Wildlife rehabilitation is the practice of caring for sick, injured, or orphaned wild animals with the goal of releasing them back to the wild. This review demonstrates the complexity of considerations rehabilitators and veterinarians face while trying to optimize the welfare of wildlife in need of care and rehabilitation. The welfare of animals, including the thousands of wild animals presented for rehabilitation each year in the United States, is of increasing concern to the public. Almost all wildlife rehabilitation in the United States requires practitioners to have a federal or state permit and is privately funded. The process of rehabilitation is inherently stressful for wildlife and maintaining the individual animal’s welfare at the center of the rehabilitation process requires deliberate, timely, and humane decision making. The welfare of wild animals can be improved by preventing human-related causes of admission, providing much-needed resources and support for those animals in rehabilitation, further developing evidence-based wildlife rehabilitation methods and welfare measures, increasing engagement of the veterinary profession, harmonizing regulatory oversight with standards of care, training, and accountability, and raising public awareness regarding the steps that can be taken to mitigate the number of wild animals in need of rehabilitation.Each year in the United States, thousands of sick, injured, or displaced wild animals are presented to individuals or organizations who have either a federal or state permit that allows them to care for these animals with the goal of releasing them back to the wild. The purpose of this review is to demonstrate the complexity of considerations rehabilitators and veterinarians face while trying to optimize the welfare of wild animals in need of care and rehabilitation. The process of rehabilitation is inherently stressful for wildlife. Maintaining an animal’s welfare during the rehabilitation process—from initial contact and tria+ge to the animal’s euthanasia, release, or captive placement—requires deliberate, timely and humane decision making. The welfare of wild animals can be improved by preventing human-related causes of admission, providing resources and support for wildlife rehabilitation ; further developing evidence-based wildlife rehabilitation methods and welfare measures, attracting more veterinary professionals to the field, harmonizing regulatory oversight with standards of care, training, and accountability, and increasing public education. The welfare of animals, including wildlife, is of increasing concern to the public. In the United States (US), it is estimated that hundreds of thousands of sick, injured or orphaned wild animals are presented for rehabilitation annually   as well as in other countries, wildlife rehabilitation, translocation, and reintroduction programs may be an important component of conservation programs . CompareIncreasingly, zoos and aquaria in the US are engaging in native wildlife rescue and rehabilitation. The Association of Zoos and Aquariums (AZA) and its member zoos and aquariums partner with regulatory authorities, and often local wildlife rehabilitators, in rescue events to improve conservation outcomes and animal welfare and engage people to care about wildlife. This provides opportunities for cross-training between zoological and rehabilitation facilities and additional support for rehabilitators. Zoos and aquariums are also frequent placement sites for non-releasable wildlife.In the US, wildlife rehabilitation generally requires a permit, and depending on the taxa, a variety of federal, state, and local agencies have regulatory oversight. Federal agencies oversee the rehabilitation of migratory birds, marine mammals, sea turtles, and federally endangered and threatened species, while states  regulate the rehabilitation of other wildlife within their borders ,45.The Animal Welfare Act (AWA) enacted in 1966 is the only federal law in the US that regulates the treatment of animals in research, exhibition, transport, and by dealers . CurrentIndividual states have taken various approaches to wildlife rehabilitation. Some states do not address the practice at all, whereas in other states, it is not permitted, and violation may result in prosecution. These states prefer to “let nature take its course”. In some states where wildlife rehabilitation is not permitted, there may be a process to place a limited number of high-profile “orphans” in zoos or other appropriate organizations. These orphans would otherwise starve to death or inappropriately interact with people. Regardless of the level of regulatory oversight, there are a finite number of outcomes for admitted wildlife: euthanasia or death-in-care, successful rehabilitation and release to the wild, or permanent placement in human care. Despite treatment and rehabilitation, ultimately, many wild animals cannot or should not return to the wild. These animals could be considered for captive placement into educational, foster, or breeding programs. There are regulatory as well as significant ethical considerations regarding captive placement, as there are limited numbers of suitable facilities and suitable candidates.Due to the lack of harmonization of permitting and/or reporting requirements between regulatory agencies, the number of wildlife rehabilitators and the number of species and animals admitted for rehabilitation in the US on an annual basis are not known. Based on federal permit numbers, organizational memberships and a dated phone survey, there are several thousand wildlife rehabilitators ,48,49. MIn the early 20th century, native bird populations were declining due to over-hunting, poaching and the millinery trade. In 1918, the Congress passed the Migratory Bird Treaty Act (MBTA), which officially made it a crime to “pursue, hunt, take, capture, kill,” or “sell” a migratory bird or any of its parts, including nests, eggs, and feathers . The BalThe Code of Federal Regulations (CFR), Title 50 Fisheries and Wildlife, Chapter 1 United States Fish and Wildlife Service (USFWS), Department of the Interior defines and describes human activities related to migratory birds such as taxidermy, import/export, scientific collection, and rehabilitation. Covered birds include all wild birds native to North America, more than 1100 species . AdminisMinimum Standards for Rehabilitation  [CFR Title 50 Part 21.76 sets out migratory bird rehabilitation policies including age and experience of the applicant and enclosures based on criteria set out by the NWRA/IWRC n, 2000) ,54. If tA condition of a migratory bird rehabilitation permit is completion of an annual report . This reAs to the scope of migratory bird rehabilitation, currently, the Migratory Bird Program has 1750 rehabilitation permits. While the total number of permits issued are tracked, the number of migratory birds, species admitted for rehabilitation and their outcomes are not tabulated except for eagles. The Raptor Center (TRC) looked at the 2011 annual reports for Region 3, which comprises eight states in the Midwest. Of the 1385 rehabilitation permits issued by USFWS that year, 254 permits were issued from Region 3. An analysis of 216 annual reports received showed that approximately 45,000 migratory birds were admitted for rehabilitation that year . AnotherChelonia mydas), leatherback (Dermochelys coriacea), loggerhead (Caretta caretta), flatback (Natator depressus), hawksbill (Eretmochelys imbricata), olive ridley (Lepidochelys olivacea), and Kemp’s ridley turtles (Lepidochelys kempii). These species are globally threatened, having been negatively impacted by habitat loss, fishery interactions, vessel collision, pollution, disease, and unusual weather events [There are seven extant sea turtle species, including green  share responsibility for sea turtle conservation ,63,64,65After sea turtles were ESA-listed, recovery plans were developed . The plans propose criteria by which sea turtle species are considered for delisting and describe activities that promote recovery. All the US sea turtle recovery plans recommend the existence of programs to manage stranded dead, ill, and injured sea turtles. The Sea Turtle Stranding and Salvage Network (STSSN) was formally established by NMFS in 1980 to document the strandings of sea turtles along the coastal US and the US Caribbean. NMFS employees participate directly in stranding response and transportation of stranded turtles in some situations and oversee mortality investigations. However, the number of NMFS employees who are assigned to this work is small relative to the number of strandings and associated resource needs. Thus, permitted private partners contribute most of the resources to respond to and document stranded sea turtles, determine causes of morbidity and mortality, and inform conservation management and recovery. While NMFS coordinates the networks, it is the participating local organizations that respond to stranded turtles, collect scientific data, transport sick and injured turtles to rehabilitation facilities, arrange veterinary care, and educate the public about sea turtle conservation. These facilities must follow the USFWS standards of care . Annual As to the scope of sea turtle rehabilitation, as of March 2022, there were 58 facilities in 21 US states and territories permitted by USFWS to provide long-term veterinary care, rehabilitation, and release for sea turtles. Release rates vary among stranding events and regions, ranging from approximately 30 to 80% ,70,71,73In the US, marine mammals are federally protected under the Marine Mammal Protection Act (MMPA) and the ESA, and their captive care is directed under the AWA. For the purposes of this publication, marine mammals include species of the order Cetacea , Pinnipedia  and Sirenia (manatees and dugongs) and select species of the families Mustelidae (sea otters) and Ursidae (polar bears). The MMPA was enacted in 1972 and prohibits, with certain exceptions, the “take” of marine mammals in US waters and by US citizens on the high seas, and the importation of marine mammals and marine mammal products into the US. As amended in 1992, the MMPA established the Marine Mammal Health and Stranding Response Program (MMHSRP), commonly called the Marine Mammal Stranding Network. The MMPA makes it illegal for anyone except agents or authorized members of the response program to respond to marine mammals in need of care and stipulates that people stay at least 50 yards away from any marine mammal, whether ill or not. The ESA, enacted in 1973, provides a program for the conservation of threatened and endangered plants and animals and the habitats in which they are found. The law also prohibits any action that causes a “taking” of any listed species of endangered fish or wildlife. “Take” as defined under the ESA means “to harass, harm, pursue, hunt, shoot, wound, kill, trap, capture, or collect, or to attempt to engage in any such conduct”.While the work of the MMHSRP is chiefly about the assessment, rescue and rehabilitation of stranded marine mammals, the effort advances scientific missions, allows for disease surveillance, provides for the early detection of such things as harmful algal blooms and serves as an incubator for advancing medical technologies, management, and conservation programs. Data amassed, and tissues cryopreserved, are the basis of research led by rehabilitation organizations and allied academic institutions. Furthermore, the care of patients that are part of healthy populations provides excellent models for the care of individual patients from populations of endangered species . Management of sea otters and polar bears is overseen by the USFWS, and all other marine mammal species are overseen by NOAA Fisheries . CertainAs to the scope of marine mammal rehabilitation, 2019 is the latest year for NOAA Fisheries’ national overview report. In 2019, there were 7719 confirmed marine mammal strandings in the US under NOAA Fisheries jurisdiction . These animals were responded to by more than 120 organizations, authorized and overseen by the same agency. This is slightly higher than the 13-year (2006–2018) average of 6365. Of these, 73% were pinnipeds , 23% were small cetaceans , and 4% were large whales  .Regulations on dealings with terrestrial animals, including wildlife rehabilitation, are determined by the states and can vary considerably. In some states, wildlife rehabilitation is not permitted on any animal, and in others, it may be limited to only native species or “non-nuisance” species. When wildlife rehabilitation of native terrestrial animals is allowed, establishments such as state departments of fish and game, natural resources, animal health and/or agriculture oversee these activities and often have permit requirements. Accessing a state’s website is the best way to determine its requirements. Requirements may include submitting an application, paying a fee, passing a test, and/or inspection by a wildlife official such as a conservation officer. In some cases, a specialized certification may be required for some species, such as large carnivores and cervids.If regulation does not specify if and/or when invasive or non-native species may be rehabilitated, it is often left to the individual centers or rehabilitators to decide whether they will accept particular species for care, euthanasia or not at all. The NWRA/IWRC Wildlife Rehabilitator’s Code of Ethics, the facility’s mission, and/or the rehabilitator’s value system may determine policies regarding invasive and non-native species ,80. The rehabilitation of rabies vector species (RVS) may also be influenced by the area’s department of health. Rabies vector species vary by region based on the prominent viral strain in the area; the most prevalent strains in the US are the raccoon, skunk, bat, and fox strains. When a RVS species is allowed to be rehabilitated, focused training and facility inspection are often required to protect the wildlife rehabilitators working with these animals. For wildlife rehabilitators working with RVS, the Centers for Disease Control and Prevention (CDC) recommends rabies pre-exposure prophylaxis (PrEP) . The emeVenomous and poisonous herptile species are not commonly presented for care, but several species exist in North America. Thus, a rehabilitator working with reptiles and amphibians must be able to identify a potentially venomous or poisonous animal and possess the skill and personal protective equipment to safely handle and treat these animals or have a resource for someone who can. The NWRA retains a small list of wildlife rehabilitators who possess permits to work with these animals. While protection of human health and animal agriculture are important considerations in wildlife rehabilitation, these policies can create an issue with how best to handle species that cannot be admitted for rehabilitation. Often these animals end up in untrained hands with poor outcomes for the animals and the public.As to the scope of terrestrial wildlife rehabilitation, based on federal permit numbers, organizational memberships and a dated phone survey, there are several thousand wildlife rehabilitators in the US ,48,49, aIn wildlife rehabilitation, there are two major categories of standards of care: policy requirements promulgated by regulatory agencies and best practices promulgated by professional groups. The permitting federal agencies and most of the permitting state agencies have minimum requirements and/or policies for rehabilitation that must be adhered to as a condition of the permit issuance (see above). The rigor of these requirements or policies varies widely and can be obtained from the appropriate regulatory authority. States may be more restrictive, but not less restrictive than federal requirements and/or policies. Federal and most state regulatory agencies require rehabilitators to have a “relationship” with a licensed veterinarian (veterinarian of record (VOR)); again, the rigor of this relationship varies widely. Licensed veterinarians are not required to obtain a federal or state rehabilitation permit to euthanize wildlife or temporarily possess and stabilize wildlife. However, within 24–48 h of stabilization, the veterinarian should transfer the patient to a permitted rehabilitator. Other federal and state acts must be adhered to during the process of wildlife rehabilitation regarding the handling and use of controlled substances and the prescribing and use of anesthetics, antimicrobials, vaccines, hormones and other substances . This isNot all wildlife admitted for rehabilitation require veterinary intervention. For example, healthy, displaced juveniles generally do not require veterinary care. In a study to characterize wildlife rehabilitation across seven northern states, the majority of rehabilitators said that a veterinarian saw 25% or less of the animals admitted; only 15% said that all animals admitted were seen by a veterinarian . The majStandards for Wildlife Rehabilitation. Currently in its 5th edition, this publication sets standards of care for ill, injured, and displaced wildlife [As for best practices, the IWRC and NWRA jointly established wildlife . While pWildlife rehabilitation continues to evolve into two distinct phases, the veterinary phase and the reconditioning and release phase. The veterinary phase, or clinical wildlife medicine, is a developing specialty within the veterinary field for the diagnosis and treatment of individual, free-living wildlife ,36,62,84The primary goal of rehabilitation is to release a healthy animal back to the wild that can survive and thrive in its natural habitat at the same level as its conspecifics. The prognosis for the animal to be restored to this level of health should be the major consideration in the decision to treat to release. When there is no realistic expectation for this outcome, a secondary option, euthanasia or captive placement, is considered.Some wildlife rehabilitation facilities have a policy to only “treat to release”; such facilities do not “treat to place.” This policy may be based on ethical grounds or on a lack of mechanisms to place animals . It shouBeginning in 1989, CFR Title 50 Part 13.41 set forth that “any live wildlife possessed under a (USFWS) permit must be maintained under humane and healthful conditions” . CFR TitThe majority of the standards of care fall under Standard Conditions (e) and include facilities, dietary requirements, disposition of birds, and reporting and record-keeping requirements. Reporting requirements include notification of the USFWS if the rehabilitator has reason to believe that a bird has been poisoned, electrocuted, shot, or otherwise subjected to criminal activity. Notification of state or local authority is required if sickness, injury, or death of any bird occurs due to an avian virus or other contagious diseases or public health hazards if an agency is currently collecting such information. Unfortunately, most wildlife rehabilitators lack the resources for laboratory testing for toxicities or disease or radiology for projectiles, resulting in significant underreporting of these conditions .Federal regulators have additional policies regarding imprinting or habituating birds, federally threatened and endangered migratory bird species, and specific requirements for the disposition of dead birds, their parts, and feathers. There are also additional policies regarding outcomes, including temporary captive placements for the purpose of fostering juveniles or with permitted falconers for the purpose of reconditioning.Standards for Wildlife Rehabilitation, the NWRA publishes Principles of Wildlife Rehabilitation [Outside of regulatory policies, there are a variety of sources for avian best practices. Many professional organizations have guidelines, standards of care, and/or publish evidence-based science. In addition to litation . Both thlitation ,91. The Veterinary management and rehabilitation of sea turtles has become quite sophisticated, and details of medical and surgical management of various conditions have been described . RecommeIn the rehabilitation facility, sea turtle wellbeing is maintained by providing adequate space, appropriate temperature, consistent light cycles, excellent water quality, excellent nutrition and dietary variety, excellent medical care, and environmental enrichment. Clearly, one cannot reproduce the freedom and habitat complexity of the oceanic environment for such highly migratory and athletic species . HoweverSea turtle rehabilitation programs have the same disposition options as those relevant to other taxa. In almost all scenarios, sea turtles are released to the wild, which offers them a chance to reproduce and contribute to the population of their species. Euthanasia and permanent captivity remove the turtles from the reproductive population; thus, these options are only elected in extreme circumstances as discussed in Only entities that hold a letter of authorization from NOAA Fisheries may rehabilitate marine mammals that fall under the auspices of the MMPA. The care of these species is guided by the 2022 NMFS Standards for Rehabilitation Facilities . The capA marine mammal “strands” when it is out of its normal habitat. Whales, dolphins, and porpoises (cetaceans) are considered stranded when they are found dead, either on the beach or floating in the water, or alive on the beach and unable to return to the water. Seals and sea lions (pinnipeds) are considered stranded when they are found dead on land or in the water, or are in need of medical attention. Because healthy pinnipeds come on land to rest, expert assessment may be needed to determine whether they need help. Live-stranded animals usually need medical attention or professional assistance to return to their natural habitat . Marine mammal rehabilitation has the same dispositions as non-marine mammal wildlife rehabilitation. While rehabilitation time varies, a patient may not be kept in rehabilitative care for more than 6 months without specific authorization. NOAA Fisheries and a veterinarian must approve a release to the wild or a captive placement. Euthanasia must be guided by AVMA guidelines.https://www.aazv.org/, accessed on 5 April 2023), the International Association of Aquatic Animal Medicine (IAAM)  and the AZA  are offered as sources of information.There exists a robust body of literature around rehabilitative care of marine mammals, namely pinnipeds, small cetaceans, and otters. Rehabilitators are referred to 2022 NMFS Standards for Rehabilitation Facilities ,96,97. Ihttps://www.fisheries.noaa.gov/west-coast/marine-mammal-protection/deterring-nuisance-pinnipeds (accessed on 15 April 2023)).Guidelines are provided and legal and acceptable measures are allowed for the safe “hazing” of healthy animals from areas where they pose a threat to themselves, people, other animals, or property. Relocating marine mammals, such as those that may be hauled out on docks in busy marinas, may well avert harm or injury, and prevent the need for active rehabilitation. This is a complicated reality for rehabilitators who are called on to care for ill or injured marine mammals and who also may be sought when marine mammals are seen as nuisances. The best response in such instances is for the responder to refer the inquiring party to NOAA’s website for deterring nuisance pinnipeds . On average, each year, 1.2 million birds are banded, and 87,000 encounters are documented. An “encounter” is any observation of a previously banded bird, living or dead. Banding is inexpensive and relatively passive. Unfortunately, the encounter rate of rehabilitated birds is low (5–8%), requiring a large number of birds to obtain statistically significant results. The third strategy for post-release monitoring is radio telemetry that uses Very High Frequency (VHF) transmitters or the Global Positioning System (GPS), cellular or satellite tracking. Telemetry allows for more detailed monitoring, but has a variety of limitations including cost, loss of the transmitter, and technological malfunctions. The transmitter must not impair any of the bird’s activities. Unfortunately, there is limited data from post-release monitoring of rehabilitated birds. The majority of articles come from oiled bird studies because some of the monetary damage assessments to the responsible party can be used for post-release monitoring ,130,131.https://www.fws.gov/migratorybirds/pdf/policies-and-regulations/templatevetletter.pdf, accessed on 28 March 2023). The bird must be examined by the veterinarian within 30 days of the proposed transfer. The veterinarian’s statement must fully describe the medical or behavioral condition of the bird, why it renders the bird non-releasable to the wild, and why the bird is suitable for life in captivity. If the bird is being transferred, the veterinarian must provide the name of the veterinarian or animal care professional whom they consulted with at the receiving facility.Avian rehabilitators may place non-releasable live birds that are suitable for use in educational programs, foster parenting, research projects, or other permitted activities with persons permitted or otherwise authorized to possess such birds with prior USFWS approval . WheneveIf the bird’s conditions meet the euthanasia criteria above (blind or has amputations), but the bird is not euthanized, there are additional requirements. First, the veterinarian must explain why the bird is not expected to experience long-term complications in captivity associated with the above-described injuries and/or ailments. Second, the veterinarian must express a commitment to provide medical care for the bird for the duration of its life, including complete examinations at least once a year, or, if the bird is being transferred, the name of the veterinarian who will provide such care. Third, an appropriate placement must be available. Lastly, the USFWS must specifically authorize the continued possession, medical treatment, and rehabilitative care of the bird . It is unclear from 50 CFR 21.76 how this process should proceed in acute medical situations. Often, a decision is made by a rehabilitator and/or a veterinarian to proceed with medical or surgical treatment that will render the bird non-releasable prior to determining the bird’s emotional suitability for captivity, placement availability, or authorization from the USFWS.Captive placement has the most regulatory requirements, as it should, because it has a significant impact on animal welfare. The natural life span of many avian species runs into decades or longer in captivity. Captive placement of an individual with physical or behavioral issues can lead to a poor quality of life for a lifetime. Many non-releasable birds are placed in zoos or nature centers, either as exhibit birds or for use as ambassador animals. Live ambassador animals are thought to engender public empathy, and by doing so, help promote ways in which people can act in relation to wildlife. It is likely that more birds are captively placed than any other rehabilitated taxa. The Raptor Center evaluated the placement of native birds via the 2017 Education (Live) permits. Nationally, there were 701 Live permits, with 162 in Region 3 (Upper Midwest). The Region 3 permits contained 1250 birds of which the vast majority were raptors. These birds came almost exclusively 89%) from dedicated wildlife rehabilitation organizations. It should be noted, however, that there are hundreds of facilities  that are exempt from these permits, except in the case of eagles, so the actual number of native bird placements on an annual basis is much higher [% from deBeginning in February 2024, the AWA will expand to include some avian species. Wildlife rehabilitators who also maintain birds under the USFWS Migratory Bird Special Purpose Possession—Education (Live) and who have more than four raptors for exhibition will be required to apply to USDA APHIS AC for an exhibitor’s license . Not all regulators allow for captive placement and not all facilities engage in captive placement, often based on ethical grounds. Disabilities that lead to a non-releasable status can also preclude a high quality of life in human care. Increasingly, avian facilities are only accepting those birds with the physical, psychological, and behavioral capacity to have a life-long high-quality welfare ,120. The International Association of Avian Trainers and Educators (IAATE) recently published a position statement titled Selection Considerations for Non-releasable Birds that states that “at a minimum, non-releasable birds used in education should be able to eat on their own, maintain good feather condition with regular preening, bathing, and the absence of fractious behavior, exhibit species-appropriate behaviors, have full mobility, and willingly engage with trainers” . The posThe decision to place a bird should be made after rehabilitation for release to the wild has failed; except for very rare circumstances, birds should not undergo rehabilitation for the purpose of placement. All medical issues should be resolved, and a current physical exam performed to ensure that the bird’s physical condition is stable and will not result in any pain or discomfort. Next, the bird should be evaluated by an experienced avian behaviorist to determine emotional and behavioral adaptability to captivity. Finally, a suitable facility with all the resources required to support this individual bird must be available ,119. BirWildlife in Education: A Guide for the Care and Use of Program Animals [https://assets.speakcdn.com/assets/2332/ambassador_animal_evaluation_tool.pdf, accessed on 28 March 2023) and the Ambassador Animal Resource and Information Center . IAATE also has resources .There are best practice resources for non-releasable birds placed into educational programs. The NWRA published a book titled  Animals . In addi Animals . The AZAAn excellent review of welfare considerations for sea turtles in human care was recently published , and a nThe welfare of sea turtles in general will be improved by ongoing efforts to reduce anthropogenic injury and illness. If interactions by boat strikes and fisheries could be reduced, many fewer sea turtles would require hospitalization. Technological methods such as turtle excluder devices and ropeless fishing technology offer hope; public outreach methods in certain regions at certain times  could reduce boat-strike injuries ,9. During initial recovery of sea turtles from a stranding location, welfare considerations include prevention of further injury and pain, provision of proper environmental temperature, limitation of physiologic stress, and efficient transfer to an authorized rehabilitation center. In general, only personnel from permitted agencies should physically interact with a stranded sea turtle, although good Samaritan interventions may occur. Initial efforts may include moving a turtle away from drowning risk and away from temperature extremes and intense sunlight. Responders must be cautious to support injured appendages and shell fractures to prevent further injury and pain. Planks of wood, blankets, etc., may be used cautiously to support the turtle from below to relocate it. In some cases of “debilitated turtle syndrome,” turtle bodies may become so fragile that they are at risk for catastrophic injury during handling  and extreme care is needed ,94. TempSeveral conditions may lead to a decision to euthanize a sea turtle or to retain it under permanent human care, including spinal injuries, total blindness, multiple limb amputations, chronic buoyancy disorders, and severe fibropapillomatosis. Consent for euthanasia or permanent human care must be acquired from official regulatory agencies (such as USFWS). When euthanasia is elected, multimodal methods are recommended, generally including general anesthesia, followed by intravenous pentobarbital, and one or more adjunctive methods such as intrathecal or intracranial lidocaine, and intravenous or intracardiac potassium chloride (to end cardiac function that can otherwise often persist for hours) ,142,143.Upon completion of rehabilitation, the wellbeing of sea turtle patients is enhanced by efficient release to the wild. USFWS requires sea turtles be examined by a veterinarian to ascertain good health prior to release, and then, unless other circumstances prevent it, turtles must be released within two weeks of medical clearance . DelayedThe choice of release location for rehabilitated sea turtles may also affect their welfare. The ocean is a dangerous place, and it is impossible to select a release location with no risk. Retrospective and prospective studies are needed on the long-term fate of sea turtles after release from various locations. It is intuitive that areas of high fishing intensity and vessel traffic should be avoided when possible. However, other factors that may affect long-term fate, such as proximity of release sites to breeding areas or seasonal foraging areas, have not yet been studied. After release, long-term survival of sea turtles is difficult to determine in the oceanic environment, relying largely on subsequent happenstance. While not an absolute requirement, many rehabilitation facilities acquire USFWS authorization to insert passive integrated transponders (PIT) and/or flipper tags prior to release, following federal specifications for turtle size and tag size ,74. HoweWhile sea turtles are robust, and can often survive with chronic conditions, the wellness of the turtle as well as long-term resource allocation must be considered. A decision toward permanent human care implies a decades-long commitment to meeting the complex environmental, nutritional, and wellness needs of a large marine animal . While tConcurrent growth in human populations along US coastlines, growing, sometimes resurgent, populations of some species of marine mammals, and dynamic and altered ocean, bay and estuary environments are placing humans and marine mammals in closer proximity and presenting risk. This urban: wildlife interface introduces the opportunity for an increased number of negative human interactions with challenging and sometimes fatal results for marine mammals . Entanglement of whales, dolphins, seals, sea lions and otters occur in the ropes, lines, nets, and other equipment that are part of fisheries, both commercial and recreational. These entanglements can often prove fatal for the marine mammals involved. While quickly fatal entanglements are tragic, sublethal entanglements that leave an animal to tow entangled lines or to endure slowly tightening constrictions of the neck or limbs are a welfare concern of heightened proportion due to the suffering they cause. The state of fisheries themselves have bearing on the state of welfare of marine mammals. Consistent and healthy stocks of forage fish are relied on by marine mammals and influence the success of trans-oceanic migrations, the rearing of the young, and more. Fish stocks are impacted by dynamic pressures such as oceanic temperature increase relative to climate change. Notably, a multiple-year occurrence of anomalously warm water in the ocean around California’s Channel Islands impacted the ability of nursing California sea lions to successfully forage for cold-water feed fish while nursing their young. Some dependent pups are left unattended on beaches while females forage offshore. The pup is vulnerable to predation, wave action, and more during such periods. Unfortunately, well-meaning members of the public visiting coastal beaches encounter these pups, mistakenly perceive them to be abandoned, and remove them from the beach, effectively orphaning them and requiring their rehabilitation. While a marine mammal stranding is an obvious concern for the animal, the situation also poses risks to the public, pets, and property as the occurrence is often on busy and heavily trafficked beaches and waterfronts. A response to a stranded marine mammal is, in fact, a public service performed by responding parties in addition to one driven by animal welfare interests. The risk of disease transmission, injurious bites, and property destruction is very real, and even the act of bringing an animal into care puts a responder at risk. For this reason, intensive classroom and field training is typically conducted for paid staff and volunteer team members of network organizations.By examining stranded marine mammals, NOAA Fisheries and stranding network partners can better understand causes of mortality and factors that affect marine animal health. Other goals of stranding response efforts are to facilitate humane care out of animal welfare concerns, assist in the recovery of protected species by returning them to the wild whenever possible, and help identify population threats and stressors of marine life, especially stressors that can be prevented or mitigated.When appropriate, based on a patient’s grave clinical prognosis or at the discretion of the attending veterinarian, euthanasia may be performed. When this is decided, the process must be guided by best practices and specifically by AVMA guidelines for euthanasia .Euthanasia of any marine mammal, whether in a rehabilitation facility or on a beach, warrants extreme care for the welfare of the individual animal, for the professionals attending to the animal, and for those who may be witness to the procedure. For pinnipeds, the procedures are often straightforward, and animals can typically be removed from public locations prior to euthanasia. For small and large cetaceans, the situation can be quite different; the animals are often in highly visible locations and are not easily relocated. The best methods of euthanasia that optimize animal welfare are yet evolving. Because of this, much attention has been placed on both the methodology of euthanasia for cetaceans, especially for large cetaceans, and the consideration of public perception for these procedures ,151,152.To bring a marine mammal into rehabilitation presents its own welfare considerations. The assessment at the stranding location alone can cause stress, as the animal, e.g., a pinniped, is closely observed, cordoned off from other risks of people and pets and otherwise constrained. Capture is inherently stressful and can cause injury, and transportation adds an additional overlay of stress and risk, which can be sustained for an hour or more. Arrival at a rehabilitation facility begins yet another suite of activities, each of which, while necessary to assess, diagnose, treat, or care for the animal, is, by its nature, stressful. While in care, marine mammals are at risk of becoming habituated to people to the point that they manifest behavior that is inconsistent with their wild cohorts, putting them at risk for negative human interactions when released. Care environments can be devoid of novel and enriching stimuli for what are often neonate and juvenile individuals. Emerging practices of environmental enrichment and research in enrichment practices can facilitate the development of physical skills and mental and behavioral development in marine mammal patients under care that will better prepare them for release ,154,155 Prior to release, and usually at the time of admission, tags with unique alphanumeric markings are placed on patients, and in certain circumstances, satellite tags are placed on individuals representing endangered species or for those animals of significant interest from a medical or other perspective. Care must be taken in choosing a release site as patients are optimally released in areas that are deemed safe and at which there are conspecifics. Following successful rehabilitation, upon clearance from attending veterinarians, and with approval of NOAA Fisheries, marine mammals may be released to the ocean. Such release is conducted under the care of professional staff to ensure the safety of the animal and attending personnel.Data are limited on animal behavior, activity, and survivorship following rehabilitation. While satellite monitoring of some marine mammal species  has demonstrated success, there is need for further study. Meanwhile, anecdotal information, such as patient re-sightings with dependent pups, offers positive, if limited, insights ,157. When a marine mammal patient is deemed to be non-releasable by the attending veterinarian, based on a determination that while otherwise healthy, the patient would likely not be able to thrive in the wild , captive placement is considered at an accredited zoo or aquarium in a process overseen by NOAA Fisheries. In such cases, consideration is paid to the quality of care and habitat provided by the receiving institutions. This includes a consideration of the presence or absence of animals of the same species, gender distribution, personnel training, environmental enrichment capacity, facility accreditation, and more. Professionals in the field of wildlife rehabilitation may have the opportunity to prevent displacement or unnecessary capture of a wild animal prior to its admission. Many good Samaritans mistake a healthy juvenile for an orphan or misinterpret natural behavior as concerning and interfere with the animal. Additionally, the actions of many humans, such as using certain rodenticides or allowing a pet cat to roam freely outdoors, can be disruptive and even fatal to wildlife. The number of wild mammals killed by domestic cats in the US each year was estimated in 2013 to be between 6.3 billion and 22.3 billion . It is iFor animals that cannot or should not be returned to the wild, there are two possible outcomes: euthanasia or placement in a permanent captive care situation for the sake of education, fostering, and/or breeding. When euthanasia is deemed the appropriate course of action, it should be performed as soon as possible to delay further suffering and stress. The process should be performed by a veterinarian or by trained personnel following the most current AVMA guidelines .The maintenance of an animal’s welfare during the rehabilitation process poses a challenge since simply by being able to be captured, transported, and held in captivity, the animal’s wellbeing declines. Even in cases when the species and nature of injury or illness are familiar and the animal’s physical needs can be appropriately met, maintaining a high-quality mental state of the animal often has limitations. Having an environmental set up that is quiet with a lower level of human-related noises and reduction in sensory stimuli may reduce the stressors . Our intWildlife rehabilitation requires animals with previously limited or non-existent human contact to be looked at, restrained, and manipulated by human caretakers. In some cases, these interactions include a painful or unpleasant component as is often necessary during a physical exam, wound management and fracture immobilization and diagnostic testing. Despite the necessity of these interactions for the ultimate, hopeful positive outcome of return to the wild, they yield a temporary negative impact on the individual’s welfare that is almost always unavoidable . To combThe nature of wild animals is often to mask injury or illness, so welfare assessment is further limited by this innate trait. Remote monitoring and telemetry systems improve animal welfare by monitoring stress levels, behavior, assessing response to therapy, or as a diagnostic tool, without needing to restrain the animal ,165,166 Enrichment in rehabilitation may also reduce the distress a wild animal experiences in captivity. The goals of enrichment programs in rehabilitation include increased diversity and range of expressed normal behaviors and reduced frequency of abnormal behavior. The benefits of intentional enrichment programs in wildlife rehabilitation can encourage natural behaviors and increase quality of life while an animal is in temporary captivity. There are generally five accepted categories of enrichment. All these categories are customized for each individual depending on the stage in rehabilitation and natural history. The enrichment categories are: social, cognitive, sensory, nutritional, and the physical habitat/enclosure design including cage furniture ,168. Finally, the use of masks, costumes, and/or puppets during the care of orphaned wild animals may likewise reduce stress associated with overt human contact and reduce the chance of habituation or imprinting  169,170170.When considering an animal for release, important criteria need to be evaluated before the animal is returned to the wild. First, the animal must be fully recovered; second, the animal must be able to survive in its natural environment, which entails finding food, water, shelter, and mates and defending itself from potential predators. In sum, terrestrial animals should be able to survive in the wild at the same level as their conspecifics . FinallyPreparation for release should include reviewing the animal’s original recovery location, preferred natural habitat, seasonal activity, and the effect the newly returned animal will have on the ecosystem to which it is returning. Some species have such a high site fidelity that they should only be returned to their specific point of origin. In fact, in some states, the release of reptile and amphibian species is forbidden if the natural point of origin is unknown. Additionally, due to concern for emerging infectious and highly fatal diseases ravaging many reptiles and amphibians, these animals may have additional restrictions regarding releasing them back to the wild . Ironically, the period of transport for release can still have significant negative effects on the welfare of the animal even though the rehabilitation process is effectively over. Transport of wild animals has been documented to increase the risk of infectious disease , and a rThere are two main types of release options: soft and hard. Age and species usually dictate what type of release is best. Soft releases are selected when the animal needs support for a period of time after it returns to the wild, as may be the case with animals that were originally presented as orphans. The animal is often kept in an enclosure at the release site for a determined amount of time. When ready, the enclosure is opened to allow the animal to leave but it is left in place, often with supplemental food. The animal then has the option to explore its new surroundings while still having some of its physical needs such as food, shelter, and safety provided with the important benefit of minimizing ongoing human contact. A hard release occurs when the animal is released into an appropriate habitat with no ongoing support after release.Journal for Nature Conservation found that by simulating advanced populations in five species of terrestrial animals commonly encountered in rehabilitation settings, rehabilitation efforts have the potential to support recovery and stabilization of populations in some cases [Post-release evaluation of terrestrial animals from rehabilitation settings is often limited but is a growing area of needed research. When performed, this research may use radio telemetry, GPS collars or passive integrated transponders (PIT) tags, among other methods. Results are often highly variable based on the species, reason for admission to rehabilitation and location of release, but a recent study in the me cases .When assessing “success” following the release of a wild animal, one is often focused on the individual rather than the population. Measurements may include long-term survival, integration into the local population if appropriate for the species and successful reproduction of the individual, but success should also focus on the maintained health status of the population and environment into which the animal has been released. A decline in the health of a local population or ecosystem as a result of introducing a rare or novel disease or parasite in the released animal, for example, would likely not be considered successful, despite the survival of the single animal .Most wild animals presented to wildlife rehabilitation facilities are not suitable for placement. This may be due to permanent and often painful deficits from the severity of the initial sustained injury or illness, the animal’s inability to comfortably perform natural behaviors in captivity, and/or a mental status that will not allow for a good quality of life in permanent captivity. Even for animals that may be candidates for placement, there are often no suitable placement options available. For terrestrial animals, restrictions for placement are usually at the state level.For placement, the animal should be medically evaluated and should “pass” a behavioral test to gauge its comfort level in captivity. Animals need to be fully healed from any injuries before placement is pursued. Applications from the placing facility and from the receiving facility need to be approved by state regulators, and most states require a letter from the VOR stating why the particular animal is not releasable and would make a good candidate for placement. Facilities placing animals need to be convinced that the receiving facility has the expertise and resources to care for, maintain and train the particular animal. Receiving facilities need to conduct due diligence to be assured that the animal is the “right animal for the job.” The receiving facility should be able to meet the current welfare needs of the animal, including diet and space needs as well as anticipate and be prepared to manage any future problems associated with the original illness or injury that would negatively impact the animal’s wellbeing.Hundreds of thousands of wild animals are presented for rehabilitation in the US every year, their lives interrupted. Increasingly, our society recognizes the concept of animal welfare and understands that humans negatively impact the welfare of animals, including wildlife. Many also acknowledge our moral obligation to promote good animal welfare. This paper discusses the intersection between wildlife rehabilitation and wild animal welfare. It presents a comprehensive overview of the practices of wildlife rehabilitation in the US for multiple taxa, and the complexity of considerations rehabilitators and veterinarians face while trying to optimize the welfare of wildlife in need of care and rehabilitation.The goal of wildlife rehabilitation is to release an animal back into the wild, a resumption of its life’s path. However, this oft-stated goal ignores reality. In fact, we have no idea what this “second chance” holds for all but a few of the animals that are released. This goal also neglects the reality of the other outcomes of rehabilitation—euthanasia, death in care, and captive placement. Maintaining the individual animal’s welfare at the center of the rehabilitation process requires deliberate, timely and humane decision making. Wildlife rehabilitation is costly in terms of animal welfare, financial resources, and people’s time and wellness. Improving the welfare of wild animals before, during, and after rehabilitation requires strengthening and harmonizing regulatory policies, investment in evidence-based science in rehabilitation, clinical wildlife medicine, and post-release monitoring, as well as forward-thinking and inclusive ethics, including the following items.Preventing Anthropogenic Causes of Admission. Increasing numbers of animals are being admitted due to anthropogenic causes related to human activities ,116. ExaProviding Resources and Support for Rehabilitation. In the US, wildlife rehabilitation is largely paid for by private individuals and donations. The adoption of established best practices may be limited by physical and/or financial resources. Having public funding at the federal, state, or county level specifically directed to wildlife rehabilitation would allow for improved care and outcomes of wild animals. Although urban wildlife management is a growing field within state fish and game departments, a dedicated revenue stream for wildlife rehabilitation has yet to be identified ,175.Continued Development of Evidence-based Wildlife Rehabilitation Methods and Welfare Measures. The advent of electronic database programs specific to wildlife rehabilitation ,178,179 Greater Engagement of the Veterinary Profession. Wildlife welfare would benefit greatly from increased veterinary involvement in wildlife rehabilitation. All animals need a rapid and thorough assessment within the first few hours of presentation. Only veterinarians can provide diagnostic testing, surgical care, access to analgesics, the most humane euthanasia methods, and recommend captive placement. There is training available for veterinarians and veterinary students ,182.Harmonization of Regulatory Oversight with Standards of Care, Training, and Accountability. Federal and state regulatory policies should be reviewed and updated on a regular schedule. These reviews should incorporate the most recent evidence-based science and enhance standards of rehabilitative care, training, and accountability to ensure optimal welfare for wildlife. Increasing harmonization of wildlife rehabilitation policies within federal programs, between state programs, and between federal and state programs would also improve wildlife welfare by ensuring prompt care and allowing for more data aggregation. Ongoing collaborations between federal and state level regulators, wildlife rehabilitators, and clinical wildlife veterinarians help prepare for and mitigate the challenges ahead for wildlife and rehabilitation.Increasing Public Education. Interactions between urban wildlife and the public are increasing. Stimulating interest in local wildlife to prevent conflict, countering misinformation about animal behavior, and providing tools for successful coexistence with wildlife is another strategy to reduce the number of animals in need of rehabilitative care ,183,184.


Importantly, β-ARs are an established target in the treatment of glaucoma. Moreover, β-adrenergic signaling has been associated with the development and progression of various tumor types. Hence, β-ARs are a potential therapeutic target for ocular neoplasms, such as ocular hemangioma and uveal melanoma. This review aims to discuss the expression and function of individual β-AR subtypes in ocular structures, as well as their role in the treatment of ocular diseases, including ocular tumors.β-adrenoreceptors (ARs) are members of the superfamily of G-protein-coupled receptors (GPCRs), and are activated by catecholamines, such as epinephrine and norepinephrine. Three subtypes of β-ARs (β The research was performed from 13 November 2022 to 23 January 2023 with the following inclusion criteria: all studies, written in English, and published after 1948. Studies reporting on the role of β-adrenoceptors in non-(peri)ocular tissues were excluded. The reference list of all selected articles was reviewed for further identification of potentially relevant studies.Catecholamines, such as adrenaline and noradrenaline, act via α- and β-adrenoceptors (ARs) to regulate central physiological functions, such as blood pressure, heart rate, and contractility, as well as metabolic and central nervous system functions . ARs areubfamily . The purThe theory on the existence of specific receptors binding drugs or intracellular molecules for regulation of cellular mechanisms was first introduced by John Newport Langley in the early 1900s . The hyp1-, β2-, and β3-ARs) can be distinguished based on pharmacological studies [4-AR turned out to be a specific affinity state of the β1-AR [1-AR has high affinity to adrenaline and noradrenaline, and is expressed predominantly in the heart, brain, and adipose tissue. A lower affinity to noradrenaline is found in the β2-AR subtype, which is more involved in relaxation of vascular and other smooth muscle cells, and further metabolic mechanisms of catecholamines [3-AR is abundantly expressed in adipose tissue, but is also expressed in the eye [Binding of an agonist to a β-adrenoceptor causes a dissociation of Gα-GTP and Gβγ subunits with consequent activation of adenylate cyclase, and production of the second messenger cyclic adenosine monophosphate (cAMP). This leads to activation of downstream effectors, such as cAMP-dependent protein kinase (PKA) and cAMP-dependent phosphorylation of gated ion channels ,7. Among studies . The prehe β1-AR . The β1-olamines . The β3- the eye ,12. β-AR the eye . In this2-ARs have been detected in corneal epithelial cells [2-ARs in corneal re-epithelialization [2-ARs, and consequent increase in extracellular signal-regulated kinase (ERK) phosphorylation, corneal epithelial cell migration and corneal wound healing was enhanced [2-AR antagonists by modulating the expression of Ki67, and phosphorylation of ERK1/2 in the limbal and regenerated corneal epithelium [2-AR in homeostasis of the corneal epithelium. However, the definite role of β-AR signaling in corneal wound healing remains to be examined in further studies.Adrenergic nerve fibers originating from the superior cervical ganglion innervate the surface of the cornea. In adult human corneas, these nerve fibers could be identified by sodium-potassium-glyoxylic acid-induced fluorescence ,15. Bothal cells ,21,22. Tlization ,24,25. Senhanced . Recentlithelium . These cConjunctival functions, such as secretion of mucous substances from goblet cells, and mobilization and attraction of conjunctival eosinophils, seem to be controlled by sympathetic nerves. These functions might be involved in the pathophysiology of common conjunctival diseases, such as allergic conjunctivitis and dry eye disease ,29,30,312-ARs was also demonstrated [1- and β2-ARs could be detected in conjunctival goblet cells in developing rats [3-AR subtype was only seen on epithelial and goblet cells of the human conjunctiva, but not in conjunctival cells of the mouse [1- and β3-ARs on cell membranes and in intracellular compartments. The β2-AR was detected only intracellularly under normal culturing conditions. Furthermore, immunofluorescence microscopy was performed, and β1- and β2-AR subtypes, but not β3-ARs, were detected in IOBA-NHC cells [1-AR was observed in all epithelial cell layers of human conjunctival biopsy specimens of patients with vernal keratoconjunctivits, suggesting an involvement of the autonomic nervous system in pathogenesis of the disease [2-AR agonists, such as salbutamol and terbutaline, reduced microvascular permeability, and exerted anti-inflammatory effects in allergic conjunctivitis [In cell culture experiments with primary human conjunctival epithelial cells, the presence of βnstrated . Using fing rats . The β3-he mouse . In the HC cells . In biopHC cells . Notably disease . Furtherctivitis ,36. Thes2C-, β1-, and β3-ARs was detected in acinar cells of the rat lacrimal gland [1- and β2-ARs to secretion in the lacrimal gland was suggested [1-ARs were the predominant AR subtype in the glands of Wolfring [2-ARs was suggested to stimulate lipid synthesis [The lacrimal gland is composed of acinar cells, myoepithelial cells, and ductal cells . With phal gland . Howeveral gland ,41,42,43uggested . NeverthWolfring . In epitynthesis . These fynthesis ,39. Base2-ARs has been shown [2-ARs was detected in pharmacological and radioligand binding studies in cultured human trabecular cells and in trabecular meshwork from human eyes [2-AR after adrenaline and noradrenaline stimulation was demonstrated in studies on monkey and human eyes [2-ARs increases outflow facility in the trabecular meshwork.In sections of the trabecular meshwork, predominant expression of βen shown . Moreoveman eyes ,49,50. Aman eyes . Furtherman eyes . In summ1- and β2-ARs was reported [2-ARs coupled to adenylate cyclase were detected in the ciliary process epithelium in various species, including humans [2-AR antagonists to β-ARs on ciliary processes in radioligand binding studies, and potent lowering of intraocular pressure in various species, β2-ARs are a pharmacological target in glaucoma treatment [3-AR agonist, BRL37344, revealed that the β3-AR may be involved in choroidal endothelial cell invasion and elongation [2-AR may participate in the regulatory process of VEGF and IL-6 expression in cells of the choroidal endothelium and other cells, indicating that blockade of these receptors may attenuate formation of choroidal neovascularization [The uvea consists of three parts: the iris, the ciliary body, and the choroid. In the human ciliary body, abundant expression of βreported ,55,56,57reported ,59. β2-Ag humans ,61,62. Ag humans . Vasocong humans . Due to reatment ,66,67,68reatment . In humareatment . Sympathreatment . Experimongation . Moreoverization ,72.The retina is a complex neuronal multilayer processing visual information to the brain . Six maj2), carbon dioxide (CO2), nitric oxide (NO), and hydrogen sulfide (H2S) [The supply of the retina with oxygen and other nutrients is ensured by two different vascular beds, both originating from the ophthalmic artery, the retinal circulation, and the choroidal circulation . Choroidde (H2S) ,80,81. Ade (H2S) . A potende (H2S) ,84. In sde (H2S) .1-, α2-, and β-ARs [1-, α2-, and β-ARs, have been detected in retinal tissue, including endothelial cells of retinal blood vessels [The predominant catecholamine in the retina is dopamine, synthetized and released by dopaminergic amacrine (DA) cells ,86. A pond β-ARs . It is i vessels ,92,93,94Sympathetic neurotransmission presents a possible mechanism to regulate expression of inducible nitric oxide synthase, angiogenic growth factors, and the number of pericytes in the retina, as described below ,96,97.β-ARs were detected in the outer nuclear layer, the outer plexiform layer, the inner nuclear layer, and the inner plexiform layer of the rat retina . In the 1-AR and β2-AR binding sites have been detected [3-ARs have also been found in rat retinal blood vessels [3-ARs in the inner capillary plexus of the mouse mid-peripheral retina [3-ARs induced cell migration and proliferation of retinal vascular endothelial cells [1- and β3-ARs has been reported in human retinal endothelial cells [In bovine retinal vessels and in the neural retina, βdetected . Functio vessels . Immunohl retina ,102. Intal cells . Furtheral cells . Due to 1- and β2-ARs is protective against retinal angiogenesis, and can enhance blood-retinal barrier function [1- and β2-ARs [2-AR by ICI 118,551 in a mouse OIR model also resulted in decreased retinal levels of proangiogenic factors, and reduced pathogenic neovascularization, suggesting that β2-AR blockade may be a potential way to block retinal angiogenesis [During stress conditions, such as hypoxia, catecholaminergic responses from the cardiovascular system are enhanced leading to activation of β-ARs . Under hfunction . Anotherd β2-ARs . Selectiogenesis . Howeverogenesis ,105,106.ogenesis . Modulatogenesis .These contradictory findings lead to the suggestion that the effects may be mediated through diverse regulatory mechanisms depending on the retinal disease and the experimental setting.2-AR desensitization that downregulates expression of β2-ARs in the retina, which in turn exerts an inhibitory effect on VEGF expression in OIR [Previous studies reported that the nonselective β-ARs agonist, isoproterenol, can cause agonist-induced βn in OIR .3-ARs were shown to have an impact on neovascularization processes of various retinal vascular diseases [3-AR immunoreactivity in engorged retinal tufts, upregulation of β3-ARs in response to hypoxia indicated that activation of β3-ARs also plays an important role in pathologic angiogenesis [3-AR gene in the hypoxic retina, supporting the hypothesis that β3-ARs may participate in the angiogenic response in hypoxia [3-AR antagonists, L-748,337, and SR59230A [3-AR agonist, CL316243 [3-ARs, which are not coupled to a G protein receptor kinase are resistant to agonist-induced desensitization [3-AR may represent an attractive therapeutic target for the treatment of ischemic retinal diseases.βdiseases . For exaogenesis . Further hypoxia . Due to  hypoxia ,112. DowSR59230A . FurtherCL316243 . In additization ,116. Theβ-AR stimulation was also shown to increase human and mouse pericyte survival under diabetic conditions . In supp1-AR agonist xamoterol [Moreover, β-ARs are involved in regulation of inducible nitric oxide synthase (iNOS) expression . Activatamoterol . An overInfantile hemangioma is a benign vascular tumor, which typically occurs as a cutaneous lesion in early childhood with a high incidence of approximately 4–5% of infants ,120. It The therapeutic effect of β-blockers on infantile hemangioma was found by chance, when patients were treated with β-blockers for cardiopulmonary indications, and regression of hemangioma was observed . Since tA randomized controlled trial found that combination of systemic and topical β-blockers in the early proliferative stage is significantly more effective than systemic treatment only . There aChoroidal hemangioma is a benign vascular tumor located at the ocular posterior pole. It can be distinguished between circumscribed choroidal hemangioma (CCH) and diffuse choroidal hemangioma. Circumscribed choroidal hemangioma emerge sporadically and are not associated to local or systemic abnormalities. CCHs are solitary, well-demarcated lesions located posterior to the equator . They arThe pathophysiology of choroidal hemangioma Is not fully understood. Choroidal hemangioma is supposed to be a congenital vascular hamartoma that is composed of choroidal vessels. Histological studies revealed that choroidal hemangiomas are nonproliferative tumors with tumor growth by venous congestion rather than cell proliferation ,166. In Retinal hemangioblastoma (RH) is a main component of ocular manifestation of Von-Hippel Lindau (VHL) disease. A national study with 64 genetically tested participants with RH revealed VHL as the underlying cause in 84% of the cases . VHL dis2-AR antagonist, ICI-118,551, in hemangioblastomas in VHL disease [Histological studies revealed that RH are composed of abnormal capillary-like fenestrated channels surrounded by vacuolated stromal cells, and tumorlet-like cells expressing stem cell markers . A loss  disease . Hence,  disease . Moreove disease . These sConjunctival hemangiomas are benign vascular tumors located at the bulbar conjunctiva that can grow sessile or pedunculated . CongeniConjunctival hemangiomas consist of vascular channels lined by endothelial cells with positive immunostaining signals for CD31 and CD34, but with negative signals for the smooth muscle marker desmin . There a3-AR is associated with a lack of response to propranolol [As described above, non-subtype-selective β-blockers represent an effective method to treat different types of ocular hemangioma, and these effects have been discovered by chance. Expression of all three subtypes of β-ARs in hemangiomas has been demonstrated . Howeverpranolol .2-ARs, and induce vasodilatation via endothelial vasorelaxing factors, such as nitric oxide [Early effects of propranolol may be due to intralesional vasoconstriction caused by decreased release of nitric oxide . Catechoic oxide . Throughic oxide ,205. Cliic oxide ,205. HowIntermediate effects of propranolol treatment could be explained by a reduction in proangiogenic factors, such as vascular endothelial growth factor (VEGF), matrix metalloproteinases (MMPs) or proangiogenic cytokines, such as interleukin-6 (IL-6) . CatechoLong-term effects of β-blockers in hemangiomas may be explained by induction of apoptosis in endothelial cells during the proliferative stage ,215,216.Uveal melanoma is the most common primary intraocular malignant tumor in adults. In Europe, standardized incidence rates of 1.3–8.6 cases per million per year were reported . HoweverA variety of clinical trials on targeting β-ARs to treat ocular hemangiomas/hemangioblastomas has been conducted. The trials are listed in In cutaneous melanoma, β-ARs were found to be a new target for inhibition of tumor growth and dissemination . In diff1- and β2-ARs in all patients with uveal melanoma, and a higher expression was found in more aggressive epitheloid cells [3-AR in melanoma growth and vascularization was identified in cutaneous melanoma cells of mice [3-ARs in uveal melanoma remains unclear. In cutaneous and uveal melanoma cell lines, potent anti-proliferative effects of propranolol have been shown in a dose-dependent manner [Immunohistochemical studies demonstrated expression of βid cells . The epiid cells . A possi of mice . Howevert manner . Decreast manner . Expresst manner . Furthert manner . This prt manner . Other at manner . Likewist manner . It is kt manner . Activatt manner . Via thet manner ,239. By 2-ARs in primary cutaneous (FM-55-P), primary uveal , and metastatic cutaneous (A375) melanoma cells, and found cell line-dependent differences in β2-AR expression with higher expression levels in primary uveal melanoma cells. Furthermore, the authors could show that, through adrenaline treatment, a stimulation of melanoma cell proliferation and activation of MMPs was induced. Especially, uveal melanoma cells showed higher migration rates in comparison to cutaneous melanoma cells [Janik et al. analyzed expression of βma cells . Patientma cells ,242.These findings indicate that blockade of β-ARs could be a potential therapeutic tool to treat uveal melanoma. However, further experimental and clinical examinations are necessary in this field. A scheme of potential effects of propranolol treatment on uveal melanoma growth is demonstrated in To expedite the clinical use of β-blockers in ocular diseases and tumor treatment, the intracellular signaling pathways, such as inflammatory, redox, and cell death signaling routes, modulated by activation or blockade of individual β-AR subtypes, remain to be characterized in more detail. Since different ocular tissues are equipped with various β-AR expression patterns, as well as with different intracellular signaling molecules, conclusions regarding individual β-AR functions cannot be generalized, but need to be confined to a specific cell type . Since t2-AR by timolol plays a central role in glaucoma therapy. β-ARs are also involved in new blood vessel formation and tumor growth. In different ocular types of hemangioma, such as periorbital infantile hemangioma, choroidal hemangioma, retinal hemangioblastoma, and conjunctival hemangioma, different studies on therapeutic administration of β-blockers have been introduced, reporting promising results. Furthermore, expression of β-ARs and inhibitory effects of β-blockers in uveal melanoma cells have been reported. From a clinical point of view, the specific role of each β-AR subtype in tumor growth and treatment needs to be pursued further to tailor specific therapeutic approaches.Disturbed β-AR signaling is discussed in the pathophysiology of some ocular surface diseases, such as dry eye disease or disturbed corneal wound healing. Moreover, β-ARs are involved in the regulation of outflow facility of the trabecular meshwork, and in the regulation of aqueous humor formation in the ciliary body. Therefore, antagonism of the β


Aortic size tends to increase with aging but the extent of this dynamic process has not been evaluated in long-term longitudinal population-based studies. We investigated the incidence of new-onset aortic root (AR) dilatation and its principal correlates among middle-aged adults over a 25-year time period.A total of 471 participants with measurable echocardiographic parameters at baseline and after a 25-year follow-up were included in the analysis. Sex-specific upper limits of normality for absolute AR diameter, AR diameter indexed to body surface area (BSA) and to height were derived from healthy normotensive PAMELA participants.New AR dilatation occurred in 7.4% (AR/BSA), 9.1% (AR/height) and 14.6% (absolute AR), respectively. According to the AR/height index, the risk of new dilation was similar in men and women. As for echocardiographic parameters, baseline AR diameter emerged as a key predictor of AR dilation, regardless of the diagnostic criteria and the 10-year change in LVMI was positively associated to new AR/height dilatation. No significant relationship was observed between baseline office and ambulatory systolic/diastolic blood pressure or their changes over time with incident AR dilatation. Baseline and the 25-year change in 24-h pulse pressure were negatively related to new AR dilatation.The incidence of AR dilatation from mid to late adulthood occurs in a small but clinically relevant fraction of participants and is unaffected by both office and out-office BP. It is significant related to baseline AR diameter and to the 25-year change in LVMI. Our data suggest that echocardiography performed in middle-aged individuals of both sexes may identify those at increased risk of future AR dilatation; moreover, preventing LVH may reduce the risk of progressive AR enlargement.The online version contains supplementary material available at 10.1007/s00392-022-02086-z. Transthoracic echocardiography is routinely used to measure the diameter of aortic root (AR) and proximal aortic segments and thus to identify aortic aneurysms or dilatations in genetic (i.e. Marfan’s and Ehlers-Danlos syndromes) or atherosclerotic diseases . Unlike The PAMELA Study was performed in an original sample of 3,200 subjects aged from 25 to 74 years representative of the population of Monza  for sex, age and other characteristics. The study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics committee of the San Gerardo University Hospital, Monza . Informed consent was obtained from all subjects., participants were invited to the outpatient clinic of the San Gerardo University Hospital of Monza in the morning of a working day (Monday to Friday), following an overnight fast and abstinence from alcohol and smoking. Data collection included medical history, weight, height, abdominal circumference, standard blood examinations, office, home and 24-h (h) ambulatory (A) blood pressure (BP) and transthoracic echocardiography. Office BP was measured three times with the subject in the sitting position, using a mercury sphygmomanometer and taking the 1st and 5th Korotkoff sounds to identify systolic (S) and diastolic (D) values, respectively. An oscillometric device was used in the third survey (Takeda TM-2430 A&D). In order to measure ABP, subjects were fitted with an ABP monitoring device  set to obtain automated oscillometric BP and heart rate (HR) readings every 20 min over 24 h. Subjects were asked to pursue their normal activities during the monitoring period, holding the arm still at time of BP readings, going to bed not later than 11.00 p.m. and arising not before 7.00 a. m. Subjects were also asked to self-measure BP at home from the arm contralateral to the one used for ABP measurement, using a validated semiautomatic oscillometric device , and a cuff size appropriate to the individual’s arm circumference, at approximately 7 pm and 7am. Advise was given to stay for 5 min in the sitting position and to avoid smoking and alcohol consumption for at least half an hour before measurements. On either occasion, one home BP measurement was obtained at the first survey and two BP measurements at the remaining ones (see below). Participants were again contacted from 2001 to 2003, i.e. after a mean time interval of 10.8 ± 0.5 years and from 2017 to 2018, i.e. after a mean time interval of 25.3 ± 0.5 years; those willing to be re-examined were asked to attend the San Gerardo Hospital for a second and third set of data collection. The same procedures used for the first survey were used for the following surveys except for the doubling of the morning and evening home BP measurements.As described in detail elsewhere , partici3-LVIDd3]) + 0.6 and normalized to body surface area. (BSA). LV hypertrophy (LVH) was defined as LV mass index (LVMI) equal to or higher than 115 g/m2 in men and 99 g/m2 in women [2, 2.3 cm/m in men and 3.4 cm, 2.2 cm/m2, 2.2 cm/m in women, respectively [Transthoracic echocardiographic data were collected according to standard procedures, as previously reported . In briein women , 19. AR ectively . An addiectively , i.e. > Statistical analysis was performed by SAS System . In each subject, BP and heart rate at the three office visits, morning and evening home values as well as 24 h values were separately averaged. Only participants with a qualitatively adequate echocardiogram and without AR enlargement, significant cardiac valve disease including bicuspid aorta, > 1 + valvular regurgitation, any degree of valvular stenosis or presence of prosthesis were included in the present analysis.The rate of incidence of new-onset AR was calculated I) for absolute AR diameter, II) AR indexed to BSA; III) AR indexed to height. In each subject, the changes (Δ) of clinical variables were calculated between the final and baseline evaluation. Incident aortic dilatation was also calculated according to the cut-off values reported by the British echocardiography guidelines . CompariFactors independently related with new-onset AR dilatation were identified by a logistic multivariable model (with stepwise selection) where baseline AR, age, sex, BMI, office, home, 24-h SBP, DBP and pulse pressure (PP), LVM index, plasma glucose, serum cholesterol, antihypertensive treatment and their changes over time (i.e. Δ) were considered as independent variables.AR diameter cut-offs associated with risk of developing AR dilatation were calculated by Youden index. Sensitivity, specificity, negative and positive predictive value were also calculated. Logistic models were used to calculate odd ratio  of each cut-offs. A p < 0.05 was considered statistically significant.Participants and/or the public were not involved in the design, conduct, reporting or dissemination plans and results of this research.The present analysis included all participants without AR dilatation at baseline and with measurable echocardiographic variables of interest in the three surveys. From the original sample of the PAMELA population (3200 subjects), 2051 subjects participated in the first survey, 1412 participated in the second survey and 562 participated in the third survey  heart rate showed a progressive reduction, whereas compared to the first survey, indexed mean absolute and indexed AR diameters showed little or no changes at the second survey and a consistent increase at the third survey . This is also shown for the AR indexed diameters in Fig. Table  ± 0.17 cm/m), baseline 24-h PP (43.0 ± 4.9 mmHg) and 25 year change of LVMI (6.1 ± 1.81 g/m2) were the independent predictors of AR dilatation also with the adoption of the British echocardiography guidelines.Logistic models with stepwise selection were constructed to identify baseline (i.e. at the first survey) factors independently associated to incidence of AR dilatation after 25 years. As shown in Table p = 0.56). This was also the case for new-onset AR/BSA and AR/height dilatation.To further explore the association between changes in BP over time and the incidence of AR dilatation, we divided the participants with AR dilatation into four subgroups: with persistently normal office BP , normal in two out of three visits, normal in one out of three visits, and persistently elevated. We did the same procedure for BP control according to ambulatory criteria (i.e. average 24-h BP < 130/80 mmHg). This categorical classification did not reveal significant differences in AR dilatation (AR/BSA and AR/height) among the various groups (data not shown). Finally, we explored the potential association between abnormal circadian BP pattern and incident AR dilatation. We found that the prevalence of the non-dipping pattern  at baseline in participants who developed non-indexed AR dilatation did not differ from participants with persistently normal AR diameter .This ma ± 4 to 30.7 ± 4 mm, p < 0.001) [Several other aspects of our study deserve mention. The first aspect is that available data on the incidence of AR dilatation in population-based samples based on follow-up periods greater than 10 years are limited to few studies. The Framingham Heart Study investigators tracked absolute AR diameter changes over 16 years in 3506 individuals in mid to late adulthood and showed that the increase in AR diameter was related principally to older age, male sex, body size and higher diastolic BP . The aut< 0.001) . Unlike < 0.001) . Althoug< 0.001) . FinallyA third aspect concerns again the importance of indexing aortic diameter to measures that help reducing interindividual body size differences. In addition to the above-mentioned advantages, in our study, the use of AR/height index highlighted an independent correlation between the occurrence of AR dilatation and LVMI increase. In this context, the correlation between AR dilatation and age is consistent with previous observations on the age-related combined arterial and ventricular remodeling , 27, pro. It is worth noting that our findings refer only to AR diameter and that they may not reflect the age-related changes of other aortic segments. Last, technical aspects, such as AR diameter measurement based on M-mode echocardiography and use of ultrasound devices with different technical characteristics during the 25-year study, may have influenced our results.Our study has some limitations. Our results refer to a population sample with an initial age of 40 years, with a low prevalence of obesity, diabetes, hypertension and LVH. Thus, extrapolation to populations at cardiovascular high-risk and prevalence of cardiovascular disease should be done with caution. Furthermore, the non-attendance at follow-up of older participants and those who had cardiovascular events may have determined an underestimation of incident AR dilatationThe present study is the first to provide 25-year longitudinal data on AR dilatation from mid to late adulthood. The incidence of AR dilatation was almost negligible after 10 years of follow-up while became more evident after 25 years; patients exhibiting aortic dilatation ranged from 7 to 15% depending on the diagnostic criteria used to define dilatation. Baseline and changes in office and out-of-office BP failed to show any relationship with AR dilatation; only baseline high-normal values of AR diameter and LVMI increase during the observation period were independent predictors. From a clinical perspective, these findings support the view that echocardiographic examinations performed in middle-aged individuals may be help to identify those at increased risk of future AR dilatation; moreover, measures aimed at preventing LVH may contribute to reduce the risk of progressive AR enlargement.Supplementary file1 (DOCX 16 KB)Supplementary file2 (DOCX 15 KB)Below is the link to the electronic supplementary material.


We also confirmed that GANs could enhance resolution and sensitivity of deep tissues without the ground truth.Acoustic-resolution photoacoustic microscopy (AR-PAM) enables visualization of biological tissues at depths of several millimeters with superior optical absorption contrast. However, the lateral resolution and sensitivity of AR-PAM are generally lower than those of optical-resolution PAM (OR-PAM) owing to the intrinsic physical acoustic focusing mechanism. Here, we demonstrate a computational strategy with two generative adversarial networks (GANs) to perform semi/unsupervised reconstruction with high resolution and sensitivity in AR-PAM by maintaining its imaging capability at enhanced depths. The b-scan PAM images were prepared as paired  and unpaired (for unsupervised CycleGAN) groups for label-free reconstructed AR-PAM b-scan image generation and training. The semi/unsupervised GANs successfully improved resolution and sensitivity in a phantom and i Owing to the rapid development of high-performance lasers, advanced acoustic sensors, and powerful computing systems, the PAI technology can provide dual image contrast by merging the strengths of optical and acoustic imaging. When a nanosecond pulsed laser is illuminated on a target sample, the biological molecules absorb the specific laser energy and generate broadband acoustic waves via thermoelastic expansion. These emitted acoustic waves are acquired using acoustic transducers, and 2D/3D images are generated through reconstruction algorithms. PAI enables maintenance of ultrasonic resolution even through deep biological tissues owing to the relatively low scattering of acoustic wave4. Based on molecular absorption at specific wavelengths, PAI provides information on structures, such as vascular network, lipids, and collagen8, in addition to functional information, such as concentration of hemoglobin, oxygen saturation, blood flow rate, and metabolism rate15. The superficiality of PAI thus contributes to advancement from basic science to clinical translation18.After the first discovered in 18805. Contrarily, PAM provides superior lateral resolution and sensitivity in shallow areas using a focused laser or acoustic beam along with fast lasing and scanning techniques6.Additionally, PAI can provide multiscale images selectively based on laser focusing and acoustic capture configuration. PAI systems are mainly of two types: photoacoustic tomography (PAT) and photoacoustic microscopy (PAM). PAT enables deep-tissue imaging with multielement transducers and a broad laser beam illumination for enhanced real-time acquisition speed without mechanical scanning approaches21. Based on the relatively less focused acoustic beam, PAM provides enhanced depth penetration via acoustic-resolution PAM (AR-PAM) for depths over 1 mm and up to several millimeters. Unfortunately, lateral resolution with the acoustic focusing approach is relatively poorer than that of OR-PAM because of the intrinsic acoustic resolution limitation7.PAM is often categorized by designing a tightly focused laser beam to achieve high lateral resolution called optical-resolution PAM (OR-PAM), which produces sharp lateral resolution and reconstruction of time-resolved photoacoustic signals from up to 1 mm depth22. E. Vienneau et al. reported a dual orthogonal ultrasonic transducer that allowed dual-view signals23 in nonlinear operations, which could affect quantitative measurements of the concentrations of molecules, such as blood oxygenation. M. Mozaffarzadeh et al. applied the double-stage delay-multiply-and-sum (DS-DMAS) algorithm to conventional AR-PAM combined with SAFT24, which incurred a higher computational complexity. Another popular solution was to use signal filtering techniques, such as FIR filtering25, empirical mode decomposition (EMD)26, and wavelet-based27 filtering to enhance acoustic signals or integration with OR-PAM systems to show the overlay of blood vessels (captured by AR-PAM) and smaller microvessels (captured by OR-PAM) with well-quality lateral resolution while maintaining imaging depth28. However, the signal filtering techniques used in AR-PAM scanning require repeated recording from the same position or the use of Fourier transform that slows the speed of real-time monitoring. Integrating the OR-PAM configuration has shown that there is no actual enhancement of the resolution.To enhance the image quality and resolution of AR-PAM while maintaining its deep imaging capability, Tang et al. proposed a dual-focused synthetic aperture focusing technique (DF-SAFT) for AR-PAM that improved the signal intensity by two times, but the performance was highly dependent on fabrication skill31, such as enhancing contrast with reconstruction under low-illumination laser34, removing artifacts with recovery of the out-of-focus regions38, and super-resolution transformation39. J. Kim et al. introduced a 3D U-Net to reconstruct high-density super-resolution images from fewer 3D label-free localizations of OR-PAM40, but this approach required downscaling to sparse localization because of limitations with the GPU memory. Another research, Z. Zhang et al. presented a deep CNN network that provided model-based iterative enhancement algorithms to recover the latent clean image from its degraded counterparts42. Most studies use convolutional neural networks (CNNs) that learn to minimize the Euclidean distances between the predicted and ground-truth properties , but decision-making with CNNs requires expert knowledge43. To distinguish classification of the output image while simultaneously training a generative CNN to minimize loss, the recently proposed generative adversarial networks (GANs) can solve the generative modelling problem46, which specifically designed for synthesize new data instances that resemble a given dataset. In particular, several researchers have tried to use GANs: S. Cheng et al. introduced an approach with Wasserstein GAN and gradient penalty (WGAN-GP) as the training network to transform low-resolution AR-PAM images to match high-resolution OR-PAM images obtained at the same depth47. Unfortunately, only the 2D maximum amplitude projection (MAP) images were processed here, which limits the accuracy of the depth area information. Y. Zhou et al. reported application of a conditional adversarial network (cGAN) to suppress the side lobes and enhance the depth of focus of a Bessel beam from a Gaussian beam48. However, these reported models required manual pairing, which causes difficulties.Because of these limitations, deep-learning methods that generally enhance PAI performance are regarded as new solutionsIn this study, we used the dual AR/OR-PAM system for data mining with a customized agar phantom embedded with three hairs and in vivo mouse ear with manual labeling for conditional GAN (cGAN) and automatic labeling without paired samples for the cycle-consistent GAN (CycleGAN). These two-representative semi/unsupervised GANs were compared for their performance enhancing abilities of low-resolution AR-PAM b-scan images to match the high-resolution OR-PAM b-scan images by measurement of the sensitivity parameters and differences in lateral/axial profile similarities. We further applied these GANs to a deep AR-PAM system to demonstrate quality enhancement with better lateral resolution at a greater depth without the validation from the OR-PAM configuration.To demonstrate enhanced depth imaging with the proposed GANs and image quality improvement in a well-prepared phantom Fig. a, we useTo demonstrate the in vivo experiment, we treated a mouse following the process described in the method section. We used AR- and OR-PAM modes in turn to quickly scan the mouse ear under anesthesia. In Fig. By applying the deep AR-PAM system alone, we conducted the same experimental process without the ground truth. We used the deep AR-PAM system to scan a side of the mouse’s back skin under anesthesia. In Fig. By demonstrating the proposed unsupervised and semi-supervised GANs for enhanced depth and image quality improvement in a customized phantom Fig. a, we pro49. These indicate that the GANs recovered complex patterns with blurring and noise under the skin and produced good effects by increasing the SNR.From the in vivo experiment with mouse ear monitoring by the dual AR/OR-PAM system, as shown in Fig. To improve the process without the ground truth images, we used the deep AR-PAM system without OR-PAM to scan a mouse’s side back that showed the twisted vascular structures and changes based on depth. Here, the proposed GANs still produced better reconstruction and lateral resolution than the original AR (~ 190% sharper). We measured the lateral profiles at three ROIs in Fig. c–e, whic47, b-scan image training48, or 3D training40. These results were verified by in/ex vivo animal experiments with random samples.In most experiments with GANs, researchers have only selected the best results they could show because the GANs are difficult to control while maintaining good conditions. To compare the differences between the cGAN and CycleGAN based on training time (epochs), we chose in vivo experiments that showed significant bias efficiency over the rest for the original OR . In Supplementary Fig. This study still has some major limitations. First, the model has a lot of parameters that should be carefully optimized cause the stability of the GAN is highly dependent on network architecture . Second, there were many factors that affected the choice of GPU . Lastly, there is a limitation with the dataset in that it does not cover all scales of biostructures  and the dataset was prepared on the limited system specification with our own experiment configuration. To deal with current issues, we are going to design a translation standard which can combine our current GANs following different transducers configuration in simple GANs models. The GANs can be used to replace manual enhancing and reduce the processing time to be able for research and commercial PAM system.50.We prepared a dual-mode AR/OR-PAM system as shown in Fig. 7 as shown in Fig. 2. The generated ultrasound waves were detected with a single-element 5 MHz ultrasonic transducer  which immersed in the water bath. The axial and lateral directions were 150 μm and 590 μm, respectively.We configured the deep AR-PAM systemFigure As experimental materials, we prepared three samples: a custom-designed three hairs embedded phantom, a mouse ear, and a mouse back skin. These samples were prepared to monitor the sensitivity and structure of the objects and their effects on the training models.51, a white polylactic acid frame (30 × 50 mm) with three top and three bottom holes was designed to fix three long black hairs at linearly increasing depths from the surface to 5 mm. The frame was internally filled with 5 g of 1.5% concentration mold jelly 52 mixed with 3 g of milk and 100 mL pure water. We cooked this mixture at 80 °C for 5 min and stirred well until the Agar dissolved completely. A solid formed stably after 15 min in the refrigerator and maintained under a wet towel.A phantom embedded with three hairs was prepared to show different imaging depths and resolutions between the AR- and OR-PAM images. Using a 3D printer 53. The energy of the laser pulse was ~ 5 mJ/cm2. Each sample was prepared and imaged separately. We scanned the mouse ear vasculature in both AR- and OR-PAM modes because the size of the mouse ear was small enough (10 × 10 mm2) for fast scanning. For the side-back skin of mouse scanning (100 × 50 mm2), the deep AR-PAM system required a longer scanning time (~ 20 min) owing to the mechanical scanning speed and laser repetition rate7. Hence, we divided the volume into four vertical stacks and displayed each b-scan image with 300 × 512 pixels to acquire the 3D volume.We prepared two in vivo samples for this study: a mouse ear for use with dual AR/OR-PAM scanning and a side-back skin of a mouse for deep AR-PAM scanning. Two healthy six-week-old male BALB/c mice  were used according to the instructions of laboratory animal protocols approved by the Institutional Animal care and Use Committee of Chonnam National University Hwasun Hospital (CNU IACUC-H-2020-35). The mice each weighed ~ 20 g and were anesthetized by intraperitoneal injection of a cocktail of Ketamine/Xylazine at 70/15 ratio. After removing the surface soft hairs, we placed the experimental mouse on a small animal table. For better acoustic coupling in vivo animal experiment, we used the ultrasound gel . An isoflurane system  was used for gaseous anesthetization, and a temperature table was used for short-term in vivo observations to maintain stable body condition of the mouse54. To clarify the opposing relationships between AR-PAM and OR-PAM b-scan images, we applied different pixelwise contrastive GAN models, cGAN and CycleGAN, to learn the mapping functions. This was to prove the effectiveness of unsupervised learning with different datasets (labeled in cGAN and label-free in CycleGAN) in AR-PAM for enhancement of the intended sample  and uncertain sample (mouse’s back skin without ground truth).Typical GAN models have been used in various imaging tasksThe schematic of the GANs consists of competing between generators and discriminators to create and discern in the network. In both cGAN and CycleGAN, we used ResNet-9 blocks Fig. a in the To replicate a probability photoacoustic amplitude distribution, the GANs were subjected to get the minimax task Eq.  between 55 to learn features from AR-PAM b-scan images paired with OR-PAM b-scan images. In Fig. P. Isola et al. proposed the modified cGAN56. Inspired by the CycleGAN structure, we denoted the data distribution as two groups of PAM images with expected values of original AR-PAM b-scan images  at focusing depth position.https://arriveguidelines.org).The authors confirm that all methods are based on relevant guidelines and regulations, and that research has been conducted in accordance with the guidelines of ARRIVE. (Supplementary Information 1.Supplementary Video 1.Supplementary Video 2.Supplementary Video 3.Supplementary Video 4.Supplementary Video 5.Supplementary Video 6.Supplementary Video 7.Supplementary Video 8.Supplementary Video 9.Supplementary Video 10.Supplementary Video 11.