Health care workers (HCW) often perform, promote, and advocate use of public funds for animal research (AR); therefore, an awareness of the empirical costs and benefits of animal research is an important issue for HCW. We aim to determine what health-care-workers consider should be acceptable standards of AR methodology and translation rate to humans.P < .05 considered significant.After development and validation, an e-mail survey was sent to all pediatricians and pediatric intensive care unit nurses and respiratory-therapists (RTs) affiliated with a Canadian University. We presented questions about demographics, methodology of AR, and expectations from AR. Responses of pediatricians and nurses/RTs were compared using Chi-square, with Response rate was 44/114(39%) (pediatricians), and 69/120 (58%) (nurses/RTs). Asked about methodological quality, most respondents expect that: AR is done to high quality; costs and difficulty are not acceptable justifications for low quality; findings should be reproducible between laboratories and strains of the same species; and guidelines for AR funded with public money should be consistent with these expectations. Asked about benefits of AR, most thought that there are sometimes/often large benefits to humans from AR, and disagreed that “AR rarely produces benefit to humans.” Asked about expectations of translation to humans , most: expect translation >40% of the time; thought that misleading AR results should occur <21% of the time; and that if translation was to occur <20% of the time, they would be less supportive of AR. There were few differences between pediatricians and nurses/RTs.HCW have high expectations for the methodological quality of, and the translation rate to humans of findings from AR. These expectations are higher than the empirical data show having been achieved. Unless these areas of AR significantly improve, HCW support of AR may be tenuous. Biomedical animal research (AR) involves some harm to sentient animals including distress , pain, and early death -3. AR isThere is a growing literature that raises concerns about the empirical practice of AR in at least two domains. First, the methodological quality of AR is often poor in both experimental design and animal welfare aspects -12. AR pempirical practice of AR, and how AR could be improved to increase the level of support. We found that HCW have high expectations for the methodological quality of, and the translation rate to humans of findings from AR.Since most AR is funded by public money through government and charitable granting agencies, it is important to know the public perception of, and the level of public support for AR. Surveys of the public find that the majority are ‘conditional acceptors’ of AR; they accept the practice because of the promise of cures and treatments for life-threatening and debilitating human diseases, so long as animal welfare is at least minimally considered and protected . To our All pediatricians and pediatric intensive care unit nurses and respiratory therapists (RTs) who are affiliated with one Canadian University were e-mailed the survey using an electronic, secure, survey distribution and collection system  . A coverWe followed published recommendations . To geneThe background section stated: “In this survey, ‘animals’ means: mammals, such as mice, rats, dogs, and cats. It has been estimated that over 100 million animals are used in the world for research each year. There are many good reasons to justify AR, which is the topic of this survey. Nevertheless, some people argue that these animals are harmed in experimentation, because their welfare is worsened. In this survey, ‘harmful’ means such things as: pain, suffering , and early death. This survey is about how AR should be performed. We value your opinion on the very important issue of the methodology of AR.”We presented demographic questions, 15 questions that asked respondents “about the methods of AR that are commonly discussed by animal researchers”, 4 questions that asked the respondent “to consider what you think the benefits to humans are as a result of AR”, and 8 questions that asked the respondent “for your opinions about what you expect from AR paid for with public funds .” Response choices included scales of “strongly agree, agree, undecided, disagree, strongly disagree”, “nearly always, often, sometimes, not often, almost never”, and “5-20%, 21-40%, 41-60%, 61-80%, over 80%” depending on the type of question. All the questions are shown in the Tables The study was approved by the health research ethics board 2 of our university (study ID Pro00039590) and return of a survey was considered consent to participate.P ≤ .05 after Bonferroni correction for multiple comparisons considered significant.The web-based tool (REDCap) allows anonymous survey responses to be collected, and later downloaded into an SPSS database for analysis. The proportions of respondents with different answers were expressed as percentages. The responses of the two predefined groups, pediatricians and pediatric intensive care unit nurses/RTs, were compared using the Chi-square statistic, with Forty-eight responded, but only 44/114 (39%) gave responses to more than the demographic questions. Demographics are given in Table The majority of respondents agreed that: anesthetic use should be monitored during surgery (100%), pain should be monitored after this surgery even over-night (91%), and experimenters in a research study should have similar training on the procedures involved (97%) Table . The majMost respondents believe that discoveries from AR sometimes or often lead to a treatment for human disease directly (77%) or indirectly (84%), and that researchers sometimes or often claim large benefits from AR (91%) Table . The majThe majority of respondents think that drugs tested on animals should correctly predict the following for humans at least 41% of the time: adverse reactions (69% of respondents), disease treatment (62% of respondents), carcinogenicity or teratogenicity (74% of respondents), and treatment of stroke, severe infection, cancer, brain or spinal cord injury (59% of respondents). The majority also expected that replication of AR findings in second laboratories or other strains of the animal should occur at least 61% of the time (95% and 68% of respondents respectively). The majority agreed that misleading (in terms of human benefit and/or harm) animal experiments should occur at most 40% of the time (86% of respondents). Finally, when asked to “assume drugs studied in animals accurately predict effects in humans less than 20% of the time. If this were true, it would significantly reduce your support for animal research”, 40% disagreed (Table Sixty-nine of 120 (58%) responded; 52 (75%) nurses and 16 (25%) RTs. Demographics are given in Table The majority of respondents agreed that: anesthetic use should be monitored during surgery (98%), pain should be monitored after this surgery even over-night (96%), and experimenters in a research study should have similar training on the procedures involved (96%) Table . The majMost respondents believe that discoveries from AR sometimes or often lead to a treatment for human disease directly (84%) or indirectly (88%), and that researchers sometimes or often claim large benefits from AR (97%) Table . The majThe majority of respondents think that drugs tested on animals should correctly predict the following for humans at least 41% of the time: adverse reactions (85% of respondents), disease treatment (82% of respondents), carcinogenicity or teratogenicity (89% of respondents), and treatment of stroke, severe infection, cancer, brain or spinal cord injury (88% of respondents). The majority also expected that replication of AR findings in second laboratories or other strains of the animal should occur at least 61% of the time (92% and 83% of respondents respectively). The majority agreed that misleading (in terms of human benefit and/or harm) animal experiments should occur at most 40% of the time (84% of respondents). Finally, when asked to “assume drugs studied in animals accurately predict effects in humans less than 20% of the time. If this were true, it would significantly reduce your support for animal research”, only 6% disagreed , carcinogenicity and teratogenicity, and disease treatments. The majority thought misleading AR results should occur no more often than 20% of the time. If translation from AR to humans was to occur <20% of the time, most would be less supportive of AR. Finally, most respondents expect that AR findings should be reproducible between laboratories and between strains of the same species. There are important implications of these findings for public and HCW acceptance of AR Table .Table 5Previous public surveys have generally asked only whether people support AR for human benefit, and not asked people to evaluate the details of their expectations of AR. For example, the Eurobarometer asks “scientists should be allowed to experiment on animals like dogs and monkeys if this can help sort out human health problems”; in 2010, 44% of Europeans responded ‘agree’ and 37% ‘disagree’ . In the Some qualitative research also suggests there is conditional public acceptance of AR based on a utilitarian analysis of costs  and benefits (to humans) ,50. Thisand e,f,g]. In complex systems [e.g. animals], very small differences in initial conditions  can result in dramatic differences in response to the same perturbation   -58. Therperty d] -62. ThusThis study has several limitations. Response rates for pediatricians and nurses/RTs were 39% and 58% respectively; thus we cannot rule out biased participation in the survey. Statements presented needed to be short and concise, and this may have left out important details that would have influenced the understanding of and response to the text. The moderate sample size from one University limits the generalizability of our results. Nevertheless, this is the first survey we are aware of that asks any group not just to consider whether they support AR; rather, to consider in detail the expectations for the methodology and translation of AR. Strengths of this study include the rigorous survey development process, and the inclusion of the most common critiques of the empirical practice of AR. Future study should determine the generalizability of our results.We found HCW respondents had high expectations for the methodological quality of AR, and the translation of findings from AR to human responses to drugs and disease. These expectations are far higher than the empirical data show having been achieved. This disconnect between HCW expectations of AR and the empirical reality of AR suggests that if HCW were better informed they would likely withdraw their conditional support of AR. Improved methodological quality is an achievable goal if this is prioritized by researchers, reviewers, editors, and funders. Whether methodologically optimal AR can achieve better human translation to meet HCW expectations is an open question.


Augmented reality (AR) is an environment-enhancing technology, widely applied in the computer sciences, which has only recently begun to permeate the medical field. Gastrointestinal endoscopy—which relies on the integration of high-definition video data with pathologic correlates—requires endoscopists to assimilate and process a tremendous amount of data in real time. We believe that AR is well positioned to provide computer-guided assistance with a wide variety of endoscopic applications, beginning with polyp detection. In this article, we review the principles of AR, describe its potential integration into an endoscopy set-up, and envisage a series of novel uses. With close collaboration between physicians and computer scientists, AR promises to contribute significant improvements to the field of endoscopy. Augmented reality (AR) is the process of superimposing computer-generated objects and data over existing, real structures . This diet al. utilized AR in 33 patients, using representations of the cerebral arteries to assist with arteriovenous malformation resection and aneurysm clipping [Despite its long history, AR has only recently made its debut in medical practice, being applied primarily to navigational surgery . This inclipping , 12. Finclipping .et al. used AR to improve the accuracy of trocar placement for cholecystecomy [et al. integrated laparoscopic ultrasound imaging in order to interrogate the liver, gallbladder, biliary tree, and kidneys below the visible surface [et al. reported a successful cholecystectomy using AR-based stereoscopic images with the da Vinci robot [et al. generated AR images from transrectal ultrasound to perform a radical prostatectomy [et al. performed partial hepatic resections in nine patients, with AR used to display vascular and biliary anatomy [et al. completed a pancreaticoduodenectomy using AR to highlight important vascular structures [Although few studies in general surgery have taken AR to the operating room, environments have been developed for both laparoscopic and open procedures . López-Mystecomy , while K surface . Volonteci robot , and Simatectomy . The dem anatomy , and Marructures .et al. described an AR system that successfully registered CT data with upper endoscopy imaging in 24 patients with GI tumors [et al. developed an image registration (IR) system to assist with natural orifice transluminal endoscopic surgery (NOTES) [et al. found that easy transluminal access to the kidneys, gallbladder, liver, and pancreas could be achieved with close AR guidance, raising the possibility of novel endoscopic surgical procedures [The role of AR in gastrointestinal (GI) endoscopy to date has been limited to localization of GI tumors and novel transluminal surgical approaches. Kranzfelder I tumors . Azaguryocedures .While the potential of navigational surgery is exciting, we believe that GI endoscopy is fertile ground for a vast array of new applications, including detection and identification of polyps, performance tracking, pathology scoring and more. In this article we review the components of AR systems, elaborate on our proposed applications for AR, highlight technical challenges, and outline a path towards innovation.Important to the fundamentals of AR are the technologies of image processing and computer vision. Image processing refers to the deconstruction of image data into a series of parameters or properties relating to the image. ‘Computer vision’ refers to high-level image processing in which a computer deciphers the contents of an image or sequence of images and uses this information to make intelligent decisions . In esseThere is a wide variety of AR set-ups and applications but they have in common a data input source, a processor, and a display . The inpThe basic hardware set-up for an endoscopy procedure is well suited to adaptation to an AR environment because high-definition video capture is already a core capability of most modern endoscopy units. A high-definition camera, at the distal tip of the flexible endoscope, supplies image data to a camera control unit (CCU), where the data are processed and formatted for output to high-definition monitors. The CCU is further connected to a quick-swap memory unit and a computer or keyboard to accept user input. An added central processing unit can be inserted in series between the CCU and monitor, to enable image processing and computer vision capabilities. This will house the signal processing, image analysis, and decision-making capabilities of the system prior to modified high-definition output with graphical overlay.Colonoscopy is the ‘gold standard’ for early detection of colorectal adenomas , and morIncreasing adenoma detection rate improves the preventive efficacy of colonoscopy and polypectomy , 47. DurSeveral hardware innovations have sought to address the first problem through expanded visualization of the colonic lumen. This includes the Third Eye retroscope camera, designed to identify polyps hidden behind folds in the bowel wall , and fulAugmented reality represents an important opportunity to improve adenoma detection rate. By aggregating a large volume of polyp images from colonoscopies, it is possible to implement machine-learning and computer vision algorithms to assist with polyp detection. This would be the first line of approach to innovation within the context of the high-resolution video data that is routinely acquired during colonoscopy. Using AR, the endoscopist could enjoy real-time visual assistance with polyp detection in the form of overlaid images on the primary HD monitor or on an adjacent one. In more advanced iterations, suspected polyp type may also be displayed, with color-coding or other visual information used to represent the level of confidence in the analysis .Figure Several important features of this technology can be highlighted. First, modern processing capabilities would enable an AR system to function in real time during a colonoscopy. Second, flat or isochromatic polyps, which might be visually occult to the less experienced or fatigued endoscopist, would be parsed by the AR system in identical fashion to any other polyp; that is to say that, while humans have a tendency to “see what they want to see” , an AR sFinally, AR is complementary to contemporary hardware innovations designed to improve adenoma detection rate through expanded visualization. Indeed, the Third Eye and FUSE systems involve additional cameras and force the operator to scan multiple screens displaying live video. As mentioned above, AR could draw the endoscopist’s eye, in real time, to lesions that give rise to concern, effectively creating ‘extra sets of eyes’ on all aspects of the video data.The American Society for Gastrointestinal Endoscopy recommends that all neoplastic polyps in the colon be resected ; howeverThere are several imaging techniques used during gastrointestinal endoscopy, which could benefit from integration with augmented reality. Endoscopic ultrasound, for example, can be used to characterize organs or masses adjacent to the gastrointestinal tract. Structural images obtained using EUS could be registered in order to display a spatial projection of the object in the live colonoscopy video feed. Registration of EUS images for computer-guided navigation has previously been described , and couAs governmental and insurance regulations surrounding accountable care take form, there will be ever-increasing pressure for endoscopists to track their polyp detection rates and other performance metrics; thus another critical aspect of endoscopic AR could be to track, test, and validate polyp detection performance. This could assist in quality reporting for the purpose of tracking and improving outcomes in gastrointestinal endoscopy . TrackinAccurate estimation of the size of various anatomical structures during endoscopy has the potential to greatly enhance current practice. In the arena of polyp detection, determining the size of a given polyp plays a critical role in dictating follow-up intervals for a patient’s next surveillance colonoscopy. Recent data suggest that, with visual estimation, substantial variations in recorded polyp size occur, leading to incorrect surveillance intervals in 10% of cases , 66. To Standardized endoscopic scoring systems exist for many disease states of the gastrointestinal tract. Unfortunately, their utility is often limited by subjective interpretation and impracticality in the clinical setting. Inflammatory bowel disease (IBD) scoring systems, for example, are used to assess disease response in clinical trials but have been difficult to apply in clinical practice , 69. AR-Wireless capsule endoscopy is indicated to interrogate portions of the small bowel that are not easily accessed through standard endoscopy—usually to search for obscure gastrointestinal bleeding or signs of Crohn’s disease. Recent advances have granted some degree of control over maneuvering the capsule whilst it is in the gastrointestinal tract. This has been accomplished through techniques such as electro-stimulation and by using magnetic fields , 73. AltWhile no software platforms to integrate augmented reality into endoscopy are currently available, this is an active area for research and innovation. We have only just entered into an era in which computer vision technology may surpass human ability in terms of facial and object recognition . The comConflict of interest statement: NM, JC, KT, and TB are advisors to EndoLayers.


Health systems strengthening is becoming a key component of development agendas for low-income countries worldwide. Systems thinking emphasizes the role of diverse stakeholders in designing solutions to system problems, including sustainability. The objective of this paper is to compare the definition and use of sustainability indicators developed through the Sustainability Analysis Process in two rehabilitation sectors, one in Nepal and one in Somaliland, and analyse the contextual factors (including the characteristics of system stakeholder networks) influencing the use of sustainability data.Using the Sustainability Analysis Process, participants collectively clarified the boundaries of their respective systems, defined sustainability, and identified sustainability indicators. Baseline indicator data was gathered, where possible, and then researched again 2 years later. As part of the exercise, system stakeholder networks were mapped at baseline and at the 2-year follow-up. We compared stakeholder networks and interrelationships with baseline and 2-year progress toward self-defined sustainability goals. Using in-depth interviews and observations, additional contextual factors affecting the use of sustainability data were identified.Differences in the selection of sustainability indicators selected by local stakeholders from Nepal and Somaliland reflected differences in the governance and structure of the present rehabilitation system. At 2 years, differences in the structure of social networks were more marked. In Nepal, the system stakeholder network had become more dense and decentralized. Financial support by an international organization facilitated advancement toward self-identified sustainability goals. In Somaliland, the small, centralised stakeholder network suffered a critical rupture between the system’s two main information brokers due to competing priorities and withdrawal of international support to one of these. Progress toward self-defined sustainability was nil.The structure of the rehabilitation system stakeholder network characteristics in Nepal and Somaliland evolved over time and helped understand the changing nature of relationships between actors and their capacity to work as a system rather than a sum of actors. Creating consensus on a common vision of sustainability requires additional system-level interventions such as identification of and support to stakeholders who promote systems thinking above individual interests. Health systems strengthening is becoming a key component of development agendas for low-income countries worldwide. As a means to achieve this, systems thinking provides perspectives on how health systems can be assessed , recogniThe structure of social networks influence individual actors’ capacity to respond to the needs of the system as a whole , 10. It In this paper, we build on previous work to introduce systems thinking among local stakeholders of the physical rehabilitation system in Nepal and Somaliland . AlthougDuring this process, the concept of sustainability is upheld as normative . The proAlong with convening and observing sustainability analysis workshops in each setting, we sought to analyse the contextual factors and the characteristics of the social networks to identify the influences affecting actors’ decisions about using sustainability data or not , 33.The objective of this paper is to compare the definition and use of sustainability indicators developed through the SAP in two rehabilitation sectors, one in Nepal and one in Somaliland, and analyse the contextual factors (including the characteristics of system stakeholder networks) influencing the use of sustainability data.is used where it is important to understand the meaning and interpretation of human social arrangements such as hospitals, clinics, forms of management or decision making”. In real-life contexts, multiple case study designs are known to be appropriate for understanding and interpreting complex causal links in natural setting interventions [In order to capture social phenomena such as management decisions and interactions between individuals, an in-depth qualitative research approach was adopted. According to Fitzpatrick and Boulton ( p. 107),ventions , 36. We Stakeholder network analysis was used to map key stakeholders in the physical rehabilitation system and identify network characteristics. The analysis was conducted in both Nepal and Somaliland in 2010 and again in 2012, and is detailed elsewhere , 38. In The SAP is a participatory method based on systems thinking, which combines the Process Analysis Method five-step approach , 48 withIn both Nepal and Somaliland, the SAP method was implemented during a three-day workshop sponsored by Handicap International in 2010 with key stakeholders involved in the physical rehabilitation system. Participants were purposively selected by Handicap International and Naspir, the national federation of rehabilitation providers in Nepal, and by the two national rehabilitation providers in Somaliland. The first list of participants was shared with and reviewed by the two investigators (KB and JP) in relation with the diversity within the rehabilitation sector. The participants included representatives from the Ministry of Health and/or Ministry of Social Affairs, regional health authorities, selected rehabilitation professional staff , rehabilitation centre managers, representatives of disabled people’s organizations, and representatives of international donors and non-governmental organizations (NGOs) involved locally.Two-year follow-up workshops were held in both countries in 2012. All the organisations that were represented at the first workshop were represented at the follow-up workshop. After the SAP was reviewed, workshop participants discussed key events that influenced the sustainability of the rehabilitation sector during the intervening 2 years. Sustainability indicators were re-measured, where possible, and participants reflected on progress toward self-defined sustainability goals.a useful tool for assessing causality in situations where temporal sequencing, particular events, and path dependence must be taken into account” .Information was collected from key informants on experiences with collecting and analysing sustainability information by individual actors/organisations. Interviews were conducted in private and participants were assured of confidentiality to encourage participants to share potentially sensitive issues and insights. Interviews were recorded. Transcripts and notes from each interview and group discussion were read in their entirety before coding line-by-line to identify and label ideas and meanings conveyed in each small section of text. These codes were then grouped and labelled to reflect broader themes within the data. Further additions and revisions to the coding framework were made on a continual basis as higher level constructs were generated, through reviewing emerging themes and interpreting them in relation to stakeholder network analysis findings.The structure and properties of the physical rehabilitation stakeholder networks in Nepal and Somaliland are described and compared elsewhere . Key notIn 2010, the social network of rehabilitation actors in Nepal (56 actors) was over twice as large as the network in Somaliland (22 actors) and there were substantial differences in the types of actors involved in service delivery and system governance. Notably, in Nepal, three ministries – the Ministry of Health and Population, the Ministry of Women, Children and Social Welfare, and the Ministry of Peace and Reconstruction – were directly involved in governance of physical rehabilitation services. In Somaliland, the Ministry of Public Health was solely in charge of rehabilitation services, although the Ministry of Labour and Social Affairs coordinated broader disability issues.Country differences in the involvement of local NGOs and disabled people’s organisations were also marked. In Nepal, local organisations provided services and directly managed rehabilitation centres. These organisations included professional associations  and disabled people’s organisations. In Somaliland, a much narrower range of actors delivered rehabilitation services. Specifically, two non-profit rehabilitation organisations (Disability Action Network (DAN), supported by Handicap International and the Somaliland Red Crescent Society (SRCS), supported by the International Red Cross Movement) were responsible for the entirety of rehabilitation service delivery. Both organisations in Somaliland were based in the capital city with complementary networks of partners and providers in the periphery. Disabled people’s organisations were not involved in service delivery, nor did they play a role in advocating for rehabilitation resources.In 2010, Somaliland’s system stakeholder network was four times more centralised than in Nepal. Nepal’s network density was twice as great as in Somaliland  . In Somaliland, the rehabilitation providers were concerned about the centralisation of services in the capital and the provinces being underserved due to insecurity outside Hargeisa, the capital, restricting both expansion of service provision and the ability of patients to complete referrals made from the periphery.In terms of service provision, both stakeholder groups recognized that limited numbers and poor geographic distribution of rehabilitation professionals would threaten the sustainability of their system. In Somaliland, network members emphasized the need for professionals to provide services outside the capital city by analysing human resources regionally. In Nepal, stakeholders took a more systemic view and identified the total number of rehabilitation professionals who needed to be trained in the country over the next few years as a sustainability indicator.In terms of organisational and financial capacity, Somaliland network stakeholders placed strong emphasis on the financial autonomy of rehabilitation service providers  and team management . One of the two service providers in Somaliland was approaching the end of a funding cycle and had no certainty that international support would continue after the end of 2011. Hence, sustainability indicators specified diverse funding sources and reduced financial risk. Some actors identified the emergence of donors in the domestic and diaspora private sector as an attractive means to diversification, particularly as trust in the political will of the Ministry of Health  to intervene in rehabilitation was low.In Nepal, organisational and financial sustainability goals are aimed at integrating physical rehabilitation into national policies and other programmes . Mainstreaming physical rehabilitation into other social or health systems represented an opportunity to secure resources that were not available in an isolated rehabilitation system. Furthermore, network members in Nepal recognised the need to associate with actors outside their system working at the community level to increase coverage of services.In terms of community capacity, the choice of sustainability indicators in both countries reflected the level of cohesiveness between the rehabilitation services and community-based organisations. In Somaliland, network members defined “community” as the users of the rehabilitation services, and community participation was described in terms of the financial capacity of users to pay for services  or participation of users in the planning of rehabilitation services .In Nepal, the “community” was defined as the population living in areas served by rehabilitation centres rather than only existing or potential service users per se. Community capacity was perceived as the capacity of community organisations to organise themselves  and integrate disability and rehabilitation into their activities at decentralised levels .In terms of the enabling environment, in both countries, workshop participants recognised the importance of securing political commitment at the national level to develop and implement disability-related policies  and allocate public financial resources to the rehabilitation sector .The system stakeholder network in Nepal became slightly less decentralised but denser over the 2 years from 2010 to 2012 with the appearance of seven new actors  Figure .In Somaliland, the major change was the disappearance of the relationship between the two principle brokers of the network (DAN and SRCS). Between the two SAP workshops, the two service providers never formally met to discuss the management of rehabilitation services or the governance of the system. The system became divided into two sub-systems with each service provider at the centre and surrounded by collaborative organisations. In spite of this system fracture, in 2012, the stakeholder network remained highly centralised (61%) and low in density (0.1) Figure .Figure 3At 2-year follow-up there was little progress towards the local vision of sustainability and almost no use of the self-defined sustainability indicators in Somaliland. System stakeholders individually had not attempted to undertake organisational measurements or use this type of information in decision making, although most still perceived the indicators to reflect their vision of sustainability of the system. During this period, no organisation took the lead to coordinate follow-up of the SAP. Handicap International had sponsored the workshop and was perceived as the “owner” of the initiative by some actors, who expected the international NGO to continue the lead role. Handicap International, however, was reluctant to maintain active involvement in coordination since their overall objective was to hand-over their support to rehabilitation services to local partners. The Ministry of Public Health had limited capacity and political will to fulfil this role. The two main service providers continued to be focused on the survival and viability of their own individual organisations. Several system stakeholders characterised sustainability as “unachievable” under current conditions in Somaliland, without sufficient funding and support from the Ministry of Public Health. In Somaliland, by 2012, apart from a tax break on land for rehabilitation facilities, there was still no government funding allocated to provision of physical rehabilitation services.In Nepal, all the sustainability indicators had been measured. A couple of months after the first workshop, Handicap International offered to take the lead and designate one of their project coordinators as the sustainability officer in charge of leading the sector on these issues. Through the initiative of this international organisation, an independent national consultant was commissioned in 2011 to collect further data to verify the estimates provided during the first workshop. During this assessment, 10 indicators could not be measured because of unclear definitions  and/or lack of existing data collection mechanisms. For example, data on the number of districts that have elaborated an action plan on disability is not routinely collected in information services primarily set-up to measure health service and human resource performance. Accessing this type of information would require comprehensively surveying district health offices and organisations, probably via field visits, which was beyond the financial resources available for the measurement exercise conducted in 2011. Data on the remaining indicators were published in a report circulated to the main actors of the rehabilitation sector .Although it is difficult to attribute the precise causes involved, evidence from qualitative discussions with stakeholders suggested that the SAP, through this process of discussion, consensus-building, data collection, and information-sharing, appeared to support the advancement of system sustainability in Nepal by encouraging the participation of major stakeholders outside the rehabilitation sector in sustainability initiatives between 2010 and 2012. Following the first workshop and lobbying from the main actors of the network, three Nepalese Ministries  agreed to invest funds to improve the functioning of the rehabilitation system as a whole instead of targeting specific rehabilitation centres. Their initial idea of creating new rehabilitation centres in the country was changed after the rehabilitation actors presented their vision of the sustainability of the sector. As a result, the Ministries agreed to support existing facilities and initiatives.In contrast, in Somaliland, systems thinking and action was observed during this period only in an area peripheral to rehabilitation service delivery, on disability mainstreaming initiatives to increase government involvement in wider disability programming in the social sector . This inIn this study, we compared the selection and use of self-defined sustainability indicators in two countries to analyse the influence of contextual factors and social network structure on the development of physical rehabilitation systems. Our assumption was that baseline differences between the two networks as well as the nature of relationships between actors would influence the way the sustainability indicators would be defined and used during follow-up.The definition of key sustainability indicators was implicitly influenced by network characteristics and actors’ perception of their own system. In Nepal, the stakeholder network at baseline was decentralized and dense with a wide diversity of stakeholder types. Here, vision of the future system was inherently systemic, including concerns about the coverage of services but also how the actors of the system work together and how integration of new actors could increase system impact. The actors of the rehabilitation sector who were interviewed recognised the importance of creating unity between all the actors of the network in order to more effectively negotiate with national authorities and donors. Hence, a national body representative of all the rehabilitation providers (NASPIR) was created in Nepal. In Somaliland, where the stakeholder network was centralized in the capital city and low in density (few stakeholders and brokers), the vision of the future physical rehabilitation system was constructed around the two rehabilitation facilities, positioned at the very centre of the system, and mainly represented their perspective. They focused on the extension of the services towards the provinces.Social network analysis provides tools to identify knowledge brokers, i.e., individuals who create links between different groups within a system, such as between users and researchers, which was the case of the two rehabilitation centres in Somaliland . The broThe use of self-defined sustainability indicators by the system was also influenced by individual actors’ survival strategies. At 2-year follow-up, the dense network in Nepal became even stronger as actors prioritised integration of services for organisational growth and survival and was an enabling factor in the utilisation of sustainability indicators . The emeAs this study demonstrates, adopting a system thinking approach involves at least three elements. First, it is essential to understand the choices and decisions being made by individual actors; second, to understand the positions of actors within the system, recognising that the choices of some actors have disproportionate influence on the system as a whole; third, it is important to understand the wider context affecting changes in the system over time , recognising that systems are dynamic, social entities which are in constant mutation or adaptation .The highly centralised structure of the social network in Somaliland had potential to help rapidly diffuse information between actors, which might be very useful in contexts of emergency . However, the rupture of relationship between the two central actors of the network completely disrupted the functioning of the rehabilitation sector and lead to the non-use of sustainability data in a sector that became the sum of dispersed actors. In Nepal, the cohesion between actors was maintained thanks to the role of a local champion and the injection of additional funds in the sector. The network remained very dense and decentralised and actors there appear to be gradually building a systemic vision of their sector, which takes account of data such as sustainability indicators for planning and negotiation purposes. These findings suggest that using sustainability indicators for a health system requires cohesion within the system between all (or most) actors, as well as an understanding, by actors, of the benefit of a collective vision for the sector. Contextual factors, such as the availability of funding for activities that primarily benefit the system rather than individual actors or organisations, can also support this. Further research is needed to analyse the different strategies that are required for health system interventions to alter the characteristics of social networks in social contexts for a collective good.


Androgens signal through the androgen receptor (AR) to regulate male secondary sexual characteristics, reproductive tract development, prostate function, sperm production, bone and muscle mass as well as body hair growth among other functions. We developed a transgenic mouse model in which endogenous AR expression was replaced by a functionally modified AR transgene. A bacterial artificial chromosome (BAC) was constructed containing all AR exons and introns plus 40 kb each of 5' and 3' regulatory sequence. Insertion of an internal ribosome entry site and the EGFP gene 3’ to AR allowed co-expression of AR and EGFP. Pronuclear injection of the BAC resulted in six founder mice that displayed EGFP production in appropriate AR expressing tissues. The six founder mice were mated into a Sertoli cell specific AR knockout (SCARKO) background in which spermatogenesis is blocked at the meiosis stage of germ cell development. The AR-EGFP transgene was expressed in a cyclical manner similar to that of endogenous AR in Sertoli cells and fertility was restored as offspring were produced in the absence of Sertoli cell AR. Thus, the AR-EGFP transgene under the control of AR regulatory elements is capable of rescuing AR function in a cell selective, AR-null background. These initial studies provide proof of principle that a strategy employing the AR-EGFP transgene can be used to understand AR functions. Transgenic mice expressing selective modifications of the AR-EGFP transgene may provide crucial information needed to elicit the molecular mechanisms by which AR acts in the testis and other androgen responsive tissues. Androgens are a class of steroid hormones that regulate prostate function, bone density, cardiac health, muscle mass, hair growth and fertility. Androgens diffuse through the plasma membrane and act via the intracellular androgen receptor (AR) to alter gene expression and intracellular signaling pathways in target cells. The two major functional androgens in mammals are testosterone and dihydrotestosterone (DHT). Because of the high levels of testosterone produced locally by the Leydig cells within the testis, this form of androgen is the major regulator of testis functions and the male reproductive tract. In most other tissues, the lower concentrations of testosterone present allow DHT to be the major acting androgen because DHT has a 10-fold greater affinity for AR than testosterone.2+] levels  levels . The res] levels , neurons] levels , prostat] levels , macroph] levels  as well ] levels ).In males, testosterone is essential for proper sexual differentiation and the maintenance of spermatogenesis, which is the progression of germ cell development into mature sperm , 9. FuncThe molecular mechanisms are beginning to be known by which androgen and AR regulate processes in Sertoli cells that are required to maintain spermatogenesis. For example, there is reduced expression of at least three tight junction components of the BTB  in the absence of AR expression in Sertoli cells . AR alsoin vivo model system in which endogenous AR expression is replaced with specific AR mutants that selectively activate either the classical or non-classical testosterone pathways would provide an essential tool for dissecting the molecular mechanisms by which AR acts. As a first step in achieving this goal, we produced transgenic mouse strains that co-express AR and EGFP from a BAC insert containing the complete mouse genomic AR gene and 40 kb each of 5’ and 3’ flanking sequences. The AR-EGFP transgene is expressed in testosterone responsive tissues and is correctly expressed in AR positive cells of the adult testis. Furthermore, we report the first transgenic rescue of AR function as the AR-EGFP transgene is able to restore complete spermatogenesis and fertility in a SCARKO mouse that lacks endogenous AR expression in Sertoli cells.Studies of SCARKO mice and testosterone depletion experiments have revealed specific processes in Sertoli cells that require testosterone action. However, little information is available regarding the specific functions of classical versus non-classical androgen signaling in regulating critical spermatogenesis processes. An All mouse procedures were approved under protocol IS00000296 titled Nongenomic Androgen Signaling in Sertoli Cells by the University of Pittsburgh Institutional Animal Care and Use Committee (IACUC), which operates under protocol A3187-01 from the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC).Three BACs containing all or parts of the gene encoding AR were obtained from the BACPAC Resource Center (BPRC) at Children's Hospital Oakland Research Institute . One BAC [mAR BAC-192 (RP24-352D1)] contains the entire gene  for AR. The other two BACs contain either 5’ [mAR BAC-185 (RP23-316P7)] or 3’ [mAR BAC-196 (RP23-102O11)] exons of the AR gene and either extensive 5’ or 3’ flanking regions . The BACNotI and XbaI sites that were engineered into the primers so the amplicon could be directionally subcloned into the multiple cloning site of pBluescript at NotI/XbaI (r) gene was amplified using PCR with primers that had AvrII/NotI and BamHI/EcoRI/ClaI sites added and inserted into AvrII and ClaI sites of the targeting cassette replacing 383 bp of AR sequence between the AvrII and ClaI sites. This process produced 598 bp and 276 bp homologous arms for recombination on both sides of the ampr cassette. Another 543 bp PCR fragment from the 3’ end of mAR BAC-185  floated on distilled water. The recombination cassette DNA was then electroporated into SW102 cells containing mAR BAC-185 and recombined as above. The resulting mAR BAC-236 (236 kb) was assessed using PFGE and Southern blotting to ensure correct recombination occurred.The 3’ end of the recombined BAC . Immunoreactive signals from western blot films were scanned with an Epson 1600 Expressions scanner using Epson Scan software.To prepare whole cell lysates, mouse tissues from decapsulated testes, epididymis, heart, liver, kidney, muscle and brain were isolated and homogenized in enhanced lysis buffer  with a protease inhibitor cocktail, rocked for 15 min at 4°C and then pelleted  to remove cell debris. Cell lysates were fractionated by SDS-PAGE, transferred to polyvinylidene difluoride (PVDF) membranes, and incubated with polyclonal primary rabbit antibodies EGFP,  at 1:1000, followed by horseradish peroxidase (HRP)-conjugated second antibody. The antigen-antibody complex was visualized with Millipore ImmobilonTestes were isolated and weighed followed by fixation in Bouin’s solution or 4% paraformaldehyde and parrafifin embedded. For analysis of testis morphology, Bouins fixed tissue sections (5 μm) were stained with a periodic acid-Schiff staining kit  followed by staining with hematoxylin. For analysis of EGFP or AR expression, paraformaldehyde fixed sections were probed with either primary goat antisera against GFP  or polyclonal primary rabbit antisera against AR . For EGFP imunodetection, biotin conjugated mouse anti-goat secondary antisera  was added and bound antibodies were detected as described by the kit instructions using DAB staining solution. Slides were counterstained with hematoxylin. For AR immunodetection, secondary donkey anti-rabbit antisera with conjugated cy3 fluor was added followed by staining with DAP1. A charged coupled device (CCD) video camera system was used to capture images of tubule cross-sections.260). Quantitative PCR (q-PCR) was performed on isolated testis genomic DNA using the Applied Biosystems 7500 Fast Real-Time System (Life Technologies Carlsbad CA) and SYBR Green PCR Master Mix (Life Technologies Carlsbad CA). For each reaction, 100 ng of genomic DNA and a final primer concentration of 250 nM each were used. Primers were designed to obtain amplicons of 212 or 245 bp. A common forward primer (Common AR For1) was used that anneals to the extreme 3’ end of the sequences flanking the AR gene that are present on the X-chromosome and in BAC-236E (t (cycle threshold) values were determined from triplicate samples in two separate experiments.Genomic DNA was extracted from testis samples using the DNeasy Blood and Tissue kit  according to the manufacturer’s instructions with the following modification: an additional 15 min incubation with RNase (10 mg/mL) was included after tissue digestion in lysis buffer. DNA concentrations were obtained using spectrophotometric determination (ODBAC-236E . Two revt (cycle threshold) using the EΔCt method, where E is the efficiency of the reaction and ΔCt = Ct (target gene) − Ct (control gene) (see Bio-Rad user bulletin #5279 at http://www.bio-rad.com). Because the amplification efficiencies for the 2 PCR products are approximately equal, the number of copies of BAC sequences can be normalized to the number of copies of endogenous AR sequences, which are present in one copy in the genome. The calculated copy numbers were reported after rounding to the nearest whole number. This method of determining the copy number requires small amounts of DNA and is found to be accurate for low (<10) transgene copy numbers [Prior to performing copy number assessments, dilutions of genomic DNA from mice containing endogenous AR sequences and BAC-236E were used to generate standard curves that provided the amplification efficiencies for the amplicons derived from endogenous flanking AR sequences and BAC-236E. The amplification efficiencies were found to be equivalent (within 0.5%). For each transgenic mouse strain, the relative number of copies of endogenous AR sequences and BAC-236E derived sequences were determined from their respective C numbers .Results were analyzed by ANOVA with Newman-Keuls PLSD at a 5% significance level utilizing GraphPad Prism 4.3 .Sal1 restriction site to the 3’ end of mAR BAC-196. A SalI fragment consisting of the region extending from the second intron of AR through the 3’ end of mAR BAC-196 that included the targeting cassette was excised and recombined with mAR BAC-185 to produce mAR BAC-236 containing the entire AR gene plus extended 5’ and 3’ flanking sequences  containing overlapping regions of the AR gene locus were recombined to create a new BAC denoted mAR BAC-236 that encompasses the entire 167 kb of the genomic mouse AR gene plus 40 kb each of 5’ and 3’ flanking sequence . Specifiequences . To faciequences .The mAR BAC-236E construct was used for pronuclear injection and 9 founder mice containing germ line transmitted mAR BAC-236E were obtained. Whole testis extracts were assessed by western blot for expression of EGFP as a marker for expression of the mAR transgene. These studies showed that six of the nine lines of transgenic mice expressed EGFP in the testis . EGFP al+/y) were mated with female mice in which either one or both alleles of AR had flox sites flanking exon 4  [The mouse lines that expressed the AR-EGFP transgene were mated with mice expressing the Cre recombinase driven by the Sertoli cell specific AMH promoter (AMH-Cre). The resulting male double transgenic mice  . GenotypIndividual AR-EGFP founder transgenic mouse strains were investigated further to determine the extent of the rescue phenotype. The AR-EGFP transgene strain 2464 permitted complete rescue of SCARKO mice with testis weights similar to that of its wild type siblings . StrainsTo confirm that the AR-EGFP transgene was expressed in the testes of the transgenic mice in a cell-specific manner, adult testis tissue sections were probed for the expression of EGFP. In mice containing the AR-EGFP transgene, EGFP immunoreactivity was observed throughout the cytoplasm of Sertoli cells surrounding germ cells as well as in Leydig cells that are known to produce AR . Immunofin vivo model in which an AR transgene is capable of rescuing AR function in a Sertoli cell specific AR null background. These results prove that the mAR BAC-236E construct has sufficient regulatory sequence to not only direct expression of AR in Sertoli cells in vivo but express AR in a stage specific manner so that spermatogenesis and fertility can be restored.As shown previously , 29, hisWe report the first transgenic rescue of AR function. We obtained 9 founder mice from our pronuclear injections, of which 6, expressed EGFP from the AR-EGFP modified BAC transgene. This number of successful founders suggest that the transgene is insulated from copy number and position effects due to its size. Because the AR gene is required for fertility and is hemizygous (present only on the X chromosome) in males, the crosses required to produce rescue mice in males were made more complex. Nevertheless, we found that expression of the AR-EGFP transgene from 5 of the founders was capable of restoring spermatogenesis in SCARKO mice that lack Sertoli cell-specific endogenous AR expression. The rescue of testis size and sperm production varied for the 5 mouse strains. One founder strain was found to rescue testis size to normal levels and reestablished full spermatogenesis in nearly all seminiferous tubule cross sections. Other strains were capable of restoring full spermatogenesis but not in all tubules. The mechanisms responsible for the variable rescue of spermatogenesis are not yet understood. However, it is possible that the extent of rescued spermatogenesis may be related to the expression levels of the AR-EGFP transgene and the capability of each mouse strain to express the transgene in Sertoli cells in a stage specific manner. In wild type rodents, AR is expressed at high levels during stages VI-VIII during the cycle of the seminiferous epithelium but AR expression is difficult to detect during other stages . Also, iThe AR-EGFP transgene was able to restore male fertility in otherwise infertile SCARKO mice as evidenced by the pups produced by the mating of mice derived from the 2457 founder strain in the SCARKO background. However, pups were not produced in 2 of 8 matings and small litter sizes were observed for two other matings. The lower fecundity of some of the matings may be explained by the intermediate rescue phenotype displayed by the 2457 strain that displayed a testis size that was restored to only 68% of wild type. Follow up studies will include evaluation of the 2464 line of rescue mice to assess the fertility of mice that had testis size and spermatogenesis restored to wild type levels.There have been no previous reports of transgenic animals that successfully express AR using limited 5’ regulatory sequences. The use of larger transgenes for AR was first reported by Sopher et al. in which a 450 kb YAC containing the 100 kb human AR gene was modified by homologous recombination to add 100 CAG repeats to exon 1 of the human AR gene. This AR transgene successfully recapitulated X-linked spinal and bulbar muscular atrophy (SBMA) in mice . This apFurther evidence that substantial 5’ regulatory sequences are required for correct AR expression was provided by Ye et al. . These iThe importance of correct temporal and cell-specific expression of AR transgenes was shown previously using a model in which expression of an AR transgene was driven by the Sertoli cell-specific Androgen Binding Protein (ABP) promoter in the presence of an intact endogenous AR gene . In thisIn the adult testis, expression of the AR-EGFP transgene is confined to the seminiferous tubule stages where endogenous AR is expressed. The restriction of AR-EGFP transgene expression to stage VI-VII Sertoli cells provides additional evidence that the transgene is expressed under tight regulation in a manner similar to that of endogenous AR. This timing and proper regulation of AR expression in the transgenic rescue mice is likely due to the use of the extensive genomic AR transgene and its intronic and associated 5’ and 3’ flanking regulatory regions (40 kb each). The lack of any effect on testis size in the presence endogenous AR expression plus the AR-EGFP transgene also suggests that expression of the AR-EGFP transgene is not affected by copy number and may be under strict physiological feedback control due to the large AR flanking regions that are associated with the AR-EGFP transgene. This proposed feedback control of AR transgene expression may explain the similar levels of spermatogenesis rescue that occur in the presence of 1 to 3 copies of the transgene. The proper expression profile of the AR transgene in Sertoli cells suggests that the transgene mimics the endogenous AR gene in other AR expressing tissues. To this end, the gross morphological structure of the Leydig cells and peritubular cells of the testis appeared normal, which suggests again that AR-EGFP transgene did not affect endogenous AR function in these cells. Finally, normal AR male mice with the transgene were functionally fertile and did not show any abnormalities.in vivo mutational analysis in the context of androgen signaling in the testis. Using the strategy that we have validated, additional transgenic mouse strains can be created in which endogenous AR is replaced with mutated AR constructs that can selectively activate only the classical or non-classical signaling pathway. In the future, these transgenic models could be used to study the functions of each testosterone-mediated pathway that are required to maintain spermatogenesis. These transgenic mice also may allow the identification of molecular and cellular mechanisms by which testosterone acts in Sertoli cells to maintain fertility and to facilitate the design of therapies to cure specific defects in testosterone-mediated signaling that cause infertility. Furthermore, transgenic mice incorporating the AR transgene or mutated versions of the AR transgene could be used to test male contraceptive candidates based on the blocking of specific testosterone dependent processes during spermatogenesis. Finally, the AR transgene and mutated derivatives may be used to study testosterone regulation of physiologically important processes in other tissues including oocyte development, sexual differentiation, heart function, muscle mass maintenance and prostate tumor progression.The purpose of producing this mouse model expressing the AR-EGFP transgene was to provide proof of principle that the functional domains of AR protein could be studied through S1 FigNotI-XbaI fragment at the 3' end of the mAR BAC-196 was amplified using PCR. A 383 bp AvrII-ClaI fragment was removed by restriction digestion and a 1220 bp PCR product containing the ampr cassette with flanking AvrII and Cla1 sites from pBluescript SK- was inserted into the 1176 bp fragment. BamHI and EcoRI sites were introduced into the amp cassette PCR product. The recombination arm  was amplified by PCR and inserted into the BamHI and EcoRI sites. The amplification of the recombination arm introduced a SalI site into the cassette allowing the removal of the 3’ SalI fragment in a later step. The final targeting cassette was named pAR2.6-Amp.A 1176 bp (PDF)Click here for additional data file.S2 FigThe Galk cassette was amplified using primers with 50 bp overhangs that were homologous to regions within exon 8 of AR This GalK cassette was selected for using minimal media plates containing galactose. The IRES-EGFP cassette was amplified by PCR using primers having the same 50 bp overhangs. Selection for BACs in which the Galk cassette was replaced by the IRES-EGFP cassette was performed on 2 deoxy galactose (DOG) plates .(PDF)Click here for additional data file.S1 TableAll oligonucleotides are 5’ to 3’.(PDF)Click here for additional data file.S2 TableUnderlined sequences are GalK or IRES.EGFP specific whereas other sequences are AR specific. The GalK cassette was then replaced with an IRES.EGFP cassette that did not have a PolyA sequence so that the mRNA produced would use the polyadenylation signal of AR. This recombineering step produced mAR BAC-236E. All oligonucleotides are 5’ to 3’.(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.


The aim of this work was to study the androgen receptors (AR) expression in pituitary pars distalis (PD) of male viscachas in relation to growth and reproductive cycle. AR were detected by immunocytochemistry and quantified by image analysis. Pituitary glands from fetus, immature, prepubertal, and adult viscachas during their reproductive cycle were used. In the fetal PD, the immunoreactivity (ir) was mainly cytoplasmic. In immature and prepubertal animals, AR-ir was cytoplasmic (ARc-ir) and nuclear (ARn-ir) in medial region. In adult animals, ARn-ir cells were numerous at caudal end. AR regionalization varied between the PD zones in relation to growth. In immature animals, the ARn-ir increased whereas the cytoplasmic expression decreased in relation to the fetal glands. The percentage of ARc-ir cells increased in prepubertal animals whereas the nuclear AR expression was predominant in adult viscachas. The AR expression changed in adults, showing minimum percentage in the gonadal regression period. The variation of nuclear AR expression was directly related with testosterone concentration. These results demonstrated variations in the immunostaining pattern, regionalization, and number of AR-ir cells throughout development, growth, and reproductive cycle, suggesting the involvement of AR in the regulation of the pituitary activity of male viscacha. Androgens are important steroidal hormones involved in the sexual development and reproduction of male mammals. They are essential during development and for the maintenance of secondary male characteristics, initiation and continuation of spermatogenesis, and gonadotropin regulation . TestostSeveral studies have demonstrated that the pituitary gland is an androgen target tissue and serves as a potential site of androgen feedback on hormone secretion , 6, 7. TSome species use environmental cues, the most important being the photoperiod, to synchronize their endogenous cycles and to reproduce at the optimal time of the year . The pitLagostomus maximus maximus), is a hystricomorph rodent of seasonal reproductive patterns. The annual reproductive cycle of adult male animals has three periods: reproductive (summer and early autumn), gonadal regression (winter), and gonadal recovery (spring). Previous studies have demonstrated that the gonadal regression period is characterized by an increase of the secretory ability of the pineal gland [Our experimental model, the viscacha , using traps placed in their burrows. In San Luis, in summer, the light phase is up to 14 h light daily (14L : 10D) with an average temperature of 25°C. In winter, the light phase decreases to 10 h (10L : 14D), and the average temperature is 10°C. In spring, the light phase increases to 12 h (12L : 12D), and the average temperature is 15°C.n = 4) and late (n = 4) pregnant females classified on the basis of fetal weight and crown-heel length [n = 4) and prepubertal  according to body weight and light microscopy observations of testes [A total of eight fetal male pituitary glands were collected. The fetal pituitaries were collected from fetuses at mid- , following the design used in previously reported studies [The animals were intramuscularly anesthetized with a combination of ketamine  and xylazine  at a dose of 12 and 0.4 mg/kg, respectively. The blood was collected for cardiac puncture for the evaluation of serum hormone concentration and quickly sacrificed by intracardiac injection of Euthanyle . The brain was rapidly exposed and the pituitary processed for light microscopy. The pituitaries were fixed in Bouin's fluid, embedded in paraffin, and sagittally sectioned  guidelines for the use of experimental animals. Moreover, the Biodiversity Control Area of the San Luis Ministry of the Environment (Argentina) approved a study protocol for conducting scientific research within the territory of this province (Resolution number 45.PBD.2014).2O2 in water for 20 min. Nonspecific binding sites for immunoglobulins were blocked by incubation for 20 min with normal serum diluted in PBS containing 1% bovine serum albumin, 0.09% sodium azide, and 0.1% Tween-20. Sections were incubated with the primary antibody: 6 hours in a humidified chamber at 20°C with rabbit polyclonal anti-human (h) AR . After rinsing with PBS for 10 min, immunohistochemical visualization was carried out using the Super Sensitive Ready-to-Use Immunostaining Kit , which was used as follows: sections were incubated for 30 min with diluted biotinylated anti-IgG and, after being washed in PBS, were incubated for 30 min with horseradish peroxidase conjugated streptavidin and were finally washed in PBS. AR immunoreactivity cells (AR-ir cells) were visualized using freshly prepared solution with 100 μL 3,3′-diaminobenzidine tetrahydrochloride chromogen in 2.5 mL PBS and 50 μL H2O2 substrate solution. In all cases, two experiments for controlling the specificity of the primary antibody were made: (i) omission of primary antibody and (ii) adsorption of primary antibody with a homologous antigen. No positive structures or cells were found in these sections. Rat prostate was used as positive control . Antigen retrieval was performed by microwaving the sections for 6 min (2 × 3 min) at full power in a sodium citrate buffer . Endogenous peroxidase activity was inhibited with 3% H control .μm2 (reference area) was defined on the monitor; distance calibration was performed using a slide with a micrometric scale for microscopy . The morphometric study was carried out as follows: 3-4 regularly spaced serial tissue sections (100 μm each) from a pituitary were used, and microscopic fields were examined under a 40x objective. In each section, 25 microscopic fields were randomly selected throughout the PD, five from each region  or end  of the PD. In each image, the percentage of immunoreactive (-ir) cells in the PD  was obtained according to the formula A/(A + B) × 100. Each image contained approximately 250–280 cells. The number of immunoreactive cells (A) and the number of nuclei in immunonegative cells (B) were counted.A computer-assisted image analysis system was used for the morphometric analysis as previously reported , 41. The∗ Medical IMMULITE∗ 1000 Immunoassay Analyzer .Blood samples were incubated at 4°C for 30 min and centrifuged at 5,000 g for 5 min, and serum was removed. Testosterone serum concentrations were measured using the total testosterone test as previously reported by Chaves et al. . This teP value of less than 0.05 was considered statistically significant.The results were expressed as mean ± the standard error of the mean (SEM) for all morphological data sets. The data were analyzed by nonparametric test procedure, the Kruskal-Wallis test, for overall significance of group, followed by the Mann-Whitney test for individual comparisons, using the Infostat 2008 version. A AR-ir cells were studied by immunohistochemistry on each zone of the pituitary PD parenchyma of fetal, immature, prepubertal, and adult male viscachas. The AR-ir cells were observed throughout the PD parenchyma, and immunohistochemical results revealed their presence in the nuclei (ARn-ir) and in the cytoplasm (ARc-ir).P > 0.05)  and the percentages of ARc-ir cells did not differ significantly among different zones of the PD  than in the ventral region and at the rostral and caudal ends .In fetal pituitary from late pregnancy, ARc-ir cells were found around blood vessels or among other pituitary parenchymal cells. The percentage of ARc-ir cells in the dorsal region was significantly higher  in different PD zones  whereas the lowest percentage was found at the caudal end . No variations were observed in the percentage of ARc-ir cells throughout the reproductive cycle (P > 0.05).In order to study the seasonal expression of AR, the three periods of the reproductive cycle were analyzed. The total percentages of ARn-ir and ARc-ir cells obtained from three periods of the reproductive cycle of adult male viscachas were statistically analyzed . The perP < 0.001; The testosterone serum concentrations of immature animals were below the detection limit (<0.20 ng/mL) and too low to reliably reveal differences from the other groups. The testosterone serum concentrations of prepubertal and adult viscachas in the gonadal regression period were lower than those in adult viscachas during their reproductive and gonadal recovery periods  the regionalization and immunostaining pattern of pituitary AR vary in relation to growth; (2) the AR expression was mainly cytoplasmic (ARc-ir) in fetuses; (3) a significant increase of ARc-ir cells was observed in prepubertal animals; (4) the nuclear AR (ARn-ir) expression increased in relation to development, reaching the highest values in adult animals; (5) the ARn-ir cells varied seasonally with maximum values during the reproductive period and minimum values during the gonadal regression period; (6) the amount of ARn-ir cells was directly related to testosterone serum concentration.There are still many questions about the relations of AR expression with the pituitary development and hormone secretion. Wu et al.  did not Some reproductive characteristics of female viscachas, such as polyovulation, implantation, and offspring birth, have been described in previous studies. Gestation lasts approximately 154 days, an unusually long period for a rodent, and it finishes in spring when the survival probabilities are ideal for the mother and offspring , 43, 44.The hypothalamic-pituitary-gonadal axis is activated during puberty when hormonal modulation of sexual behavior is organized . During In immature male viscachas, a significant increase of nuclear AR and an important decrease of cytoplasmic AR were observed. In prepubertal viscachas, more nuclei and cytoplasm appear, and there is a clear increase of ARc-ir cells mainly in the medial region, suggesting that more cells begin to express AR in preparation for sexual maturation. In adult, the immunostaining is mainly nuclear and regionalized at the caudal end of PD, suggesting that AR are active in the PD cells. The present results demonstrate variations in the regionalization and immunostaining pattern of AR during sexual maturity, probably in relation to the contribution of androgens reaching the different pituitary zones through blood vessels. Thus, the nuclear AR expression in pituitary PD of viscacha varies according to gonadal development and testosterone serum concentration. Our results are in agreement with those found by Wood and Newman , suggest Lagostomus.Bibliographic reports referring to seasonal variations of RA are scarce. Lu et al.  have recIn conclusion, our results demonstrate that AR expression is related to the pituitary histophysiological changes during the growth and sexual maturity of male viscacha. Moreover, they are likely to participate in the seasonal reproduction control in adulthood. However, the precise action mechanism of AR in the pituitary viscacha needs further study.


However, the specific effects of combined Cl+β1 blockade during MU are unknown.Clenbuterol (Cl), a β2-adrenoceptor (AR) stimulation via Cl (2 mg/kg/day) alone, and in combination with β1-AR blockade using metoprolol ((Met), 250 mg/kg/day), on whole heart/cell structure, function and excitation-contraction (EC) coupling in failing (induced by left coronary artery (LCA) ligation), and unloaded ) failing rat hearts. Combined Cl+Met therapy displayed favourable effects in HF: Met enhanced Cl's improvement in ejection fraction (EF) whilst preventing Cl-induced hypertrophy and tachycardia. During MU combined therapy was less beneficial than either mono-therapy. Met, not Cl, prevented MU-induced myocardial atrophy, with increased atrophy occurring during combined therapy. MU-induced recovery of Ca2+ transient amplitude, speed of Ca2+ release and sarcoplasmic reticulum Ca2+ content was enhanced equally by Cl or Met mono-therapy, but these benefits, together with Cl's enhancement of sarcomeric contraction speed, and MU-induced recovery of Ca2+ spark frequency, disappeared during combined therapy.We studied the chronic effects (4 weeks) of β2-AR simulation +β1-AR blockade therapy is likely to be a safe and beneficial therapeutic HF strategy, but is not as effective as mono-therapy in enhancing myocardial recovery during LVAD support.Combined Cl+Met therapy shows superior functional effects to mono-therapy in rodent HF, but appears inferior to either mono-therapy in enhancing MU-induced recovery of EC coupling. These results suggest that combined β The use of LVADs in end stage HF continues to expand 2+ handling HF-associated remodelling is a complex series of whole heart, cellular, molecular, structural and functional changes affecting various pathways, including EC coupling, the extracellular matrix (ECM), apoptosis and adrenergic receptor signalling 2-adrenoceptor (AR) agonist Cl was employed alongside conventional HF pharmacotherapy, primarily as an agent to prevent myocardial atrophy. Cl's ability to induce physiological cardiac hypertrophy “Combination therapy” describes a strategy in which pharmacotherapy is combined with LVAD support, to enhance LVAD–induced positive remodelling and/or combat LVAD-induced “negative” remodelling BTR remains controversial, but the clinical success of combination therapy  justifies continued scrutiny of this strategy. Therefore, the search for other novel pharmacological strategies aimed at enhancing LVAD-induced functional recovery continues, and we have recently shown the pacemaker current inhibitor ivabradine and Met to show positive effects on myocardial size, EC coupling and the ECM during mechanical unloading in rodent HF 1-AR signalling 1-/β2-AR signalling indicates distinct and often opposing physiological and pathological actions on cardiac structure and function 1-AR signalling associated with pro-apoptotic and maladaptive remodelling 2-AR signalling with cardio-protective actions, improved function and myocyte viability 1-AR blockade (Met) and β2-AR stimulation (fenoterol) has shown synergistic beneficial remodelling effects in rodent HF 2-AR stimulation (Cl) demonstrates positive effects during MU in rodent studies 2-AR stimulation and β1-AR blockade remain unstudied within this setting.The efficacy of β-AR blockade in HF therapy is established 2-AR stimulation (Cl) and β1-AR blockade (Met) on reverse remodelling during MU in a rodent HF model. In particular, we have examined the drug effects on whole heart/cell size, function, EC coupling and T-tubule structure in a rat model of established HF, 12 weeks post LCA ligation, and during MU using HATx.In this study, we report for the first time the chronic effects of combined βThe investigation conformed to the Guide for the care and Use of Laboratory Animals published by the US National Institutes of Health . Experiments were approved by the Harefield Heart Science Centre ethics review panel.(For extended methods please refer to 2 (2–3 L/min), intubated and mechanically ventilated via rodent ventilator, and a left thoracotomy performed. The pericardium was opened and heart visualised, the proximal LCA was identified and a suture (6–0 prolene) placed underneath the artery and lightly tied. Heamostasis was achieved and the chest closed. Sham operated animals underwent the identical protocol but the suture was not tied. Acute 24 hour mortality was 25% in the LCA ligation group and 0% in the sham group. Male syngeneic Lewis rats  were used for all experiments to avoid the need for immunosuppression following subsequent heterotopic abdominal transplant (HATx) in the mechanical unloading (MU) groups. 1 week after surgery LV dimensions and function (ejection fraction (EF)) were assessed by transthoracic echocardiography (TTE) , as previously described 2 (2–3 L/min) to provide adequate sedation but minimal cardiac depression. Experiments were not performed in non-failing hearts as these show an exaggerated atrophic response during unloading compared with failing hearts HF was induced via permanent LCA ligation as previously described 2 (2–3 L/min), and self-ventilated throughout the procedure. The failing heart was removed from the donor animal under cardioplegic arrest, and the donor aorta anastomosed to the recipient abdominal aorta and the donor pulmonary artery to the recipient inferior vena cava. Total ischaemic time was <40 minutes and operative mortality <4%, with no deaths occurring in any of the treatment groups during the 4 week treatment period.In order to test the effects of pharmacotherapy (Cl and/or Met) in combination with MU, failing hearts (12 weeks after LCA ligation) were transplanted into the abdomen of syngeneic male Lewis rats (weight ∼250 g) as previously described Recipients were then assigned to 4 treatment groups: (1) Cl  2 mg/kg/day via subcutaneous infusion using osmotic minipump , (2) Met  250 mg/kg/day in drinking water, (3) Met 250 mg/kg/day +Cl 2 mg/kg/day via drinking water and osmotic minipump respectively, (4) no drug treatment . Animals in Met-treated and MUHF group also received an osmotic minipump containing normal saline. All groups contained 8 animals and treatment duration was 4 weeks. At end of treatment Cl levels in blood were measured as previously described in vivo heart function was measured via repeat TTE.In order to test the effects of Cl and Met in non-transplanted failing hearts, HF animals were randomly allocated to 4 treatment groups: (1) Cl 2 mg/kg/day, (2) Met 250 mg/kg/day, (3) combined Met+Cl treatment, (4) no drug treatment . Drug administration was via drinking water and osmotic minipump, exactly as described above. At the end of the treatment period We have compared the effects of Met during mechanical unloading with those of ivabradine in a previously published study in vivo HR and arrhythmia studies. Animals underwent continuous 24 hour ECG recording at weekly intervals during the treatment period. In vivo ECG recordings were acquired using Dataquest ART 3.1 software , and offline HR/arrhythmia analysis performed using ECG-Auto 2.4 software .4 animals per treatment group received implantable ECG telemetry transmitters  on day 0 of the 4 week treatment period, for Adequacy of anaesthesia during all procedures was monitored by loss of reflexes, degree of muscle relaxation and respiration rate. Respiration rate and body temperature were monitored throughout all procedures and body temperature maintained at 37°C, via heating mat. Analgesia was provided by subcutaneous (SC) injection of vetergesic (buprenorphine), just prior to skin incision. For LCA ligation and implantation of telemetry device a single dose of 0.03 mg/kg was utilised, and for heterotopic abdominal heart transplant both the donor and recipient received a single dose of 0.05 mg/kg. Post-operative condition of the rats was monitored, and repeat SC injection administered as required (0.01–0.05 mg/kg) every 12 hours during the first week post-operative week. At the end of the 4 week treatment period animals were sacrificed via schedule 1 cervical dislocation. In each group, 4 hearts were utilised for acute cellular experiments, and 4 for histological analysis.LV cardiomyocytes were isolated by standard enzymatic digestion for 8–10 minutes using collagenase  and hyaluronidase  2+ handling in field stimulated cardiomyocytes was performed using an inverted microscope (TE 200), Ionoptix system  and the Ca2+ sensitive, single-excitation dual emission fluorescent dye Indo-1 AM . In addition, sarcoplasmic reticulum (SR) Ca2+ uptake (SERCA) and sodium-calcium exchanger (NCX) contribution to Ca2+ extrusion were assessed using rapid caffeine application as previously described Simultaneous assessment of sarcomere shortening and Ca2+ current were studied and normalized to cell capacitance, as previously described Cells were studied using a MultiClamp 700A (Axon Instruments) in whole cell patch configuration. Current-voltage relationships for L-type Ca2+ levels were assessed using the fluorescent dye Fluo-4 AM  and the same confocal microscope as previously described Cell volume and t-tubule organisation were studied using the membrane binding dye di-8-Anepps  and the Zeiss Axiovert microscope  with an LSM 510 confocal attachment. In addition, local changes in CaStatistical comparison of data was performed using one-way analysis of variance followed by Bonferroni post-hoc test for individual significant differences, or Student's t-test where appropriate. All statistical analyses were performed using Prism 4 software  and P<0.05 was considered significant. Data are expressed as mean ±SEM [n], where n is the number of cells unless otherwise specified. All of the experiments were performed using a minimum of four animals, unless otherwise stated.There was no difference in average HR between sham-operated and HF group at any time point (mean HR (bpm) at 4 weeks: sham 332±4.8 vs. HF 329±5.5; P>0.05). HR of transplanted failing hearts (MUHF) was significantly lower (∼20%) than that of non-transplanted failing hearts at all time-points (LCA ligation induced LV dysfunction after 12 weeks (time of randomisation), characterised by reduced LVEF (%): sham 83.3±0.8 vs. HF 37.6±0.7; reduced LVFS (%): sham 48.3±0.7 vs. HF 16.6±0.5; and LV chamber dilatation (diastolic diameter (cm)): sham 0.60±0.03 vs. HF 0.98±0.04, P<0.001 for all parameters.In-vivo function of transplanted failing hearts was not assessed, but that of non-transplanted hearts was assessed via repeat TTE at the end of the 4 week treatment period. Cl improved contractile function of non-transplanted failing hearts, and improvement in LVEF was enhanced further by combination with Met .LCA ligation caused impaired cardiomyocyte contractility and deranged Ca2+ leak, secondary to altered RyR function, is one mechanism contributing to reduced SR Ca2+ load in HF 2+ sparks are complex entities regulated by multiple factors, and represent a measure of diastolic SR Ca2+ release 2+ spark frequency was increased in HF group compared to sham group suggesting increased SR Ca2+ leak ((sparks/100 µm/sec): sham 0.80±0.17 2+ spark frequency (sparks/100 µm/sec): MUHF 0.82±0.13 2+ current and t-tubule density  and β1-AR blockade (Met) to improve depressed whole heart function in HF, to a greater degree than either mono-therapy. Cl's improvement in LVEF was enhanced by Met, but the mechanisms underlying this superiority appeared independent of cellular EC coupling. Met and Cl mono-therapy displayed distinct EC coupling effects. Met's effects were more favourable than those of Cl, with lack of evidence supporting additive or synergistic benefit, and more suggestive of selected antagonism during combined therapy. Met alone, and in combination with Cl normalised speed of sarcomeric contraction, relaxation, Ca2+ release and Ca2+ transient amplitude, and enhanced SR Ca2+ content; whereas Cl mono-therapy improved SR Ca2+ content to a lesser degree than Met, and normalised deranged Ca2+ spark frequency, L-type Ca2+ current and t-tubule density, positive effects that were lost during combined MetCl therapy. Of note, undesirable Cl-induced hypertrophic/chronotropic effects were prevented during combined therapy.The second part of this study showed combined β2-AR stimulation +β1-AR blockade is likely to be a safe and beneficial HF therapeutic pharmacological strategy and C) either the strategy of β2-AR stimulation or β1-AR blockade is likely to be superior to that of combined β2-AR stimulation +β1-AR blockade in enhancing functional recovery during LVAD support. This is, however, in contrast with the current clinical experience with β1-AR blockade in patients with LVADs and could be explained by the type and degree of adverse remodelling obtained in our models. It cannot be excluded that subpopulations of LVAD patients with specific, lower degrees of dysfunction may benefit from this pharmacological treatment and more clinical studies are warranted.These results suggest that A) Cl's success in enhancing functional recovery in LVAD-supported patients is unrelated to myocardial size, and reliant on positive EC coupling effects, B) combined βLVAD-mediated unloading of failing ventricles regresses cardiac and cellular hypertrophy A key question surrounding Cl is whether anti-atrophic properties contributed to enhanced recovery rates produced by the Harefield Protocol As we have previously shown 2+ handling elements adversely remodelled during HF. Recovery of SR Ca2+ content, L-type Ca2+ current fast inactivation and Ca2+ transient amplitude is seen LVAD support induces reversal/alteration in expression and function of numerous Ca2+ transient amplitude, speed of Ca2+ release and sarcomeric contraction, SR Ca2+ content, Ca2+ spark frequency, L-type Ca2+ current amplitude and t-tubule density. Drug effects on this recovery were not clear-cut but overall, the positive effects of either mono-therapy disappeared during combined therapy. MU-induced recovery of Ca2+ transient amplitude, speed of Ca2+ release and SR Ca2+ content, proven markers of recovery during LVAD support 2+ spark frequency).In the present study, MU recovered HF-induced deranged EC coupling with normalisation of Ca2-AR mRNA expression 2-AR overexpression with improved function following MU 2-AR agonists during LVAD support. Specific study of β1-AR blockade during MU is lacking. Hence, mechanisms driving Met's effects are speculative. Enhancement of MU-induced recovery of β-AR responsiveness 2+ current and t-tubule density recovery highlight potentially unwanted effects of Cl and Met, the significance of which requires further investigation.Cl's effects may originate from improved myofilament sensitivity 1-ARs promotes relative augmentation of β2: β1-ARs ratio and signalling 1-AR signalling is thought protective against heightened sympathetic stimulation, and the clinical efficacy of β1-AR blockade is proven Deranged β-adrenergic signalling is instrumental in HF pathogenesis 2-AR signalling is also considered cardio-protective 2-AR agonism may be beneficial in clinical HF is long-standing 2-AR agonist effects such as elevation of HR and arrhythmia risk shown by certain studies 2-AR agonists in HF therapy. These factors, coupled with expanding evidence supporting β-blocker efficacy 2-AR agonist tachyphylaxis, rendered the notion of therapeutic β2-AR agonism illogical and unsound. Hence, little clinical data exists regarding β2-AR agonist administration within this context, with data derived from a few small, non-randomised studies in HF patients 2-AR agonist therapy Augmented β2-AR agonism represents a beneficial HF therapeutic strategy, albeit alongside that of clinically proven β1-AR blockade 2+ release, Ca2+ transient amplitude, and SR Ca2+ was not enhanced by Cl. T-tubule disruption uncouples the normally tight relationship between L-type Ca2+ channels and RyRs, reducing EC coupling efficiency 2+ current and t-tubule density, along with RyR function . Such improvements appeared not to alter cellular contractility, but were abolished by Met.This study re-enforces the concept that β1-AR signalling broadly promoting apoptosis 2-AR signalling driving improved myocyte viability 2+ handling protein expression e.g SERCA2a or RyR 2-AR agonist tachyphylaxis is recognised following chronic administration 2-AR agonist (fenoterol) tachyphylaxis (caused by decreased β2-AR density), producing extension of fenoterol's positive functional effects. Such a process may also explain the findings from our, albeit shorter study.Additive anti-apoptotic effects of either Cl 2-AR stimulation or β1-AR blockade is likely to be superior to that of combined β2-AR stimulation +β1-AR blockade in enhancing functional recovery during LVAD support. Such findings warrant further investigation.In conclusion, our study shows that Met but not Cl prevented MU-induced myocardial atrophy, with atrophic worsening during combined therapy. Cl and Met broadly enhanced MU-induced recovery of deranged EC coupling, but this enhancement was lost during combined therapy. These results suggest that A) Cl's success in enhancing functional recovery in LVAD-supported patients is unrelated to myocardial size and reliant on positive EC coupling effects and B) either the strategy of β2-AR simulation (Cl) +β1-AR blockade (Met) displays superior functional effects to either mono-therapy, and undesirable Cl-induced hypertrophic/chronotropic effects are prevented by Met. Mechanisms underlying this superiority appeared independent of cellular EC coupling and require further investigation. These results suggest that this pharmacological strategy is likely to be safe and beneficial in HF therapy.In the second part of this study, we show for the first time in a model of established rodent HF that combined βMet was used in this study to facilitate comparison with previous studies using this rodent HF model. Bisoprolol and not Met is used in the Harefield Protocol. As such, findings from this study may not be directly applicable to this specific clinical scenario.Methods S1A detailed description of Material and Methods is provided. The file also contains Figure S1 showing the ventricular ectopic (VE) rate in sham, failing and treated failing hearts.(DOC)Click here for additional data file.


Hip fractures are the leading cause of hospitalization for injuries among the elderly population and have a substantial impact on both the patient and the healthcare system. The care pathway for a person with a disability goes through a set of integrated and multidisciplinary activities and care interventions. Starting with timely surgery in the acute phase, followed by adequate rehabilitation assistance and ending with territorial assistance.The aim of the study is to evaluate the association between early surgery and access to rehabilitation of elderly patients with hip fracture.We identified elderly patients hospitalised for hip fracture, between 1 January 2012 and 31 October 2012 using Hospital Information System (HIS) of the Lazio region. The outcome considered was the access to rehabilitation within 60 days from the date of hospital discharge. Rehabilitation access was derived from Admission/Discharge Rehabilitation Report (ADRR), for hospital rehabilitation, and from Residential Rehabilitation Information System (RRIS).We considered clinical variables, residence and level of education as potential risk factors of the outcome. The factors significantly associated with outcome  were selected by a bootstrap stepwise procedure and their Risks Ratio (RR) were estimated through a multivariate regression model.We selected 5,030 patients aged 65+ hospitalised for hip fracture in Lazio region, 59% with an access to rehabilitative care within 60 days from discharge. The access to rehabilitation was less likely for older patients , for patients with a longer acute event hospital stay  and for patients with senile dementia . By contrast, the probability was higher for patient who had surgery within 48h  and for residents in Rome (adjusted RR=1.24p<0.001).The level of education seems to be negatively associated with the outcome (adjusted RR=0.79 p<0.05 for patients with high level), probably because patients with a higher level of education could more easily obtain access to private services.Despite the fact that following hip fracture rehabilitation should involve all patients, in the Lazio region the percentage of access to rehabilitation within 60 days from hospital discharge is 59%. Access to rehabilitation is strongly dependent on age, clinical characteristics and acute hospital care; in particular, waiting time for surgical treatment is strongly correlated with access to rehabilitation. This could suggest that the care pathway evaluation should take into account the association between the acute and rehabilitation phase.