To conduct qualitative interviews to evaluate and refine the Itch Diary (ID) and weekly version of the PBC-40 in patients with itching associated with primary biliary cholangitis (PBC).Twenty adults with self-reported PBC diagnoses and recent/ongoing itching of at least moderate intensity participated in face-to-face qualitative combined concept elicitation (CE) interviews and cognitive interviews after completing the morning and evening versions of the ID and weekly version of the PBC-40. These questionnaires were evaluated to confirm saturation of concepts of interest and cognitively test the English language versions of the measures in patients with PBC in the US and Canada. Transcripts were organized into descriptions of PBC-related symptoms and symptom-related impacts using a structured coding framework. Two waves of interviews were conducted; revisions made after wave 1 were further tested in wave 2.Interview results confirmed the relevance of concepts presented in the PBC-40 and ID to patients’ experiences. Saturation of concept was achieved. Itching-related signs and symptoms (46%) were the most commonly expressed symptom concept in the CE interviews followed by energy-related (14%) and additional signs/symptoms (13%). Several changes to the ID were made in response to cognitive interview results. Changes to the PBC-40 included adaptations from British to North American English, and the appropriateness of a 7-day recall period was confirmed.Relevance of the symptom and impact concepts in the ID to measure PBC-related itch were confirmed. Adaptation of the PBC-40 to a weekly recall period and for North American English was successful. Primary biliary cholangitis (PBC), previously known as primary biliary cirrhosis, is a chronic autoimmune disease of the liver in which intrahepatic small bile ducts are selectively destroyed by inflammatory cells , 2. The Itching (pruritus) is an extrahepatic symptom of PBC that occurs in an estimated 55% of patients . In a suItch is a sensation that cannot be directly observed, and is only known to the individual, making its assessment subjective and difficult to quantify . PatientThe Patient Reported Symptoms Questionnaire (“Itch Diary”) was developed to meet the need for a PBC-specific itch PRO measure and was based on key concepts in the PBC-40 , 12. TheAn integral part of developing quality-of-life measures is to conduct qualitative patient interviews and such interviews are recommended by the FDA for the development of PRO measures , 16. TheWhile the qualitative sample of ten patients in the previous study at times may be acceptable for rare conditions, it was still unclear whether the list of symptoms identified in the earlier study was complete in terms of the key concepts relevant to people with PBC, and whether the content was transferrable to other countries and cultures. Therefore, we conducted additional qualitative interviews in patients with PBC to confirm saturation of the concepts of interest and the applicability of these concepts within the US and Canada. In-person qualitative interviews consisting of concept elicitation (CE) and cognitive portions were conducted in the US and Canada to collect data for confirming content relevance, instrument feasibility, and saturation of concept for the Itch Diary and the weekly version of the PBC-40. Interviews lasted for about 90 min, beginning with the CE portion of the interview, then completion of questionnaires of interest  as well as some additional secondary questionnaires , followed by the cognitive portion of the interview.Men and women aged 18 to 80 years who self-reported a diagnosis of PBC were recruited via local and national patient groups. Participants were required to have ongoing itching of at least moderate intensity  during the previous 8 weeks. Individuals with a self-reported history of liver disease of other etiology or reporting other highly symptomatic medical conditions  were excluded. As PBC predominantly affects women , 4, a maStudy recruitment was conducted via advertisements to members of patient groups in the US and Canada: PBCers and the Canadian PBC Society. The study was conducted in compliance with Health Insurance Portability and Accountability Act regulations, and all participants provided written informed consent. The study was approved by an Institutional Review Board .Demographic and basic comorbidity data were collected from each participant. Those who were willing, signed a Medical Information Release Waiver giving their consent for additional limited medical information to be obtained from their medical provider. Information collected from the medical provider, when authorized, included date of PBC diagnosis, current and past medications for itching/pruritus, previous history of liver transplant (yes/no), and, when possible, the most recent bilirubin and alkaline phosphatase lab results.Interviewers were experienced in interviewing techniques for PRO measurement development and in conducting one-on-one interviews across a wide range of therapeutic areas. Training for the interviewers included an instruction session specific to the study, review of the interview guide, and observed practice interviews.Interviews were conducted in 2 waves of 10 participants each. Revisions to the PRO measures were made in response to participant input from the first wave, and then tested in the second wave. During the CE portion of the interviews, participants were asked open-ended questions in order to elicit spontaneous reports of the patient’s experience, followed by probing questions designed to more fully explore participant experiences with PBC symptoms  and the impact of PBC on emotions, daily activities, social functioning, sleep, etc. During the cognitive portion of the interviews, participants were asked a series of structured and semi-structured questions designed to obtain feedback on the PRO measures. Topics covered were the participants’ interpretation of the individual items, how they regarded the fit and adequacy of the response scales and recall periods, and how comfortable they were with the terminology used. Participants were also asked to discuss the relevance of PRO items and whether any other important concepts were missing and should be considered for addition.PRO measures of interest were administered to each participant on paper between the CE and cognitive portions of the interview. The PROs of main interest were the Itch Diary (AM and PM versions) and a weekly version of the PBC-40 that used a 7-day recall period as opposed to the original 4-week recall period . The PBCTranscripts from the CE portion of the interview were organized into descriptions of PBC-related symptoms and symptom impacts by developing a coding framework and coding dictionary. Two coders were used during the study. Inter-rater agreement was evaluated by independent dual coding of 2 of the 20 transcripts and comparing the coding differences and expressing coding consistency by percentage of agreement in the codes assigned.A saturation table was used to track symptoms and impacts from the concept elicitation portion of the interviews. The transcripts were ordered chronologically, based on interview completion date, and then grouped into four groups of five transcripts each. Concept saturation was then evaluated by comparing the codes in each new transcript group with the codes in the previous group to identify newly appearing information. When new codes no longer appeared, no new information was being obtained; this is called “saturation of concept” meaning no further information is likely to be gained by continuing interviews in the same population.Descriptive statistics were generated for demographic and concept rating data. Information for the cognitive portion of the interviews was collected into a summary grid that organized and related the response from each participant to the item that was queried. Difficulties in understanding the item content were also noted in the cognitive summary grid.Of the 20 participants interviewed in the study, the mean age was 57.4 years and 90% were women Table . Most  duration of disease of 9.6 (6.0) years. Twelve of these 14 participants (86%) had received ursodeoxycholic acid (past or present) to treat their PBC. Only 4 reported they were currently using medication to relieve their itching.Patients who were screened but did not fully meet the eligibility criteria were considered on an individual basis to determine whether the particular criteria deviation might lead to confounding or conflict with the study design or objectives. Four of these subjects were authorized to participate despite not meeting one or more eligibility criteria, one subject cancelled the interview and the three remaining subjects were interviewed and included in the analysis. Of those included, 1 patient reported average itching of level 3 (just below the threshold of 4), although this patient reported having itching above a 4 at times during the recall period; 1 patient had been diagnosed with hepatitis C, but had been declared cured after treatment for the infection; and 1 patient reported infrequent minor flare-ups of eczema but was not experiencing eczema during screening or the interview for the current study.Saturation of concept (the point where no new concepts emerged) for the Itch Diary was reached by the end of the third transcript group . Inter-rater agreement in assignment of codes between the 2 raters was high at 94% and 95% for the 2 dual-coded transcripts.The CE portion of the interviews confirmed relevance of the concepts presented in the items of the PBC-40 and Itch Diary (both AM and PM versions). Itching-related signs and symptoms were reported by all patients, and was the most commonly expressed symptom subdomain when assessed by symptom concept code frequency  followed by energy-related concepts  and additional signs and symptoms  Table . SpecifiDuring the CE portion of the interview, participants were given the opportunity to spontaneously report symptoms related to their PBC before follow-up probing questions were asked. The symptom concepts most often mentioned spontaneously were “fatigue”  and “itching on the legs” . Other common spontaneously reported symptom concepts were “itching on the arms” , “itching on the side” , and “itching on the back” . Follow-up probing questions did not notably increase the reports related to itching, although the number of participants reporting weakness, gastrointestinal signs and symptoms, cognitive signs and symptoms, and pain and discomfort were markedly increased Table .Table 3SDuring the interview, participants were asked to refer to the symptoms they had described and identify the top 1 or 2 symptoms that were the worst for them. The symptoms most commonly chosen were itching  and fatigue . Participants identified the most common location for itching as legs (75%), followed by arms (65%), torso (60%), and feet (50%).A series of questions was asked during the CE portion of the interviews to explore the impact of PBC on patients’ lives and the language used by participants to describe these impacts. Changes in daily performance was the most commonly expressed impact subdomain when assessed by impact concept code frequency  followed by emotional functioning  and sleep difficulties  Table .Table 4SWithin these subdomains, the most frequently cited concepts overall were “scratching until skin is raw/infected” (38 expressions by 16 participants [80%]) in the additional impacts subdomain, “difficulty staying asleep” (38 expressions by 16 participants [60%]) in the sleep difficulties subdomain, “social activities limited” (37 expressions by 14 participants [70%]) in the limitations to relationships and social functioning subdomain, “limitations at work” (34 expressions by 15 participants [75%]) in the changes to daily performance subdomain, and “needing to change their diet” (32 expressions by 15 participants [75%]) in the limitations to lifestyle and activities subdomain.Similar to the symptom concepts, participants were allowed to spontaneously report impact concepts related to their PBC before follow-up probing questions were asked. The impact concepts most often mentioned spontaneously were “limitations at work”  and “difficulty staying asleep” . Other common spontaneously reported impact concepts were “needing to change their diet”  and “scratching until skin is raw/infected” . Follow-up probing questions increased the number of participants reporting most of the impact concepts  selected “general function/daily routine” and 2 participants (10%) selected “reduced sleep quality.”The cognitive portion of the interviews resulted in several changes to the Itch Diary and PBC-40 Table . During Further revisions were made to question stems and the response scale after wave 2. These changes included additional wording changes to 1 of the items in the AM and PM diaries, as well as changes to the response options of these diaries; additionally, the NRS versions of select items were added back into the measure for use in an upcoming clinical trial for testing against the VRS versions.Three of the item revisions in the PBC-40 during wave 1 were to adapt from British English in the original version to terms more common in North America . During wave 2, additional revisions were made to 3 items to further clarify their meaning. For example, 1 item asked about aching in the long bones of the patient’s arms and legs. Some participants had trouble responding because they experienced aching in the arms, but not in the legs or vice versa. Therefore, the stem was changed to “arms or legs” to make it clearer that participants could respond if they had aching in only one location.There were no major issues or revisions made to the PGIS-Itch, PGIC-Itch, or 5-D Itch measures; the PGIS-Itch and PGIC-Itch were determined to be appropriate anchor measures.During wave 1, the response options were revised for 1 item  in the Weekly Gastrointestinal Diary. No further revisions were made to this questionnaire during wave 2.The mean (range) time to complete the PRO measures during wave 1 of the interviews was 57.4 (38–111) seconds for the AM Itch Diary, 88 (39–100) seconds for the PM Itch Diary, and 252.8 (167–407) seconds (4.2 [2.8–6.8] minutes) for the PBC-40. The mean (range) time to complete the PRO measures during wave 2 of the interviews was 46.5 (28–75) seconds for the AM Itch Diary, 89.8 (47–120) seconds for the PM Itch Diary, and 291.9 (161–420) seconds (4.9 [2.7–7.0] minutes) for the PBC-40.Qualitative interviews of 20 North American participants with PBC confirmed the overall content of the Itch Diary and weekly PBC-40 measures in terms of symptoms and disease impact. Qualitative patient interviews are recognized as being an integral part of developing quality of life measures and are recommended by the FDA for the development of PRO measures to support label claims , 16. ItcWhile scratching is a behavior that can be directly observed by others, and is an activity that leaves visible evidence by way of marks and bleeding, itching is a symptom and can only be described by the individual experiencing this sensation. As demonstrated in the current study and others, PBC-related itching can be burdensome for patients , 7. HoweIn addition to the Itch Diary, the current study assessed an adapted version of the PBC-40 using a recall period of “past 7 days” instead of “last 4 weeks.” Although the cognitive interviews identified minor issues in the PBC-40, any major changes needed to be discussed and approved by the original developers. After consulting with the original developers, revisions were mainly limited to making minor grammatical changes, such as changing terms mainly used in British English to terms more commonly used and understandable to North Americans.A limitation of this study is that eligibility was based on self-reported diagnosis of PBC. However, of the 14 participants with additional medical information provided by their physician, all 14 had confirmed PBC. Another limitation is that the patient population was enriched for patients with moderate to severe PBC-related itch. Thus, the applicability of the Itch Diary to the generalized PBC population, or in other conditions, is unknown at this time.Qualitative interviews confirmed the relevance and comprehension of the Itch Diary and the weekly version of the PBC-40 as fit-for-purpose measures to assess the relevant symptoms and impacts associated with PBC and PBC-related itching, and to support the content validity of the measures. Adaptation of the PBC-40 to a weekly recall period and for North American English was successful. These measures will be useful in evaluating the efficacy of new treatments for PBC-related itch, although further work is needed to fully evaluate the psychometric properties of these measures.


The neural crest (NC) is a multipotent embryonic cell population that generates distinct cell types in an axial position-dependent manner. The production of NC cells from human pluripotent stem cells (hPSCs) is a valuable approach to study human NC biology. However, the origin of human trunk NC remains undefined and current in vitro differentiation strategies induce only a modest yield of trunk NC cells. Here we show that hPSC-derived axial progenitors, the posteriorly-located drivers of embryonic axis elongation, give rise to trunk NC cells and their derivatives. Moreover, we define the molecular signatures associated with the emergence of human NC cells of distinct axial identities in vitro. Collectively, our findings indicate that there are two routes toward a human post-cranial NC state: the birth of cardiac and vagal NC is facilitated by retinoic acid-induced posteriorisation of an anterior precursor whereas trunk NC arises within a pool of posterior axial progenitors. The neural crest (NC) is a multipotent cell population which arises in the dorsal neural plate/non-neural ectoderm border region during vertebrate embryogenesis. Studies utilising chick and amphibian embryos have indicated that different levels of BMP, WNT and FGF signals, emanating from the mesoderm/non-neural ectoderm, orchestrate NC induction and specification . This ocAn attractive approach for studying human NC biology and modelling NC-associated developmental disorders (neurocristopathies) involves the in vitro differentiation of human pluripotent stem cells (hPSCs) toward NC cells. Conventional protocols to obtain NC from hPSCs are based on the production of a neurectodermal intermediate, via TGFβ signalling inhibition, which is subsequently steered toward a NC fate, usually through stimulation of WNT activity combined with the appropriate levels of BMP signalling . These sCdx and Hox gene family members, Tbx6 and Nkx1-2  and WNT/FGF/NOTCH/RA signalling pathway components, known to be expressed at high levels in the late PS/TB regions in vivo   percentage of T+SOX2+ cells. The resulting cultures resemble embryonic posterior axial progenitors, including NMPs, both in terms of marker expression and developmental potential . To inte7, HES7) . A large7, HES7)  and we f7, HES7) . The upr7, HES7)  suggest We next sought evidence that might point to links between trunk NC and human axial progenitors. The RNAseq analysis revealed that a considerable number of genes known to mark the neural plate border and early NC in vivo  were also significantly upregulated in axial progenitors , a findiA number of both in vivo and in vitro studies have pointed to an optimal level of low/intermediate BMP signalling acting as an inducer of a NC/border character in conjunction with WNT and FGF . We therWe reasoned that if posterior axial progenitors with NC/border features correspond to pioneer trunk NC precursors then they should be competent to generate definitive trunk neural crest when placed in an appropriate culture environment. We have recently reported a protocol for the efficient generation of anterior cranial NC cells from hPSCs involving the combined stimulation of WNT signalling, TGFβ signalling inhibition and moderate BMP activity via the parallel addition of BMP4 and the BMP type one receptor inhibitor DMH1 We next tested the developmental potential of human axial progenitor-derived trunk NC cells. To this end, we grafted trunk NC cells derived from a human induced PSC (iPSC) line carrying a constitutive ZsGreen fluorescent reporter  in or onSince elevated BMP signalling appears to coincide with the acquisition of an early NC/border character by human axial progenitors  we also low fraction we detected  members compared to untreated NC suggesting a posterior cranial and vagal/cardiac NC character   cell populations from hPSCs rely on the caudalisation of an anterior ectodermal precursor via treatment with RA and/or WNT agonists . Therefofindings  RA-treatharacter . Howeverneration . Further1, SOX8)   . We thustrunk NC  and therTo further discern the identity of posterior NC subtypes induced either via RA treatment or an axial progenitor intermediate as well as identify unique associated molecular signatures we carried out analysis of the transcriptomes of NC cells arising under these conditions as well as those of their precursors using microarrays . We founFOXC1 and 2 , cardiaC1 and 2 ; PDGFRa   and vag eLife. Your article is being evaluated by three peer reviewers, and the evaluation is being overseen by a Reviewing Editor and Sean Morrison as the Senior Editor.Thank you for sending your article entitled "Human axial progenitors generate trunk neural crest cells" for peer review at As you will see from the reviewer comments below, there was enthusiasm for the technique, but there were two significant issues that preclude us from making a decision.A) There was a concern about the quantification of the day 3 NMP cell fates. Based on the data, there is a significant possibility that there are pluripotent stem cells present at day 3, and the reviewers feel that a more robust quantification of these cell fates is necessary. Although this could be done using FACS based approaches, as an alternative, a high content imaging-based quantitation could be used, assuming there is not too much clustering of the cells in 3D. Carefully tracking cells at day 3 is important, as some of the pitfalls may include the possibility of pluripotent stem cells left at day 3  which could change the interpretation of the study.B) An in vivo functional readout of the derived cells is required to establish the true importance of the technique. For example, you could transplant the cells into chick embryos to test their capacity to form neurons that express sympathetic markers in sympathetic ganglia, as has been done in prior studies .eLife generally asks that all revision experiments be performed within 2 months. Given this, we would like you to examine the list of all reviewer comments below as well as the comments discussed above, and decide if you think this is feasible within an approximately 2 month period. If so, then the editors and reviewers invite you to respond within the next two weeks with a detailed action plan and timetable for the completion of the additional work. We plan to share your responses with the reviewers and then issue a decision.We recognize that these experiments can be complex and time-consuming, and Reviewer #1:The study by Tsakiridis and colleagues combines the induction of a posterior neuromesodermal progenitor (NMP) fate with the induction of a posterior (trunk) neural crest (NC) identity and the derivation of trunk NC-derived sympathoadrenal progenitors (SAPs). Overall, the study is well designed and clearly written. However, protocols to trigger NMPs from mouse and human PSCs are already very well established across the field. Furthermore, two groups  have already provided some evidence of NMP-derived neural crest derivatives, in particular SAP derived chromaffin-like cells. Nevertheless, the current study goes beyond those published studies by focusing more on developmental approaches and by looking in more detail at the transitions from NMP induction to NC specification. In particular, the study presents detailed analyses of HOX gene induction and global gene expression, and it achieves a protocol to generate trunk NC and SAP induction at high efficiencies.Beyond the potential concerns about novelty mentioned above, there are additional issues that should be addressed including better quantification of the various subpopulations observed and the need to more clearly demonstrate the lineage relationship of NMP and subsequent NC identity. Furthermore, the characterization of the resulting SAP-like neural crest derivatives is quite limited and the trunk identity of the resulting neurons highly dependent on a few reagents such as HOXC9 antibodies which are not validated very well in the current study.1) IHC vs Flow based quantification. The study relies very heavily on the use of immunocytochemical (ICC) analysis including double and triple stains. Such analysis can be tricky as it does not allow for an easy representation of the different levels of expression among positive cells and, for many of the Figures presented here, it lacks quantification. This issue could be addressed by using intracellular flow, where both quantification and representation of levels across cells can be shown more easily. For example, it would be important to have proper quantification and presentation of expression levels for the various panels in Figure 1 as well as Figure 2B among others.2) Lack of a true lineage analysis. The authors argue lineage-relationships based the co-expression of certain markers rather than any true genetic lineage mapping or at least cell sorting based analyses. The study could be much more convincing by including genetic fate mapping data, ideally using a hPSC reporter line such as a reporter line for T. At the minimum, the authors should present a more detailed quantification of marker expression as detailed under #1 and discuss potential pitfalls of their current approach .3) BMP/LDN experiments. The authors present data to show that endogenous BMP signaling seems to be important for the expression of early NC markers in day 3 NMP cultures. Indeed, LDN treatment greatly reduces several of those markers. However, this gets back to the question of lineage relationships and whether NMPs indeed are fully plastic as suggested or whether a subset is already committee to NC lineage and therefore no longer bona fide NMPs. Another interesting point regarding those data is that LDN also reduces CDX2 levels. This argues that BMP may not only trigger NC bias but also enable CDX2 expression and posterior patterning. Do the authors have data on LDN treated NMPs that were subsequently induced towards NC lineage by using the BMP/WNT NC protocol from day 3 onwards? Do they observe comparable potential to trigger NC after early LDN treatment?4) Persisting OCT4 expression the gene expression data in Figure 4G shows highly differential expression of OCT4 in NMP vs NMP-NC  – this suggests that there is remaining OCT4 expression either in all NMPs, or there is a subset of cells at NMP stage that retain OCT4 expression. This should be addressed by ICC or flow cytometry for OCT4. If there is a subset of OCT4+ cells at day 3, it raises concerns along the lines stated under #1 and #2 regarding lineage origin of those NC cells.5) Subtype identity of NMPs There are conceptual questions about how to define NMPs if the cells already express NC  markers at the day 3 stage prior to NC induction. It raises the issue of how many NMP subpopulations co-exist and where did they come from . For example, the authors state: "Collectively, these findings indicate that a NC/border state probably arises within multipotent posterior axial progenitors which have not committed to a neural or mesodermal fate" However, if cells already express PAX3 and SOX9, it seems tricky to argue that those cells within NMP cultures are not committed at all.6) HOXC9. The HOXC9 antibody is a critical reagent for this study. The induction of HOXC9 levels seem to vary quite a bit based on the gene expression data presented throughout the paper while the antibody seems to label a very large proportion of all the cells. It would be helpful to include some additional control assays to demonstrate the specificity of the antibody, and to include stage-matched ANCs or RA treated ANCs throughout to further validate specificity.7) HOX co-expression data. There are no data looking at co-expression of various HOX genes. This is particularly relevant as the authors conclude that NMP-derived NC is HOX6-9 while ANC is HOX1-5 . However, the gene expression data indicate that most anterior HOX genes (1-5) are expressed equally or even higher in NMP vs ANC or ANC+RA derived lineages .8) Characterization of trunk NC derivatives / SAPs A key argument regarding the importance of the current study, is the ability to efficiently generate trunk NC derivatives such as sympathetic neurons without the need for sorting. However, the characterization of those sympathetic neurons is very limited and lacks basic co-expression data (e.g. TH/DBH) or functional data such as evidence of catecholamine release or other physiological hallmarks of sympathetic neurons. Such assays are commonly presented by other studies reporting on the derivation of sympathetic neurons from hPSCs .Reviewer #2:The study by Frith and colleagues employs methods to convert human pluripotent stem cells (hPSC) into posterior/caudal neural crest (NC) cells via an axial progenitor intermediate. In addition, the authors define culturing conditions that allow efficient production of peripheral sympathetic neurons without the need for additional purification schemes, such as FACS. Overall, the authors present their work very well, with excellent command of the literature, and have advanced our knowledge for generating trunk NC cells and their derivatives that I expect will be used by researchers in the field. However, in its current form, the conclusions are over-stated and the impact of the study is lessened by the fact that similar protocols for generating trunk NC and peripheral neurons (and other NC-cell types) have recently been published (as noted by the authors). In addition, the experimental approaches are not overly innovative  and largely focus on sufficiency outcomes with no experiments addressing necessity. As one of the main conclusions from the study concerns the origin of trunk NC cells from axial progenitors, there should be some spatial data to confirm the molecular signatures. For example, transplantation of anterior versus posterior NC cells into avian embryos to show the observed transcriptional changes impact the fate of cells at different axial positions. This technique is commonly used in the field to test axial identity and NC competence  and would complement the heavy reliance on using only molecular signatures in cell culture. Indeed, many of the genes used by the authors to define axial NC identity are expressed in other cell types, particularly other neural, neuroectodermal and mesoderm derivatives that develop adjacent to NC cell in vivo. Thus, without the addition of fate mapping data in vivo, other interpretations/conclusions could be drawn on what cell types are actually generated by this new method..1) The authors should employ transplantation techniques with different progenitors derived in vitro  to show they indeed are fated/committed to specific axial lineages in vivo. For example, RA-treated anterior NC cells should largely generate cardiac/vagal cell types in vivo compared to posterior NC cells, whereas transplanted axial cells would be expected to generate multiple posterior lineages. Such data would significant enhance the impact of the study and strengthen the conclusion that human axial progenitors generate trunk NC.2) The authors identify a potential new gene involved in trunk NC development, ASLX3. The authors should perform in situ hybridization or immunohistochemistry on vertebrate animals  to localize ASLX3  to trunk NC cells in vivo. This would strengthen their argument that their approach used to classify axial identity in vitro (which is reliant on HOX gene expression) can also produce new knowledge on the genetic regulation of trunk NC in vivo.Reviewer #3:The manuscript shows that trunk-NC cells are best/most efficiently derived via an axial progenitor population, similar to the neuro-mesodermal progenitor giving rise to lower motor neurons. The authors show solid work that clearly indicate that this progenitor can be guided into producing NC cells with trunk identity. More sophisticated experiments, such as following cells over time using reporter lines to show that the NMP truly is giving rise to the trunk-NC population as well as in vivo work showing the same thing would definitively prove this interesting hypothesis. Therefore, some of the conclusions should be worded a bit more carefully. Nevertheless, this is the first report (to my knowledge) of NC cells derived from NMPs with trunk NC character and it seems a highly efficient one as well. Thus, I think this is an important report that should be published.- To strengthen the conclusion in subsection “A BMP-dependent neural crest signature in human axial progenitor cultures”  it would be useful to see what percentage of all cells are posterior axial progenitors committed to become trunk-NC. For example, by doing triple stained intracellular FACS analysis of T, CDX, SOX9.- In Figure 3D, the authors show that the axial progenitor can give rise to paraxial mesoderm. This could be strengthened by showing that these PXM cells can further be differentiated into definitive mesodermal cell types.- Do the SOX9+ cells early at day 3 also express SOX10? . Why does SOX9 come on before SOX10 and does SOX10 stay on?- Can you maintain this trunk-NC cells as progenitors and expand them in culture, if that is possible it would make this technique even more useful.-The RNA-seq/microarray data suggests that DMBX1 and LHX5 versus HES6 could serve as markers to distinguish anterior from trunk NC cells. This notion should be strengthened by staining to show the same at the protein level.- Figure 5C: 80% of Phox2B-GFP- cells are ASCL1+? What are these cells? Maybe they are younger cells that have not yet turned on Phox2B, which would mean that at day 18 there is a mixture of cells in terms of developmental stage. Could that be showed more?- Scale bars are missing in all pictures.- Discussion section: This should be stated more carefully, since the transcriptome analysis is done in in vitro generated cells. Without confirming in vivo data, one cannot definitively prove that axial progenitors are converted into posterior NC in embryogenesis. [Editors' note: the authors’ plan for revisions was approved and the authors made a formal revised submission.]As you will see from the reviewer comments below, there was enthusiasm for the technique, but there were two significant issues that preclude us from making a decision.A) There was a concern about the quantification of the day 3 NMP cell fates. Based on the data, there is a significant possibility that there are pluripotent stem cells present at day 3, and the reviewers feel that a more robust quantification of these cell fates is necessary. Although this could be done using FACS based approaches, as an alternative, a high content imaging-based quantitation could be used, assuming there is not too much clustering of the cells in 3D. Carefully tracking cells at day 3 is important, as some of the pitfalls may include the possibility of pluripotent stem cells left at day 3  which could change the interpretation of the study.low fraction and no OTX2+T-VENUS+ co-expressing cells. This indicates minimal contamination of our NC-potent T+ axial progenitors with pluripotent cells. We have included these data in new Figure 3—figure supplement 3. However, to eliminate completely the possibility that pluripotent cells may be the source of trunk NC cells following re-plating of axial progenitor cells in NC-inducing conditions we also flow- sorted and tested the NC potential of T-VENUS+high cells which we found to be NANOG negative . Our new results show that indeed these cells exhibit the potential to generate SOX10+ neural crest cells, a proportion of which (approximately one third) is also positive for HOXC9 . This implies that T+high axial progenitors are the source of at least half of the trunk HOXC9+SOX10+ NC cells we obtain following differentiation of bulk unsorted human axial progenitor cultures . We speculate that the rest is likely to arise from the T+low fraction, which was not included in the sorted population. These new data also suggest that the production of trunk NC from T+ progenitors does not occur via “posteriorisation” of an OTX2+ anterior NC progenitor entity  since the very few OTX2+ cells we detected in our axial progenitor cultures were all T-VENUS negative and therefore were not included in the sorted population that gave rise to SOX10+HOXC9+ cells upon plating in NC-inducing conditions . We have discussed these findings in the relevant Results section .The presence of OCT4 in hPSC-derived axial progenitors/NMPs is not unexpected as it has been shown to be expressed within non-pluripotent cells in the posterior of the early somite mouse embryo including NMPs  and has a role in the regulation of axis elongation . Thus, to investigate the persistence of pluripotent cells, we examined the presence of cells expressing more definitive pluripotency markers such as NANOG  and OTX2  in axial progenitor cultures derived from a T-VENUS reporter hPSC line . Following quantification, we found a small  NANOG+T-VENUS+B) An in vivo functional readout of the derived cells is required to establish the true importance of the technique. For example, you could transplant the cells into chick embryos to test their capacity to form neurons that express sympathetic markers in sympathetic ganglia, as has been done in prior studies .We have now verified the functionality of the axial progenitor-derived trunk NC cells using the following assays:(i) We show that trunk NC cells differentiated from a fluorescent reporter human iPSC line available in our lab  and transplanted into/on top of the dorsal neural tube of chick embryos behave similarly to embryonic NC by exhibiting migratory behaviour (6/6 grafted embryos) and colonising the DRG where they were found to upregulate markers indicative of their location (3/6 grafted embryos). These data are shown in new Figure 3D and Figure 3—figure supplement 2A-C.(ii) We show by ELISA that axial progenitor-derived sympathetic neurons produce catecholamines (dopamine and norepinephrine) at levels similar or higher to the ones described previously for in vitro derived sympathetic neurons . These data are shown in new Figure 5G.(iii) We confirm that axial progenitor-derived sympathetic neurons exhibit an action potential firing profile similar to the one described previously for in vitro derived sympathetic neurons . These data are shown in new Figure 5F and Figure 5—figure supplement 1D.We recognize that these experiments can be complex and time-consuming, and eLife generally asks that all revision experiments be performed within 2 months. Given this, we would like you to examine the list of all reviewer comments below as well as the comments discussed above, and decide if you think this is feasible within an approximately 2 month period. If so, then the editors and reviewers invite you to respond within the next two weeks with a detailed action plan and timetable for the completion of the additional work. We plan to share your responses with the reviewers and then issue a decision.Reviewer #1:The study by Tsakiridis and colleagues combines the induction of a posterior neuromesodermal progenitor (NMP) fate with the induction of a posterior (trunk) neural crest (NC) identity and the derivation of trunk NC-derived sympathoadrenal progenitors (SAPs). Overall, the study is well designed and clearly written. However, protocols to trigger NMPs from mouse and human PSCs are already very well established across the field. Furthermore, two groups  have already provided some evidence of NMP-derived neural crest derivatives, in particular SAP derived chromaffin-like cells. Nevertheless, the current study goes beyond those published studies by focusing more on developmental approaches and by looking in more detail at the transitions from NMP induction to NC specification. In particular, the study presents detailed analyses of HOX gene induction and global gene expression, and it achieves a protocol to generate trunk NC and SAP induction at high efficiencies.Beyond the potential concerns about novelty mentioned above, there are additional issues that should be addressed including better quantification of the various subpopulations observed and the need to more clearly demonstrate the lineage relationship of NMP and subsequent NC identity.We have included new quantification data showing the correlation between T  and NC/border markers such as SOX9, SNAI2 and PAX3 using two different hPSC cell lines (MasterShef7 and the H9-derived T-VENUS line)- see data in new Figure 2C and Figure 2—figure supplement 1B.Furthermore, the characterization of the resulting SAP-like neural crest derivatives is quite limited and the trunk identity of the resulting neurons highly dependent on a few reagents such as HOXC9 antibodies which are not validated very well in the current study.We have now included a number of additional functional experiments to address this issue, see our response above to point B.1) IHC vs Flow based quantification. The study relies very heavily on the use of immunocytochemical (ICC) analysis including double and triple stains. Such analysis can be tricky as it does not allow for an easy representation of the different levels of expression among positive cells and, for many of the Figures presented here, it lacks quantification. This issue could be addressed by using intracellular flow, where both quantification and representation of levels across cells can be shown more easily. For example, it would be important to have proper quantification and presentation of expression levels for the various panels in Figure 1 as well as Figure 2B among others.We have carried out a thorough quantification of the expression of different NMP/NC/border/pluripotency markers in axial progenitor cultures following immunostaining and image analysis since in our hands intracellular FACS often results in high background and relies heavily on the quality of the antibodies used. These results are now part of figure 2C, Figure 2—figure supplement 1B, Figure 3—figure supplement 3A, B; see also our responses above.2) Lack of a true lineage analysis. The authors argue lineage-relationships based the co-expression of certain markers rather than any true genetic lineage mapping or at least cell sorting based analyses. The study could be much more convincing by including genetic fate mapping data, ideally using a hPSC reporter line such as a reporter line for T. At the minimum, the authors should present a more detailed quantification of marker expression as detailed under #1 and discuss potential pitfalls of their current approach .See our response, above, to point A.3) BMP/LDN experiments. The authors present data to show that endogenous BMP signaling seems to be important for the expression of early NC markers in day 3 NMP cultures. Indeed, LDN treatment greatly reduces several of those markers. However, this gets back to the question of lineage relationships and whether NMPs indeed are fully plastic as suggested or whether a subset is already committee to NC lineage and therefore no longer bona fide NMPs. Another interesting point regarding those data is that LDN also reduces CDX2 levels. This argues that BMP may not only trigger NC bias but also enable CDX2 expression and posterior patterning. Do the authors have data on LDN treated NMPs that were subsequently induced towards NC lineage by using the BMP/WNT NC protocol from day 3 onwards? Do they observe comparable potential to trigger NC after early LDN treatment?CDX2 transcript levels following BMP inhibition during the induction of NMPs from hPSCs does not appear to be statistically significant  and we also found that the total number of CDX2 protein positive cells is not affected by LDN treatment (data not shown). Furthermore, our new data show that BMP-inhibited axial progenitors can still generate trunk NC at a similar efficiency to untreated controls . These data indicate that endogenous BMP signalling alone is not the mediator of NC potential in axial progenitor cultures and we have therefore amended our statements in the new manuscript version regarding the role of this pathway.The reviewer is raising some very interesting points. The reduction in 4) Persisting OCT4 expression the gene expression data in Figure 4G shows highly differential expression of OCT4 in NMP vs NMP-NC  – this suggests that there is remaining OCT4 expression either in all NMPs, or there is a subset of cells at NMP stage that retain OCT4 expression. This should be addressed by ICC or flow cytometry for OCT4. If there is a subset of OCT4+ cells at day 3, it raises concerns along the lines stated under #1 and #2 regarding lineage origin of those NC cells.See our response to point A, above.5) Subtype identity of NMPs There are conceptual questions about how to define NMPs if the cells already express NC  markers at the day 3 stage prior to NC induction. It raises the issue of how many NMP subpopulations co-exist and where did they come from . For example, the authors state: "Collectively, these findings indicate that a NC/border state probably arises within multipotent posterior axial progenitors which have not committed to a neural or mesodermal fate". However, if cells already express PAX3 and SOX9, it seems tricky to argue that those cells within NMP cultures are not committed at all.The question of NMP subpopulations is very interesting and requires further investigation but it is outside the scope of this manuscript. The lack of an exclusive NMP marker makes the precise definition of committed vs non-committed NMPs very challenging. Recent lineage tracing and single cell RNA-seq studies have revealed that mouse embryonic NMP subpopulations exhibit expression of more definitive lineage-specific markers such as the paraxial mesoderm gene Tbx6 . We speculate that co-expression of NC/border markers with NMP markers such as T in day 3 FGF-CHIR treated hPSC cultures probably represents a similar phenomenon. Our new T-VENUS-sorting experiments demonstrating that at least some of the T+ axial progenitors  can generate trunk NC cells supports the notion that “a NC/border state probably arises within multipotent posterior axial progenitors which have not committed to a neural or mesodermal fate”.6) HOXC9. The HOXC9 antibody is a critical reagent for this study. The induction of HOXC9 levels seem to vary quite a bit based on the gene expression data presented throughout the paper while the antibody seems to label a very large proportion of all the cells. It would be helpful to include some additional control assays to demonstrate the specificity of the antibody, and to include stage-matched ANCs or RA treated ANCs throughout to further validate specificity.We are confident about the specificity of the monoclonal anti-HOXC9 antibody we employ. We did not obtain any positive signal when we used the antibody on undifferentiated hES cells suggesting that it is specific. We also validated the antibody’s specificity by carrying out immunostaining on ETS1+ anterior NC cultures and found no HOXC9 positivity, also in line with our transcriptome analysis data. We have included these data in new Figure 3—figure supplement 1A, B.7) HOX co-expression data. There are no data looking at co-expression of various HOX genes. This is particularly relevant as the authors conclude that NMP-derived NC is HOX6-9 while ANC is HOX1-5 . However, the gene expression data indicate that most anterior HOX genes (1-5) are expressed equally or even higher in NMP vs ANC or ANC+RA derived lineages .Hoxb1 expression in E9.5 mouse embryos see Glaser et al. Development 2006 and Arenkiel et al., 2003; for Hoxc9 expression see Bel et al., 1998) and thus the presence of HOX1-5 transcripts in HOX9+ trunk NC cells is not unexpected. Alternatively, the co-expression of transcripts belonging to both HOX1-5 and HOX6-9 groups may be a result of the co-emergence of a separate population of cardiac/vagal NC cells during trunk NC differentiation. This may be due to the action of endogenous RA signalling since our microarray data revealed the upregulation of RA signalling components in axial progenitor-derived trunk NC even though no exogenous retinoic acid was added to the differentiation medium (see Supplementary file 2 in submitted manuscript). Unfortunately, we have not found reliable antibodies against HOX group 1-5 members that we can use together with HOXC9 in order to distinguish between these two possibilities. We have included a discussion of this point in the Results section in the revised manuscript (“The simultaneous presence… even though no exogenous RA was added to the differentiation medium”).The reviewer is raising a valid point. We should note that co-expression of HOX1-5 and HOX6-9 transcripts does occur in the posterior part of the mouse embryo around E9.5, both in the NMP-containing tailbud region  as well as in differentiated posterior neural/neural crest cells (e.g. for 8) Characterization of trunk NC derivatives / SAPs A key argument regarding the importance of the current study, is the ability to efficiently generate trunk NC derivatives such as sympathetic neurons without the need for sorting. However, the characterization of those sympathetic neurons is very limited and lacks basic co-expression data (e.g. TH/DBH) or functional data such as evidence of catecholamine release or other physiological hallmarks of sympathetic neurons. Such assays are commonly presented by other studies reporting on the derivation of sympathetic neurons from hPSCs .See our response to point B. We have now also included data showing co-expression of TH and DBH in axial progenitor-derived sympathetic neurons .Reviewer #2:The study by Frith and colleagues employs methods to convert human pluripotent stem cells (hPSC) into posterior/caudal neural crest (NC) cells via an axial progenitor intermediate. In addition, the authors define culturing conditions that allow efficient production of peripheral sympathetic neurons without the need for additional purification schemes, such as FACS. Overall, the authors present their work very well, with excellent command of the literature, and have advanced our knowledge for generating trunk NC cells and their derivatives that I expect will be used by researchers in the field.We would like to thank the reviewer for their positive comments.However, in its current form, the conclusions are over-stated and the impact of the study is lessened by the fact that similar protocols for generating trunk NC and peripheral neurons (and other NC-cell types) have recently been published (as noted by the authors).We have tried to be careful about our conclusions and we are happy to consider rephrasing them on a case-to-case basis if required. We disagree that the impact of our study is lessened by the publication of similar protocols  and we would like to point to reviewer 3’s comment that “this is an important report that should be published”. Apart from establishing a highly efficient method for generating trunk NC cells we believe that our manuscript also provides an insight into the basis of anterior-posterior regionalisation in the human neural crest and therefore, as reviewer 1 correctly noted, goes beyond the Denham et al., and Abu-Bonsrah et al., publications, which focus almost exclusively on improving the technical aspects of human ES cell differentiation toward posterior neural crest and its derivatives.In addition, the experimental approaches are not overly innovative  and largely focus on sufficiency outcomes with no experiments addressing necessity.We do not understand this point. We believe that the additional data we have included  provide better insight into the functionality and nature of the cell populations we are generating in vitro.As one of the main conclusions from the study concerns the origin of trunk NC cells from axial progenitors, there should be some spatial data to confirm the molecular signatures. For example, transplantation of anterior versus posterior NC cells into avian embryos to show the observed transcriptional changes impact the fate of cells at different axial positions. This technique is commonly used in the field to test axial identity and NC competence  and would complement the heavy reliance on using only molecular signatures in cell culture.In our opinion, the use of chick embryo transplantation as a comparative assay for quantifying differences in the axial identity status of heterogeneous hPSC-derived donor cell populations is very challenging to set up and possibly hard to interpret. We predict that the location of the graft site will routinely promote the selection of “compatible” subpopulations which in some cases will be minor “contaminants” co-emerging with the desired cell type during hPSC differentiation  and thus a large number of grafts will be required in order to derive statistically significant data. Further complications may arise due to the differential effect that the axial identity of the donor cells may exert on their developmental potency/proliferation in a heterotopic grafting context . In fact, we are not aware of any study where chick embryo grafting was employed to compare in a quantitative manner distinct PSC-derived cell types in terms of axial identity. However, as stated earlier we have performed grafting of axial progenitor-derived trunk NC cells into chick embryos to confirm in qualitative terms that these behave similarly to their in vivo counterparts, i.e. they exhibit NC-like migratory behaviour and colonise the DRG.Indeed, many of the genes used by the authors to define axial NC identity are expressed in other cell types, particularly other neural, neuroectodermal and mesoderm derivatives that develop adjacent to NC cell in vivo. Thus, without the addition of fate mapping data in vivo, other interpretations/conclusions could be drawn on what cell types are actually generated by this new method.1) The authors should employ transplantation techniques with different progenitors derived in vitro  to show they indeed are fated/committed to specific axial lineages in vivo. For example, RA-treated anterior NC cells should largely generate cardiac/vagal cell types in vivo compared to posterior NC cells, whereas transplanted axial cells would be expected to generate multiple posterior lineages. Such data would significant enhance the impact of the study and strengthen the conclusion that human axial progenitors generate trunk NC.See our response above.2) The authors identify a potential new gene involved in trunk NC development, ASLX3. The authors should perform in situ hybridization or immunohistochemistry on vertebrate animals  to localize ASLX3  to trunk NC cells in vivo. This would strengthen their argument that their approach used to classify axial identity in vitro (which is reliant on HOX gene expression) can also produce new knowledge on the genetic regulation of trunk NC in vivo.We agree with the reviewer that ASLX3 is a promising candidate marker of trunk NC. However, the characterisation of its expression domains in vivo will be time-consuming since we are not aware of any published antibodies or in situ hybridisation probes that could be used for ASXL3 detection and we would have to generate these reagents from scratch. Consequently, while of clear interest, we think that carrying out these in vivo experiments are beyond the scope of the current study which is focused on the generation of trunk neural crest cells by differentiation of hPSC in vitro.Reviewer #3:The manuscript shows that trunk-NC cells are best/most efficiently derived via an axial progenitor population, similar to the neuro-mesodermal progenitor giving rise to lower motor neurons. The authors show solid work that clearly indicate that this progenitor can be guided into producing NC cells with trunk identity. More sophisticated experiments, such as following cells over time using reporter lines to show that the NMP truly is giving rise to the trunk-NC population as well as in vivo work showing the same thing would definitively prove this interesting hypothesis. Therefore, some of the conclusions should be worded a bit more carefully. Nevertheless, this is the first report (to my knowledge) of NC cells derived from NMPs with trunk NC character and it seems a highly efficient one as well. Thus, I think this is an important report that should be published.We would like to thank the reviewer for recognising the importance of our manuscript. We believe that the additional experiments we carried out in response to points A and B (see above) address the reviewer’s suggestions.- To strengthen the conclusion in subsection “A BMP-dependent neural crest signature in human axial progenitor cultures”  it would be useful to see what percentage of all cells are posterior axial progenitors committed to become trunk-NC. For example, by doing triple stained intracellular FACS analysis of T, CDX, SOX9.We have included immunostaining quantification data, see our responses above.- In Figure 3D, the authors show that the axial progenitor can give rise to paraxial mesoderm. This could be strengthened by showing that these PXM cells can further be differentiated into definitive mesodermal cell types.We have added data showing the expression of PXM and early somite markers in our axial progenitor-derived PXM cells .- Do the SOX9+ cells early at day 3 also express SOX10? . Why does SOX9 come on before SOX10 and does SOX10 stay on?We did not detect any SOX10 in day 3 axial progenitors and its expression starts round days 6-7 of differentiation (d0=start of NMP differentiation) peaking round days 8-9 and then becomes downregulated till its extinction round days 10-11. The earlier upregulation of SOX9 relative to SOX10 is in agreement with in vivo expression data .- Can you maintain this trunk-NC cells as progenitors and expand them in culture, if that is possible it would make this technique even more useful.We have tried to maintain/expand trunk NC cells as spheres in the same way as we have previously reported for cranial NC  but, unfortunately, our attempts have been unsuccessful.-The RNA-seq/microarray data suggests that DMBX1 and LHX5 versus HES6 could serve as markers to distinguish anterior from trunk NC cells. This notion should be strengthened by staining to show the same at the protein level.The expression of DMBX1/LHX5 and HES6 in the anterior and trunk NC respectively has already been examined thoroughly in the chick embryo by Simoes-Costa and Bronner, 2016 and therefore we have used them in our study as “established” axial identity readouts.- Figure 5C: 80% of Phox2B-GFP- cells are ASCL1+? What are these cells? Maybe they are younger cells that have not yet turned on Phox2B, which would mean that at day 18 there is a mixture of cells in terms of developmental stage. Could that be showed more?We would like to clarify that Figure 5C shows that 80% of the cells were found to be ASCL1+ and approximately 60% were double-positive for both PHOX2B-GFP and ASCL1 and not that 80% of PHOX2B-GFP negative cells are ASCL1+. ASCL1 has been previously shown to be turned on earlier than PHOX2B, both in vivo and in vitro, during hPSC differentiation toward sympathetic neurons , so we agree with the reviewer that the remaining 20% of ASCL1+ PHXO2B-GFP- cells could be earlier progenitors indicating that day 18 cultures contain cells from different developmental stages. We have found that at later stages almost all PHOX2B-GFP+ cells are also ASCL1+ which further supports the idea that earlier cultures are developmentally heterogeneous. We have made a note of this in the Results section .- Scale bars are missing in all pictures.This has been corrected.- Discussion section: This should be stated more carefully, since the transcriptome analysis is done in in vitro generated cells. Without confirming in vivo data, one cannot definitively prove that axial progenitors are converted into posterior NC in embryogenesis.We have re-phrased to “Our transcriptome analysis data suggest that, at least in vitro, a generic trunk identity is first installed…”.Additional references:Aires R, Jurberg AD, Leal F, Nóvoa, Cohn MJ, Mallo M. Oct4 Is a Key Regulator of Vertebrate Trunk Length Diversity. Dev Cell. 2016; 38(3):262-74.Cheung M, Briscoe J. Neural crest development is regulated by the transcription factor Sox9. Development. 2003; 130(23):5681-93.DeVeale B, Brokhman I, Mohseni P, Babak T, Yoon C, Lin A, Onishi K, Tomilin A, Pevny L, Zandstra PW, Nagy A, van der Kooy D. Oct4 is required ~E7.5 for proliferation in the primitive streak. PLoS Genet. 2013; 9(11):e1003957.Yeom YI, Fuhrmann G, Ovitt CE, Brehm A, Ohbo K, Gross M, Hübner K, Schöler HR. Germline regulatory element of Oct-4 specific for the totipotent cycle of embryonal cells. Development. 1996; 122(3):881-94.Zhang D, Brinas IM, Binder BJ, Landman KA, Newgreen DF. Neural crest regionalisation for enteric nervous system formation: implications for Hirschsprung's disease and stem cell therapy. Dev Biol. 2010; 339(2):280-94.


Limb remote ischemic postconditioning (LRIP) has been reported as an effective method to reduce the induced experimental stroke damage after ischemic reperfusion (IR) injury. Studies suggest that anesthetics used during induction of ischemic stroke can reduce IR injury, which could affect the actual mechanisms of neuroprotection by LRIP. This study focuses on the comparative effects of anesthetics such as isoflurane and ketamine-xylazine on ischemic injury when used during LRIP. Adult C57BL/6 mice were anesthetized by isoflurane or halothane, and transient middle cerebral artery occlusion (MCAO) was induced through insertion of the filament. Under isoflurane or ketamine-xylazine anesthesia, LRIP was performed after 90 min of reperfusion by carrying out three cycles of 5 min ischemia/5 min reperfusion of the bilateral hind limbs for one session per day for a total of 3 days. Results showed that the use of different anesthetics—isoflurane or ketamine-xylazine—during LRIP had no effects on body weight. However, LRIP was able to improve neurological function as observed by the neurological deficit score in ischemic mice. Interestingly, the neurological deficit in the group where ketamine-xylazine was used was better than the group where isoflurane was used during LRIP. Furthermore, the LRIP was able to prolong the period of the ischemic mice on the rotarod and this effect was more significant in the groups where ketamine-xylazine was used during LRIP. Moreover, LRIP significantly attenuated the infarction volume; however, this effect was independent of the anesthetic used during LRIP. From these results, we conclude that ischemic mice that were subjected to LRIP under ketamine-xylazine anesthesia had better neurological deficit outcomes after stroke. Cerebral ischemia, the most common acute cerebrovascular disease, has been recognized as one of the leading causes of mortality and disability in the world. Our previous studies have documented that either preconditioning or limb remote ischemic postconditioning (LRIP) is useful for reducing ischemic reperfusion (IR) brain injury ,2, but tet al. *100 ,33.p values < 0.05. For repeated measures of NDS and rotarod differences were analyzed by two-way ANOVA with subsequent group comparisons by non-parametric Wilcoxon test by using JMP Pro software. Data are expressed as means ± SD.Statistical significance was estimated by Student’s t-test for unpaired observations between two groups or by one-way analysis of variance (ANOVA) followed by Newman-Keuls multiple comparison tests. Results are considered statistically significant at After MCAO, the body weights of mice from each experimental group decreased from day 1 to day 3; however, no significant difference was observed between the groups. For NDS, in the comparison between cohorts 1 and cohort 2 that used isoflurane during MCAO as well as during LRIP, a significant (p < 0.05) improvement was observed in cohort 2 on day 1 and day 3. The comparison between cohort 3 and cohort 4 that used halothane during MCAO and ketamine-xylazine during LRIP showed significant improvement in NDS in cohort 4 at all post-MCAO time points . Of interest, the comparison of cohort 2 (group with isoflurane during LRIP) and cohort 4 (group with ketamine-xylazine during LRIP) revealed a significantly (p < 0.05) improved NDS in cohort 4 at day 2 .LRIP treatment and the use of isoflurane significantly (p < 0.01) increased the duration of time on the rotarod as compared to the ischemic group on day 1, while there was a beneficial trend on day 2 and 3 post MCAO. Similarly, mice in cohort 4 using ketamine-xylazine in the LRIP treatment group spent a significantly (p < 0.001) greater amount of time on the rotarod than mice from cohort 3 on all 3 days post MCAO. Interestingly, there was also a significant difference between cohort 2 and cohort 4 mice at day 2 (p < 0.05) and day 3 . Similarly, we did not use ketamine-xylazine during MCAO because one of our exclusion criteria includes incomplete occlusion of the MCA, which is primarily determined by a change in CBF and secondarily by circling behavior during MCAO and after reperfusion. This testing is more precise when a gaseous anesthetic such as halothane or isoflurane is used because such anesthetics can cause animals to wake up a short time after the anesthesia is discontinued. With injectable anesthetics such as ketamine-xylazine, however, mice will take a longer time to wake up, and we may not be able to determine the circling behavior precisely.It has been reported that LRIP effectively reduces the ischemic injury of the brain , heart , or otheThe body weight changes (as reported in Cerebral infarction usually occurs after the blockage of the cerebral artery in the brain, which potentially affects the brain and neuronal function. To quantify the infarct volume, we performed TTC staining. TTC staining is one of the methods used to analyze the viable tissue, notably at earlier time points. From the TTC results, we found that LRIP was able to reduce the infarct volume after MCAO, irrespective of the use of isoflurane or ketamine-xylazine. This suggests that there was no effect of isoflurane or ketamine-xylazine use during LRIP on brain infarct volume. It is important to note that LRIP resulted in a significant decrease in functional deficits in cohort 4, where ketamine-xylazine was used during LRIP as compared with cohort 2, and where isoflurane was used during LRIP. However, there was no difference in infarction volume between cohorts 2 and 4. This raises the question of why we were not able to see similar disparities in functional and anatomical outcomes. It has been observed in various preclinical and clinical studies that often functional outcomes are not fully translated into anatomical betterment or vice versa ,40; howeSome limitations of our study are that we have not investigated the neuroprotective mechanism of LRIP or how anesthetics may affect LRIP-related stroke outcomes. Although anesthetics are widely used during a range of procedures, their exact mechanism, and overall outcomes are still not completely understood. Some studies have suggested that isoflurane has some anti-cell death properties –43. KetaIn conclusion, our study suggests that the induction of LRIP is neuroprotective in ischemic mice and reduces the infarct area, motor function, and NDS. We have not observed any significant difference in infarct area when comparing isoflurane with ketamine-xylazine used during MCAO and LRIP, but there were differences in NDS and rotarod scores using these anesthetics. Hence, we conclude that LRIP confers neuroprotection against ischemic stroke, and the use of either ketamine-xylazine or isoflurane may affect the outcomes to some extent.


Physical activity plays a significant role in the managing of type 2 diabetes and is essential in reducing morbidity and mortality associated with diabetes mellitus. A number of factors influence non-adherence to physical activity: social, personal, environmental and economic factors. Diabetes research conducted in Botswana has focused on behavioural change, treatment adherence and nutrition. The physical activity levels of type 2 diabetes patients and associated factors are not known.The aim of this study was to assess the physical activity levels (PALs) and factors associated with physical activity in type 2 diabetes mellitusThe study was conducted at a public clinic in Gaborone, Botswana, in 2017.An observational cross-sectional study was conducted at a public clinic in Gaborone, Botswana. An interview-administered questionnaire was used to assess the PALs and factors associated with physical activity in type 2 diabetes mellitus patients. Data were captured on Excel and exported to SPSS software version 25 for analysis. Chi-square test, Fischer’s exact test and Pearson’s moment correlation examined the relationship between participants’ characteristics and their engagement in regular exercise.r = −0.085) and between sitting time (sedentary time) and PAL (−0.098).The majority of the study participants had low PALs (54.7%). The results showed a non-significant negative correlation between age and PAL (Most type 2 diabetes mellitus patients had low PALs. Health-promoting activities are needed to promote physical activity and thus prevent complications associated with physical inactivity. The secondary complications include retinopathy, traumatic amputations, neuropathies, nephropathy, pregnancy-related problems and hypertension.19 Diabetes research conducted in Botswana has focused on behavioural change and adherence to treatment and nutrition. The physical activity levels of type 2 diabetes mellitus patients and associated factors are not known. The aim of this study was to assess the physical activity levels (PALs) and factors associated with physical activity in type 2 diabetic patients at a public clinic in Gaborone, Botswana in 2017.Diabetes is sixth in the top ten causes of death in Botswana, and the WHO country profile report for 2016 revealed a diabetes prevalence of 5.6% (20–79 years) and a diabetes-related mortality of 4%  in Botswana.An observational cross-sectional study design with an analytic component was implemented.The study site was a public clinic in Gaborone, which is one of the two national centres of excellence for diabetes management in Botswana. The centre is managed by nurses and doctors trained in diabetes management. The clinic is an extension of the tertiary hospital. It is a catchment area for both urban and rural diabetes mellitus patients and at the same time serves as a primary health care centre for the local city residents. The clinic attends to approximately 100 diabetes patients per day. The patients come every 3 months for regular check-ups. Most of the patients who attend the clinic are in the low- to middle-income bracket as those belonging to the high-income bracket usually attend private hospitals. The clinic also serves a few expatriates.20 Gaborone covers 169 km2 with a population density of 370/km.20 The dominant languages spoken in Botswana, particularly in Gaborone, are Setswana and English, with a few people speaking Kalanga.Gaborone is the capital and commercial city of Botswana. It has a total population of 231 592 people, accounting for 11% of the total population.Type 2 diabetes patients aged between 18 and 65 years who attended the clinic were recruited into the study. The study was conducted from 29 May 2017 to 30 August 2017. Systematic random sampling method was used for sampling. Each day the clinic sees about 100 diabetes patients, of which approximately 80 have type 2 diabetes. The researcher took 27 patients per visit per week for the first 4 weeks and 26 patients per visit per week for the last 4 weeks of the study. Each day, the first participant was chosen at random and thereafter an interval of two (3rd person) patients was implemented to get the targeted the number. G-Power software was used for calculating the sample size, with the type 1 error being set at 0.05 and the statistical power at 80%. The critical F-value was set at 2.65 and the medium effect size was set at 0.23. The calculated sample size was 212.11 Age was categorised into three groups young, middle-aged and old-aged according to reviewed literature.21 Young was between 18 and 35 years, middle-aged was between 36 and 55 years and old-aged was 56+ years. The researcher added the type of medication and exercising habits to the socio-demographic section to capture all the necessary patient information. Anthropometric measures to assess nutritional status were recorded, that is, height and weight. The BMI (Body mass index) was calculated (weight [kg]/height2 [m2]) and used to rate the nutritional status according to WHO criteria. The World Health Organization classifies BMI into four categories, underweight < 18.5, normal BMI 18.5–24.9, overweight 25.0–29.9 and obese BMI ≥ 30.22A standardised interviewer administered questionnaire was used to assess the PALs and factors associated with physical activity in type 2 diabetic patients. The first section of the questionnaire covered socio-demographic information. The researcher adapted a validated questionnaire to assess socio-demographic status such as age, sex, number of people in the household, level of education, marital status and occupation.23 A separate IPAQ question assessed the time spent sitting to measure sedentary activity but was not part of the total physical activity score.24 It assessed the time spent sitting on a typical day, and weekend day, and the time spent sitting during travel.24 The last section covered the barriers and motivators to physical activity as reported by the patient.25The second section was an adopted questionnaire from the International Physical Activity Committee (IPAQ), which measured the PAL.The questionnaire was pretested with 13 patients to ensure that it was user-friendly. The questionnaire was translated into Setswana by a qualified translator. The translations were back translated into English to ensure that they are compatible with the original questionnaire. Permission to conduct the study was obtained from the hospital manager. Qualified nurses screened the patients and those that met the inclusion criteria and agreed to participate were referred to the research assistant.p-value < 0.05 was considered to be statistically significant.The data were captured into Microsoft Excel and then exported into SPSS version 25. Descriptive statistics were summarised using frequencies, while chi-square and Fischer’s exact statistics were used to determine the associations between participant’s characteristics and PAL. Where the cell frequencies are less than 5, the Fischer’s exact test is used. The Pearson’s product-moment coefficient was calculated to find the correlation between PAL and participant’s characteristics. A 24 For moderate PAL, the criteria are five or more days of any combination of walking, moderate-intensity or vigorous-intensity activities, and achieving a minimum total physical activity of at least 600 MET-minutes per week.24 Low PAL is the lowest level of physical activity and is below 600 MET per week.The level of physical activity was analysed by taking into consideration the metabolic equivalent of task (MET)-minutes per week indicators and the days of physical activity. To calculate MET-minutes per week per participant, the following formula was used:  × (time of activity in minutes per day) × (days of activity per week) = MET-minutes per week. The overall PAL was categorised as high, moderate or low. High PAL refers to seven or more days of any combination of walking, moderate-intensity or vigorous-intensity activities achieving a minimum total physical activity of at least 3000 MET-minutes per week.Ethical clearance was obtained from the University of KwaZulu-Natal Biomedical Research Ethics Committee (BE540/16). Permission to conduct the study was obtained from the Hospital Ethics Committee (PMH 5/79(286-4-2017)) and from the Botswana Ministry of Health (HPDME 13/18/1(863)). All patients who agreed to participate in the study signed an informed consent form. Questionnaires and signed consent forms were filed separately to ensure anonymity of the questionnaire. Participation was voluntary, and participants were informed that they could withdraw from the study without prejudice. The anonymous questionnaires were labelled with unique numbers to ensure confidentiality.One hundred and seventy patients participated in the study. More than half of the participants had low PAL (54.7%) with only 14 participants (8.2%) having high PAL . The revr = −0.085) and between sitting time (sedentary time) and PAL (−0.098). There was no statistical significant association between PAL and the following socio-demographic factors: sex, marital status, level of education, employment status, dwelling place and BMI . The results are similar to other studies that used the short IPAQ form although different to another study that used the long IPAQ form like in this study. Low PAL was found in Western Nigerians (62%) and in a medically underserved community in America (62.9%) with type 2 diabetes mellitus.27 The study that used a long form found 31% of type diabetes mellitus patients to be physical inactivity.28This study assessed PALs and the factors associated with physical activity in type 2 diabetes mellitus patients at a clinic in Botswana. Physical activity is a chief cornerstone in the management of type 2 diabetes although diabetes patients still remain inactive.28 A study conducted in America also reported a similar trend.27 Type 2 diabetes patients were shown to be sedentary for a minimum of 5.8 h per day (SD 4.5) in the past week.27The average sitting time (sedentary) of the participants in the study was 5.8 hours per day (SD: 5.4) in the past week. Similar results were reported in Nigeria in which type 2 diabetes patients were sedentary for a minimum of 4.8 h per day in the past week.r = −0.098). The results are similar to those of a study conducted in Malaysia which reported a weak and negative relationship between PAL and sedentary lifestyle (r = −0.175) among diabetes patients.11In this study, PAL was weakly and negatively related to sedentary lifestyle  had low PAL. The results in this study are in contrast to the results found in other studies, employed type 2 diabetes mellitus patients were shown to be significantly more physically active than their unemployed counter parts either intentionally or incidentally.p > 0.05). The findings of this study concur with the results of the study in Malaysia, which did not find any significant association between BMI and PAL.11 In contrast, studies in Ireland and America found that those with normal BMI participate more in physical activities than those with high BMI.34 This could be attributed to the barriers mentioned by those with high BMI such as physical discomfort and embarrassment.34 On the contrary, some studies have revealed that those with high BMI have high PAL because of their understanding of the effect of weight on diabetes mellitus.7Low PALs were reported mostly among the obese (50%) and overweight (60%) participants in this study. However, this study did not find any significant association between BMI and PAL . This is in contrast with studies that reported that married people have higher levels of physical activity as compared to their single, widowed and divorced counterparts.35Social support in the form of a partner or spouse plays a major role in patient’s decision to engage in physical activity.33 The same pattern has been found in the general population.36 The difference in PAL between men and women might be because of the difference in social obligations between them; men are expected to be masculine as breadwinners, while women are usually restricted around the house.33 This study did not find any significant association between gender and PAL.Men have a higher physical activity level compared to their female counterparts.28 A number of urban people have cars and tend to lead a more sedentary lifestyle, while in the rural areas people are more involved in activities that require manual work such as farming.9 In this study, a larger proportion of both urban (56.1%) and rural (54.8%) dwellers had low PALs. There was no association identified between dwelling place and PAL in this study. Similarly, a study in Ireland, Western Europe, did not find any significant relationship between PAL and dwelling place.34Environmental factors such as dwelling place has an effect on physical activity.11 Lack of time, health problems, discomfort, fear of hypoglycaemia, laziness, cost of gym and unsafe environments are perceived barriers for physical activity among diabetes mellitus patients.37 This study found that the absence of exercising space and no one to exercise with were the common reported barriers to physical activity.In this study, the common motivators to physical activity were the need to be healthy (73.3%), wanting to look good (66.9%), liking to exercise (57.1%) and wanting to lose weight (50.3%). These findings are comparable to a Malaysian study that reported the perceived motivators to exercise being: the willingness to be healthy, a realisation of the importance of exercise, love of exercise and daily routine, with the willingness to exercise having the highest score.The reliance on self-reporting data is a limitation of the study as participants may have over- or under-reported in the questionnaire which is prone to social desirability bias. The study was conducted in one clinic and the results may therefore not be generalisable to patients in other clinics. The study failed to reach the desired minimum sample size because of fluctuations in the number of patients seen per day within the 3 months study period decreasing the study power and increasing the margin error. Some variables were also missing completely at random which might have affected the results.Further studies need to be conducted on adherence to physical activity after demonstration and giving type 2 diabetes mellitus patients exercise programmes. Objective measures such accelerometers should be incorporated into the assessment of PALs to reduce social desirability bias found in self-report PALs. Case-control studies comparing the PALs of patients with diabetes mellitus and those without diabetes need to be performed. A qualitative study to get an in-depth understanding of the highlighted barriers and motivators to physical activity is needed.Physical activity needs to be incorporated in the assessment of type 2 diabetes mellitus patients and should be consistently evaluated in the same way as glycaemic control. A multi-disciplinary approach to diabetic management should consistently encourage and emphasise the importance of physical activity in the management of type 2 diabetes mellitus. There is necessity for a resident physiotherapist, or exercise specialist, who can tailor patient-specific exercises, the same way dieticians create patient-specific diets.This study revealed that type 2 diabetes mellitus patients at a public clinic in Gaborone have low PALs. The desire to look good and liking exercise were the common motivators to physical activity. Absence of exercising space and no one to exercise with were the common reported barriers to physical activity. Health-promoting activities are recommended to promote physical activity, especially in the older patients, and thus prevent complications associated with physical inactivity among patients with type 2 diabetes mellitus.