The average preparation time amounted to 162 (±34) s. The surgical procedure itself took on average 9 (±2) min. There was no statistically significant difference between the three surgeons. Quantification of the usability of HoloLens revealed a mean SUS score of 80.4 (±6.8), indicating an “excellent” usability level. In conclusion, this study implies the suitability, practicality, and simplicity of AR image guidance systems such as the HoloLens in apicoectomies and advocates their routine implementation.Implementation of augmented reality (AR) image guidance systems using preoperative cone beam computed tomography (CBCT) scans in apicoectomies promises to help surgeons overcome iatrogenic complications associated with this procedure. This study aims to evaluate the intraoperative feasibility and usability of HoloLens 2, an established AR image guidance device, in the context of apicoectomies. Three experienced surgeons carried out four AR-guided apicoectomies each on human cadaver head specimens. Preparation and operating times of each procedure, as well as the subjective usability of HoloLens for AR image guidance in apicoectomies using the System Usability Scale (SUS), were measured. In total, twelve AR-guided apicoectomies on six human cadaver head specimens were performed ( Cone beam computed tomography (CBCT) was developed by Arai and colleagues in 1997 with the intention of providing a more compact modification of computed tomography (CT) scans, specifically to be used in dentistry . In addiAugmented reality (AR) has the potential to overcome these limitations. AR is a transformative technology that expands the users’ understanding and awareness of their environment through the incorporation of virtual content into reality ,16. In tThe HL is a see-through head-mounted AR display that enables users to perceive and manipulate virtual objects within their physical environment. Compared with conventional image guidance systems, the HL circumvents the surgeons’ need to branch their attention between monitor and patient, providing real 3D visualization of patient-specific image data with a stereoscopic display. It is also comparably cheap and has a very slim form factor. That said, the application of the HL in dentistry has been limited up to now. Despite the proposition of several systems for 3D visualization of dental data ,27,28, tIn the context of dental procedures involving the craniofacial region, there has been a notable development in the form of a practical image guidance system based on the HL. This system, proposed by Gsaxner et al., provides several advantages, including simplified assembly, and eliminates the need for external hardware or infrastructure ,31,32. IThe current study aims to evaluate the potential benefits of incorporating such an AR guidance system using dental CBCT scans in apicoectomy procedures.Three experienced surgeons carried out four guided apicoectomies each on human cadaver head specimens to evaluate the potential benefits of an HL-based AR guidance system. Each specimen underwent a prior CBCT scan. The maxillary lateral incisor teeth, as well as the entire facial skeleton and skin surface, were segmented for visualization in AR. These data were precisely overlaid with the specimen before the surgery.The current study was carried out using tissues of human cadaver head specimens provided by the Division of Macroscopic and Clinical Anatomy at the Medical University of Graz. All cadaver specimens were preserved using Thiel’s method, a high-quality embalming technique used for advanced surgical training and anatomical research purposes ,34. ProtIn order to generate an objectively comparable and standardized sample size, this study followed strict inclusion and exclusion criteria. As a first step, all specimens were screened for their eligibility using an intraoral examination performed by an experienced maxillofacial surgeon. An apicoectomy was performed on the right and left maxillary lateral incisors. The primary inclusion criterion for further involvement was anatomical integrity of the maxilla with a dentition including all maxillary incisors and canines. Further inclusion criteria were maturity of the specimens  and complete preservation of soft and hard tissues. In the following step, all included specimens underwent a standard dose CBCT scan  23 × 27.5 mm, voxel size 0.400 mm, slice thickness 1 mm). Succeeding the CBCT scan, the application of pre-defined exclusion criteria  resulted in the exclusion of further specimens from this study, as shown in An AR image guidance system based on the second version of HL  was implemented. The HL 2 features a self-localization algorithm, which maps the surroundings of the user and localizes the device within. This enables users to position virtual content at specific locations within the mapped environment. Users can effortlessly explore said virtual objects by adjusting their viewpoint, resulting in an intuitive and immersive experience. Furthermore, the HL 2 is equipped with a time-of-flight (ToF) depth sensor, capable of capturing an exact 3D representation of the patient in the physical space. The distinct nature of human faces and the relative rigidity of facial structures can be exploited to automatically match the 3D representation of the patient captured by the HL 2 with a 3D model obtained from pre-interventional CBCT imaging. Once registered, imaging data are displayed automatically, overlaid onto the patient. The application is then controlled with a virtual user interface to alternate between different visualization modes, including 3D surface structures and planar slices. All structures are individually displayable, and their transparency and brightness can be adjusted using sliders. In the following sections, the AR system is explained in further detail.Patient registration is fully automated in the system used, eliminating external navigation systems, markers, or arduous assembly procedures ,32. In sThe AR system is controlled entirely with a virtual user interface (UI). Post-patient selection, fully automatic registration is initiated by examining the patient. The position and orientation of virtual content are continuously updated to account for patient movement, as well as to achieve a more precise alignment. However, in cases where a satisfactory alignment has been achieved and the patient is not expected to move , the position and orientation of the virtual content can be established. Further manual refinement of the automatic registration is possible, which may be desirable to account for perceptual misalignments due to the individual anatomy of the user or to account for soft tissue deformations between the pre-operative CBCT scan and the intervention. The UI enables the user to switch between different registration modes, select different anatomical structures to visualize, and adjust various parameters related to content positioning and visualization. The UI is conveniently locked to the users’ left hand, enabling them to bring it up anywhere simply by looking at their flat hand. Closing of the left hand locks the UI to its current position in the room.HL 2 supports several means for adapting the visualization. Anatomical structures can be switched on and off, and the user can interactively modify several parameters to adapt the visualization to their individual preferences. These parameters include options to modify brightness, transparency, and other relevant visual settings. The complete digital workflow is illustrated in www.randomizer.at accessed on 27 June 2023) by an individual not involved in treatment or evaluation.Following the selection of human cadaver head specimens, the allocation of each tooth  to one of three experienced surgeons was performed using a digital randomization software   was used. The SUS is a validated, standardized questionnaire that classifies the ease of use for a wide variety of products and services such as applications or hardware. This measuring tool consists of ten items on a 5-point Likert scale . Althougp-value of <0.05 was considered statistically significant. All statistical analyses were performed using the statistical Python package pingouin. To additionally visualize this study’s data, tables and boxplots were utilized. All specimens were anonymized before their use with the Division of Macroscopic and Clinical Anatomy at the Medical University of Graz. The recorded data were collected in a case report form. The collection, transfer, and storage of the human cadaver specimens’ image data within this study was carried out in accordance with legal regulations.Descriptive and analytical statistics were used to analyze the gathered data, which were presented as either mean ± standard deviation (SD) or median and interquartile range (IQR). Analytical statistics included the Shapiro–Wilk test, to test the collected data for normality, followed by an ANOVA, and the Kruskal–Wallis-H test, to analyze whether there were statistical differences between users regarding setup time, operation time, and SUS score. For all calculations, a n = 12) from six human cadaver head specimens . Each specimen underwent two apicoectomies in the maxilla, one on the left and one on the right side (24 teeth). All teeth showed no apical radiolucency in the preoperative CBCT scans. Each of the three experienced surgeons resected four maxillary lateral incisors, which had at least one adjacent tooth on both the mesial and the distal aspect to imitate structures of interest. No damage to structures of interest  during the procedure was observed. Throughout this study, the application encountered a singular instance of failure resulting from overheating, thus necessitating the interruption of this study until the issue was resolved using a power cycle. This incident incurred a downtime of approximately 5 min.The final sample consisted of 12 teeth .In total, the preparation times ranged from 75 to 195 s, with an average of 162 s ± 34 s. The preparation time included automatic patient registration as well as manual adjustments (if necessary). The mean operating time was 9 ± 2 min from the first incision to the final wound closure. The fastest operating time recorded was 5 min, while the longest duration required for an apicoectomy was 13 min. Differences regarding the mean operating times among the three surgeons were not statistically significant  and dynamic navigation (DN). SN uses presurgical fabricated templates to provide guidance during the osteotomy and root-end resection. Compared with the free-hand technique, this approach has demonstrated a more precise method for accessing the apical portion of the root ,9,47. HoWith a head-mounted semi-transparent display in front of the users’ eyes, AR can solve this attention deficit with in situ visualization . PreoperAccording to reviews by Farronato et al. and Joda et al., the predominant focus of AR in the field of dentistry lies within the context of maxillofacial surgical procedures ,21. OtheBosshard et al. conducted a study to assess the accuracy of AR-assisted apicoectomies compared to template-guided apicoectomies using the HL 2 as a see-through head-mounted display. A favorable level of comparability between the two approaches could be shown, indicating that AR performed well in terms of medically relevant trueness and precision. Referencing the author, even a trend toward improved accuracy regarding angular deviation in the resection could be shown. Nevertheless, with respect to the time and effort required, AR assistance was found to necessitate more time subjectively due to the technical preparation involved . HoweverWhile the presented HL 2 prototype application demonstrated its feasibility in the field of dentistry when using CBCT scans for AR-guided dental surgery, it has certain shortcomings that require attention. The current patient registration system lacks support for non-rigid registration; hence, the pre-interventional CBCT scan is assumed to perfectly align with the patient’s exact appearance in situ. While the face is generally a rigid structure, this assumption may not always hold true, especially in situations like swelling, where facial features can be subject to change. In addition, structures in the lower jaw may sometimes not be reliably registered due to its mobility. The position of the mandible may differ from the pre-interventional scan, leading to small inaccurate alignments and tracking of lower jaw structures during the dental procedure. Non-rigid registration could overcome this issue. However, this is a complex process that requires real-time physics-based modeling of soft tissue properties. A further limitation is the current streaming-based approach, which incurs additional latency. The system’s run-time performance becomes heavily reliant on the connection speed and processing capabilities of the companion PC, thus requiring sufficient infrastructure. As a result, users may experience delays or disruptions in the AR application’s functionality. An optimized local processing-based solution, potentially available in the near future, could be a viable alternative to mitigate these concerns. The HL 2 hardware, however, cannot cater to increasing hardware requirements.Since the adoption of AR systems in dentistry, their potential applications have witnessed exponential growth . NeverthAR-guided apicoectomies using the HL 2 relying on CBCT scans received an excellent rating for system utilization from three experienced surgeons. The integration of AR guidance systems into routine clinical practice is becoming more accessible, especially when relying on diagnostic CBCT scans. High peri-interventional CBCT imaging rates coupled with the presence of hard tissue-bound or well-defined structures requiring protection render this novel technology highly appealing in this specialty. As technology continues to progress, AR systems are poised to revolutionize the field of dentistry, establishing new possibilities for enhanced patient care and treatment procedures.To further prove the advantages of the AR technology in clinical routine research, the use of a prospective split-mouth design is imperative to compare the AR-guided to the non-guided method.


Important advances in diabetic retinopathy (DR) research and management have occurred in the last few years. Neurodegenerative changes before the onset of microvascular alterations have been well established. So, new strategies are required for earlier and more effective treatment of DR, which still is the first cause of blindness in working age. We describe herein gene regulation through Lnc-RNAs as an interesting subject related to DR. Long non-coding RNAs (Lnc-RNAs) are non-protein-coding transcripts larger than 200 nucleotides. Lnc-RNAs regulate gene expression and protein formation at the epigenetic, transcriptional, and translational levels and can impact cell proliferation, apoptosis, immune response, and oxidative stress. These changes are known to take part in the mechanism of DR. Recent investigations pointed out that Lnc-RNAs might play a role in retinopathy development as Metastasis-Associated Lung Adenocarcinoma Transcript (Lnc-MALAT1), Maternally expressed gene 3 (Lnc-MEG3), myocardial-infarction-associated transcript (Lnc-MIAT), Lnc-RNA H19, Lnc-RNA HOTAIR, Lnc-RNA ANRIL B-Raf proto-oncogene (Lnc-RNA BANCR), small nucleolar RNA host gene 16 (Lnc-RNA SNHG16) and others. Several molecular pathways are impacted. Some of them play a role in DR pathophysiology, including the PI3K-Akt signaling axis, NAD-dependent deacetylase sirtuin-1 (Sirti1), p38 mitogen-activated protein kinase (P38/mapk), transforming growth factor beta signaling (TGF-β) and nuclear factor erythroid 2-related factor 2 (Nrf2). The way Lnc-RNAs affect diabetic retinopathy is a question of great relevance. Performing a more in-depth analysis seems to be crucial for researchers if they want to target Lnc-RNAs. New knowledge on gene regulation and biomarkers will enable investigators to develop more specialized therapies for diabetic retinopathy, particularly in the current growing context of precision medicine. Diabetic retinopathy (DR) is a neurovascular disease caused by hyperglycemia. Retinal degeneration initially included glial and neural cells ,2. The eAlthough some advances in diabetic retinopathy management have occurred, the disease is still the first cause of blindness in working age ,7. FurthIn hyperglycemia, reactive oxygen species (ROS) are overproduced in the retina and oxidative stress begins the disruption of the mitochondria, which leads to retinal capillary cell apoptosis . In vitrOxidative stress can be both a cause and a consequence of the metabolic alterations that hyperglycemia brings about through the mechanisms mentioned above  ,9. BesidNrf2 is a transcription factor in charge of regulating the expression of several antioxidant enzymes, and it plays a very important role in the protection of the retinal vasculature from oxidative stress. Furthermore, NRF2 is involved in mitochondrial homeostasis. However, a chronic state of hyperglycemia and oxidative stress overloads the NRF2 defense mechanism: the activity of NRF2-associated antioxidant enzymes was reduced in in vitro models , glutathione peroxidase (GPx) and catalase in diabetic patients). This NRF2 deficiency leads to NOX2 activation and the production of ROS .NRF2 also regulates the inflammatory response induced by diabetes by regulating the expression of NF-κB and COX2. A decrease in NRF2 levels leads first to a release of inflammatory cytokines because of the induction of NF-κB and then to vascular cell apoptosis thanks to the overexpression of proapoptotic factor Bax or TNF-α, both associated with NF-κB .The loss of vascular cells is well characterized in early DR; in particular, the presence of “pericyte ghosts” has been related to apoptotic changes. Pericyte death leads to the formation of acellular capillary microaneurysms, the first clinically evident lesion in the examination of a diabetic eye . There aCaspases are enzymes of the group of cysteine proteases that are involved in inflammatory processes and apoptosis. Caspase-2, -8, -9 and -10 oversee the initiation of programmed cell death, and caspase-3, -6 and -7 are the “executive” caspases . The intThe accumulation of ROS induced by hyperglycemia increases the pore permeability in the mitochondria and the release of cytochrome C and activates other proapoptotic factors to initiate cell death through the caspase family . High prSirtuin 1 (SIRT1) is a deacetylase that plays a key role in the regulation of cell proliferation, apoptosis and inflammation, and it has an antiapoptotic effect by acting on P53. SIRT1 decreases mitochondrial damage by deacetylation of the protein P53, so it is unavailable to bind to BCL-1 family proteins like BAX, which would later translocate to the mitochondria and activate the caspase pathway in retinal pigment epithelial (RPE) cells. There are other factors inhibiting the process of apoptosis, such as protein kinase B (Akt), cyclooxygenase 2 (Cox-2) and myeloid leukemia cell differentiation protein (Mcl-1). Akt is a protein kinase involved in cell cycle modulation, and it interacts with a PI 3-kinase to promote cell survival. Furthermore, the activation of the PI 3-kinase/Akt pathway and the increase in the cytoplasmic levels of Cox-2 in RGC, RPE cells and the ciliary body epithelium were associated with higher cell survival rate. Cox-2 stimulates the synthesis of prostaglandins and the expression of Mcl-1 via the activation of the PI 3-kinase/Akt system .1 alpha (MIP-1α), IL-6 and macrophage colony-stimulating factor (M-CSF). Shelton et al. ..30].HOTTIP promotes retinal inflammatory processes and inhibits proliferation and tube formation on vascular endothelial cells by activating p38-MAPK . H19 oveTGF-β: Increased levels of TGF-β have been discovered in the aqueous humor of the eyes of patients with diabetes compared with normal eyes . HoweverH19 overexpression prevented glucose-induced EndMT through TGF-β signaling via a blockade of the MAPK–ERK1/2 pathway. BAMBI, a type 1 TGFβ receptor antagonist, is downregulated in retinal endothelial cells under diabetic conditions and is regulated by miR-20b-5p and circDNMT3B: the decrease in BAMBI expression was reversed with a miR-20b-5p inhibitor, and overexpression of circDNMT3B alleviated the reduced expression of BAMBI under HG conditions .In DR, MEG3 expression decreases in the serum of DR patients and HG-treated RPE cells, while MEG3 overexpression reduces VEGF and transforming growth factor-β1 (TGF-β1) expression. MEG3 knockdown also increases the proliferation, migration and tube formation of retinal endothelial cells in vitro .In ARPE-19 cells cultured in an HG environment in vitro, increased expression of MIAT significantly upregulated the expression of TGF-β1 and reduced cell viability, but TGF-β1 inhibition did not completely prevent endothelial cell damage associated with Lnc-RNA-MIAT overexpression. Also, a significant correlation between plasma TGF-β1 levels and Lnc-RNA-MIAT levels in patients with diabetic retinopathy has been described .Suppressing OGRU could improve miR-320 expression, exerting protective effects against HG-induced aberrant expression of VEGF and TGF-β1, inflammatory response and ROS production in Müller cells .Nrf2: Nuclear factor erythroid 2 related factor 2 (NRF2) is a transcription factor in charge of regulating the expression of several antioxidant enzymes, and it plays a very important role in retinal vasculature protection from oxidative stress .MALAT1 activates Keap1′s transcription, thus impeding Nrf2 nuclear movement and inhibiting the transcription of the antioxidant response enzymes in HRECs, in vivo and in retinal microvessels from human donors with diabetic retinopathy .MEG3 directly targets miR-93, and overexpression of MEG3 alleviates HG-induced apoptosis and inflammation through the miR-93/Nrf2 axis in RPE cells [PE cells . HG-induced inflammatory response and oxidative stress in vitro were markedly mitigated by OGRU knockdown through restraining IκBɑ/nuclear factor kappa beta (NF-κB) and improving nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathways, respectively, in Müller cells. Sox2OT knockdown plays a neuroprotective role in diabetes-related retinal neurodegeneration by regulating the oxidative stress response in RGCs and diabetic mouse retinas. Sox2OT knockdown plays an antioxidative role via regulating NRF2/HO-1 signaling activity.JAK/STAT3: MEG3 ameliorated cell viability and decreased HG-induced apoptosis and inflammatory factors in hRMECs by negatively regulating miR-19b and thus targeting the SOCS6-mediated JAK2/STAT3 signaling pathway .Circ_0005015 overexpression sponges miR-519d-3p, thus releasing the repressive effect of miR-519d-3p on STAT3, MMP-2 and XIAP in endothelial cells, which play important roles in many cellular processes such as cell growth, vascularization and apoptosis .TPTEP1 is downregulated in HG-stimulated HRVECs, and its overexpression reduced viability, migration and angiogenesis. Moreover, TPTEP1 suppressed phosphorylation and nuclear translocation of STAT3 and thereby downregulated VEGFA mRNA and protein levels .TXNIP: TXNIP is implicated in the maturation of IL-1β and in the inflammasome activation during DR, finally leading to oxidative stress, inflammation and apoptosis. c-Myc promotes MIAT expression, and MIAT represses TXNIP’s degradation, which leads to the release of proinflammatory factors from Müller cells .circ_0084043 upregulated TXNIP expression to activate the Wnt/β-catenin signal pathway by targeting miR-128-3p, thus promoting HG-induced RPE cell injury .XBP1: XBP1 is a transcription factor that is activated by ERS and is involved in several cellular processes such as apoptosis and inflammation. XBP1 was found to be decreased in HG-treated hRMECs, and its knockdown promoted HG-induced EndMT and angiogenesis. H19 might regulate the inflammatory processes of ARPE-19 cells under high-glucose conditions via upregulating the expression of XBP1 by sponging XBP1-suppressor miR-93 during ERS . XBP1 isIn this review, we have pointed out how Lnc-RNAs can be involved in diabetic retinopathy development. Despite efforts made by researchers, a lot of work still needs to be conducted to understand Lnc-RNAs’ influence on diabetic retinopathy. How Lnc-RNAs affect complex pathophysiological processes in the retina such as the onset and advance of diabetic retinopathy are questions of great relevance. In other medical diseases, such as asthma, Lnc-RNAs were discovered to be important post-transcriptional regulators . In addiAfter ischemic stroke, Lnc-RNAs have been reported to stimulate apoptosis, angiogenesis and inflammation ,93,94,95Targeting MALAT1 is a novel treatment strategy against cancer. Literature data on cancer indicate aberrant expression and dysregulated MALAT1 in several oncological diseases (citation). MALAT1 is an adverse prognostic marker in stage I lung adenocarcinoma and squamous cell carcinoma patients, and its expression is also associated with metastasis ,97. MALAMALAT1 is overexpressed in hepatocellular carcinoma (HCC) primary samples and cell lines , and itsSignificant advances have been also achieved in developing therapeutic reagents for drugging oncogenic Lnc-RNAs in tumor cells, and novel approaches are being addressed to design and develop small molecules targeting Lnc-RNAs. Targeted genetic deletion of MALAT1 by zinc finger nucleases, as well MALAT1 therapeutic targeting by synthetic oligonucleotides, including siRNAs and the newly developed LNA gapmeR ASOs, have established the oncogenic role of this Lnc-RNA and its druggability for therapeutic purposes .In this review, we show that Lnc-RNAs MEG3 and MALAT1 are involved in five and three relevant pathways of diabetic retinopathy, respectively. Therefore, both are good candidates to study. Among the 21 human investigations shown in To date, we have not seen a report or a registered interventional clinical trial on diabetic retinopathy targeting Lnc-RNAs in the literature. New therapies will probably become more important in the context of personalized medicine, such as adjuvant therapies targeting specific Lnc-RNAs. Further research is needed to analyze the role of Lnc-RNAs in diabetic retinopathy.


In endoscopic transsphenoidal skull base surgery, it is often difficult to accurately determine the location of a tumor and its surroundings on imaging because the lesion remarkably displaces the geography of normal anatomic structures. We aimed to create a novel augmented reality (AR) navigation system that could compensate for this displacement effect. We created a precise three-dimensional computer graphic (3DCG) model that we superimposed on a visual image of the actual surgical field and displayed on a video monitor during endoscopic transsphenoidal surgery. Surgeons evaluated its efficacy using a five-point scale in 15 consecutive patients with sellar and parasellar tumors. The average score overall was 4.7 , and the AR navigation system was considered as useful as or more useful than conventional navigation in certain patients. This system has the advantage of facilitating an immediate 3D understanding of the lesion and surrounding structures.In endoscopic transsphenoidal skull base surgery, knowledge of tumor location on imaging and the anatomic structures is required simultaneously. However, it is often difficult to accurately reconstruct the endoscopic vision of the surgical field from the pre-surgical radiographic images because the lesion remarkably displaces the geography of normal anatomic structures. We created a precise three-dimensional computer graphic model from preoperative radiographic data that was then superimposed on a visual image of the actual surgical field and displayed on a video monitor during endoscopic transsphenoidal surgery. We evaluated the efficacy of this augmented reality (AR) navigation system in 15 consecutive patients with sellar and parasellar tumors. The average score overall was 4.7 [95% confidence interval: 4.58–4.82], which indicates that the AR navigation system was as useful as or more useful than conventional navigation in certain patients. In two patients, AR navigation was assessed as less useful than conventional navigation because perception of the depth of the lesion was more difficult. The developed system was more useful than conventional navigation for facilitating an immediate three-dimensional understanding of the lesion and surrounding structures. For more than two decades, endoscopic transsphenoidal surgery (ETS) has been widely accepted as a safe approach for pituitary tumors, and its indication has recently been expanded for tumors in various skull base regions ,2,3,4,5.To compensate for this situation, neuronavigation is used for complex neurosurgical procedures ,15,16,17Several studies have suggested the possible application of this technology for clinical use in endoscopic skull base surgery ,28,29,30In this study, we developed a unique AR navigation system, which applied detailed three-dimensional computer graphics (3DCG) created from the preoperative image and using the intuitive real-time adjustment method. The aim of this study was to evaluate the efficacy of this augmented reality (AR) neuronavigation system during ETS for parasellar tumors, based on the 3DCG model created from the preoperative radiographic data.TM, Medtronic, Minneapolis, MN, USA), in which the target point was indicated on 2D radiographic images.We evaluated the efficacy of AR navigation using a 3DCG model in 15 consecutive patients who underwent ETS for sellar and parasellar tumors. Details of the patient characteristics are summarized in CT and MRI with gadolinium enhancement were acquired before surgery in all patients and were used to create the 3DCG VR model. CT was performed with a 320-slice CT scanner  using the following parameters: collimation, 0.5 mm; tube voltage, 120 kVp; tube current (volume exposure control), 100–500 mA; rotation time, 0.75 s; reconstruction section width, 0.5 mm; reconstruction interval, 0.5 mm; number of slices, 640; and voxel size, 0.43 mm × 0.43 mm × 0.5 mm. MRI was performed with a Signa 3.0-T system . Imaging parameters for gadolinium-enhanced T1 weighted images were as follows: repetition time, 7.0 ms; echo time, 3.2 ms; slice thickness, 0.7 to 1.4 mm; field of view, 22 cm; matrix size, 352 × 256; flip angle, 13 degrees; voxel size, 0.63 mm × 0.85 mm × 0.7 mm; and timing, 4 min after contrast injection. The imaging parameters for Fast Imaging Employing Steady-State Acquisition (FIESTA) were as follows: repetition time, 5.6 ms; echo time, 2.7 ms; slice thickness, 0.4 to 0.8 mm; field of view, 20 cm; matrix size, 320 × 320; flip angle, 50 degrees; voxel size, 0.63 mm × 0.63 mm × 0.4 mm; and timing, just after contrast injection. Imaging parameters for 3D time-of-flight magnetic resonance angiography (TOFMRA) were as follows: repetition time, 26 ms; echo time, 2.7 ms; slice thickness, 0.5 to 1.0 mm; field of view, 20 cm; matrix size, 384 mm × 224; flip angle, 20 degrees; and voxel size, 0.63 mm × 0.89 mm × 0.5 mm.The reconstruction method for creating 3DCG models from clinical 2D images has been reported previously ,28,29. BTM, KARL STORZ Endoscopy Japan K.K., Tokyo, Japan) were displayed on the main monitor and also output simultaneously to a video mixer . The video data of the surgical field and the 3DCG image in the portable tablet computer were separately input into the video mixer, and those 2 images were superimposed on the second monitor for preparation of the topographic adjustment.We performed endoscopic skull base surgery while facing images of the surgical field displayed on two 43-inch monitors, of which the second monitor was used for AR navigation. The optical images obtained by a 4K rigid neuroendoscope . Preoperative MR imaging showed an intrasellar tumor with suprasellar extension a. After A 50-year-old woman had a hypoglossal nerve palsy due to a chordoma and underwent ETS. Preoperative MR imaging showed a tumor at the craniovertebral junction a. A skulA 75-year-old woman had right visual disturbance due to recurrent meningioma. Preoperative MR imaging showed a tumor around the right optic nerve a. In a pIn this report, we present the preliminary results of our novel AR navigation system in ETS. The average score overall indicated that the surgeons considered AR navigation to be more useful than conventional navigation for certain patients. On the other hand, in two cases it was evaluated that AR navigation was not as useful as conventional navigation, though by only one resident in each case. The main complaint involved perception of depth; there might be individual differences among surgeons regarding recognition of the virtual image as the ‘real’ situation, depending on their previous experience with similar cases in ETS.Compared to the conventional navigation system that uses infrared light or magnetic field guidance for topographic adjustment , our AR In the conventional navigation system, a single point is indicated on the radiographic image. Thus, we must verify the anatomical location from the radiographic images presented on the monitor in the multiple sections and simultaneously estimate the margin of error, including the shifts in the x, y, and z directions. Even if there is an apparent displacement between the actual anatomy and the indicated point on the radiographic image, there is no automatic method to detect and correct the discrepancy. Therefore, to successfully use the conventional navigation system, we must bear in mind the possibility of a minor shift and readily correct it based on comparison of the anatomical topography in the surgical field and the 2D radiographic images on the navigation monitor. Our AR navigation system embraces those human abilities to correct the technological error. The accuracy and efficacy of our AR navigation system largely rely on the subjective involvement of humans, much more so than the conventional navigation system. Because our AR navigation applies a high-quality 3D image, the sensing capability of displacement in the navigation is much higher than that of conventional navigation, which can be easily corrected and may reduce the margin of error to a minimum.Although the anatomic structures in the sphenoid sinus are generally identical and easily identifiable during ETS, the AR navigation system is particularly useful for revealing the anatomic structures hidden behind the bony structure or the tumor in the surgical field. We often hesitate to resect tumors with a similar appearance or color to the surrounding normal anatomy, even if it has been recognized by conventional navigation as a “tumor”, considering the possible margin of error. In contrast, our AR navigation system enables comprehensive recognition of the target lesion in relation to the surrounding landmark anatomies, which enables direct assistance for surgical decision-making.Our AR navigation system may be more useful in young neurosurgeons who do not have significant experience with endoscopic transsphenoidal surgery. In the case of the sellar tumor (presented in the illustrative case 1), for the neurosurgeons who have certain experience with endoscopic transsphenoidal surgery, the tumor extension in the sella and position of the displaced internal carotid artery are readily recognized in the surgical field by reference to the 2D MRI. The AR navigation is used only for confirmation of their outlooks. In contrast, young neurosurgeons are less confident in identification of such “distorted” anatomies, and the AR navigation is useful for them to verify location of these important anatomies. Simultaneously, for young neurosurgeons, the AR navigation is also useful for surgical education to reconstruct the image of the surgical field from the preoperative 2D MRI in their mind and retrospectively evaluate the accuracy of their outlook of the surgical field. Furthermore, in ETS for complex skull base lesions, our system is useful, regardless of the surgeon’s experience. In the illustrative case 2, the AR navigation successfully disclosed the area of tumor extension in the retropharyngeal space, contributing sufficient exposure of the tumor. In the illustrative case 3, it was difficult to distinguish the tumor from the displaced optic nerve using the color or the consistency. Because we could clearly distinguish the optic nerve from the recurrent tumor on MRI, the AR navigation created with 3DCG from the preoperative MRI clearly indicated it as a “tumor”, and we could successfully resect the tumor.On the other hand, as limitations, it is very difficult to objectively evaluate the margin of error, which is one of the major disadvantages associated with our AR navigation. Additionally, to accurately superimpose the 3D image on the surgical field, we always require identifying the multiple anatomic landmarks. Thus, when the tumor extensively invades into the skull base regions, destroying or harboring the characteristic anatomical landmarks, it will be difficult to accurately superimpose the 3DCG, hampering its use in surgical navigation. The present AR navigation system has some issues that must be addressed. It takes more than one hour to create the 3DCG models with precision, depending on tumor location and the number of critical structures involved in the tumor ,33. ThusIn this study, we report the preliminary results of our novel AR navigation system during ETS for sellar and parasellar tumors. The resulting 3DCG images were identical to structures in the surgical field. Surgeons considered the present system more useful than the conventional navigation system for facilitating an immediate 3D understanding of the lesion and surrounding structures. Our AR navigation system facilitates intuitive comprehension of the skull base anatomy during ETS and helps not only young neurosurgeons in ETS for basic sellar lesions but also experienced neurosurgeons in ETS for complex skull base lesions. While further refinement of the image fusion techniques is essential, the preliminary results of this study suggest efficacy of our AR navigation in ETS for pituitary and parasellar tumors.


There is a large and growing unmet need for rehabilitation – a diverse category of services that aim to improve functioning across the life course – particularly in low- and middle-income countries. Yet despite urgent calls to increase political commitment, many low- and middle-income country governments have dedicated little attention to expanding rehabilitation services. Existing policy scholarship explains how and why health issues reach the policy agenda and offers applicable evidence to advance access to physical, medical, psychosocial, and other types of rehabilitation services. Drawing from this scholarship and empirical data on rehabilitation, this paper proposes a policy framework to understand national-level prioritization of rehabilitation in low- and middle-income countries.We conducted key informant interviews with rehabilitation stakeholders in 47 countries, complemented by a purposeful review of peer-reviewed and gray literature to achieve thematic saturation. We analyzed the data abductively using a thematic synthesis methodology. Rehabilitation-specific findings were triangulated with policy theory and empirical case studies on the prioritization of other health issues to develop the framework.The novel policy framework includes three components which shape the prioritization of rehabilitation on low- and middle-income countries’ national government’s health agendas. First, rehabilitation lacks a consistent problem definition, undermining the development of consensus-driven solutions which could advance the issue on policy agendas. Second, governance arrangements are fragmented within and across government ministries, between the government and its citizens, and across national and transnational actors engaged in rehabilitation service provision. Third, national legacies – particularly from civil conflict – and weaknesses in the existing health system influences both rehabilitation needs and implementation feasibility.This framework can support stakeholders in identifying the key components impeding prioritization for rehabilitation across different national contexts. This is a crucial step for ultimately better advancing the issue on national policy agendas and improving equity in access to rehabilitation services. There is a growing unmet need for rehabilitation – a category of interventions aimed at improving functioning and reducing disability . RehabilOne in three persons may require rehabilitation services within their lifetime ; howeverThe critical importance of rehabilitation in improving quality of life has led to its inclusion in international policy frameworks, including the Declaration of Alma Alta  and the But despite increasing global recognition, rehabilitation receives little attention from most governments in LMICs, with even fewer nations implementing adequate policy to support the advancement of rehabilitation services in national health systems , 7–9. ThTheories from the social sciences explain how issues are elevated to the policy agenda and can help to understand the prioritization of rehabilitation in LMICs . Agenda-Kingdon’s multiple streams and broader policy scholarship emphasizes key factors that influence prioritization. These include the extent to which there is consensuses on the nature of the problem and its solutions (problem definition) –15, the Health policy scholarship applies agenda-setting theory to understand how and why specific health issues rise on national or global agendas. The Shiffman and Smith framework on determinants of political priority for global health initiatives is a commonly utilized framework for health issue prioritization . OriginaWe identified no previous research on rehabilitation’s prioritization on national or sub-national health agendas in LMICs. A 2016 review on the governance of rehabilitation found little evidence on agenda-setting factors and called for additional research .The objectives of this study are to (1) identify the key factors that shape rehabilitation’s prioritization across LMIC national health systems, and (2) to distill these into a novel policy framework to guide future research and practice. In this paper, we analyze key informant interview data, complemented by a purposeful selection of published and gray literature, to identify the key factors that shape prioritization of rehabilitation. Guided by existing health policy scholarship and agenda-setting theory, we then synthesize these factors into a policy framework specific to the prioritization of rehabilitation in LMIC national health systems. The framework provides a path forward for rehabilitation stakeholders to better understand and address the critical factors that can advance prioritization of rehabilitation, a key first step to improving equitable access to rehabilitative services.Following a commonly utilized definition in the health policy process literature developed by Shiffman 2007), prioritization is defined in this study as concern for the issue, the enactment of policies that advance consensus-based solutions, and the consistent application of public funds aligning with the unmet need 07, prior.We use two sources of data to understand the prioritization of rehabilitation in national LMIC health systems. Our primary data source was qualitative key informant interviews (KIIs) with transnational and national rehabilitation stakeholders. We complemented these interviews with our second data source, which was a purposeful review of peer-reviewed and gray literature on governance and policy for rehabilitation. The KIIs were the foundation of our analysis and were the key data source used to develop the framework. The rehabilitation-specific literature aided in the initial coding of the interview data by highlighting potentially applicable themes across a range of countries and contexts, providing additional theoretical and empirical evidence to support interview themes, and providing more country case examples to further enrich the interview data.We complemented the KII data and rehabilitation-specific peer-reviewed and gray literatures with theories from the agenda setting phase of the policy process, which informed our analysis. A thematic synthesis methodology was used to abductively analyze both the KIIs and the rehabilitation-specific literature against the policy theory to develop the policy framework. Thematic synthesis is utilized to integrate findings of qualitative research and is appropriate for the generation of interpretive constructs . We repoSociological theory directs us to the interaction between agentic and structural factors that shape the policy prioritization process , 37. AgeTo deepen our understanding of how health issues are prioritized and apply these insights to rehabilitation, we reviewed scholarship from public administration and policy on governance, advocacy, and the agenda setting phase of the policy process , 38–42, Following the recommendations of thematic synthesis , we compWe reviewed titles and abstracts for relevance in the order they were provided by the search engine, downloading the full text of a paper if the title or abstract appeared relevant, and then reviewed the full text to determine relevance of the paper against pre-determined inclusion and exclusion criteria Table . The incWhen a relevant paper was identified per the inclusion and exclusion criteria, an initial data extraction was completed. This allowed us to build an early conceptual understanding of the sampled literature and informed our iterative and non-exhaustive search process.We continued searching for and reviewing identified literature until we reached a point of conceptual saturation, where additional papers were adding little to no new information to our initial data extraction , and wheA purposive, maximum variation sampling approach was utilized to sample key informants with extensive research and/or practice experience in rehabilitation and health systems. KIIs with 65 health systems and rehabilitation stakeholders, representing 47 countries across all WHO regions and all country income levels, were conducted via Zoom from February 2020 and April 2021.We purposefully sampled key informants (KIs) based on their perceived ability to contribute key insights on rehabilitation and the health system, including governance, policy, and leadership and based on access via the research team’s professional networks. Guided by the principle of maximum variation, we also sought to include KIs working in different professional capacities, including national LMIC government officials, rehabilitation health care providers, health professional associations, academic researchers, representatives from non-governmental or civil society organizations, leaders of disability peoples’ organizations, and international intergovernmental organizations Table . We inclSampled participants were contacted via email and invited to participate. KIs with a non-response or refusal were not included. All KIs had extensive professional experience working in rehabilitation and health systems, either at the national level in an LMIC or at the global level in an international organization or research institute.Data was iteratively reviewed during the data collection process, and we stopped contacting new participants when we reached a point of saturation – in other words, gaining little new insights during new interviews. We considered all interviews equally when conducting the analysis, so as not to preference any one perspective in our analysis.A semi-structured interview approach was utilized. KIs were asked about their definition of and perspectives on rehabilitation, leadership, governance, policy, and political factors shaping rehabilitation, rehabilitation’s role in the health system, and how to strengthen rehabilitation in their context. Interviews were conducted in English and Spanish by three interviewers, all of whom had experience working in rehabilitation in health systems and formal training in qualitative research methods. Interviews were assigned to the interviewer based on language; all interviewers in English were done by a single researcher, and a second researcher joined the first interview conducted. All interviews in Spanish were conducted by a different researcher who was a native Spanish speaker. English recordings were recorded in full, and the recording was transcribed verbatim. Spanish recordings were translated into English and transcribed in full. Participants were assigned an interview number in chronological order of the completion of the interview to maintain anonymity.The study was deemed exempted, non-human subjects research by the Institutional Review Board of the author’s institution. Oral consent was confirmed prior to conducting each interview.We took an iterative, abductive approach to data analysis, following the steps of thematic synthesis . Themes For example, literature data emphasized that rehabilitation lacks a common understanding across stakeholder groups. This finding was initially extracted as an ‘agentic feature’ — any factor largely within an actor’s power to control or act upon— influencing rehabilitation in step one. In step two, a review of empirical cases and policy theory emphasized the importance of ‘problem definition’ to prioritization, which aligned with the inductively identified challenge of ‘lack of common understanding of rehabilitation’. In step three, we reassembled the extracted data into a new theme labeled ‘problem definition’, which captured findings related to how rehabilitation was (or was not) understood and influencing factors therein. In step four, the problem definition code was utilized deductively to code KII transcripts. The interview data aligned with literature findings by helping us contextualize the initial finding through national-level examples of definitions as shared by KIs. Finally, in step five, both KIIs and the extracted data from the rehabilitation-specific literature data coded as ‘problem definition’ were analyzed against the broader policy theory and evidence from the prioritization of other health issues to consider how lack of a common definition of rehabilitation may influence prioritization in this case.This study analyzed 65 KIIs and 56 peer-reviewed or gray literature documents, representing insights from over 50 countries, regional insights from sub-Saharan Africa, Latin America, and the Caribbean, and generalized literature on LMIC experiences.We identified three components— problem definition, governance, and structural factors —shaping the prioritization of rehabilitation in national health systems. These are defined in Table These components are interconnected with prioritization at the center, as illustrated in Fig. Inconsistencies and complexity in the problem definition suggests internal framing contestation on rehabilitation, with lack of evidence on proposed solutions. Varied understanding of the problem results in fragmented domestic coalitions with some influence from transnational actors. These actors operate within historical legacies and existing health system structures which further shape how the problem is understood, the solutions that are advanced, and the actors involved.Below, we present a comprehensive synthesis of our finding against each component of the policy framework for rehabilitation.Existing theory instructs that an issue is more likely to advance if it has a unified problem statement , 13, if We identified two problem definition components influencing the prioritization of rehabilitation: 1) clarity on the nature of the problem and 2) agreement on required and acceptable solutions. Lack of consensus in these two areas challenges prioritization on national policy agendas.In problem clarity, barriers to prioritization include 1) a lack of common understanding of rehabilitation across stakeholder groups , 46 and  Related to the lack of a common understanding, rehabilitation is defined and communicated in many ways – including as a health service, development imperative, human rights concern, and substance abuse issue . The WHO“We have a huge burden of Chronic Disease, […] so that’s one huge problem, physical Rehabilitation. The second is the extent of injuries in our country. Also elderly. Certainly among those who’s had some incident, a stroke, an injury, or a fracture or something, to prevent contractures and get back to normal.”— S054In the biomedical definition, rehabilitation is considered within the boundaries of the health service delivery system, focused on disability resulting from a traumatic or non-traumatic cause. The definition focuses on body borne conditions, including stroke, congenital conditions, injuries, and orthopedic conditions  [“[W]e have survivors with disability, a cancer survivor, a cardiovascular survivor, etcetera, etcetera, so we will have more survivors with disability.” – S058From the biomedical perspective, understanding rehabilitation in relation to persons with disabilities (PWD) could attach stigmatizing views to the sector and/or exclude other populations in need of support , 48, 49.“[Rehabilitation is] a really holistic process. […] The aim is to rehabilitate or bring back a function that has been lost somehow and depending on what are the areas, you will rehabilitate the body, or you will rehabilitate the society or you will find tools in order to include the person in the society.” – S017In contrast to the biomedical perspective, a broader, psychosocial approach to rehabilitation includes how enabling or impeding factors from outside a person’s body influence their functioning  , 51. Thi“Until you have people reporting on spheres of disadvantage, […] you’re not going address disability, and it’s going to remain a kind of token gesture. [..] It needs to be more looking at sort of social, political and economic determinants of health” – S010Informants that articulated a psychosocial definition also used the term disability in relation to rehabilitation. However, there are differences in the breadth of disability and how the term is being utilized. The key difference is the inclusion of social and environmental factors:“One, what is impairment, and second, what is a disability. […] Impairment is an organic problem, and the disability is a social problem. Society is the one that imagines a person with some deficiency and speaks of them as disabled than deficient.” – S024The extent to which informants articulate personal, environmental, and social factors as it relates to disability also varied. One informant described this by using the term impairment versus disability:Finally, a critique among a small number of informants describing a pyscosocial definition was that rehabilitation could be misconstrued with the idea that someone with a disability needs to be rehabilitated to achieve a ‘normal’ state  .“Even a Ministry of Health… [doesn’t] realize what real rehabilitation means. […] He thinks it is sanatorium.” –S060In failing to present a common definition, policy makers and the broader population misunderstand rehabilitation and corresponding policy needs , 46. It “I was working in the Ministry, in the National Office of Rehabilitation [and…] we presented a project that did not proceed. All because of diarrhea, we had polio and some other diseases that affect children under one-year-old. Infectious disease is one of the biggest obstacles we have.” – S024Lack of clarity on the nature of the problem can impede stakeholder’s ability to convince others that the problem demonstrates a “socially credible threat” requiring prioritization. Rehabilitation stakeholders face challenges articulating loss of functioning or disability as a population health threat. Policymakers respond more urgently to threats of mortality, particularly from infectious diseases, compared to morbidity  , 56. OneMillions, millions [of dollars] in preventing malnutrition, prevention of diarrhea, but what is not taken into account is that malnourished children are going to need physical rehabilitation as well.”. – S024“Improved functioning is a prospective benefit to individuals and society; however, problems are seen as more ‘threatening’ when they entail a loss, rather than a future benefit or a change in the current status quo . This te“[The] people’s general perception, particularly the ones who are not so educated, is that it’s okay, once you’ve had a fracture, you know it heals and you’re fine. The notion of making sure rehabilitation is done to bring it back to its original [functioning] is really not that widespread”. – S054This may have resulted in a limited awareness of how rehabilitation could improve functioning further  . One infThese dynamics could result in rehabilitation being perceived as a ‘luxury’ rather than necessity , 57, furProposed solutions to strengthen rehabilitation in LMICs are often complex and unproven, which can dissuade prioritization. Most respondents argued for the integration of rehabilitation services into the health system, which aligns with existing global recommendations , but KIs“Primary care is missing on rehab. I am not aware of any primary care facility in India, which has rehab facilities. So again, that is a huge gap – my point would be not even to look at primary care” – P003There was a tension between management of rehabilitation through a horizontal approach  and other service packages) versus vertical approach . Starting with hospital-based care and strengthening referrals acknowledges current limitations with delivering rehabilitation in PHC . One informant shared:“[W]e integrate the rehabilitation programme, at health extension programme [PHC] level, that’s great. The second one is […] a health centre […] near to the deep rural community. […] These two groups, the large majority of the population […] can access.” – S002An opposing approach was to start with rehabilitation’s integration into PHC . An informant described this approach:“We don’t need another international plan of rehabilitation; we need to integrate rehabilitation in all parts of the health conditions…When we talk about international strategy of management of [non-communicable disease] NCD, we should integrate rehabilitation.” – S058Another option was to integrate rehabilitation through specific disease entry points . One informant described this solution:“We need to say in the next 10 years this is how I see rehabilitation services are integrated with good access throughout the continuum of care and this is how I'm going to start in a very implemental way, and this is how I'm going to evaluate as I move forward, and this is how I'm going to finance it.” – S031Despite divergence in the proposed solutions, nearly all informants identified limited evidence for integration into health systems as a key challenge  –61. SomeA possible enabler to generate solutions is international influence. Literature and interviews emphasized the importance of WHO guidelines and legislation from other countries to advance solutions , 62, 63.Effective governance is critical for generating collective action for advancing an issue . FragmenDomestic advocacy coalitions are often important to advancing prioritization , 26, 27;Rehabilitation’s governance is hampered by lack of a common problem definition and complicated by the multisectoral nature of rehabilitation. Both governmental and non-governmental actors influence via domestic advocacy coalitions, while transnational actors exert influence on rehabilitation’s prioritization via funding and advocacy.If you don’t develop strategy, [then] you don’t do work with other countries and provide information for policy makers to make [them] aware of the problem." – S005“Across our findings, there was a perceived need for increased advocacy and awareness raising of rehabilitation compared to other issues. One informant described the urgency of positioning rehabilitation at the policy level:In maximizing the influence of domestic coalitions, theory points to the importance of leveraging political windows when coalitions can influence the policy process . Politic“We don’t have individuals who are really driving it very hard. That’s what is needed. Once you have people take it up with the government, with the agencies, with the ministries, you get it taken care of because it's a problem that everybody is aware of.” – S013For government actors, KIs frequently suggested that political champions are needed within government ministries . One informant argued:But where do government champions come from, and do they have similar interests? Adopting a problem definition beyond a biomedical perspective makes rehabilitation a multisectoral issue, and we found evidence for this within existing governance arrangements. In many countries, policies, funding, and programming for rehabilitation are found across Ministries of Health (MOH), as well as social welfare, education, labor, transportation, and justice , 49, 62.“There is the Ministry [of Health]. Then, there are other institutions, like the national social security system […] along with another area called the Superintendency of Occupational Risks. […] there are many institutions at work […] and they cannot agree on anything” – S027There is often no guiding institution within government to coordinate proposals or champion the issue, which may challenge collective action. Informants and literature described a lack of intersectoral coordination across ministries, including unclear or overlapping programs, accountability structures, and poor communication  , 66–70. The most common recommendation from KIs on how to improve governance was generating support from MOHs . Institutionalization of rehabilitation within a MOH has been recommended to strengthen governance, and Chile illustrates this potential. A strategic alliance between the Mental Health Unit, which had technical expertise, and the Primary Health Care Division, which had more power and resources, was critical to establishing guiding leadership . With th In addition to the emphasis on government champions, national non-governmental organizations (NGOs) or civil society organizations (CSOs) participate in the policy process . The strength of domestic advocacy coalitions is context specific. Some informants argued for further capacity  , 60, whiThe ability of coalitions to influence decision-makers is partially dependent on their cohesion. In general, our data illustrated that health actors emphasize a biomedical framing while CSOs draw from a rights-based framing, which could impact the ability of coalitions to advance a common agenda.“Professional organizations, first of [all], do not have an awareness that they work for rehabilitation. The physical therapist work[s] and advocate[s] for physical therapy; the occupational therapist for occupational therapy…” – P004In the biomedical framing, the emphasis is on the burden of disease and technical capacities for service delivery. This constituency is likely to align themselves with MOH. The role of provider associations was considered important in elevating rehabilitation within the health agenda  , 67. HowIn contrast, CSOs are often focused on PWDs, seen to promote a rights-based approach , and connect national-level work to international treaties , 74, 75.“I think people with physical disabilities who are probably better able to self-advocate tend to command the large majority of resources…so the ability to advocate for resources and access resources is dominated by people who are physically disabled, [as opposed to] people with cognitive or mental illness…” – S010An additional complication is that representation of disability and rehabilitation within a single advocacy movement could be challenging. PWD movements themselves are not always representative. In the words of one informant:Tensions exist surrounding the influence of transnational actors – including governments and other donor agencies providing international development assistance and/or supporting external health expenditures, global health initiatives, international agencies, and international NGOs – in advancing rehabilitation on national agendas.“There was the Millennium Development Goals […] and for 15 years, everybody focused on them […] but there was no rehabilitation component or aspects of such a disease programs. […] You wouldn’t expect countries to give priority to rehab if, at a global level, there is no shared consensus or shared understanding that rehabilitation should be a major aspect of the programs” – S028National CSOs and NGOs were seen as connected to donor agencies providing technical and financial support, which was perceived to amplify their impact  , 78. How“You have the 2030 rehabilitation but like you know what I don’t know what else can be driving force for that? That could be or like a couple of champion countries regions that you know during the World Health Assembly […] are getting up and making statements in support or pushing for this rehabilitation to constantly be on the agenda” – S029Specific to international organizations, interview data highlighted that the WHO is a leading convening power  and suggAn example of transnational advocacy for a rehabilitative service comes from the Pan American Health Organization (PAHO). In Latin America and the Caribbean, PAHO has raised the profile of mental health services through alignment with and knowledge sharing on WHO mental health policies and programs, including knowledge sharing initiative across countries, the facilitation of specific policy initiatives, and provision of technical assistance . This su“[International organizations need to] dedicate the time to study the local manifestations that each population has. [gives ex from depression] I think that the WHO tries to do it, but to truly promote that, the WHO officials themselves or the people who [do the] work, do not stop traveling to the countryside to be close to the populations.” – S040This transnational advocacy has to be carefully translated to the national context, however. KIs at the national level described how international examples or advocacy can be perceived negatively if not suited to the specific needs of the country, dissuading prioritization by national policy makers . An informant explains:“WHO make[s] lots of guidelines, for example management of cancer, and they don’t talk about rehabilitation as part of the management of cancer. They make lots of guide[lines] [for] NCD, and they don’t talk about the [importance] of rehabilitation.” – S058Finally, despite considering WHO the main international champion of rehabilitation, it is possible that issue gatekeepers within the WHO have not adopted rehabilitation as widely as other health issues. This was alluded to by informants who pointed to the lack of inclusion of rehabilitation in global disease guidelines, which they felt directly influences the issue’s low prioritization at the national level:The first two components of our framework consider the agency of actors to advance policy change. In contrast, structural factors are the ‘rules of the game’ that constrain or enable the ability of actors to advance an issue , 65. ForIn the case of rehabilitation, national legacies and health system capacity are important structural factors shaping the issue’s policy advancement.National legacies impact an issue’s framing, perceived urgency of the problem, and service delivery arrangements. Most frequently, past or current conflict was linked to increases in disability, increased need for specific types of rehabilitation services, and the influx of foreign financial resources and NGOs. Conflict shaped prioritization of specific types of rehabilitative services and drove investments in specific areas.In Kenya, political conflict led to an increase of funding to NGOs for mental health work . In MoroSimilarly, the 2015 conflict in Eastern Ukraine resulted in an increased burden of injuries and disability, drawing attention to the lack of health system capacity to treat those impacted by conflict and leading to new rehabilitation professional training programmes to build workforce capacity . However“More widely since the polio epidemic in 1950, rehabilitation services were important, […] Specialists began to be trained, already with university knowledge. From that moment on, there started to be doctors specializing in rehabilitation.” – S038Other national legacies influencing rehabilitation include natural disasters (S029) , the rolIn contrast, economic turmoil in Zimbabwe led to an increase in mental health conditions while also reducing available financial and human resources to meet increasing need . These eA cross-cutting national legacy is the role of stigma in influencing how rehabilitation is framed and therefore, its relative importance to both policy makers and the general population. Cultural perceptions of disability, attitudinal barriers, discrimination from family members and communities, and a general dismissive attitude were highlighted by KIs  as well as in the literature , 76, 84.We identified three health systems challenges influencing prioritization which are related to rehabilitation’s historic lack of representation in the public health care delivery system.First, weaknesses in existing service provision included the lack of available or trained rehabilitation service providers, particularly in the public sector  , 86, 87,“[NGO service provider] would set up a building called community mental health service […].Three years, four years down the line, the fund comes to an end, everyone goes home” – S033Second, private providers  provide a large percentage of rehabilitative care in LMICs. This is closely linked to the role of transnational actors. Many not-for-profit service providers are funded by international development agencies and/or private philanthropy, raising concerns about sustainability and fragmentation  , 79, 81.“The private entities are doing the work that the State should be doing. […] So, the State and the Ministry of Health are not interested, because other entities are taking charge of partially solving the problem.” – S024Some KIs expressed concern that the private sector’s large role in care provision had displaced government interest and results in further fragmentation of a unified vision for rehabilitation services . An informant described this dynamic:Third, there is a lack of national-level data on rehabilitation, which is related to how data systems are structured , 89. ThiBased on an analysis of KIIs and a complementary review of literature, we identified three interrelated components – problem definition, governance, and structural factors – impacting the prioritization of rehabilitation in LMIC national health systems. Within these components, we draw attention to how rehabilitation is understood , the relative ability of both government and non-governmental actors at the national and transnational levels to work collectively, and national legacies and health systems structures that shape the decisions of rehabilitation actors. These components have been distilled into a novel policy framework for the prioritization of rehabilitation in national LMIC health systems. This framework can be used to analyze the factors influencing prioritization of rehabilitation in different national contexts and to formulate context-specific strategies for advancing access to rehabilitation services.Despite diversity across national contexts, national rehabilitation stakeholders face similar types of challenges in increasing the prioritization of rehabilitation on national agendas. Through application of the framework and triangulation with policy theory and evidence, we advance three interrelated considerations for policymakers and the rehabilitation community.Divergent problem definitions challenge prioritization. A high degree of homogeneity is associated with increased agenda-setting success . We idenIn governance, rehabilitation actors face challenges organizing collectively. This is linked both to divergent problem definitions and the unclear boundaries of the issue. Consideration of the benefits and drawbacks of a diverse versus narrow coalition should be considered by rehabilitation advocates. Evidence emphasizes the importance of shared goals and incentives within a broad coalition, including health and non-health actors , 28, 41.The importance of a guiding institution during the agenda-setting phase is mixed, and coalitions do not require government leadership , 25, 27.Institutionalizing governance for rehabilitation within MOH has been advocated for. This could support prioritization by creating a clear line of accountability and empowering a guiding agency. However, we should not assume that centralization will automatically create stronger inter- and intra-agency coordination , particuFinally, structural constraints influence governance and problem definition, particularly solution acceptability. Those advancing rehabilitation can explore how incremental health system reforms impact larger-scale prioritization. For example, pilots to advance service integration may provide a proof-of-concept and generate evidence. The emergence of effective solutions and their inclusion in international guidance was found to support prioritization of drowning , and theThese tensions point to a central question – can the rehabilitation field collectively overcome these challenges to advance a single agenda, or would smaller coalitions focused on specific conditions or services be more effective at making the case? Our findings challenge the idea that a cohesive rehabilitation community or coalition exists. Our goal is not to resolve these tensions, suggestions for which have been posed by others . We instThis study and the resulting policy framework make several contributions to the nascent evidence base on the prioritization of rehabilitation and broader policy process scholarship.Specific to rehabilitation, this study complements a 2016 realist synthesis on good policy and governance for rehabilitation . Our finMore broadly, the framework builds on existing policy theory and health issue prioritization frameworks in several ways. Existing policy theories are often developed from high-income country case studies and are non-health-sector specific. As a result, applying these theories to health issue prioritization in LMICs often excludes the influence of existing health system challenges in the agenda setting phase of the health policy process. But in this case, many of the rehabilitation-specific challenges we identify – lack of credible indicators, intractability, governance fragmentation – reflect longstanding structural factors of the health system. These directly contribute to the perception of intractability and fragmented governance arrangements, and in doing so, exert a path dependent influence on prioritization. For example, health information systems must be able to collect disability and rehabilitation-related indicators to generate credible indicators. Health workers must be trained to provide services before budgets can be allocated to provide those services. An abductive approach to analyzing our data surfaced these tensions, which may have remained “out of scope” if a pre-existing deductive policy framework for agenda setting was utilized.The most prominent health-specific framework, Shiffman and Smith, is specific to global issue prioritization , while oScholarship directs us to examine specific factors key to prioritization of an issue. In this case, the secondary data analyzed were primarily negative cases that identified challenges rather than successes. This allowed us to synthesize barriers to prioritization; however, we relied on informant recommendations and triangulation with theory to hypothesize enablers. Our literature review was a purposeful search to complement the interview data and should not be interpreted or appraised as a systematic review. It is possible that additional positive cases exist within the mental health or disability-specific literatures that were not identified through our purposeful, rehabilitation-focused search strategy.While some of the theoretical literature we draw from has been deployed in LMIC health systems, others remain more focused on high-income country settings . To overTaking a cross-country approach to developing the framework limited our ability to conduct a deeper analysis of context-specific considerations in a few ways. Extracted literature and policy theory focused largely on the national level; however, we recognize that sub-national prioritization is also critical. Our sample of KIs was largely at the international or national levels, with limited representation of local civil society organizations and no representation of patient organizations or advocacy groups. This was due to our inability to identify specific individuals to contact for virtual interviews in those stakeholder groups. Our literature search was also limited to publications in English, which may have reduced available literature from specific LMICs or regions. These limitations emphasize the need for cross-country validation, application and adaptation.To facilitate national adaptation, we therefore kept the framework as components (rather than recommendations) and focused the components on the types of challenges common to national contexts rather than specific national challenges. A key consideration that did not emerge from our KII and literature data is the plausible influence of differing epidemiological profiles across countries. In South Africa, for example, 25% of rehabilitation-related needs are related to HIV-related conditions . We woulWe identify that problem definition, governance, and structural factors are all important to understanding prioritization of rehabilitation (or lack thereof) across national contexts. Our framework builds on the existing work of health policy scholars by emphasizing the importance of problem definition and governance and adds additional emphasis on the existing health system and historical legacies as structural factors that influence agenda-setting. The corresponding policy framework outlines a foundational approach to understanding these challenges and directs actors to a set of components core to advancing rehabilitation.Application of this novel framework can support rehabilitation stakeholders in identifying the context-specific factors enabling or impeding the prioritization of rehabilitation in different national contexts. This is a critical first step for advancing rehabilitation on national policy agendas and ultimately, expanding equitable access to rehabilitative care in LMICs.


Older patients are more likely to have comorbidities than younger patients, and multiple comorbidities are associated with mortality in patients with cancer. Therefore, we hypothesized that a functional comorbidity index could predict the therapeutic effects of rehabilitation.In this study, we investigate whether the comorbidities influenced the execution and therapeutic effects of rehabilitation.A consecutive cohort of 48 patients with gastrointestinal cancer who underwent surgery between January 1 and November 30, 2020, was analyzed. Charlson Comorbidity Index (CCI) scores were calculated based on data derived from medical records. The primary outcomes were ambulation status, duration (days) from the start of postoperative rehabilitation, and length of hospital stay. We investigated the relationship between CCI scores and primary outcomes.The CCI did not correlate with the duration of rehabilitation or the length of hospital stay. Subsequently, patients with functional recovery problems were evaluated, and we identified the conditions that were not included in the list using CCI scores. Most conditions are associated with surgical complications. Furthermore, using the Clavien-Dindo classification (CDC), we assessed the clinical features of the severity of complications. We found that the length of stay and the duration to start rehabilitation were significantly longer in the patients with higher severity of surgical complications (CDC≧III) than in those with lower severity (CDC≦II).Treatment-related conditions may significantly impact the perioperative period more than the original comorbidities. In addition to original comorbidities, events related to surgical complications should be assessed to determine the therapeutic effects of rehabilitation in patients with gastrointestinal cancer.The online version contains supplementary material available at 10.1186/s12957-023-03207-2. The incidence of cancer in advanced countries has increased . SurgeryPerioperative rehabilitation has been promoted to prevent complications after surgery , 6. PeriHowever, the effect of comorbidities on rehabilitation remains controversial. Several researchers have reported that comorbidities can predict poor functional recovery or prolonged hospital stay in patients with stroke , 8, hip Multiple comorbidities affect the nutritional status after esophageal cancer surgery , induce Recently, Chan et al.  investigIn this study, we hypothesized that the CCI scores would predict the negative therapeutic effects of perioperative rehabilitation in patients with cancer. Higher scores would correlate with lower therapeutic effects because comorbidities affect training execution or training efficacy. Understanding the potential factors influencing rehabilitation is helpful for successful risk management during training. We retrospectively evaluated 48 patients with gastrointestinal cancer who underwent perioperative rehabilitation. We assessed whether the comorbidities influenced the execution and therapeutic effects of rehabilitation.We retrospectively reviewed data from 48 consecutive patients with gastrointestinal cancer who underwent curative or palliative surgery and perioperative rehabilitation. We examined the type of cancer, CCI score, ambulation status, duration (in days) to start rehabilitation after surgery, and length of hospital stay.This retrospective study was conducted at the Nara Medical University Hospital. The study protocol was approved by the hospital’s Institutional Review Board . The study was conducted in accordance with the principles of the Declaration of Helsinki and the laws and regulations of Japan. Between January 1 and November 30, 2020, 98 patients with gastrointestinal cancer were referred to the department of rehabilitation medicine in our hospital. Of the patients, 50 were excluded from the study because they did not undergo the planned surgery but underwent exploratory laparotomy, endoscopic drainage, medication, or radiation for local control due to unexpected tumor progression. Finally, we analyzed 48 consecutive patients. All patients received a comprehensive palliative intervention from the beginning of treatment , 20. TheCCI scores were calculated according to the patients’ preoperative conditions derived from medical records . Table 1The Clavien-Dindo classification (CDC) ranks the severity of surgical complication –24. It iAll the patients were admitted for surgery. The primary outcome evaluations were ambulation status, duration (days) to the start of rehabilitation post-surgery, and length of hospital stay. The level of mobility achieved during rehabilitation was defined as levels 1–5 as described by Kim et al.  with somRehabilitation was initiated after patients underwent medical examinations performed by a physiatrist. All patients are usually referred to the rehabilitation department the day after surgery or after the attending physician has confirmed that vital signs are stable. The physiatrist will assess the patient on the day of referral. The physiatrists evaluated the general condition of patients based on Gerber’s recommendation . The ambP < 0.05. Spearman’s correlation test was used to evaluate the association between the CCI scores; duration of rehabilitation after surgery; and length of hospital stay; and between the age, duration of rehabilitation after surgery, and length of hospital stay. Mann–Whitney’s U test was used to evaluate the association between the severity of surgical complications and the CCI scores, duration of rehabilitation after surgery, and length of hospital stay.Statistical analyses were performed using SPSS software . Statistical significance was set at The baseline characteristics of the patients are shown in Table Figure r = 0.014, p = 0.924). Figure r = 0.2, p = 0.172).Figure Next, we determined the condition of the patients who could not reach or exceed Level 4 after rehabilitation. Sixteen patients met the inclusion criteria. Table Perioperative rehabilitation is of utmost importance in older patients with gastrointestinal cancer. This reduces the risk of complications, facilitates early recovery after surgery, and shortens the length of the hospital stay. Based on previous studies that reported that comorbidities worsen various conditions in cancer patients –16, we hInterestingly, we evaluated the clinical course of patients who did not reach or exceed Level 4 and identified conditions that were not in the CCI list Table . Most ofCharlson et al.  designedAs a potential limitation of the CCI, Extermann et al.  suggesteOur study has some limitations. This study was retrospective in nature, had a limited sample size, and was conducted at a single hospital. In addition, patients have different oncological backgrounds at different clinical stages. However, in our study, 19 conditions on the Charlson list were not sufficient to predict the functional outcomes of perioperative rehabilitation in patients with gastrointestinal cancer. Although the CCI is a useful tool for predicting oncological outcomes, treatment-related conditions may have a greater impact. In this study, we found that “surgical complications,” which are components of cancer treatment-related conditions, are also essential factors for assessing the therapeutic effect of rehabilitation. However, further large-scale studies in older cancer populations are required, particularly during the perioperative period. As society ages, the number of cancer patients with comorbidities will increase. To improve the quality of life of these elderly patients and their families, it is essential to identify factors that influence rehabilitation.Treatment-related conditions may significantly impact perioperative rehabilitation more than the original comorbidities. As well as original comorbidities, clinical events related to surgical complications should be assessed to determine the therapeutic effects of rehabilitation in patients with gastrointestinal cancer.Additional file 1: Supplementary File 1. Patient characteristics for surgery and perioperative rehabilitation.


Capsicum annuum). Furthermore, we investigated the roles of apoplastic reactive oxygen species (ROS), including hydrogen peroxide (H2O2) and superoxide radical (O2•−), in EBL-promoted AR formation, by using physiological, histochemical, bioinformatic, and biochemical approaches. EBL promoted AR formation by modulating cell-wall-located polyamine oxidase (PAO)-dependent H2O2 production and respiratory burst oxidase homologue (RBOH)-dependent O2•− production, respectively. Screening of CaPAO and CaRBOH gene families combined with gene expression analysis suggested that EBL-promoted AR formation correlated with the upregulation of CaPAO1, CaRBOH2, CaRBOH5, and CaRBOH6 in the AR zone. Transient expression analysis confirmed that CaPAO1 was able to produce H2O2, and CaRBOH2, CaRBOH5, and CaRBOH6 were capable of producing O2•−. The silencing of CaPAO1, CaRBOH2, CaRBOH5, and CaRBOH6 in pepper decreased the ROS accumulation and abolished the EBL-induced AR formation. Overall, these results uncover one of the regulatory pathways for BR-regulated AR formation, and extend our knowledge of the functions of BRs and of the BRs-ROS crosstalk in plant development.Adventitious root (AR) formation is a critical process in cutting propagation of horticultural plants. Brassinosteroids (BRs) have been shown to regulate AR formation in several plant species; however, little is known about their exact effects on pepper AR formation, and the downstream signaling of BRs also remains elusive. In this study, we showed that treatment of 24-Epibrassinolide  at the concentrations of 20–100 nM promoted AR formation in pepper ( Adventitious root (AR) formation is a fundamental process in root biology . Ars arePhytohormones, together with many other internal and external stimuli, coordinate every step of AR formation from the first event of cell reprogramming until emergence and outgrowth . Of thes2O2) and superoxide radical (O2•−) are two typical ROS, belonging to non-radical and free radical forms, respectively -bis(4-methoxy-6-nitro) benzene-sulphonic acid hydrate} (XTT) by Oeviously .https://www.arabidopsis.org/, accessed on 15 March 2023), and the tomato PAO (SlPAO) proteins were obtained from Sol Genomics Network . All AtPAO and SlPAO sequences were used as baits for searching CaPAO homologs in the Capsicum annuum genome  in NCBI . A multiple-sequence alignment was performed using ClustalX (version 1.81) [CaRBOH genes was performed by using the same strategy as CaPAOs. The secretory signal peptides of AtPAO1, SlPAO1, and CaPAO1 proteins were predicted via SignalP-5.0 , and the transmembrane domains of CaRBOH proteins were predicted by TMHMM-2.0 program .The Arabidopsis PAO (AtPAO) proteins were obtained from TAIR database (on 1.81) . A phyloon 1.81) . Gene in−∆∆CT threshold cycle method. The CaUBI-3 gene (LOC107873556) was used as an internal control [Total RNA was isolated from 5 mm segments from the base of pepper hypocotyls at the indicated times during adventitious root formation. Total RNA was isolated using an EasyPure Plant RNA Kit  according to the manufacturer’s protocol. Residual genomic DNA was digested with Dnase I (Sigma-Aldrich). Reverse transcription was performed using 2 mg of total RNA and M-MLV Reverse Transcription System . Quantitative PCR (qPCR) was performed using the TransStart Green qPCR SuperMix (Transgen) on a LightCycler 96 machine , according to the manufacturer’s instructions. Three biological replicates were performed per gene, and three technical replicates were performed within an experiment. Samples were collected from three independent experiments. The relative transcript abundance was calculated based on 2 control ,48. The CaPAO1, CaRBOH2, CaRBOH5, and CaRBOH8 genes were cloned into pCAMBIA2300 vector. The specific primers are listed in Agrobacterium tumefaciens strain GV3101, which was further infiltrated into the 1st true leaves of 20-day-old pepper seedlings by a needleless syringe. After 3 d of infiltration, the leaves were harvested for RNA extraction, and the expression levels of target genes were detected by qRT-PCR. The leaves were also used for histochemical staining of H2O2 and O2•−.The full-length cDNAs of https://vigs.solgenomics.net/, accessed on 5 May 2023). PCR products were further cloned into TRV2 vector with restriction enzymes EcoR I and BamH I, resulting in the pTRV2 derivatives pTRV2-CaPAO1, pTRV2-CaRBOH2, pTRV2-CaRBOH5, and pTRV2-CaRBOH6. The transformed Agrobacterial cells (GV3101) were collected and resuspended in infiltration buffer  to a final density of OD600 = 1.0, and then left at room temperature for 3 to 4 h without shaking. Before infiltration, A. tumefaciens cultures containing pTRV1 and pTRV2 or their derivatives were mixed in a 1:1 ratio. The culture suspensions were infiltrated into the germinated seeds (with 0.5~1 cm of radicles) using a vacuum-assisted infiltration method [With the full-length cDNA of target gene as template, a 300 bp fragment was amplified using the specific primers listed in n method . After ct-tests were performed in Microsoft Excel. In graphs that include letters, each letter represents a statistically significant mean by Tukey’s test at a level of p < 0.05.Each datum is shown as mean ± standard deviation (SD) for at least three replicates. Two-way analysis of variance was performed in GraphPad Prism or with SPSS statistics (SPSS 19.0). Student’s EBL induced significant increases in AR number and AR length in a dose-dependent manner. EBL at 50 nM exhibited the greatest effect A–C. TreaBRz, a specific inhibitor of BR biosynthesis, was also used to determine the effect of BR on AR formation. As shown in 2O2 contents in the rooting zone increased slightly at 3 h and 6 h, and then, at 24 h, the rate of H2O2 production increased sharply. In the EBL-treated hypocotyls, the amounts of H2O2 were much more prevalent than in the control explants, while in the BRz-treated explants, the H2O2 accumulation after 24 h was significantly repressed , MDL 72,527 and 2-HEH (inhibitors of PAO), and DPI (an NADPH oxidase inhibitor) on EBL-induced AR formation were evaluated. The results suggested that MDL72527, 2-HEH, and DPI, but not AG, could significantly compromise the promoting effect of EBL on AR formation A, indica2O2 content   A,B. Likestaining C,D. ThesCaPAO1 and CaRBOH genes in EBL-induced AR formation. In a preliminary experiment, we silenced the expression of the phytoene desaturase (PDS) gene, which is used as a visible marker to monitor VIGS efficiency [TRV2:PDS plants exhibited a significant etiolation phenotype above the cotyledon code. These results indicated that the VIGS system worked efficiently under our experimental conditions, and gene silencing occurred in the epicotyls but not hypocotyls. Thereby, in our following studies, the epicotyls were used in the AR formation assay.We employed VIGS to investigate the function of the ficiency . As showCaPAO1 and CaRBOH genes in pepper. Under normal conditions, the growth of VIGS plants was comparable to that of the control plants , while high concentrations (≥1 μM) of EBL treatments suppressed AR development . These rgulliver1/sur2–7 mutants, likely by enhancing IAA biosynthesis [Moreover, BRs have been shown to regulate AR formation through crosstalk with other plant hormones. Auxin is the key hormone that induces AR formation . BRs andynthesis . Treatmeynthesis . Also, tynthesis . In addiynthesis . Howeverynthesis . During In addition to exogenous BRs, the endogenous BR signal was also involved in AR formation in pepper, based on our finding that BRz, an BR inhibitor, could significantly reduce the AR number . Accordi2O2 and O2•−, were required for EBL-induced AR formation in pepper  in a ROS-dependent manner (CYC and CDK genes directly via the TEOSINTE BRANCHED1-CYCLOIDEA-PROLIFERATING CELL FACTOR1 (TCP) transcription factor. When ROS levels increased, a disulfide bond was formed, preventing the binding of TCPs on the target gene promoter [Apoplastic ROS are engaged in plant development and responses to extrinsic signals. The low antioxidant efficiency in the apoplast allows ROS to accumulate easily . The apothe cell ,68. Oncethe cell . During ll cycle . AR initll cycle , and LBDidentity . In thist manner H. ROS mipromoter ,72. Take2O2 as byproduct. EBL induced the most significant increase in CW-PAO activity and CaPAO1 expression in the AR zone . The roles of RBOH-derived Ond so on ,32,33,77lication . Co-applormation . Of thesmergence . In peppormation , which iormation , indicatn pepper . Collect2•−, and then O2•− activated the apoplastic PAOs to amplify H2O2 accumulation. Then, PAO-generated H2O2 could open Ca2+ channels and thereby increase the activity of the Ca2+-regulated RBOH enzymes and form a RBOH-PAO amplification loop. Finally, this RBOH-PAO loop caused an ROS accumulation that surpassed a toxicity threshold, resulting in programmed cell death [AtPAO1 and AtPAO2 genes resulted in enhanced O2•− generation and RBOH activity [In plants, PAOs and RBOHs are functionally interlinked in controlled ROS production and homeostasis; however, their coordination is controversial . Gémes ell death . Howeveractivity . In thisCaPAO1 to increase apoplastic H2O2 generation. EBL also induces the expression of CaRBOH2, CaRBOH5, and CaRBOH6 to increase apoplastic O2•− generation. Then, O2•− can be transformed to H2O2. These ROS signals enter the cytoplasm and trigger signaling transduction, which further regulates the expression of LBD genes and cell cycle genes to initiate AR formation and promote AR elongation. This model helps elucidate the regulatory mechanism underlying EBL-induced AR formation. In future studies, the detailed signaling cassette behind ROS-induced AR formation needs to be identified.Taken together, we propose a model for ROS generation during EBL-induced AR formation in pepper hypocotyls . EBL sti