We present a patient with ischemic cardiomyopathy who had ventricular tachycardia (VT) with QRS morphology alternans. The electrophysiological findings, in this case, supported the occurrence of antegrade activation of the proximal His–Purkinje system during VT, with the ultimate electrocardiogram morphology dependent on fusion from intramyocardial and His–Purkinje activations. Intracardiac recording during tachycardia showed an atrioventricular dissociation during the ventricular tachycardia (VT) . What is the mechanism of the alternating QRS complexes?A 74-year-old woman with a history of inferoposterolateral wall myocardial infarction (MI) and coronary artery bypass grafting presented with incessant tachycardia. A cardiac ultrasonographic study showed an ejection fraction of 30% and moderate-to-severe mitral regurgitation. Electrocardiography revealed a beat-to-beat shift in both the QRS axis and amplitude occurring in a 1:1 fashion without change in a mean cycle length (CL) of 500 ms 1 Monomorphic VTs with alternating QRS morphologies have been observed to occur spontaneously and during programmed stimulation in human hearts.7 Bidirectional VT is one of the most commonly known prototypes of electrical alternans and is characterized by a rapid, wide-complex electrocardiogram (ECG) pattern with alternating QRS morphology and axis.9 Multiple exit sites from reentrant circuits would be associated with these changing QRS morphologies2; however, 3 more options should also be considered: whether more than one arrhythmia is occurring, whether fusion with sinus beats is occurring, or whether tachycardia originates within or in the proximity of the His–Purkinje conduction system (HPS).11 Identification of the mechanisms for these changes is important when considering the feasibility of ablation as therapy.4Multiple VT morphologies were observed in a majority (60%–80%) of post-MI patients who underwent catheter ablation.12 The phenomenon of an early invasion of the HPS may have an exit point near the preserved conduction system, especially in the septal area.16 However, the involvement of the HPS in VT associated with heart disease can be difficult to recognize in post-MI patients and has 3 distinct clinical forms as mainly re-entrant or focal mechanisms in distal HPS.16 A focal mechanism is present in up to 9% of VTs in patients with prior MI who are induced during the electrophysiology study for radiofrequency ablation.17 Furthermore, VT exhibits a focal activation pattern on the endocardial and epicardial surfaces despite a re-entrant VT.18In addition to a scar-based reentry, the subendocardial Purkinje fibers along the border zone of the subendocardial scar can play an important role in ventricular arrhythmia in post-MI patients.17 If the arrhythmia is a VT, then whether the tachycardia is automatic or re-entrant cannot be determined by a 12-lead ECG .20 Only pacing maneuvers  can identify the mechanism of arrhythmia. In Figure 3, a VT focus that arises within the interventricular septum may sometimes propagate into the HPS and exit into the ventricular myocardium fairly distant from the original focus (posterior fascicule in the current case). This can mistakenly suggest that the true site of origin is at the exit site of the heart’s normal conduction system. Furthermore, the re-entrant monomorphic VT originating from the left posterior Purkinje fibers, which is analogous to an idiopathic left VT, can develop in the acute or chronic phase of MI . Hayashi et al. described 4 patients with prior MI in whom VT involving the HPS was observed.15 Penetration of the HPS by myocardial VT, with resultant fusion from intramyocardial VT and HPS activation, could have accounted for the near-normalization of the QRS morphology during VT2.14 Bogun et al. described 9 patients with an ischemic VT, a relatively narrow QRS complex (≤145 ms), and HPS involvement but excluded bundle branch re-entry, interfascicular re-entry, and fascicular VT as a mechanism.12 In the current case, there was no clear evidence of conducted sinus beats giving rise to capture and fusion; however, the morphology of the QRS complex during VT1 exhibited a more distinctively inferior axis  than that during VT2  with a similar CL such that one re-entrant loop with varying degrees of fusion is likely .10 We performed both high- and low-output pacing to more clearly elucidate the mechanism . When tachycardias originate in the conduction system, the pace-map at the site of origin (within the conduction system) may be completely different from the VT QRS morphology because of concomitant capture of the surrounding myocardium near the conduction tissue, whereas, during tachycardia, the exit is at a distal site into the myocardium. Therefore, both high- and low-output pacing should be considered , especially when the tachycardia shows spontaneous variation in QRS duration or when multiple early sites are noted when mapping with a 3-dimensional system.10 On the other side, the pace-mapping of VT circuits may be difficult where there are multiple entrances and exits,21 and it should be kept in mind that pace-mapping at the high output may capture the myocardium of a contralateral chamber and result in spurious findings.Differentiating focal VTs from re-entrant VTs is important because the characteristics of the ablation site significantly differ depending on the mechanism.15 Bogun et al. identified a Purkinje potential at each successful ablation site.12 Radiofrequency ablation of presystolic Purkinje potentials in the para-Hisian region in the current case  resulted in the termination of the VT,15 and, from then on, the patient was non-inducible for VTs. Whether uncovering focal and re-entrant VT mechanisms routinely during ablation procedures will improve the outcomes of VT ablation is worthy of future study.20As there was no change in the CL despite QRS alternans, we considered it to be a single re-entrant loop with 2:1 penetration of the HPS by myocardial VT, with resultant fusion from intramyocardial VT and HPS activation. The precise electrophysiological factors underlying direct HPS penetration remain speculative. It is difficult to explain why this sudden change in the VT configuration occurred without any specific maneuver. The presence of spontaneous changes in the regional refractoriness of the HPS or the adjacent ventricular myocardium that may result in an antegrade or retrograde block of the HPS during the wide QRS complex VT might have caused these alternans. Another possible explanation could be the presence of 2 very near but distinct exit points of the tachycardia circuit, 1 connecting to the HPS directly and the other 1 connecting to the ventricular myocardium, resulting in a similar QRS configuration but a different width. Catheter ablation was highly effective in eliminating this VT without affecting left ventricular conduction.


Astyanax mexicanus as a model for understanding the mechanisms of hypoxia tolerance in the cave environment. Primitive hematopoiesis, which is restricted to the posterior lateral mesoderm in other teleosts, also occurs in the anterior lateral mesoderm in Astyanax, potentially pre-adapting surface fish for hypoxic cave colonization. Cavefish have enlarged both hematopoietic domains and develop more erythrocytes than surface fish, which are required for normal development in both morphs. Laboratory-induced hypoxia suppresses growth in surface fish but not in cavefish. Both morphs respond to hypoxia by overexpressing hypoxia-inducible factor 1 (hif1) pathway genes, and some hif1 genes are constitutively upregulated in normoxic cavefish to similar levels as in hypoxic surface fish. We conclude that cavefish cope with hypoxia by increasing erythrocyte development and constitutive hif1 gene overexpression.Dark caves lacking primary productivity can expose subterranean animals to hypoxia. We used the surface-dwelling (surface fish) and cave-dwelling (cavefish) morphs of  Astyanax mexicanus, which consists of surface-dwelling (surface fish) and cave-dwelling (cavefish) morphs, as a model to study adaptation to hypoxia in caves. Surface fish colonized subterranean waters in the Sierra de El Abra region of Mexico  undergoes myelopoiesis to form macrophages, and some of these later differentiate into neutrophils and microglia  gene, which encodes a transcriptional repressor of many different genes  at 36 hpf . At thisdization . The qPCt 60 hpf . In situace fish , which wace fish . Fourth,opoiesis , was comopoiesis . The rest 24 hpf , prior tt 24 hpf . These rhbb2, gfi1aa, and other hematopoietic marker genes during primitive hematopoiesis in surface fish and cavefish embryos  gene, a marker of developing blood cells  gene, a marker for macrophage differentiation  were compared in surface fish and cavefish after 14 hr of PHZ exposure  gene, the HIF1 family genes hif1aa and hif1a-like2, and the HIF1-regulated downstream gene hexokinase 1 (hk1) (hifab and hif1a-like genes were expressed at similar levels in normoxic cavefish and hypoxic surface fish (glucose-6-phosphate dehydrogenase (g6pd), lactate dehydrogenase a (ldha), and pyruvate dehydrogenase kinase 1 (pdk1) genes were not significantly increased by hypoxia in surface fish or cavefish  . In contace fish , implyincavefish . The resgfi1aa gene, a crucial regulator of hematopoiesis. In addition, we report that the increase in erythrocytes may be caused by expansion of embryonic hematopoietic domains in cavefish compared to surface fish. Finally, we provide evidence that cavefish have evolved a permanent response to hypoxia involving the constitutive overexpression of the some of the hif1 genes, which persists under normoxic laboratory conditions.We show that cavefish embryos develop more red blood cells than surface fish embryos, a classic response to hypoxia , when thhbb2, hbbe2, and gfi1aa expression was observed during embryogenesis and after larval hatching in cavefish. In zebrafish, transfusion experiments indicate that blood cells circulating during the first four days of development are primitive erythrocytes , which may promote the specification of more blood precursor cells. In future studies, it will be interesting to determine how the expansion of erythropoietic potential is controlled in cavefish.The expansion of erythropoiesis into the ALM during primitive hematopoiesis in opoiesis . Expressis  at 36 hr post-fertilization (hpf) and placed in water on a concave microscope slide. Blood flow was imaged as cells flowed through the Duct of Cuvier , 0.01 M sodium acetate (pH 4.5), 0.65% H2O2 for 15 min in the dark (Red blood cells were stained at 36 hpf with 0.6 mg mlthe dark . The stahemoglobin subunit beta-2-like (hbb2), growth factor independent 1 A transcription repressor (gfi1aa), LIM domain only 2 (lmo2), or lymphocyte cytosolic protein 1 (lcp1) genes. Genes were cloned from a 24 hpf surface fish cDNA library and 10 hpf, 24 hpf and 30 hpf cavefish cDNA libraries using the pCRII TOPO dual promoter vector  transformed into One shot Top10 cells and the following primers: hbb2 , gfi1aa , lmo2 , and lcp1 . DIG-labeled probes were made using SP6 or T7 transcription kits  from linearized plasmid or, in the case of gfi1aa, from a PCR product made with the cloned cDNA as template and the above-mentioned primers, after a RNA-polymerase promoter site was added to the 5’ end.In situ hybridizations were performed using probes to the In situ hybridizations were performed as previously described . Brieflyhbb2 probe. In a separate experiment, embryos were imaged on a Zeiss Axioskop2 microscope with a Axiocam 503 camera and measurements of tail length  were made in Image-J. After imaging, PHZ treated embryos and controls were stained with o-dianisidine and imaged for blood cell quantification as described above. Because low numbers of blood cells were present after PHZ treatment, blood cells were counted in a larger area covering the whole anterior side of the yolk in lateral view (400 pixels wide). Larvae of two separate clutches of eggs were used per morph for the latter experiment.Phenylhydrazine hydrochloride  was used to induce hemolytic anemia . At 14 hSurface fish larvae were also treated with PHZ at 34 hpf for 1 hr, rinsed extensively for 1 hr, imaged with a microscope as described above, and left for 16 hr before imaging again. At the end of the experiment, larvae were stained with o-dianisidine. The length of the post-anal tail was measured using Image-J and growth determined as the differential between the two time points. Blood cell number was determined by manual counting as described above.A hypoxia chamber  was used to create a hypoxic laboratory environment in which oxygen was reduced by nitrogen gas . At 30 hpf larvae were exposed to hypoxia (1 mg/L oxygen) for 18 hr or to normoxia (outside of the chamber). Larvae were placed individually in wells of a 24-well plate  containing 2 ml clean fish system-water. Twenty-four larvae were used per treatment for surface fish and 48 larvae for cavefish from two separate clutches of eggs per morph. To measure post-anal tail growth, each larva was imaged before placement in a well and again at the end of the treatment using a microscope as described above. Image-J was used to measure post-anal tail length  and growhemoglobin beta embryonic 2 (hbb2) and hbbe2 , actin alpha 1 (acta1b), and lsm12a. Only rpl13a did not vary over time or between surface and cavefish and was therefore used as the final reference gene. RNA was extracted from 30 embryos with Trizol (ThermoFisher), treated with RNase-free DNase I (ThermoFisher), cleaned and concentrated by phenol/chloroform extraction and precipitated with ammonium acetate and ethanol. Poly(A)-primed cDNA was made with SuperScript III First Strand Synthesis SuperMix (ThermoFisher) and used in qPCR with Takara SYBR Premix Ex Taq (Tli RNaseH Plus)  and LC480 (Roche).To compare the extent of early erythropoiesis between cavefish and surface fish, the expression of In the laboratory hypoxia experiments, RNA was isolated from 24 larvae placed in the 15 mm Netwell insert  in a well of a 12-well plate. Two hours after the start of the hypoxia treatment the larvae were collected in Trizol (ThermoFisher) within 1 min after taking them out the hypoxia chamber by removing the Netwell inserts from the wells, decanting excess water, and the larvae were immersed in Trizol. RNA was isolated for six samples of 24 larvae each with three samples per clutch of eggs. Two separate clutches of eggs were used per morph.hypoxia-inducible factor 1 (hif1aa) and its duplicate gene hif1ab, hypoxia-inducible factor 1-like  and its duplicate gene hif1alike2 , insulin growth factor binding protein 1a (igfbp1a), hexokinase 1 (hk1), glucose-6-phosphate dehydrogenase (g6pd), lactate dehydrogenase a (ldha), and pyruvate dehydrogenase kinase 1 (pdk1) were quantified by qPCR in surface fish and cavefish larvae exposed to hypoxic or normoxic conditions. We evaluated rpl13a, acta1b, endoplasmic reticulum protein 44 (erp44) and guanine nucleotide binding protein ß polypeptide 1b (gnb1b) as potential reference genes: gnb1b had Ct values closest to the Ct values of the queried genes, did not differ between normoxic and hypoxic surface fish or cavefish, and was chosen as a reference gene. RNA was extracted from 24 embryos and treated with ezDNase and cDNA was made with SuperScript IV VILO mastermix (ThermoFisher). The primers used in qPCR analysis are shown in The amounts of -(ΔΔCt), where values > 1 show an increase and values < 1 a decrease. Variation was expressed as the range of fold change 2- (ΔΔCt+stdevΔΔCt) for the upper value or 2- (ΔΔCt-stdevΔΔCt) for the lowest value.A ΔCt for each gene was calculated by subtracting the average Ct value of each reference gene. For comparison of gene expression in cavefish to surface fish over time, ΔΔCt was calculated by subtracting the average ΔCt of surface fish at 10 hpf from each ΔCt for each gene. For comparison of gene expression under normoxic and hypoxic conditions, the ΔCt of surface fish under normoxic conditions was subtracted from the ΔCt for each gene. For graphical representation, the fold change was calculated as 2hbb2, hbbe2, and gfi1aa over time was analyzed per gene with a two-way ANOVA followed by pairwise comparison with Student’s t-test . Comparisons of blood cell number in reciprocal hybridization experiments were analyzed using a Wilcoxon/Kruskal-Wallis Rank Sums test followed by Wilcoxon for each pair . Differences in tail length between surface fish and cavefish at different concentrations of PHZ were analyzed using Wilcoxon/Kruskal-Wallis Rank Sums test followed by Wilcoxon for surface fish (SF) or cavefish (CF) for each pair . Comparison of blood cell number at different concentrations of PHZ were analyzed with Wilcoxon/Kruskal-Wallis Rank Sums test followed by Wilcoxon for each pair . Post-anal tail growth at 0 and 5 mg L–1 PHZ was analyzed with a Student’s t-test  and cell number with Wilcoxon/Kruskal-Wallis Rank Sums test . Post-anal tail growth under normoxic and hypoxic conditions was analyzed with Wilcoxon/Kruskal-Wallis Rank Sums test followed by Wilcoxon for each SF and CF pair . Cell number under normoxic and hypoxic conditions was analyzed with one-way ANOVA followed by Student’s t-test . Eye diameters were analyzed by one-way ANOVA followed by Student’s t-test . Expression as ΔΔCt of genes under normoxic and hypoxic conditions was analyzed per gene by one-way ANOVA followed by Student’s t-test or when variances where unequal with Wilcoxon/Kruskal-Wallis Rank Sums test followed by Wilcoxon for each pair .Statistics were done using JMP pro 14 (SAS Institute Inc). Normal distribution of data was determined using the Shapiro Wilk test. Comparison of blood cell number was done using Wilcoxon/Kruskal-Wallis Rank Sums test . Expression as ΔΔCt of  We believe it will have a significant impact on our understanding of cavefish adaptation in particular and adaptation to low oxygen environments in general. public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: (i) Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Didier Stainier as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Misty Riddle (Reviewer #2); Rachel Brewster (Reviewer #4).Thank you for submitting your article "Cavefish adapt to hypoxia by constitutive overexpression of hypoxia-inducible factor genes and increased erythrocyte development" for consideration by eLife, at least as it stands. If, however, you feel that you can address the points raised by our referees and make a compelling response to all of the issues raised, then we would be willing to consider a revised version of your paper for possible publication. Given the concerns raised by the referees, we would be looking for additional experimental data along the lines requested to strengthen the evidence in support of the key claims.You will see that our referees find your work intriguing, but have raised a number of important concerns, and in the light of these issues, we cannot offer to publish your paper in The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:1) More data is needed to show that CF do better than SF in a low oxygen environment, especially the growth experiments need larger numbers as the results are highly variable.2) The use of a hypoxia chamber or a comparable set up is needed to alleviate concerns about the reproducibility of the hypoxic values between trials.Reviewer #2 (Recommendations for the authors):Figure 1: Why wasn't o-dianisidine staining used for the quantification? Figure 1B is not discussed in the main text. I think adding this quantification would strengthen the paper.Figure 2B: I am not really convinced that HBB2 is expanded in the PBI by the image. There does appear to be staining in the anterior part of the fish however. The expansion in the anterior portion should be mentioned rather than expansion in the PBI.Line 227. Line 358-361: The authors claim that "cavefish are less sensitive to hemolytic anemia". I don't agree with this interpretation of the data. I would interpret the results as cavefish being less sensitive to developing hemolytic anemia with increasing concentrations of PHZ because they have more blood cells. To compare the effect of reducing blood cells, the authors could compare SF treated with 1 mg/L PHZ to CF treated with 4 mg/L; based on Figure 3A, these treatment groups have about equivalent blood cells. You may conclude from the data that cavefish are more sensitive to a reduction in red blood cells; cavefish treated with 2 mg/L PHZ look equivalent to untreated SF in terms of hbb2 staining, but they have notochord defects and reduction in length. The authors should re-evaluate this experiment and add text to the results considering these points.Figure 4B: The data should be reported as total number as in figure 1F instead of as a % of untreated. This would help the reader interpret the results of the experiment. The authors should then compare the phenotypes of SF and CF when they have equivalent number of red blood cells. For example, in figure 1D is tail length of CF treated with 4 mg/L significantly different from SF treated with 1 or 2 mg/L? These treatments look like they result in a similar level of red blood cells.Figure 5B. "the results showed that axial growth was significantly reduced in cavefish in both normoxic and hypoxic conditions relative to surface fish under normoxic conditions." The result shows there is only a difference in the normoxic condition. The authors do not report the statistics comparing SF-normoxic to CF-hypoxic.Line 361: "Hypoxia treatment inhibited axial growth in SF…". The results are not significant. Authors should increase the number of biological replicates and/or length of exposure.Line 500: The authors say that 40-50% normoxic was lethal. Did they compare lethality in SF and CF embryos? I think it would be of interest to include lethality as a measurement in the study. Are there concentrations that CF can survive while SF die? This may be a more relevant measurement than growth considering whether the increase in red blood cells is adaptive.More data is needed to show that CF do better than SF in a low oxygen environment and that if you decrease HIF expression and/or erythrocyte number this advantage is eliminated. Experimentally increasing HIF expression and/or erythrocyte number in SF and showing that this gives them an advantage in low oxygen water would provide additional strong evidence that the trait is adaptive.The authors should add text to the discussion that the study only focused on one of the cavefish populations and that examining the other populations would be an interesting future direction. The authors should add "Astyanax mexicanus" to the title. There are many different species of cavefish and this may not be a general feature for how cavefish adapt to hypoxia.Reviewer #4 (Recommendations for the authors):1) The use of other assays (e.g metabolic rate under hypoxia) to test the functional relevance of increased erythrocyte production and enhanced transcription of hif1 would strengthen the conclusions.2) In other teleosts, it is well established that elongation of the embryo is driven by convergent extension movements in the axial mesoderm. It would therefore be worthwhile examining whether this process occurs differently in cavefish relative to surface fish under hypoxia.3) Hif protein levels under hypoxic conditions should be measured.4) The use of a hypoxia chamber to control oxygen levels is recommended. Reviewer #2 (Recommendations for the authors):Figure 1: Why wasn't o-dianisidine staining used for the quantification? Figure 1B is not discussed in the main text. I think adding this quantification would strengthen the paper.Thank you for this comment. We agree and have added quantification of o-dianisidine stained cells in revised Figure 1G. These results strengthen our conclusion that cavefish have more red blood cells than surface fish.Figure 2B: I am not really convinced that HBB2 is expanded in the PBI by the image. There does appear to be staining in the anterior part of the fish however. The expansion in the anterior portion should be mentioned rather than expansion in the PBI.hbb2 expression in the ALM and PLM rather than the PBI. The comment also prompted us to rewrite these results for clarification. There are two major conclusions of our experiments: One is that hematopoietic/erythropoietic domains are present in both the anterior and posterior regions of Astyanax embryos, which is not the case in zebrafish and other teleost embryos, where they are present only posteriorly, and another is that both the anterior and posterior hematopoietic/erythropoietic domains (ALM and PLM) are expanded in cavefish compared to surface fish based on expression of the hhb2, gfi1aa, and lmo2 genes. The section of the revised manuscript was rewritten to emphasize on these two conclusions. Please see Lines 140-166. We also revised Figure 2  to highlight the two major conclusions of this section of the manuscriptWe also thank the reviewer for this comment. In the revised manuscript, we have now emphasized the expansion of Line 227. Line 358-361: The authors claim that "cavefish are less sensitive to hemolytic anemia". I don't agree with this interpretation of the data. I would interpret the results as cavefish being less sensitive to developing hemolytic anemia with increasing concentrations of PHZ because they have more blood cells. To compare the effect of reducing blood cells, the authors could compare SF treated with 1 mg/L PHZ to CF treated with 4 mg/L; based on Figure 3A, these treatment groups have about equivalent blood cells. You may conclude from the data that cavefish are more sensitive to a reduction in red blood cells; cavefish treated with 2 mg/L PHZ look equivalent to untreated SF in terms of hbb2 staining, but they have notochord defects and reduction in length. The authors should re-evaluate this experiment and add text to the results considering these points.Astyanax development. We thought that this section was necessary because older literature from the zebrafish community suggested that erythrocytes are produced during early embryogenesis but are not required for oxygen transport until much later in larval development. Thus, it was necessary investigate whether red blood cells are required for early development in Astyanax. We believe that our PHZ results, which are now fortified by additional experiments, answer this question in the affirmative. In particular, the experiments in revised Figure 5D show that larval growth is suppressed in the absence of red blood cells. We feel that these results strongly support the conclusion that erythrocytes are functional and required during early Astyanax development.We agree with the reviewer, but point out that the major purpose of this section of the manuscript was to determine whether erythrocytes have a role in early Because of other concerns listed in the Reviewer’s comment immediately above, and also taking into account the recommendation to report red blood cell phenotypes by number suggested in the comment immediately below, we have repeated all of the experiments in question. The results are shown in revised Figure 5C, which compares larval tail length and red blood cell number at increasing PHZ concentration over a 28 hr time span. The findings are as follows. First, red blood cell numbers are decreased in concert with increasing PHZ concentration in both surface fish and cavefish. Second, the loss of red blood cells is more abrupt and severe in surface fish than in cavefish. Third, increasing PHZ concentrations reduce tail length in surface fish but not in cavefish, which have more erythrocytes. We interpret these results to mean that cavefish are less sensitive to hemolytic anemia because they have more blood cells, matching the conclusion of this reviewer.We could not compare PHZ-treated surface fish and cavefish with the same number of red blood cells because we could not find a PHZ concentration with closely matching erythrocyte numbers in SF and CF that would allow this comparison to be made .Lastly, we have re-written the section of the Results describing these data. Please see Lines 183-207.Figure 4B: The data should be reported as total number as in figure 1F instead of as a % of untreated. This would help the reader interpret the results of the experiment. The authors should then compare the phenotypes of SF and CF when they have equivalent number of red blood cells. For example, in figure 1D is tail length of CF treated with 4 mg/L significantly different from SF treated with 1 or 2 mg/L? These treatments look like they result in a similar level of red blood cells.We agree with the reviewer. In revised Figure 5C the data is shown as the total number of red blood cells. However, as pointed out above, we could not compare PHZ- treated surface fish and cavefish at equivalent red blood cell numbers because there was no PHZ concentration where approximately equivalent numbers could be found.Figure 5B. "the results showed that axial growth was significantly reduced in cavefish in both normoxic and hypoxic conditions relative to surface fish under normoxic conditions." The result shows there is only a difference in the normoxic condition. The authors do not report the statistics comparing SF-normoxic to CF-hypoxic.We have now repeated these results using a hypoxia chamber to better control oxygen levels, and have confirmed significantly different effects of hypoxia tolerance between SF and CF. Please see revised Figure 6. These results support the conclusion that CF are less sensitive to hypoxia than SF.Line 361: "Hypoxia treatment inhibited axial growth in SF…". The results are not significant. Authors should increase the number of biological replicates and/or length of exposure.Please see our comment immediately above. In revised Figure 6, we show significant differences in axial growth between cavefish and surface fish exposed to hypooxygenation in a hypoxia chamber.Line 500: The authors say that 40-50% normoxic was lethal. Did they compare lethality in SF and CF embryos? I think it would be of interest to include lethality as a measurement in the study. Are there concentrations that CF can survive while SF die? This may be a more relevant measurement than growth considering whether the increase in red blood cells is adaptive.Thank you for this comment. We agree and during the interval between submissions we have performed many experiments to compare survival of surface and cavefish under hypoxic (in a hypoxia chamber) and normoxic conditions. Unfortunately, none of these experiments produced interpretable data. During the course of these experiments it became clear that successful survival studies will require much more extensive preliminary work to develop appropriate conditions using many different oxygenation levels, which was not possible during the interval allowed for revising this manuscript.More data is needed to show that CF do better than SF in a low oxygen environment and that if you decrease HIF expression and/or erythrocyte number this advantage is eliminated. Experimentally increasing HIF expression and/or erythrocyte number in SF and showing that this gives them an advantage in low oxygen water would provide additional strong evidence that the trait is adaptive.Thank you for this advice. We have now performed new experiments to address these concerns using a hypoxia chamber. The results show that growth (and erythrocyte number) in cavefish is significantly less sensitive to oxygen reduction than in surface fish . Also, to further address these concerns, we have now performed an experiment in which erythrocytes were completely eliminated and this significantly reduced axial growth .The authors should add text to the discussion that the study only focused on one of the cavefish populations and that examining the other populations would be an interesting future direction. The authors should add "Astyanax mexicanus" to the title. There are many different species of cavefish and this may not be a general feature for how cavefish adapt to hypoxia.We agree with the reviewer that examining other cavefish populations would be interesting and have now added the suggested material to the Discussion. Please see Lines 314-321.Astyanax mexicanus. Therefore, in the interest of keeping an already long title in check, we have not added the genus and species name to the manuscript title.The expansion of the title information in the Summary, which would appear just below the title in a published manuscript, clearly states that the work was done in the cavefish Reviewer #4 (Recommendations for the authors):1) The use of other assays (e.g metabolic rate under hypoxia) to test the functional relevance of increased erythrocyte production and enhanced transcription of hif1 would strengthen the conclusions.Thank you for this suggestion. We agree that confirmatory information might be obtained by different assays under hypoxia conditions. However, we point out that the two assays we have conducted in the revised manuscript, tail and eye growth, using the hypoxia chamber permit the conclusion that cavefish have greater tolerance to hypoxia than surface fish, and that this phenotype is likely to be a consequence of more erythrocyte development. In the time allotted for revision, we have repeated all of the hypoxia experiments with these two criteria as assays. We agree that, with additional time, adding more assays might provide further confirmatory evidence, but would not strengthen our present conclusions beyond the present evidence.2) In other teleosts, it is well established that elongation of the embryo is driven by convergent extension movements in the axial mesoderm. It would therefore be worthwhile examining whether this process occurs differently in cavefish relative to surface fish under hypoxia.Astyanax. The experiments on hypoxia in our manuscript began at the 13-14 somite stage, considerably later than convergent extension in the developing chordamesoderm. Thus, we believe that the suggested experiments, although interesting, would have little direct significance for our current study.We agree that it would be interesting to see if hypoxia affects notochord morphogenesis. However, convergent extension movements forming the notochord occur during and shortly after gastrulation in zebrafish and 3) Hif protein levels under hypoxic conditions should be measured.Astyanax system to specifically detect, distinguish and quantify the four Hif proteins. Our manuscript is focused on hif1 transcripts, which can be detected using established qPCR procedures, and our conclusions do not go beyond the transcriptional level. We also point out that HIF system activation has been previously documented in a variety of fish species based entirely on (indirect) studies of hif expression levels. The article by Mandic et al. (2021) in our reference list reviews these studies. We believe that this approach is also appropriate for our study, particularly since our only claim is that hif transcription is overexpressed by hypoxia.We agree that this would be an excellent topic for future research. However, at present we do not have the tools  in the 4) The use of a hypoxia chamber to control oxygen levels is recommended.Thank you again for this excellent suggestion. During the interim between submissions, we purchased a hypoxia chamber and using it repeated all of our hypoxia experiments, as we have described above. The results are shown in revised Figures 6 and 7. Our basic conclusions are still the same, but this approach has permitted us to make stronger conclusions about the differential effects of hypoxia on surface fish and cavefish in the revised manuscript.


There is no consensus on quality of life (QOL) in patients with acromegaly requiring medical treatment after surgery compared with those achieving remission by surgery alone.QuaLAT is a cross-sectional study comparing QOL in surgery-only treated acromegaly patients versus those requiring medical treatment post-surgery. Patients attending clinics were identified and divided into—Group 1: patients who had surgery only and were in biochemical remission, Group 2: all patients on medical treatment post-surgery, Group 3: patients from Group 2 with biochemical control. Participants were asked to fill three questionnaires; Acromegaly Quality of Life Questionnaire (ACROQOL), 36-Item Short Form Survey (SF36), and Fatigue Severity Scale (FSS).There were 32 patients in Group 1 and 25 in Group 2. There was no difference in QOL scores between groups 1 and 2, as measured by ACROQOL , SF36v2 , or FSS . Comparison between groups 1 and 3 however showed that PCS (and 3 subdomains) was significantly better in group 3 . All three QOL scores were lower when compared with healthy controls.Medical treatment not only achieves a QOL comparable to surgery, it may also be associated with better QOL in physical subdomains. When compared with healthy controls, QOL remains worse in treated acromegaly patients compared to controls.The online version contains supplementary material available at 10.1007/s11102-021-01153-4. Acromegaly results from excessive secretion of growth hormone (GH) from tumours usually originating from pituitary somatotroph cells. The excess GH then stimulates the liver to produce insulin-like growth factor-1 (IGF-1), which causes most of the clinical manifestations of acromegaly . For anyThe first-line treatment for acromegaly is trans-sphenoidal surgery (TSS), to remove or debulk the tumour . HoweverQoL reflects the subjective perception of health and effects of a disease. QOL in acromegaly has been a subject of interest in several published studies. Various validated questionnaires have been used to assess QOL in these patients. The most popular of these is Acromegaly Quality of Life Questionnaire (ACROQOL) developed by Webb et al. in 2002, which is a validated questionnaire for acromegaly patients . SeveralWhen studies have assessed QOL in acromegaly the focus seems to be either on the effect of surgery , 12 or mWe therefore carried out a study to assess whether QOL between patients who have achieved remission post-surgery varies when compared to patients who receive medical treatment post-surgery.Quality of Life after Acromegaly Treatment) is a cross-sectional study carried out at the Department of Endocrinology, Sheffield Teaching Hospitals NHS Foundation Trust, a tertiary referral centre where all patients, aged 18 or above, with a histological diagnosis of a GH producing pituitary adenoma (diagnosed at least 6 months ago) were identified via our database. Exclusion criteria included having only non-surgical treatment for acromegaly, inability to provide informed consent, having a dire prognosis, or severe comorbidities unrelated to acromegaly which according to investigators may confound results. After exclusions, preliminary data for the remaining patients were collected to identify those who underwent trans-sphenoidal surgery (TSS) and went into disease remission biochemically (Group 1); that is achieved IGF1 levels within the age-and sex-specific reference range and a growth hormone suppressed level < 0.3 µg/L post OGTT and/or a growth hormone day curve average < 1.6 µg/L. Those who did not achieve biochemical remission after surgery and therefore required further medical treatment to control the disease were included in the other group (Group 2), irrespective of their biochemical status. There were some patients in Group 2 who had their last IGF-1 values and growth hormone levels outside the reference range. To avoid any potential confounding due to this factor, another group (Group 3) was crafted for the purpose of analysis, including only those patients from Group 2 who had controlled biochemistry.QuaLAT . The study was approved by the Yorkshire and the Humber Research Ethics Committee (REC reference 19/YH/0373).In this study we aimed to compare the quality of life in those who were treated with surgery only vs those treated with surgery and medications for acromegaly, at a single tertiary care centre.Eligible patients were approached via telephone and informed about the study. The willing participants were then posted the patient information sheet, a consent form, and the QOL questionnaires with two return envelopes to post back consent and questionnaires separately to maintain confidentiality. Phone numbers to contact the research team were provided in the patient information sheet, in case they had any additional questions or concerns.Those who did not reply back within 28 days were given another phone call to check if they are still interested in participating in the study. If the interest was expressed once again, they were given another 28 days to send the documents. If the research team was unable to contact the patients, or the documents were not received within 28 days, it was deemed that the patients were no longer interested and therefore removed from the study.ACROQOLAll the recruited patients were asked to fill three questionnaires to measure their QoL. One of these was a disease-specific questionnaire (ACROQOL) and the other two were generic questionnaires; SF36 and Fatigue severity scale (FSS). The permissions to use ACROQOL and SF36v2 were obtained prior to the study, while this was not required for FSS as it was freely available to use.ACROQOL consists of 22 questions, each having five options of responses scoring 1–5. The maximum score that can be reached is 110 (100%) which reflects the best QoL, with 22 (0%) being the lowest possible score. The score is calculated by the following equation , however the mean BMI was not provided. In case of SF-36, we selected the study reported by Jenkinson et al.  across all of the groups. The majority of patients in the medical group were on SRLs either alone (n = 16) or in combination with cabergoline (n = 1). Five patients were on cabergoline alone, and three patients were on pegvisomant treatment.There was no difference in QOL scores between our main groups 1 and 2, as measured by all of three questionnaires  . For tho6% vs 45%Data from older longitudinal studies comparing QOL in surgical and medical treatment only  have shown that QOL in both groups improves from baselines but is not different between the groups at 12 and 48 weeks , 20. In A Japanese study from 2014 also compared the QOL between surgical and medical patients . This stWe have also found that medically controlled patients have better physical scores: SF36 PCS and possibly, ACROQOL physical subdomain. This could be related to the improvement in external features of the disease, better control of physical symptoms, or perhaps due to a sense of physical well-being from frequent monitoring and reassurance. Higher proportion of men in the medically controlled group, could have been a contributing factor since QOL scores in the general population tend to be worse in females  and in aThe reason for a low QOL in acromegaly patients despite treatment is debatable, but there are several contributing factors. Despite substantial improvements in healthcare in the recent past, there is still considerable delay in diagnosis of acromegaly, with some studies estimating the delay to be around 8–10 years from the onset of symptoms . Owing tOne of the main limitations of our study is the cross-sectional design and therefore absence of longitudinal data and control group. However, the latter was countered by including QOL data from large population cohorts. Secondly, our sample size was relatively small which may have decreased the statistical power. Also, the BMI data for the patients in this study was not available to us, which could be a potential confounding factor. Long-term, large prospective interventional studies looking at effects of different therapeutic strategies on symptoms and QOL are necessary to confirm our findings.In conclusion, while surgery is superior in achieving biochemical remission in acromegaly, we have shown that medical treatment achieves similar QOL. In patients who do not achieve biochemical remission after surgery, medical treatment not only maintains QOL comparable to surgical patients in remission, but may also be associated with better QOL in physical subdomain. Secondly, compared to healthy controls, the QOL remains worse in acromegaly irrespective of the type of treatment. Strategies to diagnose acromegaly earlier and novel treatment modalities are required to improve this important patient-related health outcome.Supplementary file1 (DOCX 17 kb)Below is the link to the electronic supplementary material.


Research in primary care is essential, but recruiting children in this setting can be complex and may cause selection bias. Challenges surrounding informed consent, particularly in an acute clinical setting, can undermine feasibility. The off-protocol use of an intervention nearing implementation has become common in pragmatic randomised controlled trials (RCTs) set in primary care.To describe how the informed consent procedure affects study inclusion and to assess how off-protocol medication prescribing affects participant selection in a paediatric RCT.A pragmatic RCT evaluating the cost-effectiveness of oral ondansetron in children diagnosed with acute gastroenteritis (AGE) in primary care out-of-hours services and a parallel cohort study.Consecutive children aged 6 months to 6 years attending primary care out-of-hours services with AGE were evaluated to assess the feasibility of obtaining informed consent, the off-protocol use of ondansetron, and other inclusion and exclusion criteria.n = 325/834) of children were accompanied by only one parent. GPs prescribed ondansetron off-protocol to 34 children (4.1%) of which 19 children were eligible for the RCT. RCT-eligible children included in the parallel cohort study had fewer risk factors for dehydration than children in the RCT despite similar dehydration assessments by GPs.The RCT's feasibility was reduced by the informed consent procedure because 39.0% (The informed consent procedure and off-protocol use of study medication affect the inclusion rate, but had little effect on selection. A parallel cohort study alongside the RCT can help evaluate selection bias, and a pilot study can reveal potential barriers to inclusion. Research in primary care is essential for evidence-based health care, yet 40% of paediatric RCTs are discontinued prematurely. A pilot study can help to identify potential recruitment barriers and prevent many reasons for recruitment failure. However, based on this study, the authors further advocate: 1) reconsidering the need for written informed consent from both parents in trials where the risk to the child is low; and 2) improving communication with all stakeholders to prevent off-protocol prescribing of study medication. Finally, selection bias can be identified by using a parallel cohort study.1 However, recruiting sufficient numbers of representative participants can be difficult, especially for acute paediatric management.2 This is illustrated by the fact that 40% of paediatric RCTs are discontinued prematurely owing to poor recruitment.4 Besides that, GPs who are aware of the effectiveness of the intervention may use this intervention, which can cause selection bias.5Outcomes from RCTs are essential for GPs to provide evidence-based health care.6 Although GP involvement in case recruitment can decrease the chance of successful inclusion,1 not involving them is not always feasible and can be costly. In out-of-hours primary care centre (OOH-PC), GPs must also evaluate patients with whom they are unfamiliar, which may further decrease their willingness to recruit children into a trial and worsen the inclusion rate.1 When trials are discontinued, authors rarely report how and by whom participants were recruited, which prevents any lessons learnt being applied when planning trials in other settings.2 Ideally, authors would report the recruitment process of their trial in sufficient detail to help avoid the repetition of mistakes.5 Pilot studies can be used to uncover reasons for recruitment failure.5Primary care is not an easy place to conduct research.A pragmatic RCT was performed to investigate the cost-effectiveness of adding a single dose of oral ondansetron to care-as-usual (CAU) in an OOH-PC on the frequency of vomiting in children aged 6 months to 6 years with AGE. Despite a pilot study, child recruitment was challenging. In this report, the recruitment efforts are described, focusing on how the informed consent procedure and the use of off-protocol prescribing affected the inclusion rate and child selection, respectively.8 Follow-up was for 7 days after randomisation.In the pragmatic RCT, participants were enrolled from December 2015 to January 2018 at three OOH-PCs in the north of the Netherlands . After a pilot study (NL4700) from December 2015 to October 2016, in agreement with the medical ethical committee (METc), the primary outcome was changed from ‘referral rate’ to ‘proportion of children who continued vomiting in the first 4 hours after randomisation’ because this was considered a more patient-oriented outcome by both the METc and the parents involved. The primary and secondary outcomes of the RCT are detailed in Supplementary Box 1. The written informed consent procedure was also adapted because it could not be feasibly obtained from both parents, severely restricting inclusion. The METc agreed that children could be included from the pilot study in the amended RCT (NL5830), and a parallel cohort study was added. The parallel cohort study provides insight into the representativeness of the trial population and helps to assess the external validity in a non-invasive manner.Children aged 6 months to 6 years were included who presented at the OOH-PC with vomiting and for whom the GP diagnosed AGE. Specifically, children were included if: 1) they reported at least four episodes of vomiting 24 hours before presentation; 2) they reported at least one episode of vomiting 4 hours before presentation; and 3) written informed consent was obtained from both parents. If the child was accompanied by one parent, that parent could give written informed consent and the second parent could give oral informed consent via a telephone call in the presence of the research assistant (RA). The written informed consent from the second parent had to be sent by post.Children were excluded if: 1) they had used or been prescribed antiemetics in the previous 6 hours; 2) they had renal failure or hypoalbuminaemia; 3) they had diabetes mellitus or inflammatory bowel disease; 4) they had a history of abdominal surgery that could explain the current symptoms according to the GP; 5) they had sensitivity to 5-HT3 receptor antagonists; 6) they had a prolonged QT interval or were using QT-prolonging medication; or 7) they had previously been enrolled.Children aged 6 months to 6 years and diagnosed with AGE, but whose parents did not give written informed consent for the RCT, were asked if they were willing to participate in a parallel cohort study in which written informed consent was only needed from one parent.Parents were informed about the study by an RA before the GP consultation. If parents were interested, the RA started baseline assessments (Supplementary Box 2) and the GP then assessed the child (confirming or refuting a diagnosis of AGE and assessing the degree of dehydration) and their suitability for participating based on the inclusion and exclusion criteria. The RA asked the parents of eligible children for written informed consent .After obtaining at least one written and one oral informed consent from parents, children were block randomised  to intervention groups by a computer programme and were stratified by age (6–24 months or >24 months) and dehydration status  and 15 ml/kg for 4 hours when assessed as dehydrated. Parents were informed about the expected course, alarm symptoms, and when and how to contact their GP.Children received CAU  that com10 If the child vomited within 15 minutes, the dose was given a second time only.Children in the intervention group received CAU plus a single weight-based dose of oral ondansetron syrup (0.1 mg/kg) according to the Dutch Paediatric Formulary.Parents used a structured diary to record symptoms , oral rehydration therapy and fluid intake, medication use, adverse reactions, healthcare use, hours missed from work, and satisfaction with treatment during follow-up. The diary was to be completed every hour for the first 4 hours and daily thereafter for 7 days. Parents could return the diary on paper with the enclosed envelope. If parents did not return the diary after multiple requests, information was collected about the primary outcome by telephone.11 indicating that a difference of 21% in the proportion of children with persistent vomiting was clinically relevant. Therefore, 89 children per arm needed to be included for a power of 90% and an α of 0.05, allowing for a 10% loss to follow-up.Based on a systematic review, it was estimated that 85% and 64% of children in the CAU and ondansetron groups would continue vomiting after 4 hours,9 and degree of dehydration assessed by the GP . To evaluate the impact of the informed consent procedure on the inclusion rate, the number of children with AGE who visited the OOH-PC with only one parent was reported. To evaluate the impact of the off-protocol prescription of ondansetron on the selection of children in the RCT, baseline characteristics of RCT-eligible children included in the parallel cohort study and for whom the GP prescribed ondansetron were non-statistically compared with children included in the RCT. To understand the overall level of selection bias, the baseline characteristics of RCT-eligible children included in the parallel cohort study were statistically compared with the baseline characteristics of children included in the RCT. Continuous variables were compared with non-parametric tests (Mann–Whitney U test) and dichotomous variables were compared by logistic regression. Statistical significance was defined by a two-tailed P value of <0.05, and all analyses were performed using SPSS (version 25).The following baseline characteristics were assessed: age, sex, AGE symptoms , dehydration alarm symptoms, dehydration risk factors see ,9 and deThe eligibility of 1061 children meeting the enrolment criteria were assessed. Of these, 834 had a GP diagnosis of AGE, with 194 ultimately randomised in the RCT and 201 included in the parallel cohort study. In total, 70 children were eligible for both the RCT and parallel cohort study and were included in the study analysis .n = 325/834) of cases, only one parent accompanied the child with AGE. Adapting the consent procedure increased the inclusion rate from seven to 10 cases per month. Of the 194 randomised children, 42 were accompanied by only one parent (21.2%) and 16  did not send the second written informed consent after giving verbal permission, so were excluded (In 39.0% (excluded .GPs prescribed ondansetron off-protocol to 34 (4.1%) of the 834 children with AGE, and 19 of these were eligible for participation in the RCT. There were no clinically relevant differences in baseline characteristics compared with children in the RCT, except that GPs estimated the degree of dehydration to be almost twice as high in these 19 cases compared with those in the RCT  , but the median GP-assessed dehydration level did not differ with statistical significance (= 0.302) .Almost 40% of all children attended with one parent, making it difficult to obtain informed consent as required and, thereby, complicating inclusion. GPs also prescribed off-protocol ondansetron if they suspected more severe dehydration, but this did not correspond with known dehydration risk factors or alarm symptoms. Children in the parallel cohort study had fewer additional risk factors for dehydration compared with children in the RCT.n = 19), precluding statistical testing with the children in the RCT. It should also be noted that the recommendations are based on the data from one RCT and a parallel cohort study.The authors are aware of no prior research assessing the pitfalls of the trial recruitment process in an OOH-PC. Consecutive children were screened presenting to one of the three OOH-PC over a period spanning more than 2 years, making this study highly representative of the population. Lessons have been learnt from the pilot, and a parallel cohort study has been added in which the children included have been evaluated. However, a limitation is that the RCT-eligible children in the parallel cohort study — to whom GPs prescribed ondansetron — was small , this was without statistical significance. Among children receiving off-protocol ondansetron, the GP estimated dehydration to be almost twice that in children in the RCT (38 versus 20). Given that it was intended to assess the effect of ondansetron in children at increased risk of dehydration, excluding these may have resulted in an underestimation of the true effect of ondansetron. However, the study demonstrated no differences in risk factors or alarm symptoms of dehydration. Therefore, the level of dehydration estimated by the GP alone should be interpreted with caution.A clinically relevant difference existed in the level of dehydration estimated by the GP between groups, but owing to the small group size  during an RCT can seriously bias the outcomes. Communication with all stakeholders, to assess the barriers to protocol compliance, should be routine when performing (pragmatic) RCTs. Third, the authors recommend initiating a parallel cohort study to run alongside RCTs to ensure follow-up when parents do not want to participate in the RCT. This allows comparison of the characteristics of children who did and did not participate, and gives an opportunity to assess if the right population was included. Finally, initiating a pilot study before an RCT offers an invaluable opportunity to evaluate potential barriers to study inclusion and patient selection.