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"Take a friend by the hand as the manner is": vulnerability and fear in the London plague (1665) ANNE DUNAN-PAGE Aix Marseille Univ, LERMA, Aix-en-Provence, France

In the winter of 1664, the astrologer John Gadbury was unwell but nevertheless hard at work in his study. Like many others throughout Europe, he was enthralled by the appearance of a comet that Samuel Pepys and Isaac Newton had also seen in mid-December. Gadbury compiled letters relating to the comet from his correspondents in England and New England; he also scoured newsbooks for relevant news from abroad. His friend James Blackwell, from New York, first reported a comet in mid-November, before letters from Edinburgh, York, Oxford and Bristol confirmed its appearance. Newsbooks told similar tales from foreign parts. When other sightings occurred between 24 December and 12 January, Gadbury concluded this was not the same comet but another, one which he was prevented from observing between 8-12 January when he was mysteriously seized by 'an unhappy lameness in [his] most useful Arm', later interpreted as symptoms of the plague. Gadbury therefore knew from his own experience, indeed from his own body, what those comets meant. They were a presage of disasters to come.

The two comets were all the more sinister for being "retrograde", that is to say they moved in the opposite direction of to the zodiacal signs, and they were accompanied by reports of strange apparitions such as a black coffin which appeared in the sky at Hamburg (Gadbury,De Cometis,48). Even to the careless observer, the red colour of the first comet evoked Mars, the planet of war, and therefore prefigured bloodshed, while the paleness of the second called to mind Saturn, bringing "Famine, the Plague [and] exile" (47). The Bodleian Library copy of De Cometis contains a note in an unknown hand that confirms how these dire predictions were fulfilled:

A Bloody war w th Holland and many desp [er]ate Sea fights in y e yeares 64 65 66 & A Greate Plague in London in the yeare 1665 whereof dyed neere 100000 London Burnt the 2.3.4 of septemb r 1666. (66) In Protestant England, during plague time, there were no penitential processions or appeals to those traditional protectors against pestilence, Saint Sebastian and Saint Roch.

Instead, sermons and prayers, with monthly fasts, provided a collective response while newspapers and almanacs (such as Gadbury's), pamphlets and medical publications helped the members of London's private households to participate, behind closed doors, in a general, penitential abasement . In this article, I view early-modern vulnerability through the lens of such religious writings, for they offer a nuanced picture of the spiritual, moral and emotional predicament of sufferers, just as they reveal the enduring ability of clerical pastoral to adapt to circumstance.

In England, perhaps no other monograph concerning the plague has had a greater influence than Paul Slack's 1985 study, The Impact of the Plague in Tudor and Stuart England.

Slack viewed the last major epidemic in the English capital as the first modern one in the sense that the influence of the Church was on the wane, heralding a new confidence in science and initiating the management of crises by municipal authorities and salaried physicians who now inspired as much trust as ministers and astrologers. Part of Paul Slack's argument -relayed in part by Andrew Wear and Ole Peter Grell, and by Ernest Gilman for the literary tradition -was quantitative. He found that there were fewer "religious" works in 1665 than in the time of any previous major outburst (Slack 244;Grell,.

Slack worked on a total of 46 plague works, out of which he identified only 17 as "religious" (Slack 1985, n. 97, 401). My figures are different. This article was prepared using 80 plague texts, either medical (32) or religious (48, out of which 8 are sermons or collections of sermons).

I have assumed that a plague text qualifies as "religious" if it is predominantly concerned with sin, death and divine punishment, and that it may belong to any genre including broadside ballad, sermon, pamphlet, treatise, meditation and so on. When the authors are known, I have privileged texts written either by ministers or by authors who spoke as representatives of a particular religious group, such as the Quakers. There is no evidence that such publications competed with, rather than complemented, astrological predictions such as Gadbury's, which were sent by the ejected minister of Rye, John Allin, to his Sussex friends (Allin 27 October 1666, 19 January 1667). This is therefore the first attempt to take into consideration the variety of denominational positions on the plague, from clergy of the Church of England to Nonconformists, and from Presbyterians to Quakers, and to assess the political and pastoral implications of their writings.

"It was in the year of our Lord 1665"

This is this opening of the plague narrative by Thomas Vincent, a Presbyterian minister ejected from St Mary Maudlin, in Milk Street, included in his publication of 1667, God's Terrible Voice in the City, dealing with the plague and the fire of London . It is the first publication to present itself as a "historical" narrative of both calamities by a first-hand witness who chronicles the advance of the plague month by month.

All plague epidemics had a terrible air of familiarity in early-modern European cities . Between 1603 and 1665, London was beset by pestilence for a total of twenty-eight years (Slack 1985, 147). It was therefore called the sickness, "comprehending all other sickness in itself" (R.M. 3). Each incidence was explained in old and well-rehearsed ways, without much variation. It was widely believed, for example, that the plague was caused by venomous air which came from the entrails of the earth and infected the body, beginning with the heart (Boghurst 18-19), possibly under the influence of the stars, or from the putrefaction of organic matter in open graves, slaughter houses, marshes and dunghills (Wear, Knowledge and Practice 298-305). In a short and rather unusual publication, A Brief Treatise of the Nature, Causes, Signes, Preservation from, and cure of the Pestilence, W. Kemp exalted the virtues of vinegar as a plague panacea; regardless of whether his readers believed the disease arose from "the Corruption of the Air" or "the Corruption of the Humours", from "Poyson", the consequences of "Ill Diet" or some "Contagion" (81-82), Kemp assured them that vinegar was a palliative they could trust.

Civic authorities printed orders and bills of mortality, the religious authorities printed prayers and fast sermons, the College of Physicians printed remedies. Towns were sealed off, theatres were closed and a curfew was imposed on taverns. Those who could left London early, went far and returned late, as if heeding the ancient proverb often quoted in either Latin or English, Cede mox, recede longe, redi tarde (Boghurst 60; Harvey 16; Kemp 36). In London, the sick were not routinely sent to pest houses but were rather quarantined in their lodgings with the healthy, guarded by watchmen and cared for by nurses whose alleged cruelty became legendary (Pelling; Thorpe "Strange Woman"). It was generally observed that the poor suffered most, and that some died suddenly with symptoms while others lingered for a few days covered with carbuncles, spots (tokens), blains and buboes that had to be lanced or cauterized if there were to be any chance of survival. Mithridate and theriac (London or Venice treacle) were considered good preventatives and remedies; it was also believed that intense sweating eased the condition and that the poison could be sucked out by live fowls applied to buboes or counteracted by carrying amulets with an opposing poison such as arsenic, mercury or powdered toad. Such practices subsisted, despite many authors warning that they were at best useless and at worst dangerous (Garancières 13, Allin 24 August 1665; Herring B3r-B4r).

Rooms were alternatively aired and fumigated with herbaceous or acidic substances, while fires and cannons discharged in streets or squares were believed to dispel the miasma. Roses and vinegar were widely employed (see Thorpe, chapter 6 for the list of ingredients most often quoted) and when "abroad" in the streets one was advised to carry fragrant substances close to the nose or keep cinnamon, cloves (Herring B2r) or a coin of angel gold in the mouth (Allin 22 September 1665).

Despite the fear of contagion, people continued to go to church (Killeen) having taken precautionary measures by avoiding an empty stomach, burning perfume or better still brimstone. This was recommended by the physician Thomas Wharton who was perhaps conscious of taking the preaching trope of "fire and brimstone" at face value (Wharton 3). Even with a providential worldview it was considered a great sin, indeed "a gross, ignorant, and foolhardy confidence and presumption", to deny that God had sent natural remedies, doctors and ministers to treat bodies and souls (Form of Common Prayer, sig I2v, K2r). Since so much activity had ceased, people were encouraged to "improve" their time by reading, writing, praying and meditating if they could, whether they were at liberty or in confined houses (Clarke 17; Willis 21; Vincent A3r). Symon Patrick admitted to "study[ing] very hard" and "read [ing] many good authors", as he had little company in his Covent Garden parish (Patrick 9: 445) and he made sure to ask his listeners "within doors" to examine themselves carefully (3: 648).

And yet each plague was different. The year 1665 brought the first epidemic after the execution of a king, the creation of a Republic and the Restoration of a monarch whose court was reputed to be a great promoter of licentious pastimes. The Nonconformists believed that the King, the magistrates and the Church of England clergy had offended God by persecuting them, while the Church of England clergy argued that the Nonconformists had offended God by rebelling against their rightful rulers (Dobson, p. 24, 33-37). London was struck with a heavy hand because England had enjoyed more blessings than any other nation "before", a potent but imprecise word that was generally understood to identify a time before the Civil Wars, a sort of political Eden, when a morally upright people had enjoyed material prosperity and political stability under the guidance of a benevolent God. Plague was a direct product of human ingratitude for such blessings (Salthouse 2; Vincent 40).

Defining vulnerability

In 1665, London faced the plague to end all plagues: a just punishment for the prodigious and unprecedented sins of a murderous and ungrateful generation. But that did not mean that all were equal before it, and there is much to be gained by examining a society through those that were deemed vulnerable. Paul Slack has identified several periods in the development of medical and charitable care in London, with institutions for the mad (Bethlehem), the aged and infirm (St Thomas's hospital), the orphaned (Christ's hospital), the sick (St Bartholomew's hospital) and the poor (the Bridewell) (Slack 1997). Although the abstract term 'vulnerability' was not in use (the OED has no example before 1808) contemporaries had a rich vocabulary to express a sharply pragmatic sense of the way some people were at an elevated risk of infection.

Different categories of people were defined as weak, unguarded, unprotected, defenseless, endangered or unprepared, the last being a word whose meaning was both physical and psychological. In addition, there were risk-factors deemed conducive to the disease:

precipitants that disposed such-and-such a person to catching the plague. Some physicians recognised that to acknowledge such predispositions was a way to strengthen a contagionist approach: plague was indeed highly contagious but not everybody had a "disposition" to catch it, and those who did reacted in different ways. The only thing common to all people was the impossibility of a guaranteed immunity (Hodges 203,208).

In a Church order, rehearsing the duties of ministers, the theologian John Owen distinguishes between straightforwardly "sick people" and those "whose inward or outward conditions do expose them unto more ordinary trials in their sickness" (Owen 174, my emphasis). Sickness was an "ordinary" ordeal, in the sensed that it was shared by all, but for some the combination of external and/or internal "conditions" made it worse. Vulnerability varied according to the concatenation of such unfortunate factors that each person displayed.

Poverty was recognised to be an external or "outward" factor, for it was evident that the infection had a social bias; although some treatises emphasised the levelling aspect of the disease which seemed to kill at random, it was evident that the poor inhabitants of unclean and overcrowded tenements paid the highest price as the plague spread from the east (the famously indigent quarter of St Giles in the Fields) to the west, and from the outer to the inner parishes.

The so-called "London" plague was in fact a suburban phenomenon, with parishes outside the walls, or just inside to the north and north-east, such as St Giles Cripplegate, St Botolph Aldgate and St Botolph Bishopsgate, being the most affected (Slack 1985, 151-64;specifically on St Giles Cripplegate, Riddle, Bell 147-149). Although most authors made the connection between plague and poverty, they generally advanced medical rather than what would now be called social or economic arguments:

Poor people, by reason of their great want, living sluttishly, and feeding nastily and unwholesomly on any food they can with least cost purchase, have corrupted bodies; and of all others, are therefore most subject to this Sickness. At this present, most of those houses which are infected, are the habitations of poverty, in some obscure close place in the Suburbs; as towards St. Giles's, &c." (Kephale 57) "Inward" factors were deemed to make some people more vulnerable than others, chief among them being their balance of temperaments and humours. The most vulnerable were the sanguine, believed to be hot and humid with enlarged pores that offered an ineffective barrier against the disease. A few, unsystematic attempts were made to assess different degrees of vulnerability in terms of age or gender, notably in the case of pregnant women, "Virgins [...] ripe for marriage" and "youths", a term that could designate children and teenagers (Kephale 56). The aged, whose bodies were drier and cooler, were supposed to resist better, which might explain why some authors recommended sending "Elderly men" of the parish, together with the Ministers, to visit the poor and the sick (Humphrey Brooke 2).

Fear

The common ground between such outward and inward factors was that nothing could be done about them, or at least not immediately. One could not change one's physiological constitution, and most people could not move overnight into spacious and well-ventilated houses. To be sure, projectors had long drawn attention to the epidemiological consequences of a sprawling and dense urban environment, and had proposed medical, social and architectural solutions to improve the health of London's poor (Wear, Knowledge and Practice 280-86; Wear, "Sick Poor"; Gibbons). During the crisis itself, however, most authors tended to focus on the shortterm benefits of the six "non-naturals" in the Galenist system. These were air, sleep and watching, work and rest, repletion and evacuation, meat and drink and "the passions of the mind", by which the authors understood strong and sudden emotions such as fear, grief and anger that influenced one's health. Fear and grief routinely appear in bills of mortality that chartered the advance of the disease (Slauter; Greenberg; Sullivan) and Theophilus Garancières observed that otherwise perfectly healthy people caught the plague through their sheer inability to moderate their emotions: "The passions of the soul also (to wit) anger, grief and fear, dispose the bodies very much to [the plague]" (Garancières 15; see also Boghurt 65, Humphrey Brookes 7-8). Most publications, however, focused on fear, "of all Passions [...] the most pestilently pernicious" (Kephale 76; on fear, see Naphy and Riddle). Up to a point, fear was dependent on temperament, for it was a "natural inclination" (Dobson 14). Melancholics were at a disadvantage here because of their exaggerated fear and grief, precisely the emotions that the terrifying circumstances of plague were liable to induce. Diseased imagination caused people to die of extreme fear when seeing a painted door which signalled an infected house, for instance, or even when hearing "a Sad story" (Humphrey Brooke 8; Kemp 23-24; Lamentatio Civitatis 9, Vincent 33-35).

Religious writers had a subtle typology of fear that allowed them to conclude fear itself could be an antidote to fear, or to put it in their terms: "godly fear" was a remedy for "slavish fear" (Voice to the City 12-13; Dobson 14). The plague had come because sinners were not sufficiently terrified by their own sinfulness. Once they became so, and could repent, then their guilt would vanish and, at best, they would become less vulnerable to the plague; at worst, they would at least die a peaceful death. Authors offered numerous treatises or sermons to help people prepare (Dobson,Tatnall,. Thomas Willis wrote down prayers whose length varied according to the state of the patient, whether still "sensible" or "grow [ing] weaker" or lying "at the very point of death" (Willis 39-44).

It is important to recognise that the long lists of sins found in many religious treatises were part of a strategy that also had a medical justification: with a clear conscience, and less fear, people had a better chance of survival. We should not think in terms of medical practitioners and magistrates trying to reassure people on one side, and a pitiless clergy trying to terrify them into death-bed conversions on the other. All were perfectly aware that fear made people more vulnerable to the plague, and all considered it was their duty to assuage it. Some religious publications presented themselves as "cordials" (Brooks, Doolittle), while others imitated medical recipes: "First, Fast and Pray ; then take a quart of the Repentance of Niniveh, and put in two handfulls of Faith in the Blood of Christ, with as much Hope and Charity as you can get, and put into a Vessel of clean Conscience, then boyle it on the Fire of Love (An Unparalel'd Antidote, n.pag; also, E.N., Londons Loud Cryes, p. 8, Quarles, Londons Disease). Some people even seem to have been "addicted" to fear and to court danger (Defoe, Plague 22). Defoe's narrator stood at the brink of an open grave; Pepys followed coffins through the streets (Gilman 215-243). To be heedless of one's own safety could be construed as a suicidal tendency and therefore as the worst form of abandonment: despair of God, the unforgivable "sin against the Holy Ghost". The more pragmatic idea that exposure to infection could reduce one's vulnerability, however, began to gain ground (Boghurst 30-31). It was rumoured that those whom we would call front-line workers -the doctors, nurses, searchers, grave diggers, as well as members of the medical and clerical professions -had progressively gained immunity through their daily interaction with the sick: "[They] handle the [patient's] Sores, drawing their Breath, as they must needs, being long time in the Room…when others, passing but by a House or near a person Infected, yea, hearing but a sad Story, shall immediately sicken, goe home and die" (8). This perceived immunity was sometimes also ascribed simply to a lack of fear (Kemp 19). Standing at the edge of a grave, if it was not lethal, fortified the imagination, and one's ability to withstand the disease: "If thou believest the Pestilence cannot escape thee, thou shalt assuredly have it; thy Faith and Fancy will be certainly answered: whereas another of a generous confidence, and cleer resolution (though in the very midst of it) shall be free, and triumph over fear" (Gadbury, London's Deliverance Predicted 27).

Abandonment and quarantine

Finally, fear bred inhumane conduct and prevented "all Counsel, Reason, Charity, Truth, etc." (Boghurst 53). Most commentators presented London as a city living in an unending Sunday, in the phrase of one commentator (Vincent 36), or as an empty city: "[Plague] is the disease that is the most emptying disease; it empties houses, and it empties towns, and empties cities" (Bridge 8). The king and the court had first left for Salisbury, then Oxford. The well-to-do had left, first the very rich with country estates, then, albeit with some delay, the affluent middle class who could afford to hire lodgings out of town for an indefinite period of time. There were even reports that the poor had left and were now roaming the countryside and dying, if not of the plague then of hunger and exposure in ditches and open fields, adding even more stench to the ambient putrefaction (Vincent 33). The departure of the wealthier members of the population necessarily had direct social consequences; servants were turned out of doors, potentially swelling the menacing ranks of the vagrant population; the poor rate could not be levied, and empty houses attracted looters, creating a sense of insecurity. The intellectual elite had also departed (Grell; Wallis; Killeen). Most physicians had followed their wealthy patients out of London and the College of Physicians had symbolically shut its gates. The Inns of Courts, in turn, closed down and finally, as we shall see, it was believed that most of the clergy had left their pulpits too (Bell, chapter 4). For some authors, like the anonymous author of Lamentatio Civitatis, or the chemical physician George Thomson, flying from London increased vulnerability; it destroyed the "band of Charity" (Thomson 2) and was a crime against civil society "as Common-wealth is a body, and one member methinks should nourish another" (Lamentatio Civitatis 30). Some must have felt more abandoned than others. In the seventeenth century England adopted quarantine measures, the "shutting up of houses" (Newman; Thorpe, chapter 7). For some, this amounted to excommunicating the sick by putting them outside the congregations of the living (Shutting Up Infected Houses 4). Some authors wrote passionately against the inhumanity and barbarity of quarantine, which, in the end, was judged counterproductive because people feared it so much they tried to hide their symptoms, to flee as soon as possible or to break out of doors (J.V., Golgotha; Boghurst 27, Shutting Up Infected Houses 5; Thomson 9; Chamberlen, "Essay on the plague"; Hunter 10). A shut-up house became a 'dismal likeness to Hell" (J.V., Golgotha 11).

What was feared, exactly, during a quarantine? Surely the almost unbearable grief of seeing loved ones die before one's eyes, while perhaps awaiting death oneself. In a sense, however, mourning and loss were experienced by all, whether shut up or not. Quarantine added at least two distinctive layers of fear. First of all there was want, if not starvation (Hunter 11).

When a family was quarantined their stream of income dried; it was an even more pressing matter than usual to determine where the next meal would come from. In theory, poor relief and private acts of charity ensured that nobody would die of hunger (Form of Common Prayers K3v, Doolittle 61); in practice, however, the departure of the rich meant that collections were harder to levy and the financial burden on the poorer parishes, also the worst afflicted, was very difficult to bear.

Second, households became dependent on strangers (J.V., Golgotha 11). Nurses, "dirty, ugly, and unwholsome Haggs", were routinely accused of being cruel and dishonest, and were even charged at times with murdering their patients and selling their possessions, despite the danger that this might spread the plague (The Shutting up Infected Houses 9). Their scapegoating might have had something to do with their own vulnerability. Some were isolated old women, poor enough to accept risking their lives, even though recent work by Lara Thorpe on the parish of St Margaret's Westminster has revealed that affluent women could occasionally beome nurses (Thorpe, "Strange woman"; Pelling,[195][196][197][198]. Not all of them were old hags, and some indeed were vulnerable to sexual abuse; in November 1666 a tailor referred to as simply "Brother Bailie", a Baptist belonging to the Devonshire Square congregation in London, was summoned before his Church to answer a charge of "lascivious carriage towards his nurse".

Having lost his mother, father, wife and children to the plague, and having been visited by it himself, Bailie had persuaded his carer to share his bed for two days; this was not done (he claimed) with lascivious intent but only so that he might keep warm during the cold season.

The nurse was interrogated and the conclusion reached that if no sexual intercourse had occurred it was not because Bailie had been restrained by the "force and power of grace", as he claimed, but rather because he was too poorly to perform the act. He was excommunicated

(Devonshire Square, fols [6]v-[7]v).
What people feared most, however, was not so much bad treatment at the hands of uncaring nurses but rather the experience of dying alone with them, without loved ones, or a close network of friends who might be too timorous to visit an infected relative (Bridge 8, Doolittle 51, Featley 14). Doolittle transforms sympathy at such a time into a chief Christian duty (Doolittle 60). One year after the plague, Thomas Clarke published Meditations in My Confinement, when my House was Visited with the Sickness, a short work in verse, dedicated to his "friends" and describing his experience of a confinement which ended in the death of his two children. He pays tribute to those who visited him refused to leave either he or his family unaided. Clarke distinguishes the neighbours who shouted "how d'yees at the dore", from "real visits" which he describes as "so influential, /Yielding, refreshings to me, most essential" (Clarke 14), a "plaister" to his sores. This was not the experience of most. The prospect of dying among strangers was a frightening one, even more so since funeral rites could not be performed.

Bodies were maltreated, dragged from windows, hurled into carts, interred in pits (Riddle) and it was strictly forbidden to follow funeral processions (Form of Order K3r). Appeals to end such quarantine was motivated by compassion and a desire to end the violence done to the sick and to the dead, but it will now be clear that they also had a physiological justification. By inducing fear, they increased the danger of infection.

The battle for London souls

Since there was no competing explanation of any real force, it was commonly supposed that plagues originated in "the wrath of God provoked and incensed by the sins of any Nation or people" (A Form of Common Prayer H3v-H4r). The poor and the fearful, as well as the ignorant and the ungodly, had to be reached, and quickly, because the end of the divine scourge depended on their collective ability to repent. Three years before the plague, however, Presbyterian and Congregational ministers were ejected from their pulpits throughout London and the country for refusing to "conform" to the Act of Uniformity, rendering them incapable of addressing the London population in an official capacity, at a time of intense medical crisis, to urge repentance (Mead 40, 53-57; Rosewell 9).

The plague soon became a political matter, with each party accusing the other of having offended God either by persecuting Nonconformists or by rebelling against their rulers.

However, not all Nonconformists adopted the same strategy. The Quakers were the most vocal.

A great number of pamphlets that adopted a terrifying manner, in the form of addresses to London, were not so much directed to sinners in general, although some definitely were, but rather to magistrates, ministers, and "great persons" who were persecuting the people of God: O City for the innocent blood which hath been shed in our meetings, in your prisons, and on your pavements, and also for those souls who have been most barbarously thrust out of their native land, into exile, their cries with their wives, & tender children, are entred into the ears of the Lord God, and he hath regard unto them, and for their sakes, yea even for their sakes, is he entred into judgement with thee." (R.C., A Lamentation; also Voice to the City; Coale; Salthouse; Whitehead; for comments see The political and religious authorities were doubly guilty: they had persecuted the Quakers and prevented the population from listening to their prophetical warnings, later be mocked by Defoe as the fancies of "Enthusiasts" (Defoe 22-3). Exposing the intolerance of the Restoration regime meant revealing the Quakers' own vulnerability. The death toll of the community increased as members were imprisoned or even deported, some of them dying on infected ships (Whitehead 5, 13). Indeed, John Allin testified that meetings were routinely dispersed that summer (Allin 19, 24, 29 August 1665).

More moderate Nonconformist ministers pleaded for a truce that would allow them to preach and publish. Only later did the Presbyterians accuse "the persecutors" of being responsible for the plague (Vincent 240); on the whole, their attacks in the summer and autumn 1665 were more veiled than those of the Quakers, but that did not prevent them from breaking the law. Richard Baxter recalls what happened in his autobiography, worth quoting at some length because it shows how the plague emboldened the Presbyterians to come back to preach "openly" in London, despite the Act of Uniformity: But one Great Benefit the Plague brought to the City, that is, it occasioned the Silenc'd Ministers more openly and labouriously to Preach the Gospel, to the exceeding comfort and profit of the People; insomuch that to this Day the freedom of preaching, which this occasioned, cannot, by the daily Guards of Soldiers, nor by the Imprisonments of Multitudes, be restrained [...] It was the Plague that brought them out of their secret narrow Meetings into publick. And when the Plague grew hot, most of the Conformable Ministers fled, and left their Flocks, in the time of their Extremity: whereupon divers Non-conformists pitying the dying and distressed People, that had none to call the impenitent to Repentance, nor to help Men to prepare for another World; nor to comfort them in their Terrors, when about Ten Thousand dyed in a Week, resolved that no obedience to the Laws of any mortal Men whosoever, could justifie them for neglecting of Men's Souls and Bodies in such extremities […] Therefore they resolved to stay with the People, and to go in to the forsaken Pulpits, though prohibited, and to preach to the poor People before they dyed; and also to visit the Sick, and get what relief they could for the Poor, especially those that were shut up. Those that set upon this work were Mr. Thomas Vincent, later Minister in Milk-street with some Strangers that came thither, since they were Silenced, as Mr. Chester, Mr. Janeway, Mr. Turner, Mr. Grimes, Mr. Franklin, and some others (Reliquiae 2: 306) 1 . This was confirmed on both sides. The Presbyterian Thomas Vincent explained how Nonconformists chose to disobey in such extraordinary circumstances.

"[P]erceiving the

Churches to be open, and Pulpits to be open, and finding Pamphlets flung about the streets, of Pulpits to be let, they judged that the Law of God and nature did now dispense with, yea command their preaching in publick places, though the Law of man (it is to be supposed in ordinary cases) did forbid them to do it" (Vincent 48). Moreover, there was "such a vast concourse of people in the Churches, where these Ministers are to be found, that they cannot many times come neer the Pulpit doors for the press, but are forced to climb over the pews to them" (Vincent 50). The Church of England bishop Gilbert Burnet later recalled in similar terms the taking over of the London pulpits:

Many churches being shut up, when the inhabitants were in a more than ordinary disposition to profit by good sermons, some of the Non-conformists upon that went into the empty pulpits, and preached, and, it was given out, with very good success: And in many other places they began to preach openly, but not without reflecting on the sins of the Court, and on the ill usage that they themselves had met with." (Burnet I. 259) So, it seems, that the plague had justified the means. Such testimonies inspired some twentiethcentury commentators to call the Nonconformists "heroes" of the plague time (Bell 149; see also [227][228] who endangered both their lives and liberties. However, one should perhaps remain cautious about the suggestion of a Nonconformist watershed. How do we know, for instance, that "most of the Conformable Ministers" had fled? Which were the many vacant Churches? There were more than a hundred parishes, inside and outside London walls, in Westminster, Middlesex and Surrey and the whereabouts of the clergy in the summer of 1665 have simply not been chartered. Leading Church of England figures did stay in London, including the Archbishop of Canterbury (Gilbert Sheldon), the bishop of London (Humphrey Henchman) and Symon Patrick, who returned in July from Alstrop Spa to be in the midst of his fashionable parish of Covent Garden (9: 442-447; Moote and Moote 80, 93) and write about those conforming ministers who died of the plague (9: 583, 587). The flight of the Dean of St Paul's, William Sancroft, to Tunbridge Wells, is documented, but his assistants did stay and took over. In June, Henchman and Sancroft had drafted the A Form of Common Prayer, which was distributed throughout the country: prayers were to be said in church every Wednesday and there would be a national fast and a collection on the first Wednesday of each month.

Baxter mentioned "divers" Nonconformist ministers but cited only five by name. Six more can be gleaned from Edmund Calamy's biography of ejected ministers: William Dyer, John Mortimer,William Carflake,Gabriel Sangar,Stephen Ford and Obadiah Grew (Palmer 1. 128,235,400,424;2.309,484;see Calamy Revised). To the best of our knowledge, therefore, only a dozen or so preachers (outside the Baptists) were said by their contemporaries to have "openly" preached in London. Still according to Baxter, other ministers, such as George Cross and Timothy Roberts died of the plague, but both were already outside London and only a certain "Grunman", a German, died in the city, and that was not because he ministered to the sick but because he "was so poor, that he was not able to remove his Family" (Reliquiae II.304). Some Nonconformists did not fare much better than their Church of England counterparts. Rather surprisingly, Baxter himself left his family at Acton and departed alone to Buckinghamshire. Thomas Doolittle and Owen Stockton left as well, although the later regretted it soon afterwards, when his son died in 7 September 1665 . Doolittle had even tried to persuade Thomas Vincent not to stay in London, on the grounds that his calling was rather for the education of young men, as Vincent was then working with Doolittle in a private school at Bunhill Fields (Burden). The Nonconformists were not seeking martyrdom; they tried to determine whether God was calling them to stay or to leave and where they would be most able to fulfill their vocation.

In this context, it is useful to remember that preaching was not the only means to touch the spiritually vulnerable. Recent work has drawn attention to Puritan pastoral and cautioned against an overwhelming emphasis on sermons (Cooper,Searle,Goldie and Spurr). During the plague, ministers could first visit the sick and console the living, as they had always done (Baxter, Gildas Sylvianus 88-94). Francis Herring, whose work was reprinted in 1665, followed authors such as François Valleriole and gave advice to those "bound by religion, consanguinity" or "office" on how to visit the sick chamber, which included avoiding the breath of the patient and washing one's hands and face with rose-water and vinegar before and after the visit, "especially if he have taken his friend by the hand as the manner is" (Herring B2r-v;

Valleriole in Dean 140-141). Visiting the sick was one of the tasks that Baxter mentions; Thomas Vincent did it constantly, "and without fear" (Palmer 1: 126).

But face-to-face contact was not even necessary. Edmund Calamy, for instance, mentions John Rythers who showed "true sympathy" for the victims of the plague and of the fire (Palmer 2: 587) while still preaching in Yorkshire. Thomas Doolittle explains how distance did not prevent him from imagining the actual scenes of horror:

And though in his Providence, God hath removed me and my Family from the hearing of your Passing-Bells, and the doleful Lamentations that the ears of the there present, are filled with: Yet methinks I see the living laboring day and night, to Bury their Dead, while the other half, as yet remain, partly lamenting the Death of them departed, and partly looking for their own. Methinks I see many oftentimes viewing of their own Bodies, to see when the Tokens will appear. Methinks I see one sick and dying in one Room, and another in another; and those that yet are well, tread every step they take upon the Brink and Border of another World (Doolittle 2).

Letters were a source of pastoral advice but paper was believed to carry the infection so epistles from London would be aired, fumigated and even washed. Symon Patrick asked his friend Elizabeth Gauden, with whom he corresponded while he he was in London and she in Essex, to put his letters near the fire (9: 572). John Allin, who at the time of the plague was residing in Southwark complained of his Sussex friends' absence of trust (Thorpe, Bell, chapter 11); he knew that some of his letters were discarded, not opened, perhaps disposed of altogether (Allin 24 August, 14, 20 September 1665 2 ). With such a distrust of paper that had been in direct contact with the hands of their writers and the surfaces on which they wrote, and which had been entrusted to the post, many printed volumes presented themselves as safer means of conveying "letters". Even Patrick, when discussing his activities with Gauden would say "I preach to those who are well and write to those who are ill" (9: 577), but immediately added, "I meane, print little papers for them". Writing and printing had become interchangeable. Different categories of readers were specifically targeted. Patrick wrote a "short exhortation to those who were shut up because of the plague", in August, and "a little treatise of comfort in this sad time", in September as well as a consolatory letter to his mother, following the death of his father (9: 444; 3: 639-51; 654-674). For the poorest, there were broadsides, crammed to the very edges with godly advice to repent and live a better life. One of these, Roger Dixon's Directory for the Poor, was printed for free; another, Thomas Cocke's Advice to the Poor, was distributed to suburban parishes by order of the Duke of Albermale (Moote and Moote 108). The honour was not extended to Nonconformist works, but that did not prevent Richard Baxter from penning his own broadside, Short Instructions for the Sick, much praised by other authors (Plague Checkt,. It was meant to be pasted onto walls: "When the grievous plague began at London, I printed a halfe sheete (to sticke on a wall) for the use of the ignorant and ungodly, who were sick or in danger of the sicknes" (Baxter, Reliquiae Baxterianae I.492). Here Baxter clearly reveals that he perceives vulnerability, a heightened risk of infection, in terms of aggravating spiritual factors.

2 As libraries and record offices were shut at the time of writing, I have relied on the transcription of Allin's letters in Lara Thorpe's PhD thesis, as well as Palmer's Continuation of Edmund Calamy's work (1797), supplemented by Calamy Revised.

Next to broadsides, polemical pamphlets, often in the form of addresses to the city of London or the Nation, provided lengthier, if still affordable, forms of exhortation. For the middle-class, thick quartos invited the reader to meditate on Biblical episodes, to reflect on his or her sins and to prepare for confession. Doolittle even produced series of plague texts during and after the epidemic. This does not mean that those longer texts could not make their way down the social ladder; Thomas Willis directed a treatise to the poor, but it had to be "Communicated to them by the Rich" (A Help for the Poor).

There was, of course, one major obstacle to the printing and distributing of such works.

Richard Baxter and Matthew Mead both complained of the difficulty the Nonconformists faced in trying to have their works printed, even when they were writing in a private capacity (Mead 59). No matter what he wrote Baxter was told, in so many words, that his publications would always seem politically dangerous and would not be licensed (Baxter, Reliquiae I. 496). This caused intense frustration; denying the Nonconformists access to both pulpit and print made no sense at all in the prevailing situation but the authorities did not believe pastoral texts to be innocuous or void of political implications. It had escaped nobody that the two major plagues of the century, in 1603 or 1625, coincided with a change of rulers and that it might be the same in 1665. In October, just after the peak of the epidemic, the Oxford Parliament adopted the socalled Five-Mile Act forbidding ministers to reside within five miles of a corporation where they had preached before the early 1660s. Designed to split the affective bonds between the ministers and their flocks, while depriving the former of their livelihood and forcing them to change profession, it was a drastic measure reflecting the political anxiety that the Nonconformists inspired. Indeed, Roger Morrice interpreted it directly as a retaliation against Nonconformist preaching during the plague (Goldie 17).

Conclusion

The emotional instability of the destitute, the fearful and the despairing was perceived as both a medical and spiritual matter; it was deemed to explain why such persons were among the first victims of the plague. By reading religious and medical literature side by side, and by recognising the concerns they shared, we may bring fresh nuances to plague studies, especially in relation to 1665 when the recently ousted Nonconformists and Quakers tried draw attention to their own vulnerability but failed to react in a coordinated way. In 1665, there seems to have been relatively little scapegoating of vulnerable religious or racial communities, from the Jews to foreigners, but the authorities did prey on Nonconformists, on Quakers in particular, both during and after the plague. Rare is a pandemic that does not take its social and cultural toll. In

Thomas Vincent, John Chester, James Janeway, John Turner and Robert Franklin.

October 1665, the passing of the Five-Mile act, arguably the most punishing act of the so-called Clarendon code, was indeed a poor reward for the Nonconformists' efforts that summer and autumn; in a sense, it was also a measure of their success.

In the end -and arguably the situation was the same with physicians -the traditional structures survived the plague and there is no real indication that, after the epidemic, there was a surge of sympathy for Nonconformists and a wave of conversions. Samuel Pepys's reaction was perhaps the representative. When Daniel Milles, rector of Pepys's parish church of Saint Olave's, Hart Street, returned to London months after everybody else, Pepys was annoyed by his cowardice and incompetence but he nonetheless went back to his pew, albeit with the occasional sarcastic remarks, noticing that Milles "made but a very poor and short excuse, and a bad sermon" (Pepys, 4 February 1665/6).


Ph.D Muriel Golzio
email: muriel.golzio@ipbs.fr

PhD Elisabeth Bellard

; Bostjan

Sandrine Pelofy

François Le Guerroué

PhD Gregor Sersa

PhD Justin Teissié

PhD Maja Cemazar
email: mcemazar@onko-i.si

Richard Heller Heller
email: rheller@odu.edu

Raphael C Lee Lee
email: lee@uchicago.edu

Gillian M Tozer
email: g.tozer@sheffield.ac.uk

Tozer G Tozer

Raphael V Davalos
email: davalos@vt.edu

Frank Reidy

Bostjan Markelc

Muriel Golzio

Intravital microscopy at the single vessel level brings new insights of vascular modification mechanisms induced by electropermeabilization

Keywords: electroporation, intravital microscopy, blood vessels, vascular permeability, drug delivery electroporation, intravital microscopy, blood vessels; vascular permeability, drug delivery

Electroporation/electropermeabilization, i.e. the result of the application of electric pulses to tissues, is a physical method for delivery of exogenous molecules into cells. It is effective particularly for compounds with limited transmembrane transport. In vivo, electropermeabilization facilitates the delivery of chemotherapeutic drugs into tumor cells that is the basic mechanism of the antitumor effectiveness of electrochemotherapy. This therapy has also blood flow modifying effects in tissues. The aim of our present study was to understand and explain the effects of electropermeabilization on the dynamics (vasomotricity, permeability and recovery) of subcutaneous blood vessels towards different size of molecules. These features were measured in C57Bl/6 mice via a dorsal skin fold window chamber, using fluorescently labeled dextrans of different sizes, intravital fluorescence microscopy imaging and specific image analysis. Application of electric pulses on the skin in vivo resulted in a rapid increase in vascular permeability that gradually recovered to basal levels at different times post-treatment, depending on dextran size. Simultaneously, the immediate constriction of the blood vessels occurred that was more pronounced for arterioles compared to venules. This vasoconstriction of arterioles results in a transient "vascular lock". The increased permeability of small vessels walls whatever the dextran size associated with delayed perfusion explains the improved delivery of the intravenous injected molecules (i.e. drugs, gene delivery) into the tissues induced by electropermeabilization in vivo.

Letter of Submission

Sir or Madam, Attached you will find a manuscript entitled "Intravital microscopy at the single vessel level brings new insights of vascular modification mechanisms induced by electropermeabilization" By Bellard E, Markelc B, Pelofy S, Le Guerroué F, Sersa G, Teissié J, Cemazar M and Golzio M, we wish you to consider for publication in Journal of Controlled Release. Electrochemotherapy (ECT), i.e. the combination of the injection of a chemotherapeutic drug with the application of an external electric field called Electropermeabilization (EP), is nowadays successfully used to treat cutaneous tumors in clinics. Indeed, EP induces reversible permeabilization of cell membranes and therefore facilitates the delivery of the drug into tumor cells. As for mechanism of the antitumor effectiveness of ECT, he supposes a reversible permeabilization of the blood vessel wall to deliver the drug from blood to tissues. Blood flow modifying effects on healthy tissues and tumors have been reported with EP. However, the direct effect of EP on blood vessels morphology and functionality remains a challenge to explain in vivo.

By direct visualization at the single-vessel level using intravital fluorescence digitized microscopy imaging, we analyzed the effects of electric pulses on the morphology (diameter) and dynamics (vasomotricity, permeability and recovery) of subcutaneous blood vessels. Application of electric pulses on the skin resulted in the immediate constriction of the blood vessels underneath that was more pronounced for arterioles when compared to venules. It was followed by a rapid increase in vascular permeability that gradually recovered to basal (control) levels at different times posttreatment, depending on dextran size. Furthermore, vasoconstriction of arterioles results in "vascular lock" that lasted for at least 6 min.

This increase in permeability of vessels associated with delayed perfusion explains the efficiency of ECT protocols used nowadays.

To the best of our knowledge, this study is the first report of direct observation of EP-induced increased permeability of vessels in vivo with validated electric pulses used for biomedical applications according to the size of dextrans. The knowledge of the vascular effects of EP can lead to a better planning of the treatments that are based on EP (electrochemotherapy, electrogene therapy and DNA vaccination) for increased efficacy of treatment or for distribution of the i.v. injected molecules (i.e. drugs, siRNA, plasmid DNA).

Introduction

Electroporation/ electropermeabilization (EP), i.e. application of electric pulses to cells or tissues, induces reversible permeabilization of cell membranes under suitable conditions, thus facilitating entry into the cells of non-permeant or poorly permeant molecules [START_REF] Mir | Mechanisms of electrochemotherapy[END_REF]. Biological responses and associated techniques have a broad spectrum of biomedical applications, such as gene and cytotoxic drug delivery [START_REF] Cemazar | Electrically-assisted nucleic acids delivery to tissues in vivo: where do we stand?[END_REF][START_REF] Heller | Phase I/II trial for the treatment of cutaneous and subcutaneous tumors using electrochemotherapy[END_REF][START_REF] Mir | Systemic antitumor effects of electrochemotherapy combined with histoincompatible cells secreting interleukin-2[END_REF]. EP of tissues in humans is feasible, efficient and tolerable and its most advanced routine clinical use is electrochemotherapy (ECT). The cytotoxicity of bleomycin and cisplatin is potentiated by increased drug delivery into tumor cells by EP that results in up to 80% of complete responses in different tumor types [START_REF] Sersa | Electrochemotherapy in treatment of tumours[END_REF]. Reversible permeabilization of cell membrane due to EP is the basic mechanism of the antitumor effectiveness of ECT [START_REF] Sersa | Electrochemotherapy in treatment of tumours[END_REF][START_REF] Belehradek | Electrochemotherapy, a new antitumor treatment. First clinical phase I-II trial[END_REF][START_REF] Gehl | Efficient palliation of haemorrhaging malignant melanoma skin metastases by electrochemotherapy[END_REF][START_REF] Heller | Treatment of cutaneous and subcutaneous tumors with electrochemotherapy using intralesional bleomycin[END_REF][START_REF] Kubota | Histological evaluation of the effects of electropermeabilization after administration of bleomycin on bladder cancer in the rat[END_REF][START_REF] Sersa | Electrochemotherapy with cisplatin: the systemic antitumour effectiveness of cisplatin can be potentiated locally by the application of electric pulses in the treatment of malignant melanoma skin metastases[END_REF]. Nowadays, EP is also increasingly used for the delivery of different nucleic acids (plasmid DNA, siRNA, miRNA, shRNA) into cells in vitro and also into different tissues in vivo, including muscle, skin and tumors. This application is termed electrogenetherapy [START_REF] Cemazar | Cancer electrogene therapy with interleukin-12[END_REF][START_REF] Escoffre | Gene transfer: how can the biological barriers be overcome?[END_REF][START_REF] Golzio | Observations of the mechanisms of electromediated DNA uptake--from vesicles to tissues[END_REF][START_REF] Matsuda | Controlled expression of transgenes introduced by in vivo electroporation[END_REF][START_REF] Pavlin | Electrogene therapy with interleukin-12 in canine mast cell tumors[END_REF][START_REF] Vidic | MicroRNAs targeting mutant K-ras by electrotransfer inhibit human colorectal adenocarcinoma cell growth in vitro and in vivo[END_REF] where clinical trials with electrotransfer of IL-12 in melanoma patients showed positive results [START_REF] Daud | Phase I trial of interleukin-12 plasmid electroporation in patients with metastatic melanoma[END_REF][START_REF] Heller | Electroporation gene therapy preclinical and clinical trials for melanoma[END_REF].

In addition to the improved delivery of exogenous molecules to tissues, it has been observed that EP has blood modifying effects in tumors and normal tissues [START_REF] Gehl | Vascular reactions to in vivo electroporation: characterization and consequences for drug and gene delivery[END_REF][START_REF] Ramirez | Electrochemotherapy on liver tumours in rabbits[END_REF][START_REF] Sersa | Contrast enhanced MRI assessment of tumor blood volume after application of electric pulses[END_REF].Shortlived effects of EP on perfusion have been observed in muscles [START_REF] Gehl | Vascular reactions to in vivo electroporation: characterization and consequences for drug and gene delivery[END_REF]. Investigation of the reversible effects of EP on the cytoskeleton of cultured primary endothelial cells in vitro has shown that EP results in a loss of contractility and in a disruption of the barrier function of the endothelium by interacting with the cytoskeletal organization and integrity of cell junctions [START_REF] Kanthou | The endothelial cytoskeleton as a target of electroporation-based therapies[END_REF].

Using the combination of high voltage (HV) and low-voltage (LV) pulses protocol for plasmid DNA electrotransfer to the skin, we previously showed that EP induced a transient constriction and increased permeability of blood vessels as well as a ""vascular lock". We demonstrated that these blood flow modifying effects of EP in skin contributed to the infiltration of immune cells in the exposed area [START_REF] Markelc | In Vivo Molecular Imaging and Histological Analysis of Changes Induced by Electric Pulses Used for Plasmid DNA Electrotransfer to the Skin: A Study in a Dorsal Window Chamber in Mice[END_REF]. When combined with local injection of plasmid DNA for vaccination, this could enable the initial and prolonged contact of immune cells with encoded therapeutic proteins [START_REF] Thanaketpaisarn | Tissue-specific characteristics of in vivo electric gene: transfer by tissue and intravenous injection of plasmid DNA[END_REF].

However, for the validated protocol used in biomedical applications (8 pulses, 100 µs duration, 1300V/cm, 1 Hz), the effect of EP on drug delivery to the target tissue via blood vessels remains poorly addressed. Only one study showed that the expression of genes can be achieved after an intravenous injection of plasmid DNA and subsequent application of electric pulses to different tissues including skin in mice. Although the expression levels were lower in comparison to the levels achieved with direct injection of plasmid DNA into the tissue and subsequent application of electric pulses, they were still statistically relevant if compared to the intravenous injection only [START_REF] Thanaketpaisarn | Tissue-specific characteristics of in vivo electric gene: transfer by tissue and intravenous injection of plasmid DNA[END_REF]. However, how is the delivery of molecules modulated by the EP induced vascular lock and increased permeability of blood vessels for different sizes of molecules still remains to be explained.

In order to observe directly, at the single blood vessel level, the effects of electric pulses on the dynamics (vasomotricity, permeability and recovery) of subcutaneous blood vessels and the subsequent delivery of molecules with increasing sizes in the surrounding tissue, an in vivo analysis was performed on dorsal window chambers (DWC) of mice using intravital fluorescence digitized microscopy imaging [START_REF] Markelc | In Vivo Molecular Imaging and Histological Analysis of Changes Induced by Electric Pulses Used for Plasmid DNA Electrotransfer to the Skin: A Study in a Dorsal Window Chamber in Mice[END_REF][START_REF] Dreher | Tumor vascular permeability, accumulation, and penetration of macromolecular drug carriers[END_REF]. For this purpose, electric pulses were delivered directly to the skin. We used fluorescein isothiocyanate (FITC) labeled dextrans (FD) of different sizes to mimic different molecules and study the temporal and spatial changes in normal blood vessel morphology and dynamics after EP.

Materials and Methods

Animals

Adult female C57Bl/6 mice (Charles River Laboratories, France or Institute of Pathology, Faculty of Medicine, University of Ljubljana, Slovenia) weighing between 21-26 g were used as host for DWC preparations. All animal experiments were conducted in accordance with French procedural guidelines for animal handling and with approval from the Regional Ethical Review Committee (MP/02/36/10/10) or in accordance with the official guidelines and approval of the Ministry of Agriculture, Forestry and Food of the Republic of Slovenia (permission No.: 34401-12/2009/6).

Reagents

Fluorescein isothiocyanate (FITC) labeled dextrans (FD) (20, 70 and 2000 kDa) were purchased from Sigma Chemical Co. (St. Louis, MO). They were dissolved in phosphatebuffered saline (PBS) and washed through 10, 30 and 1000 kDa spin filters (Sartorius Stedim Biotech GmbH, Goettingen, Germany) at 3000 rpm to remove any free FITC or low molecular weight contaminants. The high molecular weight component was resuspended in PBS at 37.5mg/ml concentration and used in mice at 3.75 mg/100µl.

DWC surgery

Surgery was carried out under general anesthesia using intra peritoneal injection of Ketamine (1 mg/ml, MERIAL, Lyon, France or Narketan®, Vetoquinol AG, Ittigen, Switzerland), Xylazine (5 mg/ml, Bayer, Puteaux, France or Chanazine, Chanelle Pharmaceuticals manufacturing Ltd, Loughrea, Ireland) and Acepromazyne (0.4 mg/ml, Promace, Fort Dodge Animal Health, Iowa, USA). Briefly, animals were kept warm using heating pads and an aseptic technique was used throughout the surgical procedure. The DWC consisted of two symmetrical titanium frames (APJ Trading Co., Ventura, CA, USA). These frames sandwiched an extended double layer of dorsal skin with the use of stainless steel screws and sutures. After the implantation of the frames, one layer of the skin (12-mm diameter) was surgically removed to create an observation window. After addition of sterile PBS, a sterile glass coverslip was attached to the open frame with a stainless steel clip to cover the surgical site and provide visual access to the vascular network of the skin [START_REF] Palmer | In vivo optical molecular imaging and analysis in mice using dorsal window chamber models applied to hypoxia, vasculature and fluorescent reporters[END_REF]. After the surgery and on the following day, Profenid (Sanofi-Aventis, Paris, France) was injected, intramuscularly, to provide analgesia and to avoid inflammation (10mg/kg, 50 µl in each thigh).

Study design

EP was carried out three to 14 days following surgery, by application of eight square electric pulses, amplitude per distance 1300 V/cm, pulse length 100 µs, repetition frequency 1 Hz, generated by an electropulsator (Jouan GHT, St Herblain, France; or Cliniporator™, IGEA s.r.l., Carpi, Italy). Pulses were delivered by two parallel stainless steel rods (length 5 mm, width 1.3 mm) 4 mm apart, which were placed in contact with the intact skin on the opposite side of the cover glass. Good contact was assured by means of conductive gel (Ekogel, Egna, Italy). Mice in which no EP was applied served as a control group. FDs were injected in the retro-orbital plexus. FDs were injected either before EP to observe extravasation of FDs into the tissue, or at different times post-EP to observe the "resealing" of the vessel walls.

Intravital microscopy and image acquisition

Intravital microscopy was carried out on a customized stage for holding mice using an upright "Macrofluo" fluorescence macroscope (Leica Microsystems SA, Rueil-Malmaison, France), equipped with a Cool Snap HQ Camera (Roper Scientific, Photometrics, Tucson, AZ, USA), or a Zeiss SteREO Lumar.V12 (Zeiss, Jena, Germany) fluorescence stereomicroscope equipped with a MRc.5 digital camera (Zeiss). Animals were anaesthetized with inhalation anaesthesia (Isofluran, Nicholas Piramal India LTD, London, UK or Belamont, Neuilly sur Seine, France) throughout the experiment. Vessels were imaged by fluorescence using appropriate filters (excitation filter, BP: 480/40 nm, emission filter, BP 527/30 nm). After injection of 100 µl of FD, one stack of images was acquired before EP. Images were taken every 20 s for 2 min. The same procedure of image acquisition (every 20 s for 2 min) was performed immediately after EP. Immediately afterwards, a third stack of images was acquired in which images were taken every 2 min during 30 min. For experiments with 20 kDa FDs, due to its rapid clearance, the acquisition with 20s time interval after EP was performed during 12 min. When injection of FD was performed after EP, two stacks were acquired consecutively just after the injection one stack of 2 min (at a 20 s repetition rate) and another one of 30 min (at a 2 min repetition rate). The files were stored and analyzed off-line with image acquisition and analysis software (Metavue, Metamorph, Molecular devices, Sunnyvale, CA, USA or AxioVision, Zeiss).

Data analysis

Increase in fluorescence intensity inside and outside the blood vessels was determined as follows. The "flattened background" option of image analysis software (Metavue or AxioVision) allowed for the correction of the image of filled blood vessels (before extravasation) for a non-uniform background. After this correction, by applying a suitable threshold on the image, the image was binarized and a mask of the vessel network was obtained. The tissues outside the vessels were defined by an inversion of the binarized mask of the vessel network. From these masks, the area of blood vessels and tissues was determined. After combination of the masks with all the aligned images of the acquired stacks, we determined the variation in fluorescence intensity inside and outside the vessels at all-time points after EP. The percentage of variations was calculated to avoid the variability between mice in the initial value of tissue fluorescence intensity. The time dependence of the obtained values during the first 3 min post EP was calculated to determine the initial mean fluorescence intensity changes per minute. When injection of FDs was performed after EP, the initial mean fluorescence intensity changes per minute were determined from the 3 minutes following a normal time of filling up of blood vessels (Fig. 1). The diameter of venules and arterioles was measured with AxioVision (Zeiss) software or Image J software (National Institute of Mental Health, Bethesda, MD, USA). Vessels were categorized as arterioles if they were narrow, straight and with few branches, and the others were defined as venules [START_REF] Tozer | Mechanisms associated with tumor vascular shut-down induced by combretastatin A-4 phosphate: intravital microscopy and measurement of vascular permeability[END_REF]. To measure diameters, at least five venules (diameters between 20-250 µm) and arterioles (10-100 µm) were selected from each mouse. Diameters of vessels were determined before application of electric pulses and thereafter every 20 s for the first 2 min and every 2 min for the rest of the acquisition time (30 min).

Statistical analysis

Statistical analysis was carried out using Prism 5 Statistical software (GraphPad Software Inc., San Diego, CA). For comparison of control and EP groups, Student t-tests were used, and for multiple comparisons, a one-way ANOVA with Dunnett"s post-test. A value of p < 0.05 was considered to represent a significant difference between groups.

Results

Characterization of the DWC model: perfusion and clearance of FITC-labeled dextrans (FDs) in the blood vessels

In order to characterize if the perfusion of the blood vessels was independent of the molecular weight of the dextran, we first determined the kinetics of blood vessel filling after intravenous (i.v.) injections of FDs. Whatever the size of the dextran (20, 70 or 2000 kDa), the perfusion was completed within 120 s after the injection (Fig. 1A, Supplementary Movie S1). Therefore, in all further experiments, EP was applied at 2 min after the injection of FDs.

We also determined the clearance of FDs from the blood stream and their long-term presence in the surrounding tissue. The clearance kinetics of FDs in intact vessels were dependent on their size. The larger FDs stayed in the blood stream for longer. The half time of clearance for 20 kDa FD was 8. In order to study non-specific effects that could be induced by DWC surgery on vessel permeability, we measured the extravasation of 70 kDa FD from the blood vessels of a control mouse without EP, at different times after DWC implantation (Fig. 1C). The percentage of variation of mean fluorescence intensity in the tissues outside the vessels was very low and only slightly modified as a function of time during 15 days, indicating that implantation of DWC alone did not affect the permeability of vessels with time.

The EP-induced blood vessels permeability varies for different sizes of dextrans

In order to study the effects of EP on blood vessels permeability for different sizes of circulating molecules, we compared the extravasation of 20, 70 and 2000 kDa FDs from the blood vessels that were exposed to EP relative to control mice (no EP). The extravasation of 70 kDa FD induced by EP is illustrated in Figure 2 (Fig. 2, Supplementary Movie S2), demonstrating a rapid leakage from all exposed blood vessels.

Fig. 2.

The time course of extravasation was measured by the relative variation of mean fluorescence intensity in the tissue after EP (Fig. 3A). The relative variation of mean fluorescence intensity was corrected by the vascular area (area of blood vessels) in the observed tissues of mice exposed to EP (see Methods). For the 70 kDa FD, the increase was linearly dependent on time, as expected from the increase of vessel permeability present during the first minutes. For 2000 kDa FD, the same linear dependency was observed after the 3 min delay. However, the relative variation in mean fluorescence intensity was lower in comparison with 70 kDa FD (Fig. 3A). For 20 kDa FD, the increase in relative variation of mean fluorescence intensity reached a plateau value after 3 min (Fig. 3A).

For dextrans up to 70kDa, a high and statistically significant increase in relative variation of mean fluorescence intensity per minute was obtained immediately after EP in comparison to control mice (Fig. 3B). For the 20kDa FD, the fast clearance of dextran from the blood stream decreased the quantity of dextran available for leakage into the tissue, leading to a lower relative variation of mean fluorescence intensity per minute. In contrast, EP did not induce immediate leakage for 2000 kDa FD. Indeed, no modification of the relative variation of mean fluorescence intensity per minute was detected immediately after EP (Fig. 3B). However, after a delay (4 min), a significant increase in the variation of mean fluorescence intensity per minute, similar to the ones determined for smaller sizes of FDs, was quantified (10.9% +/-4.7 of variation per min).

Fig. 3.

EP-induced blood vessels permeability is reversible

In order to determine the duration of the EP induced permeability of blood vessels, we compared the extravasation of different FDs when injected at different times after EP (Fig. 4). The relative variation of mean fluorescence intensity per minute in the tissue quickly decreased when there was an increase in the delay interval between EP and 70 kDa FD injection. This suggested a quick partial restoration of blood vessel impermeability. However, the relative variation of mean fluorescence intensity per minute returned to the control level only one-hour post-EP (Fig. 4A). Similar data were observed with 2000 kDa FD (Fig. 4B).

We hypothesized that this delayed return to control level could be explained by a residual permeability to smaller molecules. Thus, we used intermediary times of injection (30s, 5, 10, 20 and 30 min) with 20 kDa FD, to measure more precisely the lifetime of blood vessels increased permeability. In these conditions, the relative variation of mean fluorescence intensity per minute in the tissue remained constant at shorter time intervals (30 s, 5 min and 10 min) between EP and injection of FD (Fig. 4C).

Fig. 4.

This result suggested that the quick restoration of blood vessel impermeability observed with 70 and 2000 kDa FDs was indeed partial and did not prevent the leakage of smaller molecules during the first 10 min after EP.

EP induces vascular lock in normal blood vessels

Without EP, the filling up of blood vessels was immediate (within 2 min) with no leakage from blood vessels whatever the size of FD used (Fig. 1). When FDs were injected 20 s after EP, the filling up of pulsed blood vessels was slowed down whatever the size of the dextran. (Fig. 5, Supplementary Movie S3).

Duration of EP-induced vascular lock

In order to determine the duration of the vascular lock, FDs (20 and 2000 kDa) were injected at different times after EP. The percentage of mean fluorescence intensity inside blood vessels was plotted as a function of time (Fig. 6A andD). The shape of the curves clearly depended on the delay between EP and FD injection. To quantify this duration, we calculated their time derivatives. When the amplitude of the derivative was maximal, the vessel filling up speed was maximal. The maximal value of the derivative was determined for each condition as the maximal variation of intensity per second (Fig. 6C andF). The delay between the time of injection (time 0) and time of the maximal derivative reflected the occurrence of blood vessels filling up. This delay was determined for each condition (Fig. 6B andE). For 20 kDa FD, the time needed to restore blood flow was at least 5 min (Fig. 6B). Ten minutes are needed to reach the normal rate of the blood flow (Fig 6C). In others words, these data showed that 5 min after EP, even if the blood flow was restored, its rate did not return to normal values until 10 min after EP. For 2000 kDa FD, the restoration of the blood flow was observed at least 15 min after EP. (Fig. 6E). The rates of blood flow were slightly decreased in comparison to normal value but not significantly (Fig 6F ). Therefore, the EP-induced vascular lock was detected over less than 10 min for 20 kDa FD and less than 15 min for 2000 kDa FD after EP.

Blood vessels constriction during EP-induced vascular lock

In order to characterize the EP-induced vascular lock, we measured the variation in the diameters of venules and arterioles during the first 30 min after EP. With 70 kDa FD, the increased permeability of blood vessels was associated with a constriction of both arterioles and venules. A small, but significant, constriction of venules (approx. 20%) was observed immediately after EP (Fig. 7B). The diameter of venules was totally restored 3 min after EP. Constriction of arterioles was more pronounced (approx. 65%) and mainly restored within 3 min after EP. However, the total restoration of their diameter was obtained after 8 min post-EP (Fig. 7A). The diameter of venules and arterioles in control mice were not modified during the same observation time. These results were not dependent on the FDs size (data not shown, Supplementary Movie S4). Fig. 7.

Discussion

This study presents the results of in vivo direct observations of the early events in blood vessels after EP in a DWC model in mice. Delivery of validated EP parameters used in clinical applications to normal tissue (skin) led to a rapid increase in the permeability of blood vessels for different sizes of molecules that gradually returned to basal (control) levels within 1 h post-treatment. Moreover, EP induced an immediate constriction of blood vessels that was transient and returned to control levels within 8 min.

First, we characterized the DWC model. We showed that the filling up of the blood vessels (perfusion) was completed within 2 min after injection of FDs, in the absence of EP, whatever the size of the FD. FD injection did not modify the blood flow. A longer plasma elimination half-life and total blood clearance were detected for larger FDs (Fig 1). This has already been observed in studies on children. Indeed, 2-3 hours after injection, the main part of the dextran molecules still circulating in the blood flow had a molecular weight of 30-50 kDa or more. Smaller molecules had already by this time been excreted into the urine [START_REF] Arturson | The Renal Clearance of Dextran of Different Molecular Sizes in Normal Humans[END_REF].

Our experimental setting using the DWC model in mice, allowed direct observation of blood vessels in vivo. As such, we could measure in situ the changes in blood vessels permeability after EP, in real time. Molecules (up to 70 kDa) were used as model molecules for drugs and oligonucleotides while 2000 kDa FD has a molecular size comparable to plasmid DNA [START_REF] Golzio | Direct visualization at the single-cell level of electrically mediated gene delivery[END_REF][START_REF] Paganin-Gioanni | Direct visualization at the single-cell level of siRNA electrotransfer into cancer cells[END_REF]. We showed an occurrence of vascular permeability immediately after EP for 20 and 70 KDa FDs (Fig. 2 and3). For 2000 kDa FD, this occurrence of permeability was delayed in time (4 min). Therefore, permeability was observed for all three sizes of FDs (20 kDa, 70 kDa and 2000 kDa) that in normal circumstances do not diffuse readily through vessel walls [START_REF] Takakura | Macromolecular carrier systems for targeted drug delivery: pharmacokinetic considerations on biodistribution[END_REF]. Vascular impermeability was partially restored within 10 min for all sizes of FDs, while a low level of leakage was still present after this time. The impermeability of vessels totally returned to basal values within 1h after EP (Fig. 4).

A direct consequence of these findings is that, once injected intravenously, plasmid DNA could mostly be delivered to the tissue between 3 and 10 min after EP. Now, plasmid DNA must be present during the application of electric pulses in order to be efficiently transferred into the cells because of the involvement of electrophoretic forces [START_REF] Escoffre | Gene transfer: how can the biological barriers be overcome?[END_REF][START_REF] Golzio | Direct visualization at the single-cell level of electrically mediated gene delivery[END_REF][START_REF] Cemazar | Sequence and time dependence of transfection efficiency of electrically-assisted gene delivery to tumors in mice[END_REF]. Therefore, skin transfection after intravenous plasmid DNA injection could be impeded because of this delayed vascular permeability that we observed with 2000 kDa FD. This delay explains the very low level of transfection in the skin after intravenous plasmid DNA injection reported elsewhere [START_REF] Thanaketpaisarn | Tissue-specific characteristics of in vivo electric gene: transfer by tissue and intravenous injection of plasmid DNA[END_REF].

The permeability of blood vessels for 70 kDa FD was slightly higher than for 2000 kDa FD whereas, for the 20 kDa FD, the results were slightly lower (Fig. 3). This was due to its quick clearance out of the blood stream (8.5 min) (Fig. 1B). Molecules in size up to 70 kDa could diffuse easier into the tissue after EP than much larger ones such as 2000 kDa FD. The increased vascular permeability is due to the structural changes in the blood vessels wall that are caused by a direct effect of EP on the endothelial lineage present in arterioles and venules [START_REF] Markelc | In Vivo Molecular Imaging and Histological Analysis of Changes Induced by Electric Pulses Used for Plasmid DNA Electrotransfer to the Skin: A Study in a Dorsal Window Chamber in Mice[END_REF]. Previously published results on endothelial cells in vitro showed reversible structural changes of the cytoskeleton and cell junctions concomitant with a rapid rise in endothelial monolayer permeability [START_REF] Kanthou | The endothelial cytoskeleton as a target of electroporation-based therapies[END_REF]. Nevertheless, the full restoration of blood vessel permeability, observed in our study, confirmed that no endothelial cells were irreversibly damaged by the validated EP parameters in vivo. The effects of irreversible electroporation (IRE) on blood vessels by applying stronger EP parameters than in our study (10 pulses of 100µs, 3800V/cm, 10 Hz) showed that in large vessels only the vascular smooth muscle cells were affected at day 28 post-treatment [START_REF] Maor | The effect of irreversible electroporation on blood vessels[END_REF]. However in our study we provide evidence that the application of validated EP parameters used in clinical applications does not harm small vessels (arterioles, venules and capillaries). Therefore, we provide additional proof of the safety of EP, when applied to normal tissues.

The immediate and transient constriction of blood vessels that we observed was in agreement with global observations of reduced perfusion measured by Gehl et al. using Patent Blue staining in vivo in normal muscle tissue after EP [START_REF] Gehl | Vascular reactions to in vivo electroporation: characterization and consequences for drug and gene delivery[END_REF]. A first phase of vascular response to EP was attributed to sympathetically-mediated vasoconstriction acting on smooth muscles of afferent arterioles, leading to a rapid and profound but short-lived initial decrease in blood flow. Arterioles have a thick vessel wall with a Tunica Media composed of several layers of circular smooth muscles, whereas veins and venules have a thinner layer of circular smooth muscles. Following the hypothesis of sympathetically-mediated vasoconstriction, these different morphologies should make arterioles more sensitive to EP than venules. Our measurements of blood vessel diameters provide direct evidence of the different levels of vasoconstriction of venules and arterioles Namely, the constriction of venules was minor (~20 %), a level significant only immediately after EP (1 min), and was restored to its basal level within 3 min. The constriction of arterioles was more profound (~ 65 %) and long lasting (8 min) (Fig. 7). These vasoconstrictions resulted in the "vascular lock" (Fig. 5). In the proposed model of vasoconstriction in mouse muscle, a second phase was attributed to the permeabilization of muscle fibers and endothelial cells membranes leading to increased interstitial pressure and decreased intravascular pressure, which could explain delay of perfusion in muscles [START_REF] Gehl | Vascular reactions to in vivo electroporation: characterization and consequences for drug and gene delivery[END_REF]. Additionally, we determined that the filling up of pulsed blood vessels was slightly different for various sizes of FDs (Fig 6). Furthermore, our results also demonstrate that the delivery of large molecules (2000 kDa) to the tissues was delayed (Fig 3A ,5). Indeed, when 2000 kDa FD was already present in the blood stream, the extravasation induced by EP was delayed compared to smaller FDs and began ~ 4min after EP.

To the best of our knowledge, this study is the first report of direct observation of EPinduced increased permeability of vessels in vivo with validated electric pulses used for biomedical applications according to the size of dextrans. We were able to gain an insight into the physiological processes that were altered by EP of the tissues, that had before only been the subject of speculation [START_REF] Gehl | Vascular reactions to in vivo electroporation: characterization and consequences for drug and gene delivery[END_REF][START_REF] Jarm | Antivascular effects of electrochemotherapy: implications in treatment of bleeding metastases[END_REF][START_REF] Sersa | Tumour blood flow changes induced by application of electric pulses[END_REF].

Conclusions

The increased leakage of different sizes of FDs indicated that EP-induced permeability of the vasculature could be effective for a broad range of molecules such as oligonucleotides and plasmid DNA. For small molecules (20 kDa FD) because of their rapid elimination EP has to be performed rapidly after the injection. If performed after EP, the injection has to be undertaken within 10 min. For dextrans above 70 kDa, their slow clearance and the linear increase in extravasation after EP suggest that, after injection of large molecules, EP can be applied after a longer time interval, resulting in the same amount of delivered molecules into the target tissue. Finally, the delayed permeability observed with 2000 kDa FD suggests that very large molecules such as plasmid DNA are more sensitive to vascular lock. Indeed, arteriole constriction delays 2000 kDa molecules extravasation, whereas leakage of smaller molecules is not impeded. Thus, knowledge of the vascular effects of EP can lead to a better planning of the treatments that are based on EP (electrochemotherapy, electrogene therapy and DNA vaccination) for increased efficacy of treatment or for distribution of the i.v. injected molecules (i.e. drugs, siRNA, plasmid DNA) [START_REF] Thanaketpaisarn | Tissue-specific characteristics of in vivo electric gene: transfer by tissue and intravenous injection of plasmid DNA[END_REF][START_REF] Edhemovic | Electrochemotherapy: a new technological approach in treatment of metastases in the liver[END_REF][START_REF] Ferraro | Clinical applications of DNA vaccines: current progress[END_REF][START_REF] Miklavcic | Towards treatment planning and treatment of deep-seated solid tumors by electrochemotherapy[END_REF][START_REF] Rosati | DNA vaccination in rhesus macaques induces potent immune responses and decreases acute and chronic viremia after SIVmac251 challenge[END_REF].

  5 min and for 70 and 2000 kDa FDs greater than 4 h (5h 30 min and 4 h, respectively) (Fig 1B).

Fig. 1 .

 1 Fig.1.

Fig. 5 .

 5 Fig. 5.

Fig. 6 .

 6 Fig. 6.

Figures legendsFig. 1 .

 1 Figures legends

Fig. 2 .

 2 Fig. 2. Direct imaging of fluorescence leakage from the DWC vessels into tissues after EP. 70 kDa FD was injected i.v. and blood vessels were visualized by fluorescence macroscopy at x25 magnification at the indicated times. Upper panel, no EP applied. Lower panel, eight square wave electric pulses, amplitude per distance 1300 V/cm, pulse length 100 µs at repetition frequency 1 Hz were applied.

Fig. 3 .

 3 Fig. 3. Quantification of FD leakage kinetics after EP. (A): Relative mean fluorescence intensity changes for 20, 70 and 2000 kDa FDs in DWC tissues outside the vessels as a function of time after EP (eight square wave electric pulses, amplitude per distance 1300 V/cm, pulse length 100 µs, repetition frequency 1 Hz, (n=6)), in comparison with the control group without EP (n=5). (B): Initial relative mean fluorescence intensity changes per minute, determined from the first 3 min following EP for 20, 70 and 2000 kDa FDs (EP group (n=5), control group (n=4)). *P<0.05, **P<0.01 relative to pertinent control. Error bars indicate SEM.

Fig. 4 .

 4 Fig. 4. Duration of the EP-induced blood vessel permeability. Relative mean fluorescence intensity changes per minute in DWC tissues outside the vessels. Eight square electric pulses, amplitude 1300 V/cm, pulse length 100 µs, repetition frequency 1 Hz were applied and FD injection was performed at the indicated times. No EP was performed in controls. (A) 70 and (B) 2000 kDa FD injected 20 s, 8, 15, 35 and 60 min after EP, and (C) 20 kDa FD injected 30 s, 5 10, 20 and 30 min after EP. Three to five mice were tested for each experimental group. *P<0.05, **P<0.01, *** P<0.001 relative to control. Error bars indicate SEM.

Fig. 5 .

 5 Fig. 5. Illustration of the EP-induced vascular lock. Eight square wave electric pulses, amplitude 1300 V/cm, pulse length 100 µs, repetition frequency 1 Hz were applied. 20, 70 and 2000 kDa FDs were injected 20 s after EP. DWC were observed by fluorescence macroscopy and images were acquired immediately after (0 min), 2, 4 and 6 min after FDs injection.

Fig. 6 .

 6 Fig. 6. Quantification of vascular lock after EP. Eight square wave electric pulses, amplitude per distance 1300 V/cm, pulse length 100 µs, repetition frequency 1 Hz, were applied. 20 or 2000 kDa FITC-dextran was injected at different times after EP, and compared to the control. (A, D) The fluorescence intensity as a percentage relative to the 6 min value is plotted as a function of time for 20 and 2000 kDa dextran, respectively. Derivatives of (A, D) curves, respectively, were calculated. (B, E) Delay of the occurrence of the maximal derivative was determined for each condition (B -20 kDa, E -2000 kDa FD). (C, F) Amplitude of the maximal derivative value was plotted for each condition (C -20 kDa, F -2000 kDa FD). *P<0.05, **P<0.01, *** P<0.001 relative to control. Error bars indicate SEM.

Acknowledgments

This work was supported by grants from the Région Midi Pyrénées, the ANR (Agence Nationale de la Recherche) (PCV06_136459 and ANR-06-BLAN-0260-03), the ARC (Association pour la Recherche sur le Cancer) program n° 8505, the European program (FP7 201102 OncomiR), Slovenian Research Agency (program no. P3-0003, project no. J3-0485), French-Slovenian Scientific Cooperation (PROTEUS and PICS). Research was conducted within the scope of the EBAM European Associated Laboratory (LEA) and network of the COST action TD1104. We thank the "Toulouse Réseau Imagerie" core IPBS facility (Genotoul, Toulouse, France), which is supported by the region Midi Pyrénées, the FEDER and the Grand Toulouse cluster. Proof Reading Services corrected the manuscript.

Supplementary data

Movie S1. Kinetic of blood vessels filling after intravenous injection of 70kDa FD. The perfusion was completed 120 s after the injection. Movie S2. Extravasation of 70 kDa FD induced by EP, eight square wave electric pulses, amplitude 1300 V/cm, pulse length 100 µs, repetition frequency 1 Hz, demonstrating a rapid leakage from all exposed blood vessels. Movie S3. Kinetic of blood vessels filling when 70 kDa FD was injected 20s after EP, eight square wave electric pulses, amplitude 1300 V/cm, pulse length 100 µs, repetition frequency 1 Hz. The filling up of all blood vessels was slowed down and they were filled up only at 6 min post-EP. Movie S4. Kinetic of blood vessels constriction during EP induced vascular lock, after intravenous injection of 70kDa FD. EP induced an immediate slight but significant constriction of the veins. The original size was restored rapidly after EP. The constriction of arterioles was more pronounced and was long lasting.