Intraoperative 3D imaging using cone-beam CT (CBCT) provides improved assessment of implant position and reduction in spine surgery, is used for navigated surgical techniques, and therefore leads to improved quality of care. However, in some cases the image quality is not sufficient to correctly assess pedicle screw position and reduction, especially due to metal artifacts. The aim of this study was to investigate whether changing the acquisition trajectory of the CBCT in relation to the pedicle screw position during dorsal instrumentation of the spine can reduce metal artifacts and consequently improve image quality as well as clinical assessability on the artificial bone model.An artificial bone model was instrumented with pedicle screws in the thoracic and lumbar spine region (Th10 to L5). Then, the acquisition trajectory of the CBCT  to the pedicle screws was systematically changed in 5° steps in angulation (− 30° to + 30°) and swivel (− 30° to + 30°). Subsequently, radiological evaluation was performed by three blinded, qualified raters on image quality using 9 questions  with a score (1–5 points). For statistical evaluation, the image quality of the different acquisition trajectories was compared to the standard acquisition trajectory and checked for significant differences.p < 0.001) as well as the clinical assessability of pedicle screw position (p < 0.001) highly significant with particularly strong effects on subjective image quality in the vertebral pedicle region (d = 1.06). Swivel of the acquisition trajectory significantly improved all queried domains of subjective image quality (p < 0.001) as well as clinical assessability of pedicle screw position (p < 0.001). The data show that maximizing the angulation or swivel angle toward 30° provides the best tested subjective image quality.The angulated acquisition trajectory increased the score for subjective image quality  during dorsal instrumentation of the spine. Dorsal instrumentation with pedicle screws can be considered as gold standard in surgical treatment of traumatic and degenerative spinal fractures , 2. It cAlthough pedicle screw surgery is considered very safe, and the rate of clinically relevant complications is low , the topIn addition to postoperative radiographic control, postoperative CT has been established for verification of reduction and implant position of pedicle screws .Intraoperatively, mobile 2D C-arms are usually used for imaging. However, assessment of reduction and pedicle screw location using 2D fluoroscopy is limited in some cases . Modern A recent study reports immediate intraoperative revision due to screw malposition in 7% of pedicle screws, based on intraoperative CBCT imaging . In the However, due to metal artifacts, which inevitably occur due to the inserted pedicle screws, the assessability of the CBCT images may be limited. In individual cases, this may result in insufficient intraoperative image quality to reliably assess pedicle screw position .Currently so-called MAR  algorithms are used to improve image quality when artifacts in CBCT occur due to metal implants , 16.The purpose of this study is to evaluate whether a change of the acquisition trajectory of the CBCT in relation to the pedicle screws leads to a reduction of metal artifacts and therefore to improvement of image quality and clinical assessability in spine surgery?Hence the hypothesis of this study is: By changing the acquisition trajectory of the CBCT during spine surgery, metal artifacts will be reduced and consequently image quality as well as clinical assessability will be improved.A total of 16 pedicle screws (8 pedicle screw pairs) were inserted into an artificial bone model of the spine with soft tissue sheath  in the vertebral bodies Th10 to L5. For this purpose, a CT of the artificial bone model was acquired using the Airo system  and subsequently instrumented via the dorsal approach using curve navigation  by an experienced spine surgeon. 3D datasets were created of each vertebra at 13 pedicle screw angulation angles and 13 pedicle screw swivel angles .The mobile 3D C-arm “Cios Spin”  was used to acquire the datasets.Standard acquisition trajectory: An implant angle of 0° pedicle screw angulation and 0° pedicle screw swivel is used as the standard acquisition trajectory. To achieve this acquisition trajectory, the pedicle screw pair to be examined was positioned accordingly in the beam path, resulting in different initial positions of the artificial bone model for each vertebral body.Orbital position: In this study orbital position refers to the position of the C-arm in orbital rotation. The 0° orbital position corresponds to an anterior–posterior (AP) image, the 90° orbital position to a lateral image.Angulation Fig. : AngulatSwivel Fig. : Swivel The tilt table was made of polymethyl methacrylate (acrylic glass), as this material does produce almost no additional artifacts in the ROI (region of interest). The tilt table was attached to a wooden substructure. The artificial bone model was fixed in the corresponding foam tray on the tilt table using Velcro straps Fig. .Fig. 3ExThe C-arm angulation of the “Cios spin” is limited to 15° for the acquisition of 3D scans. By using additional calibrations, extending the angulation range of the 3D C-arm is potentially possible. To extend the angulation range and to eliminate differences in image quality due to calibration the angulation was adjusted using only the tilt table.Subsequently, pedicle screw angulation was adjusted in 5° increments from − 30° to + 30° in relation to the standard acquisition trajectory. In each position, a lateral image  and an AP image  were taken to check the position of the pedicle screw pair for correct location in the iso-center of the 3D C-arm.The acrylic tilt table from the angulation experiments was now converted to a rotary table to adjust the artificial bone model in pedicle screw swivel. For this purpose, a base plate was fixed on an examination table with a carbon fiber work plate. After that the rotation plate was attached to this base plate via a screw, which served as a pivot. This rotation plate carried the artificial bone model with foam shell.Subsequently the pedicle screw swivel was adjusted in 5° increments from − 30° to + 30° in relation to the standard acquisition trajectory. In each position, a lateral image  and an AP image  were taken to check the position of the pedicle screw pair for correct location in the iso-center of the 3D C-arm.First, the 3D datasets were sorted by angle in the OsiriX Lite Viewer . Then, the metadata were removed from the 3D datasets and replaced by a blinded 5-digit code, which allowed a unique assignment of the 3D datasets in retrospect.Hereupon, in a randomized manner by 3 independent examiners, the radiological evaluation regarding the image quality was performed by means of a questionnaire.The dorsal vertebral body surface can be fully assessed.Fragments in the spinal canal can be definitively excluded.The width of the spinal column can be clearly determined.The edges of the vertebral pedicles can be fully assessed.Screw perforations of the vertebral pedicles can be clearly assessed.The width of the screws can be reliably determined in relation to the width of the pedicles.The length of the screws can be clearly determined.Ventral perforation of the screws can be reliably assessed.The image quality allows a reliable assessment from a clinical point of view.The questionnaire consists of 9 statements to be evaluated (hereafter referred to as "questions"), which cover different parts of the image quality:Category 1 Questions 1–3 consider image quality of the dorsal vertebral body and spinal canal.Category 2 Questions 4–6 consider the image quality of the vertebral pedicles.Category 3 Questions 7–8 consider screw length and the image quality of the ventral vertebral body.Category 4 Question 9 asked about clinical assessability.These questions can be divided into four categories:The mean score over the entire questionnaire (Q1–Q9) is used as a measure of “subjective image quality.” Individual questions were assessed with a score from 1 to 5, with a score of 1 representing the worst score and a score of 5 representing the best score. Radiological subjective assessment of metal artifacts on CBCT has been performed several times in the past using a 5-point scale , 18. TheThe program SPSS version 26  was used for statistical analysis. In the test series "pedicle screw angulation," the effect of the trajectory on the subjective image quality was determined using a repeated-measures analysis of variance (ANOVA). In the test series "pedicle screw swivel" the preexisting angulation of the pedicle screws was also included as a covariate in the ANOVA.p value < 0.05 is assumed to be statistically significant (corresponding to 95% significance level). A p value < 0.01 is considered highly significant.A  < 0.5—small effect; 0.5–0.8—medium effect; > 0.8—large effect.Cohen's d is used as a measure of effect size. The value is interpreted as follows:For analysis of the intraclass correlation coefficient (ICC) the software R  was used. The value is interpreted as recommended by Cicchetti : < 0.40—3D datasets were generated from 8 pedicle screw pairs in 13 pedicle screw angulation angles and 13 pedicle screw swivel angles . Thus, 103 3D datasets were created in the pedicle screw angulation study and 104 3D datasets were created in the pedicle screw swivel study.In the standard acquisition trajectory the average subjective image quality  was 2.83 ± 1.11. Due to the wide variation between the scores of the cranial and caudal spinal levels, two spine areas (Th10–L1 and L2–L5) were defined in the analysis of the datasets. Image quality in the standard acquisition trajectory of spine area 1 (Th10–L1) averaged 2.04 ± 0.89, while spine area 2 (L2–L5) achieved a mean of 3.62 ± 0.65.p = 0.728); therefore, absolute values of the angular change are used for evaluation.The direction of pedicle screw angulation has no significant effect on subjective image quality . The best subjective image quality was always achieved at an angulation of 30°. As can be seen in Fig. Subjective image quality can be significantly improved by angulation of the acquisition trajectory to the pedicle screws with increasing angle (p < 0.001). In all four categories the score is significantly improved by pedicle screw angulation ; screw length and ventral vertebral body (p = 0.001), clinical assessability (p < 0.001), spinal canal and dorsal vertebral body (p = 0.029).Over the vertebral bodies Th10—L1 (spine area 1), there is a significant increase in subjective image quality due to pedicle screw angulation . In 3/4 categories a significant increase of the score by pedicle screw angulation can be observed ; screw length and ventral vertebral body (p = 0.003); clinical assessability (p = 0.007); spinal canal and dorsal vertebral body (p = 0.209).Over the vertebral bodies L2–L5 (spine area 2), there is also a significant effect of pedicle screw angulation on subjective image quality ; therefore, absolute values of the angular change are used for evaluation.The direction of pedicle screw swivel has no significant effect on subjective image quality . As can be seen in Fig. Subjective image quality can be significantly improved with increasing angle by pedicle screw swivel (p < 0.001). Pedicle screw swivel also has a significant positive effect on all categories of the questionnaire ; screw length and ventral vertebral body (p = 0.001), clinical assessability (p < 0.001); spinal canal and dorsal vertebral body (p < 0.001).Over the vertebral bodies Th10–L1 (spine area 1) a significant improvement of the subjective image quality by pedicle screw swivel is shown . While the category "vertebral pedicle" is significantly improved by pedicle screw swivel (p = 0.025), no significant increase of the score is observed in the remaining categories: screw length and ventral vertebral body (p = 0.228); clinical assessability (p = 0.109); spinal canal and the dorsal vertebral body (p = 0.119) , there is also a significant effect of pedicle screw swivel on subjective image quality . The best subjective image quality was achieved by L5 with a pedicle screw angulation of 30° (4.63 ± 0.55).d = 1.06), especially in the thoracolumbar transition (spine area 1) (d = 1.52).The effect of changed acquisition trajectory is strongest on the vertebral pedicles (Δ = 2.28) and L4 the least (Δ = 0.77) from the changed acquisition trajectory.A view at the vertebrae examined shows: Looking at the delta between the pedicle screw angulation angle with the worst and best subjective image quality score, L1 benefits the most  in all categories of the questionnaire. In spine area 2 (L2–L5), however, there is only a significant improvement in the category of “vertebral pedicles.” Considering the effect sizes it also becomes apparent that even the significant results in spine area 2 are very weak compared to spine area 1.These different results are caused by the fact that in spine area 1 the influence of the pedicle screw angulation was still small, because they were relatively close to the 0° of the standard acquisition trajectory  in spine region 2; on the other hand, significantly stronger pedicle screw angulation angles  and correspondingly stronger effects on the subjective image quality were seen. The fact that the effect in spine region 2 is so small is also evident in the comparison of the subjective image quality between 0° pedicle screw swivel and 30° pedicle screw swivel. While in spine region 1 the subjective image quality in the baseline situation at 0° pedicle screw swivel always reached values in the lower range of the score (< 3) on average, in spine region 2 it was permanently above the score of 3 on average. Corresponding to the different baseline situation, the increase in quality over the increasing pedicle screw swivel angle turns out to be smaller. The effect size remains small.It can be assumed that the strong influence of pedicle screw angulation, as already seen in the experiments on pedicle screw angulation on the artificial bone model, has ensured the good image quality at 0° pedicle screw swivel in spine region 2 and thus reduces the possibilities for increasing the subjective image quality by pedicle screw swivel.To investigate this further, future studies would need to examine the controlled combination of pedicle screw angulation and pedicle screw swivel.A literature review did not reveal any relevant results on the influence of swivel on the image quality of 3D datasets. This is also due to the fact that common, stationary CT scanners do not offer direct adjustment options to change the swivel.In CBCT, intraoperatively, the 3D C-arm is probably already frequently run with a swivel angle over the pair of pedicle screws to be examined due to the limited space available. However, there are no measurements or experiments on the extent to which this angle influences image quality and the occurrence of metal artifacts.A possible explanation for the improved image quality due to swivel could be that with precise positioning of the 3D C-arm, the central beam no longer runs centrally through both pedicle screws but radiates between them. As a result, there is less metal in the beam path and the image quality increases. This space between the two pedicle screws depends on the size of the respective vertebral body, which means that it is not always easy to target.Compared to changing the angulation, changing the swivel of the 3D C-arm has the advantage that there is no need to intervene in the calibration of the 3D C-arm. In addition, the adjustment can be done faster and without significantly more effort than with normal intraoperative 3D imaging.The advantage of the swivel is that there is no change at the C-arm settings compared to the normal CBCT workflow. The C-arm simply has to be moved "diagonally" over the ROI.The disadvantage, however, is that the influence of the swivel on the reduction of metal artifacts is less effective than using the more complex angulation.In the "pedicle screw angulation" test series, as in the "pedicle screw swivel" test series, there is a dichotomy of subjective image quality in the starting position (standard acquisition trajectory). Consequently, there is a different potential for increase in image quality, which is the reason for a separate evaluation of two spine areas (Th10–L1 and L2–L5) with regard to significance and effect strength.In the "pedicle screw swivel" test series, the different increase potentials in the spinal regions can be explained by the influence of the present pedicle screw angulation as described above.In the “pedicle screw angulation” test series, there is no obvious covariate between the two areas. At this point it is helpful to take a look at the datasets created. In spine area 2, the occurrence of metal artifacts was also subjectively significantly minimized by pedicle screw angulation. However, due to the increasing size of the vertebral pedicles in area 2 compared to area 1, the metal artifacts in area 2 only slightly mask the pedicle cortical bone, which is why the assessability of this clinically very relevant structure remains good even without angulation adjustment, even in the presence of severe metal artifacts.The remarkable influence of pedicle screw angulation as well as pedicle screw swivel on subjective image quality and consequently on the score for "assessability from a clinical point of view" shows that changes in acquisition trajectory are of clinical relevance.Improved intraoperative image quality allows more precise detection of pedicle cortical injuries and better assessment of screw perforations. Especially in the thoracolumbar transition, which is characterized by relatively narrow vertebral pedicles compared to the lumbar spine, the increase in image quality can make a decisive contribution to the reliable assessment of screw position. The fact that most spinal injuries also occur in this region  further The intraoperative use of CBCT can significantly lower the postoperative revision rate, reduce the radiation exposure for the OR staff, and minimize potential postoperative complications. At the same time, however, there are individual cases in which the intraoperative image quality of CBCT is insufficient to reliably assess the position of the screw due to artifacts . This haDue to the significant increase in subjective image quality because of pedicle screw angulation and pedicle screw swivel, it can be assumed that the reported positive aspects of intraoperative CBCT can be further extended by optimizing image quality.Angulation as well as swivel of the acquisition trajectory leads to significantly improved image quality in intraoperative 3D imaging (CBCT) with pedicle screws in situ. Implementation of this knowledge into daily surgical practice requires little additional effort. Sufficient angulation can be achieved by tilting the 3D C-arm or the operating table and by combining with the anatomically determined pedicle screw angulation.With the Swivel, even less effort is required, as the 3D C-arm can simply be moved at an angle over the pedicle screws.For more precise adjustment of the pedicle screw angulation angles and to extend the range to 30°, a tilt table was used, which rotated the pedicle screws in relation to the 3D C-arm.In the intraoperative setting, this is not possible to this extent with the aid of the operating table; here, the pedicle screw angulation may have to be adjusted or extended via the 3D C-arm. However, this C-arm angulation is currently only possible to an extent of 15° in the case of the Siemens "Cios Spin" device used.Although the 15° 3D C-arm angulation initially appears to be a major limitation in relation to the possible quality improvement with 30° pedicle screw angulation, this problem appears less relevant on closer examination: On the one hand, many pedicle screws are already angulated in the beam path, which means that an addition of 15° should generally result in sufficient pedicle screw angulation in the beam path. Alternatively, the angle can be further increased by tilting the operating table additionally. On the other hand, C-arm angulation of up to 30° is also technically possible with the “Cios Spin” 3D C-arm. However, this is currently limited to 15° due to the lack of dynamic calibration.Due to the experimental setting, the angulated position of the pedicle screws could not be compensated for in the pedicle screw swivel experiments with the artificial bone model. Consequently, pedicle screw angulation also contributes to the image quality, but this was considered in the statistical evaluation.The results of experiments on the artificial bone model may differ from results obtained in a study on a human specimen or on a patient. Although the artificial bone model has a soft tissue sheath and was developed specifically for the medical imaging field, the transferability of results to patient use may be limited.Rater agreement was in the "fair" range for both swivel and angulation. Several factors have an influence here. Due to the relatively narrow scale with 5 points per question, even small deviations are associated with a strong decrease in the ICC.While the ICC is also frequently used for metric measurements and can reach correspondingly high values here, the present data collection is a subjective assessment of the data. Therefore, values cannot be expected to be as high as with a metric measurement.Angulation as well as swivel of the acquisition trajectory leads to improved image quality in intraoperative 3D imaging (CBCT) while keeping the iso-center constant, which is confirmed by the outstanding effect size, especially in the case of angulation in the thoracolumbar region.For the assessment of single pedicle screw pairs located in the iso-center, this study can demonstrate that in cases of unclear screw location, difficult assessment due to thin vertebral pedicles, or poor image quality, a modified acquisition trajectory can provide the decisive contribution for an intraoperative revision decision.With improved intraoperative imaging capabilities, potentially fewer revision surgery can be expected, leading to improved patient outcome.


Introduction: The P2X3 receptor (P2X3R), an ATP-gated non-selective cation channel of the P2X receptor family, is expressed in sensory neurons and involved in nociception. P2X3R inhibition was shown to reduce chronic and neuropathic pain. In a previous screening of 2000 approved drugs, natural products, and bioactive substances, various non-steroidal anti-inflammatory drugs (NSAIDs) were found to inhibit P2X3R-mediated currents.Methods: To investigate whether the inhibition of P2X receptors contributes to the analgesic effect of NSAIDs, we characterized the potency and selectivity of various NSAIDs at P2X3R and other P2XR subtypes using two-electrode voltage clamp electrophysiology.Results: We identified diclofenac as a hP2X3R and hP2X2/3R antagonist with micromolar potency . A weaker inhibition of hP2X1R, hP2X4R, and hP2X7R by diclofenac was determined. Flufenamic acid (FFA) inhibited hP2X3R, rP2X3R, and hP2X7R , calling into question its use as a non-selective ion channel blocker, when P2XR-mediated currents are under study. Inhibition of hP2X3R or hP2X2/3R by diclofenac could be overcome by prolonged ATP application or increasing concentrations of the agonist α,β-meATP, respectively, indicating competition of diclofenac and the agonists. Molecular dynamics simulation showed that diclofenac largely overlaps with ATP bound to the open state of the hP2X3R. Our results suggest a competitive antagonism through which diclofenac, by interacting with residues of the ATP-binding site, left flipper, and dorsal fin domains, inhibits the gating of P2X3R by conformational fixation of the left flipper and dorsal fin domains. In summary, we demonstrate the inhibition of the human P2X3 receptor by various NSAIDs. Diclofenac proved to be the most effective antagonist with a strong inhibition of hP2X3R and hP2X2/3R and a weaker inhibition of hP2X1R, hP2X4R, and hP2X7R.Discussion: Considering their involvement in nociception, inhibition of hP2X3R and hP2X2/3R by micromolar concentrations of diclofenac, which are rarely reached in the therapeutic range, may play a minor role in analgesia compared to the high-potency cyclooxygenase inhibition but may explain the known side effect of taste disturbances caused by diclofenac. P2X receptors (P2XR) constitute a family of non-selective cation channels gated by extracellular ATP . Seven dThe P2X3R, which is expressed in sensory neurons , plays aThe significant role of P2X3R in nociception makes P2X3R a promising target for the development of new analgesics . Howeverversus P2X2/3R  can cause irreversible inactivation of COX through acetylation of serine 530 , it appears interesting to investigate whether already approved drugs do affect the P2X3R-mediated responses. For this purpose, a screening of 2000 approved drugs, natural products, and bioactive substances was performed in a previous study of our group. In this screening, aurintricarboxylic acid (ATA) was identified as a potent P2X1R and P2X3R antagonist . An inhirine 530 .Considering the involvement of P2X3R in nociception, it is conceivable that inhibition of P2X3R by NSAIDs represents an additional mode of action besides COX inhibition. To investigate whether the inhibition of P2XR contributes to the analgesic effect of NSAIDs, we determined the potency and selectivity of various NSAIDs at P2X3R and other P2XR subtypes using two-electrode voltage clamp (TEVC) electrophysiology. The investigated NSAIDs included diclofenac, ibuprofen, flunixin, meclofenamic acid, naproxen, and flufenamic acid (FFA). The latter was chosen because it additionally plays an important role in research as a non-selective ion channel blocker .In the present study, we have for the first time shown that diclofenac is a hP2X3R and hP2X2/3R antagonist with micromolar potency. Our results strongly support a competitive antagonism through which diclofenac, by interacting with residues of the ATP-binding site, left flipper, and dorsal fin domains, inhibits the gating of P2X3R by conformational fixation of the left flipper and dorsal fin domains. In addition, a weaker inhibition of hP2X1R, hP2X4R, and hP2X7R was shown. Less potent inhibition of hP2X3R was observed for all other investigated NSAIDs. FFA was proven to significantly inhibit hP2X3R, rP2X3R, and hP2X7R.The investigated NSAIDs and most standard chemicals were purchased from Sigma–Aldrich/Merck , if not otherwise specified. ATP sodium salt and α,β-meATP were purchased from Roche Diagnostics  and Tocris Bioscience , respectively. Collagenase type 2 was purchased from Worthington Biochemical Corp .20RMVL23KVIV23S26N-hP2X1R, S15V-rP2X3R, and His-S15V-hP2X3R were the same as used in previous research  containing 90 mM NaCl, 1 mM KCl, 1 mM CaCl2, 1 mM MgCl2, and 10 mM HEPES  adjusted to pH 7.4 with NaOH and supplemented with 50 μg/ml gentamicin . The procedures followed for the maintenance of and the surgical treatment for X. laevis adults were approved by the governmental animal care and use committee of the State Agency for Nature, Environment, and Consumer Protection , in compliance with Directive 2010/63/EU of the European Parliament and of the Council on the protection of animals used for scientific purpose.Oocyte expression plasmids encoding the wild-type (wt) and N-terminally His-tagged (His-) fusion constructs of the hP2X2R, hP2X3R, hP2X4R, and hP2X7R, and the mutant His-research . In mostresearch . cRNA wa3 nl see . To expr− solution  was used to avoid bias due to calcium-activated chloride channels (CaCC) endogenously expressed in X. laevis oocytes . The agonists ATP or α,β-meATP and the investigated NSAIDs were diluted in ORi− on the day of the recording. The following agonist concentrations were used for the different P2X subtypes: 1 μM ATP ; 10 μM ATP (hP2X2R and hP2X4R); 300 μM free ATP4- (hP2X7R); and 1 μM α,β-meATP (hP2X2/3R). A peak current protocol (15V-P2X3R), and a steady-state protocol  as previously described . Calcium oocytes . For rec, pH 7.4 . The oocprotocol  was usedprotocol  was usedprotocol . The app2 for 120 ±30 min. After approximately 60 min in the digestion medium, the DRG were triturated using a glass pipette. After the full incubation time, they were triturated thrice using custom-pulled glass pipettes with decreasing tip diameters (from ∼1.1–1.2 to ∼0.3–0.4 mm). For further purification, the DRG were centrifuged at 500 G and 4°C twice for 4 minutes each, and the pellets were suspended in DMEM F12 with 10% FBS. They were subsequently separated from lighter cell fragments and myelin by centrifugation of a Percoll gradient containing a 60% Percoll and a 25% Percoll gradient for 20 min at 500 G. DRG neurons were plated on coverslips coated with poly-D-lysine (100 μg/ml), laminin (10 μg/ml), and fibronectin (10 μg/ml). Neurons were then cultured in neurobasal A medium supplemented with B27, penicillin, streptomycin, and L-glutamine and used for voltage-clamp recordings after 12–72 h in culture.The dorsal root ganglia (DRG) of pigs were sampled according to the 3R criteria for reductions in animal use, as leftovers from previous independent animal studies . For this purpose, pigs of the German Landrace breed, with an average age of 15 weeks (14.6 SD2.7) and weight of 47.3 kg (SD11.2), were euthanized using an overdose of pentobarbital 60 mg/kg body weight. Subsequently, the DRG were collected. The DRG of pigs were transferred on ice, and fine excision was performed in ice-cold DMEM F12 medium containing 10% FBS and treated with 1 mg/ml collagenase P, 1 mg/ml trypsin T1426, and 0.1 mg/ml DNAse for digestion. Then, the DRG were cut into small pieces inside the digestion medium for surface enlargement and incubated at 37°C and 5% CO2, 1 mM CaCl2, 10 mM HEPES, and 20 mM glucose (adjusted to pH 7.4 using NaOH). The liquid junction potential was corrected by −7.8 mV. Membrane currents were measured at room temperature, with a holding potential of −77.8 mV, using a HEKA EPC-10 USB amplifier. α, β-methylene ATP (10 μM) and 100 μM diclofenac were applied using a gravity-driven perfusion system during the recordings. PatchMaster/FitMaster software (HEKA Electronics) and IGOR Pro (WaveMetrics) were used for data acquisition and analysis. Signals were digitized at a sampling rate of 5 kHz. The low-pass filter frequency was set to 10 kHz. Series resistance compensation was between 2.5 and 11.1 MΩ.Whole-cell voltage-clamp recordings of DRG neurons were performed using glass electrodes with micropipette tip resistances of 1.3–3.5 MΩ, pulled and fire-polished with a Zeitz DMZ-puller. The intracellular solution contained 10 mM NaCl, 140 mM CsF, 10 mM HEPES, 1 mM EGTA, 5 mM glucose, and 5 mM TEA-Cl (adjusted to pH 7.3 using CsOH). The extracellular bathing solution contained 140 mM NaCl, 3 mM KCl, 1 mM MgClThe recorded TEVC currents were analyzed using CellWorks Reader 6.2.2  and Microsoft Excel . The displayed current traces were generated using IGOR Pro 6.21  and edited with Microsoft PowerPoint . To generate concentration–response curves, non-linear regression analysis was performed using GraphPad Prism 5 .50 values were calculated by normalizing ATP-induced responses to the control responses . The four-parameter Hill equation , and IC50 is the antagonist concentration that causes 50% inhibition of the response elicited by a given agonist concentration. The ratio between the response in the presence of a certain antagonist concentration and the control response in the absence of the antagonist is indicated as “% control current”.IIn the case of fast-desensitizing P2XR subtypes (P2X1R and P2X3R), ATP is applied five times in repetition, and the ATP-induced current amplitude in the presence (after pre-incubation) of the antagonist (fourth application) is compared to the arithmetic mean of flanking control ATP-induced current amplitudes in the absence of the antagonist (third and fifth ATP application). Since some of the investigated NSAIDs showed an enduring, potentially irreversible inhibitory effect on the current amplitude, only the preceding (third) ATP-induced current amplitude was used to calculate the control current. The typical run-down of current amplitudes between consecutive, repetitive ATP applications was considered by applying a correction factor. The correction factor was calculated as the ratio of the fourth ATP-induced current amplitude to the third amplitude Eq.  to obtaiIn the case of hP2X7R, the control current in the absence of the antagonist had to be extrapolated  presumin50 values are displayed as geometric means with 95% confidence intervals (95% CI). All other values (including % control current or % inhibition) are presented as arithmetic means ± SEM, if not otherwise stated. Values were compared using the t-test or one-/two-way analysis of variance and multiple comparison tests as indicated. Statistical significance was set at p < .05.The ICg_membed functionality (The human ionotropic cation-selective ATP receptor P2X3 was modeled based on its structure in the apo-closed state (PDBID: 5SVJ)  and embeionality  in GROMAionality . The staionality  version A pressure of 1 bar was applied semi-isotropically with a Berendsen barostat  using a + and Cl− were added to provide a bulk concentration of approximately 50 mM NaCl. The crystal structure of hP2X3R in complex with the AF-219 negative allosteric modulator (PDB-ID: 5YVE) was aligned with apo-state hP2X3 (PDB-ID: 5SVJ); one diclofenac molecule per protomer was then fitted onto the AF-219 molecule using PyMOL and placed into the apo-state structure. Seven independent systems, each of the wildtype P2X3 apo-closed state and L191A apo-closed state, were simulated for more than 200 ns each, preceded by equilibration for about 200 ns: first with restraints on all heavy atoms and lipids in the z-direction, second on all heavy atoms, and third on backbone atoms only. All trajectories that showed a stable binding of diclofenac were selected and clustered with the GROMACS tool gmx cluster using the GROMOS algorithm; the cut-off for RMSD differences in a cluster was set to 0.35 nm.Nahttps://charmm-gui.org/). The initial molecular geometry and charge assignments were further optimized using the force-field toolkit (ffTK) (via CHARMM-GUI (https://www.ks.uiuc.edu/Research/vmd/plugins/fftk) and the updated tutorial .Initial force-field parameters for diclofenac were generated according to the CHARMM generalized force field (CGenFF) , using tt (ffTK)  version t (ffTK) . The ffTt (ffTK)  5.0.3, ft (ffTK)  engine. ARMM-GUI . New bon15V-rP2X3R-mediated currents . Furthermore, we included the NSAIDs ibuprofen and naproxen in our investigations because these are used extensively in daily practice. The structural formulas of the NSAIDs investigated are shown in In a previous screening of 2000 approved drugs, natural products, and bioactive substances, various NSAIDs were found to inhibit Scurrents . These icurrents . Since p by TEVC . To valiA peak current protocol including a 30-s preincubation of the antagonist was applied  to deter15V-hP2X3 in 20KVIV23/26N-hP2X1 in 50 value of 138.2 µM  than at hP2X3R . However, it should be noted that the use of different agonists  complicates the assessment of a quantitative comparative analysis.In summary, diclofenac was shown to be significantly more potent at hP2X2/3R  as an unselective ion channel blocker to inhibit non-specific chloride conductance in the absence of divalent ions . Howeverrotocols , a free tely 33% , which s15V mutant of P2X3R desensitizes slowly  or a lower agonist potency of ATP remains unknown. A competitive mechanism of action is also compatible with our findings of diclofenac inhibition as well as FFA inhibition of the L191A/S15V-hP2X3R mutant shown in 191A/S15V-hP2X3R, showing the effects of 10 µM diclofenac or 30 µM FFA on ATP-induced currents. The initial inhibition by diclofenac or FFA at the beginning of the co-application could be overcome by prolonged ATP co-application (arrow), suggesting the competitive binding of ATP and the antagonist to the ATP-binding site. However, we did not quantify these findings because the time course of solution exchange may have varied between experiments, which may affect the accuracy and thus precise quantification.To further support the competitive nature of the inhibition and to exclude the possibility that diclofenac does bind in the negative allosteric site of hP2X3R as do other modulators  such as gefapixant (formerly AF-219)  or ATA , we examV-hP2X3R ] of the V-hP2X3R . These wV-hP2X3R , suggestV-hP2X3R  or ATA   were analyzed. Currents elicited by 10 μM α,β-meATP were found in medium-sized (∼35–60 μm diameter) porcine DRG neurons. 10 μM α,β-meATP was applied repeatedly every 3 min for 3 s duration onto cultured porcine DRG neurons . Whole-cn = 11) . Thus, dtes c.f. .50 values of 221.7 µM (95% CI: 98.9–497 µM) and 264.1 µM (95% CI: 56.9–612 µM) were determined at hP2X3R and rP2X3R, respectively  and COX2  . The effect of diclofenac besides COX inhibition has also been studied by other groups, such as Considering the involvement of hP2X3R in nociception, it is conceivable that inhibition of hP2X3R contributes to the analgesic effect of NSAIDs and represents an additional mode of action besides COX inhibition. However, plasma levels and IC tissues . When in tissues . Similar tissues . Accordi.038 µM)  than at Similar to our findings for diclofenac, Selectivity profiling of diclofenac at different P2XR subtypes showed strong inhibition of hP2X3R and hP2X2/3R and weaker inhibition of hP2X1R, hP2X4R, and hP2X7R. The rank order of its potencies is as follows: hP2X2/3R > hP2X3R > hP2X1R > hP2X4R > hP2X7R.50 337.8 μM) and COX  should be kept in mind.Diclofenac had a similar maximum inhibitory effect and potency at hP2X1R and hP2X3R. In contrast to its potentially irreversible effect on hP2X3R, diclofenac seemed to exert a reversible inhibitory effect on hP2X1R. Inhibition of hP2X1R, which is involved in inflammatory responses , might cConsidering its low potency at hP2X4R and hP2X7R, it is unlikely that inhibition of these P2X subtypes, which are involved in nociception , contrib50 76.7 µM) than at hP2X3R (IC50 138.2 µM). However, it should be kept in mind that the use of the correction factor in hP2X3R measurements and the use of different agonists at hP2X2/3 and hP2X3R imply a bias that may affect an accurate, direct quantitative comparison. Since the run-down varies between different recordings, the inhibitory effect of diclofenac on hP2X3R might be underestimated.Remarkably, diclofenac had a weak potentiating effect on hP2X2R-mediated currents. Regarding its strong inhibitory effect on hP2X3R and its weak potentiating effect on hP2X2R, a predominantly inhibitory effect on the heterotrimeric hP2X2/3 receptor could have been expected. Surprisingly, diclofenac proved to be more potent at hP2X2/3R (IChttp://www.adrreports.eu/de/). It must also be assumed that there are a high number of unreported cases. In a prospective, randomized clinical trial regarding the postoperative administration of diclofenac, 58% of patients reported an impaired taste sensation  the N190A and the G189R mutants of the negative allosteric binding site  inhibition by 30 µM diclofenac of hP2X2/3R could be overcome by increasing concentrations of the agonist α,β-meATP; 2) at Ling site , which mIn addition, our extensive all-atom molecular dynamics simulations have shown that the most common binding pose of diclofenac at hP2X3R largely overlaps with ATP bound to the open-state conformation of hP2X3R. Furthermore, we show by RMSF analysis that diclofenac when bound to hP2X3R alters the conformational flexibility of the left flipper and dorsal fin domains, crucially implicated in the ATP-induced gating of hP2X3R . Our simvia a similar mechanism of action to TNP-ATP  to diclofenac (IC50 138.2 µM). In contrast to diclofenac, FFA did inhibit rat P2X3R-mediated currents , indicating a weaker selectivity of FFA toward the human P2X3R. As FFA is usually applied transdermally, plasma levels do not exceed 180 nM even with repetitive application , which was also shown to be effective in antagonizing native P2X2/3R-mediated responses in pig DRG neurons. Considering their involvement in nociception, the inhibition of hP2X3R and hP2X2/3R by micromolar concentrations of diclofenac may contribute to the analgesic effect of diclofenac and represent an additional, although less potent, mode of action besides the well-known COX inhibition. Our results support the presence of a competitive antagonism through which diclofenac, by interacting with residues of the ATP-binding site, left flipper, and dorsal fin domains, inhibits the gating of P2X3R by conformational fixation of the left flipper and dorsal fin domains. FFA proved to inhibit hP2X3R, rP2X3R, and hP2X7R, calling into question its use as a non-selective ion channel blocker, when P2XR-mediated responses are under study.In a previous screening of 2000 approved drugs, natural products, and bioactive substances, various NSAIDs were found to inhibit S15V-rP2X3R-mediated currents . Using T


Background: While the protective effects of n-3 polyunsaturated fatty acids (PUFAs) on cardiac ischemia-reperfusion (IR) injury have been previously reported, limited data are available regarding how these fatty acids affect membrane receptors and their downstream signaling following IR injury. We aimed to identify potential receptors activated by n-3 PUFAs in IR hearts to understand the regulatory mechanisms of these receptors.Methods: We used fat-1 mice, which naturally have elevated levels of n-3 PUFAs, and C57BL/6J mice as a control group to create a myocardial IR injury model through Langendorff perfusion. We assessed the impact of endogenous n-3 PUFAs on left ventricular function, myocardial infarct size, myocardial apoptosis, and ATP production. RNA sequencing (RNA-seq) and bioinformatics analysis were conducted to identify molecular targets affected by n-3 PUFAs. Based on these analyses we then treated IR hearts of WT and fat-1 mice with an antagonist (ML221) or an agonist (apelin-13) for the predicted receptor to assess cardiac contractile function and intracellular signaling pathways. An in vitro hypoxia-reoxygenation (HR) model was also used to confirm the effects of n-3 PUFAs on the examined intracellular signaling pathways.Results: Endogenous n-3 PUFAs protected cardiac structure and function in post-IR hearts, and modulated phosphorylation patterns in the PI3K-AKT-mTOR signaling pathways. RNA-seq analysis revealed that n-3 PUFAs affected multiple biological processes as well as levels of the apelin receptor (APLNR). Consistent with a role for the PLNNR, ML221 synchronized the activation of the PI3K-AKT-mTOR signaling axis, suppressed the expression of PKCδ and phosphorylated p38α, upregulated PKCε expression, upregulated or restored the phosphorylation of myofilaments, and prevented myocardial injury and contractile dysfunction in WT IR hearts. By contrast, apelin-13 disrupted the PI3K-AKT-mTOR signaling axis in post-IR fat-1 hearts. The phosphorylation signaling targeted by APLNR inhibition in post-IR fat-1 hearts was also observed after treating HR cells with eicosatetraenoic acid (EPA).Conclusion: Endogenous n-3 PUFAs protect against post-IR injury and preserve cardiac contractile function possibly through APLNR inhibition. This inhibition synchronizes the PI3K-AKT-mTOR axis, suppresses detrimental phosphorylation signaling, and restores or increases myofilament phosphorylation in post-IR hearts. The beneficial effects observed in fat-1 transgenic mouse hearts can be attributed, at least in part, to elevated EPA levels. This study is the first to demonstrate that n-3 PUFAs protect hearts against IR injury through APLNR inhibition. According to the American Heart Association (AHA) publication from the Global Burden of Disease Study 2020, the estimated incidence of ischemic heart disease is approximately 240 million . ReperfuReperfusion injury can lead to heart failure (in approximately 25% of cases) and death (in approximately 10% of cases) in patients treated for acute myocardial infarction . It ofteA previous epidemiological study showed that Greenland Eskimos, with higher blood eicosatetraenoic acid (EPA) levels, had a lower incidence of myocardial infarction than did Danish participants . More reN-3 PUFAs, including alpha-linolenic acid (ALA), EPA, and docosahexaenoic acid (DHA), are primarily obtained from dietary sources, with a small amount of EPA/DHA converted from ALA. These fatty acids can be metabolized through enzymatic, non-enzymatic, or free radical-catalyzed pathways to produce oxylipins, exerting various functions . Their sRegarding intracellular regulation, evidence suggests that phosphorylation signaling plays a central role in n-3 PUFA-dependent cellular functions. For example, n-3 PUFAs regulate protein kinase C (PKC), mitogen-activated protein kinase (MAPK), and phosphatidylinositol 3-kinase (PI3K)/AKT to protect ischemic hearts . For insHowever, while the link between n-3 PUFAs and intracellular phosphorylation signaling has been established, limited data exist on the membrane receptors involved. Fatty acid receptor 4 (FFA4) activation was found to regulate PPARγ-dependent sympathetic innervation in n-3 PUFA-protected infarcted rat hearts . Neverthfat-1) from Caenorhabditis elegans. This enzyme converts n-6 PUFAs into n-3 PUFAs, consequently elevating the endogenous levels of n-3 PUFAs in fat-1 mice (fat-1 mice and wild-type (WT) C57BL/6J mice were bred and maintained in a specific pathogen-free (SPF) mouse facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) and licensed by the Department of Science and Technology of Guangdong Province, China (SYXK[YUE] 2021-0122). Two primer pairs were used for genotyping the transgenic mice, namely, 5′-AGTGGCCTCTTCCAGAAATG-3′ (forward) and 5′-TGCGACTGTGTCTGATTTCC-3′ (reverse) as well as 5′-CACAGTGGGTAACTGGATGC-3′ (forward) and 5′-GCTAACCATGTTCATGCCTTC-3` (reverse). The ambient temperature and humidity were 24°C ± 2°C and 40%–60%, respectively, with a 12-h light-dark cycle, and the mice had ad libitum access to water and provided a regular rodent diet. All animal experiments were conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee of the Guangdong Laboratory Animals Monitoring Institute.Transgenic mice, kindly donated by Dr. Jing X. Kang from the Harvard Medical School , were developed by expressing the n-3 fatty acid desaturase gene  and agonist (apelin-13) of a specific gene-encoding protein were used to determine the associated signaling pathways. The experimental design is illustrated in WT and fat-1 mouse hearts were ground in liquid nitrogen. Methanol (150 μL), methyl tert-butyl ether (200 μL), and 36% of phosphoric acid (50 μL) were added to heart samples and centrifuged at 4°C and 12,000 × g to extract total lipids. The extracted fatty acids were transmethylated with 300 μL of 15% boron trifluoride-methanol was added and incubated at 60°C for 30 min. The resulting fatty acids were extracted with n-hexane and analyzed using a gas chromatography-mass spectrometry (GC-MS) platform . Each integrated peak area representing a specific fatty acid was substituted into the standard curve equation to obtain the raw concentrations, which were further calibrated using the following equation:WT and 2/5% CO2; the perfusion pressure was set at 70 mmHg. A water-filled balloon connected to a pressure transducer and a PowerLab 16/35 system  was used to record hemodynamic parameters, including left ventricular (LV) systolic and diastolic pressure, heart rate, and LV end-diastolic pressure (LVEDP). The LV developed pressure (LVDP), rate of pressure product (RPP), peak rate of contraction dP/dt(max), and peak rate of relaxation dP/dt(min) were analyzed using LabChart 7 software (ADInstruments).For determining the mechanisms underlying n-3 PUFA-induced cardioprotective effects on IR injury, Langendorff perfusion  was performed to induce IR injury in mouse hearts, as previously described with modifications . IsolateFor IR injury induction, isolated hearts were subjected to 40 min of baseline perfusion, followed by 20 min of no-flow global ischemia and 60 min of reperfusion. The 20-min antagonist/agonist treatments were conducted after 20 min of baseline perfusion. The control hearts were continuously perfused for 120 min. During reperfusion, heart tissues were fixed for subsequent hematoxylin-eosin (HE) staining, apoptotic analysis, RNA-seq analysis, gene expression verification, cell signaling assay, and detection of myofilament phosphorylation.Triphenyl tetrazolium chloride (TTC) staining of reperfused mouse hearts was performed as previously described  to evaluHE staining was conducted to assess the pathological changes in the reperfused mouse hearts, as previously described . After eTUNEL assay was performed to assess the apoptosis of myocardial cells in reperfused mouse hearts. Paraffin-embedded tissues were sectioned, dewaxed, dehydrated, and mounted on slides. The tissue sections were treated with proteinase K at 37°C for 30 min. After washing with phosphate-buffered saline (pH 7.4) solution thrice, 50 μL of TUNEL assay solution  was added, and the slides were incubated at 37°C for 30 min in the dark, sealed with ProLong™ Gold Antifade Mountant with DAPI , and observed under a fluorescence microscope .ATP production was evaluated as previously described . Briefly® Reagent (Thermo Fisher Scientific). The concentration and integrity of the extracted RNA were assessed using RNA 6000 Nano Kit . The RNA-seq protocol used in this study has been previously published . After adding the index codes, the samples were clustered with a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS , and the HiSeq platform (Illumina) was used to sequence the libraries. The raw RNA-seq data were deposited in the NCBI Gene Expression Omnibus (GEO) database, the raw reads were preprocessed for quality control, and the clean reads were mapped to the reference transcriptome. The microarray GeneChip hybridization was carried out by Shanghai Applied Protein Technology Co., Ltd. .Total RNA was extracted from the myocardium using TRIzolublished . Brieflyp < 0.05. DEGs with fold change values >1.5 or <1/1.5 were considered upregulated or downregulated, respectively. A total of eight DEG datasets were generated, including the upregulated and downregulated comparisons for “WT-IR vs. WT-Con,” “Fat1-IR vs. Fat1-Con,” and “Fat1-IR vs WT-IR.” The web-accessible tool Metascape (https://metascape.org) was employed to enrich biological processes for two DEG datasets of “Fat1-IR vs. WT-IR”.The transcriptome data between groups were compared, and the fold change values between comparisons were log2-transformed. In this study, the cut-off for identifying differentially expressed genes (DEGs) was an adjusted Furthermore, Venn diagram analysis was performed to predict the key target genes of n-3 PUFAs, with modifications as per our methods . For preQuantitative reverse transcription PCR (RT-qPCR) was performed to verify the expression of the identified DEGs. After total RNA extraction, TB Green Premix Ex Taq II  was used for RT-qPCR, following the manufacturer’s instructions. The reaction was run using a Real-Time PCR system  as follows: initial denaturation at 95°C for 30 s, 40 cycles at 95°C for 5 s and 60°C for 34 s, and final extension at 72°C for 10 min. The transcript levels were normalized using β-actin as a reference. The forward and reverse primers used in this experiment were 5′-TCGTGGTGCTTGTAGTGACC-3′ and 5′-ATGCAGGTGCAGTACGGAAA-3′, respectively. The primers used for the reference gene (β-actin) were: 5′-GATATCGCTGCGCTGGTCG-3′ (forward) and 5′-CATTCCCACCATCACACCCT-3′ (reverse).Western blotting was used to determine the cell signaling pathways affected by n-3 PUFAs and identify those regulated by key genes in the n-3 PUFA-protected myocardia. The cytosolic fraction was extracted from cardiac tissues using a lysis buffer  containing 1% Triton X-100 and protease inhibitors ; phosphatase inhibitors  was used in the samples for detecting phosphorylated proteins. Proteins (40 μg) were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. After blocking with 5% skim milk, the membranes were incubated with the primary antibodies (1:2000) overnight at 4°C and then with the secondary antibodies (1:5000). All antibodies were purchased from Cell Signaling Technology . The primary antibodies included antibodies against PKCδ (#9616), PKCε (#2683), p-p38α , t-p38α (#9212), p-PI3K , t-PI3K (#4257), p-AKT , t-AKT (#4685), p-mTOR , t-mTOR (#2972), APLNR (#35623), GAPDH (#2118), and the second antibody used was anti-rabbit (#7074). The bands were detected using Immobilon Western Chemiluminescent HRP Substrate , and the band densities were analyzed using the ImageJ software (NIH).The phosphorylation levels of the cardiac myofilaments were measured as previously described  The myofTo investigate the effects of APLNR and its intracellular effectors on myocardial IR injury, we treated Landgendorff isolated hearts with ML221 , an antagonist of APLNR, and Apelin-13, a potent and selective endogenous APLNR agonist. The first set of three experiment groups was designed for the inhibitor treatment: 1) WT-Con group, WT hearts were perfused for 120 min; 2) WT-IR group, WT hearts were balanced for 40 min, subjected to ischemia for 20 min, and then reperfused for 60 min; 3) WT-IR + M group, WT hearts were balanced for 20 min, then ML221 (50 nM) was added for 20 min, followed by ischemia for 20 min and reperfusion for 60 min. After reperfusion, the hearts were subjected to TTC staining, Western blot analysis, and detection of myofilament phosphorylation.fat-1 mouse hearts were perfused for 120 min; 2) Fat1-IR group, fat-1 mouse hearts were perfused for 40 min and then subjected to ischemia for 20 min and reperfusion for 60 min; 3) Fat1-IR + A group: fat-1 hearts were perfused for 40 min, then treated with apelin-13 (50 nM) for 20 min, followed by ischemia for 20 min and reperfusion for 60 min. After reperfusion, all hearts were subjected to Western blot analysis.The second set of three experiment groups was designed for the agonist treatment. 1) Fat1-Con group: 2. The cells were divided into four groups, namely, Con, hypoxia-reoxygenation (HR), Con + EPA, and HR + EPA.H9C2 cells (iCell-r012) were employed to assess the expression of APLNR and intracellular phosphorylation signaling components following hypoxia and reoxygenation . The H9C2 cell line is commonly used to investigate the molecular mechanisms of ischemia, IR injury, and IR-preconditioning. The cells were cultured in Dulbecco’s modified Eagle medium  containing 10% fetal bovine serum  and 1% penicillin-streptomycin diabody  and incubated at 37°C and 5% CO2, 5% CO2, and 1% O2. After 24 h of hypoxia, the normal medium containing 10% FBS was replaced. The hypoxia and reoxygenation protocol was used to simulate in vivo IR conditions. In the EPA treatment groups (Con + EPA and HR + EPA), 100 nM EPA  was added, and the cells were incubated at 37°C and 5% CO2 for 6 h. Cells in the Con and Con + EPA groups were cultured continuously for 30 h at 37°C and 5% CO2 without hypoxia. The Con + EPA and HR + EPA groups were treated with 100 nM EPA at the same time. Cardiomyocytes were collected following treatment, and protein analysis using Western blotting was conducted.Treatment was initiated when the cells reached 90% confluency. The regular culture medium was replaced with a sugar-free, serum-free DMEM . The cells of the IR and IR + EPA groups were placed in a hypoxic apparatus at 37°C, 94% Nt-test was used, while for comparison between multiple groups, a one-way analysis of variance (ANOVA) was performed, followed by Tukey’s multiple comparison test. Statistical significance was set at p < 0.05.Data are presented as means ± SEM. The raw data from each group were analyzed using GraphPad Prism 8.0 software  to determine significant differences between groups. For comparison between two groups, an unpaired fat-1 hearts were approximately two times higher than those in WT hearts. Additionally, there were no differences in DHA concentrations between the hearts of WT and fat-1 mice, as shown in fat-1 mice, as indicated in fat-1 mice than those in WT mice at each time point during the 60-min reperfusion, as demonstrated in The EPA levels in fat-1 hearts exhibited higher ATP production than did WT hearts  than WT mice -activated protein kinase signal transduction. We found that IR increased cardiac troponin I (cTnI) phosphorylation levels in WT mice. However, under IR conditions,  WT mice . These fWe explored the regulatory mechanisms of endogenous n-3 PUFAs involved in preventing reperfusion injury. Using Venn diagram analysis , we founFurthermore, we observed that treatment with APLNR antagonist partially restored the LVDP, dP/dt(max), and RPP and suppressed the increase in LVEDP and dP/dt(min) in WT-IR hearts . AdditioSubsequently, we investigated APLNR-mediated signaling activation in reperfused mouse hearts. Firstly, we verified that APLNR expression was upregulated in WT hearts post-IR, an effect suppressed by ML221 . Next, MAdditionally, ML221 treatment suppressed the increased phosphorylation of p38α in post-IR WT hearts . After Ifat-1 IR hearts with APLNR inhibition, ML221 increased cTnT phosphorylation in post-IR WT hearts but did not further increase cTnI phosphorylation  was also assessed to identify the targets of APLNR inhibition in post-IR WT hearts . Similarrylation . Endogenectively .In conclusion, APLNR inhibition benefits cardiac contraction and relaxation, synchronizes phosphorylation of the PI3K-AKT-mTOR axis, and regulates contractile-associated protein kinases and myofilament phosphorylation.fat-1 hearts (fat-1 myocardia (fat-1 myocardia (fat-1 hearts (Treatment with the APLNR agonist (apelin-13) significantly reversed the heart rate, LVDP, LVEDP, dP/dt(max), dP/dt(min), and RPP improvements brought about by endogenous n-3 PUFAs in post-IR 1 hearts . These fyocardia . Apelin-yocardia . Further1 hearts . TherefoIn vitro experiments conducted on EPA-treated H9C2 cells under hypoxia-reoxygenation (simulated IR conditions) revealed alterations in cell morphology and components of intracellular signaling pathways , and the maintenance or enhancement of myofilament phosphorylation  in the hearts protected by n-3 PUFAs during IR . ConsequAPLNR is a GPCR with seven transmembrane domains, first reported in 1993 . APLNR iAPLNR is crucial for heart development, cardiac function, energy metabolism, and cell differentiation . Under pathological conditions, accumulating evidence suggests that the apelin/APLNR system has multiple roles . In termex vivo hearts or in vitro H9C2 cells, respectively. These findings are consistent with the increase in APLNR expression in reperfused hearts (approximately 15 min after ischemia) reported by We observed that the mRNA and protein expression of APLNR was upregulated in response to IR or HR conditions in Recent advances in RNA-seq have enabled the profiling of gene expression patterns in normal, diseased, or protected myocardia, allowing for the prediction and verification of novel signaling pathways and regulatory mechanisms using molecular biology techniques. In a prior study, we used RNA-seq analysis to identify that Slit2 protein reduces infarct size and maintains contractility in post-IR myocardia by inhibiting inflammatory signaling pathways . Further2+, acts as a second messenger to further activate PKC isoforms  cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) into inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG) . The latisoforms . Additioisoforms . In pathisoforms . In contisoforms . p38 MAPisoforms . In thisisoforms .fat-1 IR hearts, and this was associated with suppressed cardiac contractile function, suggesting that the cardioprotection by n-3 PUFAs is negated by APLNR activation under IR conditions. APLNR inhibition by ML221 maintained relatively lower expression of PKCδ and p-p38α and higher expression of PKCε in the WT hearts subjected to IR compared with post-IR hearts without antagonist treatment. APLNR inhibition by EPA in cells also resulted in decreased expression of PKCδ and p-p38α, indicating that APLNR inhibition regulates PKCδ and p-p38α. Furthermore, APLNR activation is linked to cAMP/PKA, PKC, and angiotensin AT1 receptor signaling , exhibited synchronized phosphorylation of PI3K, AKT, and mTOR. In protected post-IR hearts, this synchronized activation was associated with changes in myofilament phosphorylation, whereas in protected HR cells, this synchronized activation was associated with improved morphology. Therefore, EPA plays a pivotal role in APLNR inhibition-induced cardioprotection under ischemic conditions.Furthermore, this signaling axis was not fully activated in the IR hearts or HR cells with upregulated APLNR . In contrast, hearts or cells with APLNR inhibition, either through the use of an antagonist (ML221-treated IR hearts) or elevated n-3 PUFA levels (e.g., in In our previous studies, we have demonstrated that the phosphorylation state of myofilaments is sensitive to various stress stimuli in experimental models of mice, pigs, or monkeys, including conditions such as IR, cardioplegia, pressure overload, and a high-fat, high-sugar diet . In thisPKC and p38 MAPK are direct regulators of myofilament phosphorylation. PKC translocates and phosphorylates myofilaments upon activation, leading to changes in contractility in an isoform-dependent manner . ConversIn conclusion, elevated levels of endogenous n-3 PUFAs suppress the increase in APLNR expression induced by IR injury in mouse hearts. APLNR inhibition via ML221 treatment protects myocardial tissues and contractile function in IR hearts by activating the PI3K-AKT-mTOR signaling pathway. To the best of our knowledge, this study is the first to provide mechanistic insights into the role of APLNR inhibition in myocardial IR injury.ex vivo models; therefore, further in vivo and in vitro verification remains warranted regarding the signaling regulation and altered biological processes attributed to n-3 PUFAs via APLNR inhibition.This study revealed the cardioprotective effect of inhibition of APLNR in IR injury. However, whether the inhibition of this receptor will enable IR hearts to resist chronic maladaptive remodeling remains to be determined. When developing therapeutics targeting the apelin/APLNR system, further evaluation of APLNR function under pathological conditions such as ventricular pressure overload and myocardial IR is warranted. Although we verified that some signaling pathways/networks are regulated by n-3 PUFAs via ALPNR, our data are limited. As shown in the transcriptomic analysis, complex signaling networks and biological processes are targeted by n-3 PUFAs. Besides, the conclusion of this study are mainly derived from


As a result of global warming, the area of the polar pack ice is diminishing, making merchant travel more practical. Even if Arctic ice thickness reduced in the summer, fractured ice is still presenting operational risks to the future navigation. The intricate process of ship-ice interaction includes stochastic ice loading on the vessel hull. In order to properly construct a vessel, the severe bow forces that arise must be accurately anticipated using statistical extrapolation techniques. This study examines the severe bow forces that an oil tanker encounters when sailing in the Arctic Ocean. Two stages are taken in the analysis. Then, using the FEM program ANSYS/LS-DYNA, the oil tanker bow force distribution is estimated. Second, in order to estimate the bow force levels connected with extended return periods, the average conditional exceedance rate approach is used to anticipate severe bow forces. The vessel’s itinerary was planned to take advantage of the weaker ice. As a result, the Arctic Ocean passage took a meandering route rather than a linear one. As a result, the ship route data that was investigated was inaccurate with regard to the ice thickness data encountered by a vessel yet skewed with regard to the ice thickness distribution in the region. This research intends to demonstrate the effective application of an exact reliability approach to an oil tanker with severe bow forces on a particular route. There is a need for a comprehensive research of the associated severe force statistical distribution operating on the oil tanker bow area because ship-ice interaction is a complicated random nonlinear process that depends on in situ ice thickness distribution4.Oil tanker designs must be secure, sturdy, and reliable due to an increase in marine operations in the Arctic connected to oil and gas exploration and transportation3, and only limited experimental measurements5. It is crucial to take into account the valid range of ice thickness and its probability distribution, route-specific and vessel-specific, when calculating oil tanker bow forces. This study makes use of actual ice thickness measurements taken on board an icebreaker traveling along the 90's and 60's meridian systems towards the direction of the North Pole and back7. By photographing cracked ice pieces, the thickness of the ice was measured.Data on onboard measured ice thickness are seldom available in the scientific literature at this timeThe number of ships operating in the arctic area has constantly expanded as a result of the exploitation of polar resources and the creation of Arctic shipping routes. In the research of ship-ice collision, broken ice should be taken into consideration as usual polar ice. The techniques for creating shattered ice aren't finished yet, though. Now, a place for cracked ice is created using the notion of cellular automata. The Voronoi diagram utilizes the cell points to create a polygonal simulation model of shattered ice. With the use of Mean Calliper Diameter (MCD) theory, the shattered ice simulation model's veracity may be confirmed. The thickness, probability distribution, and scale for shattered ice are then individually optimized. To make the broken ice model as near to the real broken ice situation as possible, the optimized value will be optimized after each phase of optimization and verified using MCD theory. For the purpose of building the polar ice-breaking model, it might offer a precise reference meaning.This study was motivated by growing industrial interest to Arctic Ocean potential navigation areas. Authors therefore intend to contribute to the safe and reliable design of large vessels suitable for Arctic Ocean oil tanker operations. Table 8. Most extreme ice load research studies utilize classic extreme value distributions (EVD) theory9. As for engineering purposes, ice loads, estimated only from expected ice thickness distribution, but without accounting for the actual ship hull geometry, are insufficient, thus the primary target should be oil tanker bow geometry-specific force pattern. This study's primary contribution is applying modified Weibull method23, to the oil tanker route-specific ice thickness data with the goal of estimating the crucial stresses at the oil tanker bow.The oil tanker vessel used in this study is designated as Polar Class 4 (PC4), and it operates under yearly heavy ice conditions, including old ice, for example between 1 and 2 m thick. The relevance of vessel operations and transit under ice effect is growing as the Arctic is being explored more. Extreme bow force estimation for oil tankers is a crucial component of the preliminary design and has a significant impact on the vessel's overall performance while in operation. Although the ultimate limit states (ULS) are often based on precise prediction of extreme loads, the extreme values of oil tanker bow loads (and therefore huge bow forces) are closely related to the reliability of the whole vessel32. The authors of this paper wanted to focus attention on the vital subject of oil tanker transportation in the Arctic. The originality of this study comes in its attempt to examine the reliability of oil tankers during upcoming Arctic voyages; as a result, the topic posed on its own is pertinent to naval architecture in the very near future. The reliability technique used in this study is extremely general and may be easily adapted to other buildings and environmental conditions that are similar35.Apart from route-specific ice-thickness, sea ice concentration being an important factor for potential commercial navigation through Arctic Polar Regions. A 25 by 25 km grid of sea ice concentrations for Arctic Polar Regions are provided by the Sea Ice Concentration Climate Data Record (CDR). This information may be used to calculate the percentage of the ocean's surface that is covered by ice and to track variations in the concentration of sea ice. For an oil tankers vessel to operate dependably and be safer to build, proper reliability approach is required. The results of this work may be classified into two categories: statistical technique and ice collision modelling. Although there is a lot of current relevant research in the field of ice collision modellingThe icebreaker route follows a meandering rather than a straight line. This study is skewed since it uses statistical data on ice thickness that was observed along a particular Arctic route. The prejudice was brought on by particular route selections and particular seasons. Notice that the vessel carefully avoided regions of heavy, multi-year ice by taking use of extensive polynyas and orienting meridionally. Mean ice thickness was route-specific. Figure 7. In the probabilistic study of ice-induced loads on oil tankers, the ice thickness distribution reflects the ice that the vessel will actually encounter. As the distribution of ice thickness is inherently constrained on both sides, the so-called Beta probability distribution function (PDF) may be used to approximate it6; to mention few: generalised Gamma distribution, two-parametric Weibull distribution7, log-normal distribution36 – Note that some are less suited since they lack an upper cut-off limit. This study's objective was to effectively use the raw sampling empirical distribution with the following practical calculation of critical bow forces rather than to determine which ice thickness distribution matches best. The beta-distribution, which has the benefit of having bounded support, was thus chosen here as an example  is often underrepresented. The latter is a result of avoiding the areas with heavier ice. All measurements of vessel ice thickness have this bias built-in. The unbiased distribution of ice thickness is known as “region-specific”, whereas this biased distribution is often known as “route-specific”7. Note that ice thickness PDF was cut off at the right-hand distribution tail side. PDF tail cut-off value was corresponding to the ice thickness, that oil tanker cannot, or will not attempt to navigate through. Studied oil tanker was not intended to operate in ice thicker than 2 m, thus cut-off value has been set equal to PDF corresponding to presents a route-specific ice thickness, encountered by actual icebreaker, operating North of 81°N, was used in this study. Ice thickness PDF was then fitted using Beta distribution. Beta distribution PDF parameters have been fitted, using raw estimated mean along with standard deviation29,. Lu27, presented an expanded field of cellular automata to describe how groups of pedestrians move while walking. Zhao34, examined the evacuation of individuals with random and aggregation distribution using the cellular automata random model to mimic people with aggregation behaviour. Li27, created the pedestrian simulation model using the cellular automata concept. Cellular automata were utilized in the aforementioned investigations to model the disorderly population structure. It is used to examine ice regions and to build the broken ice model, taking into account its random properties.“Cellular automata” is a dynamic system with discrete time and space that meets the criteria for ice-breaking space random distribution, local interaction, and time causality. It can be used to study how simple local rules evolve into more complex global dynamics37, with proper input parameters for geometry and density of broken ice, thus developing ice-breaking creation method in the area close to the actually broken ice zone. MCD  distribution law of broken ice in the actually broken ice area was utilized, based on the theory of cellular automata. The four essential components of cellular automata are the cell, cell space, neighbour, and evolution rules34. Cellular automata exhibit homogeneity, spatial dispersion, and temporal dispersion. As a result of years of intensive study and development, cellular automata currently have important uses in physics, biology, and transportation. It is suggested that cellular automata and ice-breaking architecture be combined to generate the ice-breaking model. Tyson polygons, which may be used to randomly partition 2D polygons and 3D polyhedrons, are a collection of continuous polygons made up of vertical bisection lines linking two neighbouring point segments. The outcomes of this construction procedure are displayed in detail in Fig. This study used broken ice model implemented by Rhinoceros CAD softwareD of broken ice MCD are both taken from35, withD being broken ice equivalent diameter (m), and l being broken ice circumference (m). The probability distribution of ice fragmentation MCD, based on the actual measured values in the polar ice fragmentation zone, roughly complies with the formula for the negative exponential power function35.Due to the irregular polygon of broken ice collision that occurs when ships sail in the polar ice region, the size of broken ice should be expressed in a unified manner before establishing the ice model. MCD theory is introduced to represent the size of the broken ice model. MCD is the short form for Mean Caliper Diameter; a specific amount used to express the size of broken ice. MCD is also the equivalent diameter of broken ice. Probability distribution of broken ice MCD and equivalent diameter D of shattered ice serves as the independent variable and is a subtractive function for the MCD probability distribution function presented. Hence, the worth of Using the “sea of Okhotsk Feb 2003)” measured data as an example, the ice-breaking in the sea region at this time is 7 m. The parameter range was chosen between 1.5 and 2.5 in order to analyse the impact of variable parameters on probability. A probability distribution comparison diagram with various parameters was produced in Fig. ” measureTo find the desired probability function, we settle on a modest value of 1.8. A 40% dense area for cracking ice has been built. It is specifically noted that the equivalent diameter MCD (m) of the ice breaking is too large or too small, which will significantly affect the probability distribution value of ice breaking, due to the influence of the accumulated “difference” of the ice breaking areas under the ice breaking distribution points built by the cellular mechanism. The MCD probability value of the ice-breaking model is closer to the theoretical PDF, especially the significant variance reduction, indicating that the error data dispersion is controlled. The distribution criteria are too regular when creating the broken ice model, which is inconsistent with the random distribution of the broken ice in the real world. The broken ice is evenly scaled based on the Tyson polygon theory so that the distance between the produced fractured ice is identical. The unified scaling factor is optimized to a certain scaling range in order to address this issue. The Tyson polygon method is used to split the fractured ice blocks, and then various scale factors are applied to each broken ice block. The ideal handling of two-dimensional ice breaking and the thickness of ice breaking must be taken into account while creating the ice-breaking model. Broken ice areas of the same thickness are produced using the conventional method of constructing broken ice, which differs from the thickness of broken ice in actual seas. The ice breaking model has been quite near to the actual ice condition after a series of optimizations on the ice breaking scale, probability distribution, and ice breaking thickness. It establishes a strong base for the precision of later numerical simulation. Solid elements with eight free nodes were selected for sea ice model, specific parameters being shown in Table 39, and other scholars on the ice material. The test data well shows that the isotropic elastoplastic sea ice constitutive model can better describe the compression failure of the sea ice material. In the definition of isotropic elastic–plastic sea ice constitutive model, extrusion strength, plastic failure strain and fracture pressure are considered as the criteria for sea ice failure. The material is an elastic fracture failure model with plastic strain failure criterion. When the effective plastic strain reaches the failure strain or the pressure reaches the failure truncation pressure, the element loses the capacity to bear the stress and the deviatoric stress becomes zero, that is, the material behaves as a fluid state. For more details on failure mechanisms within material ice models see42. In this study simple LS-DYNA isotropic elastic–plastic material model with failure MAT_ISOTROPIC_ELASTIC_FAILURE was used. Note that focus of this study was reliability method , PC4 polar boats have a bow force of around 16 NMFrom a structural point of view, the integrated bow load pattern is often more relevant to fatigue, while more detailed reliability study would require areal stress distribution along with structural deformation analysis. However, this study advocates general purpose reliability method, applicable not only to an integrated bow force, but any kind of stochastic process: either load or structural response type.9. Bow force non-exceedance probability Let 21. In practice, the dependence between neighbouring bow force maxima 15. Equation  bands. Conditioning level k was chosen according to the convergence of modified Weibull functions, see Sect. 559.Since the original numerical simulation was just 90 s long, and extrapolation is typically done a few orders of magnitude down on the probability distribution decimal logarithmic scale, the authors have chosen 3 days return period purely for illustration. Bow force values with large return periods  are essential for engineering design. Figure Table Regarding experimental validation of above-reported results: this study intends to draw research attention to the near future commercial Arctic navigation safety and reliability concerns, thus analyzed oil tanker model has not been neither approved, nor specifically designed yet. Model tests would require scaled vessel model, for example of the scale 1:50, but even then, apart from being expensive, – it is too premature to decide on specific vessel design. This is not an operation icebreaker, but only a hypothetical oil tanker of PC4.Safety and reliability are key issues for any vessel navigation and operation, especially in Arctic areas. This paper has studied collision forces exerted on the oil tanker bow. Two different ice loading models have been studied: a solid layer of ice model and a broken ice model with a random distribution of ice debris, simulated using the cellular automata model. The authors advocate the accurate yet straightforward reliability approach to estimate extreme loads, acting on the oil tanker bow in a realistic random ice loading environment. Since the onboard route-specific ice thickness data is often not available, there is a need for accurate and robust statistical methods at the design stage. The authors have applied the average conditional exceedance rate method to estimate extreme bow forces with a large return period. Predictions and proper confidence interval limits were given to indicate the practical merits of the suggested approach that could be easy for both onboard monitoring tools and engineers at the design stage. Note that a similar approach can be applied to analyse extreme bow forces.This paper primarily focused on applying statistical techniques at the future vessel design stage. The bow force reported in this study was found in agreement with the one reported by IACS for the PC4 arctic vessel. The authors have performed a convergence study despite the absence of direct experiments.