Does the frequency of receiving concurrent hospice and dialysis among veterans with end-stage kidney disease (ESKD) vary by hospice payer—Medicare, Veterans Health Administration (VA) inpatient hospice, or VA-financed community-based hospice?This retrospective cross-sectional study of a national cohort of 18 420 veterans with ESKD who received hospice found that patients receiving VA-financed hospice services were more likely to receive concurrent dialysis care than those receiving Medicare-financed hospice. Irrespective of hospice payer, the VA paid for the majority of concurrent dialysis treatments with which median hospice length of stay was 43 days vs 4 days without dialysis.The findings of this retrospective cross-sectional study suggest that Medicare’s more restrictive hospice policy appears to limit access to concurrent dialysis and hospice care among veterans with ESKD and may be associated with a substantial reduction in length of hospice stay. For many patients with end-stage kidney disease (ESKD), the Medicare Hospice Benefit precludes concurrent receipt of hospice and dialysis services, forcing patients to choose between continuing dialysis or enrolling in hospice. Whether the more liberal hospice eligibility criteria of the Veterans Health Administration’s (VA) are associated with improved access to concurrent dialysis and hospice care for patients with ESKD is not known.To examine the frequency of concurrent hospice and dialysis care among US veterans by hospice payer and examine the payer for concurrent dialysis.This was a retrospective cross-sectional study of all 70 577 VA enrollees in the US Renal Data System registry who initiated maintenance dialysis and died in 2007 to 2016. Data were analyzed from April 2021 to August 2022.Hospice payer, either Medicare, VA inpatient hospice, or VA-financed community-based hospice (“VA community care”). Primary hospice diagnosis–ESKD vs non-ESKD.Concurrent receipt of hospice and dialysis services (“concurrent care”).P < .001). Regardless of hospice payer, the majority (87%) of the dialysis treatments after hospice initiation were financed by the VA, including for Medicare beneficiaries who had a hospice diagnosis other than ESKD. Median hospice length of stay was 43 days for veterans who received concurrent dialysis vs 4 days for those who did not.There were 18 420 (26%) eligible veterans with ESKD who received hospice services  individuals of other races). Most of the sample  received hospice services under Medicare and 5231 (28%) continued to receive dialysis after hospice initiation. The adjusted proportion of veterans receiving concurrent care was higher for those enrolled in VA inpatient hospice or VA community care hospice than it was for those enrolled in Medicare hospice (57% and 41% vs 24%, respectively; both In this retrospective cross-sectional study of US veterans with ESKD, a substantially higher proportion of veterans in VA-financed hospice received 1 or more dialysis treatments after hospice initiation than those enrolled in Medicare-financed hospice. Regardless of hospice payer, the VA financed most concurrent dialysis treatments. Hospice users who received concurrent dialysis care had substantially longer hospice lengths of stay than those who did not. These findings suggest that Medicare hospice policy may substantially restrict access to concurrent hospice and dialysis care among veterans with ESKD. This retrospective cross-sectional analysis compares the use of concurrent dialysis and hospice by payer among US veterans with end-stage kidney disease and examines the association with length of stay. To be eligible for hospice services under Medicare, a physician must certify that a patient is expected to live for 6 months or less if the qualifying terminal illness  follows its expected course. Under the Medicare Hospice Benefit, Medicare pays a hospice agency a per diem rate of approximately $200. The hospice agency is then expected to assume responsibility for all expenses related to the primary hospice diagnosis,3 while Medicare continues to cover the cost of care for unrelated conditions.When the Medicare Hospice Benefit was first established in 1982, it was explicitly intended to offer a less costly alternative to curative care.5—may be particularly challenging for patients with a terminal diagnosis such as end-stage kidney disease (ESKD). Because the cost of a single dialysis session far exceeds the Medicare per diem rate for hospice services,6 these patients are usually forced to choose between enrolling in hospice or continuing dialysis, a treatment without which most will survive only 1 to 2 weeks.8Unfortunately, Medicare’s approach to hospice eligibility and payment does not account for the complexities of serious illness, and it fails to recognize that some disease-directed therapies can have palliative benefits. The requirement to forfeit Medicare coverage for treatments related to one’s hospice diagnosis to receive hospice services—often referred to as Medicare’s “terrible choice”9 This commitment applies to all VA-enrolled veterans regardless of whether they receive VA- or Medicare-financed hospice services. Available data on concurrent receipt of hospice and chemotherapy or radiation therapy among veterans with advanced cancer suggest that concurrent care may improve the quality and outcomes of end-of-life care.11 To our knowledge, no study has previously examined use of concurrent dialysis and hospice services among veterans with ESKD.In contrast to Medicare, the US Veterans Health Administration (VA)—as part of its Comprehensive End-of-Life Care Initiative launched in 2009—is committed to ensuring that seriously-ill patients who are veterans have access to hospice services, regardless of whether they are still receiving disease-modifying treatment.Because most patients with ESKD are eligible for Medicare at any age, many VA-enrolled veterans have the option to receive hospice services under either their VA or Medicare benefits. In the context of the VA’s more liberal approach to hospice eligibility and payment, comparative patterns of concurrent care among veterans with ESKD receiving hospice services under Medicare vs VA could be informative. This study compared rates of concurrent dialysis and hospice use among a cohort of VA-enrolled veterans receiving maintenance dialysis for ESKD and examined which hospice payers financed dialysis treatments after hospice initiation.STROBE) reporting guideline for cross-sectional studies.12 This study was approved by the institutional review board of the Providence (Rhode Island) VA medical center and all linked Centers for Medicare & Medicaid Services data were obtained via a data use agreement with the VA Information Resource Center.This study followed the Strengthening the Reporting of Observational Studies in Epidemiology , a comprehensive national registry of patients treated with maintenance dialysis and/or kidney transplant. Additional data sources included VA enrollment files, and outpatient encounters, claims for VA-financed non-VA community care, the VA Vital Status File, Medicare enrollment files, and Medicare inpatient, outpatient, carrier, and hospice claims.As shown in Hospice services among all VA-enrolled veterans who appeared in the USRDS and died during the observation period were described; we then determined the proportion who received dialysis services after hospice initiation (concurrent care), by hospice payer. Veterans can receive hospice at a VA facility financed by the VA, or in the community, financed either by the VA (VA community care) or by Medicare. Because so few patients in the study cohort received hospice from more than 1 payer source , patients were assigned to the payer that financed their first day of hospice services. Concurrent care was defined as having at least 1 dialysis treatment after hospice initiation. We calculated the duration of concurrent care as the number of days between hospice initiation and the last day of dialysis treatment. Hospice length of stay (LOS) was defined as the number of days between hospice initiation and death.after hospice initiation , using the same categories.Demographic covariates included age, sex, and race ; other race included Asian, Native Hawaiian or other Pacific Islander, American Indian or Alaska Native. Ethnicity  was not considered. Race was ascertained using the patient files from the VA Corporate Data Warehouse and may have been recorded by self-report, proxy, or observer. Additional covariates included residence , defined by the Rural-Urban Commuting Areas system, and proximity to a VA facility, defined as the distance in miles from a patient’s residence to the nearest VAMC. Because dialysis payment source before hospice initiation might influence subsequent access to dialysis treatment , we examined rates of concurrent care by dominant dialysis payer, defined as the payer that financed the greatest number of dialysis treatments, categorized as either Medicare-financed dialysis (Medicare), dialysis at a VA facility (VA), or VA-financed dialysis in a non-VA facility in the community (VA community care). For patients who received concurrent care, we examined the primary dialysis payer after hospice initiation , both overall and after stratifying by hospice payer.First, we calculated the percentage of veteran decedents with ESKD who received hospice services, and, among hospice users, we described patient characteristics by hospice payer. We present unadjusted percentages of hospice users who received concurrent care by hospice payer, both overall and stratified by predominant dialysis payer. Also, we presented median hospice LOS, overall, by hospice payer, and by receipt of concurrent hospice and dialysis services. We used multivariable logistic regression models to compare adjusted proportions of hospice users who received concurrent care by hospice payer, both overall and stratified by predominant dialysis payer, after adjusting for age, race, sex, residence , and proximity to a VA facility. We also adjusted for fixed effects of Veterans Integrated Service Networks. Among patients who received concurrent care, we examined primary payment source for dialysis treatments Because eligibility for concurrent dialysis under Medicare is contingent on the primary hospice diagnosis, we examined the percentage of Medicare-financed hospice users who had a primary hospice diagnosis of ESKD vs a non-ESKD condition, overall and after stratification by concurrent care use. Primary hospice diagnosis was based on the principal diagnosis code listed on the first hospice claim. Because primary hospice diagnosis was missing for 43.6% of VA-financed hospice users, we focused the analyses on Medicare-financed hospice users.We also examined the unadjusted and adjusted percentages of hospice users who received concurrent care and hospice LOS after stratification by setting of Medicare hospice services to support comparisons with patients who received VA inpatient hospice. Setting of hospice care was defined using the facility type code from the first Medicare hospice claim, which was dichotomized as either home hospice or inpatient hospice.P values < .05 were considered statistically significant.All data analyses were performed from April 2021 to August 2022 using SAS Enterprise Guide, version 7.1 (SAS Institute), and Stata, version 15.0 . Statistical tests were 2-tailed and P < .001) and more were White individuals  veteran decedents with ESKD used hospice services prior to death . Almost half (46.8%) of cohort patients who did not receive concurrent dialysis had a primary hospice diagnosis of ESKD compared with only 6.3% of those who did receive concurrent dialysis. The VA was the predominant payer for dialysis treatments that occurred after hospice initiation, not only among those with a primary hospice diagnosis of ESKD  but also among those with a non-ESKD primary hospice diagnosis  KCEs to waive the requirement that beneficiaries who elect the Medicare Hospice Benefit forego their right to receive Medicare-financed curative care, most notably dialysis. All expenditures incurred by Medicare  are included as part of the Total Cost of Care, a major performance measure tied to the financial compensation that KCEs receive from Medicare. In the KCCM, utilization, spending, and quality measures are evaluated by CMMI will evaluate. If some KCEs elect to offer concurrent care, the initiative could offer a unique opportunity to examine the quality and cost of concurrent care among Medicare beneficiaries and to understand how its provision shapes the end-of-life experience and care of patients with ESKD and their families.Our findings among the veteran population highlight the importance of several ongoing Center for Medicare & Medicaid Innovation (CMMI) efforts to explore the feasibility of offering concurrent hospice and dialysis services under the Medicare program. In 2020, CMMI created the Kidney Care Choices Model (KCCM), a value-based payment model in which dialysis facilities, nephrologists, and other Medicare clinicians can partner to create Kidney Contracting Entities (KCEs) to provide care in an accountable care organization framework.27 there is strong precedent for using the follow-back design to describe patterns of end-of-life care given the challenges of prospectively identifying patients most likely to die within a certain time frame.28 Finally, the generalizability of these study findings to non-Veteran Medicare beneficiaries with ESKD or to those with other forms of health care coverage is uncertain. Nonetheless, the high prevalence of dual VA−Medicare use among veterans with ESKD means that these study findings have broader implications for the provision of concurrent care for Medicare beneficiaries with ESKD.Our study has several limitations. First, patients were not randomized to receive Medicare- or VA-financed hospice services, and the study’s observational design cannot support causal inferences on whether liberalizing Medicare hospice eligibility requirements and payment policies would increase rates of concurrent care. Second, the proportion of patients in VA-financed hospice was quite small relative to the proportion in Medicare-financed hospice, and the groups differed in some observable ways . Although we were able to adjust for potential confounders that were observable , the analyses could not account for potentially important unobserved confounders not available in source data, including social determinants of health such as caregiving and social support, detailed clinical context, and the treatment preferences of patients and families. All of these nonpolicy factors likely affect decision-making about hospice use and the timing of initiation relative to dialysis discontinuation. Third, to our knowledge, this study is the first to document rates of concurrent care among patients with ESKD, however, data comparing the quality of care for seriously ill patients with ESKD who receive concurrent hospice and dialysis vs hospice alone vs no hospice are not currently available. Fourth, our study uses a mortality follow-back design. Despite its inherent limitations,The findings of this retrospective cross-sectional study found that approximately 1 in 4 members of a national cohort of veterans with ESKD who received hospice services received at least 1 dialysis session after hospice initiation. Rates of concurrent care among the small minority of cohort members whose hospice services were financed by VA were substantially higher than those among cohort members receiving Medicare-financed hospice services. Furthermore, the VA was the primary payer for concurrent dialysis treatments, regardless of primary hospice payer and/or Medicare primary hospice diagnosis. Cohort members who received concurrent care had a hospice LOS of more than 1 month as compared with only 4 days for those who did not. Our findings among veterans with ESKD suggest that there is probably a substantial unmet need for concurrent care among the large majority of veterans with ESKD receiving hospice services through Medicare.


The hypothalamus is a central regulator of body weight and energy homeostasis. There is increasing evidence that innate immune activation in the mediobasal hypothalamus (MBH) is a key element in the pathogenesis of diet-induced obesity. Microglia, the resident immune cells in the brain parenchyma, have been shown to play roles in diverse aspects of brain function, including circuit refinement and synaptic pruning. As such, microglia have also been implicated in the development and progression of neurological diseases. Microglia express receptors for and are responsive to a wide variety of nutritional, hormonal, and immunological signals that modulate their distinct functions across different brain regions. We showed that microglia within the MBH sense and respond to a high-fat diet and regulate the function of hypothalamic neurons to promote food intake and obesity. Neurons, glia, and immune cells within the MBH are positioned to sense and respond to circulating signals that regulate their capacity to coordinate aspects of systemic energy metabolism. Here, we review the current knowledge of how these peripheral signals modulate the innate immune response in the MBH and enable microglia to regulate metabolic control. Immune cells in the brain that contribute to metabolic dysfunction could offer a therapeutic target for treating obesity. In a review article, a team led by Martin Valdearcos and Suneil Koliwad from the University of California, San Francisco, USA, discuss the evidence linking microglia and other immune cells found in the brain’s hypothalamus region to the regulation of food intake and weight gain. The researchers describe how hormones, inflammatory molecules, nutrients, and microbiome-derived factors can all impact microglia in ways that affect how the immune cells interact with the neurons responsible for metabolic control. Although much remains to be learned about which aspects of microglial function are integral to obesity promotion, the authors argue that, in principle, drugs that target these immune cells could help combat excess weight gain and related metabolic disorders. The remaining portion of the ARC may be exposed to circulating factors via diffusion, and the ARC is also open to signals from the CSF via the infundibular recess1. Thus, the MBH is a critical brain region that integrates circulating signals from the periphery to regulate systemic metabolism.In many animal species, the central nervous system (CNS) plays a key role in sensing and controlling energy status. In this context, in mammals, the mediobasal hypothalamus (MBH) in particular has emerged as a master regulator of energy homeostasis. The MBH contains the median eminence (ME) and arcuate nucleus (ARC) and is particularly positioned to sense circulating factors that regulate metabolism. The ME and the ventromedial ARC are circumventricular organs (CVOs) that lack a true blood–brain barrier (BBB) formed by endothelial cells, resulting in exposure to circulating factors from the hypophyseal portal system3. Both the immune and nervous systems have evolved to sense intrinsic and extrinsic signals to coordinate a multicellular response that contributes to the maintenance of tissue homeostasis. Neuroimmune interactions are bidirectional, and immune cells produce signals such as cytokines, neuropeptides, neurotransmitters, and hormones to modulate neuronal functions5.Recent research has revealed that the regulation of hypothalamic function involves complex interactions between neurons and immune cells within the MBH7. Furthermore, microglia also produce various neurotrophic factors to properly regulate neuronal excitability and promote the differentiation and survival of neurons9. Therefore, microglia are central players that contribute to neuroimmune interactions to maintain brain homeostasis.Microglia are specialized resident myeloid cells in the CNS, and there is growing evidence highlighting an expanding array of functions of these cells beyond their established roles in immunosurveillance and the clearance of cellular debris. For instance, microglia play a crucial role in shaping and maintaining neuronal circuits, which is a process called synaptic pruning, in which damaged or unnecessary synapses are eliminated to maintain synaptic homeostasis10. Moreover, microglial activation alone is sufficient to stimulate food intake and body weight gain in adult mice11. Hypothalamic inflammatory pathways are rapidly activated in response to the initiation of high-fat diet (HFD) feeding, far before any significant weight gain manifests, suggesting that this response is causative in the pathogenesis of obesity. As such, a better understanding of the dietary, metabolic, and immunological factors that drive microglial activation in the MBH will be critical in potentially developing new therapeutic strategies for obesity and related metabolic disorders.With respect to the hypothalamus, we showed that microglia in the MBH can sense increased consumption of saturated fats and transduce this signal to instruct local neurons engaged in controlling hunger and satiety12. In addition, circulating immune cells have been shown to respond to a HFD and can themselves be recruited to sites of damage or inflammation13.The signaling mechanisms connecting consumption of a HFD to the innate immune response in the MBH remain largely unknown, but ongoing work to elucidate connections between systemic signals and the MBH provides clues as to how such signals may be relayed Fig. . One pos15. In the brain, microglia express receptors for and have been shown to respond to neuropeptides and chemokines in addition to classical neurotransmitters16. However, vagal afferent fibers are not known to directly innervate the MBH and thus would require a more complex neuronal circuit to directly impact microglia in the MBH. Indeed, vagal afferent fibers synapse onto neurons in the nucleus tractus solitarius (NTS), which in turn has reciprocal connections with metabolic neurons in the hypothalamus17. Manipulation of the vagus nerve in mice and humans has well-known effects on metabolism, but little is known about the impact of vagal inputs on the state of hypothalamic immune cells18. Given the paucity of literature in this area, we will instead focus this review on circulating factors that could allow nutritional stimuli to induce hypothalamic innate immune responses.Moreover, peripheral neurons can receive and convey long-distance information through complex neural circuits, forming a brain–gut axis in which visceral and vagal afferent fibers relay local signals from the gut to the brain20, diffusion tensor imaging21, and proton density imaging22, have revealed a potential association between hypothalamic inflammation and BMI, central adiposity, and metabolic syndrome in humans22.While the evidence supporting the causal role of the hypothalamic innate immune response in obesity comes primarily from rodent models, several studies have suggested that hypothalamic gliosis is relevant to human obesity. For example, the activation of immune cells within a specific brain area results in increased regional water content, which can be detected by quantitative MRI techniques. Given this, multiple MRI techniques to assess brain water content, including T2 relaxation time23. Moreover, these imaging markers highlight a potential reduction in hypothalamic inflammation in obese individuals who experience weight loss following Roux-en-Y gastric bypass surgery25. Overall, these studies provide evidence supporting the clinical relevance of hypothalamic inflammation25. It is, however, important to note that MRI-based indicators of inflammation in the MBH could reflect increased activation of microglia and/or astrocytes. However, human hypothalamic samples examined postmortem showed that elevated BMI is indeed associated with specific increases in microglial soma size, along with reductions in the extent of microglial ramifications, which are both morphological hallmarks of inflammatory activation27 that underscore the importance of microglia in human energy balance.Obesity is associated with hypothalamic hypogonadism in men, and MRI indicators of hypothalamic gliosis are also inversely correlated with plasma testosterone levels29. Microglia are the most abundant myeloid cell type in the CNS and the predominant cell type within the brain parenchyma28. CNS myeloid cells express numerous overlapping markers, and thus a major obstacle in the field has been the inability to discriminate between resident “homeostatic” microglia and the variety of other myeloid cell types in the brain. New technical approaches for single-cell profiling, however, have recently overcome this obstacle and revealed remarkable functional complexity within the CNS myeloid compartment in the context of normal health and disease30. Resident microglia are yolk sac-derived cells and self-renew within the brain throughout life, and there is increasing evidence that these cells function differently in the brain than bone marrow (BM)-derived macrophages, which infiltrate the brain under conditions of CNS injury and disease32. Microglia are long-lived cells and depend highly on signals from their tissue environment to maintain identity, but there is growing evidence that even true microglia are not monolithic, exhibiting spatial heterogeneity and varying functional states dictated by anatomical location and physiological or pathological factors both in the rodent and human brain33. These findings have important therapeutic implications because they suggest that targeting a specific microglial subset or state may require strategies that are specific to a given brain region. Moreover, drug targeting of most parenchymal microglia is limited by the BBB. With this in mind, the MBH may provide an interesting therapeutic opportunity, as the ME and ventromedial ARC contain highly fenestrated blood vessels, which facilitate relatively open communication with the periphery2. Thus, myeloid cells in the MBH are potential targets that may be accessed by modulators delivered enterally, parenterally, or intranasally34. Furthermore, bloodborne  myeloid cells are recruited to the brain during pathological states that are associated with compromised BBB integrity, and these cells, once recruited into the brain parenchyma, have the capacity to gain key microglia-like characteristics35. As such, these infiltrating cells are also recognized as targets for therapeutic intervention in neurological disease37.The myeloid compartment of the CNS includes a heterogeneous population of tissue-resident macrophages, including microglia in the brain parenchyma and macrophages and dendritic cells in perivascular, meningeal, and choroid plexus compartments11. Moreover, a recent study showed that inducible nitric oxide synthase (iNOS) activation in hypothalamic LysM+ macrophages contributes to the diet-driven immune response within the MBH by increasing local vascular permeability and lipid influx38. These LysM+ macrophages apparently accumulate in the MBH in response to a HFD through local proliferation, without the contribution of BM-derived cells39. Understanding the functional heterogeneity of individual myeloid cell types in the MBH, both resident and infiltrative, and the specific signaling pathways that are regulated by diet in these populations will be essential in developing selective cell-based and/or immunotherapeutics to correct diet-induced hypothalamic dysfunction.We used detailed immunofluorescence histochemistry and BM lineage tracing to show that the HFD-induced hypothalamic innate immune response actually involves diverse myeloid cell phenotypes and functional states with unique spatial distributions within the MBH40. Hypothalamic neurons are well known to be regulated by circulating metabolic signals, and both hypothalamic neurons and glial cells have been shown to respond to lipid signals that regulate both food intake and energy homeostasis41. However, the comparative roles of the neuronal and nonneuronal compartments in this regard have not been fully elucidated. In addition to specific nutrients, this section will review the circulating hormonal, inflammatory, and microbiome-derived factors that may regulate hypothalamic immune activation in the development of diet-induced obesity (DIO).Translating knowledge of myeloid cells in the MBH for therapeutic purposes will require understanding which potential circulating factors influence the polarization of these cells. It has long been known that hypothalamic nutrient sensing is critical for the regulation of energy balance, with recent work focusing on the hypothalamic sensing of nutritional signals, especially glucose and lipids15. However, the effects of these hormones, which are derived from the gut, adipose tissues, and muscle, on hypothalamic immune activation remain poorly characterized. Here, we review what is known about hormonal regulation of the hypothalamic innate immune response.There is an expanding list of peripherally generated hormones that regulate food intake and metabolism and act on hypothalamic signaling pathways42. While previously thought to be expressed predominantly by specific neuronal populations, including POMC and NPY/AgRP/GABA (NAG) neurons, leptin receptor mRNA has also been found in astrocytes and microglia43. Initial studies showed that while both leptin-deficient (ob/ob) and leptin receptor-deficient (db/db) mice are obese, this phenotype alone is not sufficient for mice in either model to develop microgliosis in the MBH. However, ob/ob mice develop hypothalamic microgliosis in a manner similar to that of wild-type controls when fed a HFD44. Taken together, these early results suggested that dietary excess, rather than obesity per se, mediates the activation of MBH microglia and that leptin-dependent signaling in the brain is not a dominant determinant of this response. However, there is more recent evidence suggesting that microglial reactivity may also be modulated by leptin signaling. For example, providing ob/ob mice with leptin replacement is sufficient to increase microglial numbers in the MBH44. Moreover, pretreating cultured microglia with leptin potentiates their inflammatory response to lipopolysaccharide (LPS), and leptin increases the expression of tumor necrosis factor alpha (TNFα) and interleukin-1 beta (IL‐1β) in primary hypothalamic microglia45. Finally, myeloid-specific deletion of the leptin receptor led to increased weight gain in mice fed a standard chow diet, although this was predominantly due to increased lean mass46. Notably, this study was performed in a noninducible genetic model, pointing to the possibility that the resulting hypothalamic and metabolic phenotypes might be influenced by impairments in synaptic pruning by microglia during embryonic and early-life development46.Leptin is a peptide hormone secreted primarily by white adipocytes that is central to the regulation of body weight and energy expenditure47, and circulating levels of its high molecular-weight multimeric isoform are decreased in individuals with visceral adiposity and insulin resistance48. APN-deficient mice are resistant to HFD-induced obesity49. Exploration of this phenotype has revealed that APN is able to cross the BBB and stimulates food intake via activation of the two APN receptors (ADIPOR1 and ADIPOR2) in the ARC; in rats, these receptors have been shown to be expressed not only in POMC and NAG neurons but also in microglia and astrocytes51. Notably, APN-deficient mice displayed enhanced microglial proliferation and inflammatory cytokine expression in both the hypothalamus and hippocampus following LPS injection, and this hyperresponsiveness was suppressed by i.c.v. injection of globular APN52. Treatment of mice fed a HFD for 4 weeks with systemic (i.p.) APN was associated with decreased microglial activation, as indicated by morphological changes, and decreased expression of inflammatory cytokines in the hypothalamus; however, this effect was not associated with an alteration in body weight51. Whether APN acts directly on hypothalamic microglia in vivo has not been studied.Adiponectin (APN) is an adipokine that is almost exclusively secreted by adipocytes53. Several GLP-1 receptor agonists have now been approved for the treatment of diabetes and/or obesity, and the anorectic effects of GLP-1, although pleiotropic, are increasingly linked to agonism of the GLP-1 receptor (GLP-1R) in multiple brain regions54. Indeed, GLP-1R is expressed by neurons, astrocytes, and microglia56. Within the hypothalamus, GLP-1 stimulates the activity of specific GLP-1R-expressing neuronal populations, including POMC neurons. However, GLP-1R expression by hypothalamic neurons was paradoxically dispensable in GLP-1 treatment to induce weight loss or improve glucose tolerance in mice58. Intriguingly, GLP-1 has direct anti-inflammatory effects on microglia, decreasing their secretion of proinflammatory cytokines59. Systemic administration of the GLP-1 analog exendin-4 and, in a separate study, liraglutide decreased microglial activation within the MBH of mice fed a HFD60. However, whether the beneficial effects of GLP-1R agonists are at least partly mediated by the modulation of hypothalamic microglial activation remains to be studied.Glucagon-like peptide 1 (GLP-1) is a peptide hormone that is primarily secreted by enteroendocrine L-cells in the intestine61, it is notable that GLP-1-producing (PPG) neurons in the NTS were recently shown to be another major source of GLP-1 within the brain62. Ablating NTS PPG neurons did not affect body weight or food intake on a standard chow diet but markedly increased feeding behavior following a fast, when food consumption rates are typically high62. Nevertheless, whether GLP-1 produced in the periphery or in the brain is more dominant in modulating the hypothalamic response to HFD has not been assessed.Although systemic GLP-1 can cross the BBB64. While ghrelin has potent orexigenic effects that are mediated by NAG neurons in the ARC, it also has anti-inflammatory effects on microglia in vitro and in models of experimental autoimmune encephalitis65. In one study, pretreatment of mice with a single injection of purified ghrelin in its active acylated (n-octanoyl) form reduced the hypothalamic inflammatory response to a single day of HFD feeding66. Taken together, these results suggest that ghrelin signaling may play a role in disassociating hypothalamic microglial activation from the effects of dietary excess or perhaps even restraining it.Ghrelin is an orexigenic hormone produced predominantly by the gut, but it is also expressed in other tissues, including the ARC68. There is evidence suggesting that both cell-intrinsic and environmental factors impact microglial function in a sexually dimorphic manner, and these factors may help determine sex-specific differences in susceptibility to neurological disease69. Ovariectomy-induced estrogen deficiency increases microglial activation in female mice68. In contrast, both 17-α-estradiol (17αE2) and 17-β-estradiol (17βE2) have anti-inflammatory effects on microglia in vitro, and 17αE2 treatment reduced aging-associated hypothalamic microglial activation in male mice and reduced microglial activation and accumulation within the MBH of ovariectomized female mice fed a HFD71. However, data also suggest that microglia show innate sexual dimorphism, independent of hormonal inputs72. Future studies should examine how such innate elements interact with environmental  factors to functionally modulate MBH microglia.Estrogens may also serve to modulate both hypothalamic neurophysiology and the interaction between MBH neurons and local microglia. The hypothalamic microglial response to the consumption of a HFD exhibits striking sexual dimorphism in rodents, with males having higher levels of inflammatory cytokines and greater microglial activation than females74. A regimen of moderate exercise decreased the MBH microgliosis induced by the consumption of a high-carbohydrate HFD in LDL receptor-deficient mice75. Similarly, BAIBA treatment for 8 weeks lowered the hypothalamic mRNA levels of inflammatory genes and reduced the number of hypothalamic microglia in mice that had already been fed a HFD for 16 weeks, although it did not reverse weight gain73.β-aminoisobutyric acid (BAIBA) is a myokine that is increased by physical activity and was shown to mediate the benefits of exercise on obesity-related metabolic risk factors in mice but with only modest effects on weight gain itselfIn summary, there are currently no data directly implicating circulating hormonal signals in driving the mechanism by which dietary excess causes hypothalamic innate immune activation. However, several hormones may play roles in modulating this response, with leptin being potentially proinflammatory, and most others have roles in restraining hypothalamic immune activation.76. The levels of these factors can then rise in the MBH as well. In addition, the levels of other cytokines, such as TNFα and IL-1β, are known to be increased in the MBH in obesity; however, these cytokines are most likely increased due to local production rather than import from the systemic circulation. Microglia express receptors and machinery that are involved in many aspects of innate immune signaling77, raising the possibility that circulating and/or local inflammatory mediators may play a role in regulating the polarization of MBH microglia in obesity. However, microglial activation in the MBH occurs rapidly in response to a HFD, and a recent study demonstrated a response as early as 6 h after exposure to a HFD78. In contrast, although some studies have demonstrated an increase in circulating inflammatory cytokines after a single high-fat meal, a recent systematic review showed that only IL-6 was consistently increased after one high-fat meal in healthy individuals79. Overall, these studies raise some doubt as to whether circulating levels of inflammatory factors rise fast enough in response to the initiation of a HFD to represent viable sources of factors that then stimulate microglial activation within the MBH. The alternative is to consider that noninflammatory factors  alter the functional states of MBH microglia and that these effects precede any systemic inflammatory changes that may occur later on. Regardless, it seems likely that circulating cytokines and other immune-related signals may help regulate and modulate chronic diet-associated hypothalamic innate immune activation. Some potential signals are reviewed here.Obesity can induce chronic systemic inflammation and increase the circulating levels of inflammatory molecules, including LPS, interleukin-6 (IL-6), C-reactive protein (CRP), plasminogen activator inhibitor 1 (PAI-1), and serum amyloid A (SAA), in certain individuals81. Indeed, systemic administration of a low dose of LPS to mimic the levels seen upon chronic consumption of a HFD was sufficient to induce weight gain and insulin resistance in mice81. How peripheral LPS impacts brain function is unclear, as most of the brain is relatively protected against peripheral LPS, with an estimation that only 0.025% of an i.v. injection reaches the brain parenchyma82. A recent study showed that peripherally injected LPS is present, however, within CVOs, including the ME and that the transport of LPS into CVOs may be mediated by lipoproteins83. Thus, CVOs may function as sensors of peripheral LPS. Moreover, a single i.p. injection of LPS in mice induced the proliferation of microglia in hypothalamic CVOs, including the ME and ARC84. However, a recent study showed that while peripheral LPS injection caused microglial activation in lean animals, intermittent LPS injection decreased HFD-induced microglial activation and increased MBH astrogliosis85.LPS levels in the circulation can be increased by HFD consumption and remain elevated in obesity, possibly due to increased gut permeability87. Whole-body TLR4 knockout has shown variable effects on the response to a HFD in different studies, providing protection from weight gain in some but not in others90. One explanation for this discrepancy is that TLR4 deletion in different tissues may have variable effects on DIO. Intriguingly, the transplantation of TLR4-deficient BM also had variable effects on HFD-induced weight gain94. More recently, hepatocyte-specific targeting of TLR4 ameliorated hepatic steatosis and improved insulin resistance but did not affect weight, while macrophage-specific targeting of TLR4 did not protect mice from DIO, insulin resistance or hepatic steatosis95. In the hypothalamus, TLR4 targeting through stereotactic injection of TLR4 shRNA lentiviral particles into the ARC decreased microglial accumulation and body weight gain during 4 weeks of HFD feeding96. Daily intraperitoneal injection of a TLR4-inhibiting antibody to animals on a HFD decreased weight gain and the expression of inflammatory cytokines in the MBH97. However, microglia-specific TLR4 deficiency has not been examined in a mouse model of DIO. It has been previously hypothesized that TLR4 may also mediate innate immune activation in DIO by functioning as a direct receptor of saturated fatty acids (SFAs). However, a recent study demonstrated that while TLR4 may be required for SFA-induced innate immune activation, it is not an SFA receptor. Rather, TLR4 is required for priming cells for SFA-induced inflammatory responses98. The question remains whether increased LPS levels in the context of DIO are necessary for TLR4-dependent priming of microglia to drive hypothalamic inflammatory responses in vivo.Toll-like receptor 4 (TLR4) is the cell-surface receptor for LPS and is expressed most highly by myeloid cells in the CNS, including in the hypothalamus99. Interestingly, myeloid-specific depletion of the IL-6 receptor (IL-6R) causes a shift toward M1 macrophage polarization in liver, white adipose tissue, and brown adipose tissue. Moreover, this polarization shift in macrophage populations is associated with impaired glucose homeostasis100. However, the effects of chronically increased systemic levels of IL-6 are pleiotropic and include some beneficial consequences, depending on the model101. For example, IL-6-deficient mice paradoxically develop maturity-onset obesity, and central IL-6 administration was able to increase energy expenditure in this model102.IL-6 is a cytokine associated with obesity and insulin resistance. Indeed, acute IL-6 infusion is sufficient to induce insulin resistance in mice103. Moreover, CNS delivery of an antibody that inhibits IL-6 signaling blocked the anti-inflammatory effect of exercise on the hypothalamus104. Glycoprotein 130 (gp130) is the transmembrane receptor for IL-6 trans-signaling, which is engaged by IL-6 binding to soluble IL-6R. Targeting gp130 in neurons of the paraventricular nucleus (PVN) of the hypothalamus blocked the metabolic effects of central IL-6 administration103. The cellular source of soluble IL-6R that mediates IL-6 trans-signaling in the brain has not been found, but IL-6R mRNA is most highly expressed by microglia86. Interestingly, IL-6 trans-signaling was also essential in the neuroprotective effects of microglia in a model of traumatic brain injury105. IL-6 is released at high levels by exercised muscle106; however, multiple brain cell types may synthesize IL-6 locally107. Whether systemic and/or local IL-6 are involved in the regulation of hypothalamic function remains unknown. Further functional resolution of cell- and tissue-specific IL-6 signaling pathways will be important in determining why acute and chronic manipulations of IL-6, whether systemically or specifically in the CNS, produce wide-ranging and seemingly paradoxical effects on obesity.When delivered into the CNS (i.c.v. injection), IL-6 decreased feeding and improved glucose homeostasis, but deleting IL-6R in neurons did not block these effects108. Both genetic deletion and pharmacologic inhibition of PAI-1 protect against HFD-induced increases in food intake and body weight110, and this effect was found to be mediated at least in part by impaired leptin signaling in the hypothalamus109. The effects of PAI-1 on MBH immune activation have not been explored; however, PAI-1 has been shown to promote microglial migration and regulate phagocytosis111.Plasminogen activator inhibitor 1 (PAI-1) is produced by adipose tissues, and the levels are elevated in both adipose tissues and plasma in obesityIn summary, there are intriguing data suggesting that circulating immune-related factors may modulate the hypothalamic innate immune response by acting on microglia. However, detailed and systematic studies to determine how these factors might coordinately mediate hypothalamic dysfunction and the development of DIO have not yet been performed.112. Importantly, this systematic comparison was performed in male C57BL/6J mice, which are sensitive to both DIO and HFD-induced hypothalamic innate immune activation, and it is unclear how transferrable these findings are to other mouse strains. Moreover, interpersonal differences in obesity-susceptibility genotypes have been shown to interact with dietary macronutrient composition to produce an integrated obesity risk profile in humans114. Thus, rodent experiments exploring the effects of macronutrient composition on aspects of DIO must be interpreted with these key limitations in mind.The effects of specific macronutrient proportions on obesity remain a subject of controversy and ongoing investigation. Most diets used to produce obesity in rodents are high in saturated fat; however, these diets contain significant calories from sugar as well. A recent systematic evaluation of the effect of macronutrient composition on body weight regulation showed that in mice, increased dietary fat content but not protein or carbohydrate content was associated with increased food intake and weight gain, plateauing with diets in which 60% of calories come from fat117. These diets have been shown to increase the levels of hypothalamic inflammatory cytokines and promote microglial activation and also lead to increased caloric intake. This raises the question of whether increased caloric intake per se causes hypothalamic immune activation. However, calorically matched pair-feeding experiments with distinct HFDs demonstrate that consuming excess dietary fat is sufficient to cause weight gain and hypothalamic innate immune activation independent of overall caloric consumption117.Large-scale systematic evaluations of the effects of macronutrient composition on the activation state of hypothalamic immune cell types have not been performed, and most studies used standard HFDs varying from 40 to 60% of caloric intake from fat with the source of fat predominantly being either milk fat or animal lard, which are both high in saturated fat Table . Common 118. While there was no difference in adipose tissue inflammatory markers, animals on both types of high-saturated fat diets showed increased levels of inflammatory cytokine expression in the hypothalamus118. In another comparison of diets with similar macronutrient compositions, a diet with elevated levels of saturated fat caused a more rapid increase in food intake than did one with lower saturated fat content119. Moreover, in a direct comparison of two diets, one of animal origin and one containing olive oil, which is high in monounsaturated oleic acid, only the diet with animal fat caused increased inflammatory cytokine expression and myeloid cell immunostaining in the MBH, although the total fat content of both diets was matched at 36% of overall calories97. Thus, the available evidence suggests that high-saturated fat intake is required for a HFD to induce hypothalamic innate immune activation in mice. Furthermore, it was shown that even very short-term (1–6 h) exposure to excess dietary saturated fat is sufficient to induce changes in MBH microglia78. These studies suggest that the composition of consumed fat, rather total fat intake, regulates immune activation in the hypothalamus in promoting DIO. The rapidity of this response is consistent with a direct effect of dietary saturated fat.Several studies have also examined the effects of the amount and source of saturated fat in the diet on hypothalamic innate immune activation. One study compared the effects of diets that were low in saturated fat , high in saturated fat of plant origin , or high in saturated fat of animal origin  Table 118. Whil120. Another low carbohydrate, high-fat diet  suppressed both the increased microglial cell division seen in the MBH and the increased food intake produced by the consumption of a standard HFD (D12492)121. These studies suggest that very low carbohydrate intake can protect against HFD-induced MBH innate immune activation.A recent study explored the role of dietary carbohydrates in the pathogenesis of MBH innate immune activation using high-fat diets with either low or high amounts of carbohydrates. Neither of the two very low carbohydrate diets used in this study, a nonketogenic diet LCHF1) and a ketogenic diet (LCHF2), were able to induce MBH microgliosis or weight gain  are linked to specific SFAs122. In contrast, i.c.v. administration of oleic acid actually decreased food intake and hepatic glucose production compared with the control123. Hypothalamic sensing of oleic acid appears to be mediated by the regulation and sensing of intracellular long-chain fatty acyl-CoA levels by MBH neurons124. There is no known role for MBH microglia in the hypothalamic sensing of oleic acid; however, short-term (3 days) HFD feeding abolishes the metabolic and anorexigenic effects of i.c.v. oleic acid administration125. Of course, i.c.v. injection is an artificial model, but there is evidence that the hypothalamic lipid composition reflects dietary fat composition over time. Radiolabeled palmitic acid delivered enterally and lipoproteins injected i.v. were both able to rapidly enter the MBH126.Our group has explored the specificity of saturated fat in mediating the MBH innate immune response by direct gavage into the stomachs of mice. Gavage with milk fat, which is high in palmitic acid, but not olive oil reproduced the MBH microglial response observed following the consumption of a HFD enriched in saturated fat127. We showed that treating organotypic hypothalamic slices with palmitic acid but not oleic acid initiates an inflammatory response in the hypothalamic slice, which requires the presence of microglia10. Indeed, oleic acid cotreatment abrogated the proinflammatory response of these slices to palmitic acid treatment. Thus, saturated fat itself may function as an immunomodulatory signal that is delivered via the systemic circulation to mediate hypothalamic innate immune activation in DIO.Further supporting a direct role for SFAs in functionally regulating hypothalamic microglia, multiple groups have demonstrated that saturated but not unsaturated FAs have proinflammatory effects on cultured microglia128. These observations were made using germ-free (GF) mice that were maintained in a sterile environment without a gut microbiome. GF mice are leaner than specific pathogen-free (SPF) mice despite consuming more calories. Furthermore, transplanting the microbiota from SPF mice into GF mice increased body fat by 60% within 2 weeks129. Indeed, the gut microbiome regulates both host energy expenditure and feeding behavior130. In humans, obesity, by contrast, produces a gut microbiome that is less diverse and consumes less energy than that of lean individuals131. Genetically obese mice (ob/ob) have a higher proportion of intestinal Firmicutes and 50% fewer Bacteroidetes and a parallel enrichment of microbial genes involved in polysaccharide degradation than in the microbiomes of lean siblings132.The gut microbiome is increasingly accepted to be an environmental factor linked to obesity and associated metabolic disorders. The diverse gastrointestinal microbial community participates in energy extraction from food, host lipid metabolism, immune responses, and endocrine function134, indicating that microglia may continuously sense inputs emanating from the gut microbiome. For instance, short-chain fatty acids (SCFAs) are the main metabolites produced by the bacterial fermentation of dietary fiber in the gastrointestinal tract, and these SCFAs are rapidly absorbed by colonic cells. SCFAs influence gut–brain communication and brain function directly or indirectly through immune, endocrine, and vagal pathways135. Although SCFAs have been shown to protect against DIO in mice136 and overweight humans137, the underlying mechanisms are unclear. SCFAs are important regulators of innate immune responses and were recently shown to be involved in regulating microglial function. Indeed, GF mice display globally defective microglial phenotypes, leading to impaired CNS immune responsiveness. Remarkably, aspects of normal microglial homeostasis can be restored when GF mice are supplemented with SCFAs in their drinking water133. Furthermore, recent work suggests that microglial activity is directly regulated by the metabolites of dietary tryptophan metabolism produced by commensal gut flora and that this response controls a downstream inflammatory response in astrocytes138.The gut microbiome also controls the activity of CNS myeloid cells. Disruption of the gut microbiome resulted in significant alterations in microglial function in both GF mice and mice in which the microbiome was ablated by antibiotic treatment139. Microglia in the MBH are also affected by the intrinsic circadian clock, with microglial numbers and activity being increased in the dark relative to the light phase in mice under standard housing conditions140. However, it remains unknown whether any aspect of circadian regulation of microglial numbers or activity, particularly within the hypothalamus, is a function of the circadian aspects of the gut microbiome. Building on a wealth of information highlighting microglia and other CNS myeloid cell types as crucial mediators linking the gut microbiome to pathological outcomes in the context of several neurodegenerative disease models141, future studies will need to comprehensively evaluate how the composition, secretome, and circadian regulation of the gut microbiome interact to regulate hypothalamic control of energy and glucose homeostasis.The gut microbiota is also affected by circadian rhythms, with some microbes showing diurnal fluctuations in their relative abundance and activityCreER mouse strains have substantially advanced the microglial field. However, Cx3cr1 gene expression within the CNS is not restricted to microglia, as it is also expressed in nonparenchymal macrophages at CNS border locations142. Furthermore, Cx3cr1 has been shown to be expressed by long-lived peripheral tissue-resident myeloid cell types144. Therefore, the results obtained using this mouse model in reference to microglia should be interpreted with caution. Moreover, this mouse line was generated based on knock-in strategies in which the endogenous gene is replaced, and there is evidence that Cx3cr1 haploinsufficiency affects microglial function146. Thus, more specific tools to label and manipulate microglia are needed to further understand how this cell type contributes to brain health and disease.The most commonly used mouse line to manipulate microglia is based on the Cx3cr1  promoter, and Cx3cr1Sall1, Tmem119, P2ry12, and Hexb. Sall1, a conserved transcriptional regulator, emerged as a marker to distinguish bona fide parenchymal microglia from other brain myeloid cells147. However, Sall1CreER transgenic mice show prominent recombination in the neuroectodermal lineage148. Transmembrane protein 119 (Tmem119), a cell-surface protein, was also recently identified as a novel microglia-specific marker that is not expressed by peripheral myeloid cells149. However, a recent targeting approach revealed ectopic expression of Tmem119 in CD34+ vessels in the CNS and leptomeningeal pial cells150. P2ry12CreER transgenic mice have been recently described151. Since P2ry12 expression has been shown to be downregulated upon microglial activation152, this new mouse line will be particularly useful for tracking microglia during disease and development. Furthermore, hexosaminidase subunit beta (Hexb) is a highly enriched microglial gene with only minimal expression by other brain myeloid cell types, even in models of neuroinflammatory diseases. The Hexb locus has been used for the generation of two promising transgenic mouse lines: the HexbtdT and HexbCreERT2 strains153.Recent advances in RNA sequencing and other profiling technologies have revealed new insights into CNS myeloid cells, including a greater degree of heterogeneity than had been previously recognized. This information has been used to attempt to develop better mouse tools. Single-cell RNA-sequencing studies identified distinct core signature genes for microglia, such as 154, could be highly effective. Such a binary transgenic system involving complementation-competent NCre and CCre fragments whose expression is driven by distinct promoters was recently used to specifically target parenchymal microglia using Sall1NCre:Cx3cr1CCre mice and perivascular macrophages using Lyve1NCre:Cx3cr1CCre mice155 simultaneously.Despite these impressive recent advances, the use of single promoter activity could be insufficient to specifically target a desired population of microglia. To overcome this problem, a second dimension of recombination, such as using the “split-Cre” system where Cre recombinase expression is regulated by coincidental activity of two different promoters, increasing the specificity of Cre-mediated DNA recombinationSuch new and more specific tools will be necessary for further experiments to identify the specific myeloid cell populations in the MBH that are responsible for the initial and ongoing hypothalamic immune activation in response to dietary excess.The hypothalamic innate immune response is a component of DIO, and evidence suggests that myeloid cells, particularly microglia, are critical cellular drivers of this process. Recent studies have focused on the role of myeloid cells within the MBH in the pathogenesis of DIO. It is tempting to focus on myeloid cells in the MBH due to their position within hypothalamic CVOs, making them more amenable to drug targeting. However, currently available tools target microglia throughout the CNS. Thus, the specificity of MBH microglia in the pathogenesis of obesity compared to that of microglia in other brain regions, especially other CVOs positioned to sense circulating signals, remains intriguing.10. However, the precise mechanism by which microglia interact with hypothalamic neurons remains largely unstudied. New tools aimed at real-time monitoring of hypothalamic neuronal function will enhance our ability to study the effects of microglia. A recent study utilizing fiber photometry and optogenetics to probe the effects of DIO on NAG neuronal circuits revealed complex changes in NAG neuronal sensitivity and downstream pathways156. These new tools will be central to probing how MBH innate immune activation impacts these HFD-induced changes in hypothalamic neuronal circuits.How does innate immune activation in the MBH induce changes in neuronal activity to cause obesity? Hypothalamic neurons sense and integrate diverse signals to regulate energy homeostasis. For example, we showed that depleting microglia decreased SFA-induced neuronal injury and leptin resistance in ARC neurons and decreased SFA-induced hyperphagia157. Microglial phagocytosis in the context of synaptic pruning is tightly regulated. For example, the classical complement protein C1q is produced by microglia and tags synapses for elimination by microglia, both in the context of normal development and degenerative disease158. Another recent study showed that in the context of neuronal injury, microglia form specialized somatic junctions with neuronal cell bodies and that these connections are important for the neuroprotective role of microglia in brain injury159. How are these functions of microglia altered by a HFD to affect hypothalamic neurons? In addition, the heterogeneity of CNS myeloid cells has become apparent with the recent identification of disease-associated microglia (DAM) and lipid droplet-accumulating microglia (LDAM), which may play major roles in the pathogenesis of neurodegenerative diseases161. Further understanding of the heterogeneity of myeloid cells in the MBH will enhance our understanding of how microglial activation impacts hypothalamic neuronal pathways in the regulation of appetite and systemic metabolism.Furthermore, it remains unclear what aspects of microglial physiology are integral to the obesity-inducing effects of the MBH innate immune response. General phagocytic function and the secretion of inflammatory cytokines have been postulated to be involved; however, recent work has shown that microglia have complex interactions with neurons, including selective phagocytosis of presynaptic structures and the induction of postsynaptic spine head filopodiaWith extensive ongoing work to target obesity-associated hormones, systemic inflammation, and the microbiome in the treatment of obesity, it is essential that we understand how these systemic and circulating factors influence the hypothalamic innate immune response. An improved understanding of the mechanisms by which myeloid cells in the MBH are activated by a HFD and modulate neuronal function to regulate obesity will allow more targeted experiments to understand the role of systemic factors in modulating this response.


Liquid biopsy has been established as a powerful, minimally invasive, tool to detect clinically actionable aberrations across numerous cancer types in real-time. With the development of new therapeutic agents in prostate cancer (PC) including DNA repair targeted therapies, this is especially attractive. However, there is unclarity on how best to screen for PC, improve risk stratification and ultimately how to treat advanced disease. Therefore, there is an urgent need to develop better biomarkers to help guide oncologists’ decisions in these settings. Circulating tumour cells (CTCs), exosomes and cell-free DNA/RNA (cfDNA/cfRNA) analysis, including epigenetic features such as methylation, have all shown potential in prognostication, treatment response assessment and detection of emerging mechanisms of resistance. However, there are still challenges to overcome prior to implementing liquid biopsies in routine clinical practice such as preanalytical considerations including blood collection and storage, the cost of CTC isolation and enrichment, low-circulating tumour content as a limitation for genomic analysis and how to better interpret the sequencing data generated. In this review, we describe an overview of the up-to-date clinical opportunities in the management of PC through blood-based liquid biopsies and the next steps for its implementation in personalised treatment guidance. In the UK, prostate cancer is the most common cancer in men with approximately 48,500 cases diagnosed every year. With 11,855 prostate cancer-related deaths annually [Molecular profiling of solid cancers has been used across multiple cancer types to identify poorer prognosis cancers and guide treatment selection. Most studies have utilised analysis of DNA and/or RNA and/or protein on a biopsy from the primary tumour or less commonly, a metastatic lesion . There aDue to these limitations, there has been an interest in blood-based biomarkers, commonly referred to as liquid biopsies, in which we focus on this review, as an alternative or companion to solid tumour biopsies and imaging studies to better characterise tumour molecular drivers and response to treatment. Nucleic acids such as DNA or RNA as well as proteins, cells and vesicles circulate freely in human blood and can be isolated using various molecular techniques Fig. . CirculaLiquid biopsies also have inherent limitations. CTC-based mRNA and protein analyses are underdeveloped and usually unimodal (one or fewer markers studied) and limited to the characterisation of very small numbers of CTCs. Thus, CTC derived tumour genome representation can be far less than that usually derived from tumour biopsies . ctDNA aOther body fluid components, such as urine, have also been subjected to DNA extraction and NGS analyses with promising studies reported in the field –14.To maximise the reliability of molecular information obtained from a liquid biopsy, there is a need to develop a standardised workflow for the preanalytical steps. Different approaches for collecting, processing and storing blood can lead to an up to 50% variability in the amount of ccfDNA extracted . Each ofFor cfDNA analysis, one of the key preanalytical goals is to avoid genomic DNA release from lysing leucocytes: this DNA would dilute the already potentially small fraction of tumour-derived DNA and can lead to false-negative results . In ordePre-analytic processing of samples after blood draw should be carefully optimised, for example extending the time of proteinase K incubation of plasma during DNA extraction can minimise cross-linking-related artefacts due to the presence of fixatives in BCTs .Another important preanalytical goal is to achieve the highest extraction efficiency for fragments of ccfDNA below 200 bp as this range contains most ctDNA fragments. Different studies have tested several extraction methods showing differences in the percentage of short ccfDNA isolated depending on the kit used .For CTCs analysis, different preanalytical steps should be considered. Firstly, for blood collection, preservatives are needed to avoid cell lysis although results can be contradictory depending on the BCT used . New app®), which target different proteins , are capable to separate EVs but are expensive [Extracellular vesicle (EV) collection represents a particular challenge as vesicle release from blood cells  after venepuncture needs to be minimised to avoid reduction in purity. However, to date, no specific EV preservative is commercially available , 25. Widxpensive . Currentxpensive .ctDNA analysis of metastatic prostate cancer patients is able to identify prostate cancer genomic features. When the circulating tumour fraction is sufficiently high there is a high concordance with tissue findings for truncal alterations present in concurrently collected biopsies of metastases , 30. HowTechniques such as digital droplet PCR (ddPCR) and similar targeted approaches have been widely used and can capture a handful of mutant alleles at very high sensitivity (0.001%) . This caTM) and the Foundation Liquid CDx (FoundationOne®) test [However, it is the development of next-generation sequencing (NGS) what has enabled molecular characterisation of cancer at a reasonable cost. These include the possibility of whole-genome sequencing (WGS), whole-exome sequencing (WES) or targeted sequencing. Whole-genome libraries can be enriched to analyse specific DNA targets mainly by two approaches: amplicon-based or hybridisation-based method. Amplicon-based uses pre-designed primers to amplify regions of interest by PCR and covers generally a limited panel of mutations whereas the hybridisation capture method allows for larger size panels and uses biotinylated probes (oligonucleotides complementary to regions of interest) that bind to PCR-amplified DNA and are isolated by streptavidin-coupled magnetic beads. These targeted approaches have been reported to detect prostate cancer mutations in blood and are used in clinically validated tests such as the UW-Oncoplex (UW-OncoPlexe®) test , 32, 33.e®) test . The advThe overall sensitivity for detection of an alteration is dependent on the tumour content (TC) of ccfDNA. This could be generated either as a measure of the most abundant alteration detected or using a bespoke orthogonal assessment. The most effective methods for measuring TC include commonly occurring clonal events. In situations such as metastatic breast cancer where a discrete number of hot-spot mutations are commonly clonal, restricted assays have been used , 34. PatAR) [AR is typically amplified or mutated in this setting following the selective pressures from androgen deprivation therapy (ADT) [AR point mutations emerge after treatment with abiraterone (combined with prednisone) and appear to be the most commonly detected in metastatic castration-resistant prostate cancer (mCRPC), AR L702H (AR c.2105 T > A) occurs primarily in patients previously treated with glucocorticoids and AR T878A (AR c.2632 A > G) is associated with progesterone-mediated activation. Detection of AR gene alterations in circulation associate with worse outcome in patients treated with androgen receptor signalling inhibitors (ARSI) such as abiraterone or enzalutamide [TP53, RB1 and PTEN [Once prostate cancer relapses after castration, the most common genomic events that emerge involve the androgen receptor (AR) , 8, 40. py (ADT) , 8, 41. lutamide . A numbeand PTEN , 43. In and PTEN .BRCA2 (13%) [BRCA2 associate with more aggressive disease and if detected in tumour or plasma, can be used to select patients for PARP inhibition [TP53 and RB1 [Following the recent approval of PARP inhibitors for mCRPC, ctDNA assessment has been clinically approved for detection of alterations in DNA damage response genes (DDR). DDR genes are aberrant in up to 20% of mCRPC, most commonly A2 (13%) . Gene lohibition . Althoughibition . In addihibition . Finally and RB1 . These aIn conclusion, whilst ctDNA genomic analysis is now implemented in the clinical management pathway and will be increasingly used by physicians, technical improvement in current assays and combining with other data will improve the granularity of the read-out and the patient populations liquid biopsies can be applied to.Epigenetic changes cover functionally relevant changes to the genome that do not involve alterations in the DNA sequence. They are mainly involved in gene expression modification. The most well-studied epigenetic feature of cfDNA is 5-methylcytosine (5mC) at cytosine–guanine (CpG) sites that tend to cluster in CpG islands in the promoter region of genes and are widely known to be involved in gene silencing , 49. DNAGSTP1, APC, RASSF1A, with potential transcriptomic or physiologic impacts on prostate cancer [GSTP1, the most well-studied, aberrantly methylated gene in prostate cancer, is one of the earliest and most frequent events [GSTP1 silencing to have a key role in tumour development [GSTP1 hypermethylation in tissue is prognostic in localised disease. In blood, several studies that used detection of GSTP1 as a proxy for prostate tumour in blood have found this to be prognostic in relapsed patients [There are a number of recurrent DNA methylation alterations, such as e cancer . Hypermet events . Given ielopment –65. Furtpatients , 67. Anapatients , 68, 69.Whole-genome methylation assessment identifies 1000 s of regions that are differentially methylated in prostate cancer DNA compared to normal or leucocyte DNA . This inMoreover, the aforementioned neuroendocrine phenotype is characterised by unique methylation patterns that can be detected in liquid biopsies analysing cfDNA and matching prostate tumour biopsies. This introduces the opportunity of early identification and avoids unnecessary biopsies in advanced prostate cancer patients .Another potentially informative molecular feature of cfDNA is the DNA fragment length. The fragment length (a.k.a. fragmentomics) distribution of cfDNA, usually peaking at 167 bp, carries the information of nucleosome positioning which can be used to derive tissue-of-origin. As reported, cfDNA show different fragmentation patterns in tissue-specific open chromatin regions which relates to nucleosome positioning and reflects differences in sequencing coverage , 74.In addition, variability of genome-wide plasma DNA coverage related to nucleosome occupancy can be used to infer gene expression patterns: cancer histological types could be differentiated based on their transcription factor accessibility patterns and nucleosome positioning which are both tissue-specific and different between healthy and cancer individuals. This approach could also identify re-programming of regulatory pathways in real-time, for example, the transition of AR-dependent prostate adenocarcinoma to an AR-independent “neuroendocrine” sub-type with distinct activated regulatory pathways .ctDNA (derived from tumours) has been suggested to have shorter fragment length than associated non-tumour ccfDNA –78. As aThe study of RNA in liquid biopsies is hampered by the short half-life of messenger RNA in circulation. Therefore, efforts have focused on characterising RNA from isolated CTCs, extracellular vesicles and/or the study of short microRNAs (miRNA).CTCs analysis is challenging due to the low ratio of cells compared to leucocytes  but adequate isolation and/or enrichment (often expensive) can allow for DNA, RNA and protein analysis plus single-cell assessment or in vitro culture studies , 81.® (currently distributed by Menarini Silicon Biosystems Inc.) was cleared by the FDA for prognostication of patients with advanced cancer using enumeration. It uses an immunomagnetic beads-based approach targeting EpCAM  present on the surface of most differentiated CTCs arising from adenocarcinomas. CTCs isolated from prostate cancer patients can express PSA and show molecular characteristics of prostate cancer such as AR copy number gain, PTEN loss and TMPRSS2-ETS gene fusion [The number of CTCs detected in blood has been associated with treatment outcomes and overall survival in prostate cancer and other cancers . CellSeae fusion . Enrichme fusion .® has been used to detect CTC expression of AR splice variants predicted to lack the ligand-binding domain and be constitutively active. The presence of these CTC may associate with resistance to androgen receptor signalling (ARS) inhibitors. Matched cohort studies have suggested taxanes could be preferred over ARSI when CTCs expressing AR splicing variants are detected, although in the absence of data from randomised clinical trials, its challenging to differentiate the effect of CTC counts and tumour volume from accurate predictions associated with the molecular marker [Microfluidic technology can enhance affinity-based capture and can capture EpCAM-independent CTCs , 86. Mecr marker , 91. Thir marker .Extracellular vesicles (EV) are lipid bilayer-delimited particles containing DNA, RNA or proteins and naturally released by both normal and tumour cells. Exosomes are the most commonly studied, constituting 40–100 nm-sized particles released into blood (and other fluids) by the fusion of multivesicular bodies with the plasma membrane. They play an important role in intercellular communication, potentially contributing to tumour progression and metastases , 94. ExomiRNAs are characterised by greater stability in circulation and can be isolated directly from plasma or following EV enrichment. miRNAs are small single-stranded non-coding RNA (around 20 nucleotides) that function in post-transcriptional regulation of gene expression. Prostate cancer patients appear to have higher levels of specific miRNAs which may have distinct roles and associate with unique phenotypes , 100. FoPSA testing is not suitable for population-based screening due to its low specificity for prostate cancer, resulting in massive over-treatment and misdiagnosis. Multiparametric magnetic resonance imaging (mpMRI)  has impr®). In their Circulating Cell-free Genome Atlas (CCGA) study (designed to combine data from cfDNA sequencing and machine learning for the development of a population-based cancer screening programme) [TP53 gene on plasma DNA samples collected from a limited number of localised prostate cancer patients before and after local treatment suggests detection of ctDNA before surgery is associated with poor clinical outcome  [One of the major efforts in the cancer screening space has been undertaken by GRAIL, Inc  , they haogramme) . It is purvival) . Overallurvival) , 110.ctDNA has shown promising evidence of detecting clinically relevant, minimal residual disease after primary treatment in many tumour types , 112. St® Liquid CDx that was developed as a companion diagnostic for the PARP inhibitors rucaparib and olaparib in metastatic castration-resistant patients (together with other cancer types). This hybridisation-based NGS panel captures 300 genes including mutations in BRCA1 and 2.The role of liquid biopsies is better established in advanced metastatic disease where tumour volumes and circulating tumour are higher. Moreover, the clinical need for the selection of patients for targeted treatment has followed drug development pathways with current indications focused on relapsed patients. Several companies have developed multigene targeted-capture NGS assays, intended for cfDNA analysis, covering single-nucleotide variants (SNVs), insertion or deletions (indels) and copy number alterations (CNAs) to detect a range of genomic aberrations at low allelic frequencies, primarily to provide tumour mutation profiling  . More reBRCA1/2 and ATM) versus cohort 2 (12 others DDR genes) with positive results for radiographic PFS (rPFS) for patients included in cohort 1 . Rucaparib has been approved for BRCA-mutated patients progressing after a taxane chemotherapy and an AR-targeted therapy based on the results of the open-label Phase II TRITON2 study which reported and objective response rate for BRCA1/2 aberrant of 43% [BRCA2 is well-established, given the wide range of alterations in a multitude of genes involved in homologous recombination repair, uncertainty exists for the sensitivity of specific alterations and sensitivity to PARPi, primarily as many on an individual basis are rare. Collation of outcome data, sharing across trials and continued annotation post-licensing will be important to improving prediction and patient selection.Olaparib was approved based on the results of the PROfound study, a randomised Phase III biomarker-driven clinical trial, in which patients progressing after abiraterone or enzalutamide received Olaparib versus the alternative ARSI . Patientt of 43% . Even thcfDNA testing without concurrently sequencing the patient’s whole blood as control could uncommonly identify alterations in DDR genes that are presumed to be pathogenic but are clonal haematopoiesis of indeterminate potential (CHIP) variants. These false positives could lead to inappropriate treatment of patients with PARPi without the expected efficacy .AR copy number of 1.92 as the cut point with the greatest association with shorter progression-free survival and overall survival on ARSI [Different studies have also identified genomic and epigenomic features that could predict acetate abiraterone (AA) treatment response. In one study, cfDNA cytosine modification patterns from 33 prostate cancer patients treated with AA were identified and showed divergences between AA-sensitive versus AA-resistant patients and potential for predicting treatment response based on cfDNA modification variability . In anot on ARSI .Additional to patient selection, liquid biopsies have potential for real-time tracking of treatment response and early detection of progression. For example, in studies of men with high burden relapsed metastatic prostate cancer, CTC count included in a composite panel with serum lactate dehydrogenase is a surrogate of survival and captures the effect of ARSI treatment . AnotherBRCA2 or PALB2 that are predicted to restore BRCA2 function in samples collected at the progression that were not present prior to treatment [AR emerges in ~15% of progression samples [Liquid biopsies allow tracking of emerging clones on treatment. Detection of alterations may be biased by the site of metastases  and other biological factors. Nonetheless, given most prostate cancer spread involves bone and is blood-borne, most studies have shown that tumour DNA in plasma represents clinically relevant progressing metastases. In mCRPC patients treated with PARP inhibition, ctDNA studies have identified a heterogeneous landscape of sub-clonal mutations in reatment , 123. Bo samples . Another samples . Trackin samples , 125. AlA fit-for-purpose liquid biopsy assay, intended for implementation in clinical practice for prostate cancer patients, should achieve three main goals: (i) analytical validation which refers to the reliability, accuracy of measure and reproducibility of the test  , (ii) thUncertainty remains on whether a pan-cancer or a prostate-specific test is more suitable for use in clinical practice. Firstly, blood pan-cancer tests can achieve economies of scale but may not be optimised to include prostate cancer-specific genomic alterations. Secondly, detection of gene loss and allelic imbalance at different copy number states remains challenging and requires optimised approaches. Thirdly, technological improvements can improve detection and reliability of detection of ultra-low allelic frequency variants , 8.One of the main strength of blood assays is the feasibility of repeated collection which could allow earlier detection of mechanisms of resistance to inform on treatment changes or combinational therapy. Combinatorial genomic, transcriptomic and epigenomic testing on blood samples could improve the prediction of phenotype and could be integrated to maximise the potential of liquid biopsies for clinical practice.Overall, a good prostate cancer blood assay should guide one or more clinical decision points such as diagnosis, molecular characterisation and risk stratification in an early setting, detection of early relapses, treatment cessation or intensification and identification of mechanisms of resistance to current treatments in the metastatic setting.Addressing these challenges in the next few several years with the collaboration of clinicians, researchers and bioinformaticians, will help to understand who and when someone should be molecularly profiled, how to design better biomarker-driven clinical trials and overcome technical challenges and finally introduce the liquid biopsy in the clinical routine practice for prostate cancer patients.


COVID-19 pandemic has changed clinical practice significantly. Trying to avoid airway manipulation to reduce viral transmission favoured regional anaesthesia techniques over general anaesthesia when feasible. In this case report, we share the anaesthetic care of a 45-year-old male with a history of intensive care unit admission due to COVID, positive pressure ventilation, and ECMO support. The patient was diagnosed with cubital tunnel syndrome, and his treatment was successfully applied as surgical release under infraclavicular block. This case is discussed in relation to recent trends in anaesthesiology, regarding patients with COVID-19 history. COVID-19 altered anaesthesia preferences.Regional anaesthesia can provide surgical anaesthesia and/or analgesia for a great variety of patients undergoing surgery.We recommend avoiding general anaesthesia in patients with COVID-19 history.Interfascial plane blocks could be an alternative to neuraxial anaesthesia to provide analgesia.1 During this time, the disease itself and regulations to decrease transmission changed medical practice. These changes were mostly to decrease virus transmission during the medical care of patients. One of these changes was increased usage of regional anaesthesia procedures where it’s appropriate, as advised by both European and American authorities.2 One of the main goals was avoiding airway manipulations which was the main route of viral transmission, but it has other benefits as well. In patients with COVID-19 history, disease-related complications can affect the perioperative management of the patient.6 Using regional anaesthesia can be a solution to some of these problems. Peripheral,8 central nerve blocks,9 and interfascial plane blocks11 with their benefits in patients with COVID-19 history are discussed briefly. A sample case is used to better understand the situation. The case is an elective elbow surgery in a patient with a severe COVID-19 infection history requiring intensive care unit (ICU) admission and ECMO support before the operation.COVID-19 has been a part of our lives since the start of the pandemic in early 2020.This patient, a 45-year-old male without any known disease prior, was internalised because of dyspnoea due to COVID-19. During his stay, the patient required ICU admission and intubation following respiratory failure. His condition required continuous extracorporeal membrane oxygenation (ECMO) support for 14 days. He was discharged from ICU and then hospitalised after resolution of pulmonary inflammation. During his initial hospitalisation, sclerosing cholangitis and mediastinal fibrosis with superior vena cava syndrome were diagnosed, both thought to be secondary to COVID history. Patient was discharged with 0.6 mL subcutaneous enoxaparin twice daily.During follow-up, the patient required intermittent hospitalisation due to pericholedoctal abscesses formed by sclerosing cholangitis. During his hospitalisation, the anticoagulant regime continued. Patient complained of pain in the left hand and was diagnosed with cubital tunnel syndrome by a hand surgery specialist. Routine painkillers and physical therapy were added to the patient’s treatment regime. Ulnar nerve release surgery in elective conditions was offered to the patient. Upon antibiotic treatment, patient’s complaints diminished, and laboratory tests and images confirmed that periductal abscess was resolved.−1 per hour was used because of patient’s restlessness to increase patient comfort. Patient did not describe pain sensation after surgery, and the patient was discharged with the cure of his orthopaedic complaint.Following the resolution of abscess, surgery was planned with regional anaesthesia, following the informed consent of the patient. Surgery was planned with regional anaesthesia considering the patient’s COVID-19 history and comorbidities. Surgery was completed successfully with the left-sided infraclavicular block using ultrasound guidance. Following 2 mg of intravenous midazolam premedication, and sedation using propofol infusion 3 mg kg3 General anaesthetic agents could increase the risk of cardiac dysfunction in patients with COVID-19 history.12 In pulmonary effects of disease, small airway disease and restrictive pattern functional limitation are found in some patients, 6 months following acute infection, even if the patient did not require oxygen supplementation.4 Hematologic complications include the risk of thromboembolic events, and risk remains increased, and median time of event is 78 days after acute infection.5 Thromboembolic events can occur as a complication of general anaesthesia as well.12 COVID-19-related thromboembolic event risk is higher in these patients, and some of them use anticoagulants.13 In such cases, both coagulation and bleeding-related complications can occur, and anaesthesiologists should be beware of them.Patients with a COVID-19 history present a challenge for anaesthesiologists because of disease complications. These complications are important for the choice of anaesthesia modality. Palpitations, chest pain, dysrhythmia, and decreased functional capacity can be seen following acute COVID-19 infection.6 Other complications of general anaesthesia regarding cardiac function and thromboembolic events should be considered. Overall, avoiding general anaesthesia can prevent a variety of complications in patients with COVID-19 history. In postoperative settings, regional anaesthesia decreases analgesic requirement in various cases,8 avoiding side effects such as respiratory depression, dizziness, and hypotension related to postoperative opioids.Choice of anaesthesia should involve all these considerations regarding COVID-19 history, as most of these complications can flare following general anaesthesia administration. Using regional anaesthesia avoids potential complications of general anaesthesia. Regional anaesthesia allows the patient to continue spontaneous respiration and diminishes the necessity for mechanical ventilation, preventing further barotrauma to the patient with a history of prolonged intubation, such as this patient.8 Variability of nerve block choices enables anaesthesiologists to personalise the regional anaesthesia technique according to patient needs and type of surgery. In this case, the surgical area was defined as the elbow joint; therefore, an infraclavicular block was used to achieve surgical anaesthesia.The type of regional anaesthesia should be selected case-wise. Peripheral nerve blockage is a popular choice of anaesthesia for surgeries regarding extremities.8 In limited cases, visceral surgeries can be completed with neuraxial blockade only,9 decreasing the necessity of general anaesthesia and avoiding potential complications arising from general anaesthesia. Anticoagulant usage is especially important for neuraxial procedures, as it is known to increase potential neurological complications. Spinal hematoma risk persists in patients with COVID-19 history because anticoagulants are a part of suggested treatment in patient with severe COVID-19 history who require hospital admission.Central neuraxial blocks can be used as the sole anaesthetic technique for caesarean sections, lower extremity surgeries, and abdominal surgeries.11 and their usage is increasing daily.15 Yet, these interfascial plane blocks provide mainly analgesia and not anaesthesia.10 These blocks are considered as part of multimodal analgesia and are not always enough to provide total pain-free period on their own.10 However, they reduce postoperative opioid requirement.14Interfascial plane blocks offer an alternative to epidural analgesia mainly for abdominal and thoracic surgeries,3 For abdominal and thoracic surgeries, especially, interfascial plane blocks present an alternative to central neuraxial blocks and should be included in the armamentarium of anaesthesiologists to reduce postoperative opioid requirements.In accordance with The European Society of Regional Anaesthesia and Pain Therapy (ESRA)/The American Society of Regional Anesthesia and Pain Medicine (ASRA) societies recommendations, regarding post-COVID era, regional anaesthesia techniques present a great advantage for the safety of patients and help avoid general anaesthesia.