Knowledge on healthcare utilization after mass trauma is needed to strengthen the public health preparedness to such incidents. Using register-based data, this study had a unique opportunity to investigate how young survivors’ use of primary care physicians (PCP) and mental health services (MHS) changed after a terrorist attack.We examined register-based data on PCP and MHS consultations among 255 survivors  of the 2011 Utøya youth camp attack in Norway 3 years before and after the attack, and their reason for encounter with the PCP according to the International Classification for Primary Care (ICPC− 2).The PCP and MHS consultation rates (CR) were higher in female than male survivors both acutely and at long-term. The mean yearly CRs increased from 2.25 to 4.41 for PCP and 1.77 to 13.59 for MHS the year before and after the attack in female survivors, and from 1.45 to 3.65 for PCP and 1.02 to 11.77 for MHS in male survivors. The third year post-attack CRs for PCP were 3.55 and 2.00; and CRs for MHS were 5.24 and 2.30 in female and male survivors, respectively. Among female survivors, 76% consulted PCP and 12% MHS the year preceding the attack; post-attack 93% consulted PCP and 73% MHS the first year; decreasing to 87 and 40% the third year. Among male survivors, 61% consulted PCP and 7% MHS the year preceding the attack; post-attack 86% consulted PCP and 61% MHS the first year, and 67 and 31% the third year. As for PCP consultations, there was a particular increase in psychological reasons for encounter following the attack.This study indicates that it is important to anticipate an increased healthcare utilization several years following mass trauma, particularly of MHS. Both PCP and MHS practitioners played important roles in providing healthcare for psychological problems in young survivors of terrorism in a country with universal and largely publicly financed healthcare and a gatekeeping system. The healthcare utilization could be different in countries with other health systems or psychosocial care responses to mass trauma.The online version contains supplementary material available at 10.1186/s12888-022-04358-4. Following traumatic experiences, increased physiological reactivity and persisting stress may contribute to a range of long-term mental and physical health problems, such as posttraumatic stress disorder (PTSD), depression, anxiety disorders, chronic pain and aggravation of pre-existing illness –9. YoungOn 22 July 2011, a solitary right-wing terrorist committed two terrorist acts in Norway. First, he detonated a car bomb in the Oslo Government Quarter, killing eight individuals. Two hours later, he perpetrated a shooting spree at the summer camp of a political youth organization on the Utøya island. During the nearly 1.5 hour-long shooting, 69 individuals died. Most of them were teenagers. The Utøya attack is viewed as a severe trauma due to the scope of injuries and fatalities, its long duration, the young age of those concerned, and the fact that they were designated targets. Prior findings have revealed that the survivors were highly exposed to danger during the attack, and that nearly three of four of them experienced loss of close ones in the attack . Hence, n = 490) were invited to participate [Altogether, 495 survivors who had been on the Utøya island during the shooting were identified through police records. Study invitations were sent by postal mail to 490 survivors; whereas four survivors aged < 13 years and one living abroad were excluded. Semi-structured face-to-face interviews were performed by trained health personnel at three waves, 4–5 months, 14–15 months and 31–32 months after the attack. We asked for consent to linkage to register-based data at wave 3. The study had an open cohort design at waves 1 and 2 where all the eligible survivors . Every resident in Norway has a personal identification number (PIN) which makes it possible to carry out longitudinal research based on a combination of different registers. The PIN is recorded in encrypted form in the registers, and enables individual linkage of longitudinal health register data.Data on specialized health services were collected from the Norwegian Patient Register (NPR). The NPR covers activity data from all Norwegian specialist healthcare institutions. It includes records of all consultations and admissions in Norwegian government-owned hospitals and outpatient clinics, as well as consultations with private contract specialists who receive government reimbursement. In order to mend a small percentage of missing data for the reimbursed private contract specialists in NPR [s in NPR , we veris in NPR . In thisData on primary health services were retrieved from The Norwegian Health Economics Administration (HELFO) database. Primary care physicians send electronic compensation claims for all patient contacts to HELFO, which is responsible for the reimbursement through a national health insurance scheme. A claim identifies the patient, the reason for encounter according to the International Classification of Primary Care (ICPC-2), the fee codes for different types of contacts and the specialty of the practitioner. Consultations with Primary Care Physicians (PCP) covered consultations with the survivors’ regular General Practitioners (GPs) or other physicians working in general practice and primary care emergency units, including out-of-hours services. Based on the reason for encounter, we grouped the survivors’ PCP consultations into four categories considered relevant in the context of the terror attack:Psychological: All PCP consultations registered with an ICPC-2 code within chapter P “Psychological” reason for encounter.Injury: Consultations registered with the injury-related reasons for encounter listed in Additional file Administration/assault-related: PCP consultations with the ICPC-2 codes A97 “No disease”, A98 “Health maintenance/prevention” and Z25 “Assault/harmful event problem”. This category was created to assess if there could be a change in consultations with PCP after the attack due to screening assessments as part of the proactive outreach program where the PCP did not necessarily consider the survivor to have a particular diagnosis or disorder.Other: Consultations with any other ICPC-2 code than those stated above.For consultations with more than one ICPC-2 code, priority was given to the category «psychological», next “injury”, and then “administration/assault-related”.Our measurement of consultations covered actual meetings with a PCP or MHS practitioner. It did not include telephone calls, mail correspondence or laboratory visits only to perform prescribed blood tests, injections or similar.We examined differences between female and male survivors on a set of interview-based variables considered potentially relevant for the access to and/or need for health services; namely age, country origin, financial situation, living in a central or peripheral location, hospitalization after attack, exposure level, and psychiatric and somatic symptoms. Age was dichotomized according to the Norwegian age of minority into younger than 18 years and 18 years or older at the time of the attack. Furthermore, non-Norwegian origin was defined as having both parents born abroad. Previous research indicates that survivors with non-Norwegian origin more were overall less satisfied with the healthcare follow-up than survivors with Norwegian origin . The sura confidence intervals (CI) for the differences in mean yearly CRs for MHS and PCP consultations comparing respectively the first, second and third year post-attack with the year preceding the attack through bootstrap computations with 10,000 replications. The results were presented separately for female and male survivors, since gender may influence the healthcare consultation frequency and reasons for seeking healthcare at baseline.We calculated the mean yearly consultation rates (CRs) for female and male survivors in each of the 3 years preceding and following the attack  by dividing the total number of consultations for each year by the person time in the study for the same year. Person time was estimated as total number of days in a specific year, and for those who were admitted to a psychiatric hospital, we censored (i.e. subtracted) the number of days in psychiatric hospital. We only censored days in psychiatric hospitals and not in somatic hospitals, because we observed that consultations with specialized mental health services also occurred during admittance at somatic hospitals. The graphs in Figs. p-value. The percentages and means were based on the total number of responses for each item. This study was based on survivors who participated in the third interview wave of a longitudinal study of the survivors of the Utøya attack [In order to compare characteristics of female and male survivors considered relevant for healthcare utilization, we used Pearson chi-squared tests for categorical variables and independent t-tests for continuous variables. We applied a two-sided level of significance at 0.05 for the a attack . We lackCharacteristics of female and male survivors are presented in Table Altogether, 114 93%) of female survivors and 116 (88%) of male survivors consulted PCP in the 3 years preceding the attack, compared to respectively 120 (98%) and 126 (96%) in the 3 years following the attack. Furthermore, 21 (17%) of female survivors and 15 (11%) of male survivors consulted MHS in the 3 years preceding the attack, compared to respectively 98 (80%) and 86 (65%) in the 3 years following the attack. Among the 36 survivors who had consulted MHS in the 3 years preceding the attack, 33 (92%) also consulted MHS in the 3 years following the attack. Overall 35 (14%) survivors reported that they had consulted psychiatrists, psychologists or another specialized medical doctor where they did not get all or part of the treatment covered by public sources. All these survivors had publicly funded MHS consultations recorded in the registry. Figure % of femaFigure Overall, there were 1162 PCP consultations with a psychological reason for encounter, 300 with injury-related reason for encounter (20 of these were additionally registered with and therefore classified as psychological reason for encounter in Fig. This study demonstrated that both PCP and MHS practitioners played important roles in providing healthcare for psychological problems in the young survivors of a terrorist attack in Norway. The majority consulted PCP both before and after the attack, with a clear increase in psychological reasons for encounter after the attack. Few of the survivors consulted MHS before the attack, while most of them did after the attack. In general, the consultations rates for PCP and MHS were higher for female than male survivors, both before and after the attack. In accordance with findings in previous research, the levels of posttraumatic stress symptoms, depression and anxiety symptoms and somatic symptoms were also higher in female survivors both early and at long-term after the attack , 40. HenOur findings demonstrated a high need for primary care and specialized mental health services several years following the terrorist attacks. These findings are in line with previous studies, indicating that the needs for care may persist for years after trauma exposure , 42 It iIn Norway, there is universal health coverage and a gatekeeping system with a regular GP scheme . Our resApproximately nine of ten survivors had consulted PCPs in the 3 years preceding the attacks. We do not know whether these PCPs were the survivors’ regular GP, but one of the intentions of the regular GP scheme is that each inhabitant should have a regular GP to strengthen the continuity of care. Studies suggest that having a long-lasting patient relationship with a regular GP is associated with positive health-related outcomes , 46. ThiThe utilization of PCP and MHS after terrorist attacks could be different in countries with other health systems and/or different psychosocial care responses to terrorist attacks . For insWhen we looked at openly available data on PCP consultations in the general population in Norway in the similar age spans 16–19 and 20–29 years, we observed that the consultations rates were generally higher for girls and women than men, which was also observed in our study . FurtherThis study provided detailed and continuous data on young survivors’ utilization of different types of health services before and after exposure to a terrorist attack. Using register-based data, we obtained objective information on the frequency and timing of healthcare utilization, as well as the type of healthcare used. In contrast to studies based on self-reports, these data are more specific and not prone to recall bias. Another strength of this study was the sample consisting of approximately half female and male survivors of young age exposed to the same severe and potentially traumatic event. The fact that the study participants were exposed simultaneously made it possible to compare some healthcare utilization measures with openly available healthcare data from the general population in similar age groups in the same period , 53. SevOur findings indicate that the health services should anticipate and prepare for a large increase in the healthcare needs in young survivors of mass trauma. The increase is likely to be particularly high the first year, yet remain elevated several years compared to before the trauma exposure. Furthermore, the increase may be especially high for MHS, but also PCPs should expect a marked increase in consultations with psychological reasons for encounter, in particular related to posttraumatic stress symptoms, depression and sleep disorders Table . Recent In order to enlarge the knowledge base on healthcare provision after terrorist attacks and other mass casualty incidents, accurate pre- and post-event data on healthcare utilization from registers or administrative databases should be analyzed also in future studies of trauma-exposed populations. Another important step forward would be to link register-based healthcare data with self-reported data from affected individuals on e.g. their psychological and somatic symptoms, their experiences and satisfaction with healthcare, and socioeconomic factors. This could yield a more profound insight into the relationship between healthcare utilization and symptom trajectories, whether there were unmet needs and potential predictors for unmet needs. Detailed data on utilization of specialized somatic health services would also be relevant to examine.Following a terrorist attack in a country with universal health coverage and a gatekeeping system, both PCP and MHS practitioners played important roles in the provision of healthcare for psychological problems in young survivors. Most survivors consulted PCP both before and after the attack, with a marked post-attack increase in psychological reasons for encounter. The majority of the survivors consulted MHS after the attack, whereas few did before the attack. The healthcare utilization was elevated several years after the attack compared to before the attack, and increased most for MHS. It is therefore important to prepare for an increased healthcare utilization several years following mass trauma. This study was conducted in a country with universal and mostly publicly financed healthcare and a regular GP-based gatekeeping system. The healthcare utilization after mass trauma may be different in countries with other health systems or psychosocial care responses.Additional file 1.


NOTCH3 pathogenic variants that result in the stabilization of NOTCH3 and a gain-of-function. LMS presents with neurological developmental abnormalities and bone loss. We created a mouse model (Notch3em1Ecan) harboring a 6691TAATGA mutation in the Notch3 locus, and heterozygous Notch3em1Ecan mice exhibit cancellous and cortical bone osteopenia. In the present work, we explored whether Notch3 antisense oligonucleotides (ASO) downregulate Notch3 and have the potential to ameliorate the osteopenia of Notch3em1Ecan mice. Notch3 ASOs decreased the expression of Notch3 wild type and Notch36691-TAATGA mutant mRNA expressed by Notch3em1Ecan mice in osteoblast cultures without evidence of cellular toxicity. The effect was specific since ASOs did not downregulate Notch1, Notch2 or Notch4. The expression of Notch3 wild type and Notch36691-TAATGA mutant transcripts also was decreased in bone marrow stromal cells and osteocytes following exposure to Notch3 ASOs. In vivo, the subcutaneous administration of Notch3 ASOs at 25 to 50 mg/Kg decreased Notch3 mRNA in the liver, heart and bone. Microcomputed tomography demonstrated that the administration of Notch3 ASOs ameliorates the cortical osteopenia of Notch3em1Ecan mice, and ASOs decreased femoral cortical porosity and increased cortical thickness and bone volume. However, the administration of Notch3 ASOs did not ameliorate the cancellous bone osteopenia of Notchem1Ecan mice. In conclusion, Notch3 ASOs downregulate Notch3 expression in skeletal cells and their systemic administration ameliorates cortical osteopenia in Notch3em1Ecan mice; as such ASOs may become useful strategies in the management of skeletal diseases affected by Notch gain-of-function.Notch receptors are determinants of cell fate and function, and play an important role in the regulation of bone development and skeletal remodeling. Lateral Meningocele Syndrome (LMS) is a monogenic disorder associated with  Notch1, 2 and 3 and low levels of Notch4 transcripts are detected in bone cells, where each receptor acts in a distinct capacity to influence the fate of cells of the osteoblast and osteoclast lineages  produces a formazan dye, measured at an absorbance of 450 nm, upon reduction by cellular dehydrogenases. The assay quantifies viable cells and was used in accordance with manufacturer’s instructions .Cell replication was determined using the Cell Counting Kit-8 , trabecular thickness, trabecular number, trabecular separation, connectivity density and structure model index (SMI), using a Gaussian filter (σ = 0.8), and a threshold of 240 permil equivalent to 355.5 mg/cm3 hydroxyapatite Reviewers' comments:Reviewer's Responses to Questions  Comments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?  The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: Yes**********  2. Has the statistical analysis been performed appropriately and rigorously?   Reviewer #1: YesReviewer #2: Yes**********  3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.  The Reviewer #1: YesReviewer #2: Yes**********  4. Is the manuscript presented in an intelligible fashion and written in standard English?  PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes**********  5. Review Comments to the Author  Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: The authors created a mouse model for Lateral Meningocele Syndrome (LMS), a very rare disease associated with gain-of-function of the Notch3 Receptor. The mouse model showed cancellous and cortical bone osteopenia. Then, the Notch3 receptor was downregulated by weekly injections of antisense oligonucleotides (ASO) specific for Notch3. The effect on the expression of Notch3 was examined by RT-PCR. Interestingly, the LMS-phenotype in bone was partially reversed. The effect of the AOS was stronger in cortical than in cancellous bone. However, the LMS-phenotype was not as extensively reversed with ASO in comparison to the use of a Notch3 antibody. In 2020, the authors published the same mouse model, but used an antibody instead of ASO to reverse the gain-of-function effect in the LMS mouse. Thus, the paper is presenting a proof-of-principle rather than a significant conceptual advance. The observed differences between cortical and cancellous bone might be related to the higher biological availability of ASO in cortical than cancellous bone. Since a difference is seen, the question remains why the authors only examined the femora of the mice. I would a expect a stronger effect in vertebrae.All in all, the methods are adequate, the conclusions are supported by the data, and the paper is clearly written. However, I have some minor concerns which should be addressed by the authors first.General:-Refer to the mice age in weeks all the time. In several sections, months are used for age indication-17 of 53 citations are self-citations. Many of them are not necessary, especially in the method´s section of the paper. Please, diversify your references.Introduction:-The introduction section is sufficient. What about Notch3 loss-of-function? CADASIL? I recommend mentioning it in one or two sentences to give the reader an overall understanding about the function of Notch3 in pathophysiology of humans.Methods:-Why are genetic backgrounds of the mice mixed (BALB and C57/BL)?-Why are different housekeeping genes used as references for Notch3 expression  and 40,41 (page 7)-Were both femora examined in micro-ct or only one side (page 7)Results:-Usually, female mice show a more distinctive phenotype than male mice. The authors state that this was different in the Notch3-LMS mouse and give reference to their own paper.-What is the explanation for this exception? This is a feature of the mouse model that could easily be examined, if male and female mice were analyzed.-Could the different effect of ASO on cortical vs. cancellous bone be associated with blood perfusion of these areas? The ASO is i.p. injected. Is there data of its distribution available? Fluorescence imaging? If not, please also comment in the discussion section.Discussion:-Please, clarify what you had expected from the ASO in LMS-mice and what was different to the anti-Notch3 AB-When a difference is seen in cortical and cancellous bone in the femora, the effect on calvaria or spine would be interesting. Please comment why this analysis was not performed.Reviewer #2: This is an interesting report from a group that has worked for some time on the roles of Notch receptors in the physiology of bone formation and resorption and skeletal diseases. This study focuses on Notch 3 and the authors created a Notch3 knock-in mutant mouse to model Lehman Syndrome or Lateral meningocele syndrome (LMS). The gain-of-function mutation results in a truncated NOTCH3 devoid of the PEST domain, which is necessary for ubiquitination and degradation of NOTCH3 to limit its activity. The objective of the study, therefore, appears to be to assess antisense oligonucleotide (ASO) against Notch3 as a potential therapeutic for osteopenia in LMS in Notch3 knock-in mice and in bone cells derived from them. The experiments are well designed but stop short of addressing bone-specific molecular mechanisms and the authors need to better highlight the novel aspects of this work differentiating Notch3 effects from other Notch receptors. Specific suggestions are listed below.1. In the Abstract the nomenclature for the mouse strains is difficult to follow and this persists throughout the manuscript. The authors state that they created a novel mouse strain“ (Notch3em1Ecan) harboring a 6691-TAATGA mutation in the Notch3 locus, and heterozygous Notch3em1Ecan mice exhibit cancellous and cortical bone osteopenia”. Then later they refer to Notch36691-TAATGA mRNA. This was confusing until I read through the manuscript. Evidently, the heterozygous Notch3em1Ecan was used throughout the experiments because the mice homozygous for the 6691-TAATGA mutation are developmentally abnormal. Thus, the heterozygous Notch3 knock-in mutant have levels of Notch 3 activity that are sufficient to enable demonstration of amelioration of osteopenia with Notch3 ASOs. Please consider more consistent use of nomenclature here and throughout the manuscript.2. The concluding statement of the Abstract simply restates the findings without providing a conclusion about important insights resulting from the study.3. The last paragraph of the Introduction also needs some attention regarding the hypothesis to be test and concluding insights. One thing that needs highlighting is that the work is modeling LMS, where Notch3 is abnormally activated, so treatment Notch3 ASOs could normalize bone status by inhibitingNotch3 signaling. Is this interpretation correct?4. Page 6, line 2: This sentence does not seem correct: “Heterozygous Notch3em1Ecan mutants were crossed with wild type mice to create heterozygous Notch3em1Ecan”.5. Page 14: Regarding the results in Figure 6, there is an assumption made, based on previously published work, that the preferential effect of Notch3 ASO was limited to cortical bone osteopenia, without increasing BV/TV in cancellous bone, because Notch3 is expressed preferentially by osteocytes. Showing these effects in cortical versus cancellous bone in the mouse tissues after ASO treatment would enhance the mechanistic value of the work.6. Page 15: Similarly in the Discussion, direct evidence that the Notch3 ASOs were more effective in the osteocyte-rich compartment in Notch3 knock-in mutants, accounting for the preferential targeting of cortical bone, would add value to the study.7. Page 17: As in the Abstract, this single sentence does not constitute a conclusion.**********  6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.If you choose “no”, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.  Reviewer #1: NoReviewer #2: Nohttps://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.
 While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool,  21 Apr 2022April 18, 2022Gary SteinAcademic EditorPLOS ONEDear Dr. Stein:In accordance with your letter of March 30, 2022, we would like to submit a revised version of the manuscript entitled “Use of antisense oligonucleotides to target Notch3 in skeletal cells” (PONE-D-22-04880) to be considered for publication by PLOS One. Changes are outlined in this response and highlighted in the text of the paper. Reply to Reviewer #1: The authors created a mouse model for Lateral Meningocele Syndrome (LMS), a very rare disease associated with gain-of-function of the Notch3 Receptor. The mouse model showed cancellous and cortical bone osteopenia. Then, the Notch3 receptor was downregulated by weekly injections of antisense oligonucleotides (ASO) specific for Notch3. The effect on the expression of Notch3 was examined by RT-PCR. Interestingly, the LMS-phenotype in bone was partially reversed. The effect of the AOS was stronger in cortical than in cancellous bone. However, the LMS-phenotype was not as extensively reversed with ASO in comparison to the use of a Notch3 antibody. In 2020, the authors published the same mouse model, but used an antibody instead of ASO to reverse the gain-of-function effect in the LMS mouse. Thus, the paper is presenting a proof-of-principle rather than a significant conceptual advance. The observed differences between cortical and cancellous bone might be related to the higher biological availability of ASO in cortical than cancellous bone. Since a difference is seen, the question remains why the authors only examined the femora of the mice. I would a expect a stronger effect in vertebrae.All in all, the methods are adequate, the conclusions are supported by the data, and the paper is clearly written. However, I have some minor concerns which should be addressed by the authors first.General:-Refer to the mice age in weeks all the time. In several sections, months are used for age indication As recommended, we now refer to the age of mice in weeks throughout the paper. -17 of 53 citations are self-citations. Many of them are not necessary, especially in the method´s section of the paper. Please, diversify your references. Unnecessary references 38, 39, 40 and 41 were removed. Added 10 additional references from other laboratories. Introduction:-The introduction section is sufficient. What about Notch3 loss-of-function? CADASIL? I recommend mentioning it in one or two sentences to give the reader an overall understanding about the function of Notch3 in pathophysiology of humans. We now mention CADASIL in the Introduction and cite references on the function of NOTCH3 in mural vascular cells and role of NOTCH3 in CADASIL. Please see page 3, end of paragraph 2.Methods:-Why are genetic backgrounds of the mice mixed (BALB and C57/BL)? The initial screen and toxicology studies of ASOs were performed at the Korea Institute of Toxicology in BALB/c mice. The ASOs that were active and had no toxicity were chosen for the present studies. All the work presented in this paper was conducted in Notch3em1Ecan and littermate controls in a C57BL/6 background.-Why are different housekeeping genes used as references for Notch3 expression  and 40,41 (page 7) References 38, 39, 40 and 41 were removed as requested.-Were both femora examined in micro-ct or only one side (page 7) Only one side, and this is now stated in page 7, last paragraph.Results:-Usually, female mice show a more distinctive phenotype than male mice. The authors state that this was different in the Notch3-LMS mouse and give reference to their own paper. What is the explanation for this exception? This is a feature of the mouse model that could easily be examined, if male and female mice were analyzed. The reviewer is correct and we apologize for the error. We examined the published paper  and Notch3em1Ecan female mice have more pronounced osteopenia at 1 month of age than male mice. The error was corrected. The reason why male mice were chosen to test the effect of the ASOs is because the osteopenic phenotype is more persistent and consistent in Notch3em1Ecan male than in female mice. At 4 months of age, male but not female mice are osteopenic. In this study, 1 month old male mice were administered ASOs and examined at 2 months of age to ensure that a phenotype would be detected. The statement was changed to indicate that male mice were studied because the osteopenic phenotype in males was sustained for up to 18 weeks. Please see page 13, paragraph 2. -Could the different effect of ASO on cortical vs. cancellous bone be associated with blood perfusion of these areas? The ASO is i.p. injected. Is there data of its distribution available? Fluorescence imaging? If not, please also comment in the discussion section. We reviewed information on the vascular system supplying long bones as well as perfusion studies. Blood supply for the cortex of long bones is carried out by a central nutrient artery and periosteal arteries whereas metaphyseal-epiphyseal arteries irrigate the bone metaphysis rich in cancellous bone. Therefore, it is possible that Notch3 ASOs had greater access to cortical than cancellous bone. However, perfusion studies have not shown differences in perfusion efficiency in epiphysis, diaphysis or calvariae. We now present this information in the Discussion and quote appropriate references. Please see page 17, end of paragraph 1. Discussion:-Please, clarify what you had expected from the ASO in LMS-mice and what was different to the anti-Notch3 AB We now explain in the Discussion that we expected a downregulation of Notch3 expression by the Notch3 ASOs. We also provide possible explanation for the greater effect of anti-Notch3 antibodies such as greater stability or access to target tissues. It is possible that the anti-Notch3 antibody is more effective than the ASO because it targets the transmembrane domain of NOTCH3 and does not need to enter the cell to be effective. The ASO instead acts intracellularly to target the mRNA for degradation. Please see page 16, last paragraph.-When a difference is seen in cortical and cancellous bone in the femora, the effect on calvaria or spine would be interesting. Please comment why this analysis was not performed. We analyzed the effect of Notch3 ASOs in cancellous bone at L3 in wild type and Notch3em1Ecan mice and results were analogous to those observed in femoral cancellous bone. A statement to this effect was included in the Results and data for L3 BV/TV were included in the text. Please see page 14, paragraph 2. Calvariae were not examined since parameters of microarchitectural analysis of bone  are established for cancellous and cortical bone present in vertebrae and long bones and not for intramembranous bone present in calvariae .Reply to Reviewer #2: This is an interesting report from a group that has worked for some time on the roles of Notch receptors in the physiology of bone formation and resorption and skeletal diseases. This study focuses on Notch 3 and the authors created a Notch3 knock-in mutant mouse to model Lehman Syndrome or Lateral meningocele syndrome (LMS). The gain-of-function mutation results in a truncated NOTCH3 devoid of the PEST domain, which is necessary for ubiquitination and degradation of NOTCH3 to limit its activity. The objective of the study, therefore, appears to be to assess antisense oligonucleotide (ASO) against Notch3 as a potential therapeutic for osteopenia in LMS in Notch3 knock-in mice and in bone cells derived from them. The experiments are well designed but stop short of addressing bone-specific molecular mechanisms and the authors need to better highlight the novel aspects of this work differentiating Notch3 effects from other Notch receptors. Specific suggestions are listed below.1. In the Abstract the nomenclature for the mouse strains is difficult to follow and this persists throughout the manuscript. The authors state that they created a novel mouse strain“ (Notch3em1Ecan) harboring a 6691-TAATGA mutation in the Notch3 locus, and heterozygous Notch3em1Ecan mice exhibit cancellous and cortical bone osteopenia”. Then later they refer to Notch36691-TAATGA mRNA. This was confusing until I read through the manuscript. Evidently, the heterozygous Notch3em1Ecan was used throughout the experiments because the mice homozygous for the 6691-TAATGA mutation are developmentally abnormal. Thus, the heterozygous Notch3 knock-in mutant have levels of Notch 3 activity that are sufficient to enable demonstration of amelioration of osteopenia with Notch3 ASOs. Please consider more consistent use of nomenclature here and throughout the manuscript. We now qualify Notch36691-TAATGA as Notch36691-TAATGA mutant RNA expressed by the Notch3em1Ecan mutant mouse. In the Abstract and throughout the manuscript, we qualify the Notch36691-TAATGA as a mutant transcript and Notch3em1Ecan as the mouse model expressing the transcript.2. The concluding statement of the Abstract simply restates the findings without providing a conclusion about important insights resulting from the study. In accordance with the reviewer’s comment, a more appropriate conclusion was added in the Abstract and Conclusions. 3. The last paragraph of the Introduction also needs some attention regarding the hypothesis to be test and concluding insights. One thing that needs highlighting is that the work is modeling LMS, where Notch3 is abnormally activated, so treatment Notch3 ASOs could normalize bone status by inhibitingNotch3 signaling. Is this interpretation correct? We now highlight that Notch3em1Ecan is a model of LMS and of gain-of-NOTCH3 function, and we postulate that by decreasing Notch3 mRNA expression with specific ASOs one could decrease the levels of biologically active NOTCH3 NICD. Please see page 4, last paragraph. Strictly speaking, ASOs downregulate transcripts. By decreasing Notch3 mRNA, there is less active NOTCH3 NICD and less signaling.4. Page 6, line 2: This sentence does not seem correct: “Heterozygous Notch3em1Ecan mutants were crossed with wild type mice to create heterozygous Notch3em1Ecan”. The sentence was corrected. It now states, Heterozygous Notch3em1Ecan mutants were crossed with wild type mice to generate ~50% heterozygous Notch3em1Ecan mice and 50% control littermates to be characterized and administered Notch3 ASOs. Please see page 6, paragraph 1.5. Page 14: Regarding the results in Figure 6, there is an assumption made, based on previously published work, that the preferential effect of Notch3 ASO was limited to cortical bone osteopenia, without increasing BV/TV in cancellous bone, because Notch3 is expressed preferentially by osteocytes. Showing these effects in cortical versus cancellous bone in the mouse tissues after ASO treatment would enhance the mechanistic value of the work. The reviewer is correct, and we assume that the Notch3 ASO was more effective in osteocyte-rich cortical bone. Notch 3 is preferentially expressed by the osteocyte as previously published by us and others . However, as the reviewer indicates, we cannot state with certainty that this is the reason why the Notch3 ASO was more effective in cortical bone. Other possibilities, such as differential vascularization of cortical and cancellous bone as pointed out by reviewer 1, could play a role and are now included in the Discussion. Please see page 17, paragraph 1. We also indicate that we do not demonstrate that Notch3 ASOs are more effective in an osteocyte population in vivo. The experiment proposed by the reviewer would be valuable but technically quite challenging since one would need to show downregulation of Notch3 and Notch3 mutant mRNA in different bone compartments possibly by in situ hybridization and the method would not be sufficiently sensitive to detect modest differences or reductions in transcript expression. We sincerely apologize for this deficiency and state the deficiency in the Discussion. Please see page 17, paragraph 1.6. Page 15: Similarly in the Discussion, direct evidence that the Notch3 ASOs were more effective in the osteocyte-rich compartment in Notch3 knock-in mutants, accounting for the preferential targeting of cortical bone, would add value to the study. We agree with the reviewer; but as mentioned in the response to comment 5, we believe that it would be technically difficult to demonstrate a downregulation in bone tissue.7. Page 17: As in the Abstract, this single sentence does not constitute a conclusion.  We have modified the conclusion statement – “In conclusion, Notch3 ASOs downregulate Notch3 expression in skeletal cells from a mouse model of LMS and ameliorate its cortical osteopenic phenotype. Consequently, ASOs may become a useful strategy in the management of skeletal diseases affected by gain-of-Notch function”.Additional Modification As we were correcting the manuscript, we noticed some inconsistencies in the copy number of “transcripts” shown in Figure 2. To ensure absolute accuracy, we repeated the real time PCR of all the samples shown in Figure 2A, B and C and the figure was replaced with the newly generated transcript data. Rest assured that the effect of Notch3 ASO remains the same and the only change is in the value of the actual copy number.We hope you now find the manuscript acceptable for publication. Thank you for your consideration.Sincerely yours, Ernesto Canalis, M.D.Director, Center for Skeletal ResearchProfessor of Orthopaedics and MedicineAttachmentReply to Reviewer_PONE-D-22-04880.docxSubmitted filename: Click here for additional data file. 26 Apr 2022Use of antisense oligonucleotides to target Notch3 in skeletal cellsPONE-D-22-04880R1Dear Dr. Canalis,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact Kind regards,Gary SteinAcademic EditorPLOS ONEAdditional Editor Comments :Concerns raised by reviewers were adequately addressed so the manuscript is acceptable for publicationReviewers' comments: 29 Apr 2022PONE-D-22-04880R1 Use of antisense oligonucleotides to target Notch3 in skeletal cells Dear Dr. Canalis:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. If we can help with anything else, please email us at Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staffon behalf ofDr. Gary Stein Academic EditorPLOS ONE


The overvaluation of reward-associated stimuli such as energy-dense foods can drive compulsive eating behaviours, including overeating. Previous research has shown that training individuals to inhibit their responses towards appetitive stimuli can lead to their devaluation, providing a potential avenue for behaviour change. Over two preregistered experiments, we investigated whether training participants to inhibit their responses to specific foods would be effective in reducing their evaluations when these were assessed using both explicit and implicit measures. Participants completed an online session of go/no-go training with energy-dense foods that were consistently associated with either responding (go) or inhibiting a response (no-go). An ‘explicit’ devaluation effect was expected as a reduction in self-reported liking from pre-to post-training for no-go items compared to both go items and foods that were not presented during training (untrained items). An ‘implicit’ devaluation effect was then measured using the affective priming paradigm, by comparing differences in reaction times for congruent and incongruent trials  between food primes. Experiment 1 revealed conclusive evidence for small-to-medium devaluation effects both in terms of explicit ratings and priming effects. We also observed that the priming effect for no-go items was close to zero. Experiment 2 successfully replicated most of the preregistered and exploratory outcomes from Experiment 1 except for the priming effect for untrained items. Potential explanations for this discrepancy are discussed but overall, these findings provide further support for a devaluation effect of response inhibition training. To our knowledge, our study provides the first evidence that training-induced devaluation can potentially be captured by affective priming measures, but more research is needed to further assess their sensitivity before they can be used to elucidate the mechanisms of action underlying devaluation effects. Evidencalso see . Althougalso see , primingalso see . For thealso see .The use of implicit measures in applied research either as primary or secondary outcomes may complement existing evidence for training-induced devaluation effects, as these are commonly assessed using explicit self-report methods which can be associated with response bias and demand characteristics . Importapost hoc exploratory analyses of the original experiment.In Experiment 1 we tested several preregistered hypotheses regarding the effects of GNG training on explicit and implicit food evaluations. We expected stimulus devaluation to occur for foods associated with response inhibition during training. The devaluation effect was defined as a negative change in liking from pre-to post- training for two contrasts with different baselines . There were no deviations from the study protocol.1All hypotheses, as outlined below, and respective statistical tests were preregistered prior to data collection  and personal communication were assessed for eligibility in the study (see S1). Participants had to be at least 18 years old or older, speak English as their first or second language, have normal or corrected-to-normal vision and report no past and/or current history of eating disorders. Participants were not eligible to participate if they were dieting to lose weight and/or taking diet pills at the aim of the study. Participants also had to report no major hearing impairments that would prevent them from hearing the tones presented in the study. Pre-screening on Prolific specified that only individuals currently residing in the UK could participate, as the branded foods included in the study might not have been popular in other countries. A total of 120 eligible participants completed the study and received either monetary compensation (Prolific) or a prize draw entry for a £15 online shopping voucher. The study was approved by the local Research Ethics Committee at the School of Psychology, Cardiff University.A total of 140 participants recruited via Prolific (nmax) of 130 and a minimum sample size (nmin) of 50. Data collection would continue until either the selected evidential threshold for preregistered hypotheses  had been reached, or nmax had been met. For convenience in the interpretation of evidence we followed previous guidelines and considered a threshold of BF10 ≥ 10 would indicate strong evidence for the alternative hypothesis compared to the null and vice versa for BF01 ≥ 10. To assess the probability of this SBF design generating misleading or inconclusive evidence we performed a Bayes Factor Design Analysis .In this experiment we employed an open-ended Sequential Bayes Factor (SBF) design  with a mis BFDA; . FurtherPre-training ratings & stimulus selection) and APP (untrained foods). Participants then rated non-food stimuli and the most liked categories were used in the non-food prime APP blocks (manipulation check). During the training phase of the study, participants performed eight blocks of the GNG training task with a short practice block in the beginning (see Go/no-go training task).The overall study procedure is shown in Affective priming paradigm) and explicit evaluation task (see Post-training ratings) were presented in a counterbalanced order across participants. Food prime (APPFOOD) and non-food prime APP blocks (APPNONFOOD) were presented in a fixed order, such that a non-food prime block was always followed by two food prime blocks. A short practice block was also provided for the APP. In the explicit evaluation task after training, participants rated all foods  with a total of 24 trials. At the end of the study, questionnaire measures were completed (see Questionnaires) and participants were debriefed on the study aims. The study was run via Inquisit Web and was programmed using Inquisit Lab 5 . The food stimuli appeared in random order and participants rated them according to how much they liked them at the time (“How much do you like this food right now?”) on a visual analogue scale (VAS) ranging from –100 to 100, always centred at zero. The exact values on the VAS were not visible to the participants. The foods with the highest ratings were ranked from 1 to 24 and were assigned to three sets of eight foods, in a manner that ensures that the average explicit liking values were as matched as possible for all sets . As described in the previous section, randomly assigned foods were presented on go and no-go trials, with two exemplars for each food category. Therefore, each food category had 16 repetitions across blocks and specific exemplars were repeated eight times in total. Participants first completed a short practice block  with remaining stimuli from the selection process and accuracy feedback appeared after each trial. The screen background was white and food stimuli had relative dimensions according to the participant’s display resolution (40% width × 40% height). A short break was provided after four blocks of training.The affective priming paradigm (APP) was adapted from a previous study that assessed its utility as an indirect measure of food liking . It invoS4) that we considered could be “unambiguously” categorised  were included and represented by two exemplars, which were randomly assigned to interleaving blocks to be paired with either positive or negative targets. For example, in block 1 the first exemplar of a go food category was paired with a positive target and the second with a negative target, whereas in block 2 this assignment was reversed. This ensured that both food exemplars were presented as primes in both congruent (positive target) and incongruent trials (negative target). The APP included 24 positive and 24 negative targets (see egorised .NONFOOD) were presented in between two food prime APP blocks (APPFOOD) and the order was fixed across participants: one APPNONFOOD block, two APPFOOD blocks, …, one APPNONFOOD block.A manipulation check for the task as an indirect measure of liking was employed in line with previous work . In fourThe screen background for the APP was white, consistent with the GNG task design, and all words were capitalised. Participants completed 16 practice trials and the primes were foods that had not been assigned to an experimental set during stimulus selection. Feedback was provided for both speed and accuracy. After three APP blocks participants received a short break and were reminded of the main task instructions and response key assignments.Participants rated all foods from the go, no-go, and untrained conditions after training (random order) and food categories were represented by the same exemplars from the initial explicit evaluation task. Instructions for participants highlighted that some pictures may be the same as in the first rating task, but they should indicate how much they liked them at that specific time. Instructions for both pre- and post-training ratings encouraged participants to pay attention to the specific pictures depicting each food: “For instance, you may generally like a certain type of crisps but not find this particular flavour very appealing at this specific time [..]”.2).Several demographic as well as trait and state variables were recorded at the end of the study, including gender, ethnicity, hunger levels , hours since last meal  and dietary preferences . Body height and weight were self-reported to calculate the participant’s body-mass index .After answering these questions, participants completed a follow-up study questionnaire which assessed their perceived performance in the APP , number of interruptions during the study  and inclINC – medianRTCON) for each training condition . For explicit evaluations pre- and post-training, mean ratings for each condition were calculated . Difference scores of these means were finally calculated for each training condition . Negative difference scores (post – pre) would reflect a reduction in liking from pre-to post-training. Other descriptive measures of APP and GNG task performance were also recorded for data exclusions and exploratory analyses, such as the inspection of potential speed-accuracy trade-offs in the APP (see S7).For APP performance and calculated priming effects, median RTs were obtained from correct trials only, for each participant and each design cell . Medians were preferred instead of means as they are less sensitive to outliers and would indicate central tendency more accurately in the expected positively-skewed RT distributions. Priming effects (ΔRT) were calculated as the change in median RTs from incongruent to congruent trials (medianRTNOGO) lower than 0.65 would be excluded, as it has been shown that this is an important moderator for training effects (see section 3.3 in S1).Accuracy was inspected for both the GNG and APP tasks. Participants with error rates (ER) greater or equal to 0.4 from within either the set of critical food prime or non-food prime trials were excluded from all respective analyses. For the GNG task, participants who had a proportion of successful inhibitions . Shapiro-Wilk tests were performed to check for potential violations of the normality assumption and in case of violationS6).Data pre-processing and analyses were conducted in R  using RS t-tests , as showS1). Overall, the average liking ratings for the food items in each training condition were matched and the majority of participants found the foods appetitive . The percentage of successful inhibitions was very high across participants . Descriptive statistics of sample demographics and trait/state variables were recorded for 112 participants. Although the data were collected online the sample was not diverse in terms of ethnicity or background  and age . The proportion of female participants was 57.1% and the proportion of male participants was 42.9%.The final sample for Experiment 1 consisted of 113 participants. All participant exclusions can be found in the SI . This is consistent with the self-reported hours since last food intake, which were not indicative of elevated appetite, as 49.1% of participants had a meal 1–3 hours before the study and 23.2% had a meal less than one hour before. Most participants did not follow a specific diet (84.8%), while 6.3% reported a vegetarian diet, 3.6% a vegan diet, 3.6% a pescetarian diet and only 1.8% another diet . Participants’ average BMI was 24.2 kg/m2 . Almost all participants indicated that they had no prior knowledge of the study hypotheses/aims (98.2%). After study completion, 36.6% of participants reported an awareness of stimulus-response contingencies for no-go trials during training and most participants (94.6%) passed the attention check in the follow-up questionnaire.Participants were on average not very hungry at the time of the study . Liking ratings pre- and post-training have been visualised using a raincloud plot  and greater compared to the change for go foods . Similarly, there was conclusive support for H1b, as the negative change in liking ratings was greater for no-go foods after training relative to untrained foods .All results from Bayesian and supplementary frequentist statistical tests can be found in oud plot , 2019 anvery strong evidence for an implicit devaluation effect  compared to go foods . H2b also received conclusive support, as the RT priming effect for no-go foods was lower for untrained foods  relative to go foods. Finally, the manipulation check for the APP (H3) was successful, as there was extreme evidence that participants were on average faster to respond on congruent  rather than incongruent non-food prime trials .The RT priming effects (ΔRTs) for each training condition were also examined and there was fect see . H2a wasextreme evidence that participants were on average faster to respond on congruent  compared to incongruent go food prime trials  . There was also very strong evidence for a positive RT priming effect for congruent  and incongruent untrained food prime trials  . However, there was moderate evidence for the absence of a positive RT priming effect when no-go foods were examined. Participants were not faster to respond on congruent  compared to incongruent no-go food prime trials  . The absence of an overall RT priming effect for no-go foods is also illustrated in S7), indicating that the RT effects reported here could not be attributed to strategic responding.Although the difference scores for RT priming effects in each training condition were compared directly in planned paired comparisons, additional exploratory analyses were conducted to examine whether positive effects would be obtained for the two baselines (go and untrained foods). There was Preregistered analyses showed that the change in liking ratings from pre-to post- training was negative for no-go foods and greater in magnitude compared to the change in liking ratings for both go foods and untrained foods. These results suggest that the go/no-go training task employed in this study had an explicit no-go devaluation effect and indirectly replicate the robust devaluation effects observed in other preregistered studies . SpecifiTo our knowledge, this is the first study to show that the affective priming paradigm can be successfully applied as an outcome measure for stimulus devaluation after go/no-go training. It was hypothesised that the expected positive RT priming effects for foods would differ between training conditions, so that the priming effect for no-go food primes would be reduced compared to the observed effects for both go and untrained food primes. Although there is not enough evidence to infer whether the magnitude of the APP priming effects can be influenced by the strength of the primes, such as how much participants like the food items , there wpost hoc analyses .The primary aim of Experiment 2 was to directly replicate the observed devaluation effects for foods associated with response inhibition, both in terms of explicit evaluations and RT priming effects. All methods for the training task and outcome measures were identical to the original experiment with only a minor change to the number of food items participants could rate in the beginning of the study for stimulus selection. Certain questionnaires which were presented at the end of the study were removed and recruitment was expanded to include participants from Cardiff University. In addition to the main hypotheses in Experiment 1, we also tested hypotheses that were formulated from our https://osf.io/p6yk9. Note that hypotheses H1, H2, and H3 were identical to Experiment 1.All hypotheses that were introduced for Experiment 2 are listed below and the preregistered study protocol for the replication and extension of findings from Experiment 1 is available at H4. RT priming effects across training conditionsH4a. Participants will be on average faster to respond on congruent compared to incongruent go food prime trials.H4b. Participants will be on average faster to respond on congruent compared to incongruent untrained food prime trials.not be on average faster to respond on congruent compared to incongruent no-go food prime trials.4H4c. Participants will S1). All data collection procedures were consistent with those reported for Experiment 1. The sampling plan for Experiment 2 was updated according to a new analysis prior for planned Bayesian t-tests . For the replication and extension of findings in this experiment the nmax was increased to 200 (after exclusions), but data collection had to be terminated at N = 190 due to time constraints. Details for the BFDA run for Experiment 2 can be found in the SI (see S2).A total of 290 participants who were recruited via Prolific and the Experimental Management System at Cardiff UniversityS5). The study was run using the latest version of Inquisit Web at the time of data collection and therefore all scripts were updated in Inquisit 6 (https://osf.io/p6yk9). This modification does not affect any of the design parameters in the GNG task and APP, which remained the same as in Experiment 1.The overall study procedure in Experiment 2 was the same, but the questionnaires which were added in Experiment 1 as part of an assignment for undergraduate students were no longer included . The baseline liking ratings for selected foods in each training condition were matched, although the scores were slightly lower than those in Experiment 1 . Participants’ accuracy (%) in the GNG was high  consistent with performance in Experiment 1. Demographics showed that the samples in both experiments were approximately matched on ethnicity or background  and age . In this sample, the proportion of female participants was higher (76.8%), with 21.6% of participants identifying as male and 1.6% as gender-variant/non-conforming. A key difference between the samples in Experiments 1 and 2 would be that most participants for this replication were recruited from the student population at Cardiff University (67.4%).The final sample for Experiment 2 consisted of 190 participants , which was expected as 26.8% had a meal one hour before and 44.2% had a meal one to three hours before participation. Most participants were not following a specific diet (80.5%), while 9% were vegetarian, 4.74% were vegan, 3.7% were pescetarian and only 2.1% reported other diets. Participants’ average BMI was 23.8 kg/m2 . Most participants (95.2%) did not report any prior knowledge of the study aims/hypotheses. Only 30.7% of participants learned stimulus-response contingencies for no-go trials during training and 94.7% answered correctly on the attention check question.Participants’ self-reported hunger at the time of the study was not high  was greater than the change for go foods , but only moderate evidence for a slight difference in magnitude between the change for no-go foods and untrained foods . There was extreme evidence that the RT priming effect for no-go foods  was lower compared to the priming effect for go foods , but only inconclusive evidence for the absence of the expected difference in RT priming effects for no-go and untrained foods .Several hypotheses from Experiment 1 received conclusive support in this replication , but results were not consistent for the second baseline in our devaluation contrasts; that is, for untrained foods . All results from preregistered t-tests can be seen in extreme evidence that participants were on average faster to respond on go food prime trials when these were congruent  rather than incongruent . As expected, the RT priming effect for no-go foods was not observed,very strong evidence that participants were not faster to respond on congruent  than incongruent trials . Contrary to findings from Experiment 1 but consistent with the results regarding explicit devaluation, there was inconclusive evidence for the absence of an RT priming effect for untrained foods although descriptively participants were on average slightly faster to respond on congruent  relative to incongruent trials .Consistent with findings from exploratory analyses in Experiment 1, the preregistered hypotheses H4a and H4c were supported. There was The aim of Experiment 2 was to directly replicate all findings from Experiment 1 in a confirmatory manner. First, devaluation effects were observed for both the explicit and implicit measures of liking when no-go items were compared to go items and the effects were in the small-to-medium range consistent with Experiment 1. However, the devaluation effects when defined as the negative difference between no-go items and untrained items were smaller for explicit ratings and inconclusive for RT priming effects in Experiment 2.Consistent with Experiment 1, the manipulation check for the APP was successful and a positive RT priming effect as observed for go food primes. Importantly, the close-to-zero RT priming effect for no-go food primes was replicated in this experiment, which has implications for the theoretical explanations of training-induced devaluation effects. Specifically, these findings suggest that during training, negative affect is attached to stimuli that are consistently associated with response inhibition see also , which cThe present study employed a training task adapted from the go/no-go (GNG) training paradigm which has previously been associated with robust devaluation effects of appetitive stimuli e.g.,  to invesexplicit liking was greater for no-go foods than foods that did not appear during training. Preregistered analyses showed that this difference was replicated in the subsequent experiment, but the effect was considerably smaller and only when the data were corrected for outliers the effect size was similar to that reported in Experiment 1 (see S6.2). Overall, the study provided evidence for small-to-medium explicit devaluation effects of response inhibition training for appetitive food stimuli and the results presented here indirectly replicated previous findings from studies employing this GNG training paradigm  across training conditions. In both experiments we found that the RT priming effects for no-go food primes were lower compared to go food primes . There was, however, a noteworthy discrepancy between the two experiments for untrained food primes. The RT priming effect for no-go items was reduced relative to the priming effect for untrained items in Experiment 1, but there was only anecdotal evidence for the absence of this expected difference in Experiment 2. Similarly, there was inconclusive evidence for the lack of a positive RT priming effect for untrained items in Experiment 2.Yes. The evaluative categorisation task variant of the affective priming paradigm, which has been previously employed as an indirect measure of food liking e.g. see , was preOther studies have shown that ratings for both go and untrained items can decrease and this observation is often attributed to regression to the mean as stimuli with the highest ratings are included in training e.g. see . The priThere are certain limitations and considerations for future research with regards to the use of implicit measures in response inhibition training studies also see . First, Post hoc analyses in Experiment 1 supported our predictions of positive RT priming effects for go and untrained items, but also revealed an interesting trend in the RTs for no-go items, as the priming effect was diminished. In Experiment 2 we addressed these results as part of our preregistered hypotheses and successfully replicated the close-to-zero RT priming effect for no-go foods. This is an important finding of the present research as it has implications for the theoretical accounts of devaluation effects in response inhibition training studies.The RT priming effects were inspected on an exploratory basis in Experiment 1 to test whether there was evidence for the expected negative differences between the speed of responses on congruent and incongruent trials. All food items (i.e. primes) were selected to have the highest liking ratings pre-training and this would mean participants should be faster to correctly categorise positive words (congruent) rather than negative words (incongruent). S9) and in turn the degree of response facilitation in congruent trials and/or response interference on incongruent trials was also decreased  and sensitivity to experimental manipulation .In this study we report evidence that response inhibition training can lead to the devaluation of appetitive stimuli, such as energy-dense foods and building on previous findings by showing that this effect is not only observed when evaluations are assessed via self-report, but also when they are measured using implicit measures. Specifically, an https://osf.io/6bsnv/). All raw data have been made publicly available (DOI: 10.17605/OSF.IO/4DQZB).Data and analysis scripts for both experiments can be found in our project page on the Open Science Framework .s1 to s9.


As a type of local anaesthesia, retrobulbar nerve block is often used in vitrectomy, with patients remaining conscious during the operation. The increase in systolic blood pressure (SBP) caused by tension and fear during the operation—especially in patients with a history of hypertension—can negatively impact the safety of the procedure, resulting in suprachoroidal haemorrhage or retinal haemorrhage. Diazepam has a sedative effect and can relieve tension during surgery. This study aims to evaluate the efficacy and safety of diazepam for intraoperative BP stabilisation in hypertensive patients under retrobulbar anaesthesia during surgery.This single-centre, double-blind, randomised controlled and parallel clinical trial will include 180 hypertensive patients who will undergo vitrectomy with nerve block anaesthesia. Study participants will be randomly allocated in a 1:1 ratio to intervention  and control  groups. The primary outcome is the effective rate of intraoperative BP control . The secondary outcomes are the proportion of patients with SBP ≥180 mmHg at any timepoint from operation to 1 h post-operation, the change of mean systolic blood pressure and mean heart rate during operation from baseline, as well as the number of patients with intraoperative and post-operative adverse reactions within 12 weeks of surgery. The logistic regression model will be performed to compare the outcomes.This study will evaluate the efficacy and safety of diazepam for intraoperative BP stabilisation in hypertensive patients under nerve block anaesthesia during surgery. The results of this trial will reveal whether diazepam has a significant effect on intraoperative BP stability in patients with a history of hypertension who require vitrectomy. If the results of this trial are significant, a large-scale multi-centre clinical trial can be designed.ChiCTR2100041772. Registered on 5 January 2021.Chinese Clinical Trial Registry (ChiCTR) The online version contains supplementary material available at 10.1186/s13063-022-06686-y. Vitrectomy, a minimally invasive surgery for releasing vitreoretinal traction and restoring a transparent refractive stroma, has been widely used to treat vitreoretinal diseases including rhegmatogenous retinal detachment, traction retinal detachment, macular epiretinal membrane, and macular hole. This type of high-level modern ophthalmic microsurgery was developed in the early 1970s and is considered as revolutionary in the history of ophthalmic treatment . This suModern retrobulbar blockade techniques have long been considered as the gold standard for anaesthesia in intraocular surgery . HoweverSome patients who are normally nervous and prone to insomnia take diazepam tablets immediately before or on the day before the surgery to relieve intraoperative tension, which reduces fluctuations in intraoperative BP. Diazepam, also known as Valium, is a long-acting benzodiazepine. This class of drugs inhibits the central nervous system, which has the clinical effects of sedation and hypnosis; they are also used as anxiolytics and anticonvulsants . DiazepaThe present study will investigate the efficacy and safety of diazepam on intraoperative BP stability in patients undergoing vitrectomy with retrobulbar nerve block anaesthesia. In patients with a history of hypertension, we hypothesise that oral diazepam before surgery is safe and effective in maintaining a stable BP during vitrectomy.This is a single-centre, double-blind, parallel and randomised clinical trial (RCT) being conducted at Shanghai General Hospital affiliated with Shanghai Jiaotong University. The patients have a clear history of hypertension that is under control and require vitrectomy. Our hypothesis is that among patients with hypertension before vitrectomy, a greater proportion will have a stable BP during the operation with pre-operative diazepam vs the placebo. After comprehensive ophthalmic and physical examinations, the patients will be randomised into the diazepam or placebo group. The first participant was enrolled in August 2021. The projected completion date is August 2023.The study aims to evaluate the efficacy and safety of diazepam for intraoperative BP stabilisation in hypertensive patients under nerve block anaesthesia during surgery.This is a prospective, single-centre RCT in which patients will be allocated to two parallel groups. Vitrectomy and BP monitoring during the operation will be performed by experienced doctors. The protocol was developed in accordance with the Consolidated Standards of Reporting Trials guidelines , 13. TheThe RCT will enrol 180 participants who will be randomised in a 1:1 ratio to the intervention group  or control group . The patient’s history of hypertension and the first random BP measurement at admission will be recorded at the screening visit for the trial. The BP 1 h before the operation will be taken as the baseline reading. In both groups, intraoperative BP will be recorded every 5 min from the start of operation, which refers to after anaesthesia. Adverse events during treatment and at 1, 4, 8, and 12 weeks post treatment will be recorded. Participants have the right to withdraw their consent and discontinue participation at any time.Participants will be recruited by the surgeon during hospitalisation without any other form of advertising. Interested patients will be invited to discuss with a cardiologist the importance of intraoperative BP stabilisation. Participants meeting all of the inclusion criteria will be fully informed of their responsibilities and all procedures involved in the trial and will be asked to sign the written informed consent before enrolment.The inclusion criteria are as follows: (1) ≥18 years old, with no history of operations; (2) a clear history of arterial hypertension, with well-controlled BP  in the last 6 months by oral administration of a single calcium channel blocker; (3) requiring vitrectomy under nerve block anaesthesia based on relevant examinations including slit lamp, fundus photography, ocular B ultrasound, optical coherence tomography, fundus fluorescein angiography, indocyanine green angiography combined with a clinical diagnosis of rhegmatogenous retinal detachment, epimacular membrane, or macular hole; (4) no history of diabetes or family history of diabetes, and preoperative HbA1c ≤6%; (5) disease is not self-limiting and there is no refractive medium opacity or pupil narrowing that affects the fundus examination; (6) no history of coronary heart disease, cerebrovascular disease, or drinking and smoking and coagulation function is normal; (7) except for calcium channel blockers to reduce BP, no drugs use under ordinary normal circumstances; (8) body weight between 60 and 75 kg on admission; and (9) provision of informed consent.Exclusion criteria are as follows: (1) the patient voluntarily withdraws from the study or does not sign the informed consent form; (2) the patient ordinarily takes diazepam or other psychotropic drugs; (3) history of ataxia and tremor; (4) history of rash or leukopaenia; (5) allergy to benzodiazepines; (6) abnormal liver or kidney function and unstable BP; (7) insomnia, depression, central nervous system depression, or other mental diseases; (8) myasthenia gravis, attention deficit hyperactivity disorder, or chronic obstructive pulmonary disease; (9) cardiovascular events such as stroke, cerebral ischemia, or myocardial infarction within 6 months before screening; (10) existing diseases or conditions of the target eye or whole body ; (11) the patient is not using effective contraceptive measures or is planning to become pregnant within 6 months, or is pregnant or lactating; (12) the researchers decide during the course of the study that it is unsuitable for the patient to continue participating; and (13) participation in other trials.The stratified randomisation method will be adopted in allocating participants to the two groups in a 1:1 ratio using SPSS v22.0 . The stratification factors are as follows: (1) the history of hypertension ≥ 5 years and (2) the history of hypertension < 5 years. Random allocation sequence made in advance will be created and sealed in sequentially numbered opaque envelopes, which can allow randomising one at a time. Another researcher will be responsible for group allocation. All researchers including the outcome assessors, statisticians, and data analysts will be blinded to group assignment, but those providing the intervention/control will be informed by necessity. Diazepam and placebo tablets will be packaged in capsules of the same colour and shape. Before the trial, researchers will be fully trained in the randomisation procedure and made aware of their individual responsibilities. The successful implementation and maintenance of the randomisation and blinding method will be validated.According to previous retrospective data generated by our study group, the effective rate of intraoperative BP control is 80% in the intervention group and 61% in the control group; accordingly, the proportion of patients with intraoperative SBP ≥160 mmHg is 20% in the intervention group and 39% in the control group. According to the 1999 World Health Organization-International Society of Hypertension guidelines for the management of hypertension , mild hyBoth groups will include patients with a history of hypertension and good BP control who have been followed up in the cardiology department for at least 6 months. The timeline of data collection is shown in Table Patients will be given oral diazepam 2.5 mg 60 min before the operation. Electrocardiogram (ECG) monitoring will be performed during the whole procedure and BP will be measured every 5 min during and 1 h after the operation in the same ward using the same equipment.Patients will be given an oral starch tablet 60 min before the operation. ECG monitoring and BP measurement will be performed as for the intervention group.Intraoperative BP and heart rate will be recorded every 5 min . Follow-up examinations will be performed at 1, 4, 8, and 12 weeks after surgery in both groups. Furthermore, the operation record of each enrolled patient, including the length of operation, will be recorded in detail.The primary outcome is the effective rate of intraoperative BP control. Effective BP control during vitrectomy is defined as SBP during operation maintained at <160 mmHg at all timepoints. The strengths of SBP as an outcome measure are its accessibility, non-invasiveness, and reliability.The following indicators will also be used to evaluate the effectiveness of diazepam in controlling intraoperative BP stability: (1) proportion of patients with SBP ≥180 mmHg at any timepoint from operation to 1 h post-operation; (2) The change of mean systolic blood pressure measured by patients during operation performed from baseline; (3) The change of mean heart rate measured during operation performed from baseline; and (4) the number of patients with intraoperative and post-operative adverse reactions within 12 weeks of surgery. Adverse reactions include ophthalmic adverse reactions  and systemic adverse reactions .Basic demographic information and data on the history of hypertension and ocular diseases will be collected pre-intervention. BP will be recorded before and during the operation, post intervention (1 week after vitrectomy), and at the 3-month follow-up.The Data and Safety Monitoring Board, an independent advisory group, will assure the scientific integrity and ethical standards of the RCT and will be responsible for data evaluation during the study period. Researchers will be trained to collect good quality data, promote participant retention, and complete follow-up. Data analysts will be trained on data entry, coding, security, and storage. Statisticians will provide training on data assessment and statistical analysis.Long-term diazepam use is associated with adverse events such as drowsiness, dizziness, weakness, memory loss, and constipation. However, to date, there are no clinical reports of adverse reactions caused by a single dose of oral diazepam. During the trial, we will record and analyse any diazepam-related adverse events (including the type and number) in each group and the possible causes. Patients will receive appropriate intervention for any adverse events that occur during the treatment and follow-up phases. Serious adverse events will be immediately reported to the primary investigator, and affected participants will receive emergency treatment.Neither the patients nor the public were involved in the design of the study, nor will they be involved in its conduct (including outcome measurements). Results excluding personal data will be made available on request.t test will be used. All statistical tests will be two-sided with P<0.05 considered statistically significant.SPSS v22.0 will be used for data processing and analysis. Categorical variables (e.g. sex and number of patients with SBP ≥160 mmHg during the operation) are expressed as numbers and frequencies. Continuous variables (e.g. age) were expressed as the mean ± standard deviation (SD). In order to adjust for baseline BP of patients and any other variables which could have a potential impact on the outcomes, the logistic regression model will be used to analyse the primary outcome. When analysing the proportion of patients with SBP ≥180 mmHg and the number of patients with intraoperative and post-operative adverse reactions, the logistic regression model also will be used. While when comparing the change of mean systolic blood pressure and mean heart rate during operation from baseline, the This article describes the design and protocol of a study that aims to evaluate the efficacy and safety of diazepam for intraoperative BP stabilisation in hypertensive patients under nerve block anaesthesia during surgery. The trial protocol has been approved by the Shanghai General Hospital research ethics board . All study participants will sign the written, informed consent form and will be fully informed of their responsibilities as well as the procedures in the trial. The patient consent form (Chinese version) is attached as Additional file This study is currently in the recruitment phase. The first patient was enrolled in August 2021, and the study is expected to end in August 2023 considering the coronavirus pandemic. The protocol version is 1.0, dated 25 November 2020.Additional file 1. SPIRIT checklist.Additional file 2. Patient consent form (Chinese version).Additional file 3. Patient consent form (English version).


Accurate and efficient forward models of photon migration in heterogeneous geometries are important for many applications of light in medicine because many biological tissues exhibit a layered structure of independent optical properties and thickness. However, closed form analytical solutions are not readily available for layered tissue-models, and often are modeled using computationally expensive numerical techniques or theoretical approximations that limit accuracy and real-time analysis. Here, we develop an open-source accurate, efficient, and stable numerical routine to solve the diffusion equation in the steady-state and time-domain for a layered cylinder tissue model with an arbitrary number of layers and specified thickness and optical coefficients. We show that the steady-state ( Accurate quantitation of these properties from experimental measurements depend on analytical models that need to account for the structural complexity of the tissue system. Therefore, it is important to consider the optical heterogeneity of biological tissues, which are usually approximated as optically homogeneous to facilitate data analysis4. Experimentally, light propagation measurements are made by illuminating the tissue surface with either a continuous, frequency modulated, or pulsed light source and collecting measurements of the scattered light after it has propagated through the tissue medium6. Measured optical signals are translated into absorption and scattering properties of the medium by utilizing an appropriate forward model of light transport that best represents the measured data8.Optical properties can be used as indicators of pathological and physiological conditions of biological tissue11. Due to the highly scattering nature of these media, the RTE can be reduced to the diffusion equation, which gives analytical solutions in homogeneous, semi-infinite, or infinite slab geometries13. The RTE can also be solved by the Monte Carlo method which remains the gold-standard approach to calculate light transport in media with complex geometries14 but is computationally expensive15. Although parallel implementations have significantly decreased the computational time of forward Monte Carlo simulations down to several seconds19, they still broadly remain non-viable as inverse solvers to obtain optical properties from experimental measurements in real-time (< 1 s) which require thousands of forward simulations at different modelling parameters18.Light propagation in random media such as biological tissues is theoretically modeled using the Radiative Transfer Equation (RTE)20. Although Monte Carlo methods can simulate light propagation in realistic head geometries derived from magnetic resonance imaging (MRI) data16, modeling the head as layered homogeneous slabs, each with their own set of optical properties, provided similar accuracy in reconstruction of optical properties3. Further, diffuse optical measurements are applicable to various parts of the body that exhibit a layered structure . Therefore, an accurate, versatile and efficient analytical approach to model spatially and/or temporally resolved diffuse reflectance in layered media would enhance optical property reconstructions from diffuse optical measurements obtained in such layered media in vivo3.Theoretical approaches that account for structural complexity in tissues provide improved reconstruction of optical properties using diffuse optical measurements when studying brain hemodynamics24, method of images25, eigenfunctions27, or finite differences28. These methods do not give closed-form expressions directly in the spatial or time-domains for the photon fluence. Instead, the fluence in real-space is computed using numerical transforms24 or root-finding techniques26 which tend to increase numerical errors and computational costs29. For example, the integral transform approach24 solves the diffusion equation in the spatial-frequency domain which then must be inverse space-transformed (e.g. 2-D inverse Fourier) for real-space calculations. An additional inverse time Fourier transform is required for computation in the time-domain24. Both of these transforms make calculations of the steady-state and time-domain solutions difficult to compute for a wide range of optical and geometrical inputs24. Other approaches have been developed to compute geometries with large layer thicknesses and high scattering coefficients and/or spatial frequencies but rely on approximations32. Given these challenges, the fastest reported computational times for time-domain fluence in multi-layered media range between 0.5–5 s, depending on the number of layers and numerical accuracy required33. Such computational performance would preclude direct use of such layered analytical solutions for real-time analysis as optimization of multiple parameters in layered media would take several minutes34.Several methods to solve the diffusion equation for layered media have been reported in literature by using integral transforms23. Our code is open-source and well documented for ease of use. We overcome the computational difficulties noted above by modifying the solutions23 in the spatial-frequency domain for numerical stability, which allows for computation of arbitrarily sized inputs without approximations. Lastly, we use an inverse Laplace transform for better convergence in the time-domain which improved the numerical accuracy while decreasing the computational cost by several orders of magnitude36. Below we describe: (a) implementation of the numerical solutions in the steady-state and time-domain for diffuse optical reflectance and transmittance measurements in N-layered media, (b) verification of the numerical accuracy and stability of the approach in calculating photon fluence for several source-detector configurations and tissue models, and (c) validation by direct comparisons to Monte Carlo simulations of fluence in multi-layered tissue models.In this report, we present an accurate and efficient procedure for computing the photon fluence in a layered cylinder using solutions to the diffusion equation3737. The fluence can be used to calculate the diffusely reflected or transmitted intensity which are the quantities usually experimentally measured13. We assume an incident beam can be approximated by an isotropic point source located at a distance of 37. For the special case of a point source incident onto the center top of the cylinder, the fluence in the 37 bym and n is the z is the detector depth within the medium in cylindrical coordinates such that L is the sum of all the layer thicknesses is used for transmission. The extrapolated boundary is determined with a is the radius of the cylinder and A is proportional to the fraction of photons that are internally reflected at the boundary12. 37 can lead to numerical overflow for large layer thicknesses, scattering coefficients, and frequencies. To overcome these issues, we give expressions for In highly scattering turbid media such as biological tissue, the steady-state diffusion equationentclass1pt{minima35. As these Fourier integrals are slow to converge and can rapidly oscillate, the number of nodes needed for Gaussian integration methods are highly dependent on input parameters which makes the Fourier integral difficult to accurately and efficiently compute35. Instead, an inverse Laplace transform with 36. Utilizing a contour that begins and ends in the left hand plane Re 36. The corresponding solution for the time-domain fluence is thenB denotes the Bromwich path with 36. The full derivation of the time-domain solution shown in Eq.  must bem in Eq. . The numm in Eq. , the accd in Eq. . To allo13. In these validations, each layer in our tissue-model was set to have the same optical properties as the homogeneous medium along with large lateral boundaries. This allows us to precisely quantify numerical errors and determine convergence of our solutions in terms of the number of terms retained in the sum in Eq.  and 16 GB of memory. Simulations in the steady-state utilized a single core while the inverse Laplace transform in the time-domain used multi-threaded parallelism. Here, the Laplace space evaluations were evenly distributed across the 4 cores and 8 threads of the M1 chip. All benchmarks are done in double precision arithmetic using v0.8.0 of LightPropagation.jl.All the numerical routines and figures presented here were developed using the Julia programming language (v1.7.0)16. In the limit of an infinitely large number of photons used in the simulations, the Monte Carlo method is an exact solution of the RTE15. We utilized an independent open-source Monte Carlo code provided by the Virtual Photonics Technology Initiative40 to validate the layered diffusion theory model. The optical properties used were taken from literature using three biologically relevant tissue models with an isotropic emitting source at a depth of g = 0.8 for all layers. The Monte Carlo simulations used t and/or z was recorded in discrete bin widths of To validate the derived analytical solutions, the fluence is compared with results obtained from Monte Carlo simulations. The Monte Carlo method simulates the propagation of photons through the scattering medium using appropriate probability functions and random number generation13 and to Eqs.  to approximate a semi-infinite medium. As previously shown13, diffusion theory exhibits excellent agreement with relative errors . Less than 20 Laplace evaluations were needed to give absolute errors 41. The main limitation is that the function must be evaluated at very small and large values along the contour which leads to floating point errors limiting the procedure to absolute errors approaching the machine precision. Additionally, the evaluation points along the contour depend on N which can not be reused for different values of N. Therefore, for the best computational performance N must be determined before the computation.In Fig. N. A larger N improved the overall accuracy and was relatively independent of the time point N. For larger contours N are needed to reconstruct the time-domain signal over the whole time window N. However, smaller values of N are not able to reconstruct accurately over the entire time window due to the lower fluence values N reconstructs the time-domain signal over the entire window at a relatively fixed absolute error. Therefore, larger relative errors will be observed at later times when the fluence is lowest.In Fig. N based on the dynamic range of time-domain signal required. For example, 12 Laplace evaluations were typically required to reconstruct the time-domain signal with a dynamic range of 3 orders of magnitude where 24 evaluations can provide roughly 6 orders of magnitude which represent typical dynamic ranges of time-domain systems42. Increasing the number of evaluations does not decrease absolute errors once the errors reach the machine precision. Coincidentally, we have found that the numerical inversion of the Laplace transform is also limited by absolute errors approaching the machine precision, similar to the numerical computation in the spatial domain.We note that calculations in Fig. 31. In Fig. 13. We used The previous examples have focused on modest values of entclass1pt{minimaIn Fig. entclass1pt{minima24, a 3-layer model representing a skin/fat/muscle layer32, and a 5-layer brain model43 representing a scalp, skull, cerebrospinal fluid (CSF), and a gray and white matter layer.Next, we compared the solutions obtained from Eqs.  and 3)  3) to Mot in the time-domain for The optical properties considered in the 2-layer model are mentclass2pt{minim45 that showed a breakdown in diffusion theory when the mean free path approaches the thickness of the top layer. Here, a top layer thickness of 1.2 mm was used. Although the significance of these errors were not studied on the reconstruction of optical properties, the relative errors between Monte Carlo solutions are less than 0.1 for 44. Our analytical solutions utilized 44, though the choice of 13.In Fig. entclass1pt{minimaz and simulating for more photons in the Monte Carlo method will reduce the noise, however, smaller discretization will not improve agreement between diffusion and Monte Carlo where they do not asymptotically agree.In the right column of Fig. n considered in the sum in Eq. (n is fixed), which is instead dependent on the number of roots n used in the sum. Though, increasing n does not linearly increase the computational time as shown in Table In Table entclass1pt{minimaentclass1pt{minimaN  over any root of entclass1pt{minimaentclass1pt{minimaentclass1pt{minimaentclass1pt{minimaentclass1pt{minima30. Here, we have used the inverse Laplace transform36 for faster and more accurate reconstructions of the time-domain signal. We have found that 12–24 terms in the Laplace integral are needed in Eq.  of terms are required in the computation of Eq.  when \doentclass1pt{minima43. We note that solutions to the RTE in layered media have been presented which are more accurate than the diffusion approximation but, like Monte Carlo methods, come at increased computational cost45. Additionally, the situations presented in Fig. 3. The primary disadvantages of diffusion theory are the inability to correctly predict photon fluence for short time scales and source-detector separations and the requirement that 11. A recent report also indicated that the diffusion approximation could increase inaccuracies far away from the source in layered models where layer thicknesses are small compared to the mean scattering length45. We found that for a top layer thickness of 1.2 mm, these predictions were in agreement with the results reported in Fig. Finally, in addition to testing the numerical accuracy and efficiency, we have tested the physical approximation of diffusion theory compared to the Monte Carlo method by using three previously reported layered tissue models that approximate several different organ systemsIn conclusion, we have developed and verified an open-source, easy-to-use numerical algorithm to accurately and efficiently compute solutions of the diffusion equation in layered media. The absolute errors of the routine can be made arbitrarily accurate and can simulate both the steady-state and time-domain fluence 3 to 4 orders of magnitude faster than previously reported. Therefore, the routine could be used in inverse procedures to recover optical properties of measured data in real-time (1–10 Hz). It can also be employed for rapid generation of the intensity profile in layered media at multiple spatial locations and varying optical properties, as required in tomography and functional imaging applications. These solutions are also easily amendable to solve the correlation diffusion equation in layered media. An additional advantage of the routine is that the computational time marginally increases with the addition of a new layer, as a 4-layered medium can be computed within 10% of the time to compute 2-layers. This could allow for more accurate simulations in highly layered media such as the brain at little cost to total run times. Additionally, we showed good agreement between diffusion theory and Monte Carlo simulations in three separate tissue geometries of clinical interest.Supplementary Information.