But the term ‘criminal rehabilitation’ is often used without being explicitly defined, and in ways that are consistent with widely divergent conceptions. In this paper, we present a taxonomy that distinguishes, and explains the relationships between, different conceptions of criminal rehabilitation. Our taxonomy distinguishes conceptions of criminal rehabilitation on the basis of (i) the aims or ends of the putatively rehabilitative measure, and (ii) the means that may be used to achieve the intended end. We also explore some of the implications of each conception, some of the payoffs of a taxonomy of the kind we offer, and some areas for future work.It is often said that the institutions of criminal justice ought or—perhaps more often—ought not to  Such claims can be found in academic literature—for example, from criminology and penal theory.3It is often said that the institutions of criminal justice ought or—perhaps more often—ought not to This imprecision cannot be excused on the basis that, in practice, the boundaries of the concept of rehabilitation are intuitively clear, for there are, in fact, many grey zones. When prison authorities provide psychological therapies to prisoners suffering from depression, are they rehabilitating those prisoners? When a parole board requires that a paroled sex offender undergoes ‘chemical castration’, is it imposing a form of rehabilitation? Is imprisonment itself rehabilitative? The answers to these questions are, we think, not obvious.In this paper, we present a taxonomy that distinguishes and explains the relationships between different conceptions of criminal rehabilitation.Section one presents some of the reasons that a taxonomy of criminal rehabilitation (henceforth just ‘rehabilitation’) is needed. Section two illustrates some of the different ways in which rehabilitation is understood in the literature. Section three outlines five different conceptions of rehabilitation, distinguished from one another by the ends that they take rehabilitation to serve. Section four introduces means-based subvariants of the different conceptions identified in the preceding section. Section five explores some of the payoffs of our taxonomy of rehabilitation. Finally, section six identifies some areas for future work.sole or primary official function of criminal justice. We also take no view on whether, if criminal justice systems ought to rehabilitate, this rehabilitation ought to be conceived as an aspect of punishment, or as something that is done in place of or in addition to punishment. In addition, we leave it open whether traditional forms of punishment, such as incarceration, themselves qualify as instances or components of rehabilitation.We remain neutral throughout on the role that rehabilitation should play in actual or ideal criminal justice systems. Though we are sympathetic to the view that criminal justice systems ought to rehabilitate, and this partly motivates our interest in the topic, we are not committed to this view, let alone to the stronger view that rehabilitation ought to be the We will, from the outset, exclude from the category of rehabilitation all interventions that aim to prevent an individual from re-offending (i) by making it physically impossible for the person to re-offend , or (ii) purely by introducing disincentives or incentives. This is because we wish to maintain a distinction between rehabilitation and two forms of intervention with which it is often contrasted: incapacitation and deterrent punishment. However, in the interests of offering an inclusive taxonomy, we will otherwise start from a broad working conception of rehabilitation that includes all interventions that have commonly been referred to as ‘rehabilitation’, as well as some that we think are sufficiently similar to those practices that they might, without obvious error, be picked out using that label.We need a taxonomy of criminal rehabilitation in order to protect against the conflation and confusion of different conceptions of rehabilitation. Why do we need this? There are at least five reasons.First, the thought that criminal offenders ought to be rehabilitated has exerted a strong influence on the design of many criminal justice systems, including some not generally thought of as rehabilitation-focused, such as the United States’ system. This can be seen, for example, in the language used to describe parts of the criminal justice system: US prisons are, for instance, often referred to as ‘correctional facilities’, and their staff as ‘correctional officers’.Second, although pure rehabilitation theories according to which rehabilitation is the sole legitimate function of criminal justice are no longer popular in moral and legal philosophy, the rehabilitation of offenders—or something akin to it—does, as we will discuss further below, play some role in many currently influential theories, such as those defended by Robert Nozick, Antony Duff, and Victor Tadros.6are in this business, and criminal justice practitioners generally acknowledge this. Rehabilitation programmes, broadly construed, are in place in prisons in most jurisdictions in Europe and North America. The nature and purpose of such programmes vary according to type of offence and the offender's perceived needs, but include education, vocational training, psychological/behavioural interventions, and interventions addressing offenders’ addiction problems. The United Kingdom currently operates rehabilitation programmes designed to reduce offenders’ aggressive behaviour,are attempting to rehabilitate criminal offenders, we should get clear on what exactly rehabilitation comprises.Third, notwithstanding the turn against purely rehabilitative theories of criminal justice, our criminal justice systems do, as a matter of fact, continue to prominently pursue what could be aptly described as rehabilitation.Fourth, a better understanding of rehabilitation may allow us to better appraise moral objections to rehabilitation and to rehabilitative theories of criminal justice. Rehabilitation fell out of favour in moral and legal philosophy due in part to moral concerns, for example, regarding its putative failure to treat offenders as moral agents responsible for their conduct .Finally, a better understanding of rehabilitation may help us determine the extent to which rehabilitative theories are capable of overcoming the other main set of concerns that caused them to fall out of favour: empirical worries to the effect that measures taken aimed at rehabilitating offenders were of limited effectiveness .Though rehabilitation has been an influential concept in debates on criminal justice, it is often not properly defined or elucidated.Some authors are careful to distinguish between ‘reform’ and ‘rehabilitation’. As some characterise this distinction, reform seeks to alter character traits, motivations or dispositions, whereas rehabilitation aims at ‘improvement of … skills, capacities, and opportunities’.self-reform, which he appears to understand as distinct from reform simpliciter, as understood in traditional rehabilitation theories. He rejects the unqualified ‘reform’ and ‘rehabilitation’ labels since he takes these to be compatible with or to include interventions that make offenders law-abiding in ways that bypass or undermine their moral agency. His theory requires active engagement of the offender’s moral agency.25Steven Sverdlik notes that ‘[t]he history of reformist thinking about state punishment is confusing, in part because of terminological issues’.More recent literature concerned with the moral permissibility of using so-called neurointerventions, such as brain-active drugs, in crime-prevention employs various conceptions of rehabilitation and often expresses ambivalence about how it should be understood, and/or a reluctance to commit to any univocal conception. For example, Lene Bomann-Larsen refers to ‘voluntary rehabilitation programs aiming at correcting undesirable behaviour’ or ‘to change [offenders’] undesirable behavioural pattern’.29It is not just in the philosophical literature that criminal rehabilitation is often not clearly defined, or is used in ways that are consistent with divergent conceptions. Fergus McNeill notes that also in the criminological literature, ‘[b]oth as a set of concepts and as a set of practices, rehabilitation is a “tangle”’.32In what follows, we develop a taxonomy that clarifies how different conceptions of rehabilitation (broadly understood) differ and overlap with regard to the posited aims of rehabilitation, and the means via which they allow these aims to be pursued.We will start by distinguishing five conceptions of rehabilitation on the basis of their aims.Rehabilitation as anti-recidivism. An intervention I administered by a criminal justice system to offender O in response to O’s offence is an instance of rehabilitation just in case (1) it is intended to reduce the likelihood that O will re-offend, (2) other than by reducing O’s capacity to reoffend, disincentivising re-offending by O, or incentivising non-offending by O.The aim of reducing the likelihood of recidivism need not, we take it, be the ultimate goal of an intervention in order for it to qualify as rehabilitative on this conception. The ultimate goal may, for instance, be to protect third parties from harm, to promote public safety, to facilitate earlier release of the offender from prison, or simply to maximise aggregate utility. The aim of reducing the likelihood of recidivism also need not, we take it, be the immediate goal of the intervention. The intervention may, for instance, be intended to promote empathy, self-control, or introspection, with the reduced likelihood of offending being an intended effect of the realisation of that immediate aim.Consider first one rather ‘thin’, non-normative, conception of rehabilitation:rehabilitation as anti-recidivism, rehabilitation may share with incapacitation and specific deterrence the aim of preventing people from committing future crimes, so its aim is not a distinctive feature. Rather, its distinctive feature lies in how it gets there, that is, in the means used to achieve this end. Incapacitation seeks to reduce the likelihood of recidivism through rendering it physically impossible, for example, by separating the offender from potential victims, or killing the offender. Special deterrence seeks to reduce the likelihood of re-offending by disincentivising it. Rehabilitation, by contrast, employs other means: most likely, the alteration of the offenders’ intrinsic dispositions.33Note that, on The anti-recidivist conception of rehabilitation is commonly endorsed, at least implicitly, in policy documents. For example, the Ministry of Justice in the UK uses recidivism as the outcome measure for assessing the effectiveness of interventions they refer to as rehabilitative.34rehabilitation as anti-recidivism:Rehabilitation as harm-reduction: An intervention I administered by a criminal justice system to offender O in response to O’s offence is an instance of rehabilitation just in case (1) it is intended to prevent harmful conduct by O , (2) other than by reducing O’s capacity to engage in such conduct, disincentivising such conduct by O, or to incentivising less harmful conduct by O.This account requires some clarification.We might also consider a broader alternative to rehabilitation as harm-reduction, we take harmful conduct to include conduct with negative effects on the wellbeing of others; on some subvariants of the view, it might also include harm to the offender himself.First, for the purposes of Second, as our parenthetical rider indicates, the concept of harm, for the purposes of this account, will need to be restricted. Not all harms, even serious ones, are properly the target of the criminal law, and thus criminal rehabilitation. It is doubtful that we would classify an attempt to prevent an offender from cheating on his partner as rehabilitative. Moreover, even harms that are within the domain of criminal law may be too distant from the crime that has been committed to qualify as a proper target of an attempt at rehabilitation. It is, for instance, doubtful whether we would characterise an attempt to prevent a murderer from committing tax fraud as rehabilitative. Perhaps, to qualify as rehabilitation an intervention must target ‘harmful conduct’ relevantly similar to the offending behaviour of which the offender has been convicted.rehabilitation as anti-recidivism, we do not require that harm-reduction must be the immediate or ultimate goal of an intervention for it to qualify as rehabilitation on this view; it must simply be a goal.Third, as with Rehabilitation as harm-reduction seems to be deployed by Sverdlik in his defence of rehabilitative punishment. Sverdlik holds that punishment can be justified even when it does not have any general deterrent effects, because it may rehabilitate the offender—that is, reduce the likelihood that the offender will perform actions that ‘either cause serious setbacks to well-being, or pose a great risk of doing so’.36rehabilitation as anti-recidivism and rehabilitation as harm-reduction is:Rehabilitation as therapy. An intervention I administered by a criminal justice system to offender O in response to O’s offence is an instance of rehabilitation just in case it is intended to cure or ameliorate a mental deficit in O that is understood by the intervener (1) to have causally contributed to O’s past offence(s), or (2) to predispose O to further offending.‘Mental deficit’ can be understood in either of two different ways: as referring to a mental illness or disorder, or as referring to some defect in the capacities relevant for criminal responsibility, such as capacities for rational agency. The first might aptly be described as a ‘psychiatric’ understanding, since it equates the goals of rehabilitation with those of clinical psychiatry, whereas the second might be labelled a forensic understanding. There will likely be a large overlap in these two understandings, but we take it to be plausible that some mental disorders do not diminish rational capacities, and some diminishments in rational capacity to not constitute mental disorders.An alternative to rehabilitation as therapy, and especially on the psychiatric understanding of it, the aims of rehabilitation overlap with those of clinical medicine . As with standard medical treatments, the aim of curing or ameliorating the deficit may be instrumental to the further aim of benefitting the individual. However, other further aims are also possible. These may include, for example, preventing re-offending, protecting the public, or advancing the social good. If the further aims of the intervention include preventing recidivism or harmful conduct, then the intervention will qualify as rehabilitation on both rehabilitation as therapy and one or both of the accounts we offered above.On rehabilitation as therapy in mind when he wrote thatWhen a man is suffering from an infectious disease, he is a danger to the community, and it is necessary to restrict his liberty of movement. But no one associates any idea of guilt with such a situation. On the contrary, he is an object of commiseration to his friends. Such steps as science recommends are taken to cure him of his disease, and he submits as a rule without reluctance to the curtailment of liberty involved meanwhile. The same method in spirit ought to be shown in the treatment of what is called ‘crime’.37We think that rehabilitation as therapy can also be attributed to some who take themselves to be critics of rehabilitation. For example, Jean Hampton distinguishes her moral education theory of punishment from rehabilitative views by noting that her theory ‘does not perceive punishment as a way of treating a “sick” person for a mental disease, but rather as a way of sending a moral message to a person who has acted immorally and who is to be held responsible for her actions’.rehabilitation as therapy in characterising his own view as non-rehabilitative. He states that ‘[i]t is not one’s health; it is not even one’s moral health with respect to any particular matter that is sought to be achieved; it is one’s general character as a morally autonomous individual attached to the good’.39Bertrand Russell appears to have had something like Rehabilitation as moral improvement. An intervention I administered by a criminal justice system to offender O in response to O’s offence is an instance of rehabilitation just in case it is intended to morally improve O.This is a thicker conception of rehabilitation than the ones we have previously considered, which have all been ‘thin’, in the sense that they characterise the goals of rehabilitation in non-normative terms, or at least in terms that can plausibly be understood as non-normative.41It is, however, tempting to think of Hampton and Morris not as opponents of rehabilitation, but as proponents of a particular, non-therapeutic, kind of rehabilitation,rehabilitation as moral improvement as an account of the nature of rehabilitation.rehabilitation as moral improvement, or something close to it. From here on, we will accept the position of those (including Sverdlik) who characterise Morris and Hampton’s views as rehabilitative.44Hampton maintains that ‘punishment is justified as a way to prevent wrongdoing insofar as it can teach both wrongdoers and the public at large the moral reasons for choosing not to perform an offense’.rehabilitation as moral improvement too. For example, Duff appears to have something like this conception in mind when he uses the term ‘moral rehabilitation’ to describe the kinds of changes at which his preferred type of communicative punishment aims.rehabilitation as moral improvement, or something close to it—he aims to ‘resuscitate the rehabilitative approach to criminal justice’47Others have endorsed rehabilitation as moral improvement. These variants share a commitment to a specific kind of end, that is, making the offender morally better, but differ in their understanding of what becoming morally better consists in , and on what sorts of moral improvement rehabilitation may legitimately aim at . On the nature of moral improvement we can, for example, distinguish between views according to which moral improvement consists in the acquisition of more justified moral beliefs, more morally virtuous character traits, more praiseworthy moral motives, or more morally desirable actions.Whether or not they take themselves to be defending a variant of rehabilitation, those who defend the moral improvement of offenders as a legitimate goal of criminal justice understand moral improvement in different ways, and we might recognise these differences by distinguishing a number of different variants of It has been argued that Hampton and Morris have in common that ‘the psychological changes in offenders that they are interested in promoting are, roughly, these: becoming convinced that one’s action was wrong; feeling guilty for performing it; resolving not to do it again’, and Howard and Duff hold views that are similar with respect to the kinds of changes they believe should be promoted.50Morris favours a view on which rehabilitative measures may permissibly aim at a global kind of moral improvement.53On Howard’s view, the scope of moral improvement sought may be somewhat more local: offenders ought  to take measures to reduce the likelihood that they will commit further criminal wrongs by undertaking measures aimed at fortifying their moral capacities and in particular their sense of justice.54choosing to refrain from committing such offences.the immoral action by communicating to her that her offense was immoral’.On Hampton’s view, interventions should do more than merely deter ‘THE offender’ from committing certain offences; they should also provide him with moral reasons for Similarly, Duff defends what some see as a rehabilitation-based account of criminal justice aimed at moral improvement59rehabilitation as moral improvement, be proximal to some further aim, such as the promotion of offender wellbeing, the social good, the non-instrumental value of being morally good , or some combination of these. It may also be distal to some more immediate aim, such as the promotion of offender empathy, self-control, self-understanding, or introspection. Proponents of rehabilitation, as conceived in rehabilitation as moral improvement, typically assume some non-instrumental value to moral improvement.As with the aims invoked by thin conceptions of rehabilitation, the aim of moral improvement may, on Rehabilitation as restoration. An intervention I administered by a criminal justice system to offender O in response to O’s offence is an instance of rehabilitation just in case it is intended to restore O’s moral or social relationships or standing.On this conception, rehabilitation is a matter of restoring the offender’s social or moral standing in society or his social or moral relations with others, or fostering the capacities needed for such restoration. This could include social and vocational capacities as well as moral ones.moral relationships or standing. On this variant, criminal rehabilitation is in some respects akin to the payment of compensatory damages at tort law; its concern is to bring it about that the offender compensates his victim, pays off a moral debt owed to his victim, corrects the wrong committed, or restores the moral balance between offender and victim. On another variant, rehabilitation aims to restore the offender’s social relationships or repair a social injury, by, for example, helping the offender (re)establish friendships, family bonds, and relationships with others (including victims). A third, hybrid variant would understand rehabilitation as aiming at the restoration of both moral and social relationships.Our fifth and final conception of rehabilitation is:rehabilitation as restoration include Margaret Fry, who in her advocacy for penal reform emphasised the rehabilitative potential of offenders paying damages to their victims, arguing that ‘repayment is the best first step towards reformation that a dishonest person can take’.66Proponents of For dominion to be restored, what is sought is some evidence of a change in attitude, some expression of remorse that indicates that the victim’s rights will be respected in the future. Achieving such a change in attitude may entail the offender agreeing to undergo training, counselling or therapy and, as such, these may all be seen as part of reparative justice. A forced apology or obligatory payment of compensation will not suffice; indeed, it may even be counterproductive in eliciting a genuine change of attitude in the offender.68A restorative conception of rehabilitation can also be attributed to John Braithwaite and Philip Pettit, who hold that an aim of criminal justice is to restore ‘dominion’.rehabilitation as anti-recidivism and rehabilitation as harm-reduction—we introduced a condition restricting the means that could be used to achieve the intended aim. In respect of the latter three conceptions—rehabilitation as therapy, rehabilitation as moral improvement, and rehabilitation as restoration—we included no such condition. This is because in respect of the first two conceptions, such a condition was needed to distinguish rehabilitation from incapacitation and deterrence. In respect of the latter, we do not think a means-based condition is necessary to distinguish rehabilitation from other functions of criminal justice. Nevertheless, some proponents of these latter three conceptions may also wish to impose means-based constraints on what can qualify as rehabilitation—or permissible rehabilitation—and we can distinguish subvariants of these views by reference to the nature and stringency of these constraints. For example, we can distinguish between views according to which an intervention must, if it is to qualify as (permissible) rehabilitation, engage the offender’s rational faculties , views on which it may bypass the offender’s rational faculties but must engage other psychological processes , and views on which rehabilitation may bypass psychological processes entirely, acting directly on neurochemical states .In the previous section, we distinguished five different conceptions of rehabilitation on the basis of their aims or ends. In relation to the first two conceptions—rehabilitation as moral improvement. Several proponents of the moral improvement of criminal offenders endorse a requirement that attempts at moral improvement must employ means that engage the offender’s rationality. That is, they impose an ‘rationality constraint’ on the types of means that can permissibly be employed in rehabilitation; they reject as impermissible interventions that affect recipients in a way that bypasses their rationality ,initial effects of the intervention on the motivational states of the recipient are not mediated by rational processes’.70We have already hinted at such subvariants of Morris and Hampton think that the moral improvement sought by institutions of criminal justice ought to come about as a result of autonomous action on the part of the offender and ought not to bypass ‘the human capacity for reflection, understanding, and revision of attitude’.Hampton specifies that interventions that bring about the moral improvement of offenders are ‘not intended as a way of conditioning a human being to do what society wants her to do ’, but to teach ‘the wrongdoer that the action she did (or wants to do) is forbidden because it is morally wrong and should not be done for that reason’.73rehabilitation as moral improvement. However, a more fine-grained characterisation of their views would understand them as proponents of a subvariant of this conception, according to which moral improvement must be sought through rationality-engaging, and not rationality-bypassing, means.Though they reject the label themselves, Hampton, Morris and Duff can—as we have seen—be characterised as proponents of rehabilitation on rehabilitation as moral improvement. For example, Elizabeth Shaw holds that ‘[e]fforts to reform the offender should be through rational dialogue’ because ‘subjecting a person to direct brain interventions would amount to treating her as if she were a puppet, an automaton or a robot—as something less than human … In other words, it would “objectify” her’.75The distinction between rationality-engaging and rationality-bypassing interventions might be relevant to other conceptions of rehabilitation too. The requirement to engage rational faculties has advocates in recent literature on the use of ‘neurointerventions’—or interventions that act directly on the brain—for crime-prevention purposes, not all of whom are proponents of There are further distinctions that can be made between subvariants of the different conceptions of rehabilitation based on the means they take to be consistent with (permissible) rehabilitation. For example, one of the present authors (Douglas) has distinguished between perceptual and non-perceptual influences, that is, interventions whose primary motivational effects are mediated by perceptual processes, and interventions where this is not the case.79a host of psychological mechanisms’.83Jan Christoph Bublitz and Reinherd Merkel distinguish between interventions that ‘bypass’ psychological processes altogether  and those whose effects are ‘mediated…by internal processes on the part of the addressee’ .As with the distinction between rationality-engaging and rationality-bypassing interventions, requirements to engage perception or to employ indirect means may potentially be used to generate further means-based subvariants of each of the conceptions of rehabilitation we identified in Sect. Delineating the five different ends-based conceptions of criminal rehabilitation identified by our taxonomy, and further means based subvariants, has, we think, at least two payoffs.One payoff is that the taxonomy helps to define the scope of some objections to rehabilitative theories of criminal justice. Delineating different conceptions of rehabilitation makes it clear which conceptions are, and are not, susceptible to common criticisms of rehabilitation. One influential criticism of the view that rehabilitation is a legitimate function of criminal justice, and an important reason that such views have fallen out of favour in moral and legal philosophy, is the ‘theoretical objection’ mentioned above—that rehabilitation fails to treat offenders as responsible moral agents.rehabilitation as therapy, on which rehabilitation presupposes a mental deficit. Insofar as a mental deficit implies a lack of mental capacity, this view arguably presupposes that the recipient of the rehabilitation is less than fully responsible . Other conceptions of rehabilitation are not vulnerable to this objection, since they do not presuppose any mental incapacity or lack of rational agency.There are two reasons why a rehabilitative intervention might fail to treat the offender as a rational agent: (i) because the intervention has an aim that is incompatible with viewing the offender as a full or adequate rational agent, or (ii) because the intervention employs means that fail to engage the offender’s rational agency, thereby failing to treat him as a full moral agent. If the objection is based on (i), it seems to apply primarily to rehabilitation as moral improvement and rehabilitation as anti-recidivism presuppose that the target of rehabilitation is flawed in some way.86Perhaps Howard’s fortificationist view is presented as an attempt to overcome the class of objections according to which rehabilitation fails to treat offenders as responsible moral agents.rehabilitation as therapy or certain subvariants thereof, for the reasons proponents of the theoretical objection give—that it fails to treat offenders as morally responsible agents—but deny that these concerns or criticisms apply to other conceptions of rehabilitation and perhaps thereby maintain that rehabilitation (on these other conceptions) is a legitimate function of criminal justice.Our taxonomy thus clearly shows that we can reject rehabilitation, as characterised by rehabilitation as therapy) are compatible with rehabilitation being pursued through rationality-engaging means, such as engaging an offender in rational dialogue. As noted above, existing views that see moral improvement as a legitimate function of criminal justice typically impose a ‘rationality constraint’ on the types of means that can permissibly be used for rehabilitation purposes, such that the means used must not bypass the offender’s rational capacities. But, as we noted, such a constraint could also be included in other conceptions, including rehabilitation as therapy, giving defenders of rehabilitation a way of avoiding objections based on (ii).If the objection is instead based on (ii)—that the intervention employs means that fail to engage the offender’s rational agency—then whether the objection succeeds depends on which means are used to pursue it. All of the conceptions of rehabilitation that we have introduced , suggesting that literature from public health might fruitfully inform discussions of rehabilitation. Parallels between criminal justice and public health have already received some attention, but these have focussed on quarantine,A second payoff of our taxonomy is that it helps to draw links with other literatures by suggesting parallels between rehabilitation and other types of intervention. For example, on rehabilitation as therapy, for example, rehabilitation is in some respects similar to standard medical treatment, suggesting that literature from medical and psychiatric ethics—and especially on non-consensual psychiatric interventions—might be relevant to the discussion of rehabilitation. On rehabilitation as moral improvement, rehabilitation is relevantly similar to, for instance, the moral education of children, which standardly also aims at moral improvement. Again, this is a topic on which there is also some existing ethical discussion.rehabilitation as therapy, given that consent is standardly required for medical therapies, but not by some other conceptions, such as rehabilitation as anti-recidivism.There are further possible links with other literatures that have not been explored, or which warrant further attention. On Identifying connections to other literatures may also strengthen the case for rehabilitation. On any of our conceptions of rehabilitation there are, as we have suggested, practices analogous to rehabilitation outside the criminal justice context. These include, most obviously, health promotion interventions in public health, psychiatric treatments, and the moral education of children. This puts some pressure on opponents of rehabilitation to either (i) say something about why rehabilitation is inappropriate in criminal justice while these other interventions are appropriate outside the criminal justice context, or (ii) hold that these other interventions are inappropriate too. In the case of the comparison to the moral education of children, opponents of rehabilitation views could perhaps quite easily identify morally relevant differences; many accept that we can treat children in ways in which it would not be permissible to treat adults, for example, because children are yet to develop some capacities or agency or will that warrants the kind of respect we afford adults who are full moral agents, or because children have a different profile of prudential values.Identifying links to other literatures may also help us make headway towards greater clarity in discussions of rehabilitation. To some extent, existing unclarity can be attributed to the fact that different conceptions of rehabilitation invoke notions, such as mental disorder and moral improvement, that are themselves open to multiple interpretations and frequently used imprecisely. This source of unclarity remains even when different conceptions of rehabilitation are distinguished. However, our taxonomy also suggests that we may be able to mitigate some of this unclarity by drawing on conceptual work done in other areas. For example, when defining mental deficit we might derive some benefit from work in psychiatry, the philosophy of mind, and the philosophy of science; when clarifying the moral improvement conception, we might rely on work on moral education and moral bioenhancement. Once we have achieved greater clarity in regard of what these notions mean or have better defined them, we can proceed to examine the extent to which they are measurable and how.The concept of rehabilitation is often deployed in academic discussions, policy documents and legal judgments without being precisely defined, and without its extension being intuitively clear.ends that they ascribe to rehabilitation, but subvariants within these conceptions are in some cases distinguished also by the means used to achieve these ends.In this article, we have sought to bring a measure of clarity by offering a simple taxonomy of rehabilitation. We have outlined five conceptions of rehabilitation that can be discerned in the literature, as well as a number of subvariants of these conceptions. The five conceptions are distinguished from one another chiefly by the rehabilitation as anti-recidivism could differ in the breadth or range of the types of criminal (and other) behaviour that they seek to prevent. Another approach would be to specify hierarchies of aims. For example, we could distinguish between views according to which rehabilitation aims at moral improvement with the further aim of protecting the public and views according to which moral improvement is seen as the ultimate end. Finally, yet another approach would be to introduce further means-based distinctions. Perhaps, for example, an interesting distinction could be drawn between effortful and effortless means.This taxonomy is, however, just a beginning. There may be scope to broaden our taxonomy by adding conceptions of rehabilitation that we have missed. And there is certainly scope to deepen it by distinguishing variants of the conceptions that it posits. One way to achieve such a deepening would be to more finely specify the aims of rehabilitative interventions, as we began to do with interventions that aim at moral improvement, for example, by distinguishing local and global forms of moral improvement. Similarly, different variants of rehabilitation as therapy and rehabilitation as moral improvement, on which an individual who has committed a wrongshould voluntarily go to wherever he will pay the penalty as soon as possible, to the judge as if to the doctor, eager to take care that the disease of wrongdoing not become chronic and make his soul fester and become incurable … He ought not to hide his injustice but bring it out in the open, so that he may pay his due and become well, and it is necessary for him not to act cowardly but to shut his eyes and be courageous, as if he were going to a doctor for surgery or cautery, pursuing the good and noble and taking no account of the pain, and if his injustice is worthy of a beating, he should put himself forward to be beaten.93 In Plato’s case it is hard to say whether the deficit to be corrected is a moral one  or a prudential one (in which case it is perhaps closer to rehabilitation as therapy). This is unsurprising, given that ancient philosophers typically did not distinguish between prudence and morality.There are also difficult questions of application raised by our taxonomy: not all existing conceptions of rehabilitation can be neatly classified into the (overlapping) categories that it establishes. To give just one example, Plato describes a view that might be best understood as a mixture of Our taxonomy leaves much work to be done, in further specifying the preliminary conceptions of rehabilitation that it offers, in teasing out the relationships between them, and perhaps in adding further conceptions. Nevertheless, we hope that it will serve as a useful starting point for further work on the nature of rehabilitation, and that it already makes some progress towards clarifying this ambiguous concept and the messy literature that surrounds it.


The mutation-driven transformation of clinical anti-androgen drugs into agonists of the human androgen receptor (AR) represents a major challenge for the treatment of prostate cancer patients. To address this challenge, we have developed a novel class of inhibitors targeting the DNA-binding domain (DBD) of the receptor, which is distanced from the androgen binding site (ABS) targeted by all conventional anti-AR drugs and prone to resistant mutations. While many members of the developed 4-(4-phenylthiazol-2-yl)morpholine series of AR-DBD inhibitors demonstrated the effective suppression of wild-type AR, a few represented by 4-(4-(3-fluoro-2-methoxyphenyl)thiazol-2-yl)morpholine (VPC14368) exhibited a partial agonistic effect toward the mutated T878A form of the receptor, implying their cross-interaction with the AR ABS. To study the molecular basis of the observed cross-reactivity, we co-crystallized the T878A mutated form of the AR ligand binding domain (LBD) with a bound VPC14368 molecule. Computational modelling revealed that helix 12 of AR undergoes a characteristic shift upon VPC14368 binding causing the agonistic behaviour. Based on the obtained structural data we then designed derivatives of VPC14368 to successfully eliminate the cross-reactivity towards the AR ABS, while maintaining significant anti-AR DBD potency. The androgen receptor (AR) is a ligand-inducible transcription factor belonging to the nuclear receptor (NR) superfamily. The AR has a modular structure composed of an N-terminal domain (NTD), a highly conserved DNA-binding domain (DBD) and a C-terminal ligand-binding domain (LBD) . EndogenAs proliferation and survival of PCa cells are critically dependent on androgen stimulation, the AR signaling has been extensively studied as a hallmark for therapeutic targeting of PCa. This treatment usually involves some form of chemical castration to inhibit androgen synthesis , or to prevent AR transcriptional activity  ,7,8,9,10Abundant crystal structures of the wild-type AR-LBD in complex with agonist ligands are available in the Protein Data Bank (PDB) ,20,21,22Another way of bypassing the characteristic mutation-driven resistance of conventional anti-androgens that has been explored is targeting alternative drug target sites on the AR. ,37,38. TIn order to understand and eliminate this undesired partial agonistic effect, in the current study, we investigated the molecular basis of the interaction between T878A AR-LBD and the most powerful partial agonist compound VPC14368 shown in . A crystAR transcriptional activity was assayed as previously described . BrieflyThe PSA assay was performed in parallel with the previous reporter eGFP assay and using the same plates. After 3 days incubation of LNCAP cells with R1881 and the test compound, 150 µL of the media was taken from each well and added to 150 µL of PBS. PSA levels were then evaluated using the Cobas e 411 analyzer instrument (Roche Diagnostics) according to the manufacturer’s instructions.Full-length human wild type AR was cloned into pcDNA3.1 expression plasmid  with a CMV promoter. The T878A mutant (T878A-AR) was generated with the Quikchange Mutagenesis Kit  using hARWT plasmid as a template. Androgen-starved PC3 cells were seeded in 96-well plates (3000 cells/well) in RPMI 1640 + 5% CSS for 24 h followed by transfection with 50 ng of hARWT or hART878A along with 50 ng of ARR3tk-luciferase reporter construct  (100 ng 6-avidin (GLNDIFEAQKIEWHE) tag were expressed in E. coli BL21-DE3 cells co-transformed with pBirACm plasmid (Avidity), which expresses a biotin ligase leading to the biotynilation of the avidin tag on the LBD. Cells were grown in LB media at 37 °C to an OD600nm = 0.5 and then induced with 0.5 mM isopropyl-β-D-thiogalactoside. A measure of 20 µM of dihydrotestosterone (DHT) and 0.15 mM biotin were added and the cells were incubated at 16 °C for 18 to 20 h. Cells were then resuspended and lysed in buffer containing 500 mM Li2SO4, 50mM HEPES pH7.2, 10% Glycerol, 0.1% Octyl-beta-Glucoside, 20 µM of DHT, 5 mM β-mercaptoethanol, 10 mM imidazole and 0.1 mM phenylmethanesulfonyl fluoride. After sonication and centrifugation, the sample was loaded onto a Ni-NTA affinity column and subsequently eluted with 300 mM imidazole. After an overnight dialysis against 150 mM Li2SO4, 50 mM HEPES pH7.2, 10% Glycerol, 0.1% Octyl-beta-Glucoside, 20 µM of DHT and 0.2 mM tris(2-carboxyethyl)phosphine, purified proteins at 50 µg/mL were bound to the super-streptavidin sensors overnight at 4 °C. The assay was performed at 20 °C in the dialysis buffer supplemented with 5% dimethyl sulfoxide (DMSO) which was also used as our reference measurement.The direct reversible interaction between small molecules and the AR was quantified by BLI using OctetRED . The wild type and T878A-LDB in fusion with an N-terminal (His)6 tag followed by a thrombin cleavage site (LVPRG). 100 μM of VPC14368 were added into the cell culture medium before induction. After resuspension, sonication and centrifugation, the sample was first applied to a Ni2+ loaded affinity column and subsequently cleaved with thrombin to remove the tag. The protein was further purified by cation exchange chromatography. All buffers used during the purification contained 50 μM of VPC14368 in order to stabilize the protein. The purified protein, stored in buffer containing 150 mM Li2SO4, 50 mM HEPES pH7.2, 10% Glycerol, 50 μM VPC14368, 0.1% Octyl-beta-glucoside, and 10 mM DTT, was concentrated to 5 mg/mL and used for crystallization trials.The T878A-LBD of the human AR containing amino acid residues I6654-Q920 was cloned into the pET15b vector (Novagen) and expressed in fusion with an N-terminus (His)Diffracting crystals of T878A-LBD-VPC14368 complex were obtained at 22 °C in 0.1 M Hepes pH 7.5, 0.4–1 M Na citrate. Crystals were cryoprotected with 20% ethylene glycol and flash-frozen in liquid nitrogen. X-ray diffraction data were collected at the European Synchrotron Radiation Facility (ESRF) synchrotron  on ID23-1 beamline using a Pilatus 6M-F detector. Data processing and scaling were performed with XDS . CrystalAndrogen displacement was assessed with the Polar Screen Androgen Receptor Competitor Green Assay Kit as per the instructions of the manufacturer.To model the wild type (WT) AR structure, we computationally mutated the residue 878 from alanine to threonine using Molecular Operating Environment (MOE) software . The modMolecular dynamics (MD) were performed using Desmond package from Schrodinger . Both thBinding free energy calculations on the clustered MD snapshots were performed using Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) method . The eneTo circumvent mutation-driven resistance to conventional AR inhibitors, we explored an alternative site on the DBD segment of the AR and developed a series of 4-(4-phenylthiazol-2-yl)morpholines capable of selective inhibition of the receptor′s activity by disrupting its interaction with DNA . One derIt is well-documented that mutations in the ABS could render AR antagonists, such as hydroxyflutamide and R-bicalutamide, to act as AR receptor activators ,24. The free factors of 14.4 and 16.9%, respectively  located in opposing ends of the site, which also play a pivotal role in anchoring steroidal AR binders. In this structure (PDB ID: 8E1A), a clear electron density map allowed unambiguous fitting of the VPC14368 into the receptor′s ABS. The compound is accommodated in the ABS by a network of hydrophobic and Van der Waals interactions, as well as weak hydrogen bonds C,D. The The ABS is known as an enveloped pocket with notable plasticity that can adjust the volume to accommodate ligands of various sizes. To date, there are a significant number of structures of human AR LBD solved in the presence of various steroidal and non-steroidal ligands -dione, VPC14368 is a notably weaker AR binder, demonstrating binding at concentrations above 100 µM toward the target  14,24,7,724,70. To study the molecular mechanism of the agonistic switch of VPC14368, we computationally modelled the structure of the wild type (WT) structure in complex with VPC14368. First, the molecular docking of VPC14368 confirmed that the binding pose observed in the crystal structure remains largely unchanged in WT form with a slight tilting of the morpholine ring away from the native threonine 878.We then performed three replicas of 500 ns molecular dynamics (MD) simulations followed by trajectory clustering of both the mutant and WT complexes. We examined the overall dynamics of the proteins and observed that while most of the residues behave similarly in WT as in mutant complexes, helix 12 is more dynamic in the mutant form A. This iTo elucidate the mechanism of ligand-induced helix 12 shift, we superimposed representatives of the three most populated MD-derived clusters from each WT and T878A mutant AR-VPC14368 complexes. Importantly, all three representative WT structures had helix 12 positioned further away from the LBD site compared to all three mutant structures B. The moInterestingly, the computational analysis of R-bicalutamide agonistic switch in the presence of W742C and T878A mutations revealed that the W742 mutation reduces a clash with R-bicalutamide′s phenyl ring and enables the helix 12 shift towards the ligand binding cavity ,71. In cFinally, we estimated the free energies of bindings and energy terms of both complexes using the MM/GBSA method. We observed that predicted ΔG of binding was comparable: −68 kcal for agonist and −71 kcal for antagonist. However, the agonist form had a slightly stronger component of hydrogen bond interactions . An increase in hydrogen bond component is likely due to transient interactions observed through MD simulations between the oxygen of ligand′s morpholine ring and T878. Of note, this observation is contrary to the Singam et al. (2019) study where they computationally analyzed various agonists and antagonists and found that agonists tend to have a stronger hydrogen bond component while antagonists tend to increase LBD stability mainly through hydrophobic interactions . Thus, wBased on the structural information obtained from T878A AR LBD-VPC14368 complex, we attempted to design new derivatives with chemical features that would eliminate or reduce the binding of these compounds to the AR LBD while retaining their anti-AR activities through binding to the DBD. A number of derivatives were created to replace the benzene fragment in the studied 4-(4-phenylthiazol-2-yl)morpholines with fewer hydrophobic cycles containing various aromatic (VPC14291), aliphatic  and heterocyclic rings  see .The ability of VPC14368 and the above derivatives to inhibit AR transcriptional activity was evaluated using the eGFP-expressing LNCAP cells. Some of the derivatives  lost their anti-AR activity; however, others such as VPC14449, VPC14404 and VPC14435 maintained good potency against the mutated T878A form of AR, while no undesired partial agonistic effects were observed  (at 50 µM .50 = 0.15 µM) without showing the undesired partial agonist effect toward the AR. This was also confirmed in PC3 cells transfected by the T878A-AR where the AR stimulation by VPC14449 was significantly reduced (~80% reduction) at high concentrations in the absence of R1881 in comparison with VPC14368 thiazol-2-yl)morpholine (VPC14368), demonstrated partial agonistic effect on the T878A mutated form of the AR, indicating possible cross-reactivity with the ABS pocket of the receptor. In order to eliminate this undesirable agonistic effect, VPC14368 was co-crystallized with the T878A mutated form of the AR-LBD and analyzed for structural clues to the observed partial agonism. The solved crystal structure revealed a novel chemotype of AR ABS binders that expands the repertoire of previously reported nonsteroidal AR ABS ligands . We alsoMore importantly, the structure of the AR LBD complexed with VPC14368 provided a rational basis for developing derivatives of this chemical series such as VPC14449 that do not bind to the AR ABS and do not exhibit partial agonist effect towards the T878A mutated AR. Importantly, the designed derivatives maintain their anti-AR potency through direct DBD binding . Thus, t


Neonatal mouse heart can regenerate after left ventricle (LV) apical resection (AR). Since current AR rodent method is accomplished by resecting LV apex until exposure of LV chamber, it is relatively difficult to operate reproducibly. We aimed to develop a modified AR method with high accuracy and reproducibility and to investigate whether cardiac regenerative capacity could be replicated in neonatal rats. For 15% AR of whole heart weight in 1-day-old (P1) neonatal mice, a modified 10 μL pipette tip cut to 0.48 mm in internal diameter was connected to a vacuum pump working at 0.06 ± 0.005 MPa and gently kept in touch with LV apex for nearly but no more than 12 s. LV apex was resected by a single incision adjacent to the pipette tip. The modified AR method in P1 mice achieved cardiac structural and functional recovery at 21 days post resection (dpr). Data from different operators showed smaller variation of resected LV apex and higher reproducibility using the modified AR method. Furthermore, we showed that 5% AR of whole heart weight in P1 neonatal rats using a modified 200 μL pipette tip cut to 0.63 mm in internal diameter led to complete regeneration of LV apex and full preservation of cardiac function at 42 dpr. In conclusion, the modified AR rodent model leads to accurate resection of LV apex with high homogeneity and reproducibility and it is practically convenient for the study of structural, functional, and molecular mechanisms of cardiac regeneration in both neonatal mice and rats. Identifying molecules that promote cardiomyocyte proliferation and cardiac regeneration is essential to provide novel strategies for myocardial protection2. The cardiac apical resection (AR) model of neonatal mice has revealed a strong regenerative capacity of the heart within 7 days after birth4. Apical resection in 1-day-old (P1) neonatal mice induces robust cardiomyocyte proliferation and leads to cardiac regeneration with preserved cardiac function4. However, this regenerative capacity is lost in 7-day-old (P7) mice4. Based on the AR model of neonatal mice, molecules involved in postnatal cardiac regeneration have been increasingly identified which may also have the potential to promote cardiac regeneration and repair in adult hearts10. The regeneration model of neonatal mouse myocardium provides strong support for the study of cardiac regeneration in mammals.The adult mammalian heart has a very limited regenerative capacity, which causes permanent myocardial loss and insufficient repair after myocardial injury9. Besides, since the current AR rodent method is accomplished by resecting LV apical myocardium even through several incisions3, it is relatively difficult to operate uniformly. Surgical technicalities and assessment procedures can vary and may result in different regeneration outcomes12. Therefore, a more unified and easy-to-control and easy-to-perform AR method is required.Despite wide use of the AR model in neonatal mice, several issues remain unsolved. Although exposure of the left ventricle (LV) chamber has been used as an anatomic landmark for AR, the actual weight or size of the resected apical myocardium is difficult to control and be compared among different research groups. One reason is that either resected weight or resected area has been used to describe the resected part of apical myocardium13. Actually, whether and how cardiac regenerative capacity could be replicated in neonatal rats remain largely unclear.Compared to the AR model in neonatal mice, rare studies have systematically evaluated cardiac regenerative capacity in apically resected neonatal rats. It has previously been reported that P1 rats with 16–18% apically resected area had an early cardiac repair response and long-term preserved cardiac function even under hemodynamic stress. However, the larger apically resected area in neonatal rats (16–18%) led to an incomplete regenerative process associated with myocardial hypoperfusion and collagen depositions around the repaired area at 60 days post resection (dpr)In the present study, we developed a modified AR method in neonatal mice with robust cardiac regenerative response and normal cardiac function. Compared to the conventional AR method, this modified AR method showed lower variation of resected apex and higher reproducibility of surgery. Moreover, an efficient regenerative response and structural and functional recovery of the heart were observed in P1 neonatal rats using the modified AR method. An easy-to-control and easy-to-perform AR method will be useful to promote investigation of novel mechanisms and factors for cardiac regeneration and repair.The modified AR method was detailed in Methods section. A schematic flow chart was presented to illustrate the major operation process of modified AR model in P1 neonatal rodents Fig. . The tip3. The surgical AR procedure from anesthesia to chest closure should be limited within 10 min for both methods. For achieving a surgical procedure within 10 min, we observed that each operator needed to operate more mice (≈1.5 to 3 fold) when they learned and practiced conventional AR method compared to modified AR method. Then they performed formal experiments for comparison of conventional and modified AR methods. In the present study, each operator performed AR surgery in P1 neonatal mice using conventional (n = 5) and modified (n = 5) AR method, respectively. Based on the measured resected areas of LV apex, we calculated the standard deviation (SD) and relative standard deviation (RSD) in the modified or conventional AR method for each operator. Representative images of the resected apical tissue by one operator are demonstrated in Fig. To evaluate the AR operation homogeneity and reproducibility, we trained 5 operators to perform both the conventional and modified AR method. Conventional AR was accomplished by gently resecting the ventricular apex until the LV chamber was exposedWe next compared cardiac regenerative responses between our modified surgical AR method and conventional AR method. Using conventional AR method, we also observed comparable heart weight between AR and sham group at 7 dpr and 21 dpr  at 1 dpr; these factors were still upregulated at 7 dpr in the AR group compared to sham group  and 7-day-old (P7) rats, respectively. We observed that P3 rats with AR surgery had comparable heart weight, preserved cardiac function, and regenerated apex compared to sham rats at 42 dpr  was revealed to be significantly induced after AR, which further contributed to cardiomyocyte proliferative response and played an essential role for early cardiac regeneration after AR14. In the present study, we observed a significant increase of EdU, pHH3, and Aurora B-kinase positive cardiomyocytes in the apex, apical border zones, and remote zones of resected hearts at 7 dpr in the modified AR model, which was consistent to the robust cardiomyocyte proliferative response in the conventional AR model as previously reported4. Meanwhile, we observed obvious upregulation of inflammatory and immunomodulatory factors  in the modified AR mouse model at 1 dpr and 7 dpr, which was consistent to previous reports showing acute inflammatory responses after AR14. By determining some previously reported myocardial pro-proliferative factors16, we observed upregulation of Mydgf and Pitx2 in resected heart tissues with modified AR method at 1 dpr and 7 dpr, further validating molecular changes of myocardial pro-proliferative factors in this modified AR mouse model.Cardiac regenerative responses in the early phase after AR surgery play important roles in promoting cardiac regeneration and recovery13. Here, we hypothesized that the resected part of the heart may be too large to accomplish cardiac structural and functional recovery post resection in rats. By gradually reducing the internal diameter of the modified 200 μL pipette tip to 0.63 mm, we finally succeeded in establishing 5% AR of whole heart weight in P1 neonatal rats which led to complete regeneration of LV apex and full preservation of cardiac function at 42 dpr. Similar to the AR mouse model and previously reported cardiac regeneration studies18, an obvious cardiomyocyte proliferative response in the whole heart and marked upregulation of inflammatory and immunomodulatory factors  and regulation of myocardial pro-proliferative factors (upregulation of Mydgf and Pitx2 and reduced phosphorylation of YAP at Ser127) were observed in resected hearts at 1 dpr and 7 dpr. Moreover, we demonstrated that similar to AR-operated neonatal mice, AR-operated neonatal rats displayed comparable reduced heart rate and LV systolic function upon phenylephrine-induced hemodynamic stress when compared to sham rats even at 60 dpr, suggesting a long-term functional recovery of neonatal rat hearts after 5% AR surgery. Thus, using this modified AR method, we established 5% AR neonatal rat model which will provide a necessary basis for subsequent research on heart regeneration in genetically modified neonatal rats.Despite a validation of cardiac regeneration after AR in neonatal mice, whether and how cardiac regenerative capacity could be replicated in neonatal rats has previously been unclear. A previous study performing AR with 16–18% resected apical area in P1 neonatal rats as estimated by magnetic resonance imaging (MRI), reported an incomplete cardiac recovery accompanied with myocardial hypoperfusion and cardiac fibrosis at 60 dpr19.Here we further observed that both P1 and P3 neonatal rats have cardiac regenerative capacity and achieve functional and structural recovery of the heart after AR. However, this cardiac regenerative potential is lost in P7 rats, since AR caused reduced cardiac function and fibrotic tissue growth around the resected apex at 42 dpr. Moreover, hardly no evidence of cardiomyocyte proliferative response was observed around the resected apex at 7 dpr. This may be closely related to a rapid transition of cardiac myocytes from hyperplasia to hypertrophy within one week after birthIn conclusion, we develop a modified easy-to-control and easy-to-perform AR method by using a modified pipette tip connected to a working vacuum pump to accurately control the resection part of LV apex in rodents. This modified AR method with high homogeneity and reproducibility induces cardiac structural and functional recovery in neonatal rodents which can be practically applied by researchers after quick learning. A 5% AR neonatal rat model is proven to be practical to induce structural and functional cardiac regeneration with minimal fibrosis of LV apex. We think that the modified AR method in rodents will prove to be useful for structural, functional, and molecular investigations of cardiac regeneration and repair in mammals.Eight-week-old C57BL/6J mice and Sprague-Dawley rats were purchased from Cavens Lab Animal  and maintained in the specific-pathogen-free (SPF) laboratory animal facility of Shanghai University . Their offspring was used to establish AR models in neonatal rodents. All animal experiments were conducted in concordance with the Guidelines for the Care and Use of Laboratory Animals for biomedical research published by the National Institutes of Health  and approved by the Committee for the Ethics of Animal Experiments of Shanghai University.The modified AR method was conducted and evaluated in P1 neonatal C57BL/6J mice. For an AR of 15% weight of the whole heart, a modified 10 μL pipette tip was prepared by cutting its tip to 0.48 mm in internal diameter using a sharp blade. A formula was given to calculate the distance cutting from the pipette tip  and weighed, in order to validate the 15% AR of whole heart weight. At 7 dpr and 21 dpr, the heart weight (HW), body weight (BW), and heart weight/body weight ratio (HW/BW) were measured. Echocardiography was performed at 21 dpr. Heart tissues were harvested for histological analysis (7 dpr and 21 dpr), immunofluorescent stainings for cardiomyocyte proliferative markers (7 dpr), and molecular analysis for markers of inflammatory and myocardial proliferative responses (1 dpr and 7 dpr).The modified AR method was conducted and evaluated in P1 neonatal Sprague-Dawley rats. A modified 200 μL pipette tip was prepared by cutting its tip to 0.63 mm in internal diameter using a sharp blade, which achieved a 5% AR of whole heart weight in P1 neonatal rats. Except for a slightly larger incision (0.6 to 0.8 cm-long) for entering the chest cavity and visualizing the heart and a modified 200 μL pipette tip with its tip cut to 0.63 mm in internal diameter, the other procedures were the same as those described for the modified AR method of neonatal mouse heart. The precise position where a pipette tip attaches to the apex is shown in Supplementary Fig. Quickly after AR surgery, resected and intact hearts were rinsed with PBS and weighed. The weight of resected LV apex was proven to be 5% of whole heart weight in P1 neonatal rats by 3 independent experiments. At 7, 14, 28, and 42 dpr, the HW, BW, and HW/BW ratio were measured. Echocardiography was performed every 2 weeks from 14 to 42 dpr. Heart tissues were harvested for histological analysis , immunofluorescent stainings for cardiomyocyte proliferative markers (7 dpr), and molecular analysis for markers of inflammatory and myocardial proliferative responses (1 dpr and 7 dpr).To explore when neonatal rats lose cardiac regenerative capacity after birth, we further performed AR surgery in 3-day-old (P3) and 7-day-old (P7) rats, respectively. P3 rats underwent AR procedure using the same modified 200 μL pipette tip cut to 0.63 mm in internal diameter. While P7 rats underwent AR procedure using a modified 200 μL pipette tip cut to 0.57 mm in internal diameter for a smaller proportion of resected LV apex due to a higher mortality induced by AR surgery at day 7 after birth. Echocardiography was performed at 42 dpr, followed by heart tissue harvest and histological examinations. Cardiomyocyte cell cycle activity was determined by EdU, pHH3, or Aurora B and α-actinin co-stainings in heart tissues at 7 dpr.Rodents were anesthetized with inhaled isoflurane, and echocardiography was performed using the Vevo 2100 Imaging System . M-mode long-axis echocardiograms were obtained at the papillary muscle level to measure the LV ejection fraction (EF) and fractional shortening (FS) based on 3 consecutive cardiac cycles.Whole heart tissues were fixed in 4% paraformaldehyde (PFA) and then embedded in paraffin. The 5-μm-thick cardiac longitudinal sections were stained with Hematoxylin-Eosin  and Masson’s trichrome staining  according to manufacturer’s instructions. Tissue stainings were observed and photographed under a biological microscope . HE staining and Masson’s trichrome staining were analyzed to evaluate the recovery of LV apex and whether fibrotic tissue was present or not after AR.20.For EdU staining in heart tissues, rodents were intraperitoneally injected with 50 mg/kg EdU  at 1 day and 3 days before sacrifice. Whole heart tissues were harvested and embedded into optimal cutting temperature compound (OCT), and longitudinally cut into 7-μm-thick frozen heart sections. Sections were fixed with 4% PFA, permeabilized with 0.5% Triton X-100, and then blocked with 5% bovine serum albumin (BSA) for 1 h at room temperature. Sections were incubated with the primary antibody for α-actinin  at 4 °C overnight. After wash three times in PBS, sections were incubated with corresponding secondary antibody for 2 h at room temperature. EdU staining was performed using Cell-Light Apollo 567 Stain Kit  according to the manufacturer’s instructions. For pHH3 (or Aurora B) and α-actinin co-immunofluorescent staining, sections were fixed, permeabilized, and blocked as described above, and then incubated with primary antibodies for pHH3  or Aurora B  and α-actinin  at 4 °C overnight. After washing three times with PBS, sections were incubated with corresponding secondary antibodies for 2 h at room temperature. Nuclei were counterstained with Hoechst. Immunofluorescent staining images were viewed and taken under a confocal microscope . The number of EdU, pHH3, or Aurora B positive α-actinin-labeled cardiomyocytes per field was determined in the apex, apical border zones, and remote zones of resected hearts and intact sham hearts. The symmetrical and asymmetrical Aurora B positive cardiomyocytes were determined as previously reportedTotal RNA was isolated from resected and intact hearts using Trizol RNAiso Plus kit . RNA concentration was determined by Nanodrop 8000 (Thermo fisher), and reverse transcription of RNA was performed using RevertAid First Strand cDNA Synthesis Kit . Quantitative polymerase chain reactions were performed using SYBR Green reagent  on Roche LightCycler480 PCR System (Roche Diagnostics). Primer sequences used in this study are listed in Supplementary Table Total protein was extracted from resected and intact hearts using RIPA lysis buffer supplemented with Pierce protease and phosphatase inhibitor . Equal quantities of total protein were separated by SDS-PAGE gels and transferred to PVDF membranes. Membranes were blocked with 5% non-fat milk and then incubated with primary antibody for p-YAP1-S127 , followed by incubation with corresponding HRP-conjugated secondary antibody. Protein blots were developed using enhanced chemiluminescence kit. After, membranes were stripped and incubated with total-YAP1 antibody . GAPDH  was used as an internal control for protein loading. Protein band intensity was analyzed using ImageJ software. The phosphorylation level of YAP was determined by the ratio of p-YAP (S127) to total-YAP.For comparison of long-term cardiac function under hemodynamic stress, rats were treated with selective α1-adrenoceptor agonist phenylephrine to induce a state of hemodynamic stress at 60 dpr followed by cardiac function measurement. Before phenylephrine treatment, sham and AR rats were anesthetized with inhaled isoflurane, and cardiac function was evaluated at baseline using echocardiography (Vevo 2100 Imaging System). Tail cuff blood pressure was measured using non-invasive Tail-Cuff blood pressure device  including systolic blood pressure (SBP), dilated blood pressure (DBP), and mean blood pressure (MBP). Then rats were intraperitoneally injected with 25 mg/kg phenylephrine , followed by measurement of tail cuff blood pressure every 2 min. For rats whose SBP failed to increase over 40% after 5 min of phenylephrine injection, another injection of 12.5 mg/kg phenylephrine was given to them. Cardiac function was measured once SBP increased over 40% from baseline. Cardiac function under hemodynamic stress was also measured in sham- and AR-operated mice at 42 dpr after a single intraperitoneal injection of 300 μg/kg phenylephrine once SBP increased over 40% from baseline.t test was used for comparisons between two groups. Two-way ANOVA test followed by Tukey post hoc test was performed to compare multiple groups. Relative standard deviation (RSD) analysis was performed for evaluation of reproducibility of modified and conventional AR methods. Differences were considered statistically significant with P < 0.05.All data were analyzed using SPSS20.0 or GraphPad Prism8 and presented as means ± SD. An independent-sample two-tailed Student’s Further information on research design is available in the Supplementary MaterialSupplementary Video 1Reporting Summary