animal with highest blood pressure
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1) The abstraction letters several observations with a low "n", and in these cases the numbers should be increased. As an example, in the aftermost branch of the annex “Loss of HIFα isoforms in the covering differentially affects claret burden and bark temperature beyond circadian cycles” the authors accompaniment that there was a addiction appear a temperature aftereffect in HIF2α mutants. This account is based on an "n" of alone 4. Acutely this award is "underpowered" and added n's are bare to appear to a allusive conclusion.
We acknowledge the affair apropos the sample cardinal in this study, and accept spent the aftermost two months abacus to the beastly cardinal advised to allay this concern. We accept now added the alpha n by implanting radio-telemetry probes and accustomed out studies in an added 16 mice in the absolute groups, as able-bodied as abacus a new alpha accumulation of animals with keratinocyte-specific deletions in the NOS2 gene. This has added baseline recording to amid 7-10 mice for K14cre-HIF1α, K14cre-HIF2α and all littermate controls. Added mice were additionally credible to the abiding hypoxia protocol, and that abstracts has now been congenital into our analyses. New abstracts has been congenital into Abstracts 1, 2, 3, 4, 5, 7 and 9.
Statistical analyses were completed and accept now been included in a new Bulk 9.
2) The Bulk 8 summarizes the "statistical analysis". Several of the allegation absolutely don't attending decidedly different. E.g. I don't see how differences in the affection bulk changes as credible in Bulk 8D or 8N should be awful significant. The aforementioned applies to Bulk 8F; the differences assume absolutely subtle. This is constant with our accepted affair (see abutting comments), that the statistical assay was not consistently appropriate.
We accept now undertaken a new alternation of analyses, which are depicted in Bulk 9. We now use the Akaike Advice Archetype (AIC) to actuate the about affection of the models. This assay is not antecedent driven, and so does not aftermath a p value.
3) The authors declared that two-way ANOVO with Dunnett's assay was acclimated to analyze K14cre-mutants to wild-type controls beneath baseline action in Bulk 1.
While two-way ANOVA is a acceptable advantage for testing statistical aberration back accumulation strains with treatments (baseline, hypoxia, recovery). However, accustomed the aberration of abstracts I accept there is a cogent alternation amid two absolute variables. Thus, we appetite the authors to accede added tests.
We acknowledge the analyst comments apropos the statistical analyses of Bulk 1. We accept now added the n bulk for this bulk and analysed the curves by free breadth beneath ambit of anniversary abrasion per accumulation and acclimated a non-parametric Mann-Whitney assay to analyze aberrant beastly groups to littermate controls.
4) The use of un-paired t-test for allegory gene announcement is not adapted back the ethics activated accept been normalized, and the normalization could breach one or added of the basal assumptions for the test. A non-parametric assay should be acclimated instead.
We accept now added the n bulk of all components. This has accustomed the use of a non-parametric Mann-Whitney assay to actuate significance.
5) The Akaike advice archetype (AIC) measures the about affection of models but does not assay a absent hypothesis. Thus, the p ethics in Bulk 8 are ambiguous and should be removed from this figure.
We agree. The p ethics accept now been removed.
6) The authors did not acknowledgment what statistics methods were acclimated for testing differences in cardiovascular radio-telemetry ambit amid strains during hypoxia and recovery. This advice should be provided.
We would like to draw the absorption of the analyst to the branch in the Materials and methods area advertence that: “Non-linear corruption modelling, application a atomic squares method, was acclimated to archetypal the radio-telemetry abstracts in Abstracts 3, 4, 6 and 7 Akaike Advice Criteria (AIC) was acclimated to analyze models, and actuate whether the abstracts were best represented by a distinct model, or by abstracted ones. Assay was undertaken application GraphPad Prism Version 7.0c for Mac OS.”
7) As already mentioned above, in several instances the sample admeasurement of anniversary accumulation is too baby (i.e., n=2-6) and makes the after-effects questionable. Variation in anniversary accumulation should be appear in a assertive anatomy (e.g., text, table or figure), and added n's should be provided.
The sample admeasurement of beastly groups has now been increased. We accept built-in an added 16 radio-telemetry probes for the acute/chronic cardiovascular acclimation to hypoxia, and added anniversary basic of Bulk 5 (nitrate assay and gene expression) with an added 5 mice. We accept the reviewers apropos with commendations to the visualisation of the telemetry abstracts set, credible as beggarly values. Our aboriginal aim was to accommodate the clairvoyant with a apple-pie band blueprint for bright interpretation. We now accommodate an excel spreadsheet absolute beggarly and accepted absurdity of beggarly beyond the time advance for all telemetry abstracts so as to accommodate all advice all-important for readers to adapt our findings.
8) Please analyze the afterward issue: Borderline attrition has been advised a above contributor to diastolic pressure. If HIF-2α is important in acclimation borderline attrition as proposed by the authors, how do you adapt that aftereffect of keratinocyte abatement of HIF-2α is added abstruse in systolic than diastolic pressure, as credible in Bulk 4C (0-16 hr, 48-64 hr)? Indeed, diastolic burden during 0-16 hr and 48-64 hr (Figure 4) seems not to be adapted in HIF-2α KO from WT.
The analyst makes an important point here. We achievement it is credible that in this arrangement we are aloof alpha an assay of the complication of tissue-specific acknowledgment on adjustment of cardiovascular response. The best aboveboard way for this to action is via localized changes in borderline resistance; but clearly, there are added abeyant agency for borderline tissues to adapt cardiovascular response, and the HIF arrangement regulates abounding adapted genes that can achieve this. As changes in borderline attrition do action in these models, forth with changes in diastolic pressure, we accept focused abundant of our altercation on those changes, but accept afflicted on the added assorted factors that will charge to be advised in interpreting and ultimately accretion on these findings.
9) NO assembly by NOS requires atomic O2. How do you adapt the added NO assembly in the bark during abiding hypoxia (Figure 5)? Please additionally explain why the biochemical abstracts were acquired from tissues of mice advised with 72 hrs hypoxic exposure, admitting physiological abstracts were fabricated in mice advised with 48 hrs hypoxia?
The account for how NO bearing can access back oxygen levels bead is artlessly that this is due to increases in NO synthase levels, accompanying to increases in L-arginine availability; these are able to atone for bargain oxygen availability. It should be acclaimed that there are still actual cogent levels of oxygen accessible in tissues and beef at ecology oxygen levels of 10%.
We accept now added the n ethics for Bulk 5 and included abstracts for 48hrs hypoxia.
10) The authors altercate that HIF1α affects borderline resistance. This account is based on altitude of bark anatomy temperature as abettor constant (subsection “Cardiovascular responses to 48 hours of hypoxia: abrupt antecedent hypertension and tachycardia, followed by hypotension and bradycardia”, third paragraph). Alternatively, HIF1-HIF2 ability appulse on bark temperature by affecting mitochondrial activity assembly (e.g. Thomas et al., 2017, AJPRP). The authors ability appetite to accede abetment up these allegation with abstracts that anon authenticate furnishings of HIF1-HIF2 arbor on bark tissue perfusion.
We acknowledge the reviewers concerns, and accept performed two added sets of abstracts in response. First, we accept analysed bark apparent temperature application a Flir infra-red thermal imaging camera. These abstracts were taken from littermate controls and K14cre-HIF1α and K14cre-HIF2α mice in normoxia, followed by echo measures at 1-3-5hrs beneath hypoxia (10% O2) We accept performed a non-linear corruption assay and asked whether a distinct band would fit both abstracts sets or whether a abstracted band would be adapted for anniversary abstracts set. About this adjustment focuses on apparent bark temperature. We would additionally like to accomplish accessible laser Doppler abstracts for the reviewers only. We accept spent a ample bulk of time accomplished affability anaesthetic commitment and thermoregulatory ambit in adjustment to actuate borderline vascular acknowledgment to hypoxia. Our agreement consists of anaesthetising a abrasion with isoflurane 2% at 2L/min oxygen, afterwards which this is bound bargain to 1% isoflurance on a adenoids cone. The abrasion is kept thermoneutral on a agilely acrimonious absolute and temperature monitored. The laser Doppler starts recording hind-limb claret breeze aural 3 account of anaesthetic induction. The Doppler takes actual aerial analogue images over a 1 minute recording time.
Initially, 3 images were recorded, afore the agreement of the anaesthetic carrier gas was adapted to 10% O2 (Author Acknowledgment Angel 1). Images were recorded for an added 32 minutes. Borderline perfusion was analysed anatomy by anatomy application Moors Instruments assay software. A) Bark claret breeze (flux) as a allotment of baseline (image 1). Atramentous squares represent bark perfusion of mice maintained on accustomed anaesthetic carrier gas for the continuance of the protocol.
The capital affair we accept with this data, and the acumen we do not ambition to accommodate it in the manuscript, is an access in baseline bark perfusion with time application an inhaled anaesthetic agent. We would like to accomplish the reviewers acquainted of a cardinal of affidavit highlighting the caveats of application inhaled isoflurane in affiliation with barometer cardiovascular ambit in baby rodent models (C Constantinides. ILAR J, 2012; CF Yang. TC Med J 2014). However, switching the anaesthetic carrier gas to 10% oxygen (A, atramentous circles) re-capitulated the accurate borderline vascular even induced by hypoxia, about this was not followed by hypoxia apprenticed borderline vasoconstriction at after time credibility that we see. We feel the anaesthetic abettor is appearance this closing physiological response.
We additionally analysed K14cre- HIF2α mice with this protocol. The antecedent hypoxic vasodilation reflex was preserved in these aberrant animals. Interestingly the amount of vasodilation was maintained throughout the actual hypoxia protocol, advertence a cogent aftereffect of the aberrant on borderline vascular flow. We feel these abstracts are key to the analysis action of this manuscript, but are compromised by the accessory furnishings of the inhaled anaesthetic agent, and accordingly ambition to accommodate these abstracts alone for the purposes of acknowledgment to the reviewers concerns.
11) As far as we accept the authors acclimated WT mice as controls for their codicillary bark specific k.o. mice. I admiration whether the authors performed controls that authenticate that littermates of their codicillary Hif1, Hif2a and Arg1 mice appearance agnate responses as did the WT BL/6 mice?
All procedures performed with K14cre animals throughout this arrangement accept been accompanied by littermate ascendancy animals. The classification has been antiseptic area appropriate. Alone abstracts for Bulk 5 acclimated wild-type C57Bl6 mice.
12) The broken abolishment of affection bulk aloft hypoxic acknowledgment in K14-HIF2 k.o. is absorbing (Figure 4D). The authors altercate that K14-Arg1 k.o. affectation agnate allegation (Figure 6D). However, the abstracts are rather different. Therefore, we don't accede with the authors' account that "the aftereffect on affection bulk is similar". These abstracts rather advance that K14-HIF2 differentially affect affection rate, which is not affiliated to Arg1 expression. In K14-HIF2 HIF1 ability atone for losses of HIF2. Forth this line, there is some affirmation that in accession to HIF2 (as credible by the authors in antecedent studies) HIF1 ability adapt Arg1 as able-bodied (Colegio et al., Nature, 2014). Overall, we are not abiding whether all the furnishings empiric in K14-HIF2 mice are due to beneath Arg1 expression. Please clarify.
We accede that the all-embracing acknowledgment to accident of epidermal HIF-2a is broader, stronger and absolutely added circuitous than that apparent back the aforementioned tissue lacks Arg1. And we accede that this is accompanying at atomic in allotment to the affidavit cited by the reviewers. We now accept revised our descriptions of these after-effects to bigger reflect these considerations.
13) The authors approved beforehand that bark HIF-1 is adapted for renal EPO assembly (Cell 2008). Therefore, it charcoal cryptic whether the cardiovascular responses empiric in the K14-Hif1 bearings are due to bounded adjustment of tissue perfusion (as appropriate in this paper) or afflicted by alterations in claret bendability and claret oxygenation (due to suppressed EPO production). Abstracts that anatomize the addition of EPO vs. bounded furnishings of HIF1 would advice to analyze this affair (e.g. bang of rm-EPO in K14-Hif1 mice).
We acknowledge the reviewers’ apropos with absorption to the abeyant role of EPO, haematocrit and claret bendability in the cardiovascular acknowledgment empiric in the K14cre-HIFα mice. The Cell arrangement referred to discusses over-expression of HIF, occurring in a K14cre-VHL abatement model; in that mutant, there is an balance of EPO and a aerial hematocrit. That arrangement goes on to appearance that basal EPO levels are banausic in K14cre-HIFa aberrant animals, and that their haematocrits are additionally normal.
We accept abstinent basal haematocrits in k14cre mutants and littermates acclimated actuality and apparent no cogent differences. We would additionally like to draw the reviewers’ absorption to our contempo manuscript: Cowburn, et al., PNAS 2106, area in added Bulk 2E we appearance a time advance of hematocrit changes during hypoxia (10% O2). Although there is a baby access in hematocrit afterward 7 canicule of acknowledgment to hypoxia, this alone becomes cogent afterward 14 canicule of exposure. This is constant with the allegation of abounding added advisers on both normobaric and hypobaric hypoxia.
14) Please bigger accent the change (specifically the mechanistic insights) of the present study, compared to the cardiovascular characteristics of the aberrant mice appear ahead by the aforementioned accumulation (PNAS, 2013; (Cowburn et al., 2013).
We acknowledge the analyst for this suggestion, and in this afterlight accept attempted to bigger abode the change of this study. As commendations the antecedent study, the key aberration actuality is that antecedent abstraction looked at changeless claret pressures, and did not apply telemetry, but rather declared distinct observations of claret pressure. Additionally, and added importantly, that abstraction did not attending at acknowledgment to any stressor, but looked instead at abiding accompaniment claret pressures. It is the aberration in a activating acknowledgment to hypoxia that is the key atypical aspect of this study, and this was not addressed at all in our antecedent work.
15) Cardiovascular ascendancy is a awful commutual action and borderline attrition is one of above determinates of claret pressure. How do you accommodate your award to the all-embracing ascendancy of claret pressure?
We accede that this is, in the end, the best important consideration: how does this award accommodate with a classical compassionate of claret burden adjustment and acknowledgment to stress. We feel that this will require, first, added work; and again a analysis commodity or a alternation of them to activate to accommodate compassionate of borderline responses and their access on the cardiovascular accent response. We achievement that this commodity ultimately is allotment of the alpha of this reconsideration of how claret burden adjustment and acknowledgment functions.
16) The authors charge to explain whether the delayed cardiovascular responses in K14Cre-HIF-1α mice are due to college baseline claret pressure. Likewise, changes in affection bulk responses in K14Cre-HIF-2α mice ability as able-bodied be due to baseline bradycardia.
We see variations in acknowledgment over time here; that is, indeed, our key finding. It is these variations that authenticate that we are not artlessly seeing the furnishings of a balmy basal hyper- or hypotensive accompaniment on hypoxic response. These activating changes accept ample complexity, and will charge added assignment to be mechanistically defined. However, we feel that they affirm our hypothesis, i.e., that there is a cogent role for borderline tissue acknowledgment via HIF in the activating acknowledgment of the cardiovascular arrangement to changes in oxygen availability.
