PBMCs under PHA stimulation exhibited the immune modulation function of hUCMSCs, suppressing T cell activation and proliferation, regulating Th cell subsets , increasing secretion of anti-inflammatory factors  and decreasing secretion of inflammatory factors . In conclusion, we believe that stem cells may treat AS through the following mechanisms: 1) participate in the regeneration of damaged tissues and cells after intravenous infusion into patients; 2) have the ability to aggregate in the affected synovium, thereby promoting the repair of damaged joint tissues; 3) exhibit unique immune modulation functions by effectively inhibiting T lymphocyte proliferation and activation function through the addition of autologous and allogeneic MSCs to peripheral blood lymphocyte culture systems; 4) effectively inhibit the release of inflammatory mediators by exerting chemotaxis, thereby reducing inflammation in patients and improving clinical symptoms  maturation. symptoms , 47, 48.The safety and efficacy of mesenchymal stem cell therapy for autoimmune diseases, such as Systemic Lupus Erythematosus (SLE) and Multiple Sclerosis (MS), have been confirmed through multiple studies –51. SeveBased on the above survey research, we conducted a preclinical study of hUCMSCs for the treatment of AS. We established a PGISp model by using a proteoglycan adjuvant. After three intraperitoneal injections of proteoglycan, some or all of the limbs and soles of the PGISp mice became thickened, red and swollen, and lameness, scoliosis, and arched back were formed. CT scanning and pathological examination showed blurred edges of spinal joints, sclerosis in the proximal joint area, mild narrowing of the joint space, and symptoms such as necrosis and dissolution of chondrocytes in the fibrous annulus of the intervertebral disc, and the formation of visible and obvious cracks. The blood levels of pro-inflammatory factors such as TNF-α and INF-γ were increased, as well as the spleen coefficient and thymus coefficient, and the proportion of Th1 cell subsets in the spleen was increased. However, 40% of the mice did not show or showed extremely mild symptoms. Mice treated with hUCMSCs had milder symptoms, less swelling of limb joints, milder spinal joint lesions such as lumbar spine and sacroiliac bone, lower levels of pro-inflammatory factors in blood, lower spleen coefficient, and decreased proportion of Th1 in the spleen, while the proportion of Th2 cells increased.The thymus and spleen are important immune organs in the body, with the thymus being the central immune organ and the spleen being the peripheral immune organ. Changes in thymus and spleen indices are believed to reflect overall immune function, and abnormal increases in these indices are closely related to immune system overactivation. In the research and exploration of autoimmune diseases such as Rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) –54, immuTo study the survival time and distribution position of hUCMSCs in mice, we conducted a tissue distribution study in normal mice using oxine labeling to track 89Zr-labeled human umbilical cord mesenchymal stem cells (hUCMSCs) after tail vein injection. Micro-PET/CT scans were performed at five time points: 1 hour, 24 hours, 72 hours, 168 hours, and 240 hours  and is currently in the recruitment stage of clinical trials.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Dongguan Changan Hospital Ethics Committee (2019507150102035). The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by Shanghai Institute of Pharmaceutical Industry (SYXK(沪)2019-0027).NQ, HWW, and HW designed, directed and supervised the entire study. CJ, QLv, and QLiu performed most of the experiments; FX, ZW, and HPZ analyzed all results; DS and HYZ wrote the manuscript. All authors contributed to the article and approved the submitted version.


Background: Autism Spectrum Disorder (ASD) can be identified by a general tendency toward a reduction in the expression of low-band, widely dispersed integrative activities, which is made up for by an increase in localized, high-frequency, regionally dispersed activity. The study assessed ASD children and adults all possessing retained primitive reflexes (RPRs) compared with a control group that did not attempt to reduce or remove those RPRs and then examined the effects on qEEG and brain network connectivity. Methods: Analysis of qEEG spectral and functional connectivity was performed, to identify associations with the presence or absence of retained primitive reflexes (RPRs), before and after an intervention based on TENS unilateral stimulation. Results: The results point to abnormal lateralization in ASD, including long-range underconnectivity, a greater left-over-right qEEG functional connectivity ratio, and short-range overconnectivity in ASD. Conclusions: Clinical improvement and the absence of RPRs may be linked to variations in qEEG frequency bands and more optimized brain networks, resulting in more developmentally appropriate long-range connectivity links, primarily in the right hemisphere. Autism spectrum disorder (ASD) is a complex neurodevelopmental syndrome characterized clinically by language impairment, lessened social interaction, varied cognitive deficits, and behavioral stereotypes ,5,6,7,8.Individuals with ASD frequently struggle with interaction and social communication and demonstrate repetitive or restricted interests or actions. ASD subjects may also have unique learning, movement, and attention styles. These attributes can make life quite difficult.Although the methods and experimental paradigms employed in these investigations differed, quantitative EEG (qEEG) has been used to evaluate autistic children, and it has revealed several consistently aberrant EEG characteristics in these individuals ,12.Quantitative EEG approaches may be able to identify and quantify ASD-related dysfunctions in certain brain regions and in the regulation of neuronal activity. Asymmetry of the hemispheres, functional connectivity, and spectral power have attracted the most attention as potential ASD EEG indicators. In their review of resting-state EEG investigations in ASD, Wang and colleagues  identifiCantor et al.  and CantOne early indicator of delayed or aberrant cortical maturation, and consequently, and other neurobehavioral disorder including ASD, may be retained primitive reflexes (RPRs) . SuckingHowever, to our knowledge, the mechanism by which primitive reflexes are suppressed in neurobehavioral disorders has not yet been established. We hypothesize that making use of these reflexes enhances sensory input and feedback to the nervous system, which in turn encourages synaptogenesis and neuroplasticity in more rostral and complex brain regions ,27. The The goal of the current study was to evaluate autistic children before and after an intervention, compared to a control group, using qEEG spectral and functional connectivity analysis, with an approach that can inhibit preserved basic reflexes .Male and female participants were recruited from the Institute for Neurology and Neurosurgery in Havana, Cuba, tested and treated in the Clinical electrophysiology laboratory. Demographic data of the participants was recorded and included age, gender, Apgar score, birth weight, gestational age, and whether the birth was natural or by Caesarian section. Also collected were the IQ, overall health, grade level, and sidedness of the participants. Three age groups included 10 ASD participants each  reflecting different normative stages of development into adulthood. Additional selection criteria may be found in https://www.researchgate.net/publication/372345066_Cognitive_Effects_of_Retained_Primitive_Reflex_Reduction_by_Lateralized_Stimulation-Data, accessed on 19 July 2023).The participants included 50 males and 10 females whose mean age was 15.8 (S.D. 7.21). The groups’ characteristics can be found in a data depository at , or brain surgery, nor did they have any neurologic abnormalities, outside those specifically linked to autism. None of the participants demonstrated any genetic disorder, metabolic illness, vascular disorder, or history of cancer, and they could not be breastfeeding or pregnant. Participants were free of drugs or in drug treatment recovery. Control group participants demonstrated no manifestations of any RPRs.The Institute of Neurology and Neurosurgery Ethics Committee and the IRB for the University of Haifa approved the research projects. The relatives or responsible parties of the study participants gave their informed consent.Participants were studied in a facility with a temperature range of between 24 and 26 °C, with noise reduction, and dimmed lights. Participants under the age of majority had a parent or guardian in loco parentis present during all recording sessions, and in all cases, so too was the clinician in charge and the EEG technologist. To improve teamwork, participants were situated in comfortable chairs and familiarized with the room and experimental set prior to the experimental session.The following reflexes were tested clinically and included the Asymmetric Tonic Neck Reflex (ATNF), Symmetric Tonic Neck reflex (STNR), Spinal Galant, Babinski, Palmer Grasp, Rooting, and Tonic Neck Reflex (TNR). RPRs were defined as the presence of two or greater RPRs.The following standardized testing was performed: Wechsler Individualized Achievement Testing-III (WIAT-III) , WechsleAll children in Cuba with ASD are routinely tested by brainstem auditory evoked potentials to rule out auditory impairment. Cranial nerve function was also included as part of the evaluation protocol, as was the case in the patients evaluated in this report.EEG was recorded from 19 standard locations over the scalp according to the 10–20 system: Fp1, Fp2, F3, F4, F7, F8, T3, T4, C3, C4, P3, P4, T5, T6, O1, O2, Fz, Cz, and Pz. Gold-cup scalp electrodes applied with collodion were fixed, after careful skin cleaning, using a conductor paste and connected to the input box of the digital EEG system . Leads were employed, using linked ears as a reference. Technical parameters were: gain 20,000, pass-band filters 0.1–70 Hz, “notch” filter at 60 Hz, a noise level of 2 µV (mean root square), sampling frequency 200 Hz, and electrode–skin impedance never higher than 5 KΩ. To make eye movement artifacts easier to spot in the EEG records, electrodes were positioned across the superior and inferior rims. For each experimental portion, experts visually reviewed the recordings to choose artifact-free EEG segments with a total duration of no less than 65 s. These segments were afterwards converted to an ASCII file and saved for additional quantitative analysis.Every single one of the 19 leads’ EEG values underwent off-line pre-processing consisting of (a) removing the sequence’s mean value from the EEG values to lessen the impact of the D.C. component of the time series; (b) applying a nonlinear median filter (three-points window) to weed out outliers or abnormally high amplitude values ; (c) conqEEG samples contained in the previously specified ASCII files were imported by a custom-tailored Matlab version 7.10.0.499 R2010a software application . The software included several steps, including the estimation of the power spectral densities (PSD) for each EEG lead, the calculation of several spectral indices and coherence, and the output of these results to a Microsoft Access database.Activity in the left anterior region was evaluated by recordings obtained through the EEG leads Fp1, F3, and F7. The right anterior area was composed of the Fp2, F4, and F8 derivatives. Activity over the central left region was evaluated by activity recorded from the C3 and T3 leads, and right central region activity was reflected in activity recorded from C4 and T4 leads. The posterior left region activity was reflected by the P3, O1, and T5 leads, and a posterior right region activity was integrated with the collective activity of P4, O2, and T6 leads; a midline region was defined that included the Fz, Cz, and Pz leads.The first 12,288 samples of the EEG values of each EEG lead were submitted to a spectral analysis implemented by the Welch periodogram method, using a Hann window to avoid a leakage effect as much as possible. This approach produced 23 successive windows out of the 1024 samples (5.12 s), which overlapped every 512 samples. The estimated PSD findings for each discrete spectral frequency were then averaged to get the global smoothed spectrum for each EEG lead. This procedure had a spectral resolution of 1/5.12 s, or roughly 0.1953125 Hz. The D.C. or zero frequency and the first six discrete frequencies were eliminated, and the remaining discrete frequencies were subjected to integration within the ranges chosen for the various EEG bands. We selected 12 for the delta EEG band, which ranges from 1.17 to 3.5 Hz; 22 for theta, 3.5 to 7.5 Hz; 19 for alpha, 7.5 to 11 Hz; 21 for sigma, 11 to 15 Hz; 53 for alpha, 15 to 25 Hz; and 154 for gamma. The conventional method was also used to measure the PSD for the EEG in each band in normalized units, determining the percentage of the PSD corresponding to the total PSD of the entire spectral range ,45.The theoretical analysis of graphs was performed employing the timing matrix of all possible electrode combinations. We used programs developed in our laboratory employing MatLab (R2008b) for these analyses. The parameters that were evaluated were the mean route length  and the clustering coefficient , which both measure the effectiveness of communication in a network. The level of connectivity that the two neighbors or the local subnet share is reflected in the local efficiency. The global efficiency shows the degree of connectedness between any two nodes, and the global connectivity reflects the degree of global connectivity between each channel and the other metrics.For the statistical processing, we used the Statistica v. 8.0. program . By using log-natural transformations, the absolute PSD values were given a normal distribution, which was then verified using the Shapiro-W Wilk’s test. Based on our prior experience, normalized PSD data did not require adjustment to achieve normalcy distributions .A permutation test implemented in Matlab was used to assess functional connectivity (FC), calculating the differences between the two groups’ synchronization matrices. Each matrix element in the FC symbol represents the edge strength or functional relationship between the associated groups, with the rows and columns standing in for nodes. The assembling of the element matrices into the global coefficient matrix is plainly shown by the connection matrix.t-test for independent samples was applied. A significance of p < 0.05 was set in all cases.To analyze the differences between the controls and patients, the Comparing the control vs. the autism groups before the treatment, we found significant increments of the delta, theta, alpha, and gamma bands, with reduced activity of alpha frequencies.Nonetheless, the most interesting finding was when ASD patients were compared before and after treatment, represented in Comparing the control vs. the autism groups before the treatment, we found a significant increment of the delta, theta, alpha, and gamma bands, with reduced alpha frequency activity. This pattern is known as the “U Shape” in ASD patients. Nonetheless, the most important findings were related to the comparison before and after treatment.The most common method for characterizing resting EEG involves decomposing oscillatory patterns into frequency bands with physiological features. Clinically important EEG frequency ranges typically span from 0.3 to 70 Hz. We concentrated on five frequency bands for the purposes of this paper: delta, theta, alpha, alpha, and gamma.Clinical and cognitive neuroscience are becoming increasingly interested in these historically recognized frequency bands, which are considered to control a variety of brain activities. The event-related slow waves seen during activities that assess attention and salience are thought to be caused by delta . Most ofAccording to some experts, this U-shaped profile in ASD could be partially explained by aberrant gamma-aminobutyric acid (GABAergic) tone in inhibitory circuitry, which can affect the brain’s functional and developmental plasticity ,52.Dendritic GABAergic inhibitory dysfunction has been connected to gamma band activity observed in EEG seizure recordings ,53,54. HDuring normal brain development, synaptogenesis allows for ongoing changes in both short- and long-range neural circuitry, weakening functional connections between nearby brain regions, while bolstering connections between various regions of the brain . DevelopResearchers may be able to evaluate the changes in brain function between people with and without ASD by using qEEG coherence to look at electrical connection patterns. Our findings, particularly that of a high left-to-right qEEG functional connectivity ratio, indicate long-range underconnectivity and short-range overconnectivity with concurrent aberrant lateralization in ASD. Based on these results, we believe that ASD is characterized by a general trend toward the underexpression of low-band, widely distributed integrative processes, which is offset by localized, high-frequency, regionally specialized, and segregated processes.Hence, clinical improvement and the disappearance of RPRs in our cases might be due to a new balance in qEEG frequency bands and a more optimized organization of the brain networks, improving long-range connectivities, mainly in the right hemisphere.


Cardiovascular diseases are responsible for a significant morbimortality ratearound the world. Due to the characteristics of their work, health careprofessionals, including veterinarians, are more prone to present this typeof pathology.To determine the level of cardiovascular risk using different scales in agroup of veterinarians.A descriptive and cross-sectional study of 610 Spanish veterinarians wasconducted to assess cardiovascular risk scores, including 14 overweight andobesity scales, six fatty liver scales, six cardiovascular risk scales, fouratherogenic indices, and three metabolic syndrome scales.The prevalence of obesity among women was 7.95%, and 17.53% among men.Hypertension was present in 15.23% of women and 24.68% of men. Dyslipidemiaaffected 45% of women and 58.64% of men. The prevalence of metabolicsyndrome according to the International Diabetes Federation criteria wasslightly over 10% while 10.90% of women and 14.93% of men showed moderate tohigh values on the Registre Gironí del Cor scale.There is a moderate to high level of cardiovascular risk among veterinariansin this group. According to data from the World HealthOrganization (WHO), each year more people die from this disease than from any otherpathology.3 theDyslipidemia Obesity and Cardiovascular Risk in Spain (DORICA),4 or the Spanish Cardiovascular RiskEquation (ERICE)5 in Spain. Otherscales quantify the risk of suffering a fatal cardiovascular event over a timeperiod, as is the case with the Systematic Coronary Risk Evaluation (SCORE)scale.6 In recent years,other direct risk scales have been developed based on the aforementioned scales,such as the heart age and vascular age scales. In addition to the scales that assessdirect CVR, there are others that do so indirectly, such as scales for overweightand obesity, atherogenic indices, metabolic syndrome, or fatty liver disease. Allthese scales will be discussed more extensively in the methodology section.There are many scales for assessing cardiovascular risk (CVR), some of which quantifyit directly by determining the risk of suffering a cardiovascular event over a timeperiod, usually 10 years, as is the case, for example, of the Registre Gironídel Cor (REGICOR),7 but to the bestof our knowledge, no study has been conducted with veterinarians. As a result oftheir work in the care of suffering animals, many of these professionals are at riskof compassion fatigue and occupational stress,8 which is an important trigger for cardiovasculardisease.Many studies have determined CVR in both the general and the workingpopulationThe objective of this study was to determine the level of CVR in Spanishveterinarians and the influence of age, sex, and tobacco consumption on thisrisk.A descriptive, cross-sectional study was performed on 649 Spanish veterinariansbetween January 2019 and December 2019. Thirty-nine of them were excluded , thus610 veterinarians remained. A total of 302 (49.51%) were women (mean age 42.51years), and 308 (50.49%) were men (mean age 43.35 years).Participants were selected among workers who attended periodic occupational medicalcheckups in companies in different regions of Spain.Consent to participate in the study and to use of the data forepidemiological purposes.No previous cardiovascular events.Following standardization of measurement techniques, healthcare professionalsfrom the different occupational health units that participated in the studyconducted anthropometric, clinical, and analytical assessments.The following parameters were included in the assessment:Weight (in kg) and height (in cm) were determined with a SECA 700 scaleand a SECA 220 measuring rod.Abdominal waist circumference (in cm) was measured with a SECA 200 tapemeasure. For the waist-to-height ratio, the cut-off point was set at0.50.Blood pressure was measured in the supine position with a calibratedOMRON M3 automatic sphygmomanometer and after a 10-minute rest period.Three determinations were made at 1-minute intervals, obtaining the meanof the three. Hypertension was considered when the values were equalto/higher than 140 mmHg systolic or 90 mmHg diastolic blood pressures orif the worker was receiving antihypertensive treatment.Blood glucose, total cholesterol and triglycerides were determined by peripheralvenipuncture after fasting for at least 12 hours. Glycemia, total cholesteroland triglycerides were determined by automated enzymatic methods. HDL wasdetermined by precipitation with dextran sulfate Cl2Mg and LDL was calculatedusing the Friedewald formula (provided that triglycerides were less than 400mg/dL). All the above values are expressed in mg/dL. Friedewald’ formula:The following were considered abnormal values: 200 mg/dL for cholesterol, 130mg/dL for LDL, and 150 mg/dL for triglycerides or if they were under treatmentfor any of these factors.Blood glucose values were classified according to the criteria of the AmericanDiabetes Association (ADA) and are considered to be diabetes at 126 mg/dL or ifindividuals are receiving hypoglycemic treatment.2 or more.Body mass index (BMI) was calculated by dividing weight by height in meterssquared. Obesity was considered to be 30 kg/m9 Equation CórdobaEstimator Body Fat (ECORE-BF),10 Palafolls’s formula,11 Deurenberg’s formula,12 relative fat mass,13 visceral adiposity index (VAI),14 dysfunctional adiposityindex,15 bodyroundness index,16 bodysurface index (BSI),17conicityindex,18 body shapeindex (ABSI),19 and normalizedweight-adjusted index (NWAI).20 To measure insulin resistance we used the followingindices: triglyceride glucose index, triglyceride glucose index-BMI,triglyceride glucose index-waist.21 For cardiometabolic risk: waist triglycerideindex,22cardiometabolic index,23 fattyliver index,24 hepaticsteatosis index (HSI),25Zhejian University index (ZJU),26 fatty liver disease index (FLD),27 BARD scoring system,28 Framingham steatosis index,29 and lipid accumulationproduct.30 Thedifferent scales are shown in We used the following scales to estimate overweight and obesity: ClínicaUniversitaria de Navarra Body Adiposity Estimator (CUN BAE),The atherogenic indexes determined were:Total cholesterol/HDL (considered as high > 5 in men and > 4.5 inwomen), LDL/HDL and triglycerides/HDL (high > 3);Triglycerides/HDL (high >3);Total cholesterol-HDL (high >130).Cardiometabolic indicators:31Hypertriglyceridemic Waist Phenotype;Waist > 102 cm (men) > 88 cm (women); andTriglycerides > 150 mg/dL or treatment of hypertriglyceridemia.32Metabolic syndrome was determined using three models:National Cholesterol Educational Program Adult Treatment Panel III (NCEPATP III), which considers metabolic syndrome when three or more of thefollowing factors are present: waist circumference is greater than 88 cmin women and 102 in men; triglycerides > 150 mg/dL or specifictreatment for this lipid disorder; blood pressure > 130/85 mmHg; HDL< 40 mg/dL in women or < 50 mg/dL in men or specific treatment isfollowed, and fasting blood glucose > 100 mg/dL or specific glycemictreatment.The International Diabetes Federation (IDF) model, which considers thepresence of central obesity necessary, defined as a waist circumferenceof > 80 cm in women and > 94 cm in men, in addition to two of theother factors mentioned above for ATP III .The JIS model, which follows the same criteria as NCEP ATPIII but thewaist circumference cut-off points start at 80 cm in women and 94 cm inmen.33 ischaracterized by high triglyceride concentrations (> 150 mg/dL), low HDL(< 40 mg/dL in men and < 50 mg/dL in women), and normal or slightly highLDL. If LDL values are high (> 160 mg/dL) we speak of lipid triad.Atherogenic dyslipidemia3 andassesses the risk of suffering a cardiovascular event over a 10-year period. Itcan be applied between 35 and 74 years of age. The risk is considered moderateat 5% or above and high at 10% or above.6REGICOR is an adaptation of the Framingham scale for the Spanishpopulation6 to determine the risk of suffering a fatalcerebrovascular event over a 10-year period. It can be calculated between 40 and65 years of age. Moderate risk is defined at 4% and high risk at 5% orabove.The SCORE scale for low-risk countries is used in Spain4 which was conducted in a very large Spanishpopulation base. It estimates the risk of suffering a fatal or non-fatalcerebrovascular event over a 10-year period. The tables are applied to peoplebetween 25 and 64 years of age. To calculate risk one assesses age, sex,smoking, diabetes, systolic and diastolic blood pressure, total cholesterol, andHDL-c. To classify the level of risk with the DORICA tables, the cut-off pointsrecommended by the authors determine that risk is moderate when it is between 10and 19%, high from 20%, and very high if it exceeds 39%.DORICA. This scale is based on the DORICA study,5It estimates the risk of suffering a fatal or non-fatal cerebrovascular eventover a 10-year period. The tables apply to individuals between 30 and 80 yearsof age. To calculate risk, age, sex, smoking, diabetes, systolic blood pressure,antihypertensive treatment, and total cholesterol are taken into account. Toclassify the level of risk with the ERICE tables, the cut-off points recommendedby the group responsible for the study were used: risk is considered moderate ifit exceeds 5%, moderate-high if it is between 15 and 19%, high if it is between20 and 39%, and very high if it exceeds 39%.ERICE is based on seven population-based cohort studies conducted in differentgeographical areas of Spain.34 we need age, sex, HDL-c, total cholesterol,systolic blood pressure values, antihypertensive treatment, smoking, anddiabetes. It can be calculated from the age of 30 years.To calculate vascular age with the Framingham model35 the variables analyzed were age, sex, systolicblood pressure, smoking and total cholesterol. As with the scale from which itis derived, it can be calculated in individuals between 40 and 65 years ofage.In turn, to calculate vascular age with the SCORE modelAn interesting concept applicable to both vascular ages is avoidable lost lifeyears (ALLY), which can be defined as the difference between biological age andvascular age.A smoker is considered to be any person who has regularly consumed at least onecigarette/day  in the lastmonth, or has quit smoking less than 12 months ago.2 test(with correction of Fisher’s exact statistic when conditions required) andStudent’s t test for independent samples. For multivariateanalysis, binary logistic regression was used with the Wald method, withcalculation of the odds ratio and the Hosmer-Lemeshow goodness-of-fit test.Statistical analysis was performed with SPSS version 27.0, and statisticalsignificance was set at 0.05.A descriptive analysis of the categorical variables was conducted, calculatingthe frequency and distribution of responses for each of them. For quantitativevariables, the mean and standard deviation were calculated, and for qualitativevariables, the percentage was calculated. The bivariate association analysis wasperformed using the XThe study was approved by the Research Ethics Committee of the Illes Balearshealth area no. IB 4383/20. All procedures were performed in accordance with theethical standards of the institutional research committee and with the 2013Declaration of Helsinki. All patients signed written informed consent documentsprior to participation in the study.Clinical variables show statistically higher values in men, except for totalcholesterol, LDL cholesterol, and AST. Smoking prevalence is high, especially amongwomen. The complete data are presented in In this study, men showed higher mean values on all scales except those measuringbody fat percentage, which are higher in women. The rest of the scales analyzed wereoverweight and obesity, metabolic syndrome, fatty liver, and CVR scales as well asatherogenic indices that showed higher mean values in men. The data are presented inAccording to our analysis of the prevalence of high scores on a variety of CVR-related scales, in most cases this values were higher among men, while the mostunfavorable values were found only in women with some scales that assess body fat,such as CUN BAE, ECORE-BF, Relative Fat Mass and Deurenberg’s formula, metabolicsyndrome with the IDF criteria, Lipid triad, and atherogenic index totalcholesterol/HDL. The complete data can be found in In the multivariate analysis using binary logistic regression, being male, aged 50years and above, and tobacco consumption were established as covariates. We found that the prevalence of altered values of the different scales that directlyor indirectly assess CVR is moderate to high, with men generally suffering themost.Since we haven’t found any studies in the literature that measure the level of CVR inveterinarians, we will compare our results with those of other health careprofessionals.36 and showed, aswe did, a high prevalence of these conditions. Another study involving youngerhealth care workers37 showedsimilar results, and a third study,38 including different healthcare professionals, such asphysicians and dentists, also found similar results. A more recent study,39 found the prevalence of obesity inemployees of a teaching hospital in Brazil to be 55.5%, similarly to our study.Numerous national studies have assessed the prevalence of arterial hypertension,dyslipidemia, and obesity in health professionals, the first of which focused onmedical personnel40 As in ourstudy, this study showed a high prevalence of atherogenic indices.According to a study involving almost 2000 Mexican health care workers, thedeterminants of atherogenic indices were male sex, increasing age, increasingwaist-to-hip ratio, overweight and obesity, and working as a physician. Theoccupational category of the physician added risk factors such as stress and adversepsychosocial working conditions, which can potentiate cardiovasculardisease.37 154 health careworkers were found to have moderate and high values on the Framingham scale of 10.3and 1.3%, respectively, which are in line with our findings. Compared to our study,this study had higher prevalence of hypertension (33%), overweight and obesity(66%), and dyslipidemia (33%).In another studyAmong the strengths of the study are the large sample size, more than 700veterinarians, the wide variety of scales used, including 14 scales that assessoverweight and obesity, 6 for fatty liver, 6 for CVR, 4 atherogenic indices, and 3for metabolic syndrome, and to our knowledge, it is the first study that addressesthe level of CVR in veterinarians, which could make it a reference for subsequentstudies in this group.The most important limitation is that it was conducted in a specific geographicalarea, which makes it difficult to extrapolate the results to other countries.The prevalence of scales related to CVR in veterinarians can be consideredmoderate-high, especially if we take into account that the average age of theseworkers is not very high.