Within-near-clade PCE: the ‘Russian doll’ (RD) model This model aims at exploring the level of genetic exchange, not among the near-clades, but rather, within each of them [16].

As a matter of fact, many authors consider that there is more recombination within the near-clades than between them [17].

The RD model takes as null hypothesis that within the near-clades, recombination is potentially random (not just that occasional recombination can occur), because the expectations for random recombination are well known (linkage equilibrium) [18].

The working hypothesis is that the near-clades each exhibit a miniature picture of the whole species, with the two main features of the PCE model, namely (i) strong LD, or non-random association of genotypes occurring at different loci, and (ii) lesser near-clades [19].

The RD approach simply consists in testing the PCE model within each of the near-clades [20].

Ramírez & Llewellyn attempt to invalidate RD by favouring the null hypothesis [21].

However, the examples they advance are either weak or misleading [22].

Some of them are even counterexamples of what the authors try to demonstrate [23].

In the case of Giardia, the only case cited by the authors to falsify RD is not based on a population genetics approach, but rather on the sequencing of a few genes [24].

The conclusions of this article are based on observed discrepancies between the phylogenetic trees based on three loci for seven strains [25].

This is a possible indication that some genetic exchange may go on (although other explanations are possible), but says nothing about the frequency of recombination [26].

Moreover, with such a limited set of data, the risk of statistical type II error (impossibility to reject the null hypothesis of random recombination, not because it is proven, but because the test lacks power) is high [27].

Lastly, this study does not concern a whole Giardia ‘assemblage’ (= near-clade), but rather, the subassemblage A2 of the assemblage A [28].

The mere existence of this subassemblage, which shows that assemblage A is subdivided into discrete units, is in itself evidence supporting RD [29].

Two of the examples cited about Leishmania do not concern intra-near-clade recombination but rather, the whole species [30].

They have therefore nothing to do with the RD controversy [31].

Moreover, the ‘widespread genetic exchange’ that the examples are supposed to evidence is actually selfing/inbreeding, which is considered by us and many, if not most, authors working on pathogens population genetics as a particular case of PCE [32].

Lastly, the tests used to infer selfing/inbreeding in Leishmania are based on the hypothesis that these parasites are diploid, while convergent results rather favour that Leishmania exhibits widespread aneuploidy [33].

This bias has been underlined by us, as well as by Ramírez et al. [ 34].

Another study, repeatedly cited by Ramírez & Llewellyn, is actually an illustrative counterexample of what they try to demonstrate, and a talking illustration for RD [35].

By high-resolution sequencing, Rogers et al. evidence a monophyletic subdivision (near-clade) within Leishmania donovani/infantum [36].

They do find indication of hybridization within this near-clade [37].

However, what Ramírez & Llewellyn omit to mention is that Rogers et al. clearly state that ‘extensive LD between chromosomes is consistent with rare recombination and supports clonality as the primary reproductive mode’ and that there is ‘mainly clonal reproduction in the parasite population’ [38].

The data presented do not make it possible to know whether there are lesser near-clades within this monophyletic subdivision [39].

However, they do show that clonality is the dominant mode of reproduction within this near-clade, which supports a RD pattern [40].

The studies cited concerning Trypanosoma cruzi are not strong evidence against RD [41].

The first one is consistent with recombination in a small subpopulation of the TCI near-clade in Ecuador [42].

However, as we have shown, this ‘domestic’ population is poorly defined (33% of selvatic strains are included in it, while the ‘selvatic’ clade comprises 12% of domestic strains) [43].

The risk of type II error is high, due to very limited sampling (only 12 truly ‘domestic’ strains) [44].

Additionally, the ‘selvatic’ clade shows strong LD and clonality, which favours the RD hypothesis [45].

Observing a lack of congruence between mitochondrial and nuclear phylogenies indicates at best limited introgression, as it can be observed even between different biological species [46].

Another study has postulated ‘substantial recombination’ in Brazilian strains of the TCII near-clade [47].

However, this study relies on limited sampling and is subject to type II error [48].

There is a strong contradiction between the hypothesis of ‘substantial recombination’ (with lack of LD) and the evidence for clear structuration of these populations, even in sympatry, as shown by STRUCTURE analysis [49].

A recently published study deals with TCI T. cruzi populations isolated from selvatic triatomine bugs in Bolivia [50].

The data are compatible with frequent genetic exchange, however, as the authors assert their data are prone to type II error and should be corroborated by more extended sampling [51].

While focusing on these disputable examples consistent with the null hypothesis, Ramírez & Llewellyn omit to cite the many cases presented by us, which strongly support the RD model, including in T. cruzi, Leishmania, Toxoplasma, Giardia and Cryptococcus [52].

This is all the more astonishing, since some of the best examples of RD patterns were found in the authors’ own data [53].

As a matter of fact, convergent results show that, within T. cruzi, the near-clade TCI is subdivided into several smaller near-clades that are widespread and stable in space and time, including in sympatry [54].

These ‘mini-near-clades’ have been corroborated by several different molecular markers, including the mini-exon gene, the cytochrome B gene, multilocus PCR-RFLP and SSUrDNA [55].

Contrary to what the authors state, within Colombian TCI strains, there is LD [56].

As a matter of fact, the p values for the LD test and the Ia index of association (another LD test) are 4 x 10^-4 and 0.037, respectively [57]. As noted by Tomasini et al., this is evidence for LD, and not for the opposite, as wrongly concluded by the authors [58].

Similarly, within TCI selvatic strains, strong LD evidences ‘widespread clonality, infrequent recombination’ [59].

Lastly, strong evidence for a RD pattern within Argentinian TCI strains has been recently published [60].

We do not state that the RD pattern is presently verified in all pathogens. But we do argue that it has been strongly supported by many data, especially in T. cruzi, Leishmania infantum, Cryptococcus neoformans, Giardia and Toxoplasma gondii, in each case when genetic and population samplings are enough and adequate [61].


Conclusion We argue that the PCE hypothesis is not just an artefact, and that it is the most parsimonious model for many, if not most pathogens, including viruses, bacteria, parasitic protozoa and fungi [62].

Our conclusions are based on a large set of published data, dealing with bacteria (48 species), yeasts and fungi (nine species), parasitic protozoa (21 species) and viruses (11 species or categories) [63].

These data are easily accessible to any reader wanting to verify our proposals [64].

If looking for recombination in pathogens amounts to ‘searching for a needle in a haystack’, it obviously means that recombination is severely restricted; in other words, that the PCE model is supported [65].

When it is not, like in some populations of Plasmodium falciparum, or in Helicobacter pylori, one has no trouble finding clear indications for recombination without searching in a haystack [66].

Until now, the use of more resolutive markers has reinforced the PCE and RD hypotheses [67].

As an example, near-clades and RDs in Toxoplasma gondii are better evidenced by a set of highly resolutive markers than by more conventional tools such as multilocus sequence typing [68].

Similarly, whole-genome sequencing in Neisseria meningitidis uncovers the existence of deep phylogenies that were not apparent with multilocus sequence typing [69].

As a matter of fact, contrary to what Ramírez & Llewellyn state, lack of resolution should favour the null hypothesis (random recombination) rather than PCE, even when highly resolutive markers make it possible to find ‘the needle in the haystack’ [70].

Concerning the amount and frequency of recombination, we have called for discarding vague, subjective assertions, such as ‘widespread genetic exchange’, ‘more frequent than expected’, which amount to a conflict between the supporters of the half full and the half empty bottle [71].

We have proposed, rather, to use a clear-cut PCE criterion relying on the notion of a ‘clonality threshold’ [72].

If, within a given species, a growing phylogenetic signal is observed as more relevant data are obtained, it means that restricted recombination efficiently counters the effects of genetic exchange, and that the near-clades that subdivide the species will tend to diverge more and more [73].

This PCE-specific signal should be looked for, first at the level of the entire species [74].

Only once the near-clades have been clearly evidenced by this approach, and are fully recognized by most authors (which is still the case for a limited number of species only), RD patterns should be looked for within them, but only in a second round [75].


Tibayrenc and Ayala raised several interesting objections to an opinion piece we recently published in Molecular Ecology [1].

Our piece examined the value of an alternative perspective to their theory of predominant clonal evolution (PCE) on the prevalence and importance of genetic exchange in parasitic protozoa.

In particular, our aim was to establish whether population genetic signatures of clonality in parasites were representative of true biological/evolutionary processes or artefacts of inadequate tools and inappropriate or inadequate sampling.

We address Tibayrenc and Ayala’s criticisms and make a detailed response.

In doing so, we deny the consensus that Tibayrenc and Ayala claim around their views and dismiss much of the language which Tibayrenc and Ayala have introduced to this debate as either arbitrary or inaccurate.

We strongly reject accusations that we misunderstood and misquoted the work of others.

We do not think the PCE provides a useful framework for understanding existing parasite population structures.

Furthermore, on the eve of the population genomic era, we strongly urge Tibayrenc and Ayala to wait for the forthcoming wealth of high-resolution data before considering whether it is appropriate to refine or re-iterate their PCE hypothesis.


In our opinion, the discovery of major genetic subdivisions in parasitic protozoa is the result of the collective efforts of hundreds of researchers over the last 40 years and, yes – the self-evident result of (i) genotyping the same conserved molecular markers across studies; (ii) finding that intermediate genotypes between major subdivisions are exceedingly rare.

It is categorically not one of the ‘main achievements’ of the PCE.

In our review, we merely wished to point out that if genetic exchange occurs, it is more likely to happen within these subdivisions than between them.


Later in their critique, Tibayrenc and Ayala cite a population genomic study of Neisseria meningitidis as evidence of ‘deep phylogenies’ in pathogen populations [5], presumably to further bolster the PCE model.

Careful reading of this commendable study (and personal correspondence with its authors) reveals precisely the pattern of population diversity we highlighted: deep subdivisions with little or no exchange between them; intense lateral exchange of genetic information within them.

In still yet another recent review, Tibayrenc and Ayala conveniently dismiss this finding as ‘invisible sex’ [6], regardless of the considerable genetic diversity present among strains involved and the possible fitness benefits associated (virulence factors, antimicrobial resistance, etc) [see also Table 1 in [1]].

Importantly, Budroni et al. ’s findings absolutely contradict the expectations of Tibayrenc and Ayala’s much discussed and little supported ‘Russian doll’ hypothesis [7].


In the second section of their comment, Tibayrenc and Ayala go on further to defend their ‘Russian doll’ theory, suggesting that the data we present in support of ‘sex-within subdivisions’ is ‘weak’ and ‘misleading’ [4].

In fact, we presented the available data in a balanced fashion, including examples where evidence of genetic exchange was not uncovered [8], as well as where it was [9].

Crucially we focused on studies that examine diversity among large ‘minimally subdivided population samples’ in space and time – that is, where one could have reasonable confidence about the population genetic parameters derived from them.

It is hardly fair to call this ‘misleading’.

We did not cite the 2013 article they mention that contained an error in a P-value interpretation [10]; however, we accept that there was an error for which correction has been submitted.


Tibayrenc and Ayala suggest we furthermore ‘omit to cite the many cases presented by us [them] that support the Russian Doll model’ [4].

We invite the reader to review these ‘many cases,’ as we have performed – most are simply not convincing.

Almost all the studies they cite have sample sizes an order of magnitude lower than ours and those that we cite, as well as low numbers of conserved genetic markers used for genotyping.

In one case, Tibayrenc and Ayala totally misconstrue the study findings: Minning et al. 2011 found decoupling between copy number variation and DTU identity ‘strongly suggestive of genetic exchange playing a major role in T. cruzi population structure and biology.

’ [ 11] – the opposite meaning to that portrayed by Tibayrenc and Ayala [12].


The overall picture that emerges from existing data – including studies cited by ourselves and Tibayrenc and Ayala – is a spectrum of within-subdivision population structures, some sexual, some occasionally sexual and some clonal.

The Russian doll hypothesis is valid as a hypothetical model.

However, the statistical tools on which it is based are highly sensitive to anything but completely random associations between loci at a population level.

Thus, only truly panmictic populations – rare enough in sexual species let alone non-Mendelian or parasexual pathogens – will depart from the RD model as stated [4].

We believe that a model that ignores the shades of grey in population structure between different pathogens and pathogen populations is not fit for purpose.

It is a lovely metaphor, but it is an extremely poor summary of the data.


We stand accused of using ‘vague and subjective’ assertions in our opinion piece [4].

This is a shame, as in our review, we set out to try and define some of the increasingly impenetrable lexicon developed by Tibayrenc and Ayala to describe PCE [7, 12, 6].

Specifically, we attempted to define the meaning of the term ‘evolutionary scale’ as Tibayrenc and Ayala use it.

However, we did not explore the opacity the recently invented term ‘clonality threshold’ [13, 4] which, although pseudo-quantitative, is in fact equally as vague as a phrase we used – ‘widespread genetic exchange’ [1] which was the target of their comment in this context.

Even the word ‘predominant’ in ‘predominant clonal evolution’ is open to wide interpretation.

Another term used by Tibayrenc and Ayala that concerns us is Avises’s so-called congruence principle [4].

We can find no reference to this in the book they cite [14].

However, where the word ‘congruence’ is used in Avise 2004; it describes a means of intervalidating genetic distance measures because they give the same tree topology, not intervalidating topologies from different genetic markers because they support clonality as Tibayrenc and Ayala suggest [4].


We hope Tibayrenc and Ayala will join us in agreeing that there is no ‘one size fits all’ when it comes to making assertions about something as complex as pan-pathogen population genetic structure.

Vague language is appropriate to phenomena that are incompletely understood.

However, the use of language that implies precision where there currently is none is highly misleading.


A final concern mirrors one raised in a similar exchange earlier between Rougeron et al., and Tibayrenc and Ayala regarding Leishmania population genetics [15].

The energy and enthusiasm which Tibayrenc and Ayala have applied to their ‘clonal hypothesis’ over the years has given rise a large body of self-authored and co-authored work (including no less than eight reviews since 2012).

In their criticism of us, Tibayrenc and Ayala suggest that many of the instances of genetic exchange we cite are ‘considered by us [them] and many, if not most, authors working on pathogen population genetics as a particular case of PCE.

’ In line with Rougeron et al., we argue this consensus does not in fact exist.

There is a large (and rapidly growing) body of work around the PCE; however, it is almost all authored, co-authored or edited by Tibayrenc and Ayala.


In our review, we highlight the importance of population genomic data in providing definitive measures of population-level genetic exchange in protozoan pathogens [1].

We cited a recent study that demonstrated multiple genetic exchange events in L. infantum at a disease focus in Turkey [16].

By also citing an earlier study that used 14 microsatellite markers on the same strains but found no specific evidence of genetic exchange, we aimed to demonstrate the relatively improved power of genome-scale sequencing approaches [17].


In their comment, Tibayrenc and Ayala suggest we ‘omit to mention’ that there is a lot of clonal reproduction going on in this population.

Far from it, we clearly state the estimated frequency of meiotic events: approximately 1 per 7.5 x 10^4 mitotic events [16].

We would further highlight that the data produced by Rogers et al. 2014 were consistent with not just a single out-crossing event, but multiple later genetic exchange events among the progeny of this cross.


Population genomic studies to establish parasite mating systems should be subject to the usual caveats around study design that we discuss in our review: minimally spatio-temporally subdivided samples, no lumping together of subdivisions, etc [1].

However, if these caveats can be satisfied, the precision that accompanies the estimation of recombination rate, as well as the estimation of the likely exchange mechanism (via interpretation of the genomic distribution of break points, mosaics, aneuploidies, loss of heterozygosity, etc), will be unprecedented.


We are amazed to find that Tibayrenc and Ayala have chosen to re-iterate their views now, in advance of the majority of the data on which any clear understanding should be founded.


Evidence of genetic exchange in our view does not depend on demonstration of absolute panmixia.

Tibayrenc and Ayala insist that the predominant clonal evolution model is not incompatible with presence of recombination, or recombination of crucial impact (‘but on an evolutionary scale’) or frequent selfing. And yet, the impact of hybridisation and genetic exchange is overwhelmingly irrefutable, with multiple examples both from experimental and from natural populations, with profound and recent epidemiological impact, including the emergence of epidemic clones.

We therefore prefer an approach that is unencumbered by entrenched adherence and justification of the unnecessarily complicated and conceptually outdated ‘predominant clonal evolution’ hypothesis.


Knee osteoarthritis (OA) is a leading cause of disability, 1-4 with no approved agents proven to alter the disease course.

Joint replacement surgery remains the only definitive for end- stage disease, with knee OA accounting for 97% of all knee replacement (KR) surgeries, the numbers and costs of which are rising each year.

5 Thus, there is an urgent need for new effective treatment options for knee OA.


Bone plays an important role in OA pathophysiology and is considered an important treatment target.

Since mechanical factors play a central role in OA development, 6-8 and bone adapts readily to mechanical stresses, 9 it is essential to understand the long-term effects of modulating bone remodelling via antiresorptive agents on the course of OA.

Trials of antiresorptive agents have had conflicting results.


For bisphosphonates, trials have focused primarily on medium-term and short-term structural endpoints, such as joint-space width on radiograph and bone marrow lesions on MRI, with the majority not finding a protective effect.

10-1214 Observational studies have also focused primarily on structural changes, but their interpretation is limited by potential confounding and selection bias.

1617 Additionally, efficacy of bisphosphonates in an animal model of OA suggests that it varies based on stage of disease at which it is initiated.


While bisphosphonates may have beneficial articular cartilage effects, there are theoretical concerns about their long-term effects related to decreasing bone turnover.

Because of its high bone turnover state, OA bone may be relatively hypomineralised and weaker, and thus more easily deformed, 1920 but may also have increased stiffness overall due to increased subchondral bone thickness and volume.

21 Whether bisphosphonates could increase stiffness and interfere with bone adaptation to mechanical stresses, which could be detrimental in OA in the long term, requires clarification before further pursuit of this as an OA treatment.


The most clinically relevant long-term sequela of knee OA is end-stage disease leading to joint replacement surgery.

If bisphosphonates could reduce the risk of KR, it would suggest that these agents have a beneficial effect on the disease process, and in turn, can prevent or delay a costly surgical intervention.

On the other hand, if long-term bisphosphonate use has a negative impact on bone's mechanical properties, then this class of drugs should not be pursued for further development for OA management.

We therefore evaluated the relation of bisphosphonate use to KR surgery.


The Health Improvement Network (THIN) is a UK general practitioner (GP) electronic database that has anonymised health data on >12 million patients.

22 The information available in THIN is collected by GPs as part of routine patient care, which is deidentified and integrated into a central database for research purposes.

THIN contains information on demographic factors, referrals, hospitalisations, laboratory test results and prescriptions ordered by GPs, including the dose, strength and formulation of medication.

Diagnoses and test procedures are recorded with read codes.

Prescriptions written by GPs are recorded Ann Rheum Dis.

Author manuscript; available in PMC 2019 February 13.

automatically in the database as drug codes, with the use of a coded drug dictionary (Multilex).

Quality is checked regularly and the information from this database has been found to be representative of the UK population.


Eligible subjects of the current study consisted of women aged 50-89 years during 2003 to 2012 who were enrolled in THIN for at least 1 year without a diagnosis of knee OA prior to inclusion in the study sample.

They were also required to have at least one GP visit or prescription during the year prior to their knee OA diagnosis.


We excluded subjects who were on bisphosphonates prior to their knee OA diagnosis, on non-bisphosphonate antiresorptive agents or on bisphosphonates that are prescribed only for Paget's disease or metastatic disease in the UK (clodronate, tiludronate); those with a KR prior to a knee OA diagnosis; and those with rheumatoid arthritis.

We also excluded subjects deemed unlikely to be a candidate for KR surgery, including subjects with body mass index (BMI) >40 kg/m 2 , history of joint infection, high-risk cancer (pancreatic, oesophageal, gastric or metastatic cancer) or comorbidities with poor prognosis (end-stage renal disease on dialysis and severe pulmonary disease requiring supplemental oxygen).

In our main analyses, we also excluded individuals with incomplete covariate data for BMI, alcohol and smoking status, with sensitivity analyses conducted in which we used multiple imputation to enable inclusion of such subjects.


We conducted a sequential (ie, time-stratified) propensity score-matched cohort study to examine the relation of bisphosphonate initiation to the risk of KR among older women with incident knee OA.

We divided the study period (1 January 2003-31 December 2012) into ten 1-year cohort accrual blocks.

We propensity score-matched each bisphosphonate initiator with a non-initiator within each cohort accrual block using a greedy-matching algorithm.

23 The propensity score for initiating bisphosphonates was computed using logistic regression within each cohort accrual block.


For bisphosphonate initiators, the date of the first bisphosphonate prescription was considered the index date.

Non-initiators were assigned an index date randomly within the cohort accrual block.

The covariate assessment period for the propensity score modelling was prior to the index date.


Variables included in the propensity score model were: sociodemographic factors (age, sex, BMI, smoking status, alcohol use and socioeconomic status), knee OA characteristics (duration and analgesic use), potential indications for bisphosphonate (osteoporosis, osteopaenia and fractures (hip, wrist, vertebral, fragility and multiple fractures over time)), bone mineral density assessment, glucocorticoid use, falls and fall risk, comorbidities (see table 1), healthcare usage (GP visits and hospitalisations) and medications (see table 1).

Covariate balance was assessed with standardised mean differences, all of which were <0.1.


The first bisphosphonate prescription was identified using Multilex drug codes in THIN which have been used in prior studies.

2425 We focused on the bisphosphonates approved for use in osteoporosis in the UK, which include etidronate-calcitonin combination therapy, ibandronate, pamidronate, risedronate and zolendronate.


Trials of bisphosphonates in knee OA have not been able to examine the risk of KR surgery due to the longer duration of follow-up required to examine this clinically relevant endpoint.

Interestingly, though the clinical scenario and mechanisms are distinct from that of OA, bisphosphonates have been demonstrated to reduce the risk of revision surgery after joint replacement, potentially through antiresorptive effects minimising periprosthetic bone loss and osteolysis.

30 Nonetheless, while there are potentially beneficial effects of bisphosphonates and other bone-modulating agents on bone and cartilage, 38 study results in OA have been conflicting to date.


We previously demonstrated in a spinal OA trial that alendronate reduced spinal osteophyte and disc space narrowing progression, 39 and an observational study also noted less bone pathology on knee imaging by MRI (bone marrow lesions and subchondral bone attrition) and knee pain.

16 A trial of risedronate with 1-year follow-up suggested symptom benefits, and minimal non-significant structure benefits based on joint-space width on radiograph. 10 However, a subsequent larger trial with 2-year follow- up failed to demonstrate any symptom or structure (joint-space width) benefits, 11 and a small subset (<5%) of this trial that had MRIs demonstrated no difference in bone marrow lesions between treatment and placebo.

12 Analysis of prevalent bisphosphonate users in the Osteoarthritis Initiative (OAI) cohort demonstrated no significant difference in joint-space width versus non-users 17 ; this may be partly related to the prevalent drug user study design used and analytic approach.

40 More recently, a trial evaluating intravenous zoledronic acid demonstrated significant differences in bone marrow lesion volume and pain between treatment and placebo arms at 6 months, but not at the 12-month follow-up.

14 Taken together, these studies have raised questions about whether potential benefits are being missed by use of relatively insensitive radiographs.


Additionally, another concern is whether there are potential adverse longer term effects of bisphosphonates on OA pathology, such as may arise from inhibiting bone turnover, which may in turn lead to increased bone stiffness due to increased mineralisation and subsequent theoretical adverse biomechanical consequences.


Our findings indicate that bisphosphonates do not appear to have long-term adverse effects on the course of knee OA, and may in fact reduce the risk of KR surgery, suggesting that the clinical course may be less severe and/or have a slower rate of progression.

In the OAI, bisphosphonate use was associated with a slower trajectory of joint-space width loss and of MRI-based three-dimensional bone shape change, an imaging biomarker predictive of OA onset.

4142 Thus, there does not appear to be a reason for bisphosphonates to be avoided in those with knee OA, and in fact, their use may be beneficial, supporting the ongoing investigation of bone modulation as an OA therapeutic target.


Potential limitations of this study deserve comment.

Because THIN does not have bone density results, there is a concern for residual confounding by indication since individuals with lower bone mineral density are more likely to be on a bisphosphonate and are also more likely to have a lower BMI.

We attempted to address this by including factors in the propensity score that are likely reflective of bone mineral density and risk of osteoporosis.

The diagnostic code for osteoporosis and osteopaenia were not as frequently recorded as one would expect for individuals on bisphosphonates, likely reflecting the fact that GPs do not always record the condition for which bisphosphonates are being prescribed.

In a manual review of patient profiles, we noted that all patients on bisphosphonates without an Ann Rheum Dis. Author manuscript; available in PMC 2019 February 13.

osteoporosis code (including those who had an osteopaenia code) were universally on full- strength doses of a bisphosphonate, usually with ≥1 bone mineral density test and/or ≥1 fracture suspicious for an osteoporotic fracture.


Further, the code for osteoporosis and/or osteopaenia often occurred well after initial bisphosphonate prescription.

Additionally, existing published literature provides some reassurance that bone mineral density status is unlikely to importantly affect our study results.

Low systemic bone mineral density has either been associated with increased risk of knee OA progression or no increased risk.

43-46 Based on such data, one would expect bisphosphonate initiators, who presumably may have lower bone mineral density, to have potentially either no or increased risk of knee OA progression.

In contrast, we found that bisphosphonate initiators experienced a protective effect even while accounting for BMI.

Additionally, vitamin D, which is commonly taken as over-the-counter medication without prescription, particularly in multivitamins, is not reliably recorded within THIN in a standardised fashion, and was low but well balanced between the two groups in our study sample.

Nonetheless, their use is unlikely to importantly confound our results given the lack of association of vitamin D with knee OA in two recent rigorously conducted clinical trials.

4748 Though effects on KR cannot be determined from these trials, some studies have suggested that joint-space width may be predictive of KR surgery.


We also examined the relation of incident bisphosphonate use to hip fracture to determine whether we could replicate the findings from existing published data; such a replication would suggest that the propensity score model likely adequately captured most important confounders.

In this analysis, we found the HR to be similar to the effect noted for clinical fractures in the Fracture Intervention Trial.

49 Finally, we performed a sensitivity analysis to address the degree of unmeasured confounding that would have to be present to nullify our results.

For bone mineral density and/or vitamin D to result in a fivefold alteration in the risk of KR while also having a prevalence differential of 30% between the two groups is highly unlikely.

Thus, at minimum, it appears that bisphosphonates do not have a detrimental effect and may indeed have a potential beneficial effect.


Secular trends precluded robust active user comparator analyses, particularly as many women initiated oestrogens prior to knee OA diagnosis.

Further, these results are primarily of relevance to older women; whether there should be different biological effects in middle- aged women or in men is not clear, but cannot be extrapolated from this study.

Our approach to addressing this clinically important question also


Subjects were followed from the index date until KR, death, loss-to-follow-up (e.g., transfer out of GP practice), age >90 years, last date of data collection by GP or end of study (31 December 2013).

To examine the relation of incident bisphosphonate use to KR surgery, we conducted Cox proportional hazards regression using age as the time scale with an intention- to-treat approach, additionally adjusting for the covariates used in the propensity score model.


To explore the possibility of residual confounding, we performed additional sensitivity analyses.

In the first, we re-ran the above analyses without including BMI in the propensity score to determine indirectly how much of an influence BMI has on the effect estimate.

We also did not include alcohol or smoking so that our model was not limited to those with complete data.

We also performed multiple imputation by imputing missing BMI, alcohol and smoking data in five blocked datasets that were each used to create propensity score- matched datasets that were analysed separately with a summary effect estimate calculated.


Second, we conducted age-matched, BMI-matched and OA duration-matched analyses, in which bisphosphonate initiators were matched 1:4 with non-initiators, adjusting for all other potential confounders used in the propensity score model.

Third, this latter analysis was repeated using active comparator drugs as the referent group: (1) oestrogen initiators; (2) initiation of any other bone-modulating agent (eg, strontium, calcitonin, raloxifene, teriparatide and oestrogen).

Fourth, to account for potential selection bias due to death or loss to follow-up (eg, individuals transferring out of their GP practice or other issue), we applied inverse probability weighting.

3435 To account for competing risk of death, we implemented the Fine and Grey approach. 36 Finally, to assess the influence of unmeasured and residual confounding factors, such as bone mineral density, we performed a sensitivity analysis using an array approach, which is helpful for exploring the effect of residual confounding over a wide range of parameter values and effects, and to examine the extent of confounding necessary to fully explain the observed effect estimate.

37 All statistical analyses were performed using SAS V.9.3 (SAS Institute, Cary, North Carolina, USA).


From among 27 295 subjects who met our inclusion and exclusion criteria, we identified 2225 bisphosphonate initiators.

From these, 2006 bisphosphonate initiators were propensity- score matched to the same number of non-initiators.

Eighty-four per cent of the bisphosphonate initiators were prescribed alendronate; the remaining were risedronate (13%), ibandronate (2%) and etidronate-calcitonin combination therapy (1%).

The mean age of the cohort was 76, and the mean BMI was 27.3 kg/m 2 .

Overall, covariate data were well-balanced among the initiators and non-initiators (table 1).

Approximately 60% of the initiators and the non-initiators appeared to have an indication for bisphosphonate use prior Ann Rheum Dis.

Author manuscript; available in PMC 2019 February 13.

Author Manuscript to the index date; however, this proportion increased to 82% among the initiators, and remained fairly steady among the non-initiators (63%) over the follow-up period.


The mean follow-up time for subjects was approximately 3 years for both groups (table 2).

During the follow-up period, 208 KR surgeries were performed overall, with 138 in bisphosphonate initiators and 170 in the non-initiators.

The mortality rate was similar in both groups (39.7 and 38.2 per 1000 person-years in the initiators and non-initiators, respectively).

The crude incidence rate of KR was 22.0 per 1000 person-years among the bisphosphonate initiators and 29.1 per 1000 person-years among the comparator group, with a crude incidence rate ratio of 0.75 (95% CI 0.60 to 0.94) (table 2).

The HR for KR related to bisphosphonate initiation was 0.74 (95% CI 0.59 to 0.93), and results did not change materially when additionally adjusted for the potential confounders used in the propensity score (adjusted HR 0.76 (95% CI 0.60 to 0.95)).


Because approximately 15% of subjects had missing values for either BMI, smoking and/or alcohol, we performed a sensitivity analysis in which we created the propensity score without including these variables.

In this analysis, with 2379 initiators of bisphosphonates and the same number of propensity score-matched non-initiators, the adjusted HR was similar to the main result (0.79, 95% CI 0.64 to 0.98).

In analyses using multiple imputation, the adjusted HR was also similar to the main result (0.72, 95% CI 0.59 to 0.89).

We also conducted a sensitivity analysis in which we matched each bisphosphonate initiator to four non-initiators by age (within 2 years), BMI (±0.5 kg/m 2 ) and OA duration (within 2 years).

The adjusted HR from this matched cohort study was 0.35 (95% CI 0.26 to 0.47).

To further address confounding by indication, we compared bisphosphonate initiation to that of an active comparator drug (oestrogen and any bone-modulating agent, respectively), matched on age, BMI and OA duration, and obtained results similar to our main results (adjusted HR 0.75 (95% CI 0.30 to 1.87).

The smaller sample sizes and lower number of KR outcomes precluded precise estimation of these effects.


When we accounted for censoring due to loss to follow-up or death with inverse probability weighting, the results remained similar to the main results (HR 0.75, 95% CI: 0.59 to 0.94).

The competing risk of death analysis also provided similar results (HR 0.76, 95% CI 0.61 to 0.86).

Finally, in a sensitivity analysis to examine the potential effect of residual confounding on our results, we found that in order for an unmeasured confounder to nullify the observed HR, that confounder would need to be associated with a fivefold difference in risk of the outcome (KR) and have a 30% difference in prevalence among bisphosphonate initiators versus non-initiators.

Such a scenario would be highly unlikely (figure 1).


In this large cohort of older women with incident knee OA derived from the general population, we found that bisphosphonate initiation was associated with a ~25% lower risk of KR surgery than those who did not initiate bisphosphonates.

Our findings were consistent across several sensitivity analyses that were performed to address potential residual confounding, missing data and selection bias, suggesting that it is unlikely for unaddressed biases to result in bisphosphonate initiation to have a detrimental effect.


With great interest, we read the recent article by Neogi et al entitled, ‘Effect of bisphosphonates on knee replacement (KR) surgery’ [1].

In the study, the authors concluded that in this population-based cohort of older women with incident knee osteoarthritis (OA), those with incident bisphosphonate users had lower risk of KR than non-users of bisphosphonates, which was further supported by another large cohort study [2].

The strengths of this study include a propensity score-matched cohort design, Cox proportional hazards regression to control for potential confounders and sensitivity analyses focused on residual confounding.

Meanwhile, the authors acknowledged the limitations of their work.

We applaud and congratulate their important work for clinical practice.

However, several important points should be further discussed.


Generally, determinants of patient preferences for KR are OA severity, the level of knee pain, disability, and the weakness of quality of life, and more importantly, their willingness and access to KR.

In the current study, authors tended to agree that reduced risk of KR has resulted from weakened OA progression after bisphosphonates use.

However, recent high-level evidence indicated that bisphosphonates neither provide pain relief and function improvement nor radiographic progression in knee OA [3][4].

Therefore, we wonder that bisphosphonates may affect the willingness and access to KR of patients with OA, thus reducing the risk of KR.


Actually, many clinical and sociocultural factors, such as racial differences (African Americans and whites), social support and an educational intervention included a decision aid, have vital important influence on patient preferences for KR [5][6][7].

More importantly, a recent study revealed that patient preferences for KR were strongly associated with knee pain severity in patients with OA with health insurance, but their inverse relationship disappeared in patients without health insurance [8].

Also, another study suggested that despite worse baseline knee pain and function, black participants had much lower adjusted risk of having total KR (TKR) than white participants [9].

Thus, these indicated that sociocultural factors, but not OA severity, may have more significant influence on the willingness and access to KR.


However, Neogi and colleagues seemed to ignore these important sociocultural factors in Cox proportional hazards regression, propensity score model and sensitivity analyses.

We strongly recommend additional analyses based on aforementioned risk factors, which may further increase the robustness and credibility of the current study.


Additionally, the authors assessed the influence of bisphosphonates use on the risk of KR in patients with OA.

However, there were no any data involving the baseline characteristics of OA severity, the level of knee pain, disability and the weakness of quality of life between both cohorts.

Moreover, the influence of the dose, route, time point or measuring instrument of bisphosphonates administration on the risk of KR was unclear, so further subgroups should be warranted.

Meanwhile, the primary outcome (the risk of KR) may be involved in many similar terms, such as TKR, revision KR, hemi-KR or KR in both knees.

As with us, some readers may be confused about the notion.

Therefore, the primary outcome should be clearly defined.


To sum up, we respect the great work done by the authors, but the study should be interpreted with the aforementioned limitations and further analyses should be performed.


We thank Dr Li and colleagues [1] for their interest in our paper.

As outlined in our paper [2], we used data from a large general practitioner electronic health records database for our study.

These data are collected and recorded as part of routine clinical care with diagnostic codes.

As such, factors such as willingness to undergo and access to knee replacement (KR), knee pain severity, disability, quality of life and others are not available in this database, and we were thus unable to examine these as potential confounders.

We were, however, able to account for socioeconomic status (Townsend Deprivation Index), and performed sensitivity analyses demonstrating that substantial residual confounding is unlikely.


The assessment of bisphosphonate exposure was based on prescription data, and alendronate was the most common bisphosphonate prescribed (84%), as described in the paper [2].

The potential mechanisms by which bisphosphonates may confer the noted effects were beyond the scope of this paper.

Nonetheless, it is not clear that bisphosphonate initiation would alter one’s willingness to undergo KR or access to KR such that a protective effect would be noted in our study.


We included both total and unicompartmental primary knee arthroplasty since either surgery would indicate substantially symptomatic knee osteoarthritis, but did not include revision surgery since the reasons for that procedure are not for severe/end-stage osteoarthritis; this is the same approach used by the other studies we had cited on this matter.

As per standard time-to-event analyses, subjects were censored at the first KR, among the other censoring factors outlined in the paper.


Animal and human studies have demonstrated that water drinking may elicit a pressor response (May & Jordan, 2011).

Jordan, Shannon, Grogan, Biaggioni, and Robertson (1999) were the first to demonstrate a potent pressor response (increase of up to 33 mmHg in systolic blood pressure) after water intake in patients with severe orthostatic hypotension (i.e. patients with autonomic failure).

Thereafter, they observed that the magnitude of the pressor response was attenuated in healthy elderly subjects and completely abolished in young healthy subjects after water intake (Jordan et al., 1999, 2000; Schroeder et al., 2002; Scott, Greenwood, Gilbey, Stoker, & Mary, 2001).


The reason for the blunted pressor response after water drinking in healthy young c 2018 The Authors.

Experimental Physiology c 2018 The Physiological Society subjects is not fully understood, but previous authors suggested that the arterial baroreflex might play a role.

This idea is supported by animal experiments, in which mice with intact baroreflexes did not respond to water intake (McHugh et al., 2010).

In contrast, water elicited a substantial increase in arterial blood pressure after inter- ruption of the afferent baroreflex arc by sinoaortic denervation (McHugh et al., 2010).


In humans, water drinking increases efferent sympathetic outflow in the absence of a pressor response in young healthy subjects (Scott et al., 2001), suggesting that baroreflex function is preserved; however, to our knowledge, this has not been tested directly.


In contrast, in patients with autonomic failure and in hypertensive and healthy older adults, water intake induces sympathoexcitation, 1318 wileyonlinelibrary.com/journal/eph Experimental Physiology.

2018;103:1318-1325. 1319 which is accompanied by a significant pressor response suggestive of impaired baroreflex function (Callegaro et al., 2007; Cariga & Mathias, 2001; Jordan et al., 1999, 2000).


Interestingly, previous studies have demonstrated that the central mechanism of osmosensation and systemic osmoregulation involves neurons of the central baroreflex regulatory regions (i.e. periaqueductal grey; Bourque, 2008).

Indeed, stimulation of the ventrolateral periaqueductal grey enhanced sympathetic baroreflex sensitivity in humans (Sverrisdóttir et al., 2014).

However, the extent to which an alteration in arterial baroreflex function may contribute to arterial blood pressure responses to water intake in healthy humans remains unclear.


Therefore, the present study was undertaken to test the hypothesis that water drinking increases the arterial baroreflex control of muscle sympathetic nerve activity (MSNA) in young healthy subjects.


To test this hypothesis, MSNA, arterial blood pressure and heart rate were continuously recorded before, during and for 60 min after ingestion of water or saline, and spontaneous arterial baroreflex-MSNA gain was calculated.


We chose 60 min of data collection based on previous studies indicating that the peak MSNA response occurs ∼30 min after water ingestion in young healthy subjects (Scott et al., 2001).


The study was approved by the Fluminense Federal University ethics committee (CAAE: 53503816.0.0000.5243).

Written informed consent was obtained from all participants, and all experimental procedures conformed to the standards set by the Declaration of Helsinki, except for registration in a database.


Ten healthy and recreationally active male volunteers with a mean (±SD) age of 24 ± 2 years, body weight of 77 ± 8 kg and height of 177 ± 6 cm participated in this study.

Subjects had to be asymptomatic, non-smokers, normotensive and non-diabetic.

None of the subjects was receiving prescribed or over-the-counter medications, and all were confirmed to have no heart disease.

The subjects were recruited through posters placed on the university campus.

Subjects were requested to abstain from caffeinated beverages for 12 h and from strenuous physical activity and alcohol for a minimum of 24 h before any experimental sessions.

Subjects were a minimum 2 h postprandial upon arrival to the laboratory.


Subjects were studied in the supine position at an ambient room temperature of ∼24 • C. Arterial blood pressure and heart rate were measured on a continuous beat-to-beat basis using finger photoplethysmography (Finometer Pro; Finapres Medical Systems, Arnhem, The Netherlands) and a single-lead ECG (PowerLab 4/25T and Laboratory Chart Pro 7 software, AD Instruments, Castle Hill, NSW, Australia), respectively.

Brachial arterial blood pressure


• What is the central question of this study?

Water drinking increases muscle sympathetic nerve activity (MSNA), and it increases arterial blood pressure (ABP) in older populations but not in young healthy subjects.

Does an increase in gain of arterial baroreflex control of MSNA contribute to maintenance of ABP after water drinking in healthy young subjects?


The gain of arterial baroreflex control of MSNA was increased and remained elevated 60 min after water drinking (500 ml) but remained unchanged after saline intake.

An enhancement in gain of arterial baroreflex control of MSNA contributes to the maintenance of ABP after water drinking in young healthy subjects, probably via osmosensitive mechanisms.


of the dominant arm was measured with an automated digital sphygmomanometer (EM-759-E; Omron, Kyoto, Japan) to validate Finometer measurements of absolute blood pressure in real time during data collection.

Respiratory movements were monitored using a strain-gauge pneumograph placed in a stable position around the abdomen (MLT1132 respiratory belt; ADInstruments).


Multiunit recordings of postganglionic MSNA were obtained by inserting a unipolar tungsten microelectrode percutaneously through the intact skin and positioning it into muscle nerve fascicles of the peroneal nerve of the right leg at the fibular head.

A reference electrode was placed subcutaneously 2-3 cm distal.

The recording electrode was adjusted until a site was found where nerve activity displayed a pulse synchronous pattern of spontaneous bursts, had a signal-to-noise ratio of 3:1, was increased during an end-expiratory breath-hold or Valsalva manoeuvre and was unresponsive to an unexpected loud noise or skin stroking (Vallbo, Hagbarth, Torebjörk, & Wallin, 1979).

The raw signal was amplified (×100,000), filtered (bandwidth 700-2000 Hz), rectified and integrated (time constant 0.1 s) to obtain a mean voltage neurogram (Iowa Bioengineering, Iowa City, IA, USA).

Sympathetic bursts were identified using a fully automated program (Hamner & Taylor, 2001); burst identification was then corrected by inspection by a single blinded observer (L.C.V.).

The MSNA was quantified as burst incidence (in bursts per 100 heart beats) and burst frequency (in bursts per minute).

In one of the 10 participants, we were able to obtain a quality MSNA signal only during the saline protocol.


After instrumentation, participants rested for ∼15 min to permit stabilization of all cardiovascular variables.

Thereafter, a 10 min base- line was performed, in which the subject rested quietly while all neurocardiovascular variables were continuously recorded.

The sub- jects then ingested 500 ml of bottled water (n = 9, pH 7.5; [Na + ], 1.5 mmol l −1 ; [Ca 2+ ], 3.1 mmol l −1 ) in <5 min.


The MSNA, arterial blood 1469445x, 2018, 10, Downloaded from https://physoc.onlinelibrary.wiley.com/doi/10.1113/EP087095 by Mcgill University, Wiley Online Library on [18/05/2024].

See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 1320 VIANNA ET AL.

F I G U R E 1 Original records illustrating muscle sympathetic nerve activity (MSNA, top panel) and arterial blood pressure (bottom panel) at baseline (a) and at 30 min after the ingestion of 500 ml of bottled water (b) pressure, heart rate and respiration were measured for 60 min after the completion of drinking.


In a subset of subjects (n = 6), a control visit was performed, in which 500 ml of saline was ingested and all experimental measures were performed in an identical manner to the water drinking visit.


The temperature of the water and saline was controlled at 22 • C.


The participants were advised to ingest 500 ml of water 2 h before the experiments to start the study protocol with a suitable hydration status (Teixeira, Ramos, Marins, & Ricardo, 2015; Vianna, Oliveira, Silva, Ricardo, & Araujo, 2008) and were asked to void their bladder immediately before the experiments (Fagius & Karhuvaara, 1989).


In addition, the participants' sensation of bladder filling was reported at the end of each experiment on a 0-10 scale, with zero representing a complete lack of this sensation and 10 representing an irresistible desire to void (Fagius & Karhuvaara, 1989).


Baseline MSNA, arterial blood pressure and heart rate were calculated as mean values over a 5 min period.

After consumption of water or saline, 5 min averages were calculated every 15 min.

The same segments were used to assess arterial baroreflex control of MSNA by analysing the relationship between spontaneously occurring fluctuations in diastolic blood pressure and MSNA, as previously described (Fisher et al., 2015; Kienbaum, Karlssonn, Sverrisdóttir, Elam, & Wallin, 2001; Prodel, Barbosa, Mansur, Nobrega, & Vianna, 2017; Sundlöf & Wallin, 1978; Vianna et al., 2012).

Briefly, the diastolic blood pressure for each cardiac cycle within a data collection period was grouped into 3 mmHg pressure bins.

The slope of the relationship between MSNA burst incidence and diastolic blood pressure was identified using linear regression analysis, with a minimum r value of −0.5 used as the criterion for accepting slopes (Kienbaum et al., 2001; Sundlöf & Wallin, 1978; Young, Deo, Chaudhary, Thyfault, & Fadel, 2010).

All data were weighted to account for the number of cardiac cycles within each pressure bin, thus removing bias attributable to bins containing a small number of cardiac cycles.


In addition, spontaneous cardiac baroreflex sensitivity (cBRS) was assessed using the sequence technique as previously described (Holwerda et al., 2016; Parati, Di Rienzo, & Mancia, 2000; Teixeira et al., 2018a).

The sequence technique is based on the identification of consecutive beats in which progressive increases (or decreases) in systolic blood pressure (input variable) are followed by a progressive lengthening (or shortening) in RR interval (output variable).

Briefly, sequences of three or more consecutive and concurrent beats with corresponding increases or decreases in systolic blood pressure and RR interval were identified as arterial baroreflex sequences (Cardio- Series v2.4, Brazil).

Sequences of at least three consecutive cardiac cycles were detected only when the variations in systolic blood pressure and RR interval were ≥1 mmHg and ≥1.0 ms, respectively.


A linear regression was applied to each individual sequence, and only those sequences in which r was >0.85 were accepted (Kardos et al., 2001; Zollei, Paprika, & Rudas, 2003).

The slopes of the systolic blood pressure and RR interval relationships for up and down sequences were then calculated and averaged for a measure of spontaneous cBRS.


Comparisons were made using univariate repeated-measures ANOVAs, and significant main effects were evaluated with LSD post hoc analyses when appropriate.

Statistical significance was set at P < 0.05, and values are presented as means ± SD.


Figure 1 shows an original recording of one representative sub- ject, illustrating the MSNA and arterial blood pressure responses at 1321 TA B L E 1 Muscle sympathetic nerve activity (MSNA) and cardiovascular responses to water and saline drinking baseline and at 30 min after water drinking.

Group summary data of all neurocardiovascular variables at rest and at each time point after water drinking are presented in Table 1.

After water drinking, MSNA burst frequency and burst incidence were increased within 15 min and remained elevated above baseline for the remainder of the protocol (P < 0.05), whereas mean blood pressure and heart rate remained unchanged (P > 0.05).

In comparison, after saline drinking, all neurocardiovascular variables remained unchanged from baseline throughout the protocol (all P > 0.05).

Figure 2 illustrates the changes (∆) from baseline in MSNA burst frequency (Figure 2a), MSNA burst incidence (Figure 2b) and mean blood pressure (Figure 2c) for the 60 min period after ingestion of either bottled water (grey bars) or saline (open bars).


The arterial baroreflex gain of MSNA burst incidence was significantly enhanced (i.e. more negative) within 15 min [∆−1.04 ± 1.05 (95% confidence interval −1.84, −0.23) bursts (100 heart beats) −1 mmHg −1 ] and remained elevated for the duration of the study, reaching a peak during the 30th minute [∆−2.07 ± 0.98 (95% confidence interval −2.82, −1.32) bursts (100 heart beats) −1 mmHg −1 ] after water drinking (Figure 3, grey bars).

In contrast, after saline drinking, the gain of arterial baroreflex bursts incidence was not different from baseline at any time point (Figure 3, open bars).


The spontaneous relationship between systolic blood pressure and RR interval remained unchanged after water intake (baseline, 22.2 ± 11.9 ms mmHg −1 ; 30 min, 23.6 ± 12.4 ms mmHg −1 ; and 60 min, 26.0 ± 12.8 ms mmHg −1 ; P > 0.05) and during the saline drinking (base- line, 21.6 ± 6.6 ms mmHg −1 ; 30 min, 22.8 ± 6.9 ms mmHg −1 ; and 60 min, 25.0 ± 8.0 ms mmHg −1 ; P > 0.05).

Similar results were found


The main new finding of the present study is that water drinking enhanced the gain of the arterial baroreflex control of MSNA in young healthy men.

Indeed, the slope of the relationship between diastolic blood pressure and MSNA was increased 15 min after ingestion of water and remained elevated throughout 60 min.

Furthermore, mean arterial blood pressure, heart rate and cBRS were unchanged after water intake.

In comparison, after ingestion of saline, all neuro- cardiovascular variables were unchanged.

Collectively, these findings support the concept that an enhancement in the gain of the arterial baroreflex control of MSNA contributes to the maintenance of arterial blood pressure after water drinking in young healthy subjects and that these responses are likely to be mediated by osmosensitive mechanisms.


The sympathoexcitatory effects of water drinking are well established in both animals (McHugh et al., 2010) and humans (Callegaro et al., 2007; Scott et al., 2001).

Previous reports have demonstrated that water-induced increases in sympathetic outflow were accompanied by significant elevations in arterial blood pressure in patients with autonomic failure and elderly subjects but not in young healthy subjects (Cariga & Mathias, 2001; Jordan et al., 1999, 2000).


The findings of the present study advance our understanding of how healthy young subjects are able to buffer the increases in arterial blood pressure after water drinking.

For the first time, we demonstrate that an enhancement in the gain of arterial baroreflex control of MSNA may counteract the effects of water-induced increases in sympathetic outflow to maintain arterial blood pressure.


In line with this, previous animal reports have shown that mice with intact baroreflexes did not respond to water intake, whereas water elicited a substantial increase in arterial blood pressure after interruption of the arterial baroreflex arc via sinoaortic denervation (McHugh et al., 2010).

In addition, it has been shown that patients with autonomic failure present impaired arterial baroreflex control compared with healthy counterparts (Biaggioni, 2017).

Taken together, these findings suggest that intact arterial baroreflex function is essential for the maintenance of blood pressure after water drinking.


The precise pathways mediating the improvement in arterial baroreflex-MSNA gain after water intake remain unclear, but some relevant points warrant discussion.


The pressor response to water drinking may involve osmosensitive transient receptor potential vanniloid 4 (Trpv4).

Water absorption induces a hyposmolar stimulus in the portal circulation, probably involving activation of Trpv4 (Lechner et al., 2011; McHugh et al., 2010), which triggers a sympathetic reflex (Jordan et al., 2000) and may increase arterial blood pressure.


Indeed, duodenal infusion of water [25 l (g body weight) −1 ] decreases portal osmolality in both wild-type and Trpv4 −/− mice (all had undergone sino- aortic denervation), but only wild-type mice demonstrated increases in arterial blood pressure (McHugh et al., 2010).


Collectively, these data suggest that the pressor response induced by water ingestion involves sympathetic activation and that the Trpv4 channel is an important mediator of the afferent input of the response (McHugh et al., 2010).


In addition to the well-described sympathoexitatory effects of water intake, the present finding illustrates the impact of water drinking on the arterial baroreflex.


The central mechanisms of osmosensation and systemic osmoregulation are not fully understood, but we can speculate that the hepatic portal osmoreceptors signal hyposmotic stimuli via the splenic nerve activating different brain regions, including the peri- aqueductal grey (Bourque, 2008).

Indeed, stimulation of the ventro- lateral periaqueductal grey has been shown to enhance sympathetic baroreflex sensitivity in humans (Sverrisdóttir et al., 2014); however, whether this osmoregulatory circuit plays a role in water intake- induced elevations in arterial baroreflex-MSNA gain requires further investigation.

The osmopressor response to water is apparent in patients with autonomic failure and elderly subjects but not in young healthy populations.

We reasoned that in young healthy subjects, the pressor response to water was not observed owing the fact that osmoreceptor- mediated increases in MSNA were counteracted by an enhancement in the gain of arterial baroreflex control of MSNA.

Indeed, we found that the arterial baroreflex-MSNA gain was increased 15 min after water intake and remained elevated for the remainder of the protocol.

Inter- estingly, we did not find any changes in cBRS after water ingestion.


The reason for these differential findings are unclear, but might be associated with greater changes in sympathetic outflow compared with vagal outflow after water drinking, the latter being more representative of spontaneous cBRS.

Nevertheless, there are several reports in the literature indicating differential changes in cardiac and MSNA baroreflex control after a variety of interventions (Young et al., 2010), and a lack of relationship between cardiac and sympathetic baroreflex sensitivity has been reported in healthy humans (Dutoit et al., 2010; Taylor et al., 2015).


In addition, it is important to note that the methods used to measure cardiac and sympathetic baroreflex assess different components of the arterial baroreflex system.

More specifically, baroreflex-MSNA gain does not take into account the end-organ response, whereas the cBRS gain includes the end-organ response (Parati et al., 2000).


In this sense, further investigation is warranted to clarify the mechanisms by which water drinking differentially affects sympathetic and cardiac baroreflex sensitivity.


In the present study, we used spontaneous baroreflex measures derived from MSNA burst incidence and not total MSNA.

The rationale for this approach was based on previous studies demonstrating that the gain of arterial baroreflex control of MSNA burst incidence was similar to sympathetic baroreflex sensitivity obtained using pharmacological manipulations of arterial blood pressure (Dutoit et al., 2010; van Schelven, Karemaker, Blankestijn, & Oey, 2008).


In contrast, spontaneous arterial baroreflex gains calculated from measurements using total MSNA have been suggested to be influenced more by non- baroreflex inputs (Dutoit et al., 2010; Kienbaum et al., 2001), which might limit the interpretation of such measures.


Indeed, several studies have reported weak relationships between burst area and diastolic blood pressure, whereas burst incidence consistently demonstrates strong relationships with diastolic blood pressure (Dutoit et al., 2010; Keller et al., 2004; Kienbaum et al., 2001; Ogoh, Fisher, Raven, & Fadel, 2007; Rudas et al., 1999).


It is important to note the potential limitations of the present study.

We included only men and, given existing sex differences in blood pressure regulation (Hart & Charkoudian, 2014; Joyner, Wallin, & Charkoudian, 2016; Kim et al., 2011; Teixeira et al., 2018b), our results may not be generalizable to women.

In addition, the arterial baroreflex control of MSNA was calculated based on a closed-loop measure using spontaneous fluctuations in MSNA and blood pressure only.


We recognize that open-loop assessments of arterial baroreflex gain using perturbational methods, such as the infusion of vaso- active drugs (i.e. modified Oxford), allow for a more comprehensive examination of arterial barorefex control of MSNA.

Nevertheless, the slope of the relationship between MSNA and diastolic blood pressure derived from the closed-loop spontaneous analysis method has been shown to be correlated with the slopes derived using the open-loop modified Oxford approach for assessing arterial baroreflex-MSNA gain (Dutoit et al., 2010).


Furthermore, important to our study design, we were interested in making arterial baroreflex measurements at multiple time points to characterize the response after water ingestion fully, and repeating the modified Oxford technique at baseline and then every 15 min for 60 min was not feasible, particularly as it is standard to perform at least two modified Oxford trials at each time point (Holwerda et al., 2016; Rudas et al., 1999).


In summary, the present study demonstrates, for the first time, that bottled water, but not saline intake, enhances the gain of arterial baroreflex control of MSNA in young healthy men.

These data support the concept that the lack of a pressor response after water ingestion in young healthy subjects may be attributed to water-induced increases in the gain of arterial baroreflex control of MSNA.


We read with great interest the recent study by Vianna et al. ( 2018), who should be congratulated for their novel and enlightening findings concerning the acute effect of water ingestion on arterial baroreflex function of healthy young individuals.

Aiming to provide some additional information for better understanding and extrapolation of these findings to broader populations and conditions, we would like to underscore a few methodological and technical issues, mainly regarding the estimation of baroreflex sensitivity (BRS).


One crucial parameter, especially when assessing haemodynamic and baroreflex parameters and their responses to various interventions, is the body position [1], [2].

Subjects in the sitting, supine or other positions may exert different blood pressure (BP) and heart rate values, even without any intervention.

Furthermore, BRS changes between different body positions are unclear, because the existing published data are controversial.

Thus, it would be relevant to know whether the findings of this study remain the same for subjects who ingest water in postural positions other than supine.

More specifically, the subjects in this study ingested 500 ml of bottled water probably in the sitting position, but all measurements were performed in the supine position.

It might be meaningful to explore the same BRS and BP changes after water ingestion in the sitting or standing positions, which might be more indicative to daily habits.


In the study by Vianna et al. ( 2018), mean arterial pressure (MAP) was found to be unchanged up to 60 min after water ingestion.

Although the classic formula, MAP = DBP + 0.33 × PP (where DBP is the diastolic blood pressure, PP the pulse pressure and ⅓ = 0.33 the form factor), has been extensively used, it should be kept in mind that other formulae exist (i.e. the formula using a form factor of 0.412 instead of 0.33) [3].

Alternative calculation of MAP might yield superior associations with target organ damage [3] or more accurate estimations of MAP compared with direct intra-arterial recordings.

Furthermore, several automated oscillometric blood pressure devices provide MAP values using their own embedded algorithms.

Of course, given that the same calculation of MAP was used before and after water ingestion, it is unlikely that MAP measured using another formula or form factor would change this outcome.

However, it is important to know how exactly the MAP values are calculated, in order to be able to extrapolate or interpret the results in comparison to other studies.

Still, the pressure response to water ingestion is controversial, because previous studies have found the opposite results, i.e. a significant increase [4].


Finally, algorithms for BRS calculation used in the literature vary greatly in technical, computational and methodological perspectives.

It is known that the sequence method is characterized by strict threshold criteria, with no evidence about the optimal threshold values.

Altering these thresholds can have a great impact on the computed BRS values.

In this study, the R–R interval threshold was chosen as 1 ms, which is much lower than the widely used threshold of 4 ms.

Although there is no gold-standard R–R interval threshold, it should be kept in mind that altering these threshold values can lead to different BRS estimations.

For this reason, it could be suggested that BRS should be estimated by more than one method.

For example, using frequency domain algorithms, which may be more robust in specific circumstances, in addition to the time domain method that was followed in this study, might provide more robust results concerning BRS responses.


We are happy that our study [6] has received the attention of others and pleased to have the opportunity to respond and debate the merits of our approach.

In their Letter to the Editor, Papaioannou et al. [ 3] raise some interesting points regarding the methods of our study and how they relate to the estimation of cardiac baroreflex sensitivity, which we would like to address and provide further clarification.

We are aware and in agreement with the statement by Dr Papaioannou and colleagues that body posture is a critical factor when assessing neural cardiovascular function at rest and during perturbational conditions.

In this regard, our group has recently demonstrated that both cardiac and peripheral sympathetic outflow to postexercise ischemia (i.e. muscle metaboreflex activation) can be modulated by mobilization of central blood volume [1, 4].

Whether the arterial baroreflex responses to water drinking are dependent on the body posture is unknown.

However, this was not part of our study question.

As we have clearly stated in the Methods section, subjects were studied in a supine position, which is a standard position for human neural cardiovascular control studies and the majority of studies on water drinking.

It is important to clarify further that subjects ingested the 500 ml of water or saline in this position using a flexible straw; therefore, there was no positional change.

This approach also minimizes limb movement and muscle activation, which would jeopardize the muscle sympathetic nerve activity recordings.

In our study, mean arterial pressure remained unchanged after ingestion of either bottled water or saline.

Of note, this is a common finding in young healthy subjects [2].


With regard to mean arterial pressure calculations, Dr Papaioannou and colleagues commented on the importance of describing how mean arterial pressure was derived, because the use of different formulae might limit the extrapolation and interpretation of our findings.

With all due respect, we disagree, and we would like to provide the following points for consideration.

Firstly, blood pressure was measured on a beat-to-beat basis using a valid and reliable technique (i.e. finger photoplethysmography).

In this sense, mean arterial pressure was calculated by a time-weighted integral of the instantaneous pressures derived from the area under the curve of the pressure–time waveform of the entire cardiac cycle.

Secondly, our interpretation has emerged from a study where the subjects participated in both experimental (water drinking) and control (saline drinking) trials, and we used the same blood pressure measurement before, during and after each intervention.

Therefore, we are confident that our results and interpretations are valid and reliable and would stand regardless of the way in which mean arterial pressure is calculated.

In their letter, Dr Papaioannou and colleagues also pointed out the lack of standardization in the literature with regard to the use of the sequence technique for the estimation of cardiac baroreflex sensitivity.

We agree that there are no absolute or agreed upon ideal parameters for analysis (i.e. systolic pressure and R–R interval thresholds).

However, in terms of their concern regarding the R–R interval threshold used in our study, we re-analysed our data using a threshold of 4 ms, as suggested in their letter, and the results remained unchanged.

Furthermore, the assessment of agreement between 1 and 4 ms threshold provided a high and significant intraclass correlation coefficient (0.938; P < 0.05).


Lastly, Papaioannou et al. [ 3] advocate the use of frequency domain analyses to address the research question further.

It is important to mention that our hypothesis was focused on the sympathetic branch of arterial baroreflex function, for which we had direct muscle sympathetic nerve activity measurements.

Although we would welcome the use of frequency analyses to explore cardiac baroreflex sensitivity further, we are confident in our findings using the sequence technique.

In summary, our data provide new insights on the potential contribution of the sympathetic baroreflex in the maintenance of arterial blood pressure after water drinking in young healthy subjects.

Furthermore, independently of the mathematical approach, our findings indicate a minimal role of the cardiac baroreflex in the regulation of arterial blood pressure after water ingestion.

Nevertheless, we acknowledge there are no perfect analyses and always welcome debate on different ways to evaluate data, as provided by Dr Papaioannou and colleagues.


Regulation of microtubule (MT) 1 distribution is of critical importance for many cellular processes, including directed cell motility, cell division, and establishment of cell polar- ity.

In the developing nervous system and during nerve re- generation, axon growth depends on assembly of polarized MT arrays; however, axon guidance depends on the coor- dinated action of both MT assembly in the distal axon shaft and actomyosin-based lamellar motility in the neu- ronal growth cone (Lin et al., 1994; Tanaka and Sabry, 1995; Letourneau, 1996).

In fact, dynamic interactions be- tween MTs and the actin cytoskeleton appear to be a pre- requisite for directional cell motility in general.


Neuronal growth cones (GC) have two distinct domains created by the largely discrete localization of F-actin and MTs.

The central domain (C-domain) has a high density of MTs and organelles and is separated from the F-actin-rich peripheral domain (P-domain) by an interface referred to as the transition (T) zone.

F-actin-based motility in the P-domain has been shown to result from constitutive plus- end-directed actin assembly into networks and filopodia at the leading edge and the concurrent action of myosin motors capable of translocating actin networks away from the leading edge centripetally towards the central do- main-a process referred to as "retrograde F-actin flow" (Forscher and Smith, 1988; Mitchison and Kirschner, 1988; Cramer et al., 1994; Tanaka and Sabry, 1995; Lin et al., 1996; Mitchison and Cramer, 1996).


Several lines of evi- dence suggest that F-actin networks and retrograde flow pose a barrier to MT advance and MT-dependent or- ganelle transport into the P-domain.

For example, clear- 1034 ance of F-actin networks subsequent to cytochalasin expo- sure results in rapid MT and organelle advance into the P-domain (Forscher and Smith, 1988; Waterman-Storer and Salmon, 1997); furthermore, recent evidence suggests that retrograde F-actin flow can translocate MTs from pe- ripheral F-actin domains and decreases MT plus-end as- sembly rates in newt lung cells (Waterman-Storer and Salmon, 1997).

Finally, MT advance and retrograde flow are inversely correlated during GC interactions either with native target substrates (Lin and Forscher, 1995) or Ig CAM-coated bead substrates (Letourneau et al., 1987; Lin et al., 1996; Suter et al., 1998).


Abundant and long-standing evidence implicates pro- tein kinase C as a regulator of cytoskeletal organization and neurite outgrowth.

For example, treatment with acti- vators of PKC such as phorbol dibutyrate (PDBu) or 12- O -tetradecanoylphorbol-13-acetate (TPA) result in neurite extension in embryonic ganglia explants (Hsu et al., 1989), neuroblastoma cells (Fagerstrom et al., 1996), and NGF- treated PC12 cells (Hundle et al., 1995), as well as changes in filopodial and lamellipodial protrusions in rat cerebral neurons and affect MT distribution in SH-SY5Y neuro- blastoma growth cones (Rosner and Fischer, 1996).


Con- versely, PKC inhibition can result in reduced rates of neu- rite outgrowth as well as dramatic changes in growth cone structure, sometimes referred to as "GC collapse" (Bixby, 1989).

Given that several well-known PKC substrates (e.g., GAP-43 and MARCKS) interact with and affect F-actin, previous studies have typically invoked PKC regu- lation of F-actin structure and motility to explain these ef- fects (Meiri et al., 1988; Hartwig et al., 1992; He et al., 1997; Dent and Meiri, 1998).


Microtubule-associated pro- teins (MAPs) such as MAP1B and MAP2 and Tau (Gor- don-Weeks, 1993; Liu et al., 1999) are also abundant PKC substrates in axons, and phosphorylation by PKC tends to inhibit their MT assembly-promoting activity (Mori et al., 1991); however, the mechanism by which MAP or tau phosphorylation promotes increased neurite outgrowth appears to involve complex interactions between multiple signaling pathways (Goold et al., 1999) and is not well un- derstood.


In this report, we describe a novel mechanism by which PKC regulates MT advance by controlling assembly of a highly dynamic pool of MTs normally residing in the T zone.

PKC activation promotes MT invasion deep into the P-domain, where MTs are rarely found under control con- ditions by a process that increases the lifetime of MT growth events.


Primary culture of Aplysia bag cell neurons and video-enhanced differen- tial interference microscopy (VE-DIC) was performed as previously de- scribed (Forscher et al., 1987).

Appropriate stock solutions of cheleryth- rine chloride, calphostin C, bisindolylmaleimide 1, Gö-6976, Ro32-0432, PDBu, 4- ␣ -phorbol, 1-oleoyl-2-acetyl- sn -glycerol (OAG), nocodazole, taxol, vinblastine (Calbiochem) and other drugs were prepared in DMSO and stored in Ϫ 20 Њ C. Fresh dilutions were made before each experiment in artificial sea water containing 2-3 mg/ml BSA as an amphipathic vehi- cle to aid drug solubilization in the high salt medium.

PDBu was used rather than phorbol-12-myristate-13-acetate (PMA or TPA) since it is bet- ter characterized in Aplysia (Sossin and Schwartz, 1994) and there was no observable difference between the actions of PDBu and PMA.

We mea- sured the absorption profile of the various PKC activators and inhibitors (data not shown) and have found that most of these PKC-related drugs absorb light in the green range.

When VE-DIC was performed with white or green light, phototoxic effects were greater in the presence of these drugs.

Therefore, VE-DIC was performed with 650-750-nm light except for calphostin C, since this compound is actually activated by 450-650-nm light (Gopalakrishna et al., 1992).


After each pharmacological experiment, cells were fixed and stained for F-actin and microtubules as described (Forscher and Smith, 1988), with the following modifications.

F-actin was labeled with alexa 568 or 594 phalloidin.

For MTs, alexa 488-labeled secondary antibody was used.

The alexa dyes (Molecular Probes) were brighter, more photostable, and gave better separation of emission spectra relative to FITC and rhodamine.


Retrograde flow of F-actin in lamellipodia was measured using Con- A-coated or polyethylenimine-coated 500-nm silica beads (Bangs Labo- ratories) in the presence of 3 mg/ml BSA.

Verification of beads as F-actin markers in this system, ionizing radiation-laser trapping and measure- ment of bead displacement were as previously described (Lin and For- scher, 1995).


MT density in the P-domain was measured by counting the number of mi- crotubules spanning 75% of the P-domain as defined by distal and proxi- mal borders of F-actin bundle labeling (see Fig. 3).

Data was normalized between growth cones by GC area and expressed as the number of MT/ 100 m 2 ; i.e., MT density.


Handling of Aplysia, dissection of abdominal ganglion, Western blots, and quantification of immunoblots were as previously described (Nakhost et al., 1998).


In Aplysia, the pleural-pedal ganglia are symmetrically paired on either side of the animal.

In all experiments, the ganglion on one side of the animal was used as a control for the corresponding one on the other side.


The ganglia were desheathed to facilitate penetration of pharmaco- logical agents and transferred to resting medium (the same as dissecting medium, but with 460 mM NaCl, 11 mM CaCl 2 , 55 mM MgCl 2 ; as de- scribed by Sossin and Schwartz, 1994).

The ganglia were incubated in ei- ther 1 M 4 ␣ -PDBu (control) or 1 M 4 ␤ -PDBu (experimental) for 1 h at 15 Њ C (physiological temperature of Aplysia).


The ganglia were then washed twice with microtubule stabilization buffer (0.1 M MES, 1 mM EGTA, 0.5 mM MgCl 2 ) containing 1 mM GTP and 10% glycerol (RGG buffer) and homogenized in 120 l of RGG buffer containing protease in- hibitors (20 g/ml aprotinin, 5 mM benzamidine, and 0.1 mM leupeptin) and 1% Triton X-100.


The samples were centrifuged at 15 Њ C for 1 min at 800 g to remove any unhomogenized debris.

The resulting homogenates (100 l) were centrifuged at 100,000 g for 30 min.

Supernatants were iso- lated and added to 20 l of sample buffer.


Pellets were either resuspended in sample buffer or resuspended in 120 l RGG plus protease inhibitors and divided into two 50- l fractions.

Subsequently, one fraction from each of the experimental and control ganglia was incubated with 100 nM no- codazole (in RGG plus protease inhibitors), while the other fraction was incubated with vehicle.


The samples were incubated at 15 Њ C for 15 min, and then centrifuged at 15 Њ C for 30 min at 94,000 g .

The supernatants were removed and added to 12 l of sample buffer.

The pellets were resus- pended in 72 l of sample buffer.


The samples were loaded onto 9% SDS- polyacrylamide gels and analyzed by immunoblotting with antitubulin an- tibody (clone DM 1A; Sigma-Aldrich).


Microtubule dynamics were assessed using the recently described fluores- cent speckle microscopy (FSM) technique (described in Waterman- Storer, 1998; Waterman-Storer and Salmon, 1998).

Neurons were injected with ف 1 mg/ml rhodamine-labeled tubulin (Cytoskeleton Inc.) in injection buffer (100 mM Pipes, 1 mM MgCl 2 , 1 mM EGTA) and allowed to re- cover for ف 60 min in L15-artificial sea water medium (Forscher et al., 1987) supplemented with 3 mg/ml BSA.

Under these conditions, MT 1035 speckles could be clearly discerned in distal axonal regions and used as fi- duciary marks for assessment of MT growth, shrinkage, pause, and trans- location rates and lifetimes.


MT image sequences were obtained using a Nikon Eclipse TE300 microscope equipped with a Photometrics Quantix 57 back-illuminated frame transfer cooled CCD camera and filter wheels (Ludl Inc.) for changing both excitation and emission wavelengths and rapid switching between fluorescence and DIC imaging modes.

Meta- morph control software (Universal Imaging Corp.) was used for instru- ment control and image analysis.

Images were recorded every 10 s using 300-500-ms integration times for fluorescent MTs and 50-ms for DIC.


Contrast of the speckles was enhanced by processing the fluorescent im- ages sequentially with the following spatial filters: unsharp mask, low pass, laplace edge enhancement, and a final low pass.

For DIC-fluorescent overlays, a threshold look-up table was applied to MT images before com- bining with the DIC image to clear low level background noise.


Representative examples of microtubule dynamics and the structural re- sponse of the growth cone to PKC activation are viewable as Quicktime movies.

In the single-channel microtubules dynamics examples, the open arrow follows the distal MT end and the closed arrow is an internal refer- ence mark.

The other examples are dual channel DIC-microtubule movies with MTs pseudocolored in green.

All movies are labeled according to the corresponding figure except the example of MT buckling.

Online supple- mental materials can be found at http://www.jcb.org/cgi/ content/full/152/ 5/1033/DC1.


Fig. 1 illustrates the typical C-, P-, and T-domain struc- tures of a growth cone under control conditions (see For- scher and Smith, 1988), note the relatively low density of MTs penetrating into the P-domain.

Fig. 2 shows the ef- fects of PKC activation on growth cone structure and the underlying MT and actin cytoskeleton.


PKC activation with 1 M PDBu (Sossin and Schwartz, 1994) resulted in relatively minor acute ( ف 5 min) morphological effects at the level of VE-DIC imaging, with the exception of rapid attenuation of T zone ruffling (not shown).

After 30 min in PDBu (Fig. 2 B), C-domain organelles appeared to have advanced slightly (A and B, dotted line) and attenuation of ruffling persisted.


Visualization of F-actin (Fig. 2 C) af- ter 30 min in 1 M PDBu revealed an intact P-domain F-actin structure, including well-defined F-actin bundles; however, F-actin hotspots corresponding to ruffling in the T zone were notably absent (Fig. 2 C).

Surprisingly, immu- nofluorescent visualization of ␤ -tubulin revealed that a striking advance of MTs into the F-actin-rich P-domain had occurred during PDBu treatment (Fig. 2 D).


This ro- bust advance or "invasion" of the MTs into the P-domain was unexpected, occurred as early as 5 min after 1 M PDBu application, and with doses of PDBu as low as 10 nM for 30 min (data not shown).

Invading microtubules were mostly unbundled, straight, and, in many cases, ran along or near F-actin cables (Fig. 2, C and D, arrowheads; see also Fig. 4, C and D).


Treatment with 1 M 4- ␣ -phor- bol (Fig. 2, I-L), an inactive phorbol-ester analogue, had no observable effects on growth cone motility or structure (Fig. 2, E-H).


To determine whether these effects were due to PKC activation, we preincubated cells with 10 M bisindolyl- maleimide 1 (Bis 1), a specific PKC inhibitor that interacts with the ATP binding site, for 15 min, and then added 1 M PDBu and 10 M Bis 1 for an additional 30 min.

Es- sentially all the morphological (Fig. 2, I and J) and cyto- skeletal (K and L) changes observed during PDBu treat- ment were absent in the presence of Bis 1; note that only a few MTs invade the P-domain (Fig. 2 L) and actin-based ruffling in the T zone is still clearly present (J and K).

In 1036 addition, treatment with 10 M Bis 1 alone for 45 min had little or no effect on growth-cone motility or cytoskeletal structure (Fig. 2, M-P).

In summary, Bis 1 did not affect basal GC motility, morphology, or cytoskeletal structure, but completely inhibited PDBu-mediated changes in these attributes.


Microtubule invasion levels (shown in Fig. 3) were quantified by determining the density of MTs spanning 75% of the P-domain, the latter defined by F-actin label- ing (see Materials and Methods; Fig. 3, inset).

Under con- trol conditions, there were 0.9 Ϯ 0.2 MT/100 m 2 .

After 30 min of 1 M PDBu treatment, MT density increased to 6.7 Ϯ 1.2 MT/100 m 2 .

In addition, chronic activation of PKC with lower doses of PDBu (e.g., 40 nM for 16 h) had simi- lar effects on MT distribution (data not shown).


In con- trast, after inhibition of PKC with 10 M Bis 1 for 45 min, there were 0.7 Ϯ 0.1 MT/100 m 2 (Fig. 3); i.e., similar to control levels.

Importantly, PDBu did not increase MT density above control levels in the presence of 10 M Bis 1.

Treatment with 1 M 4- ␣ -phorbol for 30 min also had no effect on the number of MTs invading the growth cone.

Treatment with 10 M OAG resulted in a less robust but statistically significant advance of MTs (3.1 Ϯ 0.8 MT/100 m 2 crossed the 75% threshold), as expected from its lower potency relative to PDBu.


In summary, activation of PKC was found to significantly increase the density of MTs found in the distal P-domain.

This effect was not present with inactive phorbolester analogues and was completely prevented by specific PKC inhibitors such as Bis1 (Figs. 2 and 3) as well as 10 M Gö-6976 or 10 M Ro32-0432 (data not shown).


There is evidence that P-domain F-actin and the process of retrograde flow itself inhibits MT advance and plus-end assembly (Forscher and Smith, 1988; Waterman-Storer and Salmon, 1997).

Thus, a possible mechanism for PDBu- mediated MT advance was a change in retrograde F-actin flow.


Using a laser trap-assisted flow-coupled bead assay to quantitatively assess F-actin flow rates as previously de- scribed (Lin and Forscher, 1995), we found no evidence for a decrease in retrograde flow of F-actin in growth cones subsequent to PDBu treatment.


Fig. 4 (A and B) il- lustrates an example of a retrograde flow assay before and after treatment with 1 M PDBu for 30 min.

Note that flow actually increased slightly after 30 min in PDBu (6.2 Ϯ 1.9 m/min) compared with controls (4.9 Ϯ 1.5 m/min) in this experiment; however, this increase was not significant over all experiments done ( n ϭ 6; 7.9 Ϯ 7.0% increase in retrograde flow after PDBu).


The presence of intact F-actin structure as well as MT extension was verified by post- experiment fixation and MT immunofluorescence, note that MT advance often occurred along lines defined by F-actin cables (Fig. 4, C and D, arrowheads).

These results indicate that PKC-mediated MT extension is not due to at- tenuation of retrograde flow.


In addition, experiments to investigate changes in the gel exclusion properties of the P-domain using microinjected FITC-dextrans indicate that there is little change in the functional pore size of the P-domain after PDBu treatment, suggesting that simple al- teration of the physical properties of the cytomatrix are not likely to account for the observed effects (our unpub- lished observations, see Discussion).


To address whether MT assembly was involved, PDBu ef- fects were assessed under conditions where MT dynamics were damped with low concentrations of nocodazole or taxol.

We specifically chose conditions previously shown to inhibit plus-end MT assembly without significantly re- ducing polymer mass (Bamburg et al., 1986; Tanaka et al., 1995; Rochlin et al., 1996) or, alternatively, to stabilize MTs (Letourneau et al., 1987; Jordan and Wilson, 1998b).


Fig. 5, A-D, illustrates the effects of exposure to 100 nM nocodazole on growth-cone structure and the cytoskele- ton.

Similar effects were observed after exposure to 1 M taxol (Fig. 5, E-H).


Note retraction of the C-domain (Fig. 5, A and B, E and F, dotted lines) and depletion of MTs from the T zone (D and H) observed after a 30-min expo- sure to either nocodazole or taxol.

Interestingly, despite essentially complete separation of C and P cytoplasmic do- mains and the underlying microtubule retraction, retro- grade F-actin flow continued unabated (data not shown) and P-domain F-actin remained relatively unperturbed in both cases.


These results suggest that dynamic MTs are not necessary for maintenance of retrograde F-actin flow, but are a prerequisite for MT advance out of the C-domain into more distal regions.

In agreement with these results, persistent growth-cone motility and absence of P-domain MTs were reported after treatment of Xenopus neurons with nanomolar levels of vinblastine (Tanaka et al., 1995).

Note also the relatively high level of diffuse tubulin labeling in nocodazole (Fig. 5 D) when compared with taxol-treated growth cones (Fig. 5 H), where diffuse tubulin labeling is low, as expected from the decrease in critical tubulin con- centration.


We then tested whether PDBu-mediated invasion re- quires dynamic MTs by treating growth cones with taxol or nocodazole at or below the levels illustrated above.

In the absence of PDBu, all of the MT drugs examined (including vinblastine and colchicine; data not shown) decreased the density of MTs in the T zones and P-domains (as in Fig. 5, A-H) in a dose-dependent manner.

The effects of nocoda- zole were examined quantitatively since this drug was found to be highly potent and its mode of action is re- ported to be somewhat less complex and more readily re- versible than the others (Jordan and Wilson, 1998b).


Cells were treated with 0, 10, 25, or 100 nM nocodazole Ϯ PDBu and P-domain MT density was assessed (Fig. 5 I).

In the absence of PDBu, distal axonal MT's (i.e., in the T zone and P-domain) were highly sensitive to nocodazole, as pre- viously reported (Bamburg et al., 1986), with inhibitory ef- fects at doses Ͼ 10 nM. In the presence of 1 M PDBu, clear and highly significant ( P Ͻ 0.001) inhibitory effects on MT extension were observed at nocodazole doses as low as 10 nM and inhibition of the PDBu response was es- sentially complete in 100 nM nocodazole.


The extreme no- codazole sensitivity observed after PDBu treatment sug- gests that MTs assembled subsequent to PKC activation may be highly dynamic (Jordan et al., 1992; Rochlin et al., 1996; Challacombe et al., 1997; Jordan and Wilson, 1998a).

Low doses of vinblastine or taxol (Fig. 5 I) also inhibited PDBu-mediated MT advance.

In summary, these results indicate that MT assembly is a necessary component of PDBu-mediated MT advance.


To assess PDBu effects on microtubule assembly using an independent methodology, biochemical experiments were performed on Aplysia central nervous system tissue.

De- tecting changes in soluble tubulin biochemically in the face of high baseline levels was not possible due to signal-to- noise problems.

However, given the apparent high sensitiv- ity of newly assembled MTs to nocodazole, we tested whether PDBu would increase the nocodazole-sensitive MT pool as an alternative approach.

Experiments were per- formed on paired nervous systems from the same animal treated with either 1 M PDBu or 1 M of the inactive iso- mer 4-␣ phorbol.

Ganglia were lysed in microtubule stabili- zation buffer, and free tubulin and microtubules were sepa- rated by sedimentation.


We found a small but significant increase in the percentage of tubulin found in the pellet af- ter PDBu treatment (Fig. 6, A-C).

To determine whether this increase was due to assembly of nocodazole-sensitive microtubules, we assessed the amount of tubulin released from pellets by 100 nM nocodazole.

Nocodazole induced a significant increase in the amount of tubulin released from pellets prepared from PDBu-treated ganglia (Fig. 6, D-F); furthermore, the amount of tubulin released by nocodazole approximately matched the increase in tubulin sedimented after PDBu (Fig. 6 C).

Paradoxically, treatment with low doses of nocodazole alone resulted in a small decrease in the amount of tubulin released from the Triton-insoluble pellet (Fig. 6, D and E).

Similar effects have been reported in Xenopus extract preparations (Canman and Bement, 1997); however, this effect is not well understood and more- over was not observed in the presence of PDBu.


Overall, these biochemical results are consistent with PDBu increas- ing assembly of a dynamic (i.e., nocodazole-sensitive) pool of microtubules and support our in vivo findings that PDBu results in nocodazole-sensitive MT advance.


To directly address the mechanism of the PKC effects de- scribed above, we assessed microtubule dynamics before and after treatment with PDBu using FSM.

This relatively new microtubule marking technique (Waterman-Storer et al., 1998; Waterman-Storer and Salmon, 1998) permits rapid assessment of the critical dynamic parameters of MT behavior, including assembly, disassembly, and transloca- tion (sliding) rates as well as MT lifetimes in each state.


Fig. 7 shows DIC-MT overlays illustrating the distribution of MTs before (A) and after (B) PDBu exposure with re- spect to growth-cone structure.

Fig. 7 C is another growth cone in PDBu illustrating extended undbundled MTs typi- cally observed after PDBu treatment.

Note the spatial overlap between MTs and actin cables that extend into filopodia (Fig. 7 C, arrowheads), as observed in fixed cells (compare Fig. 4, C and D).


Representative MT behavior under control conditions is illustrated in the time-lapse im- age montages shown in Fig. 7, D and E. MTs underwent stochastic bursts of growth and shortening (green arrow- heads indicate the penultimate plus-end speckle) superim- posed on relatively short MT translocations in both direc- tions, as indicated by speckles maintained within the body of MTs over time (red arrowheads).

Under control condi- tions, microtubule growth, shrinkage, and sliding activity occurred within the transition zone with only occasional MT excursions into the P-domain.