This cohort study examines patients with gastrointestinal cancer to evaluate the characteristics of acquired resistance to immune checkpoint inhibitors. What are the characteristics and prognosis of acquired resistance to immune checkpoint inhibitors in gastrointestinal (GI) cancer?In this cohort study of 1124 patients with advanced GI cancer, a high frequency (46.4%) of acquired resistance (AR) was found. Oligoprogression was the most common pattern of AR progression, and lymph nodes were the most susceptible site for AR.Given that large-scale retrospective analyses of patients with GI cancer treated with immune checkpoint inhibitors are lacking, this study will provide useful information and a deeper understanding to clinicians regarding AR in patients with advanced GI cancer. With the expanding use of immune checkpoint inhibitors (ICIs) in gastrointestinal (GI) cancer, the occurrence of acquired resistance (AR) has gradually emerged. However, the progression patterns and survival of patients with AR to ICIs are still unknown.To explore the characteristics and prognosis of AR after ICI therapy in patients with advanced GI cancer.This cohort study screened patients with advanced GI cancer treated with ICIs between January 14, 2016, and December 31, 2020, at Peking University Cancer Hospital. Initial response was defined as complete response, partial response, or stable disease longer than 6 months as assessed by Response Evaluation Criteria in Solid Tumors version 1.1. Progression was also based on Response Evaluation Criteria in Solid Tumors version 1.1. Progression or death after the initial response was defined as AR. Oligoprogression of AR was defined as 2 or more disease sites progression. The current status of AR in GI cancer and the patterns of AR and its prognosis were evaluated. The site of AR and subsequent management were also assessed. Data were analyzed from June to August 2021.Patients in the cohort were treated with mono-ICI or combination therapy.Kaplan-Meier analyses and log-rank tests were conducted for overall survival analyses. Univariate and multivariate Cox analyses were conducted to determine the prognostic implications of each variable.P < .001). Lymph nodes (101 patients [58.4%]) appeared to be the most common site of AR. Management after AR was mainly systemic therapy (96 patients [55.5%]).Of the 1124 patients who received ICIs, 373 (33.2%) patients  achieved an initial response, and 173 (46.4%) patients  developed AR. Almost all patients (167 patients [96.5%]) developed AR within 24.0 months. Progression patterns of AR were most commonly oligoprogression (122 patients [70.5%]) rather than polymetastatic progression (38 patients [22.0%]) and were associated with a good prognosis (38.5 vs 14.0 months; hazard ratio, 0.37; 95% CI, 0.18-0.74; Oligoprogression was the most common pattern of AR progression, and lymph nodes were the most susceptible site for AR. Further study will be needed to determine the most favorable management for AR. Several studies have attempted to characterize the progression of AR to ICIs in other solid tumors on the basis of clinical data analysis. For example, a 2018 study by Gettinger et al11 identified and evaluated 26 patients with NSCLC who developed AR to ICI therapy. This study revealed that lymph nodes were the main progression site of AR and showed different follow-up management strategies for patients with AR. In another study on AR in NSCLC, Xu and colleagues12 found that the major progression pattern of AR was oligoprogression. They also concluded that local radiotherapy combined with continuous immunotherapy would benefit this group of patients. Similar findings have been reported in melanoma by Pires da Silva et al,13 who identified an association between the size and site of individual metastases and patterns of response, progression, and clinical outcome.Although ICIs have shown great success in many types of solid tumors, most patients who initially respond to immunotherapy develop acquired resistance (AR) over time, thus limiting the durability of immunotherapy. In the evaluation of some clinical trials, the rate of AR varied among tumor types , especially in GI cancer, where the rate was as high as 42% to 71%.However, the clinical features and survival outcomes of AR to ICIs in patients with GI cancer are still unknown. Therefore, we performed a retrospective cohort study to comprehensively describe the clinical characteristics and prognosis of AR in advanced GI cancer.14 Written informed consent was given by each individual before the treatment. The study enrolled patients with esophageal cancer, gastric cancer or esophagogastric junction (EGJ) cancer, intestinal cancer , hepatobiliary-pancreatic cancer, and neuroendocrine tumors. Patients were divided into 2 different ICI therapy cohorts. Mono-ICI was defined as programmed cell death protein 1/programmed cell death 1 ligand 1 (PD1/PD-L1) inhibitor or PD1/PD-L1 inhibitor combined with cytotoxic T-lymphocyte–associated protein 4 inhibitor or transforming growth factor–β inhibitor therapy. Combination therapy was defined as ICI therapy combined with chemotherapy and/or targeted therapy.Patients with advanced GI cancer receiving treatment with ICIs in the Department of GI Oncology, Peking University Cancer Hospital & Institute, between January 14, 2016, and December 31, 2020, were screened in this large retrospective study. This study was exempted by the institutional review board at the Peking University Cancer Hospital and was performed according to the Declaration of Helsinki.Initial response was considered achieving complete response (CR), partial response (PR), or stable disease more than 6 months after ICI therapy. Progression was defined as an increase in the size of the primary tumor and or the appearance of a new lesion. AR was defined as progression or death after initial response. Imaging scans of individual patients during ICI therapy were reviewed by trained radiologists, and responses were determined according to Response Evaluation Criteria in Solid Tumors version 1.1. Patient progression sites were captured, and follow-up management after AR was recorded.The pattern of progression was determined using predefined characteristics at the time that any tumor site met the criteria for the progression of disease, whichever occurred first. Oligoprogression and polymetastatic progression of AR were defined as progression in less than 2 or more than 3 disease sites, respectively. Original progression was defined as the progression of lesions which were originally involved before immunotherapy.P < .05 was considered to be significant. Statistical analyses were performed with GraphPad Prism statistical software version 8.0 (GraphPad Software) and R statistical software version 3.6.2 .Progression-free survival (PFS) was defined as the time from the first dose of ICIs to radiological disease progression or death from any cause. Overall survival (OS) was defined as the time from the first dose of ICIs to death from any cause. The time to AR was defined as the time from the first dose of ICIs to AR. OS was estimated using a Kaplan-Meier survival analysis and 2-sided log-rank test. The association of each variable with survival of patients with AR was completed by univariate and multivariate Cox regression analysis. All 1124 patients with GI cancer who received ICIs at Peking University Cancer Hospital & Institute between January 14, 2016, and December 31, 2020, were included in the total cohort . In addiOf the 373 patients, a total of 173 patients (46.4%) were included in the AR cohort after the initial response, most of whom were patients with CR or PR whose disease had progressed (145 patients [83.8%]). A total of 28 patients (16.2%) progressed from stable disease more than 6 months. The median OS for patients with AR was 20.9 months  , the median time to AR was 7.3 months  and almost all patients (167 patients [96.5%]) developed AR within 2 years A. The 1-We also characterized the AR in different primary foci. The frequency of AR differed among patients with GI cancer D. Among We next analyzed the patterns of progression in patients with AR and the association with clinical outcomes. Patterns of progression were predefined to assess the heterogeneity among them.Oligoprogression was more common than polymetastatic progression in the AR cohort, occurring in 122 patients (70.5%). Polymetastatic progression occurred in 38 patients (22.0%), and 13 patients died without radiographic progression. The proportions of oligoprogression and polymetastatic progression were similar in immunotherapy cohorts. In the mono-ICI cohort, oligoprogression and polymetastatic progression occurred in 49 patients (72.1%) and 12 patients (17.6%), respectively. In the combination cohort, oligoprogression and polymetastatic progression occurred in 73 patients (69.5%) and 26 patients (24.8%), respectively A.P < .001) (P < .001) and the combination therapy cohort   B. In theP = .02) C and 3D.P < .001) (eTable 1 in the P = .002)  showed progression to the original lesion (eTable 2 in the P < .001) . A total of 36 patients (20.8%) had limited lymph node progression, 58 patients (33.5%) had organ-only progression, and the largest proportion of patients (66 patients [38.2%]) had mixed progression. Patients with mixed progression had much lower OS than patients with lymph node or organ-only progression  B and C.We further compared the progression sites according to the different primary tumors. Lymph nodes remained the most predominant AR site in esophageal and gastric or EGJ cancer, with 30 patients (75.0%) and 57 patients (62.0%), respectively. The peritoneum (6 patients [33.3%]) was the main site of progression for intestinal cancer, whereas the main site of progression for hepatobiliary and pancreatic cancers was the liver (9 patients [42.9%]) D.Finally, we counted the treatment patterns of these patients with AR. A total of 149 patients (86.1%) had available follow-up treatment information , followed by those who maintained the original immunotherapy (22 patients [12.7%]). Local therapy involved interventional therapy and radiotherapy; 16 patients (9.2%) and 7 patients (4.0%) received radiotherapy and interventional therapy, respectively. We tried to compare the associations of systemic vs local therapy with long-term survival benefits. The median OS for patients who received systemic therapy was not reached, and the median OS for patients who received local therapy was also not reached. There was no significant difference in OS between the 2 groups.In this cohort study, we reviewed a large cohort of patients receiving immunotherapy and comprehensively described the frequency, progression patterns, and sites of AR in GI cancer. We found oligoprogression was the most common pattern of AR progression, and lymph nodes were the most susceptible site for AR.7 estimated the rate of AR in many types of solid tumors. For example, the rates of AR were approximately 32% to 64% in NSCLC, 35% to 54% in head and neck squamous carcinoma, and 12% to 60% in melanoma.7 In GI cancer , the rate of AR ranged from 41% to 71%. In the present study, we confirmed the high rate (46.4%) of AR in GI cancer, which was consistent with a previous study. Notably, the rate of AR in intestinal cancer (26.5%) appeared to be much lower than that in other GI cancers. This phenomenon may be associated with the fact that most of the patients with intestinal cancer (61 of 68) in our cohort had microsatellite instability-high/dMMR tumors. Furthermore, we also noticed that the median time to AR was shorter in GI cancer than in other cancers. In our cohort, the time to AR was approximately 7 to 8 months for either mono or combination ICIs or different primary sites, and most patients developed AR within 24.0 months.15 Accumulating evidence suggests that dynamic cross-talk between cancer cells and the immune microenvironment is associated with the occurrence of AR.7 Although the current mechanisms of AR are summarized as defects in tumor antigen presentation, neoantigen depletion, tumor-mediated immunosuppression or exclusion, and additional inhibitory checkpoints, the exploration of the mechanism is still far from sufficient.20 Much work remains to be done to provide patients with personalized response strategies and to improve prognosis with an increased understanding of the mechanism of AR.A previous study21 In the present study, we classified the progression patterns of AR into oligoprogression and polymetastatic progression on the basis of findings from previous studies. Consistent with studies on other solid tumors, we found that the pattern of AR was predominantly oligoprogression and was associated with a good prognosis. To distinguish the AR progression patterns of different ICI therapies, we also validated the definition and survival in mono-ICI and combination therapy cohorts. The results confirmed the reliability of this definition approach. Given the limited number of similar studies available, more clinical data are still needed to support these results.The progression patterns of AR have been previously reported in melanoma and NSCLC but, to our knowledge, have not been explored in GI cancer.25 Peritoneal metastasis is the most common form of metastasis for dMMR intestinal cancer, while hepatobiliary-pancreatic cancer is dominated by liver metastasis.28A notable finding in this study was that a large proportion of patients with AR had lymph node progression; however, this was not observed for all GI cancers. For intestinal cancer , the most common site of AR was the peritoneum, while the main site of AR for hepatobiliary-pancreatic cancer was the liver. This may be due to the inherent metastatic heterogeneity and metastatic preference of the cancer, as lymph nodes are one of the most common sites of metastasis in esophageal and gastric cancers, especially in advanced stages.15 have shown that in melanoma, different patterns of progression of AR were associated with OS with combination immunotherapy, while in patients with advanced NSCLC with AR, studies have suggested that local treatment and/or continuation of ICIs after AR may be an effective option. This revealed that there was also great variability in the management of different cancers after AR. In the present study, we found that systemic therapy was the predominant treatment for patients with AR in GI cancer, with chemotherapy being the mainstay. Considering the inclusion of multiple GI cancer species in our cohort and the inherent heterogeneity of GI cancer, it was difficult to conclusively identify the most favorable management for these patients. More clinical retrospective studies are still needed to further explore the most favorable management for patients with GI cancer who experience AR.At the end of the study, we tried to explore the treatment that would be most beneficial for the long-term survival of patients with AR. Previous limited studiesOur study has limitations. Because this was a retrospective study, the potential selection bias from a single center may affect the clinical application of our results. In addition, the treatments after AR were complicated and diverse, and we have not yet obtained the most beneficial treatment for these patients. More large-scale clinical studies are needed to further validate this definition and the reliability of the results.In summary, to our knowledge, this was the first retrospective study on the clinical characteristics and prognosis of AR in advanced GI cancer. Our findings showed a high prevalence of AR in GI cancer, oligoprogression was the predominant AR pattern with a good prognosis, and the most common sites of AR were concentrated in the lymph nodes. The most beneficial treatment for patients with AR needs further exploration. In particular, we performed a more detailed subgroup analysis for different ICI therapies and primary sites, aiming to provide more accurate information on the characteristics of AR in different patient populations. Our study may provide useful information and a deeper understanding to clinicians regarding AR in patients with advanced GI cancer.


Background: Medicine requires the brightest minds, regardless of gender. Women working in the health sciences have time and again demonstrated the value of their technical training, communication skills, emotional support, and ability to provide understandable explanations to their patients. The objective of this work was to carry out a historical review of the main female authors linked to classic sensorimotor neurorehabilitation techniques throughout the nineteenth and twentieth centuries, as well as female authors linked to eponymous tests or assessments, exposing their scientific trajectory and main contributions to the field of neurological rehabilitation. A literature review was conducted. The databases of Physiotherapy Evidence Database (PEDro), Scopus, CINAHL Medical Science, Medline through EBSCO and PubMed were used to obtain the biographical information of each author, searches of papers were limited until August 2021 in English and Spanish languages. Seventeen female authors were identified who linked to the main rehabilitation techniques or approaches described for neurological rehabilitation and for scales or tests with an eponymous origin as an example of female contribution on neurorehabilitation. Biographical information based on the computerized search in the electronic databases showed 57 potentially relevant articles. Of those articles, 43 were subsequently excluded. Fourteen articles were used to show their contribution to neurorehabilitation. This paper demonstrates the influential role of women in the history of sensorimotor neurorehabilitation throughout the nineteenth and twentieth centuries, linked to the methods, techniques, concepts, or approaches used in physical therapy or occupational therapy. Medicine requires the brightest minds, regardless of gender. The incorporation of women into the health sciences has helped to humanize them, thanks to their unquestionable technical training, their communication skills, emotional support, and their ability to provide understandable explanations to their patients . HoweverGradually, medical specialties have ceased to differentiate, although some remain known colloquially as “feminized”  and others as “masculinized” . This phenomenon has been described as “the illusion of choice” with regard to medical specialties, where it is precisely gender bias that exists in their choice . DespiteThe lack of opportunities for women in leadership positions works like a “leaky pipeline” because despite having the knowledge and preparation, they simply get lost in the process and do not reach the top. This phenomenon is also often linked to the difference in the number of publications authored by women doctors who dedicate most of their time to the clinic rather than to research, though the lack of opportunities that academics have to publish compared to their peers has also been described ,6. HowevAs such, the objective of this work was to carry out a historical review of the prominent female authors linked to classic sensorimotor neurorehabilitation techniques throughout the nineteenth and twentieth centuries, as well as female authors linked to test or assessments described as eponymous, exposing their scientific trajectory and main contributions to the field of neurological rehabilitation.A literature review was conducted. For this paper, searches focused on female authors who were linked to classic sensorimotor neurorehabilitation techniques or approaches described in proprioceptive neuromuscular facilitation; Bobath concept; Brunnstrom technique; Rood Method; Perfetti Method; Task-oriented motor learning; Body-weight–supported treadmill training; Constraint-induced movement therapy; Muscle strengthening programs; and physical reconditioning. Information about psychological rehabilitation, pharmaceutical or other medical interventions was excluded.Biographical information and a brief contribution to neurorehabilitation were described for each author by a computerized search of the following electronic databases: CINAHL Medical Science, Medline through EBSCO, PubMed, Physiotherapy Evidence Database (PEDro) and Scopus. The search was limited to papers published until August 2021 in English and Spanish. The following keywords were used: Seventeen female authors were identified who were linked to the main rehabilitation techniques or approaches described for neurological rehabilitation and for scales or tests with an eponymous origin, based on The order of the authors is not subject to any implicit meaning:Muscles, Testing and Function” has become one of the main textbooks for physical therapy students. Florence came to link muscle function with posture and pain. Drawing primarily on her extensive work researching people with polio, Florence’s book set a new standard for musculoskeletal assessment and treatment [Florence Peterson Kendall was a physical therapist considered the “mother” of physical therapy. Florence spent many years treating polio patients at Children’s Hospital in Baltimore. In the 1940s, she was a supervisor of physical therapy for the Maryland State Department of Health, specializing in polio patients. Her book “reatment . KendallLucille Daniels and Catherine Worthingham were two physiotherapists who co-authored two of the most important books in rehabilitation: Muscle Testing (1946) and Therapeutic Exercise (1957). Daniels was an associate professor and director of the Division of Physical Therapy at Stanford University School of Medicine. Worthingham worked in the field of corrective physical education. In 1939, she was an assistant professor and director of physical therapy at Stanford University. She would later become a director at the National Foundation for Infantile Paralysis. In 1950, Worthingham was the first physical therapist to hold a doctoral degree (in anatomy). These two women systematized and defined the most commonly accepted method of evaluating muscle strength against the rater´s resistance using a 0 to 5 scale ,14,15. CAnne G. Fisher is an internationally recognized expert in occupational therapy. She was a pioneer in the application of the Rasch analysis in occupational therapy, and she developed the Occupational Therapy Intervention Process Model . One of Cynthia C. Norkin was a physical therapist, an associate professor emerita and director of the School of Physical Therapy at the College of Health and Human Services, Ohio University, Athens, Ohio. She held a doctoral degree (in education) from Boston University in 1984. She authored the most important book about the measurement of joint motion in rehabilitation, which is commonly used in people with neurological disorders. She systematized the assessment of range of motion using universal goniometers and inclinometers .Mary E. Tinetti is the Gladys Phillips Crofoot Professor of Medicine and Public Health, as well as Chief of Geriatrics at Yale School of Medicine . ThroughMeasuring balance in the elderly: development and validation of an instrument”. Her research interests include disability and fall prevention as well as health services studies examining quality of care and outcomes. She is eponymously associated with the Berg Balance Scale [Katherine Berg is a Canadian physical therapist. She holds a doctoral degree, the thesis of which was titled “ce Scale , used toce Scale ,24.Gait Analysis: Normal and Pathological Function”, the classic text on gait analysis. Dr. Perry was a visionary pioneer in the field of rehabilitation sciences. She was honored with “Woman of the Year for Medicine” by the Los Angeles Times in 1959 [Jacquelin Perry was an orthopedic surgeon and leading authority on gait analysis. She was born in Denver, Colorado, USA on 31 May 1918, and died in Downey, California on 11 March 2013. When Perry found herself obliged to stop operating for health reasons, she followed the advice she was accustomed to giving to her patients: accommodate your new reality. With a prior qualification in physical therapy and already interested in human movement, her own accommodation of reality was to set up a gait laboratory at Rancho Los Amigos National Rehabilitation Center in California. Perry first established a national and then an international reputation as one of the leading authorities on the biomechanics of walking and other forms of movement. During the 1980s, Perry was among the first to closely study what became known as post-polio syndrome . In 1992 in 1959 .Margaret S. Rood, applied neurophysiological research findings to children and adults with motor impairments. The Rood method proposes achieving greater control of voluntary movement and posture based on the physiological differences of motor units. According to Rood, selecting the sensory inputs that reach the body can influence the structures of the CNS and improve muscle activity for greater control of movement and posture. In line with this physiological basis, different types of muscle fiber can be stimulated depending on the intensity of the applied stimulus. The Rood method includes different techniques to facilitate movement and normalize muscle tone, as follows: facilitating techniques ; and normalizing techniques  . Rood waAnna Signe Sofia Brunnstrom was born in Stockholm, Sweden, on 1 January 1898. She died in Darien Convalescent Center in Darien, Connecticut on 21 February 1988 . The metBerta Bobath was a German physiotherapist. She was born in Berlin on 5 December 1907. Initially, she studied at the “Anna Hermann” gymnastics and dance school and, after completing her training, she remained at this school as an instructor until 1933. In 1938, she went to Germany and following that, took up residence in London. In London she began to work as a gymnastics instructor. In 1941, she gained employment at The Princess Louise Hospital for Children and studied to become a physiotherapist. She married the physician Karel Bobath in 1941 and, from the neuroscience available at the time, together they developed the Bobath Concept. This is an approach to the assessment and treatment of individuals with impaired function, movement, and postural control due to a central nervous system injury. The Bobath Concept is based on the modulation of abnormal reflex activities and the facilitation of normal movement patterns through key control points, always directed to a specific functional objective. The treatment is globalizing, and part of an approach whose first step is the analysis of the norm. They both taught and provided training in this concept for different rehabilitation professionals throughout the world. It is the most popular approach for treating patients with neurological disorders in the Western world. Berta Bobath passed away on 20 January 1991 .Margaret Johnstone was born in 1919, eventually becoming a physiotherapist and Founder of the Johnstone Approach. Johnstone graduated from the Royal Infirmary in Edinburgh in 1943. She proposed learning as a lifelong process, reflected in her work of searching for methods of recovery for stroke patients in their rehabilitation process. Initially, she accumulated experience with orthopedic patients, children and polio patients. In 1966, Margaret started to use inflatable air splints as a simple way to treat upper limbs in people with stroke. Margaret paid specific attention to keeping the hemiplegic limb out of compensatory patterns and the detrimental movements of the low-motor stroke patient to a minimum . This coJean Ayres was an occupational therapist who developed a theory of sensory integration, which she believed to be basic to children’s ability to be successful in everyday activities, and was an advocate for individuals with special needs. She developed the psychometric properties of tests of various aspects of sensory integration, and invented equipment to be used in practice. Ayres inspired occupational therapists to become researchers both by role modeling and by teaching graduate students. She authored several books that are used worldwide .Margaret Knott was a physical therapist who became known worldwide for teaching the proprioceptive neuromuscular facilitation (PNF) technique, first described for polio, multiple sclerosis and rheumatic fever patients. Dr. Kabat, Director of the Kabat-Kaiser Institute , together with physiotherapist Margaret Knott and later with Dorothy Voss in 1953, expanded and developed the PNF method nationally and internationally. In 1956, Knott and Voss published the first textbook on PNF. Margaret Knott passed away in 1978. The philosophy and basic principles of PNF, together with the specific spiral and diagonal patterns, are the essential ideas linked to PNF. All activities within PNF intervention are directed towards a functional objective and are relative to the environment in which the function is to be achieved ,33. PNF Janet Howard Carr and Roberta Barkworth Shepherd are two Australian physiotherapists who, in 1984, based on studies of movement science, neurophysiology and learning theories, proposed a new approach to the rehabilitation of stroke patients. According to Carr and Shepherd, the goal of rehabilitative treatment should be relearning oriented to specific tasks, forcing the use of the paretic side. Their principle was simple and intuitive: “one learns what one practices” ,35. ActiPrinciples of Occupational Therapy”, published in 1947. She served as secretary from 1952 to 1956 and president from 1957 to 1962 of the World Federation of Occupational Therapists. She was an active clinician at the Curative Workshop of Philadelphia and an academic educator at the University of Pennsylvania School of Allied Health Professions [Finally, Clare S. Spackman was a pioneer in occupational therapy. She was the author and co-editor, alongside Helen Willard, of the first book in occupational therapy, “fessions .The role of women in medicine has progressively increased over time, starting from a highly competitive and predominantly male context, and moving to the distinguished collegiate physicians of present day. The notable interest of women in medical careers was promoted at the end of the nineteenth century, within the framework of a democratization process, such as the fight for the right to female suffrage, equal working conditions, and the right to education, among other social conquests ,38.The origins of neurological rehabilitation are potentially linked to polio epidemics. Elizabeth Kenny (1886–1952), a proponent of early mobilization, used humerus heat and therapeutic exercise in patients with poliomyelitis, whose treatment allowed the development of physiotherapy in the twentieth century, not only from movement therapy, but also through physical agents such as electricity, light, and heat . FurtherContemporary with Elizabeth Kenny was the British woman Olive Frances Guthrie Smith (1883–1956), a physiotherapist at St Mary’s Hospital in London, previously trained during the First World War in the “Almeric Paget (Military) Massage Corps,” an institution that would treat the injuries of wounded soldiers. Olive Guthrie Smith was practically the inventor of suspension therapy, used in the treatment of neurological patients in the first half of the twentieth century, which lost its place in rehabilitation services over time .In the 1950s, a major conceptual shift in neurological physical therapy was evident as the neurophysiological or neurofacilitation approaches were developed, mainly linked to female researchers and therapists. The focus changed from muscle to non-muscle elements. Techniques were primarily directed at the nervous system, with movement facilitation by stimulation of the nervous system. Major influences were the work of the Bobath Concept and that of Kabat, Knott, and Voss (PNF). Other therapists also developed their ideas for therapy around this time, including Rood, Ayres, and Brunnstrom. These approaches are often referred to as eponymous as they were named after their originators , all of Neurological rehabilitation has changed over the past decades as scientific and technological developments have offered a greater understanding of brain reorganization and the mechanisms of motor control, performance, impairments, and adaptations, owing to the many female researchers described in this paper. Current scientific research is leading to a changing focus in clinical interventions, with emphasis on optimizing motor performance through task-oriented exercise and training, strength training, and taking the influence of the environment into account. In this context, to achieve successful rehabilitation, increased emphasis needs to be placed on challenging, motivated, and meaningful task training to promote learning, as many of the female authors such as Carr and Shepherd highlighted, and as described in the Results section of the present paper .The influential role of women in the history of neurorehabilitation is unquestionable, as most of the classic methods, techniques, concepts, or approaches used today in neurorehabilitation  were desTo achieve these objectives, it is important to know the historical contributions of the pioneers and contemporary prominent authors on neurorehabilitation, and to promote gender equity at all levels including clinical, teaching, research, and management, since an underrepresented situation at multiple levels for females in key areas of neurorehabilitation has been documented ,43,44,45This historical review presents several limitations. First, many other female researchers have significantly contributed to the development of neurorehabilitation to its present stage. It has been presented an approach regarding the primary contributions from the developmental to contemporary period to neurological rehabilitation. Conversely, no aspect related to psychological rehabilitation has been addressed in this review. Finally, the language restriction of the literature reviewed must be noted as other important authors could be missing in this review.The role of women in medicine has been progressively increasing over time, starting from a highly competitive and predominantly male context, and moving to the distinguished collegiate physicians of present day. A historical review of the prominent women authors linked to sensorimotor neurorehabilitation throughout the nineteenth and twentieth centuries was presented, exposing their scientific trajectory and main contributions to the field of neurological rehabilitation. In this context, the influential role of women in the history of neurorehabilitation is undeniable, given that many of the classic methods, techniques, concepts, and approaches used in neurorehabilitation were described and/or developed by female researchers linked to physical therapy and occupational therapy.


Androgen-deprivation therapy (ADT) via targeting androgens/androgen receptor (AR) signals may suppress cell proliferation in both prostate cancer (PCa) and bladder cancer (BCa), yet its impact on the cell invasion of these two urological cancers remains unclear. Here we found targeting androgens/AR with either the recently developed antiandrogen Enzalutamide (Enz) or AR-shRNAs led to increase PCa cell invasion, yet decrease BCa cell invasion. Mechanistic dissection revealed that suppressing androgens/AR signals could result in differential alterations of the selective circular RNAs (circRNAs) as a result of differential endogenous AR transcription. A negative autoregulation in PCa, yet a positive autoregulation in BCa, as a result of differential binding of AR to different androgen-response elements (AREs) and a discriminating histone H3K4 methylation, likely contributes to this outcome between these two urological tumors. Further mechanistic studies indicated that AR-encoded circRNA-ARC1 might sponge/alter the availability of the miRNAs miR-125b-2-3p and/or miR-4736, to impact the metastasis-related PPARγ/MMP-9 signals to alter the PCa vs. BCa cell invasion. The preclinical study using the in vivo mouse model confirms in vitro cell lines data, showing that Enz treatment could increase PCa metastasis, which can be suppressed after suppressing circRNA-ARC1 with sh-circRNA-ARC1. Together, these in vitro/in vivo results demonstrate that antiandrogen therapy with Enz via targeting AR may lead to either increase PCa cell invasion or decrease BCa cell invasion. Targeting these newly identified AR/circRNA-ARC1/miR-125b-2-3p and/or miR-4736/PPARγ/MMP-9 signals may help in the development of new therapies to better suppress the Enz-altered PCa vs. BCa metastasis. While the androgen receptor (AR) may play key roles to increase the tumor cell proliferation in several sex hormone-related tumors including prostate –6, bladdCircular RNAs (circRNAs), noncoding forms of RNA, are widely expressed in many tissues with distinct functions to influence the development of several diseases including tumor progression , 21. FunThe circRNAs are produced as a result of endogenous host gene transcription, and sequence analysis indicated that the AR locus can produce four circRNAs from a combination of exons. The linkage of these AR gene-coded circRNAs to the differential effects on the cell invasion of prostate cancer (PCa) vs. bladder cancer (BCa) in response to androgen-deprivation therapy (ADT) with the antiandrogen Enzalutamide (Enz) treatment (ADT-Enz), however, remains unclear.Here we found that targeting androgens/AR with ADT-Enz or AR-shRNA led to increase AR-encoded circRNAs expression in PCa cells, yet suppress those circRNAs in BCa cells. The consequence of such differential alteration of the AR-encoded circRNAs may then increase PCa cell invasion, yet decrease BCa cell invasion. Further mechanistic analysis revealed that Enz/AR/AR-encoded circRNAs signaling might function via sponging/altering the expression of miR-125b-2-3p and/or miR-4736 to influence the metastasis-related PPARγ/MMP-9 signals to impact the PCa vs. BCa cell invasion.Early studies indicated that AR could enhance BCa metastasis , 8 yet sHere we first focused on the potential role(s) of AR-encoded circRNAs on the AR’s differential alteration of the PCa vs. BCa cell invasion, as these AR-encoded circRNAs are most closely related to the functional entity of AR. A database (Circbase) search indicated that the AR locus produces four circRNAs , one (ARE1) located on the 1-kb upstream of AR gene and another (ARE2) located in the intron 3 . We wereTogether, results from Fig. To study the consequences of differentially regulating the circRNA-ARC1 expression induced by targeting androgen/AR in the PCa vs. BCa, we applied the matrigel-coated transwell invasion assay, and results revealed that decreasing fAR (shAR) led to increase cell invasion in both PCa C4-2 and CWR22Rv1 cells Fig. , yet incImportantly, interruption approaches via adding shRNA to suppress the circRNA-ARC1 led to partially reverse the AR-shRNA-increased cell invasion in PCa C4-2 and CWR22Rv1 cells Fig. , while pTogether, results from Fig. Next, to dissect the mechanism of how AR-modulated circRNA-ARC1 can influence the PCa vs. BCa cell invasion, we focused on miRNAs, since early studies indicated that circRNAs might function as a miRNAs sponge or miRNAs reservoir to alter the breast or colorectal tumor progression .Through bioinformatics analysis of existing databases  and published literatures, we found that circRNA-ARC1 might be able to interact with several miRNAs  via PROMO website in the 1-kb 5′ promoter region of the MMP-9 gene Fig. . We thenConsistent with the direct regulation of the MMP-9 by PPARγ, we also confirmed that increasing AR via adding AR-cDNA in both BCa T24 and TCC-SUP cells significantly increased the expression of MMP-9 Fig. . In contTogether, results from Fig. To further demonstrate that AR-modulated circRNA-ARC1 may function via sponging/altering the miR-125b-2-3p/miR-4736 to regulate PPARγ/MMP-9 signals, we applied the interruption approach, and results revealed adding miR-125b-2-3p or miR-4736 led to reverse the AR-enhanced expression of PPARGC1B and PDK1 as well as MMP-9 in BCa cells Fig. . SimilarIt is worth noting that adding either miR-125b-2-3p or miR-4736 can result in repression of both PDK1 and PPARGC1B expression to a different degree in both PCa and BCa cells. Indeed, a survey of potential miRNA target genes with a relaxed criterion in target selection in the database of Targetscan indicated that both miRNAs can target both genes albeit with different predictive stringencies.Interestingly, human clinical sample surveys using TCGA datasets revealed that AR has a positive correlation with PPARγ, PDK1, and PPARGC1B , with (Groups 2 and 4) or without (Groups 1 and 3) intra-peritoneal (i.p) injection of Enz (30 mg/kg). Tumor growth and metastases were evaluated weekly with the In Vivo Imaging Systems (IVIS) analysis in a noninvasive manner.To validate the above results from in vitro studies of PCa and BCa cell lines in vivo, we applied an orthotopic PCa xenograft mouse model. We generated CWR22Rv1 cells with luciferase expression with added sh-circRNA-ARC1 (Groups 3 and 4) to suppress circRNA-ARC1 expression or with vector control (Groups 1 and 2) and implanted these cells into the anterior prostate lobes of nude mice  may function via differentially altering the AR-encoded circRNA-ARC1 to increase PCa metastasis, yet decrease BCa metastasis, not only may help treatment to improve the ADT-Enz therapy efficacy via reducing those adverse effects, it may also represent the first evidence to link AR-encoded circRNAs to the differential anti-AR therapies. Interestingly, a recent study also found that AR might suppress its transcription in a LSD1-dependent manner , and ADT signals , AR-coac signals , or TGFß signals .The precise mechanisms at the molecular level of how differential complexes may guide AR to bind to the distinct AREs located on different regions of the AR gene remain unclear. Our data showed that AR preferred to bind to the ARE1 in BCa cells, yet preferred to bind to the ARE2 in PCa cells. These differential bindings of AR to the different AR loci resulted in a positive autoregulation in BCa cells vs. a negative autoregulation in PCa cells, which was monitored by H3K4me2 levels. It is likely the tumor origin of tissues as well as cellular signaling networks in the PCa vs. BCa are different so that AR may associate with different co-factors/co-repressors to form distinct complexes that result in binding to different AREs in the AR locus, and consequences of such differential binding may then result in a positive autoregulation in BCa cells vs. a negative autoregulation in PCa cells. Indeed the expression of AR protein in PCa vs. BCa cells is also different with much higher AR protein detected in the PCa cells.The human clinical data indicated that ADT is an effective therapy in suppressing the recurrence of AR-positive BCa cells , with it2 environment at 37 °C.The human PCa cell lines, C4-2 and CWR22Rv1, the BCa cell lines, T24, TCC-SUP, UMUC3, TCC-5637, and 293T, were originally purchased form American Type Culture Collection . All PCa cells were cultured in RPMI 1640. All BCa cells were cultured in DMEM, all the media contained 10% FBS, penicillin (25 units/ml), and streptomycin (25 µg/ml) while cells were cultured in the humidified 5% COTo generate AR overexpressing or AR knocked-down stable cell populations, C4-2, CWR22Rv1, T24, TCC-SUP, UMUC3, or TCC-5637 cells were infected with lentiviral vectors, pWPI-AR/pWPI-Vec or pLKO1-sh-AR/pLKO1-scr. The lentiviruses were produced in 293T cells with the psAX2 packaging plasmid and pMD2G envelope plasmid together with the transfer plasmid. After 48h transfection, virus supernatants were collected, concentrated, and frozen at −80 °C for later use. Enz and PPARγ-Antagonist III treatments were performed by culturing cells with 10 µM of each drug for 48 h.The transient transfection was performed using the Lipofectamine 3000 (Invitrogen) reverse transfection protocol according to the manufacturer’s instructions.GAPDH (6c5), AR (N-20) antibodies, and PPARγ-Antagonist III were purchased from Santa Cruz Biotechnology. PPARγ, PDK1, PGC-1β, and MMP-9 antibody were purchased from One World Lab. Anti-mouse/rabbit secondary antibody for Western Blot was from Invitrogen. Normal rabbit IgG was also from Santa Cruz Biotechnology. The PPARγ-Antagonist III and Enz stock concentrations were 10 mM.2), 1-μl murine Ribonuclease Inhibitor (40 units/μl), and 1-μl Superscript III (Life Technologies) were added and this cDNA reaction was incubated at 25 °C for 10 min, 50 °C for 50 min, 55 °C for 10 min, and 85 °C for 5 min. Then 0.5-μl cDNA reaction was used as the template for qPCR and fraction resistance was computed as 2∧(RNase R Ct - Mock Ct).Total RNA was isolated by TRIZOL lysis followed by PureLink purification of the aqueous phase (Life Technologies). Total RNA at 2 μg was treated in a 10-μl reaction with 0 units (mock treatment) or 20 units of RNase R (Epicentre) in 1×RNase R buffer, 1-unit/μl murine Ribonuclease Inhibitor (New England Biolabs), and incubated at 37 °C for 1 h. Then 1 μl 1-mM EDTA, 1 μl 10-mM each dNTP, and 1 μl 100-µM random hexamer were added and the RNA denatured at 65 °C for 5 min and placed on ice. Then 4-μl 10X buffer  using the Gibson ligation system. PLKO-shRNAs were constructed following the standard protocol. Briefly, oligos were annealed and ligated with EcoR I + Age I digested PLKO vector. The junction of circARC1 was used as a template to design shRNAs.The pLVTHM-miR-125b-2-3p, pLVTHM-miR-4736, pLVTHM-miR-4725, pWPI-OE-ARC1, pLVTHM-shARC1, or pLKO.1-shAR, the psAX2 packaging plasmid, and pMD2G envelope plasmid were transfected into 293T cells using the standard calcium-chloride transfection method for 48 h to get the lentivirus supernatants. The lentivirus supernatants were collected and concentrated by density gradient centrifugation, then frozen in −80 °C for later use.For RNA extraction, total RNAs were isolated using Trizol reagent (Invitrogen) and 1 µg of total RNA was subjected to reverse transcription using Superscript III transcriptase (Invitrogen). The qRT-PCR was conducted using a Bio-Rad CFX96 system with SYBR green to determine the mRNA expression level of a gene of interest. Expression levels were normalized to the expression of TATA box binding protein TBP RNA. Primers used for genes of interest were listed in Supplementary Table Cells were lysed in RIPA buffer, and proteins (30 µg) were separated on 8–10% SDS/PAGE gel and then transferred onto PVDF membranes . After blocking membranes, they were incubated with appropriate dilutions of specific primary antibodies, and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies and visualized using the ECL system . The antibodies used were listed in Supplementary Table 5 cells/well, and the bottom chambers contained mixed conditioned media (CM) with 10% FBS, and then incubated for 24 h at 37 °C. Following incubation, the invaded cells attached to the lower surface of the membrane were fixed by 4% paraformaldehyde and stained with 1% crystal violet. Cell numbers were counted in five randomly chosen microscopic fields (100×) per membrane.The invasion capability of PCa and BCa cells was determined using the Transwell assay. Briefly, for invasion assay, before seeding the cells, 100 µl of Matrigel  was dissolved in 1.5-mL serum-free DMEM and applied to upper chambers of 8-µm-pore-size polycarbonate membrane filters , and the transwells were incubated at 37 °C for 5 h. Harvested cells were then seeded with serum-free media into the upper chambers at 1 × 10The human PPARGC1B/PDK1 3′UTR containing wild-type or mutant miRNA-responsive elements were cloned into the psiCHECK™-2 vector construct  downstream of the Renilla luciferase ORF. Luciferase activity was measured by the Dual-Luciferase Assay (Promega) according to the manufacturer’s manual.Cells were cross-linked with 4% formaldehyde for 10 min followed by cell collection and sonication with a predetermined power to shear chromatin to 200–500-bp DNA fragments. Then, cell lysates were pre-cleared sequentially with normal rabbit IgG and protein A-agarose. Anti-PPARγ antibody was used for immunoprecipitation. For the negative control, IgG was used in the reaction. Specific primer sets were designed to amplify a target sequence within the human MMP-9 promoter, and PCR products were identified by agarose gel electrophoresis.2 EDTA, 1-mM EGTA, 1% NP-40, 1% sodium deoxycholate, 2.5-mM sodium pyrophosphate, 1-mM beta-glycerophosphate, 1-mM Na3VO4, and 1-µg/ml leupeptin) supplemented with RNase inhibitor (1.5 µl). The supernatant was incubated with 250 nM of anti-sense oligos overnight at 4 °C. Streptavidin Agarose beads were incubated with the supernatant for 2 h. The complex was centrifuged at 3000 rpm, and the beads were washed five times with RIPA lysis buffer. The RNA was extracted using Trizol (Invitrogen) according to the manufacturer’s protocol and subjected to RT-PCR analysis.The culture cells were lysed in RIPA lysis buffer  according to the manufacturer’s protocol and subjected to RT-PCR analysis.n = 32 for each group): (1) Vector (DMSO); (2) Enz; (3) pWPI+shARC1; and (4) Enz+shARC1. The transduced CWR22Rv1 cells suspended in media were mixed with Matrigel  and injected into the anterior prostates (1 × 106 cells/AP) of these nude mice. Tumor development and metastasis were monitored by Luminescence Imager  once a week. When the tumors become palpable 2 weeks after implantation, the shARC1 and vector mice were randomly assigned into two groups, respectively (32 mice per group), receiving various therapies by i.p. injection 3 times/wk/4 wks (Enz at 30 mg/kg or DMSO every other day). The mouse body weights were monitored weekly as well as tumors by the IVIS system for another 2–3 weeks. At the end of the treatment, mice were sacrificed and tumor growth and metastases at lymph nodes as well as to distant organs were analyzed. The animal experiments were approved and supervised by the University Committee on Animal Resource of University of Rochester Medical Center.Six-to-eight weeks old nude mice were purchased from NCI and divided into four treatment groups (Tissues were fixed in 10% (v/v) formaldehyde in PBS, embedded in paraffin, and cut into 4-μm sections and used for H&E staining and IHC staining with specific primary antibodies against AR/MMP-9. To enhance antigen exposure, the slides were treated with 1 × EDTA at 98 °C for 10 min for antigen retrieval. The slides were incubated with endogenous peroxidase blocking solution, and then were incubated with the primary antibody at 4 °C overnight. After rinsing with Tris-buffered saline, the slides were incubated for 45 min with biotin-conjugated secondary antibody, washed, and then incubated with enzyme conjugate HRP-streptavidin. Freshly prepared DAB  was used as substrate to detect HRP. Finally, slides were counter-stained with hematoxylin and mounted with aqueous mounting media. Positive cells were calculated as the number of immunopositive cells × 100% divided by total number of cells/field in ten random fields at ×400 magnification.t-test was used to test the difference in metastatic foci numbers among the four groups of mice. Linear correlation analyses were performed to determine the correlation between the gene expression levels. P < 0.05 was considered statistically significant.Data were expressed as mean ± SEM from at least three independent experiments with each data point in triplicate. Statistical analyses involved the Tukey’s test with SPSS 17.0 . Chi-square test was used to analyze the number of mice with metastases between two groups. Exact Fisher Supplementary Figure 1Supplementary Figure 2Supplementary Figure 3Supplementary Figure legends


Nerve-glia (NG2) glia or oligodendrocyte precursor cells (OPCs) are distributed throughout the gray and white matter and generate myelinating cells. OPCs in white matter proliferate more than those in gray matter in response to platelet-derived growth factor AA (PDGF AA), despite similar levels of its alpha receptor (PDGFRα) on their surface. Here we show that the type 1 integral membrane protein neuropilin-1 (Nrp1) is expressed not on OPCs but on amoeboid and activated microglia in white but not gray matter in an age- and activity-dependent manner. Microglia-specific deletion of Nrp1 compromised developmental OPC proliferation in white matter as well as OPC expansion and subsequent myelin repair after acute demyelination. Exogenous Nrp1 increased PDGF AA-induced OPC proliferation and PDGFRα phosphorylation on dissociated OPCs, most prominently in the presence of suboptimum concentrations of PDGF AA. These findings uncover a mechanism of regulating oligodendrocyte lineage cell density that involves trans-activation of PDGFRα on OPCs via Nrp1 expressed by adjacent microglia. Oligodendrocyte precursor cell (OPC) proliferation and differentiation is greater in white matter than gray matter. Here, the authors show regulation of OPC proliferation in white matter involved trans-activation of PDGFRα on OPCs via Nrp1 expressed by adjacent microglia. The developmental expansion of this population is critically dependent on platelet-derived growth factor AA (PDGF AA) acting on their alpha receptor (PDGFRα)6. The most characterized and established role of OPCs is their ability to self-renew and generate the correct number of myelinating oligodendrocytes (OLs) needed for optimal network function. Hypomyelinating or dysmyelinating mouse mutants with reduced OLs or myelin are accompanied by increased OPC proliferation8. In the mature CNS, a demyelinated lesion rapidly elicits OPC proliferation12. However, the molecular and cellular mechanism by which such feedback signals promote OPC proliferation is not known.Oligodendrocyte precursor cells (OPCs), also known as NG2 glia or polydendrocytes, are widely distributed throughout the developing and mature central nervous system (CNS)14, and those in white matter proliferate more in response to PDGF AA15. While both cell intrinsic and extrinsic factors influence the differential OPC behavior16, heterotopic transplantation of 300-μm3 pieces in slice cultures suggests that the greater proliferative response of OPCs in white matter to PDGF AA is imparted by their local pericellular microenvironment15.While OPCs are evenly distributed throughout the CNS, the rate of OPC proliferation and OL differentiation is greater in white matter than in gray matter18 as potential candidates. Since heparin and HSPGs are known to affect a variety of growth factors, we chose to explore the role of Nrp1 as a molecule with more specific targets. Nrp1 is a type 1 transmembrane molecule, which was first discovered in the developing Xenopus optic tectum19 and subsequently shown to be expressed on developing murine axons20 and regulate axon pathfinding by binding to class III semaphorins21. In endothelial cells, Nrp1 binds vascular endothelial cell growth factor (VEGF) and modulates signal transduction through VEGF receptor (VEGFR)22. Here, we show that Nrp1 is expressed on ameboid and activated microglia in the developing and demyelinated corpus callosum and promotes OPC proliferation by activating PDGFRα on OPCs in trans.To determine the mechanism underlying the differential response of OPCs in gray and white matter to PDGF AA, we searched the literature for a potential co-receptor for PDGF that could differentially regulate the proliferative response of OPCs to PDGF AA in gray and white matter. We identified heparan sulfate proteoglycans (HSPGs), and neuropilin-1 (Nrp1)15  NG2cre;Z/EG double transgenic mice15 Fig. . In the 15 Fig. . By cont15 Fig. . Anti-Nr15 Fig. , but the15 Fig. . We did 15 Fig.  compared22. Nrp1 immunoreactivity was also detected on axons in the dorsal spinal cord  into the corpus callosum of 2- to 3-month-old mg-Nrp1-cont and mg-Nrp1-cko mice Fig. . Nrp1 wa12 of an early proliferative response in the region immediately surrounding the core demyelinated lesion. These observations indicate that loss of Nrp1 on activated microglia significantly compromised the early proliferative response29 of OPCs to acute demyelination.At 3 and 7 dpl, we pulse-labeled LPC-injected mice with EdU and examined the effect of microglial Nrp1 deletion on OPC proliferation in the lesion. EdU incorporation into EYFP-negative, NG2+ OPCs at the lesion site in mg-Nrp1-cko was 1.9-fold lower than that in mg-Nrp1-cont lesions at 3 dpl and 3.2-fold lower at 7 dpl Fig. . OPC proTo examine whether the reduced proliferation of OPCs in mg-Nrp1-cko mice affected subsequent events in myelin regeneration, we examined the lesion at 14 dpl. Quantification of OLs in and around the lesion at 14 dpl revealed that the density of CC1+ OLs in mg-Nrp1-cko lesion was reduced to 63% of that in mg-Nrp1-cont lesion Fig. . To asseWe next examined the impact of Nrp1 deletion from activated microglia on remyelination at 28 dpl. MBP immunofluorescence intensity in the lesioned corpus callosum of mg-Nrp1-cko mice was lower, at 61% of that in mg-Nrp1-cont mice Fig. . There wThe above experiments revealed that Nrp1 on microglia is critical for PDGF AA-mediated proliferation of OPCs in the white matter and for the proliferative response of OPCs to acute demyelination. We next examined whether excess Nrp1 could augment the proliferative response of OPCs. Exogenous Nrp1 was added to slice cultures from P8 NG2cre;Z/EG mice in the form of soluble Nrp1-Fc fusion protein. Nrp1-Fc consisted of the extracellular domain of rat Nrp1, which had 98% amino acid identity with mouse Nrp1, fused to the Fc region of human immunoglobulin IgG1 in a homodimeric fusion protein construct. Addition of Nrp1-Fc to slice cultures in the presence of 50 ng/mL PDGF AA led to a significant increase in OPC proliferation in the cortex at 0.4 and 2 µg/mL of Nrp1-Fc Fig. . By contTo determine whether the ability of exogenous Nrp1-Fc to augment PDGF-induced proliferation in gray matter was mediated by PDGFRα, we incubated slice cultures with function-blocking goat antibody to mouse PDGFRα in the presence of PDGF AA. In the absence of Nrp1-Fc, addition of 1 μg/mL of anti-PDGFRα antibody reduced OPC proliferation to 13.3% in gray matter and to 22.5% in white matter Fig. . In the Since slice cultures contained different cell types, the above effects of Nrp1-Fc on OPCs could have been mediated by direct effects of Nrp1-Fc on OPCs or by indirectly altering signaling pathways in microglia or other cell types. To resolve this, we added Nrp1-Fc to purified dissociated OPC cultures and examined their proliferation in response to PDGF AA. OPCs were immunopanned from P2-4 mouse neocortex, and 2 μg/mL Nrp1-Fc was added together with 0 to 50 ng/mL PDGF AA Fig. . ImmunopNext, we determined whether Nrp1-Fc could augment PDGFRα activation on OPCs exposed to limited amounts of PDGF AA. PDGFRα activation was assessed by immunoblotting for tyrosine phosphorylation on PDGFRα after treating OPCs for 30 min at 37°C with 15 μg/mL PDGF AA with or without 2 μg/mL Nrp1-Fc. Immunoblots of protein extracts from the treated cells were incubated with rabbit and goat antibodies that recognized phosphorylated PDGFRα and total PDGFRα, respectively. We detected a significant increase in the level of phosphorylated PDGFRα in Nrp1-Fc-treated cells compared with those treated with PDGF AA alone Fig. . This inTo further examine the interaction between Nrp1-Fc and PDGFRα on OPCs, we performed co-clustering experiments by incubating dissociated OPCs with control-Fc or Nrp1-Fc for 30 min at 4°C or 36°C and stained for Fc and PDGFRα, as well as Olig2. After incubating OPCs with control-Fc consisting of human IgG Fc dimer at 4°C or 36°C or with Nrp1-Fc at 4°C Fig. S, PDGFRα We have shown that Nrp1 expressed by activated microglia plays a critical role in promoting OPC proliferation in early postnatal white matter tracts and after demyelination in the adult corpus callosum. Nrp1 was detected on the majority of ameboid microglia that appeared transiently in the early postnatal white matter, was not detected on resting ramified microglia, and was re-expressed on activated microglia/macrophages after demyelination. During both development and after demyelination, Nrp1+ microglia were closely apposed to OPC processes. In slice culture, deletion of Nrp1 in microglia significantly reduced PDGF AA-induced OPC proliferation in white but not gray matter. In vivo, microglial Nrp1 deletion significantly reduced OPC proliferation, and the majority of OPCs that were proliferating in P5 corpus callosum were in contact with microglia. Microglial deletion of Nrp1 had no effect in regions where microglia were ramified and did not express Nrp1, such as the cortex and the mature corpus callosum. In adult corpus callosum, Nrp1 was robustly induced on activated microglia/macrophages after acute demyelination, and loss of Nrp1 in these cells significantly reduced OPC proliferation and subsequent oligodendrocyte differentiation and remyelination. Exogenous Nrp1augmented PDGF AA-induced proliferation of cultured OPCs by increasing phosphorylation and clustering of PDGFRα. These findings support the model in which Nrp1 on microglia modulates PDGFRα-mediated proliferation of adjacent OPCs in trans Fig. .Fig. 8Pr30. They exist in different interconvertible functional states. In the normal mature brain, they exist as resting ramified microglia, or “homeostatic microglia”, with highly branched processes32 and are involved in a wide range of homeostatic regulation under physiological conditions34, as well as in their well known immune function35. In response to a variety of insults, ramified microglia transform into activated phagocytic microglia that become rounded in morphology and upregulate the lysosomal protein CD6836. Microglia also exist in the early postnatal white matter tracts as ameboid microglia with phagocytic activity before they transform into ramified microglia37.CNS parenchymal microglia originate from the embryonic yolk sac and begin to colonize the brain after embryonic day 9.5 (E9.5)Nrp1 was expressed transiently on ameboid microglia in P5-8 corpus callosum and cerebellar white matter but not on ramified microglia in the cortex and became undetectable on microglia by P30. The developmental window during which Nrp1 was detected on ameboid microglia correlated with the period during which microglia-specific deletion of Nrp1 reduced OPC proliferation. Furthermore, this coincided with the period of rapid OPC proliferation and OL differentiation.38, could trigger a response that activates their Nrp1 expression.Resting ramified microglia in the mature CNS no longer expressed Nrp1. However, within three days following acute demyelination, activated CD68+ microglia/macrophages robustly upregulated Nrp1 on the cell surface. Deletion of Nrp1 from microglia significantly reduced OPC proliferation in the lesion during the first week after demyelination and subsequently led to reduced OL differentiation and myelination. Although the signals that dynamically upregulate Nrp1 on activated microglia in the developing and demyelinated white matter remain unknown, it is possible that phagocytosis of dead OL lineage cells by ameboid/activated microglia, a process which has been previously reported39 or early postnatal proliferative region-associated microglia (PAM)40, where oligodendrogliogenesis is actively occurring prior to myelination. Intriguingly, among microglia from five different ages and from demyelinated lesions, Nrp1 transcript counts were highest in those from P4/5 brain and LPC-induced white matter lesions39http://www.microgliasinglecell.com/), whereas Nrp1 expression was lower in the homeostatic microglia in the mature CNS. Thus, the two subpopulations of phagocytic microglia not only share their transcriptome but they also regulate OPC proliferation similarly by dynamically modulating Nrp1 expression on their surface.Recent single-cell transcriptomic studies have identified a transcriptionally unique subtype of microglia/monocytic cells in the mouse brain that are found in early postnatal white matter, which are referred to as axon tract-associated microglia (ATM)41. However, beneficial effects of microglia during myelin repair have also been identified42. Their dichotomous role appears to depend on their activation state44. During normal development, ameboid microglia/ATM/PAM in the early postnatal white matter tracts play a critical role in myelination. This is mediated in part by insulin-like growth factor-1 (IGF1)45, which is highly expressed in ameboid microglia40. Pharmacological or genetic perturbation of the activation state of the ameboid microglia causes deficits in OL development and myelination47. However, the mechanisms by which microglia impart these positive effects on OL lineage cells have remained unclear.Traditionally, microglia have been associated with inflammation and deleterious effects on OLs and myelin in demyelinating lesions of multiple sclerosisOur studies have revealed that Nrp1, which is highly expressed on activated microglia in white matter, plays a critical role in promoting OPC proliferation during development and after demyelination. Developmental deletion of microglial Nrp1 did not lead to permanent myelin defects, which could be due to compensation by proliferation and migration of microglia that had escaped Nrp1 deletion. Since we only deleted Nrp1 from microglia and not from vascular cells or axons, the partial inhibition of OPC proliferation in the mg-Nrp1-cko could be due to additional effects of Nrp1 on cell types other than microglia, as well as Nrp1-independent mechanisms that also promote developmental OPC proliferation.48, and deletion of Nrp1 from microglia and macrophages slows glioma growth49.Nrp1 loss from activated microglia that appeared in the demyelinated lesion led to a severe decrease in OPC proliferation followed by decreased OL regeneration and protracted remyelination. With compromised proliferative response of OPCs after acute demyelination, additional OPCs may have to be recruited from surrounding areas such as the subventricular zone or the gray matter, which could have contributed to the delayed remyelination. Interestingly, Nrp1 is highly expressed by microglia/macrophages associated with malignant glioma50 and is critical for growth cone collapse induced by Sema3A51. Nrp1 was highly expressed on axons in E18.5 corpus callosum but the level had significantly dropped by P5. Axonal Nrp1 became undetectable in the corpus callosum after P14 and was not re-expressed after demyelination. Thus, axonal Nrp1 is not likely to be a major modulator of OPC proliferation. A related protein Nrp2 shares 44% amino acid identity with Nrp1 and binds primarily Sema3F. Semaphorins have been implicated in OPC migration during development53 and after demyelination, acting via Nrp1 and 2 on OPCs55. However, we did not detect Nrp1 or 2 expression on OPCs, or on other cells in the corpus callosum, which was consistent with RNA-seq data for OPCs56. Neither did we detect Nrp2 on microglia, also consistent with the transcriptomic studies40. Furthermore, the dramatic decrease in OPC proliferation in demyelinated lesions after Nrp1 deletion from activated microglia supports the view that microglial Nrp1 functions as a key inducer of OPC proliferation.Nrp1 is the axonal co-receptor for class III SemaphorinsIn slice cultures, blocking Nrp1 reduced PDGF AA-mediated OPC proliferation in the corpus callosum but not in the cortex. By contrast, addition of Nrp1-Fc augmented PDGF AA-mediated OPC proliferation in gray but not white matter. Nrp1 may be present in saturating amounts in white matter, whereas in gray matter, the level of endogenous Nrp1 may be insufficient to adequately activate PDGFRα on OPCs. This is consistent with the immunohistochemical detection of higher levels of Nrp1 on activated microglia in white matter. This is also supported by the ability of Nrp1-Fc to augment proliferation and PDGFRα phosphorylation on dissociated OPCs in the presence of a suboptimum concentration of PDGF AA.18. In this case, both Nrp1 and PDGFRα are expressed in cis on the same cell, similar to the role of Nrp1 in VEGF165-mediated VEGFR2 stimulation (reviewed in refs. 57). By contrast, our findings support a model of trans-activation of PDGFRα on OPCs by Nrp1 on adjacent microglia 21Lbe/J; RRID:IMSR_JAX:003920). The NG2cre mice used in this study had been generated by injecting a BAC construct that contained NG2creERTM, but one of the founders (NG2creERA) expressed cre independently of tamoxifen64 and exhibited reporter expression in a temporal and spatial pattern that was identical to NG2creBAC;Z/EG mice previously described65. Since cre in the NG2creERA line was more stable than that in the original NG2creBAC mice described in ref. 65, the slice culture experiments described here were performed with NG2creERA;Z/EG mice, which we will refer to here as NG2cre;Z/EG mice. To delete Nrp1 in microglia, we crossed Nrp1fl/+ or Nrp1fl/fl mice26 -Nrp1tm2Ddg/J; RRID:MGI:3528190) with Cx3cr1creERT2-ires-EYFP mice27 (Jackson Laboratory stock no. 021160 B6.129P2(Cg)-Cx3cr1tm2.1(cre/ERT2)Litt/WganJ; RRID:MGI:5528845) to generate mg-Nrp1-cont or mg-Nrp1-cko mice, respectively. Both males and females were used in all the experiments. Mouse genetyping primers are listed in Supplementary Table We have complied with all relevant ethical regulations for animal testing and research. All animal procedures received ethical approval by the Institutional Animal Care and Use Committee of the University of Connecticut. Mice were housed in a facility with 12:12 light:dark cycle, 50% humidity, and maintained at 73.5 °F. For slice cultures, we used NG2cre;Z/EG double transgenic mice66. The animals were perfused at 3, 7, 14, and 28 days post lesioning (dpl). To examine OPC proliferation, EdU was injected twice, 2 h apart, before sacrifice at 3 or 7 dpl.To induce demyelination, mice were anesthetized using isoflurane and their heads were placed in a stereotaxic apparatus. We injected 2 µl of 2.5% lysolecithin  dissolved in PBS into the corpus callosum of 9- to 12-week-old mg-Nrp1-cont and mg-Nrp1-cko mice, using the stereotaxic coordinates 0.3 mm anterior from the bregma, 1 mm lateral, and 1.9 mm from the surface of the skulll-lysine and 0.01 M sodium meta-periodate and postfixed in the same fixative for 2 h, after which the tissues were dissected and washed four times in 0.2 M sodium phosphate buffer, pH 7.4. The tissues were cryoprotected in 0.2 M sodium phosphate buffer containing 30% sucrose for at least 24 h, frozen in OCT compound , and 20-µm sections were cut on a Leica CM3050S cryostat. For free floating sections, 50-μm-thick coronal sections were cut with a Leica vibratome VT1000S directly after fixation and rinsing.Mice were perfused with 4% paraformaldehyde (PFA) containing 0.1 M 6 sequence was added to a cDNA clone coding for the rat PDGFα receptor extracellular domain. This cDNA was ligated into the PCEP/4 vector and transfected into 293 EBNA cells, followed by hygromycin B selection to obtain positive colonies. After establishment of confluent monolayers of the transfected cells, the secreted his-tagged receptor fragment was purified from serum-free culture supernatant by chromatography on Ni++-agarose (Qiagen). Authenticity of the purified material was confirmed by amino acid sequencing. Rabbit antisera produced against this receptor fragment were affinity-purified on a column constructed by coupling the purified receptor fragment to cyanogen bromide-activated Sepharose CL-4B (Pharmacia). Immunofluorescence tests using frozen sections of postnatal day 10 mouse brain confirmed that the affinity-purified antibody labeled OPCs in wild type specimens, but yielded no labeling in NG2 null specimens.The antibodies are listed in Table 4.5H2O , 4 ng/mL Alexa Fluor-647-conjugated azide , and 100 mM sodium ascorbate  at room temperature for 30 min. The sections were then washed three times in PBS, stained with 5 µg/mL Hoechst 33342 in PBS , and mounted in Vectashield without DAPI . To examine cell death, we applied ApopTag Red In Situ Apoptosis Detection Kit  according to the manufacturer’s recommended procedure, after treating the tissues with pre-cooled ethanol:acetic acid (2:1) for 5 min.For immunolabeling, sections were rinsed in PBS, blocked in 5% normal goat serum (NGS) or 1% bovine serum albumin (BSA) containing 0.1% Triton X-100 in PBS for 1 h at room temperature, followed by incubation in the primary antibodies at 4°C overnight. The sections were rinsed three times in PBS and incubated in the secondary antibodies at room temperature for 1 h. Then the sections were rinsed and mounted with Vectashield with DAPI . To detect EdU, immunolabeled sections were washed 3 times with PBS and incubated in the Click reaction mixture containing 150 mM NaCl, 100 mM TrisHCl pH 7.15, 4 mM CuSOTM diamond knife on a Leica Ultracut UCT microtome and stained with methylene blue/azure blue. The region of interest was selected with upright Leica DMR and Zeiss AxiovertM200 inverted microscopes, and ultrathin sections were cut with an ultra 45° Diatome TM diamond knife. Sections were collected on 100 mesh copper grids. Images were obtained using a bright field FEI Tecnai Biotwin G2 Spirit (Netherlands) transmission electron microscope operated at an accelerating voltage of 80 kV and equipped with an AMT 2k (4 megapixel) XR40 CCD camera.After perfusion and post-fixation of LPC-injected mice at day 28 dpl, brains were washed in PBS. One-hundred micrometer thick coronal slices were obtained by slicing the brains with a Leica vibratome VT1000S. Slices containing the LPC-induced lesion were selected and fixed again in 4% PFA and 2% glutaraldehyde in 0.1 M sodium phosphate buffer for 2 h. The corpus callosum within the region of interest was dissected and processed for resin embedding. Specimens were further fixed with 1% Osmium tetroxide at room temperature for 2 h. Tissue was dehydrated through a series of graded ethanol, during which 1.5% uranyl acetate was included. 100% ethanol was gradually mixed with an increasing portion of propylene oxide and finally incubated in propylene oxide. Subsequently, infiltration with Spurr’s resin was carried out with an increasing portion of resin diluted with propylene oxide. Infiltration was completed with 100% epon resin. Tissue was embedded in flat double end molds and polymerized at 70°C for 48 h. Semithin sections were cut with a Diatome67 or from P8 mg-Nrp1-cont or cko mice after 4-OHT injection at P2-3 in vivo. Forebrains were chopped into 300-µm thick slices and transferred to ice-cold dissection medium consisting of 124 mM NaCl, 2.004 mM KCl, 1.25 mM KH2PO4, 4.004 mM MgSO4 (anhydrous), 2.0 mM CaCl2⋅2H2O, 26 mM NaHCO3, 10 mM d-( + )-glucose, 2 mM ascorbic acid and 0.075 mM adenosine dissolved in water. Individual slices were separated and placed on Millicell culture inserts and maintained at air-liquid interface in a humidified incubator at 37 °C with 5% CO2 in slice media consisting of 50% Minimal Essential Medium with Earle’s Salts, 25 mM HEPES buffer, pH 7.22, 25% HBSS without CaCl2, MgCl2, or MgSO4, 25% horse serum, 0.4 mM ascorbic acid, 1 mM l-glutamine, and 1 mg/L insulin. Culture medium was changed 24 h after dissection and every other day thereafter for 9 days. On day 7, PDGF AA  was added to the cultures and the slices were incubated for 48 h. During the last 5 h of incubation, 10 µM EdU was added to the slice medium. To block Nrp1 or PDGFRα function, slices were incubated in different concentrations of goat anti-mouse Nrp1 antibody  or 1 µg/mL of goat anti-mouse PDGFRα antibody  during the last 48 h of incubation (on day 7). Nrp1-Fc fusion protein containing the extracellular domain of rat Nrp1  or the control human Fc dimer  was added to slice cultures at the indicated concentrations for the last 48 h of incubation. At the end of the incubation, slices were fixed with 4% paraformaldehyde and processed for immunofluorescence labeling and EdU detection.Slice cultures from the forebrain and cerebellum were prepared from P8 NG2cre;Z/EG mice68. The brains were scooped out from the opened skull and diced in an ice-cold D-PBS without Ca2+/Mg2+(Invitrogen) in the dish. The minced tissues were incubated in an oxygenated papain solution containing 200 units of papain , 2 mg of l-cysteine  and DNase I  on a 37 °C heating block for 30–45 min. Following tissue digestion, papain was inactivated using a low-ovo solution containing BSA , D-PBS (Invitrogen), Trypsin inhibitor . The brain tissues were dissociated by gently pipetting in low-ovo solution. Following centrifugation, the cell pellet was suspended in panning buffer consisting of 0.2% BSA and 2 mg/mL insulin in DPBS and immunopanned in 10-cm petri dishes precoated with CD140a rat anti-mouse PDGFRα antibodies for 45 min. Following 6-8 times wash with D-PBS, the adhered OPCs were trypsinized for 6–7 min, and trypsin was inactivated using 30% fetal bovine serum (FBS). Following centrifugation, the OPCs were resuspend in DMEM-Sato medium containing Dulbecco’s Modified Eagle’s Medium (Invitrogen), 2 mM l-glutamine, SATO supplements , penicillin/streptomycin, 1 mM sodium pyruvate, 5 μg/mL insulin, 5 μg/mL N-acetyl-l-cysteine , 1X Trace Elements B ,10 ng/mL d-Biotin  and 1X B-27 . Purified OPCs were plated on glass coverslips coated with poly-d-lysine  at a density of 40,000 cells/well in 24-well plates for EdU incorporation assays or on 35-mm tissue culture dishes coated with poly-l-lysine  at a density 10 × 106 cells/dish for immunoblotting. For EdU detection, the cells on coverslips were initially incubated in DMEM-Sato medium and PDGF AA for 2 days, and 10 µM EdU was added to the cells for 6 h prior to staining for Olig2, NG2, and EdU. For detecting PDGFRα phosphorylation by immunoblotting, immunopurified OPCs were incubated with DMEM-Sato medium without PDGFAA for 12 h. Then, the medium was replaced with new medium containing 2 μg/mL Nrp1-Fc or control Fc protein and 15 ng/mL PDGFAA and incubated at 37 °C for 30 min, after which the cells were promptly chilled on ice for protein extraction.OPCs from the cerebral cortex of P4-5 wild type CD1 mice were immunopanned for PDGFRα using CD140a rat anti-mouse PDGFRα antibodyFor Nrp1-Fc-PDGFRα capping experiments, immunopanned OPCs from P4-5 CD1 mice were plated on PDL coated coverslips at a density of 40,000–50,000 cells/well in 24-well plates and incubated in DMEM-Sato medium overnight. Then, 2 µg/mL control human IgG1-Fc (cont-Fc) or Nrp1-Fc was added in the presence of 15 ng/mL PDGF AA and incubated at 4 °C or 36 °C for 30 min. The coverslips were washed with cold PBS, fixed with 4% PFA, and then immunolabeled with mouse anti-Olig2  and rabbit anti-PDGFRα antibodies . Following washes, cells were incubated in Alexa488-goat anti-human Fc, Cy3-bovine anti-rabbit, and Alexa647-donkey anti-mouse antibodies.d-lysine  at a density of 40,000–50,000 cells/well in 24-well plates and incubated overnight in DMEM-Sato medium with or without 15 ng/mL PDGF AA for 2 days. Ten micromolar EdU was added to the cells 6 h prior to fixation. Fixed cells were stained for Olig2 and EdU.For OPC-microglia cocultures, we first injected 4-OHT into mg-Nrp1-cont and mg-Nrp1-cko mice from P2-3. On P5, microglia were isolated by immunopanning using rat anti-mouse F4/80 antibody (Bio-Rad). Purified OPCs from CD1 pups and microglia were plated together on glass coverslips coated with poly-Purified immunopanned OPCs were rinsed with chilled PBS containing 1x PhosSTOP  and 1x halt protease inhibitor cocktail  and harvested in 1 mL of the same buffer. The cell pellet was lysed by homogenizing in RIPA Buffer (ThermoFisher) supplemented with PhosSTOP and protease inhibitors on ice for 30 min and then centrifuged to remove insoluble matter. The cleared lysate was frozen in liquid nitrogen and stored at −80 °C. Protein concentration was measured using DC protein assay (Bio-Rad). Samples were denatured with 4X Bolt LDS sample buffer and 10X sample reducing agent  and heated at 70 °C for 10 min. Samples (30 µg/lane) and prestained molecular weight standards  were electrophoresed through 8% Bolt Bis-Tris plus polyacrylamide gels (ThermoFisher) in Bolt MOPS SDS running buffer (ThermoFisher). Proteins were transferred to Immobilon FL membranes (EMD-Millipore) using a transfer buffer containing 1.25 mM bicine, 1.25 mM bis-Tris, and 0.05 mM EDTA. Blots were blocked in Odyssey TBS  blocking buffer (LI-COR) at room temperature for 1 h. Blots were incubated at 4 °C overnight in primary antibodies diluted in TBS blocking buffer containing 0.1% Tween 20. The primary antibodies were rabbit anti-Phospho-PDGFRα  and goat anti-mouse PDGFRα that recognizes both phosphorylated and non-phosphorylated forms (R&D Systems). Blots were washed with TBST buffer containing 137 mM NaCl, 20 mM TrisHCl, pH 7.6, and 0.1% Tween 20) four times, 10 min each, and incubated with IRDye 800RD donkey anti-rabbit or IRDye 680RD donkey anti-goat secondary antibodies diluted in Odyssey blocking buffer with 0.1% Tween 20 and 0.01%SDS. Blots were washed with TBST and imaged on a LI-COR Odyssey Imager. To quantify protein expression, the density of protein bands was determined using Image Studio Lite (LI-COR).Images of fluorescently labeled tissue sections, slice cultures, and dissociated cells were acquired using Leica SP8 confocal microscope (Advanced Light Microscopy Facility) or Zeiss Axiovert 200 M with apotome. Images were analyzed with the Leica LAS X, Zeiss Axiovision, or ImageJ. For quantification, images were captured from several random fields of defined area within the cortex, corpus callosum, or the cerebellar white matter, based on DAPI label and blind to the experimental labels, and immunolabeled cells in each field were counted.z-stack images were acquired from 20-μm-thick sections using a 40X objective on a Leica SP8 confocal microscope at 1024 × 1024 pixel image size and 700 Hz scan speed. Three-dimensional images were reconstructed from the z-stacks using the 3D module of the Leica LAS X software. Each PDGFRα + OPC was marked and first examined for contact with Nrp1+ microglia by rotating the plane in all directions. Then, the EdU channel was revealed, and the OPC was scored for whether it had EdU label.To quantify the proliferative OPCs that were in contact with microglia, 0.35-μm For quantification of the degree of myelination, sections from P14 corpus callosum or demyelinated corpus callosum at 28 dpl were immunolabeled for MBP, and MBP immunofluorescence intensity was quantified on images scanned on the Leica SP8 confocal microscope. Fiji  was used to obtain the average integrated pixel density of gray values over a defined region of interest. Background values were obtained from areas devoid of signal.For dissociated OPCs, images were taken from randomly selected fields from each coverslip based on DAPI label and the labeled cells were quantified. Adobe Photoshop was used to generate image panels for the figures. Quantification data are represented as means ± standard deviations. Statistical analyses were performed using GraphPad Prism v8 and v9. The specific statistical method used to evaluate each dataset is indicated in the figure legends.Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


They play a key role in the regulation of the sympathetic nervous system along with β and α2-AR family members. While all of the adrenergic receptors bind with similar affinity to the catecholamines, they can regulate different physiologies and pathophysiologies in the body because they couple to different G-proteins and signal transduction pathways, commonly in opposition to one another. While α1-AR subtypes  have long been known to be primary regulators of vascular smooth muscle contraction, blood pressure, and cardiac hypertrophy, their role in neurotransmission, improving cognition, protecting the heart during ischemia and failure, and regulating whole body and organ metabolism are not well known and are more recent developments. These advancements have been made possible through the development of transgenic and knockout mouse models and more selective ligands to advance their research. Here, we will review the recent literature to provide new insights into these physiological functions and possible use as a therapeutic target.The α ARs are1-AR subtype cDNAs were cloned in the late 1980s and early 1990s  family of G-proteins to activate phospholipase Cβ1 (PLCβ1), resulting in the hydrolysis of membrane-bound phosphatidylinositol 4,5-bisphosphate and the cytosolic release of inositol triphosphate (IP3) and diacylglycerol (DAG)  ; Table 1ll lines  but this and Src . α1-ARs coupling .1-AR subtypes display differences in internalization resulting in spatio-temporal changes in signaling   . These Gsubtypes . As RGS2subtypes  and GPCRpathways , α1A-AR  agonism . Cirazolrylation  or can e process  leading in vivo stimulatory or inhibitory effects of the α1-AR subtypes on physiology and pathophysiology (1A-AR (1B-AR (1D-AR (1A-AR was developed (1A and α1B-AR KO mice (1-ARs were designed to either target to the myocyte using the α myosin heavy chain promoter to drive only cardiac expression of wild-type (WT) or constitutively active mutations (CAMs) in the receptor  and throughout the body in using the endogenous promoters as compared to using the α myosin heavy chain promoter which caused very high amounts of receptor overexpression, often exceeding 100-fold. The use of CAMs instead of the WT receptor results in continuously activated receptors that do not need an agonist to be present and can be representative of a chronically stimulated condition, but this is still debated. In both overexpressed and KO mouse models, there is always the possibility of changes in the expression of other genes and receptors in compensation or as a result of additional insertion or deletion of genetic material, a widespread phenomenon that is hard to decipher and under reported  mouse models were developed that were used to determine long-term ysiology . KOs of y  is a type of long-lasting synaptic plasticity that increases the strength of synaptic transmission over a long period of time  . LTP is pocampus  and therpocampus  which ispocampus . Interes and LTP . LTP stisynapses ; Table 2controls . The α1Aor tests , there w1-AR mediated LTD required co-activation with a number of partners such as β-ARs (1-ARs have been shown to induce LTD at excitatory CA3–CA1 synapses in the rat hippocampus (1-AR subtype(s) mediate LTD. This Hebbian LTD requires coincident presynaptic and postsynaptic NMDAR activity (1-AR as the paired pulse facilitation ratio did not change (Long-term depression (LTD) is also a form of long-term synaptic plasticity that can contribute to cognitive functions by increasing the flexibility of the synapse to store information , such asas β-ARs , NMDA  located on the interneuron, leading to depolarization and an increased calcium influx through calcium channels resulting in increased GABAergic transmission onto the pyramidal neurons  can also be involved in the storage and consolidation of memory . As cAMPP analog , b. Simi storage , b. α1B- in vivo ; Table 2 in vivo . α1-AR s-induced  and scop-induced  and enhae shocks .1-AR functions may change and contribute to the aging process in the loss of memory function. α1-AR protein is increased in the aging mouse brain and with improved learning, supporting a role for these receptors in age-related cognitive decline  is a widely used AD mouse model that contains three genetic mutations associated with familial AD  . This ADwith LTP . These r1-AR autoantibodies with agonistic function were found in 50% of people with dementia  and atrial naturietic factor while the CAM α1B-AR mice activated nuclear factor-kB  resulting in injury to the muscle. α species . In prec species , mitocho species , mitocho species , 5′-nucl species  or angio species . In rece glucose . Most moffectors  and are ffectors .1A-AR subtype has been shown to mediate the cardioprotective effects of α1-ARs in ischemic preconditioning. These studies have been performed in transgenic or KO mouse models as blocking one subtype is still not specific enough to perform with antagonists. The systemically expressed CAM α1A mice were inherently preconditioned against ischemia while the CAM α1B was not  after ischemia  cells  cells . While g tissues . The mecthe cell .1A- and α1B-AR mice had an increased tolerance for glucose, lower fasting glucose levels while KO mice had poor tolerance and high blood glucose after fasting  (ratio of CO2 production and O2 consumption) which indicated an increase in whole body preference to metabolize fatty acids as a substrate  while the KO mice from both subtypes preferred to burn carbohydrates and increased the RER  to regulate fatty acid oxidation and utilization  is an ubiquitous and multi-functional protein and enzyme with regulatory crosslinking functions in cell adhesion and the cytoskeleton but also has GTP hydrolyzing activities . Phenyletact TG2 . α1-AR smal mice . When thmal mice . A β3 age tissue . These rty acids  and protty acids . TG2 is s   isomer when compared to the S(+)  isomer  isomer . Imidazonephrine . While mhe α1-AR . During agonists . Furtherr α1D-AR . Structupothesis . These d1A-AR versus the α1B- and α1D-AR subtypes and with lower affinity against the α2-AR. An analog of cirazoline and an α1A-AR partial agonist, RO 115–1240 and later by the commercial product dabuzalgron, was shown to reduce stress urinary incontinence without increasing blood pressure  (A second avenue of drug development for αosteric) . Besidesosteric) .Allosteric modulators are classified by their ability to modulate function. Positive allosteric modulators (PAMs) increase a receptor’s functional response while negative allosteric modulators (NAMs) decrease the functional response. There are also neutral or silent allosteric modulators (SAMs) that bind to the receptor and display no measurable changes in function  but can 1-AR but have not been developed for clinical use  to generate NO and to reduce blood pressure (1D-AR vaccine reduced the systolic blood pressure up to 15 mmHg in the SHR group and up to 29 mmHG in the SHR + L-NAME group. This vaccine also prevented cardiac hypertrophy and fibrosis, vascular remodeling, and renal injury even better than compared to treatment with prazosin, suggesting that the antibody has blocking activity. There is one commercially available α1D-AR antagonist, BMY7378 (1-AR subtypes for therapeutic use. Because of the unique amino acid sequence used in a non-conserved region of the second extracellular loop of the receptor, vaccines against the α1D-AR subtype would be highly selective and avid to regulate the blood pressure response and avoid blocking the other α1-AR subtypes.There has been recent work in therapeutic vaccines directed at the αrtension . A vaccibehavior . The vacpressure . This α1 BMY7378 , but is 1-AR autoantibodies displayed normal cardiovascular responses to α1-AR stimulation and removal of α1-AR autoantibodies by immunoadsorption did not alter that response (However, the vast majority of autoantibodies are associated with agonistic activity resulting from a rise in intracellular calcium, and postulated to result in a vasoconstrictive effect . Howeverresponse .1D-AR appears antagonistic, several autoantibodies have been developed or discovered against the first or second extracellular loop of the α1-AR appear to be agonistic in behavior (1A-AR may be tempting, they may not be regulated by the normal desensitization and negative feedback mechanisms common in GPCRs to turn off or wane the signal, resulting in abnormal and non-physiological signaling and proliferation (1-AR have also been associated with coronary heart disease (1-AR subtypes would need to be thoroughly analyzed for off target effects.While the autoantibody against the αbehavior . While dferation . This abferation . Autoant disease , cardiac disease , pre-ecl disease , thrombo disease , AD and  disease , and pro disease . Therefo1-ARs are part of the adrenergic family of sympathetic control and have long been known to regulate blood pressure, smooth muscle contraction and cardiac hypertrophy. In recent work, α1A-AR stimulation also mediates adaptive effects and signals in the heart that lead to protective outcomes against ischemia and heart failure. They are also highly expressed in the cognitive centers of the brain and stimulation of α1-ARs, particularly the α1A-AR, can increase both short-term as well as LTP leading to increased learning and memory functions. With its ability to increase adult neurogenesis, there is a potential for α1A-AR agonists or positive allosteric modulators to treat AD and to protect the heart at the same time. α1-AR stimulation also mediates several aspects of whole-body and organ-specific metabolism to regulate glucose uptake, gluconeogenesis, glucose breakdown, lipolysis, and fatty acid oxidation for energy production. The regulation of cardiac metabolism by the α1A-AR is likely a contributing factor for its protective effects in the heart. For pharmacological interventions, it is suggested that therapeutics that focus on α1A-AR agonism be developed. To avoid the potential side effects on blood pressure, the imidazoline rather than the phenethylamine pharmacophore should be of primary focus for drug discovery. Several α1A-AR imidazoline-based agonists have been used in preclinical studies and allosteric agonists that will not increase blood pressure are now in development for heart failure and AD.αDP performed all of the research, writing, and editing of this manuscript.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.