Colony stimulating factor (CSF)-1 is a growth factor that stimulates the survival, proliferation and differentiation of mononuclear phagocytes, which has been implicated in several inflammatory diseases. This study evaluated the possible influence of age, sex, smoking, periodontitis, caries, and several systemic conditions on salivary levels of CSF-1. Four-hundred and forty-one individuals were enrolled in this study. All participants answered a health questionnaire and underwent a comprehensive oral examination. Stimulated saliva was collected and CSF-1 levels were analysed by enzyme-linked immunosorbent assay. Salivary levels of CSF-1 were significantly increased in participants over 64 years old and in non-smoking individuals, whereas no difference was observed between men and women. Individuals having periodontitis and manifest caries had significantly higher levels of CSF-1. Participants with muscle and joint disease exhibited increased CSF-1 levels as compared to those without. Age, smoking, percentage of pockets ≥4 mm, number of manifest caries lesions, and presence of tumor were associated with CSF-1 levels. Salivary levels of CSF-1 are associated with age, smoking, periodontitis, manifest caries, and the presence of muscle and joint diseases and tumors. CSF-1 might be a promising biomarker candidate in saliva of both local and systemic conditions that needs further investigation. It contains several biomolecules, such as DNA, RNA, proteins, metabolites, and microbiota, which can be applied to the early detection, risk assessment, diagnosis, prognosis, and monitoring of several oral and systemic infectious and immune diseases2. Approximately 27% of the proteins found in saliva are also found in blood3.The search for biomarkers that can accurately reflect and monitor disease states is a goal of molecular diagnostics. In this regard, saliva is emerging as a desirable body fluid that is simple, noninvasive, and inexpensive to collect. Saliva is an oral fluid whose protein composition derives mainly from salivary acinar cells, with contributions from epithelial cells shed from mucosa, blood content and tissue fluid from gingivae, and oral microorganisms8, such as higher interleukin-1β (IL-1β) levels in patients with periodontitis, and inflammatory bowel disease9. However, most biomarker candidates so far are not solely expressed in diseased tissues, and their levels are affected by genetic, physical constitution, lifestyle and medication10. In fact, genetic, clinical and lifestyle factors have been shown to influence the levels of plasma biomarkers, in which as much as 56% of the biomarker variance can be attributed to non-disease factors10. To the best of our knowledge, no such comprehensive evaluation has been done in saliva so far. Therefore, the understanding of factors influencing immune response is necessary to comprehend inter-individual variation and its consequences on health and disease11.Indeed, several studies have pointed out the utility of saliva to evaluate biomarkers associated with both oral and systemic diseases12. CSF-1 has been implicated in inflammatory diseases, such as inflammatory bowel disease13 and rheumatoid arthritis14, and cancer15. CSF-1 is also implicated in periodontal disease, as highlighted by reduced alveolar bone loss when blocking the CSF-1 receptor16. In circulation, CSF-1 levels are associated with age in healthy individuals17, gender18, and are also influenced by weight10. Circulating CSF-1 levels are proposed to be a useful biomarker for lupus nephritis, as an increase in its levels predicted recurrences of nephritis before glomerular dysfunction19. CSF-1 levels might also be a biomarker for rheumatoid arthritis20, breast cancer21 and unstable angina22. We recently identified the presence of CSF-1 in saliva as a potential biomarker candidate of periodontal disease with increased levels in periodontitis compared to healthy subjects, and correlations to clinical parameters of periodontal disease severity23. To date, the possible impact of non-disease related factors such as age, sex, and smoking on salivary CSF-1 levels are unknown. The potential association between salivary levels of CSF-1 and other oral conditions including caries, and to systemic conditions has neither been explored.Colony stimulating factor-1 (CSF-1), also known as macrophage colony-stimulating factor-1 (M-CSF), is a pleiotropic growth factor that stimulates the survival, proliferation and differentiation of mononuclear phagocytesTherefore, we hypothesized that CSF-1 levels in saliva would be affected by disease- and non-disease-related covariates, which imply considering them when trying to establish cutoffs for disease diagnostics and/or monitoring. With that in mind, this study aimed to evaluate the possible influence of age, sex, smoking, periodontitis, caries, and systemic conditions on salivary levels of CSF-1, as well as to assess a normal reference range for CSF-1 in saliva.This study analysed the influence of several disease- and non-disease-related covariates on salivary levels of CSF-1. Demographics of the cohort of 441 participants are presented in Fig. 17. Therefore, we compared the salivary levels of CSF-1 in three age categories . Mean (±SD) levels of CSF-1 were 793.0 (±511.8), 848.5 (±636.8) and 1185.0 (±868.9) pg/ml for participants <40 years old, 40–64 years old, and >64 years old respectively. Participants over 64 years (n = 90) old showed significantly higher levels of CSF-1 in comparison to participants of both the age group below 40 years (n = 150) and between 40–64 years (n = 199) old  CSF-1 levels of 873.7 (±677.6) pg/ml and males of 927.3 (±661.5) pg/ml. There was no significant difference in CSF-1 levels between female (n = 221) and male (n = 218) participants, both uncorrected and corrected for total amount of protein Fig. . Regardi23, we next investigated the relation between CSF-1 levels and periodontitis in this large cohort. Participants without periodontitis (n = 74) exhibited mean (±SD) CSF-1 levels of 802.1 (±532.9) pg/ml, while participants with periodontitis (n = 220) showed mean (±SD) levels of 973.3 (±725.2) pg/ml. Salivary CSF-1 levels were significantly increased in periodontitis compared to without  levels of CSF-1 were, respectively, 852.2 (±626.2), 945.0 (±641.5) and 1158.2 (±964.5) pg/ml for participants with no MCL (n = 299), MCL 1–2 (n = 102) and MCL ≥3 (n = 38). Salivary CSF-1 levels were significantly higher in participants with MCL ≥3 as compared to MCL 0, whereas no difference was found between MCL 1–2 and MCL 0 or between MCL 1–2 and MCL ≥3 Fig. . The dif5 and found significant correlations to the levels of IL-1β , IL-8 , and MMP-8 .We also assessed correlations between the levels of CSF-1 and other inflammatory molecules previously analyzed in saliva of this cohortTo address the possible relation between CSF-1 levels in saliva and systemic diseases we analysed CSF-1 levels with respect to several systemic conditions. Individuals having muscle and joint diseases (n = 96) showed increased levels of CSF-1 as compared to patients not having the disease (n = 335), but the difference was lost after CSF-1 correction for total amount of protein. Participants who reported tumors (n = 16) showed a trend towards increased levels of CSF-1, however this did not reach statistical significance. There was no significant difference in mean levels of CSF-1 between participants having heart disease, hypertension, bowel disease, mental illness and diabetes and participants not having the disease  CSF-1 was 763.95 (±415.55) pg/ml, ranging from 224.55 to 2218.00 pg/ml. The 95% reference range was 234.42–1862.08 pg/ml. For the normalized values, mean (±SD) was 1083.72 (±459.70) pg/mg, ranging from 405.53 to 2174.98 pg/mg. The 95% reference range was 426.57–2187.76 pg/mg. Worth noting, CSF-1 levels were detected in 100% of the saliva samples of the whole cohort (n = 439).Seeking to identify the determinants of CSF-1 levels in saliva, results from a linear regression analysis found that age, presence of tumors, percentage of PD ≥4 mm, and number of MCL were significantly associated with CSF-1 levels. Overall, these 4 variables accounted for 32.8% of the variance in CSF-1 levels  in women, but not in men30, this might account for the controversies found in the literature regarding its levels. Contrary to sex, smoking exerted a significant role in CSF-1 levels in saliva. Being a smoker was associated with an average decrease of 166.20 pg/ml in CSF-1 levels. Smoking has been related to decreased levels of other inflammatory markers in saliva such as IL-8 and MMP8/TIMP1 ratio5. Liede et al. reported lower salivary MMP-8 levels in current smokers34. Decreased flow of gingival crevicular fluid in smokers, which would lead to a lower influx of inflammatory mediators into saliva, might be one possible reason for this decrease35. Lower levels of CSF-1 in serum have been found in smoking patients with long bone fractures32, whereas others found no significant effect of smoking on plasmatic CSF-110. Together, our data indicates the need of taking age and smoking into account when relating salivary CSF-1 levels to diseases.In our study, there was no significant association between sex and CSF-1 levels in saliva. In line with our finding, no significant effect of sex has been reported on blood levels of CSF-123. Further, CSF-1 levels correlated significantly with the levels of IL-1β, IL-8, and MMP-8, which were previously analysed in participants from this cohort5. This is also in line with our previous finding of a significant correlation between the levels of CSF-1 and MMP-8 in saliva23. Blocking of the CSF-1 receptor has been shown to reduce alveolar bone loss and number of osteoclasts in experimental periodontitis in mice16. Further, in experimental arthritis in mice injections of CSF-1 have been shown to exacerbate disease whereas CSF-1 blocking alleviates disease severity14. Along with the fact that CSF-1 stimulates RANKL-induced osteoclast formation36, increased salivary levels of CSF-1 might be reflective of higher osteoclastogenic potential in periodontitis.We reported significantly higher levels of CSF-1 in participants diagnosed with periodontitis, even when restricted to patients not having manifest caries. We also found significant correlations to clinical periodontal parameters. These results confirm, in a larger group, recent findings of increased CSF-1 levels in saliva from periodontitis patients by our group37. However, when we restricted the analysis to participants not having periodontitis, there was only a trend towards higher CSF-1 levels in subjects with manifest caries , which suggest that the effects of caries on CSF-1 levels might partially be due to their periodontal condition. Dental pulp fibroblasts from dentinal caries sections immunostained for CSF-1 in about 38% of the teeth, while no immunostaining was observed in sections from non-carious teeth. Moreover, TNF-α-stimulate pulp fibroblasts produced CSF-138. We can speculate that, at least partially, CSF-1 might leak out from the carious dentin to saliva.Participants with high level of caries (MCL ≥3) exhibited higher levels of CSF-1 in saliva. Further, we found a significant correlation between salivary CSF-1 and caries risk. An association between MCL and MMP-8 has previously been reported in this cohort7. Our study found increased salivary levels of CSF-1 in participants having muscle and joint diseases, which is in line with findings of higher CSF-1 in serum and urine from patients with systemic lupus erythematosus19. Increased expression of CSF-1 in plasma has also been reported in active as compared to quiescent rheumatoid arthritis20. Our study also showed that the presence of tumors was associated with CSF-1 levels. Elevated serum levels of CSF-1 have been found in patients with breast cancer, and even higher in patients with invasive cancer and concomitant lymph node metastasis21. Increased serum levels have also been reported in patients with colorectal cancer39. Our result thus suggests that assessment of CSF-1 levels in saliva might be valuable for monitoring patients with muscle and joint diseases and tumors however, further investigations are needed.Inflammatory molecules in saliva have been investigated in relation to systemic diseases and their utility as biomarkers have been demonstrated26, thus we speculate that genetic factors might also play a role in the variation of salivary CSF-1. As saliva is a complex biologic fluid, attention to its collection method is mandatory, which can have an impact on the concentration of proteins. Generally, levels of inflammatory biomarkers are higher in unstimulated than stimulated whole saliva40. However, we have shown no significant difference in CSF-1 levels between unstimulated and stimulated saliva, either fasting or non-fasting23. We also reported that the significant differences in CSF-1 levels were lost for smoking, periodontitis, caries, and muscle and joint diseases when correcting for total amount of protein. This might be explained by the higher amount of total protein in these groups of patients, which could be a consequence of increased plasma protein leakage during the inflammatory process. Higher protein concentration in periodontitis patients has been shown, which could not be attributed to increased salivary flow rate42. On the other hand, decreased concentration of total protein has been reported in smokers43.The main limitation of this study is its cross-sectional nature and, thus, no causal claim can be done. Prospective studies would be of great value to confirm hypotheses generated in this study, as well as the addition of gingival crevicular fluid. Besides, medical conditions were determined by self-report and, then the diseases were grouped together in broader categories. Therefore, we cannot rule out that particularities from each disease might influence differently on CSF-1 levels, as well as medications used to treat them and the disease status during saliva collection, such as active/quiescent and controlled/uncontrolled. We also cannot rule out that other covariates might play a role in determining salivary levels of CSF-1. Indeed, an impact of genetic factors has been reported on the variation of plasmatic CSF-1 levelsIn conclusion, this study found that age, smoking, periodontitis, manifest caries, and the presence of tumors are associated with the salivary levels of CSF-1. Furthermore, these results showed increased CSF-1 levels in patients reporting muscle and joint diseases.44.This cross-sectional study enrolled 441 individuals  living in Skåne, Sweden, who were orally examined and provided a saliva sample. Study protocol was approved by the Research Ethics Committee at the Lund University, Sweden, and all participants gave their written informed consent. All methods were performed in accordance with the relevant guidelines and regulations. Participants were asked to answer a questionnaire regarding oral health, and data concerning age, sex, smoking habits (including use of Swedish snus), presence of diseases, and use of medication were collected. Smoking was recording as yes or no. Diseases were self-reported and grouped as heart disease, hypertension, bowel disease, muscle and joint disease, mental illness, tumor, and diabetes. Details of the sample selection and the questionnaire were described previously90.5% of the clinical examinations were performed by four dentists employed at the Department of Oral Diagnostics, Faculty of Odontology, Malmö University. Periodontal parameters included visible plaque index (PI), bleeding on probing (BOP) and probing depth (PD), all recorded in four sites on each tooth using a periodontal probe . In addition, digital bitewings and panoramic radiographs were taken for evaluation of alveolar bone loss. Periodontitis was defined as the presence of PD ≥4 mm and BOP >20%. Participants having no PD ≥4 mm and bone loss not exceeding 1/3 of the root length were selected as controls.37. Caries risk was evaluated using the Cariogram, showing the %-chance of avoiding caries45.Cariological parameters included DMFT , number of manifest caries lesions (MCL), which encompassed lesions involving dentin, as seen on bitewing radiographs and cavitated lesions on other surfaces, and caries risk. Number of MCL was grouped as previously described: MCL 0: no MCL, MCL 1–2: 1 or 2 MCLs and MCL ≥3: 3 or more MCLsStimulated saliva was collected during 5 minutes chewing 0.5 g of paraffin in a graded tube. Secretion rate (ml/min) was determined after excluding the foam. Samples were immediately frozen at −20 °C until processing. Saliva samples were then centrifuged and supernatants aliquoted and stored at −80 °C. Each aliquot was used only once for biomarker analysis. For the analysis of CSF-1, 439 samples were included.Levels of CSF-1 in saliva were determined using a commercial enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions . Samples were thawed on ice and centrifuged at 10000 rpm for 5 min prior to analysis. The detection limit of the assay was 78.1 pg/ml and the sensitivity 11.2 pg/ml. Readings were made at 450 nm with wavelength correction set to 540 nm to subtract background using a microplate spectrophotometer . Total amount of protein was determined by the Bradford assay to enable normalization of CSF-1 levels to total salivary protein.P ≤ 0.05.Data analyses were performed using Statistical Package for Social Sciences (SPSS) version 20 . Continuous variables are presented as mean and standard deviation (SD) and categorical variables as frequencies. Group comparisons were performed with Student’s t-test or ANOVA with Bonferroni post-test. Correlations between CSF-1 levels and other variables were determined by Pearson correlation coefficient. A multiple linear regression was done with CSF-1 as dependent variable and age, sex, smoking, bleeding on probing, percentage of PD ≥4 mm, number of MCL, and the systemic conditions as independent variables. Statistical significance was set at In order to establish a reference range for CSF-1 in saliva, a 95% reference range was calculated as mean ± (1.96 × SD). For that purpose, CSF-1 values were log-transformed in order to achieve a normal distribution, and then the anti-log was calculated to exhibit the reference range in pg/ml.Supplementary  Information


Despite extensive publication of clinical guidelines on how to manage musculoskeletal pain and back pain in particular, these efforts have not significantly translated into decreases in work disability due to musculoskeletal pain. Previous studies have indicated a potential for better outcomes by formalized, early referral to allied healthcare providers familiar with occupational health issues. Instances where allied healthcare providers of comparable professional characteristics, but with differing practice parameters, can highlight important social and organisational strategies useful for informing policy and practice. Currently, Norwegian chiropractors have legislated sickness certification rights, whereas their Danish and Swedish counterparts do not. Against the backdrop of legislative variation, we described, compared and contrasted the views and experiences of Scandinavian chiropractors engaging in work disability prevention and sickness absence management.This study was embedded in a two-phased, sequential exploratory mixed-methods design. In a comparative qualitative case study design, we explored the experience of chiropractors regarding sickness absence management drawn from face-to-face, semi-structured interviews. We subsequently coded and thematically restructured their experiences and perceptions.Twelve interviews were conducted. Thematically, chiropractors’ capacity to support patients in sickness absence management revolved around four key issues: issues of legislation and politics; the rationale for being a sickness absence management partner; whether an integrated sickness absence management pathway existed/could be created; and finally, the barriers to service provision for sickness absence management.Allied health providers, in this instance chiropractors, with patient management expertise can fulfil a key role in sickness absence management and by extension work disability prevention when these practices are legislatively supported. In cases where these practices occur informally, however, practitioners face systemic-related issues and professional self-image challenges that tend to hamper them in fulfilling a more integrated role as providers of work disability prevention practices. Musculoskeletal pain is a major cause of work disability with enormous socioeconomic consequences. Back pain-related disorders alone are costly and responsible for up to one quarter of days off work in European countries like Sweden  and DenmFor patients with musculoskeletal pain or other work-related problems, general practitioners (GPs) are the traditional gatekeepers to workers’ compensation through sickness certification in the majority of European countries, but studies from the UK and Scandinavia have indicated that GPs question the relevance of work-related issues to their primary healthcare provider role –8. RestrThe use of allied healthcare providers (AHP), like physiotherapists, chiropractors and manual therapists, within the field of musculoskeletal pain is gaining popularity amongst patients, especially in the working population . AHP areAs the population ages and the current health reforms focus on shifting secondary care services into the community, demands on GPs and primary healthcare continue to rise , 19. DesOccupational groups operating within similar social contexts, but with varied legislated practice parameters, provide an opportunity to observe the impact of systematic variation . More spconcerned with the diagnosis, treatment and prevention of mechanical disorders of the musculoskeletal system. Members of the respective national chiropractic associations hold a 4- or 5-years Master’s degree in musculoskeletal health, which, when followed by a 1-year internship, qualifies for the respective national board of health certifications as independent healthcare providers. In all three Scandinavian countries, chiropractors function as first points of contact for patients with musculoskeletal disorders, but under different regulations and levels of integration in the welfare systems. In Sweden, chiropractors are largely private musculoskeletal practitioners outside the national health service with limited integration into the national healthcare system, while in Denmark and Norway, the chiropractors work inside the respective national health services as AHPs.Chiropractic is a growing musculoskeletal health profession in Norway, Denmark, and Sweden. It is In Norway, chiropractors and manual therapists (i.e. physiotherapists with a degree at the masters level) received authorization to issue sickness certifications for 0–8 weeks in 2006, and since 2008, for 0–12 weeks . HoweverIn the context of changing healthcare policies and organisational structures, there is an increasing need for evaluations of the impact of role extensions, and the potential barriers and facilitators for implementation of such a change.Describe the experiences of chiropractors engaging in sickness absence management (SAM).Compare and contrast chiropractors’ integration of SAM in their model of care in a context with legislated sickness certification rights (Norway) and in two contexts without sickness certification rights (Sweden and Denmark).The objectives of this study were to:This study formed part of a two-phased sequential, exploratory mixed-methods design (the results of the quantitative phase will be reported separately) .Using a postpositivistic lens, a comparative qualitative case study  was concDuring the period of June 2015 to March 2016, we purposively sampled chiropractors across the three countries with experiences regarding SAM who were willing to share these . SpecifiAn interview guide with prompts was developed where the team drew on their experience of work disability programs as researchers and as clinicians, and on recent research on the topic. Face-to-face, semi-structured interviews were conducted where the chiropractors were asked to draw on examples of cases from their own experience . A rolliAt the commencement of each interview, demographic data  were collected. Interviews were conducted in the native language of the participants by one or two out of three interviewers from the research team at locations convenient for the participants. Each interview was audio-recorded and transcribed verbatim by the research team into computer-readable text files. During the interview phase of the project, the researchers documented their reflections about the interviews in a journal. These included notes about informal conversations prior to or after the interviews, as well as other information not captured in an audio transcript.The interviews were analysed in the language of the participants. The three Scandinavian languages have similar roots and are understood across the three countries, which enabled coding and interpretation of the transcripts in the original language. Further, the team consisted of three bilingual  team members and one trilingual (fluent in Danish/Norwegian/Swedish) team member. Quotes were translated into English by the team members in combination in a way that transferred content equivalence in translation while maintaining semantic equivalence . The resTwo members of the research team (OKL and CGN) independently coded the transcripts using content analysis before discussing the codes and categories with an experienced qualitative researcher (CM) who provided peer consultation about the qualitative data analysis process . A qualiIn instances where new topics emerged, follow-up questions were e-mailed to the previous participants who were asked to state their experience or perception regarding the newly emerged topics. This data was then incorporated into the data analysis to ensure data saturation  .In Sweden, the regional ethics committee evaluated the project and found that the study did not need ethical permission (advisory statement 2016/3:1). In Denmark, the Regional ethics of Southern Denmark gave approval for the study and declared that the study does not fall within the scope of the Medical Research Involving Human Subject Act (§14). Approval for data handling and storage covering both Denmark and Norway under the European Economic Area collaboration was granted from the Danish Data Protection Agency. Prior to the interviews, written and oral information about the study were provided to the participants. Written informed consent was obtained from all participants. All participants were advised that conversations were to be audio or video recorded, and assured of confidentiality and anonymity in reporting of the results.Twelve chiropractors participated in the study with interviews lasting between 12 and 65 min with a mean of 44 min. The descriptive participant characteristics of our sample are presented in Table Four themes emerged from our analysis: Legislation and politics shape sick leave practice, a rationale for the chiropractor as sickness absence manager, an integrated sick leave management pathway and the emergence of the chiropractor as a sick leave manager.When we talk about long-term sick leave, the GP is perhaps a good basis, also because they, they kind of have the whole package [of knowledge]. (DK4-16)When it’s related to patients within our area of expertise, then it is us who are in control, it is us who know about the course of treatment and follows the patient closely, and it is us who have the main competencies. (DK2-10)Norwegian and Danish chiropractors appeared mindful of maintaining the traditional gatekeeper role of GPs, as they consistently recognized the importance of informing the GPs regarding sick leave issues. There were, however, a variety of opinions about which patients or situations were best coordinated by the GP and which should be supervised by the chiropractor.This related mainly to situations where co-morbidity, such as mental disorder had been identified as well as long duration sickness absences.I write why the patient is here and what has been done and what would be good for the patient in the future. So, in that way, you can say it is a grey area. I do not write a sick leave certification, but they can get a document, which says I [the patient] have actually seen a chiropractor with this problem. And it [the problem] has to be rectified or I need help with this. (S4-6)Danish chiropractors, the group that occupies the legislative “middle ground”, expressed a degree of uncertainty about where the responsibility lay for SAM. In particular, the group’s perception was that current Danish legislation left most of the responsibility to the patient, but that patients were not always aware of the system. They also frequently received requests for assistance from not only patients, but also their place of work with respect to job modification, but they felt unsure whether this type of activity lay within their scope of practice. This issue is illustrated by the following passage from DK4’s interview:… a lot of citizens are perhaps downright uncertain about, how is it…who can certify sick leave and who will do it. It’s a bit of a grey area, where people are perhaps a little unsure about how the system works, and maybe it’s also an area, where we as chiropractors are a little afraid to open up in that respect. (DK4-1)By contrast, efforts to contribute to SAM in the Swedish context are characterized by uncertainty.Informal roles are furthermore observable in the Danish context as the patient functions as a messenger and the chiropractor takes on the role of arbitrator, balancing agreeing with the patient about sick leave and the need to “push” the patient back to work to prevent unnecessary absence.We are private, it is an expense for the patient, and there are many who do not consult us because of that. … they rather go to the public healthcare, and then they get the sick certification that way. (S2-4)In all three contexts, fee-for-service significantly influences SAM:This filter results in a situation where chiropractors tend not to see patients with a low socioeconomic status, who in turn, are perceived by the chiropractors to be more complicated to manage.But it’s the financial part, because you do not get a dime, and it’s actually quite time consuming…and having to write an employer, it takes a long time. You could easily see one, two, three patients instead. (DK2-4)As a consequence, the incentive to engage in complicated SAM was considered low. This was particularly observed in the Danish context:… then they [the patients] appreciate it [the chiropractors’ sickness certification rights], they also see as a quality stamp that we can do it. (NO2-3)For the Norwegian and Swedish chiropractors, sick leave certification rights were perceived as a “seal of approval” of the profession. The rights were perceived as a way of becoming fully integrated into the healthcare system, and a way of changing the scope of practice from  therapy to being recognized for diagnostic and management skills too.… if you want a card to play, which makes chiropractors someone to trust, someone who is known, and who delivers measurable results… (S3-5)andMirroring the legal status in the two countries, the Norwegians saw the sick leave certification rights as a final approval, the Swedish struggled to gain recognition and mentioned sick leave certification rights as a means to become integrated into the national healthcare system.Generally speaking, Danish and Norwegian chiropractors perceived themselves as musculoskeletal specialists due to their university degree and as such, as competent as SAM partners for patients with musculoskeletal problems. They felt comfortable in assessing musculoskeletal function and saw the assessments as integral in routine practice.I think we have a good basis for doing the evaluation, because we actually know the patients well. And we do it [assessments] already at the first visit. (DK3–18).If you look at long term sick leave and the level of workability and so on, chiropractors are not a group who are used for those kinds of assessments…. No one is educated in that system, but we refer to the GP then. (S1-4)However, despite the same level of training, the Swedish chiropractors expressed more hesitation regarding their competencies. According to a Swedish respondent:You can continue one week, maybe two weeks, and then have the dialogue with the patient all the time, during treatments too. So, like that you have more options than when you say “I’ll put you off work for three weeks. See you.” And that’s when they return [the patients]. Instead, you have a continuous dialogue about progress. (NO2-4)Across all three contexts, practitioners cited frequent patient contact as a facilitating element in SAM. They explained how this allowed them to get to know the patients through continuous dialogue and to establish trust. As a Norwegian participant explains, routine contact was also thought to provide the opportunity to optimally monitor progression and adapt plans accordingly:… a disadvantage or a limitation [of the present system], is for the patient, they have to first seek our help and then they have to go further to seek help at the GP who can give them a sick leave certification, if they assess that as necessary. So, it is both the cost, that they meet two health professionals, when it could be enough with one. (S2-7)Being able to both handle the patients’ musculoskeletal complaint and certify sickness absence was also perceived by chiropractors from all three countries as a means to prevent chronicity. Across contexts our participants argued that the risk of chronicity was a weakness of the current sick leave management system. By adding a chiropractor, the risk would reduce by shortening the chain of management. As S2 explains:It’s really an area that is important also socio-economically, and that’s why I think, at one point, we need to step up to the plate as a profession and give it the prestige that it really has, and say “listen, this means something with respect to us saving tax money.” So that Denmark becomes a cheaper country to live in, rather than saying “we focus on our own game, our own everyday life”, but takes on, perhaps something more like a societal supportive function. (DK1-37)Despite some reluctance to engage in SAM due to lack of financial incentive, Danish chiropractors expressed a sense of obligation toward society, and expressed a moral dilemma between service for the greater good or their own pocket. According to DK1:Well, it is different, right? An office worker, it might be an arm, shoulder or the neck. If the work regards heavy loads, it might be the lower back, for example. (NO3-19)And then, of course, there are the ones where stress management and all thoughts of things, where it is psychological and the work environment, or a lot of other stuff... (DK4-5)Norwegian and Danish chiropractors described a spectrum of sick leave-related conditions, ranging from uncomplicated musculoskeletal conditions to more complex cases with various degrees of psychosocial and work-related factors, whereas, the Swedish chiropractors predominantly referred to patients with few apparent psychosocial problems.Well, the patients visiting me who are in need of sick leave are very, very few…most of the patients we see either have had chronic pain for a long time, but are still working,.., or come in with more acute conditions and get symptom free quite fast. (S2–1).And then there are situations where you, ehm, again, you have to look at the whole situation. Are there any other factors in play, their employer, their workability, other than just the musculoskeletal? What is the big picture, and so on? (DK3-17)Further, the Danish and Norwegian chiropractors more often described multifaceted patient-centred and work-related action plans,whereas, the Swedish chiropractors described a focus on manual therapy as their primary tool.So really, I look at the function of people, even though they have a lot of pain, but I always ask them “Is it worse when you go to work?”…but it starts to hurt after 2 PM [mimics the patient], well, then perhaps you should ask about a shorter working day, right? Or ask if is possible for you to lay down somewhere to rest, like every other hour. Do you have an adjustable table, can you stand and sit, like. So, it’s dependent on what option people have for adjustments. (DK2-20)The chiropractors had a general approach to the patients, where emphasis was laid on functional disabilities rather than diagnoses or pain location.But if it is a person with a more active profession, which does not include heavy lifting and where it is optional to sit, stand, walk etc., then I recommend them to go to work, because if you stay home, you will become inactive and then you have problems for a longer period compared to if you stay active. (S2-2)Not only did the chiropractors discuss return-to-work, but often mentioned the importance of the patient staying at work despite some degree of pain or disability.Both Norwegian and Danish chiropractors described how early and timely communication between stakeholders was key to successful return-to-work.Then I write to the GP and state “this is my opinion” so the GP is informed, so at least there is a common ground, because if we start to say something different, then all of the sudden it becomes difficult. (DK2–18).They never write anything. (DK2-34)The standard method of communication with the GPs was via the official electronic platforms, but many felt that the communication was unidirectional, and requested more information going from the GP to the chiropractors.…as the GP, or as everybody is listed in various places is impossible to have communication with everyone. (S2-10)This is contrasted by the Swedish chiropractors, which for the most part did not have access to electronic communication platforms, and did not communicate directly with GPs.But, perhaps, I would like more collaboration with NAV [social services]. That there was a closer dialogue basically. (NO4-12)The Norwegians also described a unidirectional flow of information to the social services, whereas the Danish had very little contact or communication with the social services.It’s my consistent, positive experience with sickness certification,…. and if I was to move to another country without these rights, I would feel a little helpless and naked, I think. It would take some getting used to because it is such an important tool, because it is so closely linked to the result of what you do and the process, and how you plan a treatment course. It’s not like we’re just therapists anymore, but there is also the advice part, right? (NO3-17)A noticeable difference between the countries was that the Norwegians positively described how SAM was integral to practice. They used SAM as an integral tool and natural part of the care package that they offered their patients. They described SAM as an additional tool in the clinical tool box that they used if they felt it was relevant, and, therefore, as an add-on to clinical practice. They perceived this extra tool as essential to clinical practice, but also an instrument that lead to higher levels of involvement in their patients’ care, which in turn was perceived as challenging and personally and professionally rewarding.Not if you ask the chiropractors, because the paperwork takes up too much. And that’s where you don’t want to spend your time. It’s, it’s the bureaucratic hassle. (DK3-8)This was in contrast to most of the Danish and Swedish chiropractors, who were more hesitant about engaging in SAM. The Danes generally described SAM as a tedious process that they were somewhat reluctant to engage in.Functioning in the role of a sick leave manager is, as the previous themes indicate, influenced by the individual practitioner, the profession’s state of practice and the social systems that facilitate day-to-day implementation. Our final theme then, chronicles the experiences of chiropractors across the three case settings where these variables are present or absent.Danish chiropractors expressed some degree of ambivalence regarding embracing the SAM role. It was on one hand perceived as a natural progression of the profession, a responsibility to be taken for the greater good, as an obligation or to honour six years of free university schooling, but on the other hand as bothersome and with no financial incentive.And that [the chiropractors’ role] will slowly change, and that’s why I think there’ll be a big difference between clinics along the way. How many are willing to venture into this, and how many can’t be bothered, and there will be some who can’t be bothered…. We also know there are some [chiropractors] who won’t see chronic patients. And you can’t say that when you have six years of university schooling. Well, you just can’t. (DK2-26)For the Swedes, the lack of systematic channels of communication was an important barrier for communication and involvement.It is a waste of social resources in some way. That the patient all the time has to contact another authority to….Mostly we tell the patients they should contact the GP and then they make the call themselves. We don’t have direct communication with the GP. (S4-5)S4–11: … [SL rights] would be a good commercial for us. Yes, that would initiate an automatic dialogue.Interviewer: A dialogue with?S4–11: Other healthcare professions.Further, the Swedish chiropractors, perceived sickness certification rights as a direct platform for increasing the general communication with GPs and other healthcare professionals:Well, in respect to the Jobcenter [social services], it’s my experience that if you get hold of a specific case worker there on a specific case, then you can make it work, but otherwise it’s often, I think, that the Jobcenter has its own agenda, at least in the area where I work, where I get most of my patients from, they have an agenda, which is fastest return to work. (DK1-13)Danish chiropractors had reservations regarding engaging in SAM because of the administrative burden associated with SAM. The task was considered time consuming and bureaucratic, and lacking the necessary administrative support systems. Especially regarding the collaboration with the social services, which was considered non-existent, unsatisfactory or even adversarial. The chiropractors also reported how they perceived the case managers to have a specific agenda relating to cutting costs to a minimum.Then some jolly caseworker emails me and asks about things in relation to the [confidential] social security number and this and that in an unencrypted email. Then sometimes I write them to please don’t include full social security numbers in an open email. And with respect to the actual case, it would be better if we communicated via Status  or similar. (DK1-40)The Danish chiropractors alluded to the notion that to avoid paying for the chiropractors’ services, the social services case managers did not stick to procedure protocols.A salient issue in all three countries is the transition in self-image and adaptation in thinking and behaviour from chiropractors being manual therapists or even alternative care providers to becoming fully integrated members in the primary healthcare sector, which includes more responsibility in terms of communication, collaboration and patient management. It was quite clearly addressed by the chiropractors that they did not see the profession as united in this issue (difference of opinions), and they did not think that all colleagues delivered the same standard of care. It was clear how the Norwegians, to a larger degree, talked about embracing the role, whereas the Danes were more ambivalent, and the Swedes more hesitant.It’s still not our second nature to be part of the team now. But we’re still a little like a lonesome cowboy who’s been given a responsibility …but I know of colleagues who thinks of certification rights as a hassle. They would rather treat, examine and treat and continue their workday. But that’s why I think it’s more and more important that you embrace the role as a primary care provider. (NO2-40)You have to think about how you can make it work, what it is you accept. How do you make it work? That’s the hardest part. And then make sure you have 100% support before you start. Make sure everybody is onboard. Also those who don’t give a damn. (DK3-30)That is how it is. Then, well, I don’t think the limitations are disinterest, I just think we have not been ready for it. (S3-8)andI think it is missing, in some way, enlightenment about who we are, and where we come from and respect for the education, we actually have. That kind of enlightenment… (S4-8)I still have patients who doesn’t know that I prescribe sick leave. (NO1-35)Public knowledge of chiropractors and their scope of practice were frequently mentioned as barriers in Norway and Sweden.And in reality that’s where….if you want it to work, then you would need those round table discussions, right?....I don’t know how often they are held, but I don’t think it’s often, and again because it’s so damn expensive. (DK2-8)In Denmark, it was also mentioned that the political climate ran counter to changing the system, and that there wasn’t a willingness to provide the necessary funding for collaboration between stakeholders and for holding dialogue meetings.The final barriers, mentioned by most of the Danish chiropractors, were the patient fee and patients’ expectations of manual treatment.…I know that, at least some [patients] think whether they can afford the consultations or find an alternative… (DK4-21)But actually, some patients are not really set on paying for just a piece of advice, they think it’s strange to pay to go for longer walks. (DK2-24)andThe practitioners in this study described different levels of integration in their respective healthcare settings and how the legislation impacts the clinical encounter and the level of their involvement in SAM. In Norway, SAM was described as highly integrated in the clinical encounter and as “part of the tool box”, whereas in Denmark the participants described SAM as a pending issue and questioned “Is it worth the trouble?” In Sweden, where chiropractors usually are not part of the national health services, SAM is not integrated in routine practice, and the profession is still struggling to gain broad public recognition. In both Norway and Sweden, the participants described sick leave certification rights as a seal of approval of the profession.The sickness certification rights in Norway were negotiated as part of a larger agreement regarding reimbursement schemes and referral rights to imaging and medical specialties between the Norwegian Chiropractic Association and the Norwegian government. The sickness certification rights were included in the negotiations as an “expendable” item . Much to the surprise of many Norwegian chiropractors, the certification rights were never discussed in detail, but granted without further ado . This situation serves as an example of how professional territories are acquired by political manoeuvres rather than by virtue of the content of clinical work . It alsoThe legitimacy that sickness certification rights add to the profession was perceived differently in the three countries. Over the past 25 years, the Danish chiropractic community has raised its professional profile to that of an acknowledged contributor to local musculoskeletal healthcare. Thus, it is perhaps the first example of the chiropractic profession being accepted into mainstream healthcare as an equal partner . This waThe rationale of chiropractors as SAM partners put forward by the participants are highly linked to issues of legitimacy. Respondents based their claims on perceptions of their own and other professions’ specialist expertise to justify their part in the certification process and the role extension concept . The chiWe identified a range of barriers for the chiropractors to engage in SAM. Especially salient were organizational or systemic factors, such as the patients’ fee-for-service, which was voiced as a barrier for seeing patients with lower socioeconomic status. Affordability has been identified as a significant gap in health system coverage , and it Communication was described as an essential part of SAM, but the chiropractors described a predominantly unidirectional flow to other stakeholders and perceived this to be an important barrier for SAM. For the Swedish chiropractors in particular, lack of communication because of the absence of formal communication platforms was especially pertinent. In Denmark, we found that the lack of financial incentives and the administrative burden were important barriers for engaging in SAM. In a systematic review, Kilgour et al.  suggesteThis study used a qualitative description with in-depth, semi-structured interviews. This provided a broad perspective as well as a deep understanding of the participants’ experiences and perceptions regarding this previously uncharted topic. The study was conducted as part of a mixed-method study, and will inform a quantitative phase. Therefore, we see the present results as a first exploration of SAM among Scandinavian chiropractors. Further interviews may have provided other perspectives. A specific concern is the lack of triangulation with other stakeholders such as GPs, patients or case-workers. In the quantitative phase, we will extend the data collection to representative samples from all three countries.Healthcare systems, employment settings and work legislation vary considerably internationally, and the findings from this study may not be applicable to settings outside the Scandinavian countries. However, we have found commonalities with existing literature in the area of professional legitimacy and role extension. The chiropractic’s professional development is considered a test case with the potential to affect other AHPs with aspirations to move towards mainstream healthcare . TherefoAHPs, in this instance chiropractors, with patient management expertise can fulfil a key role in SAM and by extension work disability prevention when these practices are legislatively supported. In cases where these practices occur informally, however, practitioners face systemic-related issues and professional self-image challenges that tend to hamper them in fulfilling a more integrated role as providers of WDP practices.


Our solution involves two complementary novel MRI techniques (Glasgow Oxygen Level Dependant (GOLD) Metabolic Imaging), which when combined with a perfluorocarbon (PFC) based oxygen carrier and hyperoxia can identify penumbra due to dynamic changes related to continued metabolism within this tissue compartment. Our aims were (i) to investigate whether PFC offers similar enhancement of the second technique (Lactate Change) as previously demonstrated for the TMethods: An established rat stroke model was utilised. Part-1: Following either saline or PFC, magnetic resonance spectroscopy was applied to investigate the effect of hyperoxia on lactate change in presumed penumbra. Part-2; rats received O-PFC prior to T2*OC (technique 1) and MR spectroscopic imaging, which was used to identify regions of tissue lactate change (technique 2) in response to hyperoxia. In order to validate the techniques, imaging was followed by [14C]2-deoxyglucose autoradiography to correlate tissue metabolic status to areas identified as penumbra.Results: Part-1: PFC+hyperoxia resulted in an enhanced reduction of lactate in the penumbra when compared to saline+hyperoxia. Part-2: Regions of brain tissue identified as potential penumbra by both GOLD metabolic imaging techniques utilising O-PFC, demonstrated maintained glucose metabolism as compared to adjacent core tissue.Conclusion: For the first time in vivo, enhancement of both GOLD metabolic imaging techniques has been demonstrated following intravenous O-PFC+hyperoxia to identify ischaemic penumbra. We have also presented preliminary evidence of the potential therapeutic benefit offered by O-PFC. These unique theranostic applications would enable treatment based on metabolic status of the brain tissue, independent of time from stroke onset, leading to increased uptake and safer use of currently available treatment options. The global burden of stroke continues to escalate with an ageing population being associated with a 62% increase in global death rate between 1990 - 2013 Recent data from the global burden of disease 2013 study reveal that every year ~6.5 million people die as a result of stroke worldwide The restoration of blood flow through thrombolytic therapy by administration of recombinant tissue plasminogen activator (rtPA) to break down the clot during the first 4.5 h from stroke onset has resulted in benefit for acute ischaemic stroke (AIS) patients Overall current treatment rates for AIS remain poor with ~75% of patients being excluded from thrombolytic treatment where stroke onset time is unknown, has exceeded 4.5hours or where there is risk-benefit uncertainty. However, many patients excluded on the basis of information on stroke onset time are likely to have potentially salvageable penumbral tissue Accurate identification of the salvageable ischaemic penumbra is crucial for improving treatment decisions in AIS patients. However, the current lack of an easily applied and accurate imaging technique enabling reliable identification of salvageable penumbral tissue acutely following stroke has hampered progress in attempts to improve the management of patients.Currently, non-contrast computed tomography is used to quickly evaluate suspected stroke patients to discriminate between ischaemic and haemorrhagic stroke and to exclude stroke mimics Current advanced brain imaging techniques including MRI-based perfusion/diffusion (DWI/PI) mismatch and CT perfusion (CTP) lack accuracy and do not provide direct assessment of tissue metabolic status. Both techniques have already been applied to select patients for acute stroke clinical trials based on “penumbra” imaging. However neither technique, which rely on selecting threshold values to define the DWI lesion and the perfusion deficit, have as yet been validated and accepted for clinical use as routine for penumbra imaging. This lack of validated thresholds leads to difficulties in accurately differentiating penumbra from irreversibly damaged core and benign oligaemic tissue, destined to survive 2* signal based on the different magnetic properties of deoxy- and oxyhaemoglobin in blood  and this technique has been shown to identify a region within the perfusion deficit in rodent stroke models that displays several features of penumbra: histological evidence of normal neuronal morphology, ongoing glucose metabolism, and tissue recovery on prompt reperfusion 2 within the paranasal sinuses which caused artefacts of T2*-weighted images, due to the paramagnetic effect of oxygen. The technique has also been successfully reproduced pre-clinically by another group Our group have been developing GOLD (Glasgow Oxygen Level Dependent) diagnostic imaging, which comprises two independent but complementary MRI based techniques combined with therapeutic potential to extend the lifespan of penumbral tissue. The MRI sequences when combined with an oxygen challenge  identify the ischaemic penumbra by identifying aerobic metabolism within the hypoperfused ischaemic tissue compartment Technique 2 uses the novel concept of lactate change imaging, which can compartmentalise brain tissue based on the ability to identify tissues displaying anaerobic metabolism but having the potential for aerobic metabolism with increased oxygen delivery To increase the amount of oxygen delivered to the tissues we have continued to develop GOLD imaging using an approach where an intravenous oxygen carrier perfluorocrabon (PFC) is administered prior to OC. In addition, unlike other advanced brain imaging techniques, additional imaging time carries no penalty (“time lost is brain lost”) Perfluorocarbons (PFCs) are fluorinated hydrocarbons with respiratory gas carrying capacity superior to that of haemoglobin We have previously demonstrated enhanced detection of penumbra with T2*OC using our b-PFC combined with a lower level (40%) of inhaled oxygen 14C]2-deoxyglucose autoradiography. A secondary aim was to investigate the therapeutic potential of the enhanced oxygen delivery to the compromised penumbra by addition of intravenous PFC in combination with hyperoxia.The primary aims of the current studies were to (i) determine whether our lactate change technique could also be enhanced by accelerating the change with the addition of intravenous PFC (ii) investigate the potential of combining the metabolic GOLD imaging techniques (T2*OC and Lactate Change) within a single scanning protocol, with confirmation of ongoing glucose metabolism in regions identified as penumbra using  were analyzed from 14 timed arterial blood samples over 45 minutes by glucose oxidase assay and liquid scintillation analysis, respectively. At 45 minutes, animals were killed by intravenous injection of sodium pentobarbitone and the brains quickly dissected out, frozen  and processed for quantitative autoradiography. Coronal cryostat sections (20 µm) were exposed to X-ray film  for 3 days with a set of [14C] standards . Autoradiograms were analyzed using a computer digitized image analysis system . Quantitative optical density measurements were taken from four regions of interest  contralateral region homotopic to the penumbra and (4) contralateral region homotopic to the ischemic core. Optical density values were converted into [14C] tissue concentrations using the calibration curve derived from the set of [14C] standards. The [14C] tissue concentrations along with the plasma glucose and [14C] plasma concentrations were used to calculate local cerebral glucose utilization  in ROIs using the operational equation of Sokoloff At the end of the MRI scanning session, animals were quickly removed from the magnet, and returned to the operating theatre where a bolus of 4C2-DG wa2* signal change maps, which also approximated the slice used for lactate change maps. This enabled glucose utilization within each ROI to be determined from autoradiograms.Glucose utilization for each ROI was generated from 3 autoradiographic images covering the rostro-caudal extent of each of the two selected coronal MRI slices (2 mm) used to generate the T2* percentage signal change maps during hyperoxia and lactate spectroscopic images were generated using Image J software (http://rsb.info.nih.gov/ij/).ADC maps, CBF maps and TLactate Change OC maps were generated for a single coronal slice by subtraction of spectroscopic images acquired during baseline (normoxia) and hyperoxia with a spatial smoothing filter being applied. Aerobic Lactate Change maps represent the change in tissue lactate in response to OC following administration of O-PFC in Study 2. Anaerobic Lactate Change maps represent the change in tissue lactate in response to returning to ventilation with medical air following a period of hyperoxia.2* percentage signal change was analysed on MRI-based ROIs, which were selected according to specific features on the images; (1) Ischaemic core in caudate nucleus within the thresholded ADC lesion; (2) Its mirror contralateral region manually designated by researcher; (3) Penumbra as defined by thresholded T2* percentage signal change (4) contralateral cortex  was measured in the dorso-lateral contralateral cortex, avoiding any large veins and venous sinuses. The T2* penumbra ROI was automatically derived from a threshold: tissue with a percentage signal change ≥ the contralateral ROI mean + two standard deviations. The ischaemic core ROI was selected as a region within the thresholded ADC lesion for the corresponding slice. A representative contralateral region was manually drawn avoiding areas of high signal change on T2* maps due to the presence of large veins, meaning that the region was not always an exact mirror of the ipsilateral ROI.T2* percentage signal change maps were produced by comparing the peak signal during each level of the hyperoxia protocol (mean taken over ~2 min scanning) with the mean signal during baseline (first 3 min of scan prior to hyperoxia). The difference in signal was divided by the mean baseline signal and multiplied by 100 to give percentage signal change maps. Penumbra tissue was defined using a threshold based on the empirical rule: tissue showing a mean increase in T2* which was greater than 2 standard deviations above the contralateral cortex mean.The ROIs were selected and analysed on two coronal slices within middle cerebral artery territory. TApproximate penumbral tissue generated from DWI-PI mismatch area Figure A iii was2*signal change in penumbra, contralateral cortex and ischaemic core ROIs were assessed using a 2-tailed paired Student's t-test. Data from different groups were assessed using a 2-tailed unpaired Student's t-test. For comparison of paired data within groups, such as the change in lactate levels measured within the penumbra voxel from Study 1, a repeated measures one-way ANOVA followed by a Dunnet's multiple comparison test was performed. For comparison of unpaired data such as the magnitude of T2* signal change between ROI's, a one-way ANOVA followed by a Bonferroni's multiple comparison test was performed. For all comparisons, p<0.05 was considered as statistically significant.All data are presented as mean ± SD. Statistical analysis was performed using GraphPad Prism . Comparison of physiological variables before and during OC, and of OC-induced T2 ~1.0) induced a transient, significant increase in PaO2 of ~3.5-4.0 fold in arterial blood samples from both groups. This is consistent with previous studies using this level of hyperoxia 2 observed during OC in the group receiving saline, prior to hyperoxia.Physiological data are shown in supplementary Table 1H spectra upon changing ventilation from air to 100% oxygen in animals receiving either PFC or saline prior to hyperoxia  compared to the saline group (80.8 ± 27.3 mm3)  the magnitude of TC Figure A. When ca Figure A. Followa Figure B & 8B wi2*OC with O-PFC was similar for 50% and 100% OC . This would enable significant improvements in the management of acute stroke patients by differentiating patients with viable brain tissue (therapeutic target) irrespective of time from stroke onset, from those who have largely irreversible damage at presentation and who could therefore be exposed to additional risk from reperfusion therapy. In this paper we have presented further preclinical validation of two complementary diagnostic MR imaging techniques, which when combined with intravenous O-PFC and OC, (GOLD imaging), have the potential to identify metabolically active penumbra in rodent models of acute stroke. Further studies (unpublished) demonstrating a concomitant therapeutic benefit of O-PFC and hyperoxia supports the true theranostic potential of this novel technology, for which initial clinical trials in acute stroke patients are planned for late 2017.The first studies to metabolically map the ischaemic penumbra following acute stroke used PET Likewise CTP demonstrates a high degree of heterogeneity in the acquisition protocols and post-processing software and this lack of standardisation has resulted in the lack of established validated thresholds for the quantitative definition of the ischaemic penumbra MR perfusion and CTP were also used to select patients in clinical trials assessing endovascular therapy. In the MR RESCUE trial a lack of correlation between penumbral pattern on neuroimaging and outcome in terms of benefit from endovascular therapy was demonstrated Therefore it is clear that, at present, these techniques require further refinement and optimisation to identify potential responders to reperfusion therapy beyond the currently recommended time windows. Furthermore, as they don't provide a metabolic approximation of penumbra their clinical utility in this regard will remain open to question.The objective of the studies presented here was to advance the development of two complementary metabolically MRI based neuroimaging techniques (T2* and Lactate Change sequences combined with OC and O-PFC = GOLD imaging) for diagnostic use in the acute stroke setting to determine the presence of salvageable penumbra based on the metabolic status of this tissue.Proof-of-concept for detection of penumbra was initially established for both techniques using 100% oxygen alone in rodent stroke models It is “change” in tissue lactate to an oxygen challenge, not absolute lactate levels, that are important in assessing “metabolic integrity” . Our group previously developed the novel method of lactate change MRI, which is based on imaging changes in brain tissue lactate levels in response to hyperoxic challenges th - 1/40th the size of red blood cells (diameter of about 200 nanometers) and are therefore able to penetrate the smallest blood vessels, enabling oxygen delivery to ischaemic tissues. This along with an improved extraction ratio of oxygen from PFC emulsions when compared to haemoglobin The primary aim of Study 1 was to evaluate whether addition of a PFC improved the sensitivity and reduced the time for the Lactate Change OC, making the technique an applicable tool in the clinical setting. The data presented in Figures We next investigated the potential of combining the metabolic GOLD imaging techniques (T2*OC and Lactate Change OC) within a single scanning protocol using O-PFC. The protocol was designed such that T2*OC and Lactate Change OC imaging could be applied concurrently in the same scanning session in a way that would most likely mimic the intended clinical utility of these imaging techniques in the acute stroke setting.+/K+ ATPase and these require energy. Anaerobic metabolism of glucose only provides 2 ATPs from a molecule of glucose but with aerobic metabolism one molecule of glucose can provide additional 36 ATPs. The energy produced by anaerobic metabolism cannot sustain the membrane potentials and this lack of energy ultimately results in cell death. As long as the mitochondria are functional and able to utilise oxygen to generate ATP, the neuron will have the energy to survive. Therefore the ischaemic penumbra represents hypo-perfused tissue displaying anaerobic metabolism due to lack of oxygen, which still has the potential for aerobic metabolism and so is potentially salvageable.For lactate change imaging, the example shown in Figure 2 alone The results presented in Study 2 utilising spectroscopic imaging of lactate to map lactate change in response to hyperoxic challenge following O-PFC administration, corroborate with the results from Study 1 where b-PFC was shown to improve the possibility to detect decreases in the lactate spectral peak. Together these findings substantiate an enhancement by PFC to delineate metabolically active penumbra using this technique. Furthermore the ability to detect regions of lactate change in all animals analysed in Study 2 signifies an improvement in the sensitivity to detect lactate change, through the addition of O-PFC emulsion. Previously published findings reported a decrease in tissue lactate in only a subset of animals when using 100% O2) significantly enhanced T2* signal change in the ischaemic penumbra ROI compared with lower oxygen levels alone 2*OC signal change maps, there was no difference in the volume detected by either 50% or 100% oxygen providing further support for increased sensitivity at lower levels of inhaled oxygen with the addition of O-PFC. It was however notable from Study 2 that the volume of the region detected as penumbra during the later T2*OC at ~2.5 h post-MCAO, was significantly smaller than that detected at the earlier time point. This indicates a progression of the ischaemic damage and incorporation of penumbral tissue into ischaemic core between the two OCs when animals were ventilated with air. This provides additional validation that the technique identifies ischaemic penumbra and also that in the absence of hyperoxia, O-PFC was limited in its capacity to maintain penumbra viability.To address limitations encountered during preliminary translation of the T2*OC technique an intravenous PFC was administered prior to OC with lower oxygen levels. Following administration of our b-PFC, lower level hyperoxia  they have the capacity to reach the microcirculation beyond an occluding clot in any remaining plasma flow, or via collaterals, thereby improving delivery of oxygen to the compromised ischaemic penumbra. It has been shown that ischemia induces sustained contraction of pericytes on microvessels and thereby causes capillary constriction and obstruction of erythrocyte flow Initial evidence for the ability of PFC to support the ischaemic penumbra and offer therapeutic potential following acute ischaemic stroke comes from analysis of lesion growth in Study 1. In addition to measuring lactate, a secondary aim of this study was to quantify the area of ischaemic damage from thresholded ADC maps during the period of hyperoxia and return to normoxia. It was shown that lesion expansion during this period was significantly smaller in the group of animals receiving b-PFC when compared to the group receiving saline prior to hyperoxia. This suggests that animals given b-PFC in combination with hyperoxia could maintain aerobic metabolism because of functioning mitochondria and the resulting cell energetics would support the survival of penumbral tissue.in vivo, working concurrently to identify the metabolic penumbra in a rodent stroke model. Regions identified as penumbra with both T2*OC and Lactate Change OC techniques displaying maintained glucose metabolism. Further development of co-registration and analysis software to provide a penumbra map on clinical MRI scanners is ongoing.In summary enhancement of both GOLD imaging techniques following intravenous O-PFC and hyperoxia has been demonstrated for the first time Successful translation of GOLD imaging would enable treatment decisions and recruitment into future acute stroke trials investigating efficacy endpoints to be based on metabolic status of the brain tissue independent of time from stroke onset. This could improve the safe use and extend the availability of current therapeutic options to many more acute stroke patients. We have also uncovered preliminary evidence for a potential therapeutic benefit of O-PFC plus hyperoxia during scanning, which could offer significant advantages over current penumbral imaging techniques by slowing the progression of ongoing ischaemic damage. Further therapeutic benefit could be realised by extending hyperoxia beyond the scanning period and this is the focus of additional research by our group. Therefore, through unique simultaneous diagnostic and therapeutic application in acute ischaemic stroke, GOLD imaging technology incorporating intravenous O-PFC and hyperoxia offers new hope in improving the management of acute stroke patients.Supplementary figures and tables.Click here for additional data file.