Prior studies have shown that political affiliation affected COVID-19 vaccine hesitancy. This study re-examined the data to see if these findings hold after controlling for alternative explanations. The dependent variable in the study was COVID-19 vaccination rates in 3,109 counties in the United States as of April 2022. The study examined 36 possible alternative explanations for vaccine hesitancy, including demographic, social, economic, environmental, and medical variables known to affect vaccine hesitancy. County-level political affiliation was measured as a percent of voters in the county who were affiliated with Democratic or Republican political parties. Data were analyzed using a temporally constrained multiple mediation network, which allowed for the identification of both direct and indirect predictors of vaccination rates. Despite controlling for alternative explanations of hesitancy, there was a statistically significant relationship between the percentage of Republican supporters and rates of vaccine hesitancy. The higher the Republican affiliation, the lower the vaccination rates. It is possible that the Republican Party has played an organizing role in encouraging vaccine hesitancy and patient harm. IntroductionPolitical leaning is frequently linked to particular values and ideological frameworks, which can shape people's attitudes and beliefs on a broad range of topics, including public health and vaccination -3. For iSeveral recently published studies found a relationship between political preference and COVID-19 vaccination hesitancy. Albrecht found that Republican-leaning counties (as measured by the percentage of the residents voting for Trump) experienced lower vaccination rates for COVID-19, higher COVID-19 cases, and higher death rates . His subSince political leaning is affected by the same set of variables  as vaccine hesitancy, it is not clear if the observed differences are due to confounding in observational data. It is possible that the observed relationship between political leaning and vaccination hesitancy disappears if one statistically controls for other explanations of vaccine hesitancy. The main purpose of our study is to re-examine the effect of political leaning on vaccination rates, after controlling for a larger set of demographics, social, medical, access, and environmental determinants of vaccine hesitancy.Materials and methodshttps://archive.org/web/), U.S. Census Bureau (American Community Survey (ACS), Small Area Income and Poverty Estimates (SAIPE), and population estimates and projection), U.S. Environmental Protection Agency (EJScreen data), U.S. Department of Agriculture, Environmental Systems Research Institute , and the National Center for Health Statistics.Data were collected from multiple secondary sources, including the U.S. Census, Centers for Disease Control and Prevention, Wayback Internet Machine  regressions. This methodology allows for the identification of both direct and indirect predictors of vaccination rates. The first regression identifies the direct predictors of vaccination rates. Subsequently, the direct predictors are regressed on variables preceding in time to identify the indirect predictors of vaccination rates Figure . The metResultsTable Figure In the previous study, the following 13 variables affected vaccination rates: (1) percent of residents with American Indians or Alaska Natives; (2) percent of Asian residents; (3) percent of senior residents (≥65 years). The percentage of Black or African American residents did not directly predict vaccination rates. (4) The higher the number of vaccination providers in the county, the higher the vaccination rates. Clearly, access mattered.The following social determinants of illness affected vaccination rates: (5) percent of residents with high school completion; (6) percent of residents with some college; (7) percent of residents unemployed; (8) median household income; (9) percent of residents not proficient in English; and (10) percent of children in poverty.The following health status variables affected vaccination rates: (11) average life expectancy of residents; (12) percent of residents with a disability; and (13) premature mortality rates.These direct predictors of vaccination rate also mediated the effect of other factors, including (1) level of income inequality in the county, (2) traffic volume, (3) percent of residents with home ownership, (4) percent of children in single-parent homes, (5) percent of African American residents, (6) total population, (7) rural/urban settings, (8) severe housing costs, and (9) percent of residents employed.Given these relationships, we examined if political affiliation was predictive of vaccine hesitancy. In the LASSO regression of vaccination rates on 36 2022-measured variables, 14 variables had a non-zero relationship with vaccination hesitancy, including the 13 variables listed above and political affiliation.DiscussionOur analysis identified a variety of factors that affect vaccine hesitancy, as have a large number of previous studies. Education affected vaccine hesitancy. “Some college” and “high school completion” were positively associated with higher vaccination rates, as seen in other studies as well -14,. RacBefore we discuss the impact of political affiliation on vaccination rate, it is important to point out that vaccination rates and political affiliations were affected by the same set of covariates. Counties that had a higher percentage of Republican supporters were also likely to be (1) rural, (2) White, and (3) had a higher percentage of residents over the age of 65 years or under 18 years. These counties were also more likely to have (4) lower unemployment rates, (5) lower levels of traffic volume, (6) lower levels of housing cost burden, and (7) lower levels of median house income. Additionally, higher poverty rates positively contributed to Democratic-leaning counties rather than Republican-leaning ones. The network model statistically controlled for these covariates and identified political affiliation as a mediator of vaccination rate. Seven variables (listed above) impacted the vaccination rate through increasing Republican political affiliation. These characteristics that increased the likelihood of supporting the Republican Party, with two exceptions (median household income and older than 65 years), did not directly affect vaccination rates. In short, party affiliation was a mediator of the effect of five variables that also increased party affiliation.This study shows that party affiliation was not only an independent direct predictor of hesitancy but also a mediator of the effects of other variables. In statistics, when a variable is a mediator of the effects of other variables, it is generally interpreted that it shows the mechanism through which the indirect variables affect the outcome. There are many ways in which party affiliation may have acted as the mechanism leading to lower vaccination rates. One study has suggested that political affiliation leads to media choices that could affect vaccine hesitancy . AnotherConclusionsPolitical affiliation plays an important direct, and mediating, role in reducing vaccination rates. The direct effect of political affiliations on vaccine hesitancy remained despite the use of a variety of alternative explanations. It also appears that these affiliations played an organizing role and mediated the effects of a variety of other variables. This study, and other investigators, suggest that interventions and public health policies should be designed to specifically address the impact of political affiliation on vaccine hesitancy .


Background: Autism spectrum disorder (ASD) encompasses a group of disorders characterized by difficulties with social interaction and repetitive behavior. The condition is supposed to originate from early shifts in brain development, while the underlying processes are unknown. Moreover, a considerable number of patients with ASD experience digestive difficulties. Metalloproteases (ADAMs) are a class of enzymes capable of cleaving membrane-bound proteins. Members of this family, ADAM17 and ADAM22, have the ability to cleave proteins like the pro-inflammatory cytokine TNF-ά and glutamate synaptic molecules, which are both engaged in neuro-inflammation and glutamate excitotoxicity as crucial etiological mechanisms in ASD. ADAM17 and ADAM22 may also have a role in ASD microbiota–gut–brain axis connections by regulating immunological and inflammatory responses in the intestinal tract. Subjects and Methods: Using ELISA kits, the plasma levels of ADAM17 and ADAM22 were compared in 40 children with ASD and 40 typically developing children. All of the autistic participants’ childhood autism rating scores (CARS), social responsiveness scales (SRS), and short sensory profiles (SSP) were evaluated as indicators of ASD severity. Results: Our results showed that plasma levels of ADAM17 were significantly lower in ASD children than in control children, while ADAM22 demonstrated non-significantly lower levels. Our data also indicate that while ADAM17 correlates significantly with age, ADAM22 correlates significantly with CARS as a marker of ASD severity. Conclusions: Our interpreted data showed that alteration in ADAM17 and ADAM22 might be associated with glutamate excitotoxicity, neuroinflammation, and altered gut microbiota as etiological mechanisms of ASD and could be an indicator of the severity of the disorder. Autism spectrum disorder (ASD) encompasses a group of neurodevelopmental diseases typically identified in infancy but which may last throughout adulthood. It is mainly indicated by a lack of social engagement and communication as well as by the existence of stereotyped behaviors . ASD is a collection of neurodevelopmental disorders characterized by social and behavioral abnormalities, difficulty in verbal and nonverbal communication, and confined, repetitive behaviors and interests. The World Health Organization estimates that the global prevalence of ASD in 2022 is almost one in every 36 individuals [In addition to synapse-related genes being downregulated, microglia and immune-related genes were upregulated in autistic patients’ brains ,4. The pThe disintegrin and metalloprotease (ADAMs) family of proteins are type I transmembrane and secreted metalloendopeptidases that mediate the cleavage of membrane protein extracellular domains (ectodomains). ADAMs regulate a variety of proteins, including growth factors, cytokines, receptors, and adhesion molecules, all of which play critical roles in a variety of biological processes such as cell migration, cell adhesion, proteolysis, and signal transmission . ADAM 17 and ADAM 22 enzymes are members of the family of metalloproteases (ADAMs) that can degrade membrane-bound proteins. ADAM17 and ADAM22 cleave essential protein substrates that govern neuronal networks and immunological responses, respectively, such as the neuro-inflammatory cytokine TNF-α and glutamate synaptic molecules, all of which are involved or disturbed in ASD .Higher brain operations like learning and memory rely on finely regulated synaptic transmission and plasticity . PSD-95 The gut–brain axis denotes the bidirectional pathway between the gut and the brain . AlteratIn an attempt to link neuroinflammation, glutamate excitotoxicity, and impaired gut microbiota as etiological pathways in ASD that are regulated by ADAM 17 and ADAM 22 respectively, new evidence has revealed that TNF-ά has been shown to increase excitatory synaptic strength via AMPA receptor exocytosis and reduce inhibitory synaptic strength via GABA (A) receptor endocytosis, shifting the E/I balance towards excitation ,19.El-Ansary  proposedBecause there is no particular biomarker for ASDs, they are frequently not properly diagnosed. The discovery of possible targets for the development of diagnostic and/or therapeutic procedures must be given top priority in ASD care in the future. Numerous proteases are connected to both the regulatory mechanisms of ASD etiopathology and therapy as well as the physiological processes that underlie these disorders. In genetic animal models of ASD, the AMPA receptor positive allosteric modulator increased social interaction . These fThis information tempered our interest in measuring ADAM 17 and ADAM 22 in the plasma of individuals with ASD to emphasize the role of both proteases in neuroinflammation and unbalanced E/I neurotransmission as two known etiological mechanisms of ASD previously reported in the same cohort of participants . Figure The Autism Research and Treatment Center, Faculty of Medicine, King Saud University, and King Khalid University Hospital were the sites of this study. The King Saud University Faculty of Medicine’s Institutional Review Board authorized the study protocol following the most recent Declaration of Helsinki (Ref. No. 22/0122/IRB). Before the assessment, all participants’ parents provided informed consent. The Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, was used to diagnose ASD (DSM-IV).This study included 80 children . ASD male and female children aged 3 to 12 (mean age of 6.43 ± 2.22) who met the inclusion criteria for this study were recruited from the Autism Research and Treatment Center, Faculty of Medicine, King Saud University. The control group consisted of 40 age-matched healthy male and female children who visited King Khalid University Hospital’s pediatric clinic for normal check-ups. The study excluded anyone with an infectious condition or a mental problem. Patients who were associated with neurological disease , metabolic disorders , and autoimmune disease were excluded from the study, as metabolic diseases and autoimmunity may influence the results of plasma ADAM 17 and ADAM 22 levels. The control group consisted of 40 healthy male and female children who were matched for age and sex. They had no family relations to the ASD children, and their ages ranged from 3 to 12 (5.83 ± 3.22 years). Additionally, they did not have any clinical signs of immunological, diabetic, chronic, or neuropsychiatric illnesses. CARS is commonly used to screen for autism . It is aThe SRS is a 65-item scale for assessing the severity of autistic symptoms . It is aThe SSP is a 38-item questionnaire used to score a variety of sensory deficits . It is cACollection of plasma samples:g at 2–8 °C within 30 min of collection. The samples were stored at −20 °C or −80 °C until use. Repeated freeze-thaw cycles were avoided.After overnight fasting, blood was collected using EDTA or heparin as an anticoagulant. Samples were then centrifuged for 15 min at 1000× BAssay of ADAM17 and ADAM22 using ELISA technique:EIAab Science INC, Wuhan’s ELISA kits were used to assay both metalloproteases. The microtiter plate for these kits has been pre-coated with either an antibody specific for ADAM 17  or ADAM 22 . For each protein assay, the standards or samples were added to the microtiter plate wells first, followed by the addition of a biotin-conjugated polyclonal antibody preparation, specific for ADAM 17 or ADAM 22, and finally the addition of avidin conjugated to horseradish peroxidase (HRP) and incubated. To each well, a TMB substrate solution was then added. Only the ADAM17, or ADAM22 biotin-conjugated antibody, and enzyme-conjugated avidin-containing wells show a change of color which was measured at 450 nm wavelength using a spectrophotometer. The O.D. of the samples was then compared to the standard curve to determine the concentration of both proteins in the control and patient plasma samples. Detection limit was 78–5000 pg/mL for both. The summary of the experiment is illustrated in t-tests were used for all statistical comparisons, with a p value < 0.05 being considered significant. When the Shapiro–Wilk’s test is negative and the data are not normally distributed, the Wilcoxon–Mann–Whitney test is typically utilized. The Spearman correlations test was used to calculate the association between the ADAM 17 and ADAM 22, CARS, SRS, SSP, and age. Positive or negative correlations were reported. The ROC curve was generated using the following parameters: (a) diagnostic sensitivity, demonstrating a proportion of true positive results in all patients with the analyzed pathology; (b) diagnostic specificity, demonstrating a proportion of true negative results in all healthy individuals without such pathology. AUC equal to 0.5 means no discrimination, 0.6–0.7 acceptable discrimination, 0.7–0.8 excellent discrimination, and AUC > 0.9 means outstanding discrimination.The statistical analysis was conducted using software, and the results were presented as mean ± S.D. Independent Student’s p < 0.001) (p < 0.001) and a non-significant but still considerably lower level (−17.2%) of ADAM22 (p < 0.413) in ASD participants compared to controls. p < 0.001). ADAM 17 and ADAM 22 showed significant differences between mild-moderate and severe autistic persons enrolled in the study for CARS as a measure of ASD severity (p < 0.041 and 0.05 respectively), but not in SRS or SSP (< 0.001) . Table 3S or SSP . Table 8Transmembrane-TNF-(tmTNF-), the precursor of the pro-inflammatory soluble form of TNF-(sTNF-), is converted by ADAM17 as TNF-converting enzyme (TACE) ,29,30. AASD patients frequently experience gastrointestinal (GI) issues. Madra et al.  reportedp < 0.001) and the non-significant but still considerable 20% lower levels of ADAM22 in the plasma of children with ASD compared to controls could be attributed to the disrupted blood–brain barrier (BBB) in patients with ASD [Our study showed a significantly lower level of ADAM17 in plasma of ASD participants compared to controls. It could be suggested that the reported lower level of ADAM17 in plasma would be accompanied by higher levels in the brains of participants with ASD. In the present study, the significant decrease of ADAM 17  and ADAM22 (p < 0.05) participants in regard to CARS as the most reliable severity score of ASD. This could be explained and supported by considering the in vitro study in which ADAM17 inhibition lowers constitutive and LPS-activated TNF-α, TNFR-1, and IL-6R production by microglial cells while increasing constitutive and LPS-activated microglial phagocytotic activity. On the other hand, SRS and SSP as a measure of social interaction impairment and sensory profile, respectively show no significant changes in both measured enzymes.This study highlights neuroinflammation and glutamate excitotoxicity, as two ASD etiological mechanisms that may be exacerbated by the elevated expression and activity of brain ADAM17 and ADAM22. Reduced or inhibited levels of these targets may therefore prove exciting as therapeutic approaches for ASD.


N = 245). Hierarchical multiple regression analyses showed that quantitative job insecurity climate was associated with higher levels of loyalty and neglect, while qualitative job insecurity climate was related to higher levels of exit and lower levels of loyalty. Importantly, job insecurity climate explained additional variance over individual job insecurity in exit and loyalty. Our findings underscore the importance of addressing job insecurity in a broader context regarding one’s situation and the psychological collective climate. This study contributes to addressing the knowledge gap concerning job insecurity climate, an emerging construct in the organizational behavior literature, and its incremental impact beyond individual job insecurity. The foremost implication is that organizations need to pay attention to the evolving climate perceptions about the future of jobs in the work environment, because such perceptions are related to critical employee behaviors.Past work has extensively documented that job insecurity predicts various work- and health-related outcomes. However, limited research has focused on the potential consequences of perceived job insecurity climate. Our objective was to investigate how the psychological climate about losing a job and valuable job features  relate to employees’ exit, voice, loyalty, and neglect behaviors, and whether such climate perceptions explain additional variance in these behaviors over individual job insecurity. Data were collected through an online survey using a convenience sample of employees working in different organizations in Türkiye ( Job insecurity is among the most crucial work stressors affecting employees and organizations . Past reJob insecurity has been traditionally defined as the concerns or threats regarding the potential loss of the job itself (quantitative job insecurity) or loss of valued job features such as pay, promotion opportunities, working hours, and job content  ,8. It haIndividuals may resort to various behavioral responses in the face of unsatisfactory working conditions. The exit, voice, loyalty, and neglect (EVLN) framework argues that leaving the organization (exit), trying to affect the organization in a desired way (voice), staying loyal to the organization , or protesting by negligence (neglect) are responses employees may engage in when exposed to dissatisfying employment conditions  ,15,16. SShoss  called fResearch Question 1: How do the psychological collective climate perceptions about losing the job and valuable job features  relate to employees’ exit, voice, loyalty, and neglect behaviors?Research Question 2: Do job insecurity climate perceptions explain additional variance in these behaviors over individual job insecurity perceptions?In the following sections, we first define the job insecurity climate. Then, we review the literature on how job insecurity (climate) relates to EVLN behaviors, concluding by stating the research hypotheses and presenting the conceptual model guiding the present study.The social information processing theory  emphasizIn workplaces, individuals often work together in workgroups and teams. They share emotions, thoughts, and perceptions with others in those groups. Thus, the perception of job insecurity can be shared and exchanged through individual interactions within workgroups ,12,13 anScholars have conceptualized job insecurity climate as a psychological climate-related construct about losing a job and valuable job features ,25. SimiWhen employees face unfavorable conditions in their work situation, they may choose to leave the organization (exit), raise concerns and attempt to change the dissatisfying condition in a desired way (voice), stay and support the organization , or become slack and engage in disregardful behaviors such as lateness and absenteeism (neglect) ,26. ThesPast research has demonstrated significant associations between individual job insecurity and EVLN behaviors ,22. RegaAlthough contradictory results exist regarding the association between job insecurity and voice ,23,30, mWhen it comes to loyalty, research has generally found a negative relationship between individual quantitative job insecurity and loyalty, suggesting that job insecurity is associated with decreased commitment to the organization ,3,35. AlWhile research on the link between job insecurity and neglect is limited, past findings have yielded positive associations between organizational downsizing and the risk of long-term sick leave , as wellBased on previous research on individual job insecurity in relation to exit, voice, loyalty, and neglect—and the few studies on job insecurity climate—it would be reasonable to expect the relationship between job insecurity climate perceptions and such outcomes to be of a similar nature to those of individual job insecurity. To answer our research questions about how the climate of job insecurity relates to employee behaviors and whether such climate perceptions explain additional variance in these behaviors over individual job insecurity, we proposed the following hypotheses  is related to higher levels of exit (H1a) and neglect (H1b), and lower levels of voice (H1c) and loyalty (H1d), when tested together with individual job insecurity dimensions.Hypothesis 2. Qualitative job insecurity climate  is related to higher levels of exit (H2a) and neglect (H2b), and lower levels of voice (H2c) and loyalty (H2d), when tested together with individual job insecurity dimensions.Hypothesis 3. Job insecurity climate perceptions explain additional variance in exit (H3a), voice (H3b), loyalty (H3c), and neglect (H3d) after considering the effects of individual job insecurity.The cross-sectional study was conducted between May and September 2019 with an online survey using a convenience sampling method. The sample consisted of 245 employees working in different organizations in Turkey . All participants were assured of confidentiality and informed that their answers would only be used for this research. No incentive was provided for participation. The missing values (1.14% of the data set) were imputed using the EM algorithm based on the recommendations by Tabachnick and Fidell . EthicalThe items of all scales used in the study were translated into Turkish by a research team whose members had a good command of the English language. The back-translation from Turkish to English was then performed by a bilingual person unfamiliar with the item wordings in their original English version. After gathering the translated and back-translated information, the researchers created the proper Turkish translations for each item.Individually perceived quantitative job insecurity was assessed using the three-item subscale, and qualitative job insecurity was measured using the four-item subscale of Hellgren et al. . Sample In this study, we operationalized job insecurity climate based on the referent-shift approach . We aske2(84) = 188.62, p < 0.001, CFI = 0.96, RMSEA = 0.07, SRMR = 0.06). The four-factor representation clearly outperformed the model where all items were loaded on a single job insecurity factor (Satorra–Bentler χ2(90) = 746.79, p < 0.001, CFI = 0.74, RMSEA = 0.17, SRMR = 0.15), a two-factor model distinguishing between individual job insecurity and job insecurity climate (Satorra–Bentler χ2(89) = 735.25, p < 0.001, CFI = 0.75, RMSEA = 0.17, SRMR = 0.14), and a two-factor model distinguishing between quantitative and qualitative job insecurity (Satorra–Bentler χ2(89) = 415.75, p < 0.001, CFI = 0.87, RMSEA = 12, SRMR = 0.12), as well as a second-order model in which all four first-order factors loaded on a higher-order job insecurity factor (Satorra–Bentler χ2(86) = 217.08, p < 0.001, CFI = 0.95, RMSEA = 0.08, SRMR = 0.09).We subjected the job insecurity items to confirmatory factor analysis to ensure that the four measures represented distinct dimensions of job insecurity. The results indicated that the four-factor structure of job insecurity—consisting of quantitative and qualitative individual job insecurity, and quantitative and qualitative job insecurity climate—provided a satisfactory fit to data  = 668.85, p < 0.001, CFI = 0.94, RMSEA = 0.07, SRMR = 0.08).We conducted a confirmatory factor analysis on the items to ensure that the four measures represented distinct dimensions. The results revealed that the four-factor structure of EVLN  provided a satisfactory fit to data in the present sample (Satorra–Bentler χParticipants were asked to report their age (in years), gender , education level , organizational tenure (in years), and sector they were working in .We performed hierarchical multiple regression analyses for each behavior  to examine the relation between the two job insecurity climate dimensions  and the four behaviors. The predictors were added in two pre-determined steps. In Step 1, we entered the demographic variables that had significant bivariate correlations with the outcome variables, and individual quantitative and qualitative job insecurity into the analyses to exclude their effects on the dependent variables. In Step 2, we added the quantitative and qualitative job insecurity climate dimensions to the model. These regression analyses enabled observing the specific variance explained by job insecurity climate dimensions in the four behaviors beyond the effects of the controlled demographic variables and individual job insecurity perceptions.p < 0.01) and neglect , suggesting that participants who perceived threats to the continuity of their jobs reported higher preferences for quitting the job and protesting with neglect. Moreover, individual qualitative job insecurity was positively associated with exit  and negatively related with voice  and loyalty . These results indicate that participants who perceived threats of losing valued features of their jobs might be more prone to quit their jobs and less inclined to engage in voice and loyalty. In total, the first step accounted for 13% of the variance in exit, 11% in voice, and 16% in loyalty, while it did not explain a significant proportion of variance in neglect.In Step 2, with the addition of quantitative and qualitative job insecurity climate dimensions to the model, all six variables explained 19%, 12%, 23%, and 6% of the variances in exit, voice, loyalty, and neglect, respectively. In this step, the effects of individual quantitative job insecurity in predicting exit and neglect were no longer significant. In addition, different from Step 1, working in the private sector was associated with lower loyalty.p < 0.001) and neglect , indicating that a psychological collective climate characterized by concern about losing the current job was associated with higher levels of loyalty to the organization and protesting with neglect. Hypothesis 1 stated that quantitative job insecurity climate is related to higher levels of exit (H1a) and neglect (H1b), and lower levels of voice (H1c) and loyalty (H1d) when tested together with individual job insecurity dimensions. Thus, Hypothesis H1b was supported. As quantitative job insecurity climate was not predictive of exit and voice, and it predicted loyalty in the opposite direction, H1a, H1c, and H1d were not supported.Quantitative job insecurity climate was positively related to loyalty  and lower levels of loyalty . These results suggest that those who experienced a climate of losing valuable job features were more likely to leave their jobs and less likely to be committed to their organizations. Hypothesis 2 stated that qualitative job insecurity climate is related to higher levels of exit (H2a) and neglect (H2b), and lower levels of voice (H2c) and loyalty (H2d), when tested together with individual job insecurity dimensions. Therefore, Hypotheses H2a and H2d were supported. However, as qualitative job insecurity climate did not predict voice and neglect, H2b and H2c did not receive support.Qualitative job insecurity climate was related to higher levels of exit  and loyalty . These results indicate that job insecurity climate explained additional variance in employees’ exit and loyalty behaviors, beyond what was accounted for by individual job insecurity. Hypothesis 3 proposed that job insecurity climate explains additional variance in exit (H3a), voice (H3b), loyalty (H3c), and neglect (H3d) after considering the effects of individual job insecurity. Hence, the results provide support for H3a and H3c. However, the fact that the job insecurity climate dimensions did not account for significant increments in explained variance in voice and neglect means that we found no support for H3b and H3d.As shown in The current study investigated how quantitative and qualitative job insecurity climates were related to exit, voice, loyalty, and neglect behaviors among employees in Turkey, and whether such climate perceptions explained additional variance in these behaviors over individual job insecurity. Specifically, we hypothesized that quantitative job insecurity climate is related to higher levels of exit (H1a) and neglect (H1b) and lower levels of voice (H1c) and loyalty (H1d), and that qualitative job insecurity climate is related to higher levels of exit (H2a) and neglect (H2b), and lower levels of voice (H2c) and loyalty (H2d), when tested together with individual job insecurity dimensions . Our third hypothesis was related to the effect of job insecurity climate in explaining these behaviors beyond the effect of individual job insecurity, and stated that job insecurity climate explains additional variance in exit (H3a), voice (H3b), loyalty (H3c), and neglect (H3d) after considering the effects of individual job insecurity.Regarding the associations between quantitative job insecurity climate and EVLN (Hypothesis 1), our study revealed that quantitative job insecurity climate was associated with higher levels of loyalty and neglect; however, it did not significantly predict exit and voice after controlling for the effects of individual job insecurity and the critical control variables . These results support only H1b by suggesting that employees’ tendency to engage in negligent behaviors might be higher when they experience a collective concern about the continued existence of their jobs. On the other hand, the results did not support H1a, H1c, and H1d, as quantitative job insecurity climate was not predictive of exit and voice, and it predicted loyalty in the opposite direction .The supported positive association between job insecurity climate and neglect is in accordance with past findings indicating that threat of dismissal is associated with increased risk of long-term sick leave  and avoiIn addition, our results unexpectedly showed that the climate of quantitative job insecurity was associated with increased loyalty. Although this finding challenges the widely held belief that job insecurity and loyalty are negatively related ,3, it reOur results on the relationships between qualitative job insecurity climate and the EVLN behaviors (Hypothesis 2) revealed that, after controlling for the effects of individual job insecurity and the control variables , qualitative job insecurity climate was related to higher levels of exit and lower levels of loyalty, but did not predict voice and neglect. These findings support H2a and H2d by suggesting that employees who experience a climate of losing valuable job features are more likely to leave their jobs and less likely to be loyal to their organizations. These supported hypotheses agree with previous findings demonstrating individual qualitative job insecurity to be associated with higher turnover intention ,29 and lNotably, our results—showing a non-significant association between exit and the quantitative aspect of job insecurity climate, but a positive relationship with the qualitative aspect—are in line with Hellgren et al.’s  finding In our study, neither of the two job insecurity climate dimensions  significantly predicted voice. This result suggests that the psychological collective climate related to the future state of a job might not motivate employees to strive to change a problematic situation. Although this contradicts previous findings showing a significant relationship between individual job insecurity and voice ,31, it mIn terms of the additional variance explained by job insecurity climate in EVLN behaviors after controlling for the effect of individual job insecurity (Hypothesis 3), our study revealed that job insecurity climate explained additional variance in exit and loyalty, but not in voice and neglect, beyond what was accounted for by individual job insecurity (and the control variables education and sector), providing support for H3a and H3c. The additional explained variance in exit and loyalty of the job insecurity climate is in line with previous findings showing that the experience of job insecurity climate has been shown to predict employees’ health , job attDespite some unexpected results , the present study contributes to the literature in several ways. The first contribution of this study is that it responds to Shoss’  call forAs another contribution, the relationships investigated in this study were tested using data from a non-Western country, namely Turkey. Considering that research on job insecurity in relation to EVLN behaviors has typically been investigated using data collected from European countries ,22,23,31Taken together, our study extends the existing knowledge on the consequences of the job insecurity climate in organizational settings. Our findings should encourage future research to explore the dynamics of job insecurity climate perceptions.The results of our study point to some practical implications. Our results inform practitioners, managers, and policymakers that they need to be aware of the winds blowing in organizations while developing interventions to deal with a particular workplace stressor, namely job insecurity. It is crucial for practitioners to fully realize the form  and the nature  of job insecurity perceptions, perhaps by conducting needs assessments at regular intervals. The outcomes of job insecurity climate shown in this study  support the importance of allocating budgets for such preventive approaches in organizations. Moreover, beyond preventive practices, organizations should try to create a supportive climate for employees, and practitioners should develop adequate interventions in this regard. Such interventions may benefit from focusing on job insecurity as a collective stressor (job insecurity climate), which appears to have additional effects beyond those of individual job insecurity. To the best of our knowledge, no intervention has yet been developed for organizations in which a job insecurity climate prevails. Practitioners can work to determine whether various interventions designed for individual job insecurity perception also apply to job insecurity climate perceptions or to determine how such interventions can be modified to ameliorate the negative effects of an insecure climate.Some limitations of this study need to be mentioned. First, due to our data’s cross-sectional nature, it is not possible to make causal inferences about the associations between job insecurity and employee behavioral reactions, and thus our results should be interpreted with caution. Future research using longitudinal data may consolidate the statistical strength of the information provided by this study . Second,This study investigated how the psychological collective climate about losing a job and valuable job features  relate to employees’ exit, voice, loyalty, and neglect behaviors, and whether job insecurity climate perceptions explain additional variance in these behaviors over individual job insecurity. We found quantitative job insecurity climate to be associated with higher levels of neglect , and qualitative job insecurity climate to be related to higher levels of exit and lower levels of loyalty. These results, together with the finding that job insecurity climate explained additional variance in exit and loyalty beyond what was accounted for by individual job insecurity, demonstrate the importance of taking into account the psychological collective climate regarding job insecurity in explaining employee behaviors.


Usher syndrome type 2A (USH2A) is a genetic disorder characterized by retinal degeneration and hearing loss. To better understand the pathogenesis and progression of this syndrome, animal models such as USH2A knockout (USH2AKO) rabbits have been developed. In this study, we employed multimodal imaging techniques, including photoacoustic microscopy (PAM), optical coherence tomography (OCT), fundus autofluorescence (FAF), fluorescein angiography (FA), and indocyanine green angiography (ICGA) imaging to evaluate the retinal changes in the USH2AKO rabbit model. Twelve New Zealand White rabbits including USH2AKO and wild type (WT) were used for the experiments. Multimodal imaging was implemented at different time points over a period of 12 months to visualize the progression of retinal changes in USH2AKO rabbits. The results demonstrate that ellipsoid zone (EZ) disruption and degeneration, key features of Usher syndrome, began at the age of 4 months old and persisted up to 12 months. The EZ degeneration areas were clearly observed on the FAF and OCT images. The FAF images revealed retinal pigment epithelium (RPE) degeneration, confirming the presence of the disease phenotype in the USH2AKO rabbits. In addition, PAM images provided high-resolution and high image contrast of the optic nerve and the retinal microvasculature, including retinal vessels, choroidal vessels, and capillaries in three-dimensions. The quantification of EZ fluorescent intensity using FAF and EZ thickness using OCT provided comprehensive quantitative data on the progression of degenerative changes over time. This multimodal imaging approach allowed for a comprehensive and non-invasive assessment of retinal structure, microvasculature, and degenerative changes in the USH2AKO rabbit model. The combination of PAM, OCT, and fluorescent imaging facilitated longitudinal monitoring of disease progression and provided valuable insights into the pathophysiology of USH2A syndrome. These findings contribute to the understanding of USH2A syndrome and may have implications for the development of diagnostic and therapeutic strategies for affected individuals. The multimodal imaging techniques employed in this study offer a promising platform for preclinical evaluation of potential treatments and may pave the way for future clinical applications in patients with Usher syndrome. In RP, the photoreceptor cells deteriorate and lose function over time, resulting in night blindness and peripheral vision loss before progressing to permanent, late central vision loss. USH is the most prevalent condition of combined vision and hearing impairment, with an estimated over 400,000 people impacted globally2. USH accounts for approximately 50% of total hereditary deaf-blindness cases3. USH can be categorized into three subtypes , with each subtype decreasing in condition severity, starting with Type I. Each subtype varies in age of onset of SNHL and RP as well as in whether vestibular dysfunction is present. Type II (USH2) is the most common USH subtype, making up nearly half of all USH cases4. Individuals with USH2 experience moderate to severe SNHL at birth followed by RP onset in adolescence or early adulthood but maintain normal vestibular function. Currently, 16 USH causative genes have been identified and confirmed, with three genes, USH2A, GPR98 (USH2C), and DFNB31 (USH2D), corresponding to USH2. USH2A mutations are the most common, making up about 85% of all USH2 cases5. Although numerous clinical and non-clinical studies have investigated USH, there is currently no cure for the condition. In addition to early screening and diagnosis of USH, hearing aids and cochlear implants can help manage hearing loss. However, there are far less effective management resources for RP and no treatment options, leaving a significant need for research to identify therapies to address progressive vision loss and eventual blindness stemming from USH.Usher syndrome (USH) is an autosomal recessive genetic disorder characterized by sensorineural hearing loss (SNHL), type-dependent vestibular impairment, and retinitis pigmentosa (RP), an inherited degenerative eye disease that progressively causes severe vision loss10. Although mice share many anatomical similarities of the eye with humans, the mouse model has multiple limitations that hinder the translation of results to clinical applications and treatment development. One is that the average mouse eyeball is much smaller in size—nearly 8 times smaller in diameter—than the human eye12. However, the most prominent limitation is the significant phenotypic difference between human patients and current USH2A mutant mouse models13. This discrepancy is problematic, as the retinal degeneration phenotype is typically very weak or absent among USH2A knockout mouse models. Zebrafish models are promising in that they are being increasingly used for ophthalmic research. There are several USH2A mutant zebrafish models that exhibit early onset retinal degeneration as well as a recent model that displays slower, progressive retinal degeneration14. However, there are notable anatomical differences between zebrafish and human eyes, including variation in retinal structure and photoreceptor regeneration capacity that may pose problems in translational studies. Accordingly, the development of a larger mammalian model that more closely resembles the anatomy, physiology, and RP pathogenesis of the human eye may provide greater insight into USH2A mutation-induced RP and stimulate the advancement of clinical therapies and interventions.Animal models of USH2A have been previously developed in mice and zebrafish15. To better understand the microvasculature changes that occur over time, additional imaging modalities like photoacoustic microscopy (PAM) have recently been developed that may provide greater insight and characterization of the USH2AKO rabbit model. Photoacoustic microscopy (PAM) is a valuable imaging technique used in animal models for ocular research20. PAM relies on the photoacoustic effect, where tissue absorbs pulsed laser light and emits ultrasound waves as a result. These ultrasound waves are detected and converted into images, allowing researchers to visualize various structures within the eye. PAM offers high-resolution imaging capabilities, enabling the visualization of ocular structures such as retinal layers, blood vessels, and the choroid in great detail23. PAM is particularly useful in animal models of ocular diseases such as glaucoma, age-related macular degeneration (AMD), diabetic retinopathy, and retinal vascular disorders25. PAM can be used to track disease progression and assess the effectiveness of potential treatments. Another benefit of PAM is its ability to integrate with other optical systems to establish a multimodal imaging system. Song and Jao et al. have developed a PAM and OCT system to image the retina, melanin, and CNV in mice27. A limitation of this study lies in the fact that it can only visualize the structure of mouse eyes, which are considerably smaller than human eyes. The size of a mouse eyeball is roughly estimated to be eight times smaller than that of a human eye28. This constraint may hinder the translation of this system for clinical applications.There are very few studies investigating USH2A that involve a larger mammalian model. Recently, our group generated a successful USH2A knockout (USH2AKO) model in New Zealand White rabbits that shares resemblance with human USH2A progression and holds promise for future USH2A research40. We applied our technique to visualize different ocular pathologies including retinal vein occlusion (RVO)41, choroidal vascular occlusion42, choroidal neovascularization (CNV)46, retinal neovascularization (RNV)47, and corneal neovascularization48. The uniqueness of our system relies on the low excitation laser energy (~ 80 nJ), which is about half below the ANSI safety limit (~ 160 nJ at 578 nm)36. Our novel multimodal imaging system can provide a high aerial spatial resolution of 4.1 μm and 3.8 μm for PAM and OCT, respectively, as described previously36. The acquisition time is 65 s to achieve three-dimensional imaging with a scanning resolution of 256 × 256 pixels. By employing the advantages of the high-resolution multimodal PAM, OCT, and FA imaging system, this study aims to observe and track the progression of USH2A in the rabbit model. This imaging system can provide detailed information about the structure and function of the retina and other ocular tissues, allowing researchers to monitor changes or abnormalities associated with USH2A over time. The combination of PAM, OCT, and FM in this study offers complementary imaging modalities that can provide deeper insights into the disease progression and potentially contribute to the development of diagnostic and therapeutic approaches for USH2A and related disorders.To overcome such challenges, our group has developed a multimodal PAM, OCT, and fluorescence microscopy (FM) imaging system for application in the eyes of large animals like rabbitsen face 3D volumetric OCT images, where the entire optic nerve, RVs and EZ degeneration are clearly visualized with high resolution and contrast. Additionally, Fig. en face and volumetric OCT images provided detailed information about the retinal architecture, facilitating the identification and analysis of the EZ degeneration areas in comparison to surrounding tissues.High-resolution spectral domain optical coherence tomography (SD-OCT) imaging was employed to visualize the progression of RP USH2AKO rabbits. Figure Through segmentation of the images to extract the EZ degeneration area, the thickness of the damaged EZ region was measured using ImageJ software Fig. i. At 4 mColor fundus photography, fluorescein angiography (FA), indocyanine green angiography (ICGA), and fundus autofluorescence (FAF) imaging were performed at different time points  on both the USH2A knockout (USH2AKO) rabbit models and wildtype (WT) rabbits, which served as the control group. The color fundus photography images show normal retinal microvasculature (RVs), choroidal vessels (CVs), and the optic nerve in both the USH2AKO and WT groups  from the segmented image Fig. c using ITo enhance the visualization of the retinal degeneration, photoacoustic microscopy (PAM) imaging was performed on rabbits at 4 and 12 months  rabbit model has been utilized as an important tool for studying the pathogenesis and progression of USH2A syndrome, a genetic disorder associated with moderate to severe SNHL and RP. This animal model provides valuable insights into the understanding of the disease and the evaluation of potential therapeutic interventions. The characterization of the USH2AKO rabbit model involved a comprehensive evaluation using various imaging modalities. Color fundus photography, fluorescein angiography (FA), fundus autofluorescence (FAF) imaging, indocyanine green angiography (ICGA), spectral domain optical coherence tomography (SD-OCT), and photoacoustic microscopy (PAM) were employed to assess the retinal structure, microvasculature, and RPE and EZ degeneration.Color fundus photography and FA revealed no significant abnormalities in the retinal microvasculature or structure in the USH2AKO rabbits compared to the wildtype control group. However, FAF imaging identified RPE degeneration in the USH2AKO rabbits, which persisted throughout the observation period of up to 12 months. SD-OCT imaging demonstrated the presence of EZ degeneration, demonstrating a significant reduction in the thickness of the EZ layer in the USH2AKO rabbits compared to the wildtype controls. The EZ degeneration was found to progress over time. Additionally, photoacoustic microscopy (PAM) imaging was used to visualize the retinal structure, including the RVs, CVs, optic nerve, and capillaries. PAM imaging did not provide specific markers or evidence of USH2A syndrome in the USH2AKO rabbits likely due to its imaging of hemoglobin in vasculature, which does not change until very late in the disease course. The USH2AKO rabbit model has proven to be valuable for longitudinal studies and monitoring the progression of USH2A syndrome. The longitudinal RPE and EZ degeneration over time and the presence of retinal atrophy observed in this model closely resemble the pathological characteristics seen in human patients with USH2A syndrome.The RPE is a critical component of the retinal structure, playing a pivotal role in maintaining the health and function of photoreceptor cells. The identified RPE degeneration in our USH2AKO rabbit model may encompass various changes, including disruptions in RPE cell morphology, alterations in RPE cell function, and potential structural anomalies within the RPE layer itself. Drawing parallels between our findings in the USH2AKO rabbit model and human pathological conditions is an essential avenue of discussion. RPE degeneration is a common feature in several retinal diseases, including age-related macular degeneration (AMD), retinitis pigmentosa (RP), and various forms of Usher syndrome. We hypothesize that the RPE changes observed in our model may reflect a process akin to what occurs in some human retinal diseases. Given that USH2A mutations are linked to Usher syndrome, a genetic disorder characterized by retinal degeneration, it is plausible that our findings hold relevance to understanding the pathophysiological mechanisms at play in the human context. The connection between the RPE degeneration observed in our USH2AKO rabbit model and human pathological conditions can shed light on potential clinical implications. It may open avenues for further research into therapeutic interventions and diagnostic markers that target RPE-related degenerative processes, benefiting patients with Usher syndrome and related retinal degenerative disorders.Another advantange of the multimodal imaging system is that 3D PAM provided high-resolution images with enhanced contrast, allowing for visualization of retinal vessels, choroidal vessels, and capillaries. OCT imaging played a crucial role in visualizing the progression of ellipsoid zone (EZ) degeneration, a hallmark feature of USH2A syndrome. The measurements of EZ thickness over time using OCT provided quantitative data on the progression of degeneration and the degenerative changes observed in the USH2AKO model over the long term. This study highlights the potential of using multimodal imaging for non-invasive and longitudinal monitoring of USH2A syndrome in an animal model. The use of these techniques allows for the assessment of disease progression, evaluation of potential therapeutic interventions, and may aid in the development of novel diagnostic and treatment strategies for USH2A syndrome.. conducted a groundbreaking study that utilized optical coherence tomography (OCT) and autofluorescence for imaging USH2A-associated retinal changes49. They reported significant reduction in retinal thickness and extensive disruption in the ellipsoid zone as biomarkers of disease progression. This study collectively underscores the importance of multimodal imaging in elucidating the biology of USH2A-related retinal degeneration. The combined imaging approaches have allowed for the identification of specific markers associated with USH2A, aiding in the early diagnosis and monitoring of the syndrome. In addition, the integration of PAM with other techniques can provide insights into the mechanisms underlying USH2A progression. This study has demonstrated how multimodal PAM can reveal microvascular alterations associated with USH2A, contributing to a comprehensive understanding of the disease.Tsz Kin Ng et alFurthermore, these multimodal imaging studies have far-reaching clinical implications, such as the potential to improve early diagnosis. By identifying disease-specific markers, clinicians may be able to diagnose USH2A-related retinal conditions at an earlier stage. Understanding the biological mechanism and disease progression can inform the development of targeted therapies for USH2A patients and serve as novel biomarkers and endpoints for the development of new therapeutics. Lastly, multimodal imaging provides a valuable tool for monitoring disease progression over time, allowing for timely interventions and personalized treatment plans. These studies not only enhance our understanding of the biology of USH2A but also hold promise for improved diagnosis and treatment strategies for affected individuals. Our research builds upon these foundational studies to further advance our knowledge of USH2A-associated retinal degeneration.While our PAM imaging system delivers comprehensive anatomical information on the USH2AKO model, offering high-resolution visualization of retinal vessels, choroidal vessels, and capillaries in both 2D and 3D, it falls short in providing significant insights into the progression of Usher syndrome. This limitation can be attributed to the absence of melanin in the White New Zealand rabbits employed in this study. The absence of melanin in the RPE cells serves as a crucial diagnostic hallmark for RPE degeneration, resulting in a reduction in intrinsic photoacoustic signals. To gain a more profound understanding of the progression of USH2A syndrome, one can consider the use of pigmented rabbits.In conclusion, this study demonstrates the utility of multimodal photoacoustic microscopy (PAM), optical coherence tomography (OCT), and fluorescent imaging as promising techniques for in vivo monitoring of a reproducible USH2A knockout (USH2AKO) model. The multimodal imaging modalities allowed for the evaluation of the retinal structure, microvasculature, and degenerative changes associated with USH2A syndrome. Thus, this study provides valuable tools for the characterization and monitoring of USH2A syndrome, contributing to a better understanding of the disease and potentially leading to improved management of affected individuals in the future.All animal studies conducted in this research adhered to the ARRIVE  guidelines and followed the ARVO  Statement for the Use of Laboratory Animals in Ophthalmic and Vision Research. The experimental procedures were performed in compliance with the applicable guidelines, regulations, and ethical considerations. Prior to the initiation of the study, the research protocol (Protocol PRO00010388) was reviewed and approved by the Institutional Animal Care & Use Committee (IACUC) of the University of Michigan.15. Rabbits were selected in this study due to their similarities to human eyes and suitability for ophthalmic research. Rabbits were housed in a controlled environment with an air-conditioned room. The lighting conditions followed a 12-h light–dark cycle, providing a standard diurnal rhythm. They were given unrestricted access to water and provided with standard laboratory food to meet their nutritional requirements.In this study, six New Zealand white rabbits, including both males and females, aged between 4 and 12 months, and weighing 2.5–3.4 kg, were bred at the Center for Advanced Models and Translational Sciences and Therapeutics (CAMTraST) at the University of Michigan Medical School. The generation and characterization of the USH2AKO rabbits was reported previouslyPrior to any procedures or imaging, all rabbits were anesthetized using intramuscular injections. Ketamine  obtained from JHP Pharmaceuticals  and xylazine  from Anased®  were used for anesthesia induction. To facilitate ophthalmic examinations, pupillary dilation was achieved by administering tropicamide 1% ophthalmic and phenylephrine hydrochloride 2.5% ophthalmic solutions. Topical anesthesia was also applied using 0.5% tetracaine or proparacaine to ensure the comfort of the rabbits during the procedures. To maintain corneal hydration throughout the experiments, phosphate-buffered saline  was administered regularly, typically every minute. The vital signs of the rabbits, including mucous membrane color, temperature, heart rate, respiratory rate, and oxygen saturation, were continuously monitored before, during, and after anesthesia. A pulse oximeter  was employed to assess oxygen saturation and monitor the respiratory status of the rabbits.50. The system, as described by Tian et al., integrates the capabilities of both modalities to provide complementary imaging information36. The SD-OCT component of the system was modified from a commercially available system . The system included an additional ocular lens following a scan lens, and a dispersion compensation glass (DCG) was added to the OCT reference arm. For the PAM component, an optical parametric oscillator (OPO)  served as the illumination source. The OPO offered tunability in wavelength (405–2600 nm) and had a pulse duration of 3–6 ns. The PA signal, generated by endogenous chromophores such as hemoglobin and melanin, was detected by a custom-built needle-shaped ultrasonic transducer  with a center frequency of 27.0 MHz. The transducer was in direct contact with phosphate-buffered saline (PBS) on the rabbit conjunctiva. The PA signal was digitized using a high-speed digitizer  with a sampling rate of 200 MS/s and amplified using a low-noise amplifier  with a gain of 57 dB.Multimodality imaging combining SD-OCT and PAM allowed for simultaneous or sequential acquisition of structural and functional information with high resolution and depth of penetration. Figure 51. The PAM and OCT systems have quantified axial resolutions of 37.0 μm and 4.0 μm, respectively. The laser energy was maintained at approximately half of the ANSI safety limit, measuring around 80 nJ.The center laser wavelength for the OCT system was 905 nm, using two superluminescent light-emitting diodes with center wavelengths of 845 nm and 932 nm. The approximate bandwidth estimation is around 220 nm. The imaging depth of the OCT system was 1.9 mm. The aerial lateral resolution of the PAM system was demonstrated to be 4.1 μm, while the SD-OCT system achieved an aerial lateral resolution of 3.8 μmAll rabbits, including both wild-type (WT) and USH2AKO rabbits, underwent imaging using various techniques, including fundus photography, fundus autofluorescence (FAF), fluorescein angiography (FA), indocyanine green angiography (ICGA), photoacoustic microscopy (PAM), and optical coherence tomography (OCT). The experiments were first implemented with color fundus imaging, followed by sequential imaging using fundus autofluorescence (FAF), fluorescent angiography (FA), and indocyanine green (ICGA). Subsequently, following ICGA imaging, the animal was transitioned to the multimodal PAM and OCT imaging system. The imaging was performed longitudinally to assess progressive retinal structure, vascular perfusion, neovascularization, and the presence of RPE and EZ degeneration lesions. To facilitate the imaging procedures, a pediatric Barraquer wire speculum was placed to keep the eyelids open. Fundus photography was acquired using the Topcon 50 EX system , allowing for visualization of the posterior segment of the eye.For FA imaging, a 0.2 mL injection of fluorescein sodium solution  was administered intravenously through the marginal ear vein of the anesthetized rabbit. The FA images were acquired immediately after the injection of fluorescein sodium solution followed by every minute, for a duration of 5 to 15 min post-injection. This imaging protocol enabled the monitoring of fluorescence intensity and the detection of vascular changes.Similarly, ICGA imaging was performed by injecting 0.2 mL of indocyanine green (ICG) dye . The ICGA contrast was imaged at the same intervals as the FA imaging to assess vascular perfusion and potential abnormalities.To analyze the fluorescence intensity and surface area changes over time, the acquired images were processed using ImageJ software. Image segmentations were implemented to isolate the location of EZ degeneration . Regions of interest (ROI) were selected at the location of the segmented EZ areas, ensuring the entire margin of the EZ degeneration lesion was included. The average fluorescence intensity and surface area of these regions were determined by ImageJ. To account for background intensity noise, the average of several images from different time points was calculated and subtracted from the values obtained from the ROI. Hyperfluorescent EZ degeneration lesions were identified using the free-hand application in ImageJ, allowing for the delineation of the affected areas. For WT group, a dozen areas were selected at different location on the FAF images  which is matched with the USH2AKO group. Average signals were determined using ImageJ. By employing these imaging and analysis techniques, the study aimed to evaluate and quantify the characteristics and changes in RPE and EZ degeneration in the USH2AKO rabbits compared to WT rabbits.To minimize motion artifacts during imaging, an anesthetized rabbit's head and body were positioned on two stabilization platforms. This setup ensured stability and reduced movement during the imaging process. Additionally, a water-circulation blanket  was used to maintain an appropriate body temperature. An integrated charge-coupled device (CCD) camera provided real-time visualization, allowing researchers to precisely target the region of interest (ROI) during in vivo experiments. This visualization assisted in positioning the imaging probes accurately. The reference arm of the OCT system was calibrated to enhance image quality, ensuring accurate depth information during image acquisition. For the OCT imaging, B-scan images were acquired with a resolution of 512 × 1024 A-lines and an acquisition rate of 36 kHz. The OCT system enabled cross-sectional imaging of the retina, providing high-resolution structural information. To perform multimodality imaging, the PAM system was integrated with the OCT system. The PAM system was controlled using Matlab2019b software . PAM utilized an ultrasonic transducer in combination with optical scanning galvanometers to obtain three-dimensional volumetric PAM images. These images were acquired through raster scanning, capturing the photoacoustic signals from the tissue. The acquired PAM images were further processed and rendered using Amira software. The integration of PAM and OCT into a single system allowed for complementary information from the imaging modalities.The experimental conditions were repeated a minimum of three times, with measurements of fluorescent intensity and OCT signals taken for each condition. An ANOVA analysis was conducted to assess any notable distinctions among the treatment groups. The resulting data was represented as the mean ± standard deviation (SD), with p-values less than 0.05 indicating statistical significance.Supplementary Figures.


Ischaemic heart disease results from insufficient coronary blood flow. Direct measurement of absolute flow (mL/min) is feasible, but has not entered routine clinical practice in most catheterization laboratories. Interventional cardiologists, therefore, rely on surrogate markers of flow. Recently, we described a computational fluid dynamics (CFD) method for predicting flow that differentiates inlet, side branch, and outlet flows during angiography. In the current study, we evaluate a new method that regionalizes flow along the length of the artery.r 0.47, P 0.006; zero bias; 95% CI −168 to +168 mL/min); regionalized method .Three-dimensional coronary anatomy was reconstructed from angiograms from 20 patients with chronic coronary syndrome. All flows were computed using CFD by applying the pressure gradient to the reconstructed geometry. Side branch flow was modelled as a porous wall boundary. Side branch flow magnitude was based on morphometric scaling laws with two models: a homogeneous model with flow loss along the entire arterial length; and a regionalized model with flow proportional to local taper. Flow results were validated against invasive measurements of flow by continuous infusion thermodilution . Both methods quantified flow relative to the invasive measures: homogeneous (During angiography and pressure wire assessment, coronary flow can now be regionalized and differentiated at the inlet, outlet, and side branches. The effect of epicardial disease on agreement suggests the model may be best targeted at cases with a stenosis close to side branches.  FFR is the ratio of distal to proximal translesional pressure measurements, but it only expresses fractional and not absolute reductions in Q and it cannot diagnose coronary microvascular disease (MVD); a common but often overlooked cause of IHD.5–10Ischaemic heart disease (IHD) is the leading cause of death worldwide. IHD results from an insufficiency of coronary blood flow (Q), commonly caused by occlusive coronary arterial disease and encompasses a wide variety of clinical syndromes including symptomatic ischaemia (angina), myocardial infarction, and heart failure. Interventional treatments are effective in restoring Q, but should only be targeted at lesions that result in ischaemia. There is, however, no technique available for routine clinical use in the cardiac catheterization laboratory which directly measures Q. Over several decades, cardiologists have relied upon indirect surrogate markers, such as thermodilution-derived mean transit time, Doppler-derived flow velocity, and pressure-derived fractional flow reserve (FFR) and related indices12 The second, virtuQ™, derives Q from a computational fluid dynamics (CFD) simulation based upon the 3D angiographic anatomy and pressure wire measurements, and is the subject of this study.13 Both methods also quantify absolute microvascular resistance (Rmicro) and so provide a comprehensive evaluation of the entire coronary circulation. A limitation of the CFD method was that it only considered the main vessel and was agnostic to side branch flow.13 While this may be acceptable for computing vFFR, which simply interrogates the effect of a localized lesion, it is less so for computing Q, which is dependent upon the lesion, side branches, and microvasculature.14 In a recent study, we described a porous wall simulation method that used established morphometric scaling laws and case-specific knowledge of arterial taper to allow blood to virtually ‘leak’ out of the main vessel proportional to side branch flow.14 A limitation of this approach was that side branch flow was distributed diffusely across the entire length of the main vessel. This approach, therefore, failed to capture the haemodynamic effects of large volumes of regionalized flow loss to major arterial side branches.Two methods have been developed to quantify Q. The first, continuous infusion thermodilution (CIT), is the most established and validated method and uses the Rayflow™ infusion catheter .In this study, we describe a novel method that attempts to correlate side branch flow ‘leak’ to areas of significant bifurcation and therefore represents the pattern of flow commonly encountered in real coronary arteries. We aimed to validate this new method against CIT measurements and compare results with those from the homogeneous CFD method.supplementary material. A derivation analysis of Murray’s exponent, based upon the same raw clinical dataset as this study, has already been published.15CFD analyses were performed at the University of Sheffield, UK on retrospective clinical data collected from adult patients (≥18 years old) with suspected myocardial ischaemia with non-obstructive coronary arteries (INOCA) undergoing coronary angiography and Rayflow catheterization at the Catharina Hospital, Eindhoven, NL. Patients provided informed consent and the study was approved by the research ethics board [Medical research Ethics Committees United (MEC-U)]. All supporting data are provided in this manuscript and in the 16 and the Rayflow™ infusion catheter  and the Coroventis™  system,12 were used to quantify Q (QCIT) and Rmicro (RmicroCIT). Thermodilution measurements were taken in the proximal coronary artery, with pressure readings under saline-induced hyperaemia taken at the location of 6 cm distal to the tip of the infusion catheter.12 Pseudonymized angiography (DICOM) and physiological (pressure and flow) data were exported to the University of Sheffield for computational processing in February 2021. Full exclusion criteria have previously been published.15 In brief, cases were excluded if transferred clinical data were unsuitable for computational processing or major arterial side branches were present within 3.0 cm of the Rayflow™ infusion port. The latter criterion was used because side branches close to the infusion port can draw a significant portion of saline infusate and therefore affect QCIT and RmicroCIT results.11 A recommended correction for the partial luminal occlusion created by the infusion catheter was applied . Visual percentage stenosis was evaluated by two interventional cardiologists and quantitative coronary angiography (QCA) was used to grade 2D- and 3D-percentage stenosis.FFR was assessed in arteries of interest using standard technique , with a maximum tolerated signal drift of 2 mmHg,17 Two angiographic projections of the vessel of interest, acquired ≥30° apart and during end-diastole, were used to reconstruct coronary anatomy. Image selection and a correction for table movement between angiographic runs was manually performed, while centreline tracing and vessel border detection was performed semi-automatically from contrast gradient for both images with manual correction if required. Finally, a rigid, 3D, axisymmetric geometry representative of patient anatomy was automatically created. The inlet of reconstructed arteries corresponded to the location of invasive QCIT and aP measurement, while the outlet corresponded to the location of dP. Invasive pressure readings were used to define inlet and outlet boundary conditions. The CFD simulation was executed using standard blood parameters , modelling steady, laminar flow of a Newtonian fluid. The suitability of these assumptions has been demonstrated previously.18–20A complete description of the virtuQ method and arterial reconstruction process has been published previously.21 which relates the diameters (D) of parent vessel (PV) and daughter branches (DB) around a bifurcation:In this study, side branch flow was simulated by modelling arterial reconstructions with a porous wall boundary. This allowed flow loss from the main vessel lumen. For all circumstances, inlet flow = outlet flow + side branch flow. Side branch magnitude was inferred from taper of the main vessel using Murray’s law,x’ represents a constant whose value varies between 2.0 and 3.0.22–24 The magnitude of side branch flow  was dependent upon the value used for this proportionality constant (which was optimized in a previous study15) and the method used to distribute side branch losses . The aim of this study was to compare accuracy of the homogeneous and regional techniques against the invasively derived measures. The homogeneous porous wall method distributed side branch losses consistently along the entire length of the reconstructed vessel and was unaffected by local taper. The regional porous wall method distributed side branch flow loss proportionally to the local healthy vessel taper. This was done to better regionalize Q. In healthy vessels, regional taper occurs at branch points. Thus, this method should more accurately regionalize blood flow in the in-vivo artery. To distinguish healthy vessel taper from diameter reduction caused by atherosclerotic plaque, a stenosis detection filter was used which excluded porous wall leak in sections with downstream recovery of vessel diameter . It was therefore imperative that the inlet and outlet of reconstructed vessels were selected in healthy sections of vessel. All reconstructions were processed blind to the intra-coronary pressure and CIT results.In which ‘To assess variability in physiological results that may be introduced by subtle differences in reconstructed coronary anatomy during the manual phases of artery reconstruction, intra-operator variability was assessed for CFD-derived Q (QCFD) and Rmicro (RmicroCFD) across three repeated reconstructions of the five most and least stenosed cases.t-test, while a Mann–Whitney U test was used for non-parametric data. Correlation was quantified using Pearson’s correlation coefficient (r) and agreement was assessed with both Passing and Bablok regression and Bland Altman plots. Log-transformation was used for skewed datasets. Analyses were performed using MATLAB R2021a .Categorical variables are presented as frequency (percentage). Normally distributed continuous variables are presented as mean (standard deviation), while those with a non-normal distribution are presented as median [interquartile range]. Normality of data distribution was assessed using the Shapiro–Wilk test. For parametric data, mean values of haemodynamic parameters were compared using an unpaired n = 2) and pressure (n = 1) data, insufficient angiographic projections (n = 11), unsuitable patient anatomy (n = 3), coronary intervention performed prior to QCIT measurement (n = 2), and major side branches present ≤3.0 cm of the Rayflow catheter (n = 2). Seven patients (35%) were male, the mean age was 62 (±10) years, and the mean body mass index (BMI) was 25.2 (±3.6) kg/m2. The 27 cases included left anterior descending (LAD) arteries (n = 18), left circumflex (Cx) arteries (n = 7), and right coronary arteries (RCA) (n = 2). The mean QCIT was 219 (±61) mL/min and the median RmicroCIT was 360 [290–450] mmHg min/L. The majority of included cases contained minimal epicardial disease, with a mean FFR of 0.87 (±0.08) and only three cases meeting a clinical threshold of FFR significance (≤0.80). Median percentage stenosis assessed by operator, 2D QCA and 3D QCA was 10% [0–25%], 16% [0–31%], and 15% [0–33%], respectively (full details of cases shown in Table 1). No included cases contained diffuse epicardial disease. Using a threshold of 460 mmHg min/L,25–27 five (25%) patients had clinically significant MVD as assessed by RmicroCIT.From the original 48 cases, 27 cases from 20 patients yielded full physiological datasets. Cases were excluded due to insufficient angiographic quality (CFD of 219 (±86) mL/min. There was a statistically significant correlation between QCFD and QCIT , Passing and Bablok regression identified constant and proportional differences between techniques , the mean delta between techniques was zero and the 95% Bland Altman limits of agreement were −168 to +168 mL/min . The proportional differences between QCFD and QCIT was characterized by an increase in bias for higher flow rates, which is visually displayed by Bland Altman analysis. A significant correlation between RmicroCFD and RmicroCIT was also observed , constant and proportional differences were present , the mean delta between techniques was +30 mmHg min/L and the 95% Bland Altman limits of agreement were −210 to +480 mmHg min/L .The homogeneous porous wall boundary method disclosed a mean QCFD of 219 (±96) mL/min. The correlation between QCFD and QCIT was significant , Passing and Bablok regression identified constant and proportional differences between techniques , the mean delta between techniques was zero and the 95% Bland Altman limits of agreement were −175 to +175 mL/min . Agreement between QCFD and QCIT was not superior for either the homogeneous or regional techniques . A significant correlation between RmicroCFD and RmicroCIT was also observed , constant and proportional differences were present , the mean delta between techniques was +37 mmHg min/L and the 95% Bland Altman limits of agreement were −220 to +540 mmHg min/L . Agreement between RmicroCFD and RmicroCIT was not superior for either the homogeneous or regional techniques  (full CFD results shown in Table 1). Using a threshold of 460 mmHg min/L, seven (35%) patients had clinically significant MVD as assessed by RmicroCFD. Using RmicroCIT as gold standard measurements, the sensitivity, specificity, positive predictive value, and negative predictive value of the CFD technique was 80%, 80%, 57%, and 92%, respectively.The regional porous wall boundary method disclosed a mean QaP − dP) , FFR  and percentage stenosis assessed by 2D  and 3D QCA . This meant, for both homogeneous and regional techniques, that agreement between QCFD and QCIT improved in cases with greater disease burden assessed by pressure wire studies, 2D QCA and 3D QCA. For the regional method, Q agreement also correlated with visually assessed stenosis .Certain patient and vessel characteristics did appear to influence agreement between the CFD and invasive measurements. For homogeneous and regional porous wall boundary methods, respectively, Q agreement was significantly correlated with translesional pressure drop  and FFR , but no effect was seen for any assessment of stenosis (full results are shown in Table 2). This meant, for both homogeneous and regional techniques, that agreement between RmicroCFD and RmicroCIT improved in cases with greater disease burden assessed by pressure wire studies, but disease burden assessed by percentage stenosis was not associated with agreement.For Rmicro, both homogeneous and regional techniques correlated with translesional pressure drop (CFD variability was 7.8% (±4.2%) and 5.6% (±2.7%), respectively. For RmicroCFD, this was 2.6% [4.6–6.1%] and 3.2% [1.9–10.2%], respectively. Intra-observer variability did not differ between homogeneous and regional techniques for either QCFD  or RmicroCFD .For the homogeneous and regional porous wall boundary methods, Q15 The main aim of this work was to regionalize the side branch and main branch flow. This was achieved with no major difference in overall side branch flow compared with the homogeneous method. The new regionalized method correlated with CIT measurements, with zero bias and 95% limits of agreement of ±175 mL/min. Agreement with invasive clinical measures was suboptimal, which we believe resulted from inclusion of several INOCA cases which have minimal stenosis and pressure gradient.In this retrospective study, we have validated a regionalized porous wall boundary method for simulating side branch Q and compared the results with those from the original homogeneous method.10 and has led to the emergence of FFR as the current ‘gold standard’ evaluation of an epicardial lesion’s haemodynamic significance. However, this technique expresses percentage reduction in flow and does not quantify MVD. Combined use of FFR with other indices of coronary flow, such as coronary flow reserve (CFR) and index of microvascular resistance (IMR), allows for quantification of disease in both epicardial and microvascular compartments, but this requires additional time and hardware and is currently reserved for a relatively small number of patients. Additionally, the binary nature of treatment thresholds means different indices may conflict and all fail to quantify a direct determinant of myocardial ischaemia – absolute Q. The CIT technique addresses these issues but requires a dedicated infusion catheter and accuracy may be decreased in arteries with multiple, large bifurcations. In contrast, virtuQ may quantify Q at the inlet, outlet, and side branches of reconstructed arteries, along with all of the above-mentioned indices of flow from standard angiography and pressure wire assessment.The superiority of translesional pressure indices over standard angiography alone is well documentedin-vitro validation of virtuQ outlet Q results has been performed, reporting a bias of +2.08 ± 3.45 mL/min13 and our previous validation of inlet QCFD using the homogeneous porous wall boundary method reported zero bias between CIT results with 95% limits of agreement of ±168 mL/min.15 More data are available for the latest, monorail Rayflow catheter design. An in-vitro trial of QCIT reported a bias of −6.5 ± 15.5 mL/min12 and one animal study reported a bias of +5 ± 8 mL/min from Q measured in 12 pigs (mean Q 37 mL/min).28 A direct validation study of the CIT method with [15O]H2O positron emission tomography (PET) in 25 patients referred for coronary angiography showed a bias between techniques of −0.9 ± 35 mL/min .29The Rayflow CIT and virtuQ CFD methods quantify Q within the cardiac catheterization laboratory. Previously, one direct 29 even when accounting for the larger mean Q of patients included in the current study. Several factors may have negatively influenced agreement, the most important of which is likely to be characteristics of included arteries. QCFD accuracy is critically dependent upon agreement between simulated flow patterns and those occurring in-vivo. In healthy arteries, coronary flow is governed predominantly by Poiseuille (viscous) effects and as such, results are extremely sensitive to errors in reconstruction diameter. To put this into context, in a theoretical case taking the average outlet diameter and QCFD of cases included in this study, an error in outlet reconstruction diameter of ± one single pixel would vary outlet QCFD from −42 to +62 mL/min (assuming Poiseuille’s law with a constant pressure gradient across the artery). This error will be further magnified for inlet QCFD, when side branch flow is accounted for. Conversely, the complex flow patterns observed in diseased arteries reduce the dominance of Poiseuille effects on flow local to the disease, thereby reducing the sensitivity of QCFD to small errors in reconstruction diameter. This phenomenon is evidenced by both the correlations observed between QCFD and QCIT agreement with percentage luminal stenosis and translesional pressure gradients; both of which are markers of disease severity, and the clustering of cases without any appreciable epicardial disease at the higher error ranges. This means agreement between the two methods would likely have improved through inclusion of cases with moderate to severe epicardial disease.In the present study, we reported moderate correlation between CIT and CFD Q for the regional porous wall boundary method. The 95% limits of agreement were ±175 mL/min. These limits of agreement are larger than reported in previous studies of different datasets,29 It is therefore important to consider the limits of agreement reported in this study as an amalgamation of the errors of both CFD and CIT methods and not solely attribute them to either technique.Furthermore, the CIT method is dependent upon assumptions such as the complete mixing between saline infusate and blood prior to side branches and negligible heat loss to the vessel wall. These conditions may not be observed in patients, therefore resulting in inaccuracies of the CIT technique.total side branch flow between the homogeneous and regional porous wall boundary method was reassuring. The main advantage of the regionalized technique is that it seeks to concentrate side branch flow to the location of side branches. This may have advantages for more precise flow predictions when planning intervention just proximal or distal to significant bifurcation points. This, however, is more challenging to validate given that there are no methods that accurately predict coronary blood flow with this level of localization.The absence of any significant difference in CFD and RmicroCFD. For both porous wall boundary methods, intra-observer variability was less than 5% for outlet QCFD and 4% for RmicroCFD, which is considerably lower than previously reported results, of 10% and 11% for the same parameters, respectively.13Intra-operator variability was excellent for both QCFD and provides the first clinical, supporting evidence of the previously reported requirement for a translesional pressure drop.13 Because this was a retrospective analysis of angiograms captured at another centre, the protocols were not optimized for virtuQ processing4 which may have affected reconstruction accuracy of included cases. Also, the correction calculation to accommodate for the partial coronary arterial lumen occlusion introduced by the in-situ Rayflow catheter is currently unvalidated. Clinically, the patient cohort for which absolute Q assessment will be of value and thresholds for intervention are currently unknown and subject to ongoing research.30 Fournier et al. have previously reported a difference in hyperaemic LAD flow between healthy and mildly disease arteries of 65 mL/min.27 Given the reported error in the current study, at the current stage of development, virtuQ assessment of absolute Q in minimally diseased arteries may be of limited clinical utility. Furthermore, the porous wall boundary method assumes the inlet and outlet of reconstructed arteries are healthy, so the tool may not be applicable for patients with diffuse epicardial disease. Finally, while this CFD technique quantifies epicardial arterial Q, it cannot assess myocardial flow and may therefore be misleading in the presence of a major collateral blood supply.The number of included patients was modest and case exclusion rate was high, but this in keeping with similar retrospective computational studies and no exclusion criteria were applied on successfully modelled cases to improve accuracy. A disproportionate number of LAD arteries were included in this study. Moreover, our model of side branch flow relies on vessel taper, and the proximal RCA has less taper due to a relative lack of major side branches. For both these reasons, the model may lack generalisability for RCA modelling. Future studies will therefore aim to include more RCA’s and include posterior descending and posterior left ventricular branches. Of the included arterial cases, only two (7%) met the criteria for moderate stenosis (>40%) as graded by either 2D or 3D QCA and three (11%) met the FFR threshold of haemodynamic significance (≤0.80). As previously described, this has negative implications for the accuracy of QUsing a CFD model of coronary arterial blood flow, Q can be quantified and regionalized within the epicardial coronary arteries with flow loss from the main lumen concentrated at the location of side branches. This retrospective study showed no overall difference in performance between the regional and homogeneous techniques. Although the cases met the basic criteria for modelling, the lack of stenosis and relatively small pressure gradient negatively influenced results compared with the invasive technique. Further validation work is required, but this approach may be useful in predicting local blood flow dynamics, particularly in cases with a stenosis close to significant side branches.ztac077_Supplementary_DataClick here for additional data file.