Studies have compared the safety and accuracy of robot‐assisted techniques for inserting conventional open pedicle screws for spinal surgery. However, no relevant studies have confirmed that robot‐assisted percutaneous screw placement is better than fluoroscopic percutaneous screw placement for the treatment of thoracolumbar fractures. This study compared the accuracy and safety of TiRobot‐assisted percutaneous pedicle screw placement with those of the fluoroscopy‐assisted percutaneous technique for the treatment of thoracolumbar fractures.2 test, or Fisher's exact test.This retrospective study included 126 patients with thoracolumbar fractures who underwent percutaneous pedicle screw placement. Sixty‐five patients were treated with the TiRobot‐assisted technique and 61 patients were treated with the fluoroscopy‐assisted technique. Patient demographics, accuracy of screw placement , screw insertion angle, radiation exposure, surgical time, intraoperative blood loss, length of hospital stay, incision length, hospital expenses, surgical site infection, and neurological injury of the TiRobot‐assisted and fluoroscopy‐assisted groups were compared using Student's t‐test, Pearson χvs 20.55° ± 5.15°), larger incision length (13.86 ± 1.24 cm vs 12.77 ± 1.43 cm), and higher hospital expenses (69061.55 ± 7166.60 yuan vs 59383.85 ± 5019.64 yuan) than the fluoroscopy‐assisted group. There were no significant differences in the intraoperative blood loss, length of hospital stay, and rates of surgical site infection and neurological injury in both groups (p > 0.05). However, the TiRobot‐assisted group had significantly better surgical times, radiation times, and radiation exposure than the fluoroscopy‐assisted group (p < 0.05).A total of 729 screws were placed . In the TiRobot‐assisted group, 82.8% of screws were optimally positioned (grade A); however, the placement grades of the remaining screws were categorized as grade B (13.3%), grade C (3.2%), and grade D (0.5%). In the fluoroscopy‐assisted group, 66.7% of the screws were optimally positioned (grade A); however, the placement grades of the remaining screws were categorized as grade B (21.4%), grade C (7.6%), grade D (3.6%), and grade E (0.5%). The proportion of clinically acceptable screws (grade A or B) was greater in the TiRobot‐assisted group than in the fluoroscopy‐assisted group. Additionally, the TiRobot‐assisted group had a significantly larger mean screw insertion angle (22.27° ± 5.48° Percutaneous TiRobot‐assisted pedicle screw placement is a safe, useful, and potentially more accurate alternative to the percutaneous fluoroscopy‐assisted technique for treating thoracolumbar fractures. Traditional percutaneous pedicle screw placement has advantages of less trauma, less bleeding, less pain, and rapid recovery. However, due to insufficient exposure, the risk is high for these operations, and there is a significant learning curve, which is mainly reflected by operation time, radiation exposure and accuracy of screw placement. Compared with traditional percutaneous fluoroscopy‐assisted technique, percutaneous TiRobot‐assisted pedicle screw placement could improve the accuracy of pedicle screw placement, reduce radiation exposure, reduce operation time. Ltd., and received approval from the Chinese Food and Drug Administration in 2016. During this study, we analyzed the safety of the TiRobot for pedicle screw implantation and assessed the radiation exposure of patients and surgeons during fluoroscopy‐assisted pedicle screw placement procedures.This study was approved by the Institutional Review Board of Sichuan Provincial People's Hospital (no.289 in 2019). Patients were selected based on the following inclusion criteria: fresh single‐segment thoracolumbar fractures; indications for thoracolumbar posterior fixation; and a minimum of 6 months of follow‐up. In contrast, the exclusion criteria were as follows: pedicle dysplasia; neurological symptoms; tumors or severe osteoporosis; and incomplete data or difficulty accepting the diagnosis and treatment. This retrospective chart review included the records of 126 patients from January 2018 to August 2020. TiRobot‐assisted (65 cases) or fluoroscopy‐assisted (61 cases) treatment was chosen by patients after the surgeons presented the details of these methods. All participants provided written informed consent before enrollment.After general anesthesia, the patient was maintained in the prone position on a radiotransparent operating table. A C‐arm fluoroscopic machine was used to mark the pedicle projection of the injured vertebrae and the upper and lower vertebrae. A 2‐cm vertical incision was made in the pedicle projection area. Under the guidance of C‐arm fluoroscopy, a puncture needle was inserted to reach the outer edge of the superior articular process of the pedicle. After confirming the position of the puncture needle in the anteroposterior and lateral views using C‐arm fluoroscopy, the stylet was removed from the puncture needle and a guide wire was inserted. Then, the cannulated screw was tapped and inserted directly along the guide wire using a soft tissue expander to expand the channel. The remaining pedicle screws were similarly inserted. Subsequently, the connecting rod was installed and secured after the reduction of the injured vertebral body.We used the TiRobot system, a C‐arm X‐ray system , and internal fixation implants . The TiRobot system includes a robotic arm, optical tracking system, robotic workstation, and navigation toolkit.After general anesthesia, the patient was positioned in a manner similar to that used during the fluoroscopy‐assisted procedures. The C‐arm was used to locate the fractured vertebra segment. The percutaneous reference tracker (patient tracker) was placed in the spinous process superior to the instrumented segment. A calibrator was held by the robotic arm on the skin surface of the injured vertebra, and three‐dimensional images were acquired using C‐arm scanning. The image data were transferred to the robot workstation. Subsequently, we planned the surgery, issued the appropriate commands according to the plan, and moved the robot arm to the designated position. The sleeve guider was placed at the end of the robot arm. A 2‐cm incision was made using positioning directed by the sleeve guider. Next, a guide wire was drilled into the vertebrae. The screw placement and reduction procedures were the same as those used for the fluoroscopy‐assisted group. The TiRobot components are shown in Figures Three‐dimensional reconstruction performed with postoperative computed tomography (CT) were used to assess the accuracy of pedicle screw placement. The accuracy of screw placement was evaluated according to the Gertzbein and Robbins scalep < 0.001). When the two evaluation results differed, the final results were determined through a discussion with the first corresponding author of this article.Two senior spine surgeons who were blinded to the treatment group evaluated the accuracy of pedicle screw placement. The consistency test results were substantial  during the procedure. Additionally, the cumulative radiation exposure for the surgeon was recorded using adosimeter  attached to the surgeon's chest outside the lead apron and read by a blinded independent radiologist.We collected data regarding patient age, sex, body mass index (BMI), number of screws per case, and fracture segment. We also noted and evaluated data regarding the surgical time (from skin to skin), intraoperative blood loss, hospital stay length, incision length, hospital expenses, surgical site infection, and neurological injury.2 test or Fisher's exact test was performed. The level of statistical significance was set as α = 0.05.SPSS 19.0  was used for the analysis. Quantitative data are presented as the mean ± standard deviation. Statistical significance was tested using Student's t‐test when Gaussian distribution was expected. For categorical variables, the Pearson χ2) were treated with the TiRobot‐assisted technique. The number of screws per case in the TiRobot‐assisted group was 5.76 ± 0.42. Fracture segments included T11 (16 cases), T12 (27 cases), and L1 (22 cases). The fluoroscopy‐assisted technique was performed for 61 patients , and the number of screws per case in the fluoroscopy‐assisted group was 5.81 ± 0.38. The fracture segments included T11 (14 cases), T12 (24 cases), and L1 (23 cases). There were no significant differences in age, sex, BMI, screws per case, and fracture segments in both groups . Additionally, the mean screw insertion angle was significantly greater in the TiRobot‐assisted group than in the fluoroscopy‐assisted group . Data regarding intrapedicle accuracy are summarized in Table A total of 729 screws were placed . In the TiRobot‐assisted group, 82.8% of the screws were optimally positioned (grade A). The placement of the remaining screws was categorized as grade B (13.3%), grade C (3.2%), or grade D (0.5%). In the fluoroscopy‐assisted group, 66.7% of the screws were optimally positioned (grade A). The placement of the remaining screws was categorized as grade B (21.4%), grade C (7.6%), grade D (3.6%), or grade E (0.5%). The proportion of clinically acceptable screws (grade A or B) was greater in the TiRobot‐assisted group than in the fluoroscopy‐assisted group . The cumulative radiation dose for the surgeon was significantly less in the TiRobot‐assisted group (14.5 ± 2.48 μSv) than in the fluoroscopy‐assisted group . The cumulative radiation dose for the patient was also significantly less in the TiRobot‐assisted group (170.38 ± 43.07 cGy cm−2) than in the fluoroscopy‐assisted group . Data regarding radiation exposure are shown in Table The TiRobot required additional three‐dimensional 3D) scans of the robotic system for intraoperative registration, with an average time and average radiation exposure of 63.2 s and 68.6 cGy cmD scans op = 0.383). Patients in the TiRobot‐assisted group experienced less blood loss (57.84 ± 19.96 mL) than those in the fluoroscopy‐assisted group (60.00 ± 20.24 mL); however, this difference was not significant (p = 0.549). The operative time in the TiRobot‐assisted group (99.90 ± 22.82 min) was significantly shorter than that in the fluoroscopy‐assisted group . The incision length in the TiRobot‐assisted group (13.86 ± 1.24 cm) was longer than that in the fluoroscopy‐assisted group . Additionally, the hospital expenses were higher in the TiRobot‐assisted group  than in the fluoroscopy‐assisted group .The hospital stay length tended to be shorter in the fluoroscopy‐assisted group (5.75 ± 1.42 days) than in the TiRobot‐assisted group (5.98 ± 1.52 days); however, this difference was not significant  remains the most commonly used robot for spinal surgeryet al.et al.Hyun et al.p = 0.017). Additionally, Feng et al.p < 0.05). Similar screw placement accuracy results have been reported using the TiRobot‐assisted technique and traditional open techniquesAt the time of this research, 21 leading medical units in China were performing surgery and research studies using the TiRobot. Because of its automatic robotic arm, the TiRobot‐assisted procedure can be used for all levels of spinal instrumentation and pelvic and limb surgeriesDuring this study, patients who underwent percutaneous screw placement were categorized into two groups. The accuracy of implantation in the TiRobot group was 96.2%, which was significantly higher than that in the fluoroscopy‐assisted group (88.1%). Because a high‐precision incisional design can reduce the effects on surrounding muscles and adipose tissue, some studies have proposed that the robot can achieve a better screw insertion angleLimiting radiation exposure during surgery is important because excessive radiation can increase the risk of malignancieset al.et al.The Mazor robot is one of the most widely used robots for spinal surgery; however, it offers few advantages over the traditional open technique in terms of radiation exposureet al.Radiation exposure was mainly dependent on the evaluation method, measurement equipment, BMI of the patient, and robot typep = 0.484).During this study, the total expenses for the TiRobot‐assisted group were higher by approximately 10,000 yuan at our hospital; this may be a major limitation to the wider clinical application of the TiRobotAccording to the radiological indicators of this study, and compared with the traditional percutaneous fluoroscopy‐assisted technique, the TiRobot‐assisted technique had obviously better screw placement accuracy and screw insertion angles, which are advantages that reduce complications such as misplaced screws and strengthening the stability of the screws. Furthermore, the TiRobot‐assisted technique could reduce the surgical time and radiation exposure for surgeons and patients.This study had some limitations. The follow‐up time of this study was short, and the postoperative radiological view might not always show the smooth flow of the procedure. The clinical conditions of the patient, such as postoperative chronic pain, disability, and ability to return to employment, are equally importantTiRobot‐assisted percutaneous pedicle screw placement is a safe, useful, and potentially more accurate alternative to the fluoroscopy‐assisted percutaneous technique for treating thoracolumbar fractures. However, the TiRobot system has certain disadvantages, such as it high cost, extra preparation time, and insufficient real‐time feedback system (intraoperative fluoroscopy is still needed to confirm the position of the guidewire). In the future, reducing the cost, simplifying the robot preparation process, and optimizing the feedback mechanism are important aspects of research that must be explored.The authors declare that they have no competing interests.All authors listed meet the authorship criteria of the latest guidelines of the International Committee of Medical Journal Editors and are in agreement of this manuscript.Shu Lin contributed to the conception of the study. Jiang Hu and Liu‐yi Tang performed the experiment. Shu Lin and Fei Wang contributed significantly to the analysis and manuscript preparation. Shu Lin performed the data analyses and wrote the manuscript. Jiang Hu and Liu‐yi helped perform the analysis and participated in constructive discussions.None.


Styela plicata . Our results demonstrate the presence of immune cells in the endostyle of S. plicata, highlighting that innate immune mechanisms are highly conserved in the phylogeny of the chordates.The endostyle is the first component of the ascidian digestive tract, it is shaped like a through and is located in the pharynx's ventral wall. This organ is divided longitudinally into nine zones that are parallel to each other. Each zone's cells are physically and functionally distinct. Support elements are found in zones 1, 3, and 5, while mucoproteins secreting elements related to the filtering function are found in zones 2, 4, and 6. Zones 7, 8, and 9, which are located in the lateral dorsal section of the endostyle, include cells with high iodine and peroxidase concentrations. Immunohistochemical technique using the following antibodies, Toll‐like receptor 2 (TLR‐2) and vasoactive intestinal peptide (VIP), and lectin histochemistry (WGA—wheat‐germagglutinin), were used in this investigation to define immune cells in the endostyle of Styela plicata.Immune cells positive to TLR‐2 and VIP in the endostyle of Expression of WGA in several zones of endostyle.Use of comparative biology to improve the knowledge about immunology in ascidians. Styela plicata photographed in its natural environment; morphological staining  of a section from the endostyle; immunoistochemical localization of VIP and TLR2 in endostyle. From left to right:  Finally, serial sections (3–5 μm thick) were obtained by a rotary microtome (LEICA 2065 Supercut)  pH 7.4, and incubated in lectin solution for 1 h at room temperature (RT). After rinsing thrice in TBS, the peroxidase activity was visualized by incubation in a solution containing 0.05% 3,30‐diaminobenzidine (DAB) and 0.003% H2O2 in 0.05 mol/L TBS (pH 7.6) for 10 min at RT before dehydration and mounting.For light microscopic examination, serial sections were stained with May‐Grünvald‐Giemsa (04‐081802 Bio‐Optica Milano S.p.A.) and Alcian Blue pH 2.5‐PAS (04‐163802 Bio‐Optica Milano S.p.A) methods  and VIP . Then, the sections were washed in phosphate‐buffered saline (PBS) and incubated for 60 min with a goat anti‐rabbit IgG‐peroxidase conjugate. Peroxidase activity was determined by incubating the sections in a solution of 0.02% diaminobenzidine (DAB) and 0.015% hydrogen peroxide for 1–5 min at room temperature . To compare regularly distributed data, two‐tailed t tests were utilized, and Mann–Whitney rank‐sum tests were used to analyze non‐normally distributed data. Values of p below .05 were judged statistically significant in this order: *p ≤ .01, **p ≤ .02, ***p ≤ .03, ****p ≤ .04, *****p ≤ .05.For each sample, 5 sections and 10 fields were investigated to generate data for statistical analysis. Subjectively, the fields were chosen based on the cell's positivity reaction. The ImageJ software was used to examine each field . The TLR2 immunohistochemistry demonstrated, labeled scattered immunocytes, in the tissues surrounding the endostyle; furthermore, TLR‐2 marked numerous cells of some zones of endostyle with thyroidal and peroxidase activities (zone 5 and 8); the immune cells are often organized in strongly reactive clusters  represent the largest fraction of circulating hemocytes  (Pestarino, Our results show a marked positivity to TLR‐2 in zones 5 and 8 and in circulating immune cells. Ascidia immunocytes can synthesize and secrete humoral lectins involved in the recognition of foreign molecules and modulation of immune responses (Vasta et al., 5S. plicata, highlighting that innate immune mechanisms are highly conserved in the phylogeny of the chordates. TLR2 and VIP play in ascidians a key role in adaptive immune response, as in mammals. Therefore, this animal model allows the study of the cellular and molecular processes that orchestrate innate immune responses. This information can be translated into human immunity, with a particular impact on improving therapeutic strategies for stem cells, tissues, and organ transplantation. In addition, the immune defenses of tunicates have made them a potential source of natural drug resources with great potential for pharmacological applications.In conclusion, our results demonstrating the presence of immune cells in the endostyle of The authors declare no conflict of interest.Conceptualization, Eugenia Rita Lauriano; methodology, Alessio Alesci, Simona Pergolizzi, Patrizia Lo Cascio, Gioele Capillo, and Eugenia Rita Lauriano; formal analysis, Alessio Alesci; investigation, Alessio Alesci and Eugenia Rita Lauriano; resources, Alessio Alesci, Simona Pergolizzi, Patrizia Lo Cascio, Gioele Capillo, and Eugenia Rita Lauriano; data curation, Simona Pergolizzi, Patrizia Lo Cascio, and Gioele Capillo; writing—original draft preparation, Alessio Alesci; writing—review and editing, Alessio Alesci and Eugenia Rita Lauriano; visualization, Alessio Alesci and Gioele Capillo; supervision, Eugenia Rita Lauriano All authors have read and agreed to the published version of the manuscript.


To investigate the relationships between visual function and the retinal and choroid microstructure in idiopathic macular hole patients after surgery.  A prospective study investigated changes in the fundus structure and visual function in 16 macular hole patients before and after surgery. Patients' best-corrected visual acuity (BCVA) and retinal sensitivity were measured by an EDTRS visual chart and microperimetry (MP1), respectively. The thickness of the retina and the blood supply to the retina and superficial choroid were detected by OCTA, and the choroidal capillary blood flow density was analysed with MATLAB. The thickness of the choroid and the aperture size of the macular hole were detected by Heidelberg OCT. P < 0.05) and the central sensitivity of the retina  were significantly improved three months after surgery. The change in BCVA was significantly correlated with the basal diameter (r = 0.677 and P = 0.004), the minimum diameter (r = 0.585 and P = 0.017), the macular hole cystoid height area index (r = −0.618 and P = 0.011), the central macular hole index (r = −0.727 and P = 0.001), the peripheral macular hole index (r = −0.758 and P = 0.001), the central tractional hole index (r = −0.717 and P = 0.002), the peripheral tractional hole index (r = −0.725 and P = 0.001), and changes in the peripheral blood vessel density of the choroid capillary layer (r = 0.585 and P = 0.0017). The change in central retinal sensitivity was correlated with the change in the superficial foveal avascular zone , change in the retinal superficial peripheral blood flow density (r = −0.503 and P = 0.047), change in the deep FAZ (r = 0.599 and P = 0.014), and change in the retinal deep peripheral blood flow density (r = −0.601 and P = 0.014).  Compared with before surgery and one month after surgery, the BCVA (3 months: 0.47 ± 0.27, before: 1.02 ± 0.22, 1 month: 0.66 ± 0.27, and  The morphology of the macular hole as well as changes to the retinal and choroidal microstructure contributes to the recovery of visual function after surgery. The idiopathic macular hole (IMH) seriously affects central vision , 2. SurgIn addition to the assessment of visual acuity, microperimetry (MP1) –12 can bIn patients with the macular hole, visual function and the morphology of the retina and choroid are different from normal eyes –6. AlthoThe sample for this study comprised 16 patients who were diagnosed with the idiopathic full-thickness macular hole in the Wenzhou Medical University Eye Hospital from June 2018 to July 2019. All patients with the macular hole underwent vitrectomy combined with internal limiting membrane peeling. All patients agreed to participate and provided written informed consent. The inclusion criteria were as follows: diagnosed with the idiopathic full-thickness macular hole (Gass classification [The following instruments were used: Heidelberg OCT (Germany), AngioVue OCTA Optovue (USA), IOL-Master (Zeiss), MP-1 , comprehensive refractometer, and EDTRS visual chart. The Optical Laboratory  provided special OCTA image analysis software.Patients underwent comprehensive ophthalmologic examinations, including BCVA measured through EDTRS visual charts, a slit-lamp examination, indirect ophthalmoscopy, and intraocular pressure (IOP) measurement by using a noncontact tonometer (KT-500 Japan) at the preoperative and postoperative visits. Retinal sensitivity was measured by an MP-1 microperimetry examination –12: the OCT images were collected from 9 a.m. to 11 a.m.; clear and full-thickness choroidal images , 13–16 w∗3 mm2 area of the macula. Then, the retinal superficial or deep capillaries (IPL: inner plexiform payer to OPL: outer plexiform layer), the FAZ, and the blood vessel density of the choroid capillary layer  were measured by MATLAB, and OCTA image analysis software developed in the OCT laboratory . Pearson correlation analysis was performed to examine the relationships between the variables.P < 0.05 was considered statistically significant. SPSS 25 software was used for all statistical analyses .The data are expressed as the mean ± standard deviation (SD). The differences between the three groups were analysed by ANOVA (an independent samples P > 0.05). The basal diameter was 885 ± 255 μm (172–1210 μm), the minimum diameter was 529 ± 168 μm (117–769 μm), the DHI was 0.61 ± 0.13 (0.40–0.85), the average height of the retina within 1 mm of the hole was 419 ± 48 μm (309–514 μm), the total cystic height within 1 mm diameter of the hole centre was 610 ± 226 μm (191–1028 μm), and the MCHAI was 0.79 ± 0.54 (0.22–2.61).The sample comprised 16 patients with the macular hole  and 16 patients with normal eyes . There were no statistically significant differences in age and sex between these two groups (P < 0.05. The BCVA of fellow eyes (nonaffected eyes of the macular hole patients) exhibited no statistically significant difference compared to normal eyes, P > 0.05. The retinal sensitivity of the macular hole eyes (0°–4°) gradually increased after surgery; these changes were also statistically significant, P < 0.05. There were no significant differences between the macular hole eyes three months after surgery relative to the fellow eyes and the normal eyes, P > 0.05. There were no significant differences in peripheral retinal sensitivity (4°–10°) among the groups, P < 0.05. These findings are presented in The BCVA of the macular hole eyes gradually improved after surgery; the improvement in these eyes was statistically significant, P < 0.05; however, there were no significant differences between the postoperative retinal thickness of the hole eyes, the retinal thickness of the fellow eyes, and the retinal thickness of the normal eyes, P > 0.05. The preoperative central retinal blood density of the superficial and deep capillaries was statistically lower than the postoperative central retinal blood density, P < 0.05. The FAZ of the hole eyes in both the superficial and deep layers was significantly lower than the fellow eyes and the normal eyes, P < 0.05. These results are presented in The preoperative retinal thickness of the hole eyes was statistically higher than the postoperative retinal thickness of the hole eyes, the retinal thickness of the fellow eyes, and the retinal thickness of the normal eyes, P < 0.05; however, there were no statistically significant differences in the postoperative superficial blood flow areas of the hole eyes, the superficial blood flow areas of the fellow eyes, and the superficial blood flow areas of the normal eyes, P > 0.05. The peripheral choroidal blood vessel superficial capillary density of the hole eyes was significantly lower than that of the fellow eyes and normal eyes, P < 0.05. The preoperative subfoveal choroidal thickness of the hole eyes was significantly lower than the postoperative subfoveal choroidal thickness of the hole eyes, the fellow eyes, and the normal eyes, P < 0.05; however, there were no statistically significant differences in the postoperative subfoveal choroidal thickness of the hole eyes, the fellow eyes, and the normal eyes, P > 0.05 , the minimum diameter (r = 0.585 and P = 0.017), the MCHAI (r = −0.618 and P = 0.011), the central MHI (r = −0.727 and P = 0.001), the peripheral MHI (r = −0.758 and P = 0.001), the central THI (r = −0.717 and P = 0.002), the peripheral THI (r = −0.725 and P = 0.001), and the change in the peripheral blood vessel density of the choroid capillary layer (r = 0.585 and P = 0.0017). The change in the central retinal sensitivity was correlated with the change in the superficial FAZ (r = 0.520 and P = 0.039), the change in the retinal superficial peripheral blood flow density , the change in the deep FAZ (r = 0.599 and P = 0.014), and the change in the retinal deep peripheral blood flow density (r = −0.601 and P = 0.014), as shown in r = 0.592 and P = 0.016), the CMHI (r = −0.548 and P = 0.028), and the PMHI (r = −0.531 and v= 0.034), as shown in The change in BCVA was significantly correlated with the basal diameter (This study found that BCVA and central retinal sensitivity of the macular hole gradually improved after vitrectomy combined with internal limiting membrane stripping and tamponade. Chen et al. –12 also The correlation analysis indicated that the postoperative change in BCVA was correlated with the morphology of the macular hole and its derivative indices. The ∆BCVA was negative as it is calculated as the three-month BCVA minus the preoperative BCVA. The smaller basal diameter, smaller minimum diameter, larger MCHAI, larger central or peripheral MHI, larger central or peripheral THI, and smaller peripheral blood vessel density of the choroid capillary layer all contributed to the improvement in BCVA. Macular holes seriously damage vision, and patients with macular holes with a smaller basal diameter and smaller minimum diameter may exhibit greater improvements in visual function following surgery. This is consistent with the research of Venkatesh et al. . The preThickening of the retina in the macular hole may be attributed to the anteroposterior and tangential traction caused by incomplete vitreoretinal detachment, the presence of the vitreous cortex and epimacular glial tissues, or retinal swelling with subretinal fluid components –4. The MThe retinal thickness was decreased in the central region, upper and lower nasal regions, and temporal region after surgery. Thinning of the retina in the macular hole mainly occurs in the nerve fibre layer and ganglion cell layer. Ozdemir et al.  found thThe results of this study indicated that the change in central retinal sensitivity was correlated with changes in the superficial or deep FAZ and the retinal superficial or deep peripheral blood flow density. After the macular hole is closed, the FAZ reduces. The main reason for the decrease in the FAZ is the surgical stripping of the inner limiting membrane, which improves the closure rate of the hole. After surgery, the retinal cells migrate to fill the gap caused by the macular hole, the tissue increases in the macular hole, and the FAZ decreases , 24–26. The central superficial retinal blood flow density was significantly increased, and the peripheral superficial retinal blood flow density was decreased one month after surgery. This suggests that the month after surgery is the critical period for closure of the macular hole. The morphology and function of the central retina recovered gradually; the peripheral retinal blood flow slowly migrated to the central part of the retina, contributing to the morphological and functional recovery of the macular hole. Thus, the peripheral retinal blood flow decreased, and the central blood flow increased. The decrease in the FAZ also reflects this phenomenon. Although the retinal blood flow increased from one month to three months after surgery, this difference was not statistically significant, presumably, because the hole was basically closed one month after surgery and the morphology tended to be stable. The retinal blood flow also tended to be stable; thus, there was no need for peripheral blood flow support.This study revealed that the subfoveal, temporal, and superior choroidal thickness were greatest, followed by the inferior choroidal thickness; the nasal choroidal thickness was the thinnest. This is consistent with previous studies –8. It isZhang et al.  reportedThere are several limitations of this study that should be noted. First, the number of cases was limited. The study recruited more than 40 individuals with the idiopathic macular hole, but most of them were excluded due to the fit and poor image quality from the optical examinations. Second, the central retina and choroid could not be examined directly due to the macular hole; thus, the peripheral changes in the retina and choroid were examined. Third, because of the high loss of participants, this study could only analyse changes three months after surgery rather than the trend in visual recovery. BCVA can also represent recovery of visual function to a certain extent.The recovery of visual function is affected by many factions after macular hole surgery. The morphology of the macular hole as well as changes in the retinal and choroidal microstructure all contribute to the recovery of visual function after surgery. The MHI, THI, and MCHAI could help analyse the anteroposterior tractional forces caused by cysts, and the lower index might obtain better restoration of visual function. Macular holes with a smaller basal diameter and greater height might obtain more blood flow support from the choroid, especially from the peripheral blood vessel of the choroid, allowing better recovery and greater improvements in visual function.


Breastcancer.org experienced a sustained surge in requested pandemic‐related information and support. To characterize the pandemic‐related experiences of breast cancer patients, we surveyed the Breastcancer.org Community early in the COVID‐19 era.The COVID‐19 pandemic has disrupted medical care, increased isolation, and exacerbated anxiety in breast cancer patients. Since March 2020, Breastcancer.org Community members were invited to complete an online questionnaire regarding their experience during the pandemic. Self‐reported data on demographics, comorbidities, care disruptions, anxiety, coping ability, telemedicine use, and satisfaction with care were collected. Results were analyzed using Stata 16.0 .p = 0.021), current breast cancer diagnosis , and poorer coping ability . Moderate to extreme anxiety about cancer care disruptions was reported by 29.1%, increasing with current breast cancer diagnosis , actual delayed care , and poorer coping ability . Most utilized telehealth and found it helpful, but also expressed increased anxiety and subjectively expressed that these were less preferable.Included were 568 current and previous breast cancer patients, primarily with U.S. residence. Overall, 43.8% reported at least one comorbidity associated with severe COVID‐19 illness and 61.9% experienced care delays. Moderate to extreme anxiety about contracting COVID‐19 was reported by 36.5%, increasing with number of comorbidities (33.0% vs. 55.4%, Early in the COVID‐19 pandemic, anxiety was reported by a large proportion of breast cancer patients, with increased prevalence in those with risk factors. Attention to mental health is critical, as emotional distress not only harms quality of life but may also compromise outcomes. COVID‐related anxiety is prevalent in breast cancer patients, particularly in those with a current diagnosis, co‐morbidities, care delays and disruptions. It’s important for health care providers to measure and manage pandemic‐related anxiety to improve access to care and mental health services, and to assist in COVID risk mitigation. Support communities such as Breastcancer.org can facilitate cancer patient wellness. Thus, in order to inform patient‐targeted communications surrounding mental health and anxiety, Breastcancer.org conducted an online global survey of website visitors and online community members following the pandemic's onset in the U.S. The purpose of this study was to (1) assess the self‐reported prevalence of anxiety about contracting COVID‐19 and anxiety about care disruptions and (2) discuss the need for mental health evaluation and provide practicing oncologists with the tools to recognize these issues, ensuring their patients can be quickly referred to mental health care. Given the impact of mental health on cancer survival and the multitude of treatments available, it is crucial to recognize these symptoms, even as the pandemic cautiously eases and cancer care returns to pre‐COVID‐19 levels.At 2Breastcancer.org is a nonprofit organization that provides medical expertise and a peer‐support community to a global audience of over 20 million unique users while advancing shared patient and doctor decision‐making and producing patient‐centered research. From April 27 to June 1, 2020, Breastcancer.org conducted a retrospective cross‐sectional survey where Community Members were invited to participate in an anonymous, self‐reported online questionnaire within the discussion board section of the website  regarding the pandemic's impact upon their care and well‐being. The survey was developed by Breastcancer.org prior to the release and validation of the COVID Anxiety Syndrome Scale (see survey tool in Appendix S2). Because this was a patient‐reported prevalence of anxiety survey study, we did not assess the syndrome specifically. This study was deemed IRB‐exempt by the institutional review committee as this project does not involve “human subjects research” as defined by Department of Health and Human Services Protection of Human Subjects regulations (45 CFR 46). Informed consent was obtained prior to survey participation. The survey and data analysis were conducted in accordance with recommendations from the Strengthening and Reporting of Observational Studies in Epidemiology (STROBE) initiative. All data were patient‐reported and duplicate data or incomplete surveys were excluded.Patient‐reported unidentifiable data were collected on the following: demographics , comorbidities associated with risk of severe illness or complications from COVID‐19 infection ,p‐value of 0.05 was initially considered significant, and after Benjamini‐Hochberg correction, significance was reported accordingly. A subset of this analysis was previously presented at the 2021 ASCO Annual Meeting.Data verification with electronic medical records was not performed. Statistical analysis was utilized to evaluate patient‐specific factors modulating anxiety levels. Data analysis was completed using Stata 16.0 . Aggregate results were tabulated as frequencies and compared using chi‐square test for independence. Multivariable ordinal logistic regression was utilized to calculate associations with anxiety about contracting COVID‐19 and treatment delays. Adjusted odds ratios and 95% confidence intervals were reported. A 3In total, 568 current and previous breast cancer patients from 24 countries and 43 U.S. states completed the survey Table . Respond3.1p = 0.011), and those with two or more vs. no comorbidities . Anxiety about contracting COVID‐19 also increased with age (p = 0.04): among 35‐44‐year‐olds, 13 (26.0%) reported moderate/extreme anxiety, while 85 (45.2%) respondents aged 65 or older reported the same level of distress. However, after multiple testing correction, the relationship between anxiety and age was no longer significant.Overall, 298 (57.2%) of participants reported being slightly/somewhat anxious and 206 (36.5%) were moderately/extremely anxious about contracting COVID‐19 (Table p < 0.0001), with two or more comorbidities , aged 65 years or older , and coping fairly or not well at all  compared to those with past diagnoses, with no other comorbidities, aged 25–44 years old, and who were coping very well or well  experienced delays in oncology care that were directly attributable to the pandemic, either initiated by the patient themselves or their provider Figure . Of thosp < 0.0001). Participants experiencing care delays were significantly more likely to report moderate/extreme anxiety .Regarding anxiety surrounding breast cancer care disruptions, 285 (50.9%) reported slight/somewhat anxiety and 163 (29.1%) expressed moderate/extreme anxiety Table . ModeratThere were significantly higher odds of anxiety about care disruptions for participants with a current cancer diagnosis (aOR 2.7 (95% CI 1.9–3.9)), those who experienced a care delay (aOR 2.1 (95% CI 1.4–3.0)), and those who said they were coping fairly well or not at all with pandemic challenges (aOR 2.2 (95% CI 1.5–3.1)). Comorbidities and age group were not associated with anxiety about breast cancer care being affected by the pandemic.3.3p < 0.0001). By contrast, those with no anxiety about contracting COVID‐19 were more likely to select coping well or very well (44 [72.1%] vs 17[27.9%]) (Table p < 0.0001).Of the 564 participants who reported on their ability to cope with the challenges of COVID‐19, 87 (15.4%) stated they were coping very well, 198 (35.1%) coping well, 253 (44.9%) coping fairly well, and 26 (4.6%) not coping well at all. Participants with moderate/extreme anxiety about contracting COVID‐19 reported they were not coping at all or fairly well at significantly higher levels than those coping well or very well  Table . In sum,Respondents also provided open‐ended comments about coping. Those coping better shared themes of gratitude, faith, enjoying the outdoors, and trusting safety protocols including masking and social distancing. Those with poorer coping experiences expressed themes of sadness, isolation from family, stress, anxiety about viral contraction, and the difficulties of having cancer while enduring a pandemic Table .3.4p = 0.010).331 (58.6%) had at least one virtual appointment via telephone or video, and 253 (75.3%) of these found such appointments helpful Table . HoweverSimilarly, in their subjective comments, many respondents found virtual appointments less preferable than in‐person appointments. Those who did not find virtual appointments helpful found their care subpar, and one participant expressed that their disease progression would have been caught earlier if not for a virtual appointment Table . Nonethe4Our study confirms previously observed disruptions in breast cancer care following the onset of the COVID‐19 pandemic and further reveals significant anxiety levels among both current and previous breast cancer patients.4.1Participants currently under treatment for breast cancer or having at least 3 other comorbidities reported significantly higher COVID‐19‐related anxiety compared to those post‐treatment or without comorbidities ; formal analysis ; investigation ; writing – original draft ; writing – review and editing . Stephanie Kjelstrom: Formal analysis ; methodology ; visualization . Diana M. Martinez: Project administration ; writing – original draft ; writing – review and editing . Adam Leitenberger: Methodology ; project administration . Donna Marie‐Manasseh: Writing – review and editing . Melissa Bollmann‐Jenkins: Writing – review and editing . Ann Partridge: Resources ; writing – review and editing . Virginia G. Kaklamani: Validation ; writing – review and editing . Rowan T Chlebowski: Validation ; writing – review and editing . Sharon L. Larson: Formal analysis ; investigation ; supervision ; visualization ; writing – review and editing . Marisa Carey Weiss: Conceptualization ; investigation ; methodology ; project administration ; writing – review and editing .The effort of DM is supported by federal grant K24 DA‐20620‐05. All other authors report no funding sources.The authors have no conflicts of interest to declare. The authors were entirely responsible for the design and conduct of the study; the analysis and interpretation of the data; and preparation, review, approval, and submission of the manuscript for publication.This study was deemed IRB‐exempt as this project does not involve “human subjects research” as defined by the Department of Health and Human Services Protection of Human Subjects regulations (45 CFR 46). No identifiable information was collected from respondents. Informed consent was obtained.Appendix S1Click here for additional data file.Appendix S2Click here for additional data file.


Robotic gastrectomy is a commonly used procedure for early gastric cancer and it also overcomes the limitation of laparoscopic. However, the complications of robotic gastrectomy (RG) still need to be assessed. Our study was designed to compare postoperative complications of RG with laparoscopic gastrectomy (LG).A meta‐analysis and systemic review were prospectively collected using the PubMed, Cochrane Library, and MEDLINE database of published studies by comparing the RG and LG with gastric cancer up to December 2021. To evaluate the postoperative outcomes, odds ratios were calculated for Dichotomous data and the mean difference with 95% confidence interval (CI) was calculated for continuous data, and measured by the random‐effect model.p < 0.001), Clavien−Dindo grade Ⅲ (odds ratio (OR) = 0.60, 95% CI: 0.48−0.76, p < 0.01), and harvested lymph node . There was no significant difference between robotic gastrectomy surgery (RGS) and laparoscopic gastrectomy surgery (LGS) regarding distal resection margin (DRM), proximal resection margin (PRM), conversion rate, anastomotic leakage, and overall complications.Thirty‐two retrospective studies describing 13,585 patients (4484 RG and 9101 LG) satisfied the inclusion criteria. A statistically significant result was in blood loss (MD = −17.97, 95% Cl: −25.61 to 10.32, Having significant outcomes in Clavien–Dindo grade III, and blood loss, harvested lymph nodes are more common in RGS, and they also help in increasing the quality of life. English language full‐text article containing at least one of the following postoperative complications; blood loss, conversion rate, DRM, PRM, Clavien–Dindo grade Ⅲ, HLN, anastomosis leakage, and overall complication.2.2.6Articles about robotic or laparoscopic surgery that did not provide a comparison, evaluations that did not address complications, reviews, case reports, animal studies, and letters were all omitted.2.3We systematically explored the literature by SCOPUS, Cochrane Library, and PubMed database for articles available until December 2021. Our research work included the keywords “Robotic gastrectomy,” “laparoscopic gastrectomy,” and “gastric cancer.” Our search is limited to humans and English language articles.2.4p < 0.05), Q statistics were used to assess the treatment effects of heterogeneities, and I2 was assessed for the total variation studies.RevMan 5.4 was implemented for statistical meta‐analysis. Summative figures are arranged according to descriptive analysis and we set the confidence interval (Cl) at 95%. Outcomes are reported for dichotomous as odds ratios (OR) and 95% Cl through Mantel–Haenszel way and continuous variables as mean difference (MD) through generic inverse variance way. Continuous data, standard deviation (SD), and mean were reported in median and range. We set statistically significant at , Q statistics were used to assess the treatment effects of heterogeneity, and I2 was assessed for the total variation studies as shown in Table We set the statistical (3.3p < 0.001) as shown in Figure Meta‐analysis results showed a marked rise in the total amount of blood loss following the LG group compared with RG  as shown in Figure The overall conversion rate was 0.6% (18/2899) to open surgery (OS) in the RG group and 0.86% (55/6415) in the LG group. In this study, the conversion rate following OS was statistically not significant in 21 different trials within the two groups  as shown in Figure An overall complication has been found in multiple 32 studies. The proportion rate for overall complications was 11.8% (529/4484) in the RG group and 11.9% (1086/9101) in the LG group. The result for this study proposed a statistically significant  as shown in Figure The frequency rate of complication in the nineteen retrospective studies reported that Clavien–Dindo grade > Ⅲ in the RG group was 3.9% (112/2851) and LG group was 6.3% (320/5022). The rate is lesser in RG as compared with LG  as shown in Figure Overall anastomotic leakage was found in 24 studies. Therefore, the RG group was 1.7% (57/3275) and the LG group was 2.03% (140/6890). Our study did not show the most significant change in the anastomotic leakage  as shown in Figure Eleven out of 32 studies informed the DRM. The mean difference in the robotic gastrectomy was found at 6.69 while LG was 6.5. Our study indicated that there is no significant  as shown in Figure Following 32 studies the PRM was reported in 12. The mean distance in RG was 4.5 while LG was 4.4. There is no statistical difference seen in RG with comparison to LG group, a mean difference . However, our data showed statistically significant as shown in Figure Our study reported a raised number of the HLN in RG compared with LG  affirms that this manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned  have been explained.


Zeaxanthin is a yellow‑coloured dietary carotenoid widely recognized as an essential component of the macula. It exerts blue light filtering and antioxidant activities, offering eye health and vision benefits.This study explores the oral absorption and systemic disposition of zeaxanthin from biopharmaceutical and pharmacokinetic perspectives.In vivo intravenous (5 and 10 mg/kg) and intraportal (5 mg/kg) pharmacokinetic studies were performed to determine intrinsic tissue‑blood partition coefficient, elimination pathway, and hepatic clearance, of zeaxanthin in rats. Moreover, in vitro physicochemical property test, in situ closed loop study, in vivo oral pharmacokinetic study (20 and 100 mg/kg), and in vivo lymphatic absorption study (100 mg/kg) were conducted to investigate the gut absorption properties of zeaxanthin and assess the effects of several lipids on the lymphatic absorption of zeaxanthin in rats.Zeaxanthin exhibited poor solubility (≤144 ng/mL) and stability  in simulated gut luminal fluids. Gut absorption of zeaxanthin occurred primarily in the duodenum, but the major fraction (≥84.7%) of the dose remained unabsorbed across the entire gut tract. Considerable fractions of intravenous zeaxanthin accumulated in the liver, lung, and spleen . It was found that the liver is the major eliminating organ of zeaxanthin, accounting for 53.5–90.1% of the total clearance process (hepatic extraction ratio of 0.623).To our knowledge, this is the first systematic study to report factors that determine the oral bioavailability and systemic clearance of zeaxanthin. Simulated gastric fluid (SGF), with pH 1.2, was purchased from Wako Pure Chemical Co. . Buffer solutions  and simulated intestinal fluid (SIF), with pH 6.8, were purchased from Samchun Pure Chemical Co., Ltd. . Pooled human blood and plasma were purchased from Innovative Research, Inc. . Eight‑week‑old male Sprague–Dawley rats (240–280 g) were purchased from Koatech Co., Ltd. . Rats were individually housed in clean cages at an ambient temperature of 23 °C. The animal study protocols were approved by the Institutional Animal Care and Use Committee of the Pusan National University for scientific care and ethical procedures .Zeaxanthin (purity > 95%) and β‑carotene  were purchased from Biosynth Carbosynth . HPLC‑grade acetonitrile, methanol, dimethyl sulfoxide, and methyl g for 10 min. Next, 500 μL of the resultant supernatant was transferred to a clean microtube and evaporated using a SpeedVac centrifugation system . The resultant dried residue was reconstituted with 60 μL of a methanol/MTBE/ethyl acetate mixture , and a 30 μL aliquot was injected into the HPLC system.The zeaxanthin concentration in the samples obtained from the experiments conducted in this study was determined using a validated HPLC method. A Shimadzu HPLC system coupled with an ultraviolet‑visible (UV‑Vis) detector  was used in this study. The light wavelengths that produced the maximum UV‑Vis absorbance intensity were chosen based on the UV‑Vis absorbance spectrum measurement for zeaxanthin and the IS (1000 ng/mL in methanol solution). Chromatographic separation was performed at 30 °C using a YMC Carotenoid C30 column  connected to a C18 guard column . Gradient elution of the mobile phase consisting of deionized water , methanol (solvent B), and MTBE (solvent C) was performed at a flow rate of 1 mL/min, and the procedure was as follows : started at 10:60:30 at 0 min, ramped from 10:60:30 to 10:20:70 for 0.1 min, maintained at 10:20:70 for 5.5 min, back to 10:60:30 for 0.1 min, and maintained for 6.5 min . plasma samples (120 μL) were deproteinized with 120 μL of ice‑cold methanol/MTBE mixture  containing IS (2000 ng/mL) and 600 μL of ethyl acetate. The resultant mixture was vortexed for 10 min and centrifuged at 16,000 × The bioanalytical method proposed herein was validated by analyzing calibration standards and quality control (QC) samples according to US FDA guidelines (USA Food and Drug Administration D) was measured in buffers of varying pH values . The SGF and SIF used in the solubility and stability tests were prepared as follows: SGF was prepared by dissolving 0.32% (w/v) pepsin, 0.2% (w/v) sodium chloride, and 0.7% (v/v) HCl in DW ; SIF was prepared by dissolving 0.1% (w/v) pancreatin and 3 mM sodium taurocholate in phosphate buffer . The solubility of zeaxanthin (2 μM) was measured in buffers of varying pH values , plasma, phosphate buffer saline, SGF, and SIF. The unbound fraction in plasma (fuP) and the blood‑to‑plasma concentration ratio (BR) were determined at a zeaxanthin concentration of 2 μM. The stability of zeaxanthin (2 μM) was determined in several matrices, including buffers of varying pH values , plasma, urine, SGF, and SIF.The lipophilicity, solubility, plasma protein binding, blood distribution, and biological stability of zeaxanthin were determined as described in our previous study  was collected. Urine was collected in a 15 mL tube over a period of 24 h, in parallel with blood collection. Subsequently, the rats were euthanized by carbon dioxide inhalation, and the entire gut tract, including its contents and faeces, was removed, transferred into a beaker, and cut into pieces using scissors. Next, 200 mL of a methanol/MTBE mixture  was added to the beaker, followed by gentle stirring with a glass rod for 1 min, and 120 μL of the resultant supernatant was collected. Plasma, urine, and gut luminal extract samples were analyzed using the validated HPLC method.Rats underwent surgery for cannula implantation into the femoral vein and artery under light ether anaesthesia . For intraportal administration, the same dose of zeaxanthin was infused over 30 min into the portal vein, and simultaneously, the same volume of the vehicle solution was infused over 30 min into the femoral vein. Approximately 250 μL of blood was collected via the femoral artery at 0, 15, 30, 32, 35, 45, 60, 90, 120, 150, 210, 270, 390, and 510 min following intravenous and intraportal administration. After centrifugation of the blood sample at 3000 × g at 4 °C for 5 min, 120 μL of plasma was obtained.The femoral vein, femoral artery, and portal vein were cannulated as described in our previous studies , the loops were prepared in the same manner as the ‘in situ loop’ study. The loops were removed from the rat gut tract by carefully cutting with scissors, and transferred to a glass beaker. The zeaxanthin suspension was injected into the in vitro isolated loop. After 0 and 4 h of incubation at 37 °C, the remaining fraction of zeaxanthin in the in vitro isolated loop was measured in the same manner as for the gut luminal extract samples.After a minimal abdominal incision under light ether anaesthesia followed by sufficient washing of the contents within the gut tract, the gastric, duodenal, jejunal, ileal, and colonic closed‑loops were prepared as previously described in our previous study  was intravenously injected into the femoral vein of rats. The brain, liver, heart, lung, kidney, gut, spleen, muscle, adipose, and eyes were excised 90 min after the administration, rinsed with saline, and blotted dry with clean paper. Each weighed tissue sample was homogenized  in four volumes of ice‑cold saline/methanol mixture . The tissue homogenate was then centrifuged at 9000 × via the femoral artery at 0, 5, 15, 30, 60, 90, and 120 min. The liver and mesenteric lymph tissues were excised immediately after the last blood sampling and then processed using the same procedure as described in the in vivo tissue distribution study.Zeaxanthin was dispersed in four different vehicle systems consisting of oil phase , polyethylene glycol 400, propylene glycol, Tween 80, and saline . Following oral administration of zeaxanthin (100 mg/kg) in rats, blood samples (250 μL) were collected in heparin‑coated microcentrifuge tubes ® software  to estimate the following pharmacokinetic parameters: total area under the plasma concentration vs. time curve from time zero to infinity , total body clearance , terminal half‑life (t1/2), and apparent volume of distribution at steady state (Vss). Assuming dose‑linear conditions, Foral was calculated as the ratio of the dose‑normalized AUC between intravenous and oral administration. The peak plasma concentration (Cmax) and time to reach Cmax (Tmax) were obtained directly from the experimental data. The statistical p‑values were estimated using the unpaired t‑test for comparison between two means or one‑way analysis of variance with post-hoc Tukey’s honestly significant difference test for comparison among three or more means; p < 0.05 was considered statistically significant. Unless otherwise indicated, all numerical data were rounded to three significant figures.Non‑compartmental analysis was performed using WinNonliny = 0.0002 × x + 0.0003; where x is the nominal concentration ratio of zeaxanthin to IS, and y is the peak area ratio of zeaxanthin to IS. The correlation coefficients for the calibration curves were ≥0.9993. The validation parameters of the present bioanalytical method are listed in p = 0.718 and 0.522 for the recovery and matrix effect, respectively). Under routine experimental conditions relevant to the present bioanalytical procedures, such as being kept on the bench top and in the autosampler, frozen for storage and thawed for analysis, and stored for a relatively long period, the stability of zeaxanthin in rat plasma sample was determined at two quality control levels, namely LQC and HQC. The bias in the observed concentrations was within ±15% of the corresponding nominal values; the mean remaining fractions of zeaxanthin ranged from 89.6 to 112%, with a CV value of ≤9.14%.P (or log D) > 4.51, which coincides with the clog p-values estimated using ALOGPS 2.1 and ACD software v11.02 (8.3–10.9). P of zeaxanthin was very low, and BR of zeaxanthin was close to 1 . This indicates that zeaxanthin is a highly lipophilic compound with log ose to 1 . This invs. time profiles of zeaxanthin following intravenous administration at 5 and 10 mg/kg in rats, and the relevant parameters are listed in t1/2 of 71.4–102 min, and relatively high Vss and CL were observed, indicating an extensive tissue distribution and rapid elimination. The AU24h and AG24h values were below the detection limit, indicating negligible urinary and gut (including biliary) excretion of zeaxanthin. Notably, there were no significant differences in the pharmacokinetic parameters between the two tested doses . However, following oral administration at 50 and 100 mg/kg, the concentrations of zeaxanthin in the plasma and urine samples were below the quantitation limit, indicating extremely low oral bioavailability. Notably, considerable AG24h values of 16.3–54.8% were observed in rats, indicating variable and limited absorption of zeaxanthin from the gut. The hepatic extraction ratio (HE) of a drug can be estimated by comparing the AUC observed after intravenous and intraportal administration. vs. time profiles of zeaxanthin in rats, following intravenous and intraportal infusion over a period of 30 min at a dose of 5 mg/kg. The AUC of zeaxanthin was 243 ± 13 and 91.6 ± 14.2 μg·min/mL, following intravenous and intraportal infusion, respectively. This indicates that zeaxanthin was moderately extracted by the liver, with an HE of 0.623 [=1 − (91.6/243)].in vitro isolated (control) or in situ closed loops of the stomach, duodenum, jejunum, ileum, and colon. It is plausible that both gut luminal degradation and gut absorption of drugs occur simultaneously in the in situ closed loops, whereas only gut luminal degradation occurs in the in vitro isolated loops. Thus, the differences between the remaining fractions observed in the in situ and in vitro loops correspond to the actual fractions of drugs absorbed from the loops . In contrast, the remaining fractions in the duodenum loops differed significantly between the two states (p = 0.0119). These results indicate that the gut absorption of zeaxanthin could occur in the duodenum rather than in the other segments; the actual fraction of zeaxanthin absorbed from the duodenum loops can be calculated as 15.3%. in vivo lymphatic absorption of zeaxanthin after oral administration of four different zeaxanthin suspensions prepared with four different types of the oil phase, i.e., corn oil, stearic acid, oleic acid, and linoleic acid. The blood concentrations of zeaxanthin for 120 min were below the quantitation limit, whereas quantifiable amounts of zeaxanthin were recovered from the liver and lymph tissues. Notably, the zeaxanthin levels in the lymph were significantly higher than those in the liver. Moreover, there were no significant differences in the liver concentrations, lymph concentrations, and lymph‑to‑liver concentration ratios (indicating lymphatic transport efficiency) of zeaxanthin among the test formulations containing four different types of lipid .The present study aimed to develop an efficient bioanalytical HPLC method for zeaxanthin and elucidate the biopharmaceutical and pharmacokinetic factors that determine the gut absorption and systemic disposition of zeaxanthin in rats. Several bioanalytical conditions, such as light wavelength, stationary/mobile phase, and sample pre-treatment procedure, have been optimized to achieve acceptable chromatographic separation of analytes in biological matrix. The chemical structure of zeaxanthin contains a conjugated polyene chain that acts as a light absorber , gut availability (GF), and hepatic availability (HF). The results depicted in in vivo. The Fabs of a drug can be estimated from the AG24h values observed after oral dosing, which is valid only when the drug is stable in the gut lumen for 24 h  would remain unabsorbed in the gut following oral administration. This implies that gut absorption of zeaxanthin may be limited.The plasma pharmacokinetics of zeaxanthin following intravenous administration was linear in the tested dose range. However, the plasma concentrations of zeaxanthin following oral administration were below the quantitation limit, suggesting an extremely low oral bioavailability of zeaxanthin. Oral bioavailability can be calculated as the product of three factors: fraction absorbed from the gut  of zeaxanthin can be estimated to be 31.2–49.8 mL/kg from the observed HE of 0.623 and reported hepatic blood flow rate of 50–80 mL/kg  of zeaxanthin, it can be inferred that the liver is the major eliminating organ of zeaxanthin, accounting for 53.5–90.1% of the total clearance process. This indicates minor contributions of extrahepatic organs, including the gut, to the entire process of systemic elimination, which can reconfirm the suitability of the present in vivo metabolism study focussing on the liver.A previous study on lutein, a structural analogue of zeaxanthin, reported that large fractions of the intravenous dose were distributed into the liver, spleen, and lung (in the descending order) (Itagaki et al. stration . The hepvia two different routes: the hepatic portal system and the lymphatic system via chylomicron formation (Deming and Erdman The absorption of zeaxanthin from the gut lumen into the systemic circulation occurs via other routes, such as parenteral, pulmonary, and percutaneous routes.This study presented rat pharmacokinetic data obtained after a single intake of synthetic zeaxanthin. As these experimental conditions may not fully reflect actual clinical situations, they need to be further extended to more clinically relevant studies with long‑term multiple intakes of natural products containing zeaxanthin and other various active phytochemicals. Moreover, plasma concentration levels of zeaxanthin after oral administration were below the quantitation limit. Thus, if possible, the sensitivity of the bioanalytical method used in this study needs to be improved to assess reliably the oral pharmacokinetic profiles of zeaxanthin. All the present results taken together, it is suggested that the poor oral bioavailability of zeaxanthin can be attributed primarily to the interplay between low gut luminal solubility/stability, limited gut absorption, and hepatic first‑pass metabolism. Therefore, to enhance the oral bioavailability of zeaxanthin, the vehicle system used for the present study can be further optimized using formulation strategies to modify these factors. For instance, solubility/permeation enhancers and lipid‑based micro/nano‑colloidal systems to enhance solubility/permeability and target lymphatic pathways could serve as feasible tools for efficient oral delivery of zeaxanthin. Furthermore, the information regarding the disposition characteristics of zeaxanthin could be useful for designing formulations administered in vivo pharmacokinetic study revealed that the liver is the major organ responsible for the tissue distribution and systemic metabolism of zeaxanthin. Taken together, the poor oral bioavailability of zeaxanthin can be attributed mainly to its poor gut luminal solubility/stability, limited gut absorption, and hepatic first‑pass metabolism. To our knowledge, this is the first systematic study to report the oral absorption and systemic disposition of zeaxanthin from the biopharmaceutical and pharmacokinetic perspectives.Physicochemical compatibility, gut absorption, and systemic disposition of zeaxanthin were comprehensively assessed using relevant rat models. Zeaxanthin exhibited poor solubility and stability in simulated gut luminal fluids. Gut absorption of zeaxanthin occurred primarily in the duodenum, but the major fractions of the dose remained unabsorbed across the entire gut tract. This


Dear Editor,1A 21‐year‐old male patient affected by severe atopic dermatitis (AD) since childhood started dupilumab in January 2019 after treatment failure with cyclosporin. Serum examination at the baseline revealed high levels of IgE (6.027 IU/ml) and lactate dehydrogenase (LDH 482 IU/L). The Eczema Area and Severity Index (EASI) was 24, while the Dermatology Life Quality Index (DLQI) was 20. He reported severe pruritus  and sleep disorders. After 16 weeks of treatment, the patient's outcomes were significantly improved with NRS of 0, EASI and DLQI of 0 and 2, respectively, IgE of 831 IU/ml and LDH of 168 IU/L.In July 2020, he developed isolated paraesthesias in his left hand, followed by gait ataxia, and in September 2020 Lhermitte's sign and right lower limb paresis. Therefore, he was admitted at the Neurology clinic for further evaluation. The neurological examination revealed left upper limb downward drift and pronation, right leg downward drift and ankle clonus, paresthesias in the distal phalanges of the fingers of the right hand, hypopallesthesia in the left hand and right lower limb and positive Lhermitte's sign.Magnetic resonance imaging (MRI) of the spine showed non‐enhancing spinal cord lesions at C2, C4, C5‐C6, T1, T5‐T6, and T10‐T11 Figure . Brain MSerum Aquaporin‐4 (AQP4) and myelin oligodendrocyte glycoprotein (MOG) antibodies were absent. Antibodies to Borrelia burgdorferi, Treponema pallidum, and HIV were negative. JCV Stratify Assay® tested negative.Based on clinical history, MRI, and CSF examination, a diagnosis of relapsing–remitting multiple sclerosis (MS) was made.Due to the aggressive presentation of MS, in March 2021 the patient was started on natalizumab . As there are no data on the safety of the association with natalizumab, dupilumab was discontinued.Since then, the patient has been clinically stable, with improvement of previous neurological symptoms and no further MS relapses. Follow‐up MRIs after initiation of natalizumab showed no further evidence of disease activity. Natalizumab therapy was well tolerated. He had moderate relapses of AD managed with topical corticosteroids alone.AD is characterized by a Th2 cellular‐mediated immune response by cytokines IL‐13 and Il‐4.Recent studies have shown that IL‐4 has a negative regulatory action on the Th1 and Th17 pathway by silencing of IL‐23 in antigen‐presenting cells.α (TNFα), INF‐γ, IL‐2, and IL‐17.In MS, the main mediators of inflammation are Th1 and Th17 related cytokines, such as tumor necrosis factor‐We hypothesize that dupilumab, which leads to Th2 suppression by blocking the shared receptor subunit for IL‐4 and IL‐13,This case is the second reporting the onset of MS after initiation of dupilumab: Laageide et al. described the onset of similar symptoms just 2 months after initiation of dupilumab.Further studies are needed to better understand if dupilumab can increase the risk of MS onset or flareups. Meanwhile we recommend caution in prescribing dupilumab in patients with MS or a family history of MS.Federica Gelato and Luca Mastorino have given substantial contributions to the conception and the design of the manuscript; Michela Ortoncelli, Giovanni Cavaliere, and Paola Cavalla to acquisition, analysis, and interpretation of the data; Marco Vercellino, Chiara Bosa, Matilde Inglese, Raffaele De Palma have been involved in drafting the manuscript; Simone Ribero and Pietro Quaglino revised the manuscript critically. All authors have read and agreed to the published version of the manuscript.The authors declare no conflict of interest.


Encephalic vascular accident, or stroke, is the most common pathology of the central nervous system in humans, the second leading cause of death and physical and cognitive disabilities, in developing countries. It presents as an ischemic (more common) or hemorrhagic form. Ozone therapy has been shown to be effective in neuromodulation, neuroprotection, and nerve regeneration. The present study aimed to evaluate the effect of targeted mild ozone after inducing cerebral ischemia in vitro. Neuroblastoma lineage cells (SH-SY5Y) and canine amniotic membrane stem cells were subjected to 24 hours of hypoxia in an incubator culture chamber. The cells were evaluated by MTT assay, colorimetric assay spectrophotometry, fluorescence microscopy, and flow cytometry. Treatment with low concentrations of ozone (2–10 µg/mL), indicated a possible neuroregenerative effect at low concentrations, correlated with lower levels of apoptosis and oxidative stress compared to cells not subjected to hypoxia. High concentrations of ozone (18–30 µg/mL) promoted an increase in rate of apoptosis and cell death. We developed a novel protocol that mimics ozone therapy for ischemic stroke, using ozonized culture medium after hypoxia induction. Although more studies are needed, we conclude that ozone has a dose-dependent hormetic effect and can reverse the effect of ischemia in vitro at low concentrations. A stroke is a neurological deficit caused by an altered brain circulation. This term includes injuries caused by hemodynamic and clotting disorders in arteries or veins  and is tIn ischemic stroke, interrupting cellular oxidative metabolism reduces phosphate and glucose production, releasing neurotransmitters and increasing calcium and sodium levels. These factors lead to a reduction in neuronal metabolism and mitochondrial function; energy insufficiency; formation of arachidonic acid, prostaglandin, and leukotrienes; vasoconstriction; platelet aggregation; and poor microvasculature ,7Ozone is a gas composed of three oxygen atoms, performing a specialized function of oxidizing double bonds of organic molecules. It is a powerful oxidant that immediately reacts with hydrophilic antioxidants in plasma, such as uric acid, ascorbic acid, and albumin, producing lipoperoxides and aldehydes. Ozone exhibits a hormetic effect; it can peroxidize the erythrocyte membrane and induce a change in its negative charge, causing minimal oxidative stress. Moreover, it improves circulation and oxygen delivery in ischemic tissues, enhances cell metabolism, modulates antioxidant enzymes, induces a slight activation of the immune system, and increases the release of growth factors. Ozone also induces platelet degranulation, stimulates the neuroendocrine system, activates neuroprotective strategies, protects against apoptosis induced via oxidative stress, and increases gene repair activity. When administered in adequate concentration, no side effects are observed .Previous studies indicate that ozone therapy can be effective in neuromodulation, neuroprotection, and nerve regeneration . HoweverIt is worth noting that most studies use ozone as a preconditioning strategy before inducing hypoxia. This approach is not feasible since patients affected by this type of illness seek treatment only after the onset of symptoms and not as prevention. Our pioneering research efforts utilize ozone after hypoxia, with cells still in culture, without resuspending them for ozone treatment.in vitro effects of mild oxidative stress caused by ozone after induction of cerebral ischemia, mimicking an ischemic stroke. Ozone therapy, when applied in adequate concentrations, improves oxygenation levels and cell metabolism, promoting cell regeneration. The ozone treatment was conducted in two phases, using neuroblastoma cells and amniotic membrane stem cells.The present study aimed to evaluate the in vitro, subjected to hypoxia in an incubator culture chamber, and then treated with different ozone concentrations (2–30 µg/mL). As expected, 2,5-diphenyl-2H-tetrazolium bromide (MTT) assays showed a hormetic effect of ozone: low concentrations could promote cell regeneration, while high concentrations were harmful to cells.Neuroblastoma lineage cells (SH-SY5Y) were cultured Similar results were observed in the second step of our study, where canine amniotic membrane stem cells were subjected to the same hypoxia-ozonization protocol and evaluated by colorimetric assay spectrophotometry, fluorescence microscopy, and flow cytometry. These results indicated concentration-dependent effects and possibly neuro-regeneration at low concentrations.in vitro, low concentrations were able to reverse the damage caused by hypoxia.Although more studies are needed, our results suggest that Importantly, our study proposed an unprecedented protocol. In contrast to our study, most other studies use ozone as preconditioning. This is incompatible with what we see in clinical routine, wherein ozone is used as post-stroke therapy.The experiment was divided into two phases performed with neuroblastoma lineage cells and with amniotic membrane stem cells, carried out in Brazil. This project was submitted to the Ethics Committee on the Use of Animals of the Faculty of Animal Science and Food Engineering of the University of São Paulo on August 23, 2018 and was approved through protocol CEUA FZEA-USP n. 3722230818 and CEUA IFSC-USP n. 8689190419.2, for about 3 or 4 days, until they reached about 90% of confluence. Cells were trypsinized and counted using Thoma Chamber, with trypan blue. Approximately 1 × 10−4 SH-SY5Y cells, diluted in 200 µL of culture medium, were plated per well in a 96-well culture plate. Four wells were also plated with the same number of cells on another plate, as a hypoxia control. The plates were incubated at 37 °C and 5% CO2 for 24 h. The experiment was carried out thrice in quadruplicates. After 24 h, the culture plate was placed inside an incubation culture chamber. The completely sealed chamber was filled with about 10% CO2 + N2, and the cells were incubated for another 24 h at 37 °C. The control plate (no hypoxia group) was not subjected to the hypoxia protocol and remained incubated at 37 °C and 5% CO2 for 24 h. A difference in the color of the medium could be observed after 24 h of hypoxia.For the first step, SH-SY5Y neuroblastoma lineage cells were grown in bottles in culture medium  and incubated at 37 °C and 5% CO2, while no gas was added to another. The culture medium was removed from each well of the 96-well plate and replaced with ozonized culture medium containing different concentrations of ozone, according to the scheme below. The plate was incubated for another 24 h. In the control plate, the culture medium was replaced with normal medium, with no addition of gas.After 24 h, 1.5 mL of culture medium was mixed with 1.5 mL of ozone in a 5 mL syringe and stirred for 5 min. Each syringe contained a different concentration of ozone ; an additional syringe contained only OThe colorimetric MTT assay was performed to verify cellular metabolic activity. After 24 h, in a light-deprived environment, the ozonized medium was replaced with 200 µL of MTT (0.5 mg/mL diluted in PBS) per well. After a 90-minute incubation, MTT was removed, and 200 µL of DMSO was added per well. The absorbance was measured at 690 nm using a spectrophotometer (Multiskan Go Spectrophotometer – Thermo Fisher Scientific). The spectrophotometry results were transformed into a percentage, wherein the group not subjected to hypoxia represented 100%.The experimental design is depicted in in vitro results with neuroblastoma cells, we decided to perform the same hypoxia-ozonation protocol with canine amniotic membrane stem cells, the same cells used in a previously published study [After satisfactory ed study . These med study . After e2 for 3 days when they reached approximately 80% confluence.Following the protocol by Orlandin et al. , membranFor cell trypsinization, the medium was discarded and the bottle was washed with PBS. Tryple Express  were added. The bottle remained in an incubator for 2 min, to allow the cells to detach from the plate. Tryple Express was collected in a falcon tube, and the bottle was washed with the culture medium to help detach the remaining cells. This culture medium was collected in the same falcon and centrifuged at 1500 rpm for 5 min. Cells were picked and plated for an additional 4 days until they reached approximately 90% confluence. The trypsinization process was repeated, and the cells were counted using Thoma Chamber, with trypan blue, and harvested according to the specified assay.For each assay, cells were harvested at different concentrations, using different plates, and at different volumes. Corresponding specifications are presented for each assay. Regardless, all of them were subjected to the same hypoxia protocol, as described below. All experiments were performed in triplicate and repeated three times. In addition to the experimental wells, separate wells were set aside for negative and positive control, for each protocol.2 + 90% N2, and incubated for another 24 h. The control plate did not go through the hypoxia protocol and was incubated at 37 °C and 5% CO2 for another 24 h.After 24 h, the culture plate was placed inside a completely sealed incubator culture chamber, filled with about 10% CO2 and another without the addition of any gas (no gas group) were included.After 24 h, a 20-mL syringe filled with 10 mL of culture medium was attached to an ozone generator (Ozone Generator O&L 3.0 RM – Ozone & Life Ltda – Brazil – ref 301140366), which injected another 10 mL of the gas into the syringe. They were stirred for 5 min. Each syringe contained a different concentration of ozone: 3, 8, 12, 18, and 30 µg/mL; additionally, a syringe with 10 mL of OThe culture medium in each well was replaced by the ozonized medium at different concentrations. The plate was incubated for another 24 h. In the control plate (no hypoxia group), the culture medium was replaced by a normal medium with no gas addition.For the MTT assay, the above protocol was used. The absorbance was measured at 690 nm using a spectrophotometer (BioTek Epoch 2 microplate Spectrophotometer). The spectrophotometry results were transformed into percentage values, where the group not subjected to hypoxia represented 100%.−5 cells were inoculated in a 6-well plate in 3 mL of culture medium per well in triplicate and subjected to the hypoxia-ozonation protocol, as mentioned above. The same plate was used for evaluating SOD (Superoxide Dismutase Colorimetric Activity Kit – Thermo Fisher Scientific – ref EIASODC) and CAT  activities, as only the medium was used to perform the SOD assay. In contrast, the cells were used to perform the CAT assay. The substrates were added, and the plate was incubated according to the manufacturer’s instructions. The absorbance was read at 450 and 520 nm by a spectrophotometer for SOD and CAT quantification, respectively.For the superoxide dismutase (SOD) and catalase (CAT) quantification, 1 × 10−3 cells were harvested in a 96-well plate in 200 µL of culture medium per well in duplicate and subjected to the hypoxia-ozonation protocol, as mentioned above. Using the commercial Click-iT TUNEL  colorimetric detection kit, following the manufacturer’s instructions, the culture medium was removed and the cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate. TUNEL mixing solution (50 µL) was added to each sample and the positive and negative controls. The plate was incubated in an oven for 60 min, washed with PBS three times, and 200 µL of PBS was added for microscopic analysis. The cells were analyzed using a digital inverted EVOS microscope (Microscope EVOS™ FLc M5000 Imaging System – Thermo Fisher Scientific – ref AMEP4650) with a GFP laser (470 nm excitation/525 nm emission). The images were analyzed via fluorescence intensity quantification using ImageJ software, with intensity assignment between 0 and 200 per pixel.For the apoptosis detection, we performed the TUNEL test, where 1 × 10−4 cells were harvested in a 24-well plate in 1 mL of culture medium per well in triplicate and subjected to the hypoxia-ozonation protocol, as mentioned above. Image-iT® Lipid Peroxidation Sensor  was added, and the plate was incubated at 37 °C for 30 min in the dark. The culture medium was removed, and the plate was washed 3 times with PBS; 1 mL of PBS was added per well. As observable in Figure 15, the cells were analyzed with a digital inverted EVOS microscope, with a Texas Red laser (585 nm excitation/624 nm emission) and GFP (470 nm excitation/525 nm emission). The excitation and emission were adjusted for each probe. Cells were trypsinized and immediately read using a BD Accuri C6 flow cytometer (BD Accuri™ C6 Flow Cytometer – BD Biosciences – ref 7820018).The Image-iT® Lipid Peroxidation Kit has a lipid peroxidation sensitive agent. After oxidation, the fluorescence changes from red to green, allowing its quantification. For the lipid peroxidation assay, 2.5 × 10−4 cells were harvested in a 24-well plate in 1 mL of culture medium per well in triplicate and subjected to the hypoxia-ozonation protocol, as mentioned above. CellROX Green  reagent working dye solution was added to the culture medium of each well, followed by incubation at 37 °C for 30 min in the dark. The culture medium was removed, and the plate was washed thrice with PBS; 1 mL of PBS was added per well. The cells were analyzed using a digital inverted EVOS microscope with a GFP laser (470 nm excitation/525 nm emission). Cells were trypsinized and read using a BD Accuri flow cytometer.CellROX® Green is a fluorogenic probe that after oxidation, binds to DNA, rendering the nucleus and mitochondria fluorescent. For the ROS assay, 2.5 × 10−4 cells were harvested in a 24-well plate in 1 mL of culture medium per well in duplicate and subjected to the hypoxia-ozonation protocol, as mentioned above. The culture medium was removed, and a solution containing 10 µM of CellTracker Blue (CellTracker Blue CMF2HC (Molecular Probes) – Thermo Fisher Scientific – ref C12881) in DMSO was added to each well. The plate was incubated for 30 min at 37 °C in the dark. The solution was removed, and 1 mL of PBS was added per well. The cells were analyzed using a digital inverted EVOS microscope with a tgBFP laser (390 nm excitation/447 nm emission). Cells were trypsinized and read using a BD FACSARIA flow cytometer (BD FACSAria™ II Flow Cytometer Cell Sorter).CellTracker™ Blue fluorescent probes cross the cell membrane and are transferred to daughter cells but not adjacent cells in a population. Therefore, they are an important tool for monitoring chemotaxis and cell proliferation. As it presents a reaction mediated by glutathione S-transferase, CellTracker Blue probes have also been used for GSH quantification ,29. For p < 0.05). The results are expressed as mean ± standard error of the mean.For all of the tests, data were analyzed using Prism 5.0. Tests for normality of residuals and homogeneity of variances were performed for each variable . Data that did not meet the ANOVA assumptions were transformed. Statistical analysis of total infarct volume data was performed using the one-Way ANOVA test, a non-parametric statistic for purchasing groups, followed by Tukey’s Post-hoc test, considering the significance level (p > 0.05). Cells treated with low concentrations of ozone (2 µg/mL) showed higher rates of cell viability than those that were subjected to hypoxia but did not receive any treatment or only received oxygen (p < 0.05). This indicates that the treatment not only reversed the effects of ischemia but also promoted cell regeneration in these cells. Furthermore, cells treated with 2 µg/mL ozone were significantly similar to those not subjected to hypoxia and those treated with 5 and 10 µg/mL ozone (p > 0.05), showing that these low concentrations are safe and maintain high rates of cell viability. Cells subjected to hypoxia that received no gas or only oxygen had the same cellular viability rate as those treated with 15 and 20 µg/mL of ozone (p > 0.05). Finally, cells treated with 30 µg/mL of ozone showed a decrease in cell viability (p < 0.05), indicating that high concentrations of ozone can harm cells.As expected, the neuroblastoma cells that suffered hypoxia, but received the culture medium without the addition of any gas, presented lower metabolic activity with respect to the control cells, which were not subjected to hypoxia. These results indicate that the protocol resulted in partial cell death, although it was not statistically significant (p > 0.05). Additionally, these cells are similar to those that received no gas, received only oxygen, or were treated with 8 µg/mL ozone (p > 0.05). Cells treated with low concentrations of ozone (2–8 µg/mL) showed better cell metabolism, compared to cell metabolism noted for other ozone treatments (12–30 µg/mL) (p < 0.05), implying that these concentrations are safe and do not promote cell damage. Treating cells with 18 and 30 µg/mL of ozone harmed them, promoting cellular apoptosis compared to the cells in other groups (p < 0.05). These results corroborate the results of the experiments conducted on our neuroblastoma cell lineages.Amniotic membrane stem cells (AMSC) treated with 3 µg/mL of ozone showed no significant difference compared to cells that did not undergo the hypoxia protocol (p > 0.05). Cells that were subjected to hypoxia but received no gas or only oxygen had higher levels of SOD when compared to the group without hypoxia (p < 0.05). It is important to emphasize that the commercial kit allows the quantification of SOD-1 and SOD-2, which are intracellular and mitochondrial enzymes, and SOD-3, which is extracellular and can be detected in serum, plasma, ascites, and synovial fluids. We tried to standardize a protocol for quantifying SOD in the culture medium to mimic the serological levels of the enzyme. Nonetheless, the results are inconsistent, and we do not consider them to be representative.SOD analyses were performed on the culture medium and not on the cells. They were intended to mimic a serological quantification of SOD emitted by cells. SOD activity is associated with the cellular ability to scavenge free radicals. All ozone-treated groups had SOD levels comparable to cells not subjected to hypoxia (p > 0.05). Cells treated with 30 µg/mL of ozone had the lowest levels of the intracellular enzyme, followed by cells of the groups treated with 18 and 12 µg/mL ozone, all of which presented significant differences between each other (p < 0.05). This probably occurred due to the cytotoxic effect of ozone at high concentrations, as one of its by-products is hydrogen peroxide, a substance degraded by the peroxidase under study.We observed a significant decrease in catalase rate following the increase in ozone concentration. Catalase levels of cells not subjected to hypoxia were significantly similar to those treated with no gas, only oxygen, and 3 and 8 µg/mL ozone . Cells treated with 3 or 8 µg/mL of ozone showed higher levels of cellular apoptosis than the cells in the no hypoxia group but lower levels than the cells in other groups (p < 0.05). Cells subjected to hypoxia, but not receiving any gas, behaved similarly to those receiving oxygen (p > 0.05). Nevertheless, they exhibited a lower rate of cellular apoptosis than cells of groups treated with 18 or 30 µg/mL (p < 0.05). Cells treated with 30 µg/mL of ozone showed the highest rates of cell apoptosis compared to cells of all other groups (p < 0.05).Cells that were not subjected to hypoxia showed the lowest levels of cellular apoptosis compared to the other groups . The results have been presented in The Image-iT® Lipid Peroxidation Kit has a lipid peroxidation sensitive agent. After oxidation, the fluorescence changes from red to green, allowing its quantification. As noted, the assessment of lipid peroxidation did not reveal significant differences between any of the groups . Only the groups treated with 12 or 18 µg/mL of ozone presented reduced levels of ROS, compared to the other groups (p < 0.05). ROS levels increased again in cells treated with 30 µg/mL ozone, equaling the levels noted for the other groups (p > 0.05).According to the analyses, low concentrations of ozone (3 and 8 µg/mL) did not show changes in ROS levels, compared to groups that were not subjected to hypoxia without gas and oxygen (p > 0.05) for reduced GSH.The results presented below  show no Importantly, these CellTracker™ Blue fluorescent probes are intended to stain daughter cells to screen the cell population. Few studies have used this probe to quantify GSH, owing to the reaction mediated by glutathione S-transferase. However, we reiterate that this is not the purpose recommended by the manufacturer ,29.3 events). The data is unreliable and inconsistent. We do not recommend using the CellTracker™ Blue probe to quantify GSH in cells under the conditions discussed in this manuscript.Another critical point is that this technique was performed only in duplicate, with a very limited number of cells  were able to repair the effects of ischemia and promoted cell regeneration. This result is consistent with those of the MTT assay for canine amniotic membrane stem cells that underwent the same protocol. The group treated with 3–8 µg/mL showed improved cell metabolism. The results of TUNEL assay indicated that apoptosis rates are lower in cells treated with low ozone concentration (3–8 µg/mL). In all these trials, the harmful effects of ozone were observed at high concentrations (18–30 µg/mL), which promoted higher rates of apoptosis and cell death.An increase in SOD levels after ozone treatment has been described previously ,38,39. WIn contrast with some prior studies ,38, we oAs reported in other studies ,41, we eThe quantification of ROS levels after hypoxia revealed no difference in the control and low ozone-treated groups (3 and 8 µg/mL). The groups treated with 12 or 18 µg/mL of ozone showed a significant decrease in ROS levels, which increased again in the group treated with 30 µg/mL, still being comparable with ROS levels noted for other groups. We expected to observe an increase in ROS levels, as it is one of the by-products of ozone, and the higher its concentration, the greater should be the production of ROS .To quantify levels of reduced GSH, we used a protocol detailed in King et al.  and Casain vitro ischemia. Most studies used ozone for preconditioning cells, which were subjected to hypoxia only after treatment with the gas [Few studies have evaluated the effect of ozone as a treatment for  the gas ,39,47–52 the gas  used ozoin vitro consequences to a tissue treated with ozone therapy after suffering hypoxia.In our study, all groups were initially plated at the same cell concentration. The culture plates were subjected to hypoxia. The cells themselves were not ozonized, but the cell culture medium was. We attempted to re-create the events of an ischemic stroke, wherein brain cells do not receive the gas, and instead, receive its by-products via the bloodstream. In addition, in real life, hardly any patient has sought treatment before disease onset. Patients who have suffered from ischemia and have survived seek therapies to minimize its sequelae. A literature survey did not reveal any publication using this protocol. Therefore, our study has pioneered the protocol for mimicking the Some professionals are against the practice of ozone therapy, owing to concerns regarding the toxicity of ozone at any concentration; however, such a perspective disregards the foundations of physiology and toxicology, as well as the advances in research and science. . Ozone tOne of the recently raised hypotheses about the mechanisms of ozone therapy relates to its effect on blood cells and the extracellular matrix of the intravascular compartment . The intTo conclude, we reported a novel protocol, which involves using ozone after cell damage, as doing so is closer to the real-world scenario experienced by patients.in vitro post-hypoxia ozone treatment in cell cultures, without the need to resuspend them, simulating an ischemic stroke.It is possible to perform In vitro, low concentrations of ozone (2–10 µg/mL) enhanced the cellular metabolism of a neuronal cell line, which may be suggestive of post-hypoxia cell regeneration and reversal of cellular damage caused by hypoxia. High ozone concentrations (above 18 µg/mL) in vitro can cause cell damage.in vitro.Therefore, ozone exhibits a dose-dependent hormetic effect, and at low concentrations, is capable of reversing the effect of ischemia Although further studies are warranted, the use of ozone may be a promising therapeutic complement for treating sequelae in stroke patients. Nevertheless, the concentration of ozone is an essential factor to be considered before prescribing treatment, especially in post-hypoxia conditions, wherein the cells are already damaged.