This guideline provides evidence-based guidance on the risk-based management of cervical dysplasia in the colposcopy setting in the context of primary HPV-based screening and HPV testing in colposcopy. Colposcopy management of special populations is also discussed. The guideline was developed by a working group in collaboration with the Gynecologic Oncology Society of Canada (GOC), Society of Colposcopists of Canada (SCC) and the Canadian Partnership Against Cancer (CPAC). The literature informing these guidelines was obtained through a systematic review of the relevant literature via a multi-step search process led by information specialists. The literature was reviewed up to June 2021 with manual searches of relevant national guidelines and more recent publications. Quality of the evidence and strength of recommendations was assessed using the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) framework. The intended users of this guideline include gynecologists, colposcopists, screening programs and healthcare facilities. Implementation of the recommendations is intended to promote equitable and standardized care for all people undergoing colposcopy in Canada. The risk-based approach aims to improve personalized care and reduce over-/under-treatment in colposcopy. In 2020, the World Health Organization (WHO) announced a global strategy towards the elimination of cervical cancer as a public health problem by reducing the incidence of cervical cancer to less than 4 per 100,000 . PrioritPrimary HPV screening is superior to cervical cytology in detecting pre-invasive disease and is the topic of a complimentary guideline . Once hiThe following guideline is presented in the context of primary HPV-based screening for cervical cancer, whereby a person’s HPV status is known on entry to colposcopy. The purpose of this guideline is to provide guidance on how to incorporate high-risk HPV (HR-HPV) testing and results within our current colposcopy framework with an emphasis on risk-based management. In Canada, each province and territory administers and delivers most health services independently and needs to consider their own populations and resources. This guideline is not meant to supersede local recommendations; rather, it is meant to provide evidence to advocate for equitable resources and standardized care for all people undergoing colposcopy in Canada.Guideline committee members were recruited to participate in the development of the document with consideration given to including colposcopists from a range of Canadian provinces and practice settings . The committee convened in the fall of 2020 and developed a series of objectives to guide the document.https://www.cadth.ca/strings-attached-cadths-database-search-filters (accessed on 10 May 2021). No date or language filters were applied to the search. The search strategy is reported in full in The literature informing the guideline was located using a multi-step search process led by an information specialist. First, a search for existing clinical practice guidelines related to colposcopy for cervical cancer was designed in Ovid MEDLINE All and executed on 12 May 2021. The search combined search terms for colposcopy, cervical cancer and the Canadian Agency for Drugs and Technology in Health guidelines filter for Ovid MEDLINE  framework; this is shown in Histopathology should be reported using the two-tiered terminology described by the LAST Project: LSIL for CIN1 and HSIL for CIN2/CIN3 .p16 immunohistochemistry may be used to upgrade CIN2 to CIN3. P16 should not be used to upgrade morphologically appearing CIN1 .Recommendations:The Lower Anogenital Squamous Terminology (LAST) Standardization Project for HPV-Associated Lesions, published in 2012, recommends harmonizing cytology and pathology into two-tier reporting . CIN 1 iGuidance for the use of p16 immunochemistry is published in the LAST project. In general, when the morphologic interpretation of hematoxylin and eosin (H & E) staining is suggestive of HSIL, then positive p16 immunochemistry is confirmatory [People with a positive HPV screening test should undergo HPV genotyping and reflex cytology before referral to colposcopy .People with HPV 16/18 should be referred to colposcopy .People with HPV ‘other’ ASCUS or LSIL should have HPV testing repeated at 12 and 24 months, only referred to colposcopy if they meet other criteria or have persistent HPV “other” at 24 months .People with HPV-positive HSIL, ASC-H, AGC, AIS or cytology suspicious for invasive cancer should be referred directly to colposcopy, regardless of HPV genotype .People with immunocompromise with any HR HPV should be referred to colposcopy .Recommendations:Primary HPV-based screening with reflex cytology allows for risk stratification based on immediate and future risk of histologic HSIL+. Large population-based studies, most notably data from the Kaiser Permanente Northern California (KPNC) cohort, have examined the risk of histologic HSIL based on combined HPV and cytology results, contributing to our understanding of these risk profiles ,11. BaseTabulated risks of various combinations of cytology and HR-HPV results are presented in The transformation zone should be assessed, and the type should be documented .International Federation of Cervical Pathology and Colposcopy (IFCPC) terminology is recommended for documenting colposcopic findings .Targeted biopsies of lesions are recommended. In the setting of HSIL, or positive HPV16/18, where colposcopic impression is normal, any area of acetowhitening, metaplasia or uncertainty should be biopsied .Endocervical curettage and endometrial biopsies are contraindicated in pregnancy .Endocervical curettage is recommended with: (i) a type 3 transformation zone, (ii) HSIL/ASC-H cytology when no lesion is identified, (iii) AGC/AIS cytology, (iv) when excisional treatment has positive margins and (v) in people over age 45 with HPV 16 .Endometrial sampling is recommended in those 35 years and older for all categories of AGC/AIS or atypical endometrial cells on cytology. Endometrial sampling is also indicated in those under 35 with increased risks of endometrial cancer  or atypical endometrial cells on cytology in those of any age .For pain management for routine exam and cervical biopsies, thorough pre-procedure counseling and non-pharmacologic methods are recommended. Oral analgesics may be considered. Topical and injected analgesics are not recommended for routine exam and biopsies of the cervix .Recommendations:When the cervix is first examined, cervical mucus is removed and gross examination is performed. Using a colposcope for visualization, 3–5% acetic acid is applied to the cervix, and the transformation zone is then visualized. The transformation zone type is noted  and any When abnormal colposcopic findings are identified, IFCPC recommends documenting both the location of the lesion, inside or outside the transformation zone and location of the lesion by clock position, and the size of the lesion, by the number of cervical quadrants the lesion covers and the size of the lesion in the percentage of the cervix affected. Further findings are categorized as grade 1 (minor) or grade 2 (major), suggestive of low- versus higher-grade lesions, respectively. Findings suspicious for invasion should also be described .Targeted biopsy of all discrete lesions is recommended. Studies have shown that taking two or more biopsies improves the sensitivity of the exam and increases the detection of histologic HSIL . ColposcEndocervical curettage (ECC) is contraindicated in pregnancy. ECC is generally not required with a type 1 transformation zone. ECC is recommended for type 3 transformation zones, HSIL/ASC-H cytology when no lesion is identified, AGC/AIS cytology and on post-treatment follow-up when excisional treatment has positive margins. Furthermore, ECC should be considered in people over age 45 with HPV 16 ,21. As pEndometrial sampling is contraindicated in pregnancy. Endometrial sampling is recommended in those 35 years and older for all categories of AGC/AIS cytology, as well as when atypical endometrial cells are found on cytology. Endometrial sampling is indicated in those under 35 with increased risks of endometrial hyperplasia or cancer  and when atypical endometrial cells are found on cytology in those of any age . Pain management should be a consideration during all colposcopy exams and procedures. Significant discomfort may impede the exam and can contribute to loss of follow-up. Pain during colposcopy is multi-factorial, influenced by physical factors, psychological factors and social factors . ThorougSeveral studies have shown that application of topical analgesics to the cervix prior to biopsy and manipulation are ineffective compared to placebo ,31,32,33After initial colposcopy assessment, those with normal or LSIL histology can be discharged from colposcopy .Where HSIL is identified on histology, excisional procedure is recommended .Recommendations:The majority of abnormal Pap tests are low-grade. Most of these will spontaneously resolve, especially in the young, previously vaccinated cohort . The purThe low-grade referral pathway  addresseAfter initial colposcopy assessment, if no histologic HSIL is identified, people can be discharged from colposcopy with recommendations for 12-month HPV-based screening with their primary care provider. This is in line with the 2019 ASCCP risk-based guidelines  and is bPeople with histologic HSIL identified at the time of the initial colposcopy assessment should undergo excisional treatment ; they suPeople with evidence of histologic HSIL should undergo an excisional procedure .In cases where no lesion is identified following referral for HPV-positive cytologic ASC-H/HSIL, review by an experienced cytopathologist should be considered .In cases where no cervical lesion is identified following referral for HPV-positive cytologic ASC-H/HSIL, VAIN must be ruled out by colposcopy .In cases of discordance, where no histologic HSIL is confirmed, management depends on transformation zone type and referral cytology .For a type 3 transformation zone, excisional procedure is recommended .For a type 1 or 2 transformation zone, the preferred management for ASC-H referral cytology is surveillance. For HSIL referral cytology, excisional procedure can be considered .If surveillance without treatment is undertaken, people should remain in colposcopy at 6-month intervals with HPV testing at annual intervals until HPV is negative on two consecutive tests and histology remains normal or LSIL .If HPV remains positive despite negative colposcopy, people should remain in colposcopy for surveillance at 12-month intervals until they meet the above criteria for discharge .If, during surveillance, there is evidence of cytologic or histologic HSIL, excisional procedure is recommended .Recommendations:The high-grade referral pathway  addresseIn cases of discordant cytology, histology and colposcopy findings, the presence and type of oncologic HPV infection, adequacy of colposcopy and transformation zone type need to be considered. For people referred with HPV-positive high-grade cytology, if colposcopy is adequate and no lesion is found, a detailed examination of the endocervix and vagina should be undertaken . Random For persons where high-grade discordance persists after cytopathology review , management depends on transformation zone type and referral cytology. All people with HPV-positive high-grade referral cytology (HSIL or ASC-H) with a type 3 transformation zone and otherwise negative colposcopy exam are recommended to undergo an excisional procedure. For those with HSIL referral cytology and a type 1 or 2 transformation zone, an excisional procedure can be considered, whereas close surveillance is preferred for ASC-H referral cytology. This is based on risk estimates that show the 1-year risk of CIN3+ is higher for discordance involving cytologic HSIL/histologic LSIL at 3.9% compared to cytologic ASC-H/histologic LSIL at 1.4% . Those wFor those who do not undergo an excisional procedure, close surveillance is required with colposcopy at 6-month intervals and HPV testing at annual intervals. If HSIL cytology persists or is identified at any follow-up visit, an excisional procedure is recommended. Once HPV is negative for two consecutive annual tests, and colposcopic impression/histology remain normal or LSIL, people can be discharged back to their primary care provider for repeat HPV-based screening in 12 months .In these cases, review by an expert cytopathologist should rule out CIN3 .Findings of CIN2 in a young person with a type 3 transformation zone or CIN2 identified on endocervical curettage should undergo excisional procedure .Findings of CIN2 in a young person with a type 1 or 2 transformation zone may be managed conservatively for childbearing considerations; surveillance should include colposcopy at 6-month intervals and HPV testing at annual intervals for 3 years to allow these young people to resolve their HPV infections .If CIN2 persists, continue colposcopy at 6-month intervals with HPV testing annually. People under 30 with persistent CIN2 > 36 months or CIN3 at any colposcopy visit should have an excisional procedure .People under the age of 30 with initial CIN2 who are managed conservatively can be discharged from colposcopy once histology is normal or LSIL and HPV is negative on two consecutive annual follow-up visits .People under the age of 30 with initial CIN2 who remain HPV-positive at annual follow-up should remain in colposcopy with HPV tests at annual intervals .Recommendations:There is controversy about the reporting of HSIL into distinct CIN2 versus CIN3 categories as there is low inter-rater reliability for CIN2 among pathologists . HoweverThe conservative management of the CIN2 pathway presented here  addresseFor those who are treated conservatively, colposcopy is recommended at 6-month intervals with HPV testing annually. This allows time for regression in this young cohort. At any point during follow-up, if CIN3 is found, an excisional procedure is required. Similarly, if histologic CIN2+ persists >36 months, an excisional treatment is recommended. If HPV remains positive with normal or LSIL histology, the person should remain in colposcopy including HPV testing at annual intervals. Once histology is normal or LSIL and HPV is negative on two consecutive annual follow-up visits, the person can be discharged from colposcopy to 12-month HPV-based screening with their primary care provider. Subsequent screening then follows the ‘Post-discharge follow-up for people with SIL not treated in colposcopy’ pathway .Recommended treatment for histological HSIL is an excision procedure with a LEEP .An ablative procedure with carbon dioxide laser is acceptable when used by trained and experienced colposcopists when specific criteria are met .Cryotherapy for HSIL is not recommended .Treatment should be performed in the clinic setting with local anesthesia plus a vasopressor to the cervix .Recommendations:Histologic HSIL is treated with either an ablative or excisional procedure. Excisional procedures offer a secondary diagnostic benefit as it allows for pathologic examination to assess for cancer and margin status. Excisional techniques include the loop electrosurgical excision procedure (LEEP), large loop excision of the transformation zone (LLETZ) and cold knife conization (CKC). Meta-analyses have shown similar oncologic outcomes, including recurrence rate, margin status and residual disease between LEEP and CKC ,53. HoweAblation with either carbon dioxide laser or thermoablation have also been used to treat HSIL. When ablation is used, the transformation zone is destroyed to a depth of 7 mm, as a SIL can exist to a depth of at least 4 mm in gland crypts and 7 mm is considered a suitable safety margin . AblativWith excisional procedures, the goal of achieving negative margins must be balanced with the risk of obstetrical complication. A meta-analysis suggests cervical dysplasia is a risk for preterm birth and excisional and ablative treatments increase that risk, with increasing depths of excision/ablation increasing obstetrical risks . Across Despite this knowledge, no study has been able to determine an optimal resection volume/length for excisional techniques. A 10 mm length seems to provide adequate margins while maintaining adequate cervical length for future pregnancies where risk of preterm birth increases with depth >10 mm . TreatmeThere are very few randomized controlled trials that directly compare treatment modalities. Most of the evidence comes from systematic reviews and meta-analyses with high heterogeneity. Minor and major adverse events from any treatment are low across the studies (<1%) . The mosAnalgesia is required for any ablative or excisional treatments of the lower genital tract. The most accepted technique is para-cervical block with local anesthetic and a vasopressor. The vasopressor reduces blood loss, reduces the threshold of toxicity from the local anesthetic and has been shown to reduce pain compared to local anesthetic alone. A systematic review and meta-analysis by Gajjar et al. confirmed that intracervical injection of local anesthetic with a vasopressor provided optimum analgesia for treatment, although data were scant, underpowered and showed a high degree of variability . OptimalIt is recommended that LEEP is performed in the clinic setting. The benefits of treatment in the outpatient clinic, as opposed to the operating room, include faster access to treatment, quicker recovery and overall cost-effectiveness. A LEEP in the operating room or under general anesthesia should only be carried out in extenuating circumstances . It is rAll people undergoing an excisional procedure in colposcopy should have an HPV test of cure and cytology at 6 months post treatment, as well as colposcopic assessment and endocervical curettage if endocervical margin is positive on excision specimen .People who are HPV-negative with normal, ASCUS or LSIL cytology and histology after treatment can be discharged from colposcopy .People who remain persistently HPV-positive with cytology and/or histology that is normal, ASCUS or LSIL, regardless of genotype, should remain in colposcopy at 12-month intervals until HPV is negative .After discharge from colposcopy, people treated for HSIL should have 12-month HPV-based screening with their primary care provider. If HPV-negative, they can resume HPV-based screening at 3-year intervals indefinitely .Recommendations: The post-treatment pathway  addresseA recent review of guidelines from seven countries  showed no agreement regarding timing of the test of cure and whether one or two post-treatment tests of cure are needed to decide who can be discharged from colposcopy  A study Based on recurrence risk, people treated in colposcopy who are HPV-negative with normal, ASCUS or LSIL cytology and histology at the 6-month post-treatment visit can be discharged from colposcopy. They should undergo HPV-based screening with their primary care provider at 12 months post-colposcopy; if HPV is negative, they can transition to HPV-based screening at 3-year intervals indefinitely. At any point, if HPV is positive, they should be re-referred to colposcopy for the usual indications. People who are HPV-positive on the 6-month test of cure should remain in colposcopy with HPV testing annually until negative. All people with HPV-positive AGC or AIS cytology should be referred directly to colposcopy, regardless of HPV genotype .At time of initial colposcopic assessment, all people with HPV-positive AGC or AIS should have endocervical curettage .Endometrial biopsy is recommended for all people >35 years old AND/OR risk factors for endometrial cancer AND/OR atypical endometrial cells on cytology .People referred with AGC-NOS cytology, in the absence of endometrial pathology and histologic HSIL or AIS, may be managed conservatively with colposcopy, cytology and HPV testing at annual intervals. If all negative on two consecutive visits, they can be discharged from colposcopy to HPV-based screening at 5-year intervals .All people AGC-N or AIS should undergo excisional procedure regardless of HPV status .Following the excisional procedure for AIS/AGC-N, if margins are positive, consider re-excision .Following excisional procedure for AIS/AGC-N, in the absence of cancer, surveillance with 6-monthly colposcopy, ECC and HPV testing is recommended; if surveillance is negative ×3 years in colposcopy , people can be discharged to HPV-based screening at 3-year intervals. If HPV is persistently positive or histology shows HSIL or persistent glandular abnormalities, people should stay in colposcopy and be managed per algorithms .Hysterectomy can be considered when post-treatment margins/ECC are persistently positive for AIS and/or fertility is not desired .Recommendations:Colposcopy is less reliable for the assessment of cervical glandular lesions than for squamous intraepithelial lesions. Most glandular lesions are recognized during evaluation of abnormal squamous cytology and small lesions that originate high in the endocervical canal can easily be missed . HoweverAlthough atypical glandular cells (AGC) make up <1% of cervical cytology samples, approximately one-third of these are associated with clinically significant pre-invasive or invasive disease ,77,78,79Risk estimates for HPV-positive glandular lesions are dependent on the cytology classification, as described by the Bethesda system in 2014 . A largeThe glandular referral pathway  addresseFor people referred to colposcopy for HPV-positive AGC-N who present with a type 3 transformation zone, a diagnostic excisional procedure is required to rule out a glandular lesion not identified at colposcopy. A systematic review by Schnatz et al. found the risk of AIS and malignancy following AGC-N cytology was 13% and 21%, respectively, compared to 2.9% and 5.2% for AGC-NOS . TherefoAll people with cytology suggestive of adenocarcinoma in situ (AIS) should undergo a diagnostic excisional procedure to rule out adenocarcinoma, even when definitive hysterectomy is planned ,42,74. THistorically, CKC was considered superior to LEEP for AIS. However, more recent retrospective reviews have shown equivocal rates of residual disease and recurrence between these treatment modalities ,84. ManyAdherence to long-term surveillance following conservative management for AIS is crucial; this needs to be considered when determining best management for people with AIS. Following an excisional procedure for AIS with negative margins, when conservative management is undertaken, follow-up colposcopy with HPV testing and ECC should be carried out at 6 and 18 months, then annually for 3 further years in colposcopy. After treatment, HPV status is highly predictive of recurrent AIS. A study by Costa et al. found that HPV testing significantly predicted persistence/clearance of AIS at 6-month follow-up . In thisAll people discharged from colposcopy should have HPV-based screening at 12 months with their primary care provider .Subsequent management depends on referral cytology and HPV status.People referred with low-grade cytology who are HPV-negative on 12-month screening with their primary care provider may transition to routine HPV-based screening at 5-year intervals .People referred with low-grade cytology who are HPV-positive (regardless of genotype) at 12-month screening with their primary care provider should be re-referred to colposcopy for the usual indications .People referred with high-grade cytology (untreated) should have two negative annual HPV tests in colposcopy with colposcopic findings that are normal or LSIL before they can be discharged to 12-month HPV-based screening with their primary care provider . If HPV remains negative at 12-month post-colposcopy screening, they may transition to HPV-based screening at 3-year intervals indefinitely. If HPV is positive at 12-month post-colposcopy screening, they should be re-referred to colposcopy for the usual indications .Recommendations:The post-discharge pathway  addressePeople referred to colposcopy with HPV-positive low-grade cytology, where no histologic HSIL was found at time of colposcopy, have a low risk of subsequent HSIL within the next three years . HoweverRegarding the choice of post-colposcopy screening test, a study by Katki et al. found that for people referred with HPV-positive ASCUS or LSIL cytology, in whom no HSIL was identified at colposcopy, a single negative HPV/cytology co-test post-colposcopy reduced the 5-year risk of HSIL+ to 1.1%, compared to a single negative HPV test (2.0%) or a single negative cytology test (6.4%). A second negative HPV/cytology result in this context did not significantly reduce the risk any further . Guido eBased on these data, all people seen in colposcopy for HPV-positive low-grade cytology, in which no high-grade cytology was found at the initial visit, can be discharged to HPV-based screening with their primary care provider at 12 months post-colposcopy. If HPV is negative, they can resume routine HPV-based screening at 5-year intervals. If HPV is positive on post-discharge screening, they should be re-referred to colposcopy for the usual indications.Despite the absence of histologic HSIL on colposcopic-directed biopsy, people who are HPV-positive with high-grade cytology at time of referral remain at higher risk of HSIL, sufficient to warrant ongoing colposcopy per the ASCCP 2019 risk-based thresholds ,90. All Those under 25 should not have screening with HPV testing or cytology .If screening occurs and high-grade cytologic abnormalities are identified, indications for colposcopy remain the same, regardless of age .When a CIN2 lesion is confirmed, and CIN3 is ruled out, conservative management may be undertaken when childbearing considerations outweigh the risk of invasive disease .Recommendations:Studies have shown the highest prevalence of HPV infection is in those under the age of 25, and this population also has the highest rate of spontaneous clearance of HPV ,92. InvaRecommendationsRisk-based threshold for entry to colposcopy are the same, regardless of pregnancy .Pregnant people should be evaluated by an experienced colposcopist .Pregnant people who are HR-HPV-positive with reflex normal or low-grade referral cytology (ASCUS or LSIL) should have HPV-based screening repeated 3 months post-partum ; pregnant people who are HR-HPV positive with reflex high-grade or glandular cytology  should be seen in colposcopy within 4 weeks .Endometrial biopsy and endocervical curettage are contraindicated in pregnancy. Cervical biopsies are indicated when there is a concern for HSIL or cancer; adverse obstetrical outcomes of cervical biopsies are rare .Excisional procedures for biopsy-proven HSIL or AIS in pregnancy can be delayed until 8–12 weeks post-partum .Biopsy-proven carcinoma in pregnancy should be referred urgently to gynecologic oncology .Pregnant people should be screened according to provincial guidelines. Risk-based indications for colposcopy are the same, regardless of pregnancy. Pregnant people who are HR-HPV-positive with reflex normal or low-grade referral cytology (ASCUS or LSIL) should have HPV-based screening repeated 3 months post-partum; pregnant people who are HR-HPV-positive with reflex high-grade or glandular cytology  should be seen in colposcopy within 4 weeks.The pregnant cervix undergoes changes in appearance that may complicate colposcopic diagnosis; therefore, pregnant individuals should be evaluated by an experienced colopsocopist ,95. PregEndometrial biopsy and endocervical curettage are contraindicated in pregnancy as they may negatively impact the pregnancy. Cervical biopsies are indicated when there is a concern for HSIL or cancer ,99,100. Colposcopy is recommended for all immunocompromised people who are HPV-positive, regardless of HPV genotype .Recommendations:In immunocompromised people of any age, colposcopy is recommended for all cytology results if HPV-positive . ImmunocMenopausal people have higher rates of cervix cancer and unsatisfactory colposcopy. Consider ECC and larger excisions when indicated .Consider pre-treatment with vaginal estrogen for 6 weeks prior to colposcopy to increase the rates of a satisfactory exam in postmenopausal people .Recommendations:Colposcopy can be challenging in menopause as the transformation zone recedes up the endocervical canal (type 3 transformation zone). Abnormal lesions are more likely to involve the cervical canal and vagina . On systColposcopy providers should be aware of the barriers to access cervical cancer screening and colposcopy, including geographical, socioeconomic, cultural, physical, psychological, provider-related and system-related barriers .Colposcopy providers are encouraged to seek additional training in cultural safety and trauma-informed care .Every effort should be made to facilitate access to care for individuals from historically underserved populations, including people with mobility restrictions, obesity, members of the transgender community, immigrants, Indigenous peoples, people from rural communities and those with mental health disorders .Recommendations:Barriers to accessing cervical cancer screening and colposcopy can be geographical, socioeconomic, cultural, physical, psychological, provider-related and system-related. Data support inequitable access to primary cervical screening for wheelchair users , sex wor


Radium-223 dichloride (Ra-223) is now frequently used to treat prostate cancer that has metastasized to bone, although patient selection continues to be suboptimal for determining who will benefit most from this novel treatment modality.Seventy-nine patients with metastatic castration-resistant prostate cancer (mCRPC) were treated with Ra-223 from 2012 to 2016. The burden of skeletal metastasis was determined for each using the Bone Scan Index (BSI) as a ratio of diseased to normal bone. Clinical, laboratory, and survival data were collected and examined for associations with BSI, and treatment tolerability was assessed.P < .05). Patients with higher BSI tended toward poorer outcomes. Nearly half the patients with low baseline BSI survived 3 years or more following Ra-223 treatment. By contrast, only 20% of the patients with high baseline BSI lived for 1 year, and none lived for an additional 3. Baseline BSI was significantly associated with decreased hemoglobin, higher serum PSA and alkaline phosphatase levels, and treatment-associated reductions in platelet and absolute neutrophil counts.Chemotherapy-naïve patients were significantly more likely to complete the full course of treatment. Median follow-up was 31 months (range 0.7-38.8 months) and median overall survival was 15.4 months (range 9.5-20.6 months). Overall survival was significantly associated with findings on bone scans (Our results suggest better outcomes to Ra-223 therapy for patients who are chemotherapy-naïve and who undergo treatment earlier in the course of their disease as reflected by low BSI and concordant laboratory parameters. Patient selection methods are suboptimal for determining who will benefit most from Ra-223 treatment for prostate cancer that has metastasized to bone. This article characterizes bone scintigraphy as a potentially useful and important biomarker for patient selection and prognosis. It is not yet entirely clear who will benefit most from Ra-223 in the context of total burden of osseous metastasis, osseous metastasis to specific regions, and underlying hematopoietic function. Based on the results of this retrospective analysis in a well-defined group of patients with castration-resistant prostate cancer, a better outcome to radium 223 therapy can be expected in chemotherapy-naïve patients with a lower BSI score, making such patients optimal candidates for this approach.1In 2020, the National Cancer Institute (NCI)-sponsored Surveillance, Epidemiology, and End Results (SEER) program reported that prostate cancer was the third most diagnosed cancer (191 930 cases) and led to approximately 33 330 cancer-related deaths among men in the US. Lethality is highest in cases of metastatic castration-resistant disease, suggesting an advanced clinical state and poor prognosis. As metastatic castration-resistant prostate cancer (mCRPC) is a lethal disease, the primary goal of treatment is to increase survival and enhance the quality of life.2 Ra-223 is a calcium-mimetic that forms complexes with hydroxyapatite in areas of increased bone turnover, for example, in osseous metastases. Ra-223 emits alpha particle radiation which has a short path length and results in high energy transfer. These properties are responsible for its localized anti-tumor effect, which is mediated by double-strand DNA breaks.3 Ra-223 has a half-life of 11.4 days and emits 4 alpha particles of varying energy and detectable gamma radiation. It is excreted mainly through the intestine.4Radium-223 (Ra-223) is a first-in-class alpha-particle-emitting radiopharmaceutical currently used to treat mCRPC with overall survival benefit when used as a single agent. Ra-223 has a novel mode of action that differs from those used by chemotherapeutic agents and androgen signaling inhibitors and has a relatively benign, non-overlapping toxicity profile compared to other classes of drugs used to treat advanced-stage disease.versus 9.8 months). However, no imaging was mandated in the trial nor was this considered among outcome measures.5Ra-223 was approved for clinical use in mCRPC based on positive results from the randomized Phase III ALSYMPCA trial. In this trial, 921 patients with mCRPC with symptomatic metastatic disease limited to the skeleton were treated with either 50 kBq/kg Ra-223 (given intravenously every 4 weeks for up to 6 cycles) or a placebo. Administration of Ra-223 significantly improved overall survival (OS) which was the primary endpoint of this study; median OS was 14 months in patients who received infusions of Ra-223 and 11.2 months among those treated with placebo. The trial also revealed a tolerable safety profile. The most common adverse effects were gastrointestinal disturbances and bone pain; grades 3 and 4 hematological toxicity was detected rarely in the Ra-223-treated patient cohort. Median time to first symptomatic skeleton-related event was significantly longer in patients treated with Ra-223 . The approximate net weight of all metastatic lesions throughout the skeleton was calculated using International Commission on Radiological Protection (ICRP) publication No 23 (Report of the Task Group on Reference Man) and expressed as the total body BSI Time-Zero (BSI0). Clinical, laboratory, and survival data were obtained from the medical records to facilitate the evaluation of the potential links between pre-treatment BSI0 at the start of therapy, treatment tolerability, and outcomes. The skeleton was divided into 4 regions with the following designations: BSI Time-Zero Pelvis (BSI0P), BSI Time-Zero Vertebrae (BSI0V), BSI Time-Zero Ribs/Clavicle/Scapulae (BSI0RCS), and BSI Time-Zero Long Bones and Skull (BSI0LBS); these regional measurements were evaluated individually to determine the efficacy of therapy.The burden of pre-Ra223 treatment skeletal metastasis was determined as the Bone Scan Index (BSI) as described by Imbriaco et al.Patient demographics, clinical characteristics, and BSI measurements were reported using mean, median, and standard deviation for continuous variables and frequencies and relative frequencies for categorical variables. Laboratory parameters were reported by Ra-233 treatment number. The association between laboratory parameters and baseline BSI values was assessed using linear mixed models where each laboratory parameter was modeled as a function of selected BSI measurement, time, their interactions, and random subject effect. Tests focused on the appropriate contrasts from the model estimates were used to evaluate (a) the main effect for each BSI measurement and (b) interactions with time . All models were evaluated graphically using residual and quantile–quantile plots; transformations were applied as appropriate. Relationships were also presented graphically using line plots based on fitted regression models, in which curves for the estimated mean laboratory values over time were generated for low, average, and high baseline BSI values :Low baseline BSI (<Q1): based on the average BSI value for samples falling below the first quartile.Average baseline BSI (Q1-Q3): based on the average BSI value for the samples between the first and third quartile.High baseline BSI (>Q3): based on the average BSI value for samples falling above the third quartile.P-values less than .05 denoted statistically significant results.Seventy-nine patients with mCRPC were treated with Ra-223 from 2012 to 2016 at Roswell Park Comprehensive Cancer Center, Buffalo, NY, USA were retrospectively evaluated. The median patient age at initiation of Ra-223 therapy was 71 years (range 46-91 years). Eighty-five percent of patients were at a Gleason score of 7 or higher and 51 patients (65%) presented with serum levels of prostate-specific antigen (PSA) >20. Nearly half of patients had undergone previous chemotherapy. Forty-nine patients (62%) completed the full 6 injections of Ra-223. Patient demographic and clinical characteristics are presented in P < .05). In general, patients with higher baseline BSIs tended to have poorer outcomes (P = .036) between patients with no prior chemotherapy and a higher number of Ra-233 treatments. Conversely, no significant association was found between baseline BSI and the number of Ra-223 cycles received  as well as the potential impact of specific regional metastases on prognosis and OS. Interestingly, we detected no differential response based on region; metastatic burden alone had a similar effect on prognosis and disease regardless of its localization both during and following Ra-223 therapy.7-9 have documented a relationship between the baseline BSI and survival in subjects undergoing treatment with Ra-223. In 3 studies,8 the use of Ra-223 resulted in worse outcomes for individuals who presented with baseline BSI > 5. Although our results also showed that baseline BSI was significantly associated with prognosis, we did not focus on specific threshold values. Instead, by describing a stepwise association between BSI and prognosis, our analysis would indicate that the association between BSI and survival is more of a continuum with contrasting levels where patients do very poorly or very well, but also intermediate range measurements where mixed results are observed. Using this method, we identified significant differences in survival between the 3 groups. A study by Anand et al9 also did not propose a cutoff value for BSI, but on bivariate analysis found that ALP (and not BSI) was an indirect measure of overall tumor burden and a predictor for OS.Few previous studies2 in their study of heavily pre-treated patients, and most likely results from chemotherapy-associated marrow toxicity and lower Eastern Cooperative Oncology Group (ECOG) status in this patient subgroup. According to recommendations of the European Medicines Agency and Pharmacovigilance Risk Assessment Committee, Ra-223 treatment should be started only after the failure of 2 different systemic regimens and the presence of ≥6 osseous metastases.10 National Comprehensive Cancer Network Guidelines support use of Ra-223 as an alternative option for mCRPC with symptomatic bone metastases before or after docetaxel chemotherapy or novel hormonal therapy.11 Our results propose that Ra-223 would be most effective in chemotherapy naïve patients with lower burden of osseous metastasis.In our study, chemotherapy-naïve patients were more likely to survive following Ra-223 therapy and were more likely to complete all 6 treatments. This is similar to the findings of Alva et alPatients with high total BSI values were most likely to exhibit reduced platelet counts and significant reductions in other laboratory parameters, resulting in the discontinuation of therapy. Although >Q3 patients exhibited irregularities in all laboratory parameters, reductions in platelet counts and ANCs were much more pronounced and sudden in this group than in any of the other patient cohorts. This observation may relate both to the degree of treatment-associated hematopoietic dysfunction in addition to the higher osseous metastatic burden.2 which documented no increases in PSA associated with BSI. In the ALSYMCA study, a decline in ALP levels during Ra-223 therapy was associated with improved OS.5 While Alva et al2 did not reproduce these results, we found that serum ALP levels did not decrease in the >Q3 group but remained constantly elevated throughout. This is also compatible with the results of Arnand et al9 showing that post-Ra223 decline in ALP is associated with longer survival. In an exploratory analysis of alkaline phosphatase dynamics in the Phase III ALSYMPCA trial, Radium-223 treated patients with ALP decline from baseline to week 12 had 55% lower risk of death compared to the patients with no confirmed decline in ALP.12 Similarly, van der Doelen et al13 have recently shown that lack of ≥10% decline in ALP following the first injection of Ra-223 may be a significant early indicator of treatment resistance.Patients with higher BSI values experienced increases in serum PSA during Ra-223 therapy. By contrast, those assigned to <Q1 and Q1-Q3 groups exhibited negligible PSA shifts. These findings are not consistent with those reported by Alva et al14 An additional important implication of these findings is that they may provide added insight into the optimum timing of Ra-223 therapy, suggesting patients with mCRPC at earlier stages of osseous disease progression derive greatest benefit from Ra-223 administration.Based on our results, a poor response to Ra-223 can be predicted during treatment in patients who experience no decline in serum ALP and/or who exhibit a rapid drop in platelet counts in conjunction with increasing PSA. These findings are typical of many patients with high baseline BSI values and would suggest that re-treatment with Ra-233 would be of little value and thus inadvisable for patients in the >Q3 cohort. This conclusion is in line with the results of a recent large clinical trial comparing standard-dose, high-dose and extended-schedule (up to 12 cycles) Ra-223 treatment, which could find no advantage in terms of overall survival or other efficacy endpoints for high-dose or extended-schedule regimens.The current study and conclusions drawn have certain limitations, and to change or guide current practice these would need to be validated in a larger prospective clinical trial. We are not certain if BSI cutoffs for Q1 and Q3 could be used clinically at this early stage and given the relatively small sample size in this study this would need to be validated in an expanded cohort. Moreover, in this proposed larger trial outcome measurements should also include analysis of reduction in skeletal related events and increase in quality of life. Lastly, it is presumed that the methodology used in this investigation would be further improved though enhanced consistency derived from count-based artificial intelligence-enabled automated region of interest generation for analysis of BSI.oyac245_suppl_Supplementary_FiguresClick here for additional data file.oyac245_suppl_Supplementary_TablesClick here for additional data file.


In the test cohort, the area under the receiver operator curve (AUC) for the discrimination of acute HF vs. NSTE-ACS without HF was 0.725  for copeptin and significantly higher than for hs-cTnT at 0 h  or at 3 h . Copeptin and hs-cTnT used either as continuous values or at cutoffs optimized to yield 90% specificity for acute HF were associated with significantly higher age- and sex-adjusted risk for all-cause mortality at 365 days. The findings from the test cohort were consistently replicated in the independent external NSTE-ACS validation cohort. Conclusions: High concentrations of copeptin in patients with suspected NSTE-ACS and equivocal clinical presentation suggest the presence of acute HF compared to uncomplicated NSTE-ACS and are associated with higher rates of all-cause death at 365 days.Background: In light of overlapping symptoms, discrimination between non-ST-elevation (NSTE) acute coronary syndrome (ACS) and acute heart failure (HF) is challenging, particularly in patients with equivocal clinical presentation for suspected ACS. We sought to evaluate the diagnostic and prognostic properties of copeptin in this scenario. Methods: Data from 1088 patients from a single-center observational registry were used to test the ability of serial high sensitivity cardiac troponin T (hs-cTnT)—compared to copeptin, or a combination of copeptin with hs-cTnT—to discriminate acute HF from uncomplicated non-ST-elevation myocardial infarction (NSTEMI) and to evaluate all-cause mortality after 365 days. Patients with STEMI, those with unstable angina and either normal or undetectable hs-cTnT concentrations were excluded. The findings were validated in an independent external NSTE-ACS cohort. Results: A total of 219 patients were included in the analysis. The final diagnosis was acute HF in 56 and NSTE-ACS in 163, with NSTEMI in 78 and unstable angina having stable elevation of hs-cTnT >ULN in 85. The rate of all-cause death at 1 year was 9.6% and occurred significantly more often in acute HF than in NSTE-ACS (15 vs. 6%,  The clinical presentation of patients with acute coronary syndromes (ACS) is heterogenous, and many cases present with atypical symptoms, dyspnea, unspecific ECG abnormalities or normal ECG, and elevated cardiac troponin values. The discrimination of non-ST-elevation (NSTE) ACS from other life-threatening differential diagnoses including heart failure (HF) or pulmonary embolism is challenging when the clinical and laboratory criteria of the fourth universal definition of myocardial infarction are applied . Since oWhile biomarkers have been found useful to diagnose acute or chronic heart failure in patients presenting with suspected heart failure, there is sparse evidence on the role of additional biomarkers beyond the routine measurement of cardiac troponins among dyspneic patients presenting with suspected ACS, a setting where natriuretic peptides are not recommended routinely.Due to its tissue specificity, cardiac troponin is regarded as the preferred biomarker for the diagnosis of acute myocardial infarction (MI), but it lacks clinical specificity to allow a discrimination between the various cardiovascular reasons for acute or chronic myocardial injury including acute heart failure, myocarditis or pulmonary embolism . CardiacWhile natriuretic peptides are recommended for the diagnosis of suspected acute or chronic HF , they prGiven that hs-cTn values are almost always elevated in patients with acute HF or with NSTEMI, surprisingly little information is available as to whether copeptin may help to discriminate acute HF from uncomplicated NSTE-ACS. The measurement of copeptin together with hs-cTn has been found particularly useful to rule out NSTEMI ,14,15 ann = 6). Acute HF with or without NSTE-ACS was diagnosed using a combination of clinical signs and symptoms with a value of N-terminal pro B-type natriuretic peptide (NT-proBNP) exceeding the age-adjusted rule-in cutoff  vs. 67 (25–201) ng/L at 3 h, p < 0.001).The BACC study population consisted of 2719 patients, 1286 patients of whom fulfilled the entry criterion for the present analysis of an available assessment of copeptin. The population considered for the analysis with a diagnosis of NSTE-ACS or acute HF consisted of 288  patients as described in the consort diagram in p = 0.2).The discrimination of acute HF against type 1 MI was poor for hs-cTnT at baseline , at 3 h . Cox regression analysis for all-cause death showed that copeptin predicted 365-day mortality, either as a continuous variable, or at the cutoff that yielded 90% specificity for the discrimination of acute HF. Conversely, hs-cTnT did not predict 365-day mortality, even after log transformation used as a continuous variable or at the 90% specificity cutoff  and higher in patients with acute HF than in those with type 1 MI , y cutoff .Our data show that in situations where natriuretic peptide measurements are not recommended for routine diagnostic assessment, because the chief complaint is suggestive of ACS rather than acute HF, a highly elevated copeptin indicates acute HF rather than uncomplicated NSTEMI. The early differential diagnosis has huge therapeutic implications, and it is pivotal to start the right treatment as soon after presentation as possible. Apart from our findings on copeptin, we have shown that the ability of cardiac troponin to discriminate between myocardial injury and infarction is poor, and optimal thresholds associated with 90% specificity for the diagnosis of acute HF are situated at almost five times the upper limit of normal, i.e., around 70 ng/L. The AUCs vary between 0.460 and 0.441 for the hs-cTnT assessed at 0 h and at 3 h. In contrast, copeptin has demonstrated a better ability to discriminate acute HF, with an AUC of 0.725. The combination of hs-cTnT with copeptin further increases the AUC, albeit in neither a clinically meaningful nor statistically significant way. With regards to prognostic evaluation, we found that a higher risk of all-cause death at 365 days was best predicted by an elevated copeptin but not by hs-cTnT. In an adjusted Cox proportional hazard model, copeptin was associated with a 1% higher risk of death per 1 pmol/L increase at 365 days, which retained its prognostic information after adjustment for hs-cTnT. We validated our observations in an external NSTE-ACS cohort.Both ACS and acute HF are diagnoses that require immediate attention in the ED. Given that the differentiation between acute myocardial injury and acute MI rests on clinical criteria that suggest a context of myocardial ischemia, an accurate diagnosis may be challenging when patients present with atypical symptoms or dyspnea. In particular, emergency physicians are frequently required to differentiate between type 1 MI, type 2 MI, or an acute myocardial injury due to miscellaneous causes. Although 2D echocardiography and cardiac imaging are strongly recommended for the diagnosis and differential diagnosis of suspected ACS, there is an obvious underuse of cardiac imaging in the ED . Unless p < 0.0001).Copeptin can add important information in this regard. The 39-amino-acid-long peptide is secreted from the pituitary gland stoichiometrically with biologically active vasopressin . The relUsing data from 1967 patients presenting with chest pain, the CHOPIN trial has shown that the addition of copeptin to the initial work-up, including cTnI assessment, allowed the safe rule-out of acute MI with a negative predictive value >99% in patients with suspected ACS . CompareDue to the fast and reversible release kinetics, a dual-marker strategy for the instant rule-out of MI is recommended if copeptin and cardiac troponin are below their respective cutoffs. However, as supported by our data, elevated levels of copeptin carry independent prognostic information, and this finding is irrespective of the underlying condition, which can be extrapolated to acute HF, thus extending our knowledge to an area for which available data have been sparse.We cannot exclude a sample size error since the numbers of patients with acute HF and fatality rates were low. A power calculation was not performed given the observational nature of the study.Access to external cohorts for the validation of non-routine biomarkers is challenging. Therefore, a multicenter validation was not in our scope.In order to validate the preliminary findings of this pilot study, we validated our findings from the test cohort in an external independent NSTE-ACS cohort. Given the low event rate, Cox regression modelling restricts adjustment to only a few possible confounders. Therefore, we cannot exclude that copeptin might lose its independent prognostic value after full adjustment in a considerably larger study population. Copeptin, while recommended for the instant rule-out of MI if a high sensitivity cardiac troponin assay is not available, is still not available on a fully automated central laboratory instrument. This issue has probably hindered a broader market availability.


Determine how ashift- based schedule to accommodate more students affects learning,performance, and satisfaction with the Obstetrics and Gynecology (OBG) rotation.The study wasconducted among third year OBG medical students with a triangular convergentcross-sectional approach. A new shift-based schedule was implemented. Aftereach rotation, an online survey was conducted using a convenience sampling.Student scores on the National Board of Medical Examiner (NBME) OBG subjectexam were analyzed using paired t test. Survey data was analyzed using two sample ttest. The relationship between surveyresponses and exam score findings were described. Data from shift-schedulestudents was compared to traditional schedule students from the prior academicyear.(145)=3.69, p =.001]. A similar pattern was not seen in subsequent groups (groups4-6). Shift-schedule students alsoshowed a statistically significant improvement in their perception of learning(t (183) =-2.54, p =.012). Parallel results were seen forbelonging, manageable workload, time to study, and engaging meaningfully. Usingthis model, we increased rotation capacity from 24 to 30 students per group(20%).A statisticallysignificant improvement was seen for average NBME score for shift-schedulestudents during the beginning portion (groups 1-3) of the academic year  compared to traditional  [t Shift based scheduling allows 20% increase in capacity. Exam scores andstudent learning outcomes were similar or better than traditional schedulecontrols. Increasing numbers of learners and shortage of clinical sites require innovative strategies to address demand. Since 2002, Twenty-nine new accredited US medical schools have opened, along with 17 new schools of osteopathic medicine. Enrollment in U.S. medical schools has grown by 31% and combined with increases in enrollment at schools of osteopathic medicine, overall medical student enrollment is now 52% higher than in 2002.Increasing medical student numbers along with concomitant growth of physician assistant and nurse practitioner training programs  has compounded the need. This is not a uniquely American problem. Shortage of clinical training sites and clinical supervisors has been acknowledged around the world, particularly in Europe and Australia. Contributing factors are multiple including rising numbers of medical students, shorter hospital stays, more outpatient surgeries, and prehospitalization workups, and increasing demands on clinicians’ time.Medical education has experienced shifts in curricular organization, content, delivery, assessment, and the use of technology in response to the needs of our learners over the last decade.Additionally, there are measures and methods  that provide data to assess curriculum and student performance following curricular modifications.One such change is shortening the traditional preclinical curriculum and allowing students to enter the clinical learning environment earlier in their training trajectory. This change results in challenges and downstream implications for medical schools and clinical partners alike. A report by Kraakevik and colleaguesWhen assessing the effects of curriculum changes, student perception from their role as stakeholder provides a unique and important perspective. Makoul and colleagues-Other studies have reported the value of gauging students’ perceptions regarding a variety of education goals and outcomes including well-being, learning activity, learning environment, and long-term effect.There are many factors that influence a student’s perception of his or her clinical rotation experience. A study by Gerbase and colleaguesSeveral studies in the literature have evaluated the effect of shortening the clinical rotation length and its effect on student learning with varied outcomes.Edwards and colleaguesReece and colleaguesWhile many studies have investigated the effects of shortening overall clinical rotation length, none have evaluated a shift-based experience having each student spending fewer hours per day in the clinical learning environment to allow an increased capacity for a standard 6-week block.The objective of this study was to investigate effects of a new shift- based scheduling in the OBG rotation on capacity, student learning and performance, and satisfaction.We also wanted to compare outcomes for students who completed the rotation on the shift-based schedule to a group of control students from the year prior who had traditional shift schedules.A triangular convergent cross-sectional design was utilized to investigate the impact of shift-based scheduling for the OBG rotation. A new shift-based schedule was implemented at the beginning of the new academic year. Upon completion of each group, an online survey was conducted using a convenience sampling to assess student perception of the rotation, their learning and clinical experience. To design the survey tool and protocols, we engaged the KirkpatrickOn the last day of the rotation, students take the National Board of Medical Examiner (NBME) OBG subject exam. These scores were collected at the same time as the survey and analyzed separately to provide an objective measure of student learning.Survey data and Quantitative NBME exam scores were analyzed using t-tests. The relationship between survey and exam score findings was explored, and finally data collected from shift-schedule students was compared to the same data collected from non-shift-based schedule students from the prior academic year.The study setting was the University of Texas Health Science Center at San Antonio, Joe R. & Teresa Lozano Long School of Medicine. Following the preclinical curriculum, students embark on clinical rotations. There are eight “core” rotations: internal medicine, family medicine, surgery, pediatrics, emergency medicine, neurology, and obstetrics and gynecology. The OBG rotation is 6 weeks in length with students spending 3 weeks on obstetrics (OB) and 3 weeks on gynecology(G).Prior to July 2018, students had a traditional 12-hour day schedule (5:00am-5:00pm M-Fri). In July 2018, a new clerkship schedule was implemented. This shift-based schedule was developed based on expert opinion after discussion with stakeholders including students, residents, faculty, medical educators. The main goals of the shift-based schedule were to provide a robust clinical experience with good coverage of areas within the specialty, mitigate long work hours and lack of predictability, maintain the ability of students to meet required encounters, and maintain or improve student satisfaction with the rotation experience.Students worked fixed 8- or 9-hour shifts, equally distributed: 5:00am-1:30pm; 1:30pm-9:30pm and 9:30pm-7:00am (“night shift”). Over a week, students worked approximately 33% fewer hours compared to traditional 12-hour day schedules from the prior year.Medical students completing their obstetrics and gynecology rotation between July of 2017 and January of 2019 comprised the study population. Students were excluded if they rotated off site, were not in classes of 2019 or 2020 or declined to participate. The study was reviewed by the Institutional Review Board (IRB) of the University of Texas Health Science Center at San Antonio and designated EXEMPT.Sample Size Calculation: No sampling procedure was implemented since all eligible students were recruited for the study. The number of student eligible was 299.The outcome measures included Likert scores on survey (see below) which are reflective of student perception of their learning environment, clinical experience, team inclusion, well-being, and overall satisfaction (modified DREEM as described below). Quantitative outcomes were NBME exam scores  published in 1997 by Roff and colleaguesThe DREEM is a 50-item instrument developed to evaluate the educational environment in undergraduate medical education institutions and has been recommended as the most suitable tool available for this purpose.Our survey instrument was developed to focus on student perception of the following domains:  workload, opportunities to interact with patients, ability to complete required clinical encounters, their sense of belonging and being meaningful participants in the clinical team, learning environment, study time, and National Board of Medical Examiner (NBME) subject exam preparation. These domains were selected to represent DREEM subscales of student perception of teaching, teachers, atmosphere, social self-perception, and self-performance. The tool was reviewed by recognized medical educators and their feedback was incorporated into the final draft of the survey. The instrument was piloted with a representative group of medical students and minimal adaptations were required. Content validity was supported through expert review of survey items, literature review and our piloting and review process for the instrument. The final instrument included 30 items which were scored using a 5-point Likert scale. The Likert scale was as follows; Never (1), rarely (2), sometimes (3), often (4) and always (5) for items 6-16 . Items 17-30, for domains of academic environment, engagement and inclusion utilized a Likert scale with Strongly disagree (1), Disagree (2), neither agree nor disagree (3), agree (4), and strongly agree (5). Reliability of the instrument was established using Cronbach alpha. .This was a closed online survey using the Qualtrics platform, accessible only via the student being logged into a university password protected account to avoid multiple entries per person. Students were recruited via e-mail invitations and the class Facebook page to participate. The first paragraph of the survey instrument included information about the study, name and contact information of the Principal Investigator, the IRB protocol review number, provision that participation was voluntary, and that responses had no effect on student grading for the clerkship. No compensation was offered.Demographic information collected was limited to gender and year of graduation. 299 third- year medical students from the classes of 2019 and 2020 were invited to participate with a link sent via email upon completion of their OBG rotation. Surveys were collected between July of 2017 and January of 2019. The survey instrument contained 30 items which were distributed over 10 pages (3 items per page). A final completeness prompt allowing review of response was provided and a thank you message confirmed successful submission. Once a survey was completed it was not displayed a second time. Participation rate was 97.9% with 185 out of 189 students completing the survey.Only completed surveys were entered into the study. Incomplete surveys were excluded from analysis (n=4). Survey data (survey responses and Likert scores) were compared between traditional and shift-schedule using two sample T test with statistical significance defined as a p value of <0.05. Average NBME exam score was compared between traditional and shift-schedule groups using paired t test with statistical significance defined as a p value of <0.05. The standard deviation on the NBME scoring was 7.3 for the traditional group and 6.5 for the shift-schedule group.Analysis was performed using SAS Using the shift based-schedule, we increased the number of students from 24 to 30 within the same 6-week rotation (20% expansion).Hundred and eighty-five (185) surveys were completed (response rate 49.3%), 50.8 % were male and 49.2% were female, 93 (59.2%) were from the class of 2019 and 92 (49.7%) were from the class of 2020.(183) =-2.54, p =.012) The shift-based schedule students additionally demonstrated an increased sense of belonging  compared to traditional schedule  (t(182)=-3.11, p =.002). They also perceived having enough time to study at a higher level  than traditional shift counterparts  (t(170)=-4.27, p <.001) Shift- based students had a manageable workload  at higher levels compared to traditional schedule students  (t(166)=-3.47, p<.001). Shift-based students had higher levels of engagement  compared to traditional schedule students  (t (180), p <.001)  indicated that shift-based schedule students had a statistically significant improvement in their perception of learning  compared to traditional schedule  (t p <.001) .(145) =3.69, p =0.001]. A similar pattern was not seen in subsequent groups (groups 4-6). Post-hoc analysis was performed to investigate trends and it was determined that after group 3 (November), scores were similar between groups  with higher means  for shift-based schedule students compared to traditional  on paired t test [t n groups .The purpose of our study was to evaluate the impact of a new 8-hour shift-based schedule on learning outcomes and capacity on the OBG rotation and compare this to traditional 12-hour day historical controls.Our data demonstrates an 8-hour shift- based schedule increased capacity by 20% while maintaining or improving student learning, performance on NBME exam and satisfaction with the learning experience. Shift based students spent approximately 33% less time in the hospital and clinics compared to their traditional 12-hour shift historical controls. Despite this, shift-based students’ satisfaction, and learning outcomes were similar or improved compared to 12-hour shift historical controls. Interestingly, students with the shift-based schedule anecdotally reported increased flexibility in balancing personal and professional responsibilities.Review of the literature reveals no studies of shorter, shift- based OBG rotation schedules and their effect on student perceptions of their clinical experience, learning environment, or well-being.Most studies have focused on shortening overall rotation length and examination score outcomes  rather than the actual number of hours per day students spend in the clinical learning environment as we did.A 2004 study by MylesIn 2018, one US medical school (University of Michigan) reported that following a curricular modification which shortened all its required clinical rotations by 25%, there was no difference in NBME subject exam or year-end clinical skills exam scores. There was also no significant difference in student perception of rotation quality nor their perception of stress, well-being, or resiliency between cohorts.Strowd and colleaguesSimilarly, Reece and colleaguesA study by Talib and colleaguesThe main strength of the study is that it is the first to investigate using a shorter shift-based schedule for clinical rotations in medical education. The study included data from both survey responses as well as exam scores so outcomes could be evaluated from different perspectives. Another strength of our study was the high participation rate on the survey.The study had several limitations. First is the study design, which uses historical controls as a comparison group rather than a true randomized control design with students from the same academic year. There may be confounding variables or differences between academic years we did not account for in the dataset. The study was performed at a single institution, potentially introducing bias, and student performance data from other clinical rotations was not collected for comparison. Although adapted from the Dundee Ready Education Environment Measure (DREEM), considered the most suitable tool to evaluate educational learning environments available, our survey was not previously validated.Future directions for study include expansion of the shorter, shift-based schedule to other institutions and specialties to investigate whether outcomes are similar across a broader range of clinical learning experiences.Additionally, evaluating perceptions of nursing staff, resident physicians and faculty on the shift model and its effect (if any) on patient care delivery may add important additional information and could be a focus of future studies.Finally, we recommend studies evaluating whether faculty perceive interaction with students to be different with this model and what implications that may have on their teaching.Using shift-based scheduling model, we successfully increased OBG rotation capacity by 20%. Data from our study indicates the transition to a shorter, shift based (8-9 hours) clinical experience improved student perception of the learning environment, workload, and ability to study on the OBGYN rotation. This shorter shift-based schedule was associated with similar or improved outcomes in learning and student experience on the OBG rotation. Clerkship quality and clinical experiences were similar to traditional shift controls. Overall, students spent approximately 33% fewer hours in the clinical work environment. Shift based scheduling may allow students to better balance clinical, study and personal time. Shift based scheduling could have a major potential implication for medical education where clinical slots are at a premium.Zhu Wang PhD, Xuemei Song, Joel Michalek, PhD Stephanie Hernandez, MS and Jessica Perry, BS for statistical support. Veronica Riggs, MS4, and Keri Rowley, MS4 for assisting with survey piloting, recruitment of subjects.The author declares that she has no conflict of interest.


Immunity from rabies depends on rabies virus neutralizing antibodies (RVNA) induced after immunization; however, the influence of antibody isotype switching has not been extensively investigated. This has become particularly relevant with changes in World Health Organization (WHO) recommended rabies vaccine regimens that may influence RVNA isotype kinetics, potentially affecting the peak, and longevity, of RVNA immunoglobulin (IgG) levels. We developed rapid and reliable assays for quantifying the anti-rabies IgM/IgG class switch in human serum based on an indirect ELISA technique. The immune response was tracked in ten individuals naïve to the rabies vaccine by quantifying serum titers weekly, from day seven to day 42 post-immunization, using a serum neutralization assay and the ELISA IgM/IgG assays. The average RVNA IU/mL levels were at D0 ≤ 0.1, D7 0.24, D14 8.36, D21 12.84, D28 25.74 and D42 28.68. Levels of specific IgM antibodies to rabies glycoprotein (EU/mL) were higher, on average, at D7, 1.37, and from D14, 5.49, to D21, 6.59. In contrast, average IgG antibodies (EU/mL) predominated from D28, 10.03, to D42, 14.45. We conclude that levels of anti-rabies IgM/IgG at D28 characterize the isotype class switch. These assays, combined with serum neutralization assays, distinguished the RVNA levels in terms of the IgM/IgG responses and are expected to add to the diagnostic repertoire, provide additional information in establishing rabies vaccine regimens, both post- and pre-exposure prophylaxis, and contribute to research efforts. Rabies infection is nearly 100% fatal once symptoms appear. Post-exposure prophylaxis (PEP), including a rabies vaccination series and rabies immunoglobulin administration (in those with no previous rabies vaccination), has been shown to be nearly 100% effective in preventing disease. Eliminating human rabies deaths caused by canine rabies, as targeted by the Zero by 30 effort launched by WHO, the World Organisation for Animal Health (WOAH) and the Food and Agriculture Organization (FAO) in 2015, hinges upon successful vaccination and availability of reliable, readily accessible diagnostics to detect and monitor adequate levels of protection in vaccinated individuals and animals .Rabies PrEP vaccination regimes have varied over time. Purified cell-culture and embryonated egg-based rabies vaccines currently in use have been shown (using the 3-dose regimen) to confer both long-lived immunity and efficacy, and have been proven to be safe for usage in a large range of age groups ,6,7. ThePrEP is administered for those persons deemed a high risk of exposure, such as laboratory workers and those traveling to rabies endemic areas . StudiesVaccine effectiveness is challenged within the population by the individual’s unique immunological response to vaccination. The different factors that affect the individual populations are due to both intrinsic host factors and extrinsic vaccine factors . Some inThe ACIP updated the regimen for PrEP in 2022, from a three-dose series to a two-dose series, similar to the current WHO PrEP recommendations. The shortened vaccine regimens may influence peak levels of rabies virus neutralizing antibody (RVNA) and Ig class kinetics. Previous studies have indicated that peak levels of antibody-producing plasma cells are achieved after two vaccinations; however, memory B cell frequency increases after a third vaccination  cells  followed by incubation for 24 h. Detection of the residual virus under fluorescence microscopy was achieved by staining the acetone-fixed slides with FITC-conjugated anti-N rabies antibody . Counts of virus-detected fields were transformed to IU/mL results using the Reed and Muench formula for endpoint titer, and titer results were compared to an international RVNA standard sample titer tested alongside the samples. High-titer sera were pre-diluted in cell media to obtain a readable result within the linear range of the assay (0.1 IU/mL to 15.0 IU/mL) as defined per method validation.RFFIT, using CVS-11 as the challenge virus strain, was used to test all serum samples for the baseline rabies virus neutralizing antibody (RVNA) titer value, as previously described . The RFFT Rabies II Kit  is an indirect ELISA using rabies glycoprotein as the antigen coating the wells and a Protein A-peroxidase conjugate, referred to below as Protein A ELISA. It is used for in vitro detection and titration of anti-rabies virus glycoprotein IgG in human serum and plasma and was used following the manufacturer’s instructions as previously described [The Plateliaescribed . The optThe Protein A ELISA was modified for the detection of rabies-specific human IgM and IgG antibodies. Indirect ELISA secondary (detection) antibodies consisting of anti-human IgG  and anti-human IgM antibodies , conjugated to horseradish peroxidase (HRP), were used to detect IgG and IgM, respectively, replacing the kit’s peroxidase-Protein A. Internal standards at known rabies antibody levels and a negative control were included on each ELISA plate to assess inter-assay precision. Sample dilutions were prepared for high-titer sera to bring the antibody level within the assay range (0.625 to 20 EU/mL for IgG and 0.250 EU/mL to 8.0 EU/mL for IgM). The non-linear curve fit standard curve in Gen5 software was selected for analysis of OD readings  based on the tightest fit to the Bio-Rad proprietary standard curve analysis method  . SamplesSubject serum samples drawn at day 21 or later were screened in the Protein A ELISA to select samples with high EU/mL results to serve as anti-rabies human IgG antibody controls along with the Protein A ELISA kit control sample (4.0 EU/mL). In addition, an anti-rabies IgG internal control sample (RIgG) with a Protein A ELISA value of 20 EU/mL was serially diluted to prepare a standard curve. Serial two-fold dilutions  of anti-human IgG-HRP conjugate were used to identify the IgG-HRP conjugate dilution required to produce comparable results against the Protein A ELISA. A sample with no rabies antibody (negative control) was included to demonstrate specificity of the conjugate to the rabies antibodies. Tests for linearity, accuracy and precision were performed. Gen5 was used to construct the standard curve and for interpolation of sample data.Subject samples drawn on days 7, 14 and 21 post-vaccination were screened by RFFIT testing to identify samples with high RVNA titers, that is samples with RVNA IgM and/or IgG. Then, the samples were screened by Protein A ELISA to identify samples with low Protein A ELISA titers, that is samples with low IgG binding antibody. Screened serum samples served as the assay positive controls for ELISA development. A checkerboard experiment was used to optimize anti-human IgM-HRP conjugate assay dilution. Samples and anti-IgM-HRP conjugate were used in two-fold serial dilutions (1:2 to 1:64) against anti-IgM conjugate serial two-fold dilutions to identify the target conjugate dilution, followed by optimization experiments to develop a standard curve. A sample with no rabies antibody (negative control) was tested to ensure anti IgM-HRP conjugate specificity; no background reactivity was noted with the conjugate dilutions evaluated. Tests for linearity, accuracy and precision were performed. Gen5 was used to construct the standard curve and for interpolation of sample data.GraphPad Prism, version 9.5.0, and Gen5 software were used for the construction and evaluation of standard curves. Coefficient of variation (%) was calculated to describe the level of variability within the measured isotype titers and to determine the assay precision. CV% was calculated by the formula standard deviation/average multiplied by 100, and % recovery (%R) by the formula actual EU/mL/expected EU/mL multiplied by 100.All subject serum samples  were tested using the RFFIT, the Protein A ELISA and the developed IgG and IgM ELISAs, see The closest standard curve fit was obtained using the IgG-HRP conjugate dilution of 1:4800 against two-fold serial dilutions of the RIgG internal control sample . The RIg2 values were calculated in Excel; the R2 values for the three samples  were 0.98, 0.94 and 0.97, respectively. For accuracy, the % recovery (%R) of the measured EU/mL against the expected value (the Protein A ELISA result) was found to be 85.77%, 113.12% and 52.94% for the high, medium and low IgG positive samples, respectively. The precision of the assay, determined by calculating the CV% of duplicate results of the three samples, ranged from 5.24% to 7.76%.All subject serum samples  were tested in the developed IgG ELISA, see Four of the 10 serum samples with the highest RFFIT and low Protein A ELISA kit results were selected as the IgM control samples. The IgM positive control samples were tested in duplicate in the IgM ELISA, using IgM-HRP conjugate in dilutions starting with IgM-HRP manufacturer’s recommended dilution of 1:10,000. The observation that higher concentrations (lower dilutions) of the IgM-HRP conjugate raised the OD reading to approximately 1.8 for the lowest dilution of the sample while maintaining good curve fit led to experimenting with lower IgM-HRP dilutions with two-fold serial dilutions of sample RAE-21 (estimated ~8.0 EU/mL based on the RFFIT result), see 2 values were calculated; the R2 values for the three samples’ results were 0.95, 0.99 and 0.96, respectively. The precision of the assay, determined by calculating the CV% of duplicate results of the three samples, ranged from 3.83% to 13.28%. Accuracy was not determined due to the unavailability of a validated rabies IgM reference assay.All subject serum samples  were tested using the IgM ELISA, see The IgM to IgG ratio, using the mean EU/mL values of all subjects from each assay, displayed with the means (IU/mL) from the RFFIT assay , illustrIgM is the predominate class of human immunoglobulin present in the early stages of the vaccine-induced immune response, day 7–21 post-vaccination . The IgGIt has long been understood that rabies antibodies, particularly neutralizing antibodies, are of primary importance in immunity to rabies virus infection ,26,27. LTreponema pallidum infection leading to Syphilis, it is clear that IgM is only present in early infection, with IgG being the primary immunoglobulin detected in late infection [The rabies antibody kinetics identified in this study demonstrate similarity to published studies into the antibody response to human viral and bacterial infections. In rotavirus infection, IgM peaks in early collected serum samples and falls to low levels in the late infection-stage samples whereas IgG was present in the later days as detected by a combination of ELISA and serum neutralization methods, which is similar to the findings in our study for the response to rabies vaccination . Antibodnfection ,35,36. Enfection ,37.Purified cell-culture and embryonated egg-based rabies vaccines that are currently in use confer both long-lived immunity and efficacy, and have been proven to be safe for usage in a large range of age groups ,38,39. TBy utilizing a variety of validation parameters for evaluating the use of the ELISA method in detecting rabies-specific IgG and IgM in human sera, method conditions were optimized to measure the concentrations of both IgG and IgM for each sample and accurately determine the antibody isotype kinetics of the immune response to rabies vaccination. Investigation of the immune reaction of each individual’s response to vaccination has contributed to further our understanding of the variability of immunoglobulin class switching between individuals and the specific timing of that class switch from, IgM to neutralizing IgG, via clonal expansion through affinity maturation in the lymphoid tissue for these best fit immune cells. A limitation of this study is the low sample size; however, these results are a starting point in being able to accurately predict high versus low responders’ effective immune defense and possibly identify kinetics of rabies antibody development related to longevity, to better classify an individual’s rabies protection status. This data will also help guide future research on preventative and treatment options as well as guide further development of anti-rabies immunological assays.Future improvements, such as increased subject numbers, would aid in determining trends in non-conformant patient samples, such as is the case with samples RAE-55 through RAE-57 collected from a single patient all showing lower titration values across sampling days. Upon collection of the data from these subjects, further testing and analysis can be conducted to investigate other biologic players in the immune response, for example, cytokines, to expand our knowledge of atypical responses to rabies vaccination. This understanding would drive further research and development of more effective vaccines or vaccine schedules. In addition, further work to establish a qualified rabies IgM reference serum would improve standardization, and hence the accuracy, of rabies IgM assays. Validation of the IgM/IgG ELISA in a high-throughput automated ELISA system would also provide practical usefulness and increase the accessibility of these assays.As demonstrated, the modified rabies enzyme-immunosorbent assays (ELISAs), with anti-IgM or anti-IgG secondary conjugates paired with the appropriate standard curve samples, are quantitative, obtain an objective value from a microplate reader and display similar specificity and sensitivity to the reference Protein A ELISA. Procedural steps and test conditions were optimized to detect, with confidence, the concentrations of both IgM and IgG for each sample and to accurately determine the kinetics of the immune response to rabies vaccination. Rabies antibody IgM to IgG class switch is relevant because it provides knowledge of the antibody response, particularly the isotype kinetics, to rabies vaccination. Understanding immunization responses to the different rabies vaccine regimens and vaccine types currently and in the future will aid in establishing efficient vaccine schedules and administration as the Zero by 30 effort progresses.


We found that early exposure to an enriched environment alone was not sufficient to cause resilience against pain-induced depression-like symptoms. However, extending the enrichment after the injury prevented the development of depression and reduced mechanical hypersensitivity. On the cellular level, increased neuronal excitability was associated with the depressive phenotype that was reversed by the enrichment. Therefore, neuronal excitability in the ACC was inversely correlated to the extended enrichment-induced resilience to depression. These results suggest that the improvement of environmental factors enhanced the resilience to developing chronic pain-related depression. Additionally, we confirmed the association between increased neuronal excitability in the ACC and depression-like states. Therefore, this non-pharmacological intervention could serve as a potential treatment strategy for comorbid symptoms of chronic pain.Depression is a common comorbidity of chronic pain with many patients being affected. However, efficient pharmacological treatment strategies are still lacking. Therefore, it is desirable to find additional alternative approaches. Environmental enrichment has been suggested as a method to alleviate pain-induced depression. However, the neuronal mechanisms of its beneficial effects are still elusive. The anterior cingulate cortex (ACC) plays a central role in processing pain-related negative affect and chronic pain-induced plasticity in this region correlates with depressive symptoms. We studied the consequences of different durations of environmental enrichment on pain sensitivity and chronic pain-induced depression-like behaviors in a mouse model of neuropathic pain. Furthermore, we correlated the behavioral outcomes to the activity levels of pyramidal neurons in the ACC by analyzing their electrophysiological properties  Chronic pain affects around ten percent of the world’s population, varying from 19% in Europe, to 20–25% in the USA , making Environmental enrichment (EE) relates to the optimization and enhancement of external living conditions to promote sensorimotor, cognitive stimulation and physical exercise. EE has been shown to counteract depression-like behaviors in rodents . In precA large body of evidence in animal models suggests that the comorbidity of chronic pain and depression may be related to the involvement of specific brain areas , particuex vivo electrophysiology of layer 2/3 pyramidal neurons, we found that an extended protocol of environmental enrichment was sufficient to decrease pain-induced mechanical hypersensitivity and to produce resilience to depressive behaviors. In contrast, early enrichment had no beneficial effect. Moreover, we provide evidence of an inverse correlation between depression resilience and ACC neuronal hyperactivity, which supports a strong relation between neuronal activity and chronic pain-induced depression-like behaviors.Therefore, we characterized the effects of two types of environmental enrichment, namely, early enrichment (EEE) and extended enrichment (ExEE), on the depressive state of mice with chronic neuropathic pain, and investigated the consequences of this enrichment treatment on neuronal activity in the ACC. Using a battery of depression-like behavioral tests and All experiments were conducted in accordance to the rules of the veterinary office of the canton of Bern, Switzerland. All animals  were kept in a controlled environment  with free access to food and water and housed in groups of four. To minimize litter effect, animals were randomly distributed in groups of four per cage, upon arrival from the Janvier Labs. All experiments were conducted under low light intensity (50–85 lux).Neuropathic pain was induced using the Spared Nerve Injury (SNI) model as previously described . Brieflyt-test was used as exclusion/inclusion criterion.Mechanical hypersensitivity was performed using the electronic version of the Von Frey test . Mice weThe splash test allows to evaluate loss of self-interest in animals by controlling grooming . AnimalsTail suspension evaluates helplessness and despair symptoms of depression-like behaviors in rodents . Mice weSimilar to the tail suspension test, the forced swimming test evaluates depressive despair . Mice weAnimals that escaped any of the experimental arena were excluded from the analysis. Mice were not exposed more than once to the same experimental test.ad libitum access to food and water. Each cage was equipped with a house, a wheel for voluntary physical exercising and 4–5 toys: bridges, swings, fabric hammocks, colored or white ping-pong balls, shelters, mazes, tubes, ladders, paper braids, nestlets. Non-toxic plastic toys as well as custom-made cardboard and paper objects were used , filled with 1 cm of shredded wood bedding. Mice were housed in groups of four and provided with ere used . Additioere used .In the control group (SH), mice from the same batch were housed in a standard mouse cage (15 cm × 37 cm × 13 cm) . No toys3, 1.25 NaH2PO4, 7 MgCl2, 0.5 CaCl2, 25 glucose, and 105 sucrose). After a 30-min incubation in 30°C artificial cerebrospinal fluid , and at least 30 additional minutes of recovery at room temperature, ACC slices were transferred to a recording chamber constantly superfused with 30°C aCSF (2 ml/min). All solutions used were constantly bubbled with a 95% O2 and 5% CO2 gas mixture. Borosilicate glass pipettes were pulled at a resistance of 4–7 MΩ and filled with an internal solution containing, in mM: 130 K-gluconate, 5 KCl, 10 Na-phosphocreatine, 4 Mg-ATP, 0.3 Na-GTP, 10 HEPES, and 2 mg/ml biocytin . Layer 2/3 neurons were visually identified under infrared LED illumination  through an IR CCD camera mounted on a Leica DM LFSA microscope . Whole-cell current clamp recordings were filtered at 5 kHz and amplified, then digitized at 10 kHz and stored on hard disk. All data were acquired and analyzed using custom-made MATLAB (MathWorks) codes. Intrinsic neuronal excitability was assessed by quantifying the action potential firing frequency response to ten progressively increasing square current injections (from −300 pA to +600 pA). From this input-output relationship, the action potential (AP) threshold was estimated as the minimum current required to elicit a single action potential derived from a spline function from the input/output curve, and the input resistance Rin was calculated from the hyperpolarization △V evoked at −300 pA (I), according to the relationship Rin = △V/I.Coronal sections (300 μm) of the rostral ACC were prepared using a vibratome in an ice-cold slicing solution .After patch-clamp electrophysiology recordings were terminated, we characterized the morphology of the recorded neurons, as previously described . BrieflyN = 16, We analyzed the association between neuronal excitability and resilience to depression within a group of animals N = 16, . We z-scpost-hoc test for multiple comparisons. All other behavioral tests outcomes were analyzed by unpaired t-test, where only two groups where compared, or two-way ANOVA followed by post-hoc tests for multiple comparisons. All data are expressed and plotted as mean ± SEM. Significance threshold was set at p < 0.05.Statistical analyses were carried out using MATLAB and GraphPad Prism 9.3.1. The time course for sensory measures considering contra- and ipsilateral paws were analyzed by 2-way repeated measures analysis of variance (ANOVA) followed by the Tukey https://github.com/marioaacuna/in-vitro-ephys.All codes for the electrophysiological analysis performed in this study were written are publicly available at Long-lasting neuropathic pain has been associated with a progressive development of depression-like behaviors . We thern = 15) or standard housing (n = 16) for 6 weeks, after which animals were separated into SNI (n = 8) and sham (n = 7–8) groups. After surgery, EEE animals were placed in standard cages for another 6 weeks. We found that the EEE protocol failed to protect against neuropathic pain-induced mechanical hypersensitivity  was exposed to the extended environmental enrichment protocol . Housing control littermates were kept in standard housing (SH) conditions throughout the duration of the protocol . We found that the ExEE protocol prevented the full development of mechanical hypersensitivity in SNI animals already from post-operative day +7 (Given the lack of protective effect of the early enrichment housing prior to surgery, we designed an extended version of the enrichment protocol, where the animals where continuously exposed to the enriched environment both before and after surgery. For this protocol, a different cohort of animals (e day +7 , with a e day +7 , indicate day +7 . These rPost-hoc comparison of the electrophysiological parameters of individual animals to their depression-like state yielded a significant inverse correlation between the degree of resilience to depression and neuronal excitability (Several lines of evidence suggest that neuropathic pain leads to hyperexcitability of neurons in the ACC . Moreovetability . These dWe found that an extended environmental enrichment that starts at post-natal day 28 and continues for 18 weeks can reduce the mechanical pain phenotype and importantly unmask resilience to chronic pain-induced depression-like behaviors. Additionally, we were able to associate this resilience to depression to the excitability state of L2/3 pyramidal neurons in the ACC, a brain area within the pain matrix involved in affective and emotional processing. ACC neurons of neuropathic pain animals housed in standard conditions showed hyperexcitability as manifested by an increase in input resistance and a decrease of AP firing threshold, all of which could be reversed by environmental enrichment, suggesting a relation between cellular plastic changes and the presence of pain-induced depression-like behaviors. Our results therefore stress the importance of environmental enrichment as a potential alternative strategy to prevent the development of depressive disorders in chronic pain and indicate that the regulation of neuronal activity in the ACC might be the basis of such beneficial effects. Additionally, we present data supporting the central role of the ACC in chronic pain-induced mood disorders. Moreover, we provide novel evidence for a physical intervention that modulates the activity of ACC neurons and results in resilience to depression in neuropathic pain animals.In this study, we showed that an early exposure to an enriched environment limited to the time before the induction of neuropathic pain, failed to reduce neuropathic pain-induced mechanical hypersensitivity or behavioral symptoms of depression. However, a combination of pre-and post-operative environmental enrichment successfully prevented both the full development of allodynia and depression-like behaviors in chronic neuropathic pain mice. In contrast to previous studies , we obseOur electrophysiological data in the neuropathic pain condition show that environmental enrichment induces homeostatic plastic changes which decrease cellular excitability to balance the network architecture. In contrast, environmental enrichment has been associated with increased excitability in the healthy brain. In the hippocampus, this leads to enhanced learning and memory . In the Recent evidence suggest that voluntary exercise could be sufficient to prevent the full expression of mechanical hypersensitivity and depression-like behaviors in rodents  and humaOur study was limited to the evaluation only in male mice. It has been shown that the development of chronic pain in female mice diverges from that of male through specific mechanisms . Indeed,via modulation of activity in the ACC (Taken together, our results provide evidence of a non-pharmacological treatment against mechanical hypersensitivity and depression-like behaviors. Moreover, we confirm the association between dampened neuronal excitability in the ACC and resilience to depression in chronic neuropathic pain, suggesting a possible therapeutic intervention  the ACC  for treaThe original contributions presented in this study are included in the article/This animal study was reviewed and approved by the Veterinary Office of the Canton of Bern, Switzerland.MF, TN, and MA designed the experiments. MF performed the behavioral experiments. NRN performed the electrophysiological experiments. NEN performed the biocytin neuronal reconstructions. MF and MA analyzed data. MA wrote the manuscript. All authors contributed to the article and approved the submitted version.