A longitudinal course pilot to improve surgical resident acquisition of quality improvement skills.

PROBLEM: Despite mounting evidence that incorporation of QI curricula into surgical trainee education improves morbidity and outcomes, surgery training programs lack standardized QI curricula and tools to measure QI knowledge. In the current study, we developed, implemented, and evaluated a quality improvement curriculum for surgical residents. INTERVENTION: Surgical trainees participated in a longitudinal, year-long (2019-2020) curriculum based on the Institute for Healthcare Improvement's online program. Online curriculum was supplemented with in person didactics and small group projects. Acquisition of skills was assessed pre- and post- course via self-report on a Likert scale as well as the Quality Improvement Knowledge Application Tool (QIKAT). Self-efficacy scores were assessed using the General Self-Efficacy Scale. 9 out of 18 total course participants completed the post course survey. This first course cohort was analyzed as a pilot for future work. CONTEXT: The project was developed and deployed among surgical residents during their research/lab year. Teams of surgical residents were partnered with a faculty project mentor, as well as non-physician teammates for project work. IMPACT: Participation in the QI course significantly increased skills related to studying the process (p = 0.0463), making changes in a system (p = 0.0167), identifying whether a change leads to an improvement (p = 0.0039), using small cycles of change (p = 0.0000), identifying best practices and comparing them to local practices (p = 0.0020), using PDSA model as a systematic framework for trial and learning (p = 0.0004), identifying how data is linked to specific processes (p = 0.0488), and building the next improvement cycle upon success or failure (p = 0.0316). There was also a significant improvement in aim (p = 0.037) and change (p = 0.029) responses to one QIKAT vignette. LESSONS LEARNED: We describe the effectiveness of a pilot longitudinal, multi component QI course based on the IHI online curriculum in improving surgical trainee knowledge and use of key QI skills.

Human-Induced Pluripotent Stem-Cell-Derived Smooth Muscle Cells Increase Angiogenesis to Treat Hindlimb Ischemia.

Induced pluripotent stem cells (iPSC) represent an innovative, somatic cell-derived, easily obtained and renewable stem cell source without considerable ethical issues. iPSC and their derived cells may have enhanced therapeutic and translational potential compared with other stem cells. We previously showed that human iPSC-derived smooth muscle cells (hiPSC-SMC) promote angiogenesis and wound healing. Accordingly, we hypothesized that hiPSC-SMC may be a novel treatment for human patients with chronic limb-threatening ischemia who have no standard options for therapy. We determined the angiogenic potential of hiPSC-SMC in a murine hindlimb ischemia model. hiPSC-SMC were injected intramuscularly into nude mice after creation of hindlimb ischemia. Functional outcomes and perfusion were measured using standardized scores, laser Doppler imaging, microCT, histology and immunofluorescence. Functional outcomes and blood flow were improved in hiPSC-SMC-treated mice compared with controls (Tarlov score, p < 0.05; Faber score, p < 0.05; flow, p = 0.054). hiPSC-SMC-treated mice showed fewer gastrocnemius fibers (p < 0.0001), increased fiber area (p < 0.0001), and enhanced capillary density (p < 0.01); microCT showed more arterioles (<96 µm). hiPSC-SMC treatment was associated with fewer numbers of macrophages, decreased numbers of M1-type (p < 0.05) and increased numbers of M2-type macrophages (p < 0.0001). Vascular endothelial growth factor (VEGF) expression in ischemic limbs was significantly elevated with hiPSC-SMC treatment (p < 0.05), and inhibition of VEGFR-2 with SU5416 was associated with fewer capillaries in hiPSC-SMC-treated limbs (p < 0.0001). hiPSC-SMC promote VEGF-mediated angiogenesis, leading to improved hindlimb ischemia. Stem cell therapy using iPSC-derived cells may represent a novel and potentially translatable therapy for limb-threatening ischemia.

Activation of EphrinB2 Signaling Promotes Adaptive Venous Remodeling in Murine Arteriovenous Fistulae.

BACKGROUND: Arteriovenous fistulae (AVF) are the preferred mode of vascular access for hemodialysis. Before use, AVF remodel by thickening and dilating to achieve a functional conduit via an adaptive process characterized by expression of molecular markers characteristic of both venous and arterial identity. Although signaling via EphB4, a determinant of venous identity, mediates AVF maturation, the role of its counterpart EphrinB2, a determinant of arterial identity, remains unclear. We hypothesize that EphrinB2 signaling is active during AVF maturation and may be a mechanism of venous remodeling. METHODS: Aortocaval fistulae were created or sham laparotomy was performed in C57Bl/6 mice, and specimens were examined on Days 7 or 21. EphrinB2 reverse signaling was activated with EphB4-Fc applied periadventitially in vivo and in endothelial cell culture medium in vitro. Downstream signaling was assessed using immunoblotting and immunofluorescence. RESULTS: Venous remodeling during AVF maturation was characterized by increased expression of EphrinB2 as well as Akt1, extracellular signal-regulated kinases 1/2 (ERK1/2), and p38. Activation of EphrinB2 with EphB4-Fc increased phosphorylation of EphrinB2, endothelial nitric oxide synthase, Akt1, ERK1/2, and p38 and was associated with increased diameter and wall thickness in the AVF. Both mouse and human endothelial cells treated with EphB4-Fc increased phosphorylation of EphrinB2, endothelial nitric oxide synthase, Akt1, ERK1/2, and p38 and increased endothelial cell tube formation and migration. CONCLUSIONS: Activation of EphrinB2 signaling by EphB4-Fc was associated with adaptive venous remodeling in vivo while activating endothelial cell function in vitro. Regulation of EphrinB2 signaling may be a new strategy to improve AVF maturation and patency.

A hybrid approach for vascular control and repair of an expanding iatrogenic femoral artery pseudoaneurysm.

Surgical repair of iatrogenic femoral pseudoaneurysms in the setting of distorted anatomy, multiple prior interventions, and ongoing hemorrhage requires extensive proximal and distal dissection for control. Furthermore, profunda femoral and other arterial branch control may not always be feasible and can lead to considerable blood loss at the time of surgical exploration. We present a simple, safe, and effective hybrid approach for inflow, outflow, and branch control for treatment of a proximally located, actively expanding, iatrogenic common femoral artery pseudoaneurysm recalcitrant to multiple sessions of percutaneous thrombin injection.

Artery to vein configuration of arteriovenous fistula improves hemodynamics to increase maturation and patency.

Arteriovenous fistulae (AVF) are the preferred mode of hemodialysis access, but 60% of conventional [vein-to-artery (V-A)] AVF fail to mature, and only 50% remain patent at 1 year. We previously showed improved maturation and patency in a pilot study of the radial artery deviation and reimplantation (RADAR) technique that uses an artery-to-vein (A-V) configuration. Here, we show that RADAR exhibits higher rates of maturation, as well as increased primary and secondary long-term patencies. RADAR is also protective in female patients, where it is associated with decreased reintervention rates and improved secondary patency. RADAR and conventional geometries were compared further in a rat bilateral carotid artery-internal jugular vein fistula model. There was decreased cell proliferation and neointimal hyperplasia in the A-V configuration in male and female animals, but no difference in hypoxia between the A-V and V-A configurations. Similar trends were seen in uremic male rats. The A-V configuration also associated with increased peak systolic velocity and expression of Kruppel-like factor 2 and phosphorylated endothelial nitric oxide synthase, consistent with improved hemodynamics. Computed tomography and ultrasound-informed computational modeling showed different hemodynamics in the A-V and V-A configurations, and improving the hemodynamics in the V-A configuration was protective against neointimal hyperplasia. These findings collectively demonstrate that RADAR is a durable surgical option for patients requiring radial-cephalic AVF for hemodialysis access.

Author Correction: Inhibition of the Akt1-mTORC1 Axis Alters Venous Remodeling to Improve Arteriovenous Fistula Patency.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Induced pluripotent stem cell-derived smooth muscle cells increase angiogenesis and accelerate diabetic wound healing.

Aim: To assess the potential of human induced pluripotent stem cell-derived smooth muscle cells (hiPSC-SMC) to accelerate diabetic wound healing. Methods: hiPSC-SMC were embedded in 3D collagen scaffolds and cultured in vitro for 72 h; scaffolds were then applied to diabetic, nude mouse, splinted back wounds to assess in vivo healing. Cultured medium after scaffold incubation was collected and analyzed for expression of pro-angiogenic cytokines. Results: hiPSC-SMC secrete increased concentration of pro-angiogenic cytokines, compared with murine adipose derived stem cells. Delivery of hiPSC-SMC-containing collagen scaffolds accelerates diabetic wound healing and is associated with an increased number of total and M2 type macrophages. Conclusion: hiPSC-SMC promote angiogenesis and accelerate diabetic wound healing, making them a promising new candidate for treatment of diabetic wounds.

A Dense Fibrillar Collagen Scaffold Differentially Modulates Secretory Function of iPSC-Derived Vascular Smooth Muscle Cells to Promote Wound Healing.

The application of human-induced pluripotent stem cells (hiPSCs) to generate vascular smooth muscle cells (hiPSC-VSMCs) in abundance is a promising strategy for vascular regeneration. While hiPSC-VSMCs have already been utilized for tissue-engineered vascular grafts and disease modeling, there is a lack of investigations exploring their therapeutic secretory factors. The objective of this manuscript was to understand how the biophysical property of a collagen-based scaffold dictates changes in the secretory function of hiPSC-VSMCs while developing hiPSC-VSMC-based therapy for durable regenerative wound healing. We investigated the effect of collagen fibrillar density (CFD) on hiPSC-VSMC's paracrine secretion and cytokines via the construction of varying density of collagen scaffolds. Our study demonstrated that CFD is a key scaffold property that modulates the secretory function of hiPSC-VSMCs. This study lays the foundation for developing collagen-based scaffold materials for the delivery of hiPSC-VSMCs to promote regenerative healing through guiding paracrine signaling pathways.

A mouse model of stenosis distal to an arteriovenous fistula recapitulates human central venous stenosis.

OBJECTIVE: Central venous stenosis (CVS) is a major cause of arteriovenous fistula (AVF) failure. However, central veins are relatively inaccessible to study with conventional Doppler ultrasound methods. To understand mechanisms underlying AVF failure owing to CVS, an animal model was established that creates a stenosis distal to an AVF. We hypothesized that this mouse model will show comparable morphology and physiology to human CVS. METHODS: An aortocaval fistula was created between the distal aorta and inferior vena cava (IVC); a stenosis was then created distal to the fistula by partial IVC ligation. Sham-operated animals, AVF without venous stenosis, and venous stenosis without AVF were used as controls. Physiologic properties of the IVC, both upstream and downstream of the stenosis, or the corresponding sites in models without stenosis, were assessed with ultrasound examination on days 0 to 21. The spectral broadening index was measured to assess the degree of disturbed shear stress. The IVC was harvested at day 21 and specimens were analyzed with immunofluorescence. RESULTS: The IVC diameter of mice with an AVF and stenosis showed increased upstream (P = .013), but decreased downstream diameter (P = .001) compared with mice with an AVF but without a stenosis, at all postoperative times (days 3-21). IVC wall thickness increased in mice with an AVF, compared with IVC without an AVF (upstream of stenosis: 13.9 µm vs 11.0 µm vs 4.5 µm vs 3.9 µm; P = .020; downstream of stenosis: 6.0 µm vs 6.6 µm vs µm 4.5 µm vs 3.8 µm; P = .002; AVF with stenosis, AVF, stenosis, sham, respectively). AVF patency significantly decreased in mice with an AVF and stenosis by day 21 (50% vs 90%; P = .048). The IVC of mice with AVF and stenosis showed a venous waveform with pulsatility as well as enhanced velocity at and downstream of the stenosis; similar waveforms were observed in a human case of CVS. Downstream to the stenosis, the spectral broadening index was significantly higher compared with mice with AVF alone (1.06 vs 0.78; P = .011; day 21), and there was a trend towards less immunoreactivity of both Krüppel-like factor 2 and phosphorylated-endothelial nitric oxide synthase compared with mice with an AVF alone. CONCLUSIONS: Partial IVC ligation distal to a mouse aortocaval fistula alters the fistula diameter and wall thickness, decreases patency, and increases distal disturbed flow compared with fistulae without a distal stenosis. Our mouse model of stenosis distal to an AVF may be a faithful representation of human CVS that shows similar morphology and physiology, including disturbed shear stress.

Using the epidemiology of critical limb ischemia to estimate the number of patients amenable to endovascular therapy.

Critical limb ischemia represents the advanced stage of peripheral artery disease, a health problem with increasing prevalence. Critical limb ischemia is associated with significant mortality, limb loss, pain, and diminished health-related quality of life. Public awareness and early diagnosis are necessary for an effective treatment with early risk factor modification, smoking cessation, and exercise therapy. Herein, we present an overview of the epidemiology as well as the clinical stages of the disease, and estimate that there are 6.5 million patients with critical limb ischemia in the US, Europe, and Japan based on global population-based studies. At least 75% of these patients, accounting for approximately 4.8 million patients, are amenable to endovascular therapy.

Molecular Targets for Improving Arteriovenous Fistula Maturation and Patency.

The increasing prevalence of chronic and end-stage renal disease creates an increased need for reliable vascular access, and although arteriovenous fistulae (AVF) are the preferred mode of hemodialysis access, 60% fail to mature and only 50% remain patent at one year. Fistulae mature by diameter expansion and wall thickening; this outward remodeling of the venous wall in the fistula environment relies on a delicate balance of extracellular matrix (ECM) remodeling, inflammation, growth factor secretion, and cell adhesion molecule upregulation in the venous wall. AVF failure occurs via two distinct mechanisms with early failure secondary to lack of outward remodeling, that is insufficient diameter expansion or wall thickening, whereas late failure occurs with excessive wall thickening due to neointimal hyperplasia (NIH) and insufficient diameter expansion in a previously functional fistula. In recent years, the molecular basis of AVF maturation and failure are becoming understood in order to develop potential therapeutic targets to aide maturation and prevent access loss. Erythropoietin-producing hepatocellular carcinoma (Eph) receptors, along with their ligands, ephrins, determine vascular identity and are critical for vascular remodeling in the embryo. Manipulation of Eph receptor signaling in adults, as well as downstream pathways, is a potential treatment strategy to improve the rates of AVF maturation and patency. This review examines our current understanding of molecular changes occurring following fistula creation, factors predictive of fistula success, and potential areas of intervention to decrease AVF failure.

Carotid Endarterectomy and Carotid Artery Stenting for Patients With Crescendo Transient Ischemic Attacks: A Systematic Review.

Importance: Thromboembolic stroke attributable to an ipsilateral carotid artery plaque is a leading cause of disability in the United States and a major source of morbidity. Randomized clinical trials have demonstrated the efficacy of carotid endarterectomy and carotid stenting at minimizing stroke risk in patients with minor stroke and transient ischemic attack. However, there is no consensus on guidelines for medical management and the timing of revascularization in patients with multiple recurrent episodes of transient ischemic attack over hours or days, an acute neurological event known as crescendo transient ischemic attack. Objective: To review the management of and timing of intervention in patients presenting with crescendo transient ischemic attack. Evidence Review: This systematic review included all English-language articles published from January 1, 1985, to January 1, 2019, available from PubMed (MEDLINE) and Google Scholar. Articles were excluded if they did not include analysis of patients with symptoms, did not report the timing of intervention after crescendo transient ischemic attack, or mixed analysis of patients with stroke in evolution with patients with crescendo transient ischemic attack. The quality of the evidence was assessed with the modified rating from the Oxford Centre for Evidence-based Medicine. Observations: Patients with crescendo transient ischemic attack were found to have a higher risk of stroke or death after carotid endarterectomy compared with patients with a single transient ischemic attack or stable stroke. With medical therapy alone, a considerable number of patients with crescendo transient ischemic attack experience a completed stroke within several months and have a poor prognosis without intervention. Urgent carotid endarterectomy, typically performed within 48 hours of initial presentation, is beneficial in carefully selected patients. There have been several reports of operative treatment within the first 24 hours of presentation; however, review of these reports does not show any additional benefit from emergency treatment. Carotid artery stenting is reserved only for selected patients with prohibitive surgical risk for endarterectomy. The literature does not clearly support any additional benefit of intravenous heparin therapy over mono or dual antiplatelet therapy prior to carotid endarterectomy. Conclusions and Relevance: Crescendo transient ischemic attack is best managed with optimal medical management as well as urgent carotid endarterectomy within 2 days of presentation. Surgical endarterectomy appears to be preferred because of the increased embolic potential of bifurcation plaque, whereas stenting is an option for patients with contraindications for surgery. With ongoing advances in cerebrovascular imaging and medical treatment of stroke, there is a need for better evidence to determine the optimal timing and preoperative medical management of patients with crescendo transient ischemic attack.

Inhibition of the Akt1-mTORC1 Axis Alters Venous Remodeling to Improve Arteriovenous Fistula Patency.

Arteriovenous fistulae (AVF) are the most common access created for hemodialysis, but up to 60% do not sustain dialysis within a year, suggesting a need to improve AVF maturation and patency. In a mouse AVF model, Akt1 regulates fistula wall thickness and diameter. We hypothesized that inhibition of the Akt1-mTORC1 axis alters venous remodeling to improve AVF patency. Daily intraperitoneal injections of rapamycin reduced AVF wall thickness with no change in diameter. Rapamycin decreased smooth muscle cell (SMC) and macrophage proliferation; rapamycin also reduced both M1 and M2 type macrophages. AVF in mice treated with rapamycin had reduced Akt1 and mTORC1 but not mTORC2 phosphorylation. Depletion of macrophages with clodronate-containing liposomes was also associated with reduced AVF wall thickness and both M1- and M2-type macrophages; however, AVF patency was reduced. Rapamycin was associated with improved long-term patency, enhanced early AVF remodeling and sustained reduction of SMC proliferation. These results suggest that rapamycin improves AVF patency by reducing early inflammation and wall thickening while attenuating the Akt1-mTORC1 signaling pathway in SMC and macrophages. Macrophages are associated with AVF wall thickening and M2-type macrophages may play a mechanistic role in AVF maturation. Rapamycin is a potential translational strategy to improve AVF patency.

The potential and limitations of induced pluripotent stem cells to achieve wound healing.

Wound healing is the physiologic response to a disruption in normal skin architecture and requires both spatial and temporal coordination of multiple cell types and cytokines. This complex process is prone to dysregulation secondary to local and systemic factors such as ischemia and diabetes that frequently lead to chronic wounds. Chronic wounds such as diabetic foot ulcers are epidemic with great cost to the healthcare system as they heal poorly and recur frequently, creating an urgent need for new and advanced therapies. Stem cell therapy is emerging as a potential treatment for chronic wounds, and adult-derived stem cells are currently employed in several commercially available products; however, stem cell therapy is limited by the need for invasive harvesting techniques, immunogenicity, and limited cell survival in vivo. Induced pluripotent stem cells (iPSC) are an exciting cell type with enhanced therapeutic and translational potential. iPSC are derived from adult cells by in vitro induction of pluripotency, obviating the ethical dilemmas surrounding the use of embryonic stem cells; they are harvested non-invasively and can be transplanted autologously, reducing immune rejection; and iPSC are the only cell type capable of being differentiated into all of the cell types in healthy skin. This review focuses on the use of iPSC in animal models of wound healing including limb ischemia, as well as their limitations and methods aimed at improving iPSC safety profile in an effort to hasten translation to human studies.

Adipose-derived mesenchymal stem cells accelerate diabetic wound healing in a similar fashion as bone marrow-derived cells.

We have previously shown that bone marrow-derived mesenchymal stem cells (BMSC) accelerate wound healing in a diabetic mouse model. In this study, we hypothesized that adipose tissue-derived stem cells (ADSC), cells of greater translational potential to human therapy, improve diabetic wound healing to a similar extent as BMSC. In vitro, the characterization and function of murine ADSC and BMSC as well as human diabetic and nondiabetic ADSC were evaluated by flow cytometry, cell viability, and VEGF expression. In vivo, biomimetic collagen scaffolds containing murine ADSC or BMSC were used to treat splinted full-thickness excisional back wounds on diabetic C57BL/6 mice, and human healthy and diabetic ADSC were used to treat back wounds on nude mice. Wound healing was evaluated by wound area, local VEGF-A expression, and count of CD31-positive cells. Delivery of murine ADSC or BMSC accelerated wound healing in diabetic mice to a similar extent, compared with acellular controls ( P < 0.0001). Histological analysis showed similarly increased cellular proliferation ( P < 0.0001), VEGF-A expression ( P = 0.0002), endothelial cell density ( P < 0.0001), numbers of macrophages ( P < 0.0001), and smooth muscle cells ( P < 0.0001) with ADSC and BMSC treatment, compared with controls. Cell survival and migration of ADSC and BMSC within the scaffolds were similar ( P = 0.781). Notch signaling was upregulated to a similar degree by both ADSC and BMSC. Diabetic and nondiabetic human ADSC expressed similar levels of VEGF-A ( P = 0.836) in vitro, as well as in scaffolds ( P = 1.000). Delivery of human diabetic and nondiabetic ADSC enhanced wound healing to a similar extent in a nude mouse wound model. Murine ADSC and BMSC delivered in a biomimetic-collagen scaffold are equivalent at enhancing diabetic wound healing. Human diabetic ADSC are not inferior to nondiabetic ADSC at accelerating wound healing in a nude mouse model. This data suggests that ADSC are a reasonable choice to evaluate for translational therapy in the treatment of human diabetic wounds.

Trends of ureteral stent usage in surgery for diverticulitis.

BACKGROUND: Many believe that the use of ureteral stents in colorectal surgery for diverticulitis aids prevention and easier identification of ureteral injuries; others argue that the added time, cost, and risks of stent placement negate potential benefits. Even among providers who use stents, selective use is common. Among unclear consensus, it remains unknown if the use of stents is growing. MATERIALS: Patients in the National Inpatient Sample who underwent a partial colectomy or anterior rectal excision for diverticulitis between 2000 and 2013 were included (n = 811,071). Trends in ureteral stent use, multivariate logistic regression of factors influencing stent placement, and linear regression of length of stay (LOS) and costs associated with stent use were examined. RESULTS: Usage of ureteral stents increased from 6.66% in 2000 to 16.30% in 2013 (P < 0.0001). Rates of stent usage were higher with laparoscopic surgery (19.31% versus 12.31% open, P < 0.0001). Regression demonstrated patients in the Northeast (Midwest odds ratio (OR) 0.49 [0.37-0.66] P < 0.0001, South OR 0.60 [0.45-0.80] P = 0.0004, West OR 0.30 [0.22-0.41], P < 0.0001), and those whose admission was elective (OR 2.37 [2.08-2.69], P < 0.0001) were more likely to receive stents. Stent use was associated with an increased LOS (0.55 days, P < 0.0001) and cost ($1,983, P < 0.0001). CONCLUSIONS: The use of ureteral stents in surgery for diverticulitis has steadily increased since 2000, despite the lack of consensus of their overall benefit. Stent usage is associated with laparoscopic surgery and varies widely among regions of the country. Further studies are required to truly understand the risk-benefit ratio of ureteral stenting and to determine if its increased use is warranted.

Loss of MEN1 activates DNMT1 implicating DNA hypermethylation as a driver of MEN1 tumorigenesis.

Multiple endocrine neoplasia type 1 (MEN1) syndrome results from mutations in the MEN1 gene and causes tumor formation via largely unknown mechanisms. Using a novel genome-wide methylation analysis, we studied tissues from MEN1-parathyroid tumors, Men1 knockout (KO) mice, and Men1 null mouse embryonic fibroblast (MEF) cell lines. We demonstrated that inactivation of menin (the protein product of MEN1) increases activity of DNA (cytosine-5)-methyltransferase 1 (DNMT1) by activating retinoblastoma-binding protein 5 (Rbbp5). The increased activity of DNMT1 mediates global DNA hypermethylation, which results in aberrant activation of the Wnt/ß-catenin signaling pathway through inactivation of Sox regulatory genes. Our study provides important insights into the role of menin in DNA methylation and its impact on the pathogenesis of MEN1 tumor development.

G-protein-coupled estrogen receptor 1 is anatomically positioned to modulate synaptic plasticity in the mouse hippocampus.

Both estrous cycle and sex affect the numbers and types of neuronal and glial profiles containing the classical estrogen receptors α and ß, and synaptic levels in the rodent dorsal hippocampus. Here, we examined whether the membrane estrogen receptor, G-protein-coupled estrogen receptor 1 (GPER1), is anatomically positioned in the dorsal hippocampus of mice to regulate synaptic plasticity. By light microscopy, GPER1-immunoreactivity (IR) was most noticeable in the pyramidal cell layer and interspersed interneurons, especially those in the hilus of the dentate gyrus. Diffuse GPER1-IR was found in all lamina but was most dense in stratum lucidum of CA3. Ultrastructural analysis revealed discrete extranuclear GPER1-IR affiliated with the plasma membrane and endoplasmic reticulum of neuronal perikarya and dendritic shafts, synaptic specializations in dendritic spines, and clusters of vesicles in axon terminals. Moreover, GPER1-IR was found in unmyelinated axons and glial profiles. Overall, the types and amounts of GPER1-labeled profiles were similar between males and females; however, in females elevated estrogen levels generally increased axonal labeling. Some estradiol-induced changes observed in previous studies were replicated by the GPER agonist G1: G1 increased PSD95-IR in strata oriens, lucidum, and radiatum of CA3 in ovariectomized mice 6 h after administration. In contrast, estradiol but not G1 increased Akt phosphorylation levels. Instead, GPER1 actions in the synapse may be due to interactions with synaptic scaffolding proteins, such as SAP97. These results suggest that although estrogen's actions via GPER1 may converge on the same synaptic elements, different pathways are used to achieve these actions.

Characterization of neural estrogen signaling and neurotrophic changes in the accelerated ovarian failure mouse model of menopause.

Accelerated ovarian failure (AOF) can be induced in young mice with low doses of 4-vinylcyclohexene diepoxide (VCD), modeling the hormone changes observed across menopause. We assessed markers of synaptic plasticity in the hippocampus, anxiety-like behavior, and spatial learning longitudinally at 4 time points across the AOF model: premenopause, early perimenopause, late perimenopause, and postmenopause (POST). As others have shown, VCD administration decreased ovarian follicle counts and increased acyclicity as the model progressed to POST but with no impact on organ or body weights. The morphology of Iba1 immunoreactive microglia did not differ between vehicle- and VCD-administered mice. Hippocampal postsynaptic density 95 levels were minimally altered across the AOF model but decreased at POST in CA3b 24 hours after exogenous estradiol benzoate (EB). In contrast, hippocampal phosphorylated AKT levels transiently decreased in premenopause but increased at POST after 24 hours of EB in select subregions. Electron microscopy revealed fewer estrogen receptor α containing dendritic spines and terminals in CA1 stratum radiatum at POST. mRNA levels of most brain-derived neurotrophic factor exons (except V and VI) were lower in POST compared with ovariectomized mice. Exon V was sensitive to 24 hours of EB administration in POST-VCD. Anxiety-like behavior was unaffected at any menopause phase. Spatial learning was unaffected in all groups, but POST-VCD mice performed below chance. Our results suggest that the AOF model is suitable for longitudinal studies of neurobiological changes across the menopause transition in mice. Our findings also point to complex interactions between estrogen receptors and pathways involved in synaptic plasticity.