Adult neurogenesis alters response to an aversive distractor in a labyrinth maze without affecting spatial learning or memory.

Adult neurogenesis has been implicated in learning and memory of complex spatial environments. However, new neurons also play a role in nonmnemonic behavior, including the stress response and attention shifting. Many commonly used spatial tasks are very simple, and unsuitable for detecting neurogenesis effects, or are aversively motivated, making it difficult to dissociate effects on spatial learning and memory from effects on stress. We have therefore created a novel complex spatial environment, the flex maze, to enable reward-mediated testing of spatial learning in a flexibly configurable labyrinth. Using a pharmacogenetic method to completely inhibit neurogenesis in adulthood, we found that rats lacking new neurons (TK rats) and wild type controls completed and remembered most mazes equally well. However, control rats were slower to complete peppermint-scented mazes than other mazes, while neurogenesis-deficient rats showed no effect of mint on maze behavior, completing these mazes significantly faster than control rats. Additional testing found that wild type and TK rats showed similar detection of, avoidance of, and glucocorticoid response to the mint odor. These results suggest that spatial learning and memory in a labyrinth task is unaffected by the loss of new neurons, but that these cells affect the ability of an aversive stimulus to distract rats from completing the maze.

Adult-born granule cell mossy fibers preferentially target parvalbumin-positive interneurons surrounded by perineuronal nets.

Adult-born granule cells (abGCs) integrate into the hippocampus and form connections with dentate gyrus parvalbumin-positive (PV+) interneurons, a circuit important for modulating plasticity. Many of these interneurons are surrounded by perineuronal nets (PNNs), extracellular matrix structures known to participate in plasticity. We compared abGC projections to PV+ interneurons with negative-to-low intensity PNNs to those with high intensity PNNs using retroviral and 3R-Tau labeling in adult mice, and found that abGC mossy fibers and boutons are more frequently located near PV+ interneurons with high intensity PNNs. These results suggest that axons of new neurons preferentially stabilize near target cells with intense PNNs. Next, we asked whether the number of abGCs influences PNN formation around PV+ interneurons, and found that near complete ablation of abGCs produced a decrease in the intensity and number of PV+ neurons with PNNs, suggesting that new neuron innervation may enhance PNN formation. Experience-driven changes in adult neurogenesis did not produce consistent effects, perhaps due to widespread effects on plasticity. Our study identifies abGC projections to PV+ interneurons with PNNs, with more presumed abGC mossy fiber boutons found near the cell body of PV+ interneurons with strong PNNs.

Establishing a reproductive biorepository for basic and translational research: experience developing the reproductive subjects registry and sample repository.

PURPOSE: To report experience designing and establishing a reproductive registry and sample biorepository and to describe initial subject characteristics and biospecimens. METHODS: Beginning in December 2017, patients presenting for reproductive care at the University of Michigan were approached for study enrollment. Following consent, subjects completed detailed reproductive and health questionnaires. A variety of reproductive specimens and tissues were collected and processed for multiple downstream applications. RESULTS: Subject enrollment began in December of 2017. There are currently 1798 subjects enrolled. Female participants report a variety of reproductive disorders. Available samples include semen, sperm, follicular fluid, granulosa cells, immature oocytes, ovarian and uterine tissue, and blood samples. CONCLUSION: We report the successful establishment of a reproductive registry and sample biorepository. Furthermore, we describe methods for collection and storage of a variety of reproductive tissue processed for multiple downstream translational applications.

Neurolastin, a dynamin family GTPase, translocates to mitochondria upon neuronal stress and alters mitochondrial morphology in vivo.

Neurolastin is a dynamin family GTPase that also contains a RING domain and exhibits both GTPase and E3 ligase activities. It is specifically expressed in the brain and is important for synaptic transmission, as neurolastin knockout animals have fewer dendritic spines and exhibit a reduction in functional synapses. Our initial study of neurolastin revealed that it is membrane-associated and partially co-localizes with endosomes. Using various biochemical and cell-culture approaches, we now show that neurolastin also localizes to mitochondria in HeLa cells, cultured neurons, and brain tissue. We found that the mitochondrial localization of neurolastin depends upon an N-terminal mitochondrial targeting sequence and that neurolastin is imported into the mitochondrial intermembrane space. Although neurolastin was only partially mitochondrially localized at steady state, it displayed increased translocation to mitochondria in response to neuronal stress and mitochondrial fragmentation. Interestingly, inactivation or deletion of neurolastin's RING domain also increased its mitochondrial localization. Using EM, we observed that neurolastin knockout animals have smaller but more numerous mitochondria in cerebellar Purkinje neurons, indicating that neurolastin regulates mitochondrial morphology. We conclude that the brain-specific dynamin GTPase neurolastin exhibits stress-responsive localization to mitochondria and is required for proper mitochondrial morphology.

Ongoing neurogenesis in the adult dentate gyrus mediates behavioral responses to ambiguous threat cues.

Fear learning is highly adaptive if utilized in appropriate situations but can lead to generalized anxiety if applied too widely. A role of predictive cues in inhibiting fear generalization has been suggested by stress and fear learning studies, but the effects of partially predictive cues (ambiguous cues) and the neuronal populations responsible for linking the predictive ability of cues and generalization of fear responses are unknown. Here, we show that inhibition of adult neurogenesis in the mouse dentate gyrus decreases hippocampal network activation and reduces defensive behavior to ambiguous threat cues but has neither of these effects if the same negative experience is reliably predicted. Additionally, we find that this ambiguity related to negative events determines their effect on fear generalization, that is, how the events affect future behavior under novel conditions. Both new neurons and glucocorticoid hormones are required for the enhancement of fear generalization following an unpredictably cued threat. Thus, adult neurogenesis plays a central role in the adaptive changes resulting from experience involving unpredictable or ambiguous threat cues, optimizing behavior in novel and uncertain situations.

New neurons restore structural and behavioral abnormalities in a rat model of PTSD.

Post-traumatic stress disorder (PTSD) has been associated with anxiety, memory impairments, enhanced fear, and hippocampal volume loss, although the relationship between these changes remain unknown. Single-prolonged stress (SPS) is a model for PTSD combining three forms of stress (restraint, swim, and anesthesia) in a single session that results in prolonged behavioral effects. Using pharmacogenetic ablation of adult neurogenesis in rats, we investigated the role of new neurons in the hippocampus in the long-lasting structural and behavioral effects of SPS. Two weeks after SPS, stressed rats displayed increased anxiety-like behavior and decreased preference for objects in novel locations regardless of the presence or absence of new neurons. Chronic stress produced by daily restraint for 2 or 6 hr produced similar behavioral effects that were also independent of ongoing neurogenesis. At a longer recovery time point, 1 month after SPS, rats with intact neurogenesis had normalized, showing control levels of anxiety-like behavior. However, GFAP-TK rats, which lacked new neurons, continued to show elevated anxiety-like behavior and enhanced serum corticosterone response to anxiogenic experience. Volume loss in ventral CA1 region of the hippocampus paralleled increases in anxiety-like behavior, occurring in all rats exposed to SPS at the early time point and only rats lacking adult neurogenesis at the later time point. In chronic stress experiments, volume loss occurred broadly throughout the dentate gyrus and CA1 after 6-hr daily stress but was not apparent in any hippocampal subregion after 2-hr daily stress. No effect of SPS was seen on cell proliferation in the dentate gyrus, but the survival of young neurons born a week after stress was decreased. Together, these data suggest that new neurons are important for recovery of normal behavior and hippocampal structure following a strong acute stress and point to the ventral CA1 region as a potential key mediator of stress-induced anxiety-like behavior.

Behavioral and structural adaptations to stress.

Unpredictable aversive experiences, or stressors, lead to changes in depression- and anxiety-related behavior and to changes in hippocampal structure including decreases in adult neurogenesis, granule cell and pyramidal cell dendritic morphology, and volume. Here we review the relationship between these behavioral and structural changes and discuss the possibility that these changes may be largely adaptive. Specifically, we suggest that new neurons in the dentate gyrus enhance behavioral adaptability to changes in the environment, biasing behavior in novel situations based on previous experience with stress. Conversely, atrophy-like changes in the hippocampus and decreased adult neurogenesis following chronic stress may serve to limit stress responses and stabilize behavior during chronic stress.

Axonal ribosomes and mRNAs associate with fragile X granules in adult rodent and human brains.

Local mRNA translation in growing axons allows for rapid and precise regulation of protein expression in response to extrinsic stimuli. However, the role of local translation in mature CNS axons is unknown. Such a mechanism requires the presence of translational machinery and associated mRNAs in circuit-integrated brain axons. Here we use a combination of genetic, quantitative imaging and super-resolution microscopy approaches to show that mature axons in the mammalian brain contain ribosomes, the translational regulator FMRP and a subset of FMRP mRNA targets. This axonal translational machinery is associated with Fragile X granules (FXGs), which are restricted to axons in a stereotyped subset of brain circuits. FXGs and associated axonal translational machinery are present in hippocampus in humans as old as 57 years. This FXG-associated axonal translational machinery is present in adult rats, even when adult neurogenesis is blocked. In contrast, in mouse this machinery is only observed in juvenile hippocampal axons. This differential developmental expression was specific to the hippocampus, as both mice and rats exhibit FXGs in mature axons in the adult olfactory system. Experiments in Fmr1 null mice show that FMRP regulates axonal protein expression but is not required for axonal transport of ribosomes or its target mRNAs. Axonal translational machinery is thus a feature of adult CNS neurons. Regulation of this machinery by FMRP could support complex behaviours in humans throughout life.

Multiplex Enrichment and Detection of Rare KRAS Mutations in Liquid Biopsy Samples using Digital Droplet Pre-Amplification.

Oncology research is increasingly incorporating molecular detection of circulating tumor DNA (ctDNA) as a tool for cancer surveillance and early detection. However, noninvasive monitoring of conditions with low tumor burden remains challenging, as the diagnostic sensitivity of most ctDNA assays is inversely correlated with total DNA concentration and ctDNA abundance. Here we present the Multiplex Enrichment using Droplet Pre-Amplification (MED-Amp) method, which combines single-molecule emulsification and short-round polymerase chain reaction (PCR) preamplification with digital droplet PCR detection of mutant DNA template. The MED-Amp assay increased mutant signal by over 50-fold with minimal distortion in allelic frequency. We demonstrate detection of as few as three mutant copies in wild-type DNA concentrations ranging from 5 to 50 ng. The MED-Amp assay successfully detected KRAS mutant ctDNA in 86% plasma samples obtained from patients with metastatic pancreatic ductal adenocarcinoma. This assay for high-sensitivity rare variant detection is appropriate for liquid biopsy samples or other limited clinical biospecimens.

Adult neurogenesis affects motivation to obtain weak, but not strong, reward in operant tasks.

Decreased motivation to seek rewards is a key feature of mood disorders that correlates with severity and treatment outcome. This anhedonia, or apathy, likely reflects impairment in reward circuitry, but the specific neuronal populations controlling motivation are unclear. Granule neurons generated in the adult hippocampus have been implicated in mood disorders, but are not generally considered as part of reward circuits. We investigated a possible role of these new neurons in motivation to work for food and sucrose rewards in operant conditioning tasks using GFAP-TK pharmacogenetic ablation of adult neurogenesis in both rats and mice. Rats and mice lacking adult neurogenesis showed normal lever press responding during fixed ratio training, reward devaluation, and Pavlovian Instrumental Transfer, suggesting no impairment in learning. However, on an exponentially progressive ratio schedule, or when regular chow was freely available in the testing chamber, TK rats and mice showed less effort to gain sucrose tablets. When working for balanced food tablets, which rats and mice of both genotypes strongly preferred over sucrose, the genotype effects on behavior were lost. This decrease in effort under conditions of low reward suggests that loss of adult neurogenesis decreases motivation to seek reward in a manner that may model behavioral apathy.

Lasting Adaptations in Social Behavior Produced by Social Disruption and Inhibition of Adult Neurogenesis.

UNLABELLED: Research on social instability has focused on its detrimental consequences, but most people are resilient and respond by invoking various coping strategies. To investigate cellular processes underlying such strategies, a dominance hierarchy of rats was formed and then destabilized. Regardless of social position, rats from disrupted hierarchies had fewer new neurons in the hippocampus compared with rats from control cages and those from stable hierarchies. Social disruption produced a preference for familiar over novel conspecifics, a change that did not involve global memory impairments or increased anxiety. Using the neuropeptide oxytocin as a tool to increase neurogenesis in the hippocampus of disrupted rats restored preference for novel conspecifics to predisruption levels. Conversely, reducing the number of new neurons by limited inhibition of adult neurogenesis in naive transgenic GFAP-thymidine kinase rats resulted in social behavior similar to disrupted rats. Together, these results provide novel mechanistic evidence that social disruption shapes behavior in a potentially adaptive way, possibly by reducing adult neurogenesis in the hippocampus. SIGNIFICANCE STATEMENT: To investigate cellular processes underlying adaptation to social instability, a dominance hierarchy of rats was formed and then destabilized. Regardless of social position, rats from disrupted hierarchies had fewer new neurons in the hippocampus compared with rats from control cages and those from stable hierarchies. Unexpectedly, these changes were accompanied by changes in social strategies without evidence of impairments in cognition or anxiety regulation. Restoring adult neurogenesis in disrupted rats using oxytocin and conditionally suppressing the production of new neurons in socially naive GFAP-thymidine kinase rats showed that loss of 6-week-old neurons may be responsible for adaptive changes in social behavior.

Magnetic resonance imaging of odorant activity-dependent migration of neural precursor cells and olfactory bulb growth.

Neural progenitors or neuroblasts are produced by precursor cells in the subventricular zone (SVZ) and migrate along the rostral migratory stream (RMS) to the olfactory bulbs (OB) throughout life. In the OB, these adult born neurons either die or replace existing olfactory interneurons, playing a critical role in the stabilization of OB circuitry. Although several aspects of the addition of new neurons into the OB have been studied, it is unclear whether long-distance activity from the OB can regulate the influx of migrating neuroblasts along the RMS. In this study, iron oxide-assisted MRI was used to track the migration of neuroblasts in combination with reversible naris occlusion to manipulate odorant-induced activity. It was found that decreasing olfactory activity led to a decrease in the rate of neuroblast migration along the RMS. Removal of the naris occlusion led to an increase in migratory rate back to control levels, indicating that olfactory activity has regulatory function on neuroblast migration in the RMS. Blocking odorant activity also led to an arrest in OB growth and re-opening the block led to a rapid re-growth returning the bulb size to control levels. Furthermore, pharmacogenetic elimination of the neuroblasts demonstrated that they were required for re-growth of the bulb following sensory deprivation. Together, these results show that sensory activity, neural migration and OB growth are tightly coupled in an interdependent manner.

Adult hippocampal neurogenesis buffers stress responses and depressive behaviour.

Glucocorticoids are released in response to stressful experiences and serve many beneficial homeostatic functions. However, dysregulation of glucocorticoids is associated with cognitive impairments and depressive illness. In the hippocampus, a brain region densely populated with receptors for stress hormones, stress and glucocorticoids strongly inhibit adult neurogenesis. Decreased neurogenesis has been implicated in the pathogenesis of anxiety and depression, but direct evidence for this role is lacking. Here we show that adult-born hippocampal neurons are required for normal expression of the endocrine and behavioural components of the stress response. Using either transgenic or radiation methods to inhibit adult neurogenesis specifically, we find that glucocorticoid levels are slower to recover after moderate stress and are less suppressed by dexamethasone in neurogenesis-deficient mice than intact mice, consistent with a role for the hippocampus in regulation of the hypothalamic-pituitary-adrenal (HPA) axis. Relative to controls, neurogenesis-deficient mice also showed increased food avoidance in a novel environment after acute stress, increased behavioural despair in the forced swim test, and decreased sucrose preference, a measure of anhedonia. These findings identify a small subset of neurons within the dentate gyrus that are critical for hippocampal negative control of the HPA axis and support a direct role for adult neurogenesis in depressive illness.

Anxiolytic Actions of Exercise in Absence of New Neurons.

Physical exercise reduces anxiety-like behavior in adult mice. The specific mechanisms that mediate this anxiolytic effect are unclear, but adult neurogenesis in the dentate gyrus has been implicated because it is robustly increased by running and has been linked to anxiodepressive-like behavior. We therefore tested the effects of long-term wheel running on anxiety-like behavior in GFAP-TK (TK) mice, a transgenic strain with complete ablation of adult neurogenesis. Five weeks of running reduced anxiety-like behavior equally in both TK mice and wild type (WT) control mice on two tests, elevated plus-maze and novelty-suppressed feeding. WT and TK mice also had similar patterns of c-fos expression in the hippocampus following anxiety testing. Following testing on the elevated plus-maze, running reduced c-fos expression in the dorsal dentate gyrus and CA3 in both WT and TK mice. Following testing on novelty-suppressed feeding, running reduced c-fos expression throughout the dentate gyrus and CA3 in both WT and TK mice. Interestingly, following testing on a less anxiogenic version of novelty-suppressed feeding, running reduced c-fos expression only in the dorsal dentate gyrus in both WT and TK mice, supporting earlier suggestions that the dorsal hippocampus is less involved in emotional behavior than the ventral region. These results suggest that although running increases adult neurogenesis, new neurons are not involved in the decreased anxiety-like behavior or hippocampal activation produced by running. © 2016 Wiley Periodicals, Inc.

Adult neurogenesis: beyond learning and memory.

New neurons continue to be generated in the dentate gyrus throughout life, providing this region of the hippocampus with exceptional structural plasticity, but the function of this ongoing neurogenesis is unknown. Inhibition of adult neurogenesis produces some behavioral impairments that suggest a role for new neurons in learning and memory; however, other behavioral changes appear inconsistent with this function. A review of studies investigating the function of the hippocampus going back several decades reveals many ideas that seem to converge on a critical role for the hippocampus in stress response and emotion. These potential hippocampal functions provide new avenues for investigating the behavioral functions of adult neurogenesis. And, conversely, studies in animals lacking adult neurogenesis, which are likely to have more limited and more specific impairments than are seen with lesions, may provide valuable new insights into the function of the hippocampus. A complete understanding of the function of the hippocampus must explain its role in emotion and the relationship between its emotional and memory functions.

Anxiety- and depression-like behavior and impaired neurogenesis evoked by peripheral neuropathy persist following resolution of prolonged tactile hypersensitivity.

Pain and depression are frequently associated with and often persist after resolution of an initial injury. Identifying the extent to which depression remains causally associated with ongoing physical discomfort during chronic pain, or becomes independent of it, is an important problem for basic neuroscience and psychiatry. Difficulty in distinguishing between effects of ongoing aversive sensory input and its long-term consequences is a significant roadblock, especially in animal models. To address this relationship between localized physical discomfort and its more global consequences, we investigated cellular and behavioral changes during and after reversing a mouse model of neuropathic pain. Tactile allodynia produced by placing a plastic cuff around the sciatic nerve resolved within several days when the cuff was removed. In contrast, the changes in elevated O-maze, forced-swim, Y-maze spontaneous alternation and novel-object recognition test performance that developed after nerve cuff placement remained for at least 3 weeks after the nerve cuffs were removed, or 10-15 d following complete normalization of mechanical sensitivity. Hippocampal neurogenesis, measured by doublecortin and proliferating cell nuclear antigen expression, was also suppressed after nerve cuff placement and remained suppressed 3 weeks after cuff removal. FosB expression was elevated in the central nucleus of the amygdala and spinal cord dorsal horn only in mice with ongoing allodynia. In contrast, FosB remained elevated in the basolateral amygdala of mice with resolved nociception and persisting behavioral effects. These observations suggest that different processes control tactile hypersensitivity and the behavioral changes and impaired neurogenesis that are associated with neuropathic allodynia.

Adult neurogenesis is necessary to refine and maintain circuit specificity.

The circuitry of the olfactory bulb contains a precise anatomical map that links isofunctional regions within each olfactory bulb. This intrabulbar map forms perinatally and undergoes activity-dependent refinement during the first postnatal weeks. Although this map retains its plasticity throughout adulthood, its organization is remarkably stable despite the addition of millions of new neurons to this circuit. Here we show that the continuous supply of new neuroblasts from the subventricular zone is necessary for both the restoration and maintenance of this precise central circuit. Using pharmacogenetic methods to conditionally ablate adult neurogenesis in transgenic mice, we find that the influx of neuroblasts is required for recovery of intrabulbar map precision after disruption due to sensory block. We further demonstrate that eliminating adult-born interneurons in naive animals leads to an expansion of tufted cell axons that is identical to the changes caused by sensory block, thus revealing an essential role for new neurons in circuit maintenance under baseline conditions. These findings show, for the first time, that inhibiting adult neurogenesis alters the circuitry of projection neurons in brain regions that receive new interneurons and points to a critical role for adult-born neurons in stabilizing a brain circuit that exhibits high levels of plasticity.

Chronic cerebrospinal venous insufficiency is not more prevalent in patients with mild multiple sclerosis: a sonographer-blinded, case-control ultrasound study.

OBJECTIVES: We designed a prospective case-control study of patients with clinically isolated syndrome (CIS) and Relapsing-Remitting Multiple Sclerosis (RRMS) with an Expanded Disability Status Score (EDSS) of ≤2, compared with age-and-sex-matched healthy controls, to test the hypothesis that chronic cerebrospinal venous insufficiency (CCSVI) is more prevalent in patients with CIS or mild MS. METHODS: All subjects were examined using a Siemens Antares duplex ultrasound machine. The internal jugular, vertebral and intracranial veins were studied in subjects in both supine and sitting postures. The sonographer was blind to the subject's clinical status. Measures included the criteria proposed by Zamboni and volume flow. Presence of CCSVI was defined as ≥2 Zamboni criteria. RESULTS: Seventy patient-control pairs were recruited, with 11 males and 59 females in each group. Only one subject, a control, satisfied the Zamboni definition of CCSVI; however, 19 patients and 13 controls had abnormalities as defined by Zamboni, the difference largely caused by a higher prevalence in patients of internal jugular vein (IJV) stenosis, defined as a cross-sectional area ≤0.3cm(2). This difference disappeared with a more rigorous stenosis definition. Further analysis revealed there was IJV valve variation in seven patients and one control. CONCLUSIONS: Our findings indicate that CCSVI, as defined by the Zamboni ultrasound criteria, is not present in CIS and mild RRMS (EDSS ≤2), providing further evidence that CCSVI does not have a causal role in MS; however, we found an apparent increase in IJV variation in patients with CIS or mild MS that would warrant further investigation.

Assessing reward preference using operant behavior in male and female mice.

Many different solid food pellets are available as reinforcers for rodents in operant behavior tests. Different reward formulations have not been compared, so it is unclear whether mice show strong preferences for different rewards and whether such preferences are consistent within or across sex and background strain. Here we show that mice have strong preferences for two balanced diet food rewards over sucrose pellets, and preference for one balanced diet pellet formulation over another, in a simultaneous choice test using a low effort fixed ratio operant test. All mice, of both sexes and both CD1 and C57 background strains, showed the same strong preferences among these three types of reinforcers. In contrast, flavorings added to the reward pellets had relatively small and more variable effects on preference. The preference for balanced diet pellets over sucrose pellets was seen also in the total numbers of rewards consumed in low effort tests with food pellets or only sucrose pellets available. However, progressive ratio testing showed that mice worked harder for sucrose pellets than for the preferred balanced diet pellets. These findings indicate that reinforcers with similar and very different preference profiles are readily available and that testing with different rewards can produce different, and sometimes unexpected, results.

Economic analysis of n-butyl cyanoacrylate compared with ethylene vinyl alcohol copolymer liquid embolic embolization of brain arteriovenous malformations (bAVMs) from a US hospital perspective.

BACKGROUND: In US hospitals, the liquid embolic systems (LESs) n-butyl cyanoacrylate (n-BCA) and ethylene vinyl alcohol copolymer (EVOH) are used for brain arteriovenous malformation (bAVM) embolization to achieve presurgical devascularization. The aim of this study was to perform an economic analysis comparing four techniques for bAVM embolization based on LES, ancillary device, and angiography suite time costs. METHODS: An economic model was developed comparing the embolization costs for n-BCA, EVOH with the plug and push technique, EVOH with detachable-tip microcatheters, and EVOH with balloon microcatheters. Per procedure costs were calculated for bAVMs with one to four pedicles. Annual cohort analyses were performed to evaluate the potential impact for low and high-volume centers. Sensitivity analyses were performed to determine cost drivers. RESULTS: The analyses showed that the n-BCA technique was the least costly of the four techniques. Total per procedure costs for one to four embolized pedicles ranged from $5941 to $10,074 for the n-BCA technique, $8428 to $30,345 for the EVOH balloon microcatheter technique, $12,711 to $47,477 for the EVOH plug and push technique, and $13,900 to $52,233 for the EVOH detachable-tip microcatheter technique. Cohort analyses costs for 52 annual cases ranged from $308,953 to $523,838 with the n-BCA technique and from $722,816 to $2,716,096 with the EVOH detachable-tip microcatheter technique. CONCLUSIONS: Procedure costs associated with n-BCA are lower than those with each of the three EVOH techniques examined. Future cost analyses should compare the costs of new LES products once available.