Bronchoscopy involves exploration of a three-dimensional (3D) bronchial tree environment using just two-dimensional (2D) images, visual cues and haptic feedback. Sound knowledge and understanding of tracheobronchial anatomy as well as ample training experience is mandatory for technical mastery. Although simulated modalities facilitate safe training for inexperienced operators, current commercial training models are expensive or deficient in anatomical accuracy, clinical fidelity and patient representation. The advent of Three-dimensional (3D) printing technology may resolve the current limitations with commercial simulators. The purpose of this report is to develop and test the novel multi-material three-dimensional (3D) printed airway models for bronchoscopy simulation.Using material jetting 3D printing and polymer amalgamation, human airway models were created from anonymized human thoracic computed tomography images from three patients: one normal, a second with a tumour obstructing the right main bronchus and third with a goitre causing external tracheal compression. We validated their efficacy as airway trainers by expert bronchoscopists. Recruited study participants performed bronchoscopy on the 3D printed airway models and then completed a standardized evaluation questionnaire.The models are flexible, life size, anatomically accurate and patient specific. Five expert respiratory physicians participated in validation of the airway models. All the participants agreed that the models were suitable for training bronchoscopic anatomy and access. Participants suggested further refinement of colour and texture of the internal surface of the airways. Most respondents felt that the models are suitable simulators for tracheal pathology, have a learning value and recommend it to others for use in training.Using material jetting 3D printing to create patient-specific anatomical models is a promising modality of simulation training. Our results support further evaluation of the printed airway model as a bronchoscopic trainer, and suggest that pathological airways may be simulated using this technique. Lung cancer is the leading cause of cancer death and accounts for a disproportionate burden of cancer-related mortality . AdvanceEarly diagnosis and staging by histological study are therefore essential in guiding clinical decisions and ultimately predicting disease progression. Suspicious lung nodules are usually benign but occasionally malignant; therefore, a clinical need exists for techniques that are minimally invasive, and at the same time are of sufficient diagnostic yield.The approach to histological evaluation of a suspicious lung nodule depends primarily on its location. If proximal, a respiratory physician may obtain tissue endoscopically via the airways, using bronchoscopic biopsy of the lesion under ultrasonographic guidance. Peripheral nodules are preferentially sampled using computed tomography (CT) guided transthoracic needle biopsy by interventional radiologists .Bronchoscopy is a versatile procedure for endoscopic exploration of the airways. Beyond biopsy of suspicious nodules, the bronchoscopic arsenal includes forceps, ultrasonographic probes, endobronchial tubes and even stent insertion, offering myriad diagnostic and therapeutic applications in the field of pulmonology .Skilful bronchoscopy requires a working knowledge of bronchopulmonary segmental anatomy, as well as experience in endoscopic manoeuvres . Being aProcedural training in medical education must balance the importance of realistic training experience with risk of harm to patients by inexperienced operators. In this regard, simulation-based training with physical or virtual airway models eliminates patient risk and is recommended over traditional apprenticeship teaching –9, in whIn developing these bronchoscopic simulators, important considerations are anatomical accuracy, fidelity (the degree of representation) to actual clinical scenarios, and cost . To addrMulti-material 3D printing, by means of customized colours and haptics, offers the potential for high-precision anatomical modelling which demands differentiation of tissue subtypes , 22. TheThis study was granted institutional review board approval (IRB-2017-05-052). Following development of three patient-specific 3D printed airway models, this study evaluated its validity as an airway trainer by expert respiratory physicians, who tested the model and provided their opinion by means of a structured questionnaire.Computed tomography (CT) scans of the thorax were obtained and anonymized from patients from the Diagnostic Radiology department in Tan Tock Seng Hospital (TTSH), Singapore with SOMATOM Definition AS for 256-slice series, SOMATOM Definition Flash for 128-slice series and Sensation Cardiac 64 for 64-slice series .A 256-slice series (1.00 mm slices) from a patient with normal lung findings, a 128-slice series (3.00 mm slices) from a patient with an obstructing right mainstem bronchus tumour, and a 64-slice series (3.00 mm slices) from a patient with tracheal compression by a retrosternal goitre were segmented using OsiriX Lite  with a radiodensity-based threshold algorithm to isolate the radiolucent airways as a printable standard triangle language (.stl) mesh file. This initial model, representing the intraluminal space of the tracheobronchial tree, was then processed in Autodesk Meshmixer  where a surrounding airway wall of 2 mm could be fashioned, and the bronchi truncated at the third order of airways  was chosen as a 3D printer for its multi-material jet capable of fusing plastic and rubber . The accTo validate the printed models as airway trainers we recruited expert respiratory physicians to perform bronchoscopy and provide feedback on their experience. Consultant respiratory physicians with at least six years’ formalized training in respiratory medicine, following an accredited residency programme based on standards set by the American Council for Graduate Medical Education (ACGME) working in association with Tan Tock Seng Hospital and the Lee Kong Chian School of Medicine were identified, and recruited to the study by email invitation. Prior to the study, informed consent was obtained and documented from participants.Before attempting bronchoscopy on the 3D printed models, all participants first performed flexible bronchoscopy using a standard endoscopic trainer Karl Storz 8402 ZX  comprising a flexible bronchoscope coupled with a video monitor, with an open-mouth airway simulator model. This rubber-and-silicone airway trainer simulated both upper and lower airway anatomy, and incorporated a detachable tracheobronchial tree with an interface at the subglottic level  present in the print substrate. The first model of Shore hardness D50 Fig. a depictsFive consultant respiratory physicians  responded to the invitation. All of them accepted the terms of participation and provided responses to the survey questionnaire. From the subjective rating responses, the calculated Cronbach’s alpha of 0.83 indicated reliability of the rating component of the questionnaire.The 3D printed model was generally well received by the bronchoscopists, with positive opinions of the anatomical accuracy Fig. . All parIn this study, we developed 3D printed airways by material jetting technology and validated their efficacy as a bronchoscopic trainer. To our knowledge, this is the first multi-material print of the tracheobronchial tree. Other investigators have developed models with reasonable realism, but that require additional post-print processing, such as painting , or siliFurthermore, the capability of multi-material printing to admix two or more materials allows one to exploit desirable attributes of both materials; in this case, the strength of plastic and the flexibility of rubber in a custom plastic-rubber blend are superior to either in isolation as a choice for the luminal material. Single-material prints of flexible rubber are prone to damage, requiring support to prevent from bending during the bronchoscopy procedure , while sIt is important to note that where endoscopic simulators are concerned, the required physical characteristics differ somewhat from a surgical incision-site simulator. Being an intraluminal procedure with manoeuvres performed in “negative” space, more emphasis is placed on anatomical accuracy and user navigational experience than the consistency of the wall material. Our study questionnaire found that the anatomical accuracy of the 3D printed model was well-received by expert bronchoscopists. Our model, made at a cost of SGD~$135, offers an inexpensive modality for depicting to-scale real anatomy and a wide range of pathologies. Owing to the versatility of 3D printing, it is conceivable to design a series of anatomical and pathological models (patient specific) that may be affixed to a single commercial task trainer, sparing the cost of simulating a range of clinical scenarios. The prime advantage of 3D printed simulators is that of patient-specific modelling, which can conceptualize unique clinical situations. For instance, where variant anatomy or pathological distortion of the airways is encountered, 3D printed airway models accurately represent pathoanatomy , 33, 35,Only five expert bronchoscopists participated in our study. For comprehensive validation of the model, a larger pool of both expert and trainee bronchoscopists is required, ideally in a blinded study comparing standard bronchoscopic trainers to the 3D printed airway model.Three multi-material 3D models of the airway were developed and validated by expert bronchoscopists for simulation training of flexible bronchoscopy. One normal and two pathological patient-specific, life-sized airway models were made to represent normal anatomy, an anatomical lesion, and extrinsic tracheal compression. With minor refinements, 3D printed models shows potential for patient-specific bronchoscopic training, particularly in the setting of airway pathology.


Low-grade inflammation and insulin resistance are among the clinical features of obesity that are thought to promote the progressive onset of type 2 diabetes. However, the underlying mechanisms linking these disorders remain not fully understood. Recent reports pointed out hypothalamic inflammation as a major step in the onset of obesity-induced insulin resistance. In light of the increasing prevalence of obesity and T2D, two worldwide public health concerns, deciphering mechanisms implicated in hypothalamic inflammation constitutes a major challenge in the field of insulin-resistance/obesity. Several clinical and experimental studies have identified resistin as a key hormone linking insulin-resistance to obesity, notably through the activation of Toll Like Receptor (TLR) 4 signaling pathways. In this review, we present an overview of the molecular mechanisms underlying obesity-induced hypothalamic inflammation and insulin resistance with peculiar focus on the role of resistin/TLR4 signaling pathway. Obesity is a global epidemic triggering significant morbidity and mortality rate, through mainly interactions between genetic and environmental factors, notably sedentary lifestyle and unhealthy eating habits –3. ObesiIn rodents, it is largely documented that obesity alters secretory adipose tissue functions mainly adipokines and pro-inflammatory cytokines secretions , 17. AmoThe hypothalamus is the main brain area controlling feeding behavior and energy homeostasis implicating complex neuronal circuits that project toward several brain regions and brainstem , 29. HypAmong the peripheral signals, the anorexigenic effect of insulin in the hypothalamus is largely documented where insulin modulates food intake and glucose homeostasis , 11. InsIn rodents, HFD consumption is considered as an important nutritional factors predisposing to obesity-induced insulin resistance. HFD consumption alters hypothalamic insulin responsiveness deregulating energy homeostasis control –9. SeverEpidemiological studies in obese and insulin resistant subjects have revealed a causal link between increased pro-inflammatory markers and decreased insulin sensitivity , 62. In At the molecular levels, IKKβ/NF-kB inflammatory pathways have been described to be critical in the development and progression of hypothalamic insulin resistance. Indeed, inactivation of IKKβ/NF-kB signaling in neurons, astrocytes or microglia of the MBH protects against HFD-induced obesity, glucose intolerance and hypothalamic insulin resistance , 68–70. JNK signaling is another pathway proposed to be critical for the development of obesity associated hypothalamic insulin resistance. Obesity-dependent activation of JNK signaling was shown to impair insulin signaling . JNK proER stress and the unfolded protein response (UPR) also contribute to HFD-induced hypothalamic inflammation and insulin resistance –92. ER sInflammatory responses induced by HFD are also mediated by TLRs known to be involved in innate immunity , 97. At Even though the mechanisms underlying obesity-induced hypothalamic inflammation are still not fully characterized, it is now recognized that HFD promotes both central and peripheral inflammation through the increase of circulating levels of deleterious adipokines and proinflammatory cytokines originated from the adipose tissue –6, 17. AResistin directly binds to TLR4 in the hypothalamus, promoting the recruitment of the adaptors proteins MyD88 and TIRAP and the activation of downstream signaling pathways. The activation of Resistin/TLR4 signaling impairs hypothalamic insulin responsiveness through the alteration of the insulin-mediated insulin receptor (IR), AKT, and ERK1/2 phosphorylations. This could be attributed to the resistin-dependent downregulation of IR as well as the upregulation of the hypothalamic expression of SOCS-3 and PTP1B, known as key negative modulators of insulin signaling , 76. ResAnother suggested mechanism is that central resistin might induce hypothalamic insulin resistance through the impairment of adiponectin and FGF21 signaling known as insulin-sensitizing hormones –152. CenOther candidate for resistin downstream effects on hypothalamic insulin sensitivity could be the alteration of neuronal autophagy. In fact, the disruption of autophagy in the hypothalamic neurons is critically involved in diet-induced obesity and associated hypothalamic inflammation and insulin resistance , 154–158Obesity is associated with low grade inflammation which occurs in peripheral metabolic tissues, resulting in whole body insulin resistance, but also in the hypothalamus causing local impairment of insulin signaling and sensitivity. Our recent findings evidenced that central Resistin/TLR4 signaling pathway promotes the onset of hypothalamic inflammation and insulin resistance. Despite these evidences, further studies are needed to clarify the specific role of resistin produced within the hypothalamic neurons as compared to that produced in the periphery, and to identify the neural circuitries mediating resistin effects within hypothalamic nuclei. Indeed, it is also necessary to elucidate the role of resistin/TLR-4 signaling pathway in hypothalamic astrocytes and microglia cells, which are critically involved in the onset of hypothalamic inflammation. Further investigations are needed to clearly decipher regulatory mechanisms involved in hypothalamic resistin/TLR4 signaling in the context of obesity. This will constitute a major step to understand the molecular mechanisms underlying the onset of obesity-induced neuroinflammation, and will identify new potential therapeutic targets to overcome obesity-associated hypothalamic inflammation and related metabolic disorders.YB wrote the manuscript and produced the final version. MT contributed to the redaction and reviewed the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Objective: To investigate the feasibility of serial radiotracer-based imaging as a noninvasive approach for quantifying volumetric changes in microvascular perfusion within angiosomes of the foot following lower extremity revascularization in the setting of critical limb ischemia (CLI).Approach: A CLI patient with a nonhealing foot ulcer underwent single-photon emission computed tomography (SPECT)/computed tomography (CT) imaging of the feet before and after balloon angioplasty of the superficial femoral artery (SFA) and popliteal artery. SPECT/CT imaging was used to evaluate serial changes in angiosome perfusion, which was compared to quantitative changes in peripheral vascular anatomy and hemodynamics, as assessed by standard clinical tools that included digital subtraction angiography (DSA), ankle-brachial index (ABI), and toe-brachial index (TBI).Results: Following revascularization, upstream quantitative improvements in stenosis of the SFA (pre: 35.4% to post: 11.9%) and popliteal artery (pre: 59.1% to post: 21.7%) shown by DSA were associated with downstream angiosome-dependent improvements in SPECT microvascular foot perfusion that ranged from 2% to 16%. ABI measurement was not possible due to extensive arterial calcification, while TBI values decreased from 0.26 to 0.16 following revascularization.Innovation: This is the first study to demonstrate the feasibility of assessing noninvasive volumetric changes in angiosome foot perfusion in response to lower extremity revascularization in a patient with CLI by utilizing radiotracer-based imaging.Conclusion: SPECT/CT imaging allows for quantification of serial perfusion changes within angiosomes containing nonhealing ulcers and provides physiological assessment that is complementary to conventional anatomical (DSA) and hemodynamic (ABI/TBI) measures in the evaluation of lower extremity revascularization. One specific revascularization approach that has emerged in recent years is angiosome-guided revascularization, which is based on the concept that angiosomes, or three-dimensional (3D) blocks of tissue supplied by specific upstream source arteries, can be utilized to guide targeting of specific downstream vascular runoff territories of the foot that contain nonhealing wounds, thereby improving rates of wound healing and limb salvage.9 We hypothesized that this imaging technology and image processing approach could be further utilized to evaluate the response to revascularization in patients with CLI. Therefore, in the current study, we applied SPECT/CT imaging to assess the perfusion response to revascularization within specific 3D foot angiosomes in CLI patients with DM. In addition, our SPECT/CT perfusion results were compared to revascularization-induced changes in arterial stenosis, as assessed by conventional DSA, as well as serial changes in lower extremity hemodynamics, as evaluated by ABI and TBI.Our research team has recently developed a clinical imaging protocol that utilizes hybrid single-photon emission CT (SPECT)/CT imaging, CT-based image segmentation of angiosomes, and serial image registration, which allows for assessment of 3D angiosome foot perfusion under baseline conditions and has demonstrated potential for noninvasively detecting perfusion abnormalities in CLI patients with diabetes mellitus (DM).2) exist and provide some information related to microvascular perfusion of the foot, clinical techniques such as these are superficial in nature and do not allow for 3D assessment of angiosomes being targeted for revascularization. Therefore, in the present study, we present technological feasibility of utilizing SPECT/CT imaging to quantify serial physiological changes in 3D angiosome perfusion in a patient with DM and CLI who was scheduled to undergo lower extremity revascularization.CLI patients often undergo revascularization procedures that are directed at improving downstream foot perfusion to facilitate wound healing and limb salvage. However, standard clinical tools, such as ABI, TBI, and Duplex ultrasound, generally provide information related to hemodynamic responses to revascularization, while other conventional approaches such as magnetic resonance (MR) angiography, x-ray CT angiography, and DSA, are limited to morphological changes in the peripheral atherosclerotic lesions. While noninvasive tools such as transcutaneous oxygen pressure  with CLI, type 2 DM, and nonhealing foot ulcers of the first and second digits of the right foot underwent SPECT/CT perfusion imaging before and after clinically indicated revascularization of the lower extremity. The patient was enrolled into a recently initiated prospective clinical trial (NCT03622359) focused on evaluating the utility of radiotracer-based imaging for the assessment of angiosome perfusion responses to lower extremity revascularization. The study protocol was approved by both the Institutional Review Board and Radiation Safety Committee and was in accordance with the guidelines set forth by the Declaration of Helsinki. All patients were provided written informed consent after receiving an explanation of the experimental procedures and potential risks associated with participating in the study.99mTc-tetrofosmin) under resting conditions and underwent SPECT/CT imaging 20 min postinjection. Images were acquired at the level of the ankle and foot. The patient then returned 4 days following revascularization to undergo the same SPECT/CT imaging protocol. SPECT perfusion images were acquired using a 360° step and shoot acquisition with a 140.5 keV ±10% window, 3° projections, and 30 s per stop. Immediately after the SPECT acquisition, CT images were acquired for attenuation correction at a slice thickness of 0.625 mm, at 140 kVp, and 15 mA. All SPECT images were reconstructed using iterative reconstruction, applying corrections for attenuation and resolution loss .Perfusion imaging was performed using a conventional hybrid SPECT/16-slice CT imaging system with large field-of-view sodium iodide detectors  before and after balloon angioplasty of the right superficial femoral artery (SFA) and popliteal artery. The patient received a low-dose intravenous radiotracer injection  and patient body weight (kg) to generate standardized uptake values (SUVs). Following evaluation of SPECT SUVs for the pre-revascularization time point, serial CT images from the pre- to post-revascularization time points were coregistered . The minimal luminal diameter (MLD) and the mean reference diameter (RD), obtained from averaging a 5-mm segment proximal to the atherosclerotic lesion, were used for calculations of percent diameter stenosis [(1-MLD/RD) × 100]. Mean segment diameter represented the mean diameter of the treated arterial segment. The gain in mean segment diameter was defined as the change in mean segment diameter from baseline to the final postprocedural angiogram.Pressure waveforms as well as the ABI and TBI were evaluated at both the pre- and post-revascularization time points to assess hemodynamic changes that may occur in the lower extremity after treatment. Specifically, serial systolic blood pressures were acquired on the left and right extremities for the brachial artery, dorsalis pedis artery, posterior tibial artery, and the great toes using Doppler ultrasound.All angiographic results from pre- to post-revascularization are summarized in All angiosome perfusion results are summarized in The ABI was not assessable due to diffuse calcification of the lower extremities that resulted in noncompressible arteries. The absolute systolic pressure measured in the great toe of the right foot slightly increased from a baseline value of 27 mmHg to a post-revascularization value of 30 mmHg, while the baseline TBI value of 0.26 changed to 0.16 after revascularization .In the present report, we have demonstrated for the first time the utility of SPECT/CT imaging for quantitative assessment of serial changes in microvascular perfusion within specific 3D angiosomes of the foot following revascularization. In addition to quantifying heterogeneous perfusion responses in the foot, we demonstrated that these changes in perfusion were associated with angioplasty-induced quantitative improvements in upstream arterial dimensions, but not in agreement with traditional hemodynamic measures of the foot, thus supporting the notion that SPECT/CT perfusion imaging may offer complementary novel insight into physiological alterations that occur in specific vascular territories of the foot following revascularization.2 is the only widely utilized tool in the clinical setting for assessing foot perfusion; however, TcPO2 is superficial in nature, does not have a large field-of-view, and often does not allow for assessment of the plantar surface of the foot. In addition, while MR-based imaging approaches such as contrast-enhanced imaging,12 arterial spin labeling,14 and blood oxygen level dependent15 can quantify volumetric tissue oxygenation and perfusion of the lower extremities, these approaches are not capable of measuring perfusion under resting conditions and generally require pharmacological, reactive hyperemia, or exercise stress paradigms. Our radiotracer-based imaging approach with SPECT/CT imaging has previously demonstrated utility for quantifying regional volumetric perfusion under resting conditions in both the preclinical10 and clinical9 setting and offers a fast and straightforward imaging protocol for assessing limb perfusion in a patient population at high risk for lower extremity ulceration and amputation. In addition, SPECT/CT imaging does not require the use of intravenous iodinated contrast agents and instead utilizes 99mTc-tetrofosmin, a relatively safe agent that has no known contraindications, thus offering a safe technique for evaluating lower extremity perfusion in severe PAD and CLI patients who commonly present with impaired renal function.17 In the present case, we have demonstrated through the use of our image segmentation and registration approach, the further utility of SPECT/CT imaging for quantifying serial changes in perfusion within specific 3D angiosomes of the foot after revascularization in a CLI patient with nonhealing wounds.While a variety of noninvasive tools have been utilized to diagnose and evaluate PAD and CLI in the clinical setting, most have been limited to assessment of hemodynamics or vascular anatomy. TcPOQuantitative analysis of DSA images that were acquired before and after balloon angioplasty of the SFA and popliteal artery demonstrated treatment-induced improvements in upstream arterial diameter that were associated with subsequent downstream improvements in angiosome perfusion quantified by SPECT/CT imaging. While increased diameters of the upstream SFA and popliteal artery should theoretically result in diffuse improvements in foot perfusion that are angiosome independent, SPECT/CT imaging revealed heterogeneous improvements in foot perfusion that were angiosome dependent. Thus, the poor perfusion responses to revascularization that were quantified by SPECT/CT imaging for certain angiosomes may be due to additional downstream minor occlusions in arteries, microvasculature, or a combination of the two. For example, the present patient in this report demonstrated higher perfusion responses (12% and 16%) within the medial plantar and medial heel angiosomes, which may suggest that the supplying posterior tibial and medial plantar arteries may be absent of significant atherosclerosis. Alternatively, perfusion in the lateral plantar, lateral heel, and dorsal foot angiosomes increased by ∼2–3%, which may suggest that the supplying anterior tibial and peroneal arteries, as well as the microvasculature in these specific angiosomes, could possess varying degrees of atherosclerosis that are undetectable by conventional DSA. Therefore, in the present case, SPECT/CT perfusion imaging appears to offer complementary evaluation of regional physiological responses to revascularization that could be used in combination with conventional anatomical imaging.In addition to DSA, standard ABI and TBI measurements were attempted and acquired for comparison to SPECT/CT perfusion imaging results. However, due to diffuse arterial calcification in the present patient, ABI measurements were not possible, which is a common occurrence and problem with ABI measurements for many patients with DM. Also, while absolute measures of systolic blood pressure in the great toe of the revascularized foot did improve from 27 to 30 mmHg, TBI measurements actually decreased, further highlighting common inconsistencies and limitations of hemodynamic measurements in the setting of CLI and DM. Thus, while ABI and TBI are frequently used in the clinical evaluation of PAD patients, SPECT/CT perfusion imaging may offer additional functional assessment for angiosomes being specifically targeted for revascularization.In summary, this report demonstrates the potential utility of SPECT/CT imaging for quantifying revascularization-induced changes in microvascular perfusion within specific 3D angiosomes of the foot, which could complement standard clinical tools such as DSA, ABI, and TBI. Additional clinical studies are warranted to investigate the utility of SPECT/CT perfusion imaging as a noninvasive tool for evaluating the efficacy of the treatment in PAD and CLI patients.This is the first reported case to demonstrate the feasibility of assessing noninvasive volumetric changes in angiosome foot perfusion in response to lower extremity revascularization by utilizing radiotracer-based imaging. In addition, we show that our SPECT/CT perfusion imaging approach is complementary to conventional techniques for anatomical evaluation (DSA), and may offer further insight into the physiological changes in microvascular perfusion that occur within specific vascular territories of the foot that are being targeted for revascularization. Further application of SPECT/CT imaging in PAD patients may offer a novel noninvasive approach for evaluating perfusion response to revascularization and assist with guiding medical treatment.Key FindingsSPECT/CT imaging allows for serial assessment of the perfusion response to peripheral angioplasty within the 3D angiosome of the foot that contains a nonhealing ulcer and is being targeted for revascularization.SPECT/CT-derived changes in angiosome foot perfusion following revascularization are associated with upstream improvements in arterial diameter.SPECT/CT perfusion imaging provides 3D regional assessment of the perfusion responses to medical treatment, which could offer a more sensitive technique for evaluating local changes in tissue physiology than standard hemodynamic measures such as ABI and TBI.


Dystonia is a debilitating disease that causes abnormal, often repetitive, movements, postures or both. The pathophysiology is unknown but related to loss of neuronal inhibition, aberrant sensorimotor integration, and/or derangements of synaptic plasticity. Current treatments include pharmacotherapy, botulinum toxin injections and deep brain stimulation (DBS). The response to these treatments are often limited and carry the risk of side effects requiring alternative therapies such as non-invasive brain stimulation.We present a case report of a 65-year -old man with refractory focal ‘task-specific’ dystonia. The treatment plan included 10-daily sessions of 1 Hz, 2600 pulses of repetitive transcranial magnetic stimulation (rTMS) targeting the primary motor cortex.There were no clinical benefits noticed. Currently, there are no rTMS protocol treatments for dystonia. Publication of negative results will help in refining the optimal stimulation parameters, thus maximizing the effectiveness and reproducibility of future therapeutic protocols.The online version of this article (10.1186/s40734-019-0076-z) contains supplementary material, which is available to authorized users. Dystonic movements are typically patterned and twisting, and may be tremulous. It is often initiated or worsened by voluntary action and associated with overflow muscle activation [Dystonia refers to a heterogeneous group of disorders defined as tivation .The impairment is significantly variable and can range from a mild functional disturbance to complete and severe incapacitating motor dysfunction, resulting in a functional and socioeconomic burden.The pathophysiology of dystonia is still not clear, but at least three, non-mutually exclusive mechanisms have been proposed: loss of neuronal inhibition, aberrant sensorimotor integration, and/or derangements of synaptic plasticity . A numbeCurrently, the treatments include pharmacotherapy, Botulinum NeuroToxin (BoNT) injections and deep brain stimulation (DBS). The response to these treatments are often limited and carry the risk of important side effects. For the above reasons, alternative and non-invasive therapeutic strategies are needed.Transcranial magnetic stimulation (TMS) is a non-invasive brain stimulation (NIBS) technique that can be utilized to probe cortical excitability and perform in-vivo motor brain mapping. TMS pulses can be applied in trains (rTMS) at various frequencies. When used at low frequency, rTMS can generate inhibitory effects, while high frequency rTMS can produce excitatory responses. These effects are often described as LTD- or LTP-like, because the duration of these alterations may implicate changes in synaptic plasticity . rTMS caHere, we report a case involving a patient affected by left lower extremity dystonia refractory to BoNT, who was treated with 10 daily sessions of low frequency rTMS at our neurostimulation laboratory to alleviate his symptoms. A 65 year-old male with a history of left leg dystonia for the past three years presented to our clinic for rTMS therapy consultation. The patient is DYT1 mutation negative. The focal dystonia was ‘task specific’ and disabling. While walking forward, posturing was evident in the left lower limb with inversion and plantar flexion of the ankle with hyperextension of the knee Baseline evaluation of focal leg dystonia before 10 rTMS sessions 2 Stimulator  with the handle along the sagittal line. A total of 2600 pulses were given as continuous trains for a total of 44 min at an intensity of 90% of his left leg active motor threshold  rTMS targeting the primary motor cortex (left leg motor area of the homunculus). rTMS was delivered using a 70-mm figure-of-8 coil connected to the MAGSTIM RapidThe patient provided written consent for the TMS procedures as well as for the videotaping of the focal dystonia phenomenology. The patient completed self-assessments of Patient Health Questionnaire (PHQ-9), Clinical Global Impression (CGI) and videotaping of unassisted walking at baseline, prior to rTMS, Follow up evaluation of focal leg dystonia after 10 rTMS sessions The patient tolerated the treatment well and without adverse events. However, after the ten sessions were completed the patient presented with no clear improvement of FTSD, with no change on the PHQ-9 and CGI. Furthermore, there was no apparent change in walking as determined by comparison to the video recordings. A follow-up phone call that took place three weeks post-procedures indicated that there has been no further subjective changes in the patient’s symptoms.Here we report a case of refractory FTSD of the left lower limb showing no clinical benefit from 10 sessions of LF-rTMS over motor cortex. Prior studies utilizing rTMS for the treatment of dystonia have shown modest clinical benefits of rTMS for focal dystonia affecting upper extremities and neck , 8. We cThe current model according to which low frequency TMS and high frequency TMS are respectively associated to “inhibitory” and “excitatory” effects of the stimulated area is, admittedly, an oversimplification. Neurophysiology of motor cortex was not assessed in this patient to allow a better characterization of the patient’s cortical physiology.To summarize, here we report a case involving a patient affected by left lower extremity dystonia refractory to BoNT, who was treated with 10 daily sessions of low frequency rTMS with no clinical improvement. This is the first report of rTMS for the treatment of idiopathic adult-onset FTSD of the lower extremities. As optimal protocols for FTSD have not yet been developed. We trust that our contribution will expand the currently limited knowledge regarding the use of rTMS for refractory dystonia. Improving the current publication bias towards negative results will help in refining the optimal stimulation parameters, thus maximizing the effectiveness and reproducibility of future therapeutic protocols. Dystonia pathophysiological models may vary across dystonia subtypes and further studies must take this into account. Individual variability in TMS responses can further result in difficulties validating this method as a therapeutic tool.


GBA may be more common in cohorts treated with deep brain stimulation. Studies assessing deep brain stimulation outcomes in patients carrying mutations in specific genes are limited in size. There are reports suggesting that the phenotype associated with parkin mutations could be suitable for early surgery. In patients with LRRK2 mutations, outcomes of deep brain stimulation seem at least as good as in mutation-negative patients, whereas less favorable outcomes are seen in patients carrying mutations in GBA. Careful assessment of clinical symptoms remains the primary basis for clinical decisions associated with deep brain stimulation surgery in Parkinson’s disease, although genetic information could arguably be taken into account in special cases. Current evidence is scarce, but highlights a promising development where genetic profiling may be increasingly relevant for clinicians tailoring personalized medical or surgical therapy to Parkinson’s disease patients.Deep brain stimulation is offered as symptomatic treatment in advanced Parkinson’s disease, depending on a clinical assessment of the individual patient’s risk-benefit profile. Genetics contribute to phenotypic variability in Parkinson’s disease, suggesting that genetic testing could have clinical relevance for personalized therapy. Aiming to review current evidence linking genetic variation to deep brain stimulation treatment and outcomes in Parkinson’s disease we performed systematic searches in the Embase and PubMed databases to identify relevant publications and summarized the findings. We identified 39 publications of interest. Genetic screening studies indicate that monogenic forms of Parkinson’s disease and high-risk variants of  However, as DBS is ineffective against a number of PD symptoms and carries potential risks and side effects, careful individualized patient screening and target selection are essential for good surgical outcomes.Parkinson’s disease (PD) is a neurodegenerative disorder clinically characterized by bradykinesia, tremor, rigidity, and postural instability, as well as a range of non-motor symptoms including cognitive decline and dementia.2 The decision to offer surgery should be based on an individual assessment of the risk-benefit profile. The ideal candidates have age below 70–75 and a good levodopa response.3 Conversely, DBS is not suitable for patients with predominantly axial symptoms, cognitive impairment or active depression. Current evidence favors STN as the most effective target, although GPi might be considered in patients with pronounced dyskinesias or mild cognitive impairment. DBS targeting the ventral intermediate nucleus of the thalamus (VIM) is an option in elderly PD patients with medication-refractory tremor as the predominant symptom.4DBS targeting the subthalamic nucleus (STN) and globus pallidus pars interna (GPi) are both effective in reducing motor fluctuations in PD.5 For the common, sporadic form of PD, more than 40 genetic risk-loci have been identified through genome-wide association studies (GWAS).6 Rare and low-frequency missense variants in GBA are strong risk factors for PD, representing an intermediate between Mendelian genes and typical GWAS loci in terms of frequency and effect size.7 Evidence indicates that genetic variants causing or conferring susceptibility to PD also show correlations with clinical phenotype, contributing to the striking clinical variability observed across individual PD patients.11Over the last two decades a number of genes causing Mendelian forms of PD have been identified. It has been estimated that Mendelian PD overall accounts for 5–10% of cases, with numbers varying significantly across populations.12Given that genetic background partly determines PD phenotype it could plausibly be hypothesized that genetic profiling could be used to predict DBS outcome and help clinicians select the right patients for surgery. Ideally, genetics could provide a rationale for implantation at an earlier stage in subgroups of patients particularly well suited for DBS, and warrant caution in others, where risks and side effects are likely to outweigh the clinical benefit. Such a development would be in line with the principles of personalized medicine or precision medicine, where individualized treatment attuned to the patient’s genetic profile has been proposed as a key element.In recent years, an increasing number of publications have reported details of DBS treatment and outcomes in genetically characterized PD patients. We present a systematic review of this literature, summarize key insights and discuss the rationale for genetic screening as a clinical tool for patient selection to DBS in PD.The 39 articles identified reported studies employing principally two kinds of study design. A few studies performed genetic screening in a group of PD patients treated with DBS comparing the frequency of specific genetic variants against a group not treated with DBS. The majority of studies evaluated the efficacy of DBS in patients of a known genotype, either descriptively or compared quantitatively to a mutation-negative group. Some articles describe a combination of these study designs. Reports of few or single cases constituted about half of the included publications.20 Only one mutation-positive patient (GBA) was treated with VIM stimulation.20 A number of different outcome measures are used to assess DBS treatment response and safety; the most commonly used being the Unified Parkinson’s Disease Rating Scale (UPDRS) and levodopa equivalent daily dose (LEDD) reduction.The majority of the publications included patients who underwent DBS surgery with implantation in STN. However, a few reported patients with severe troublesome dyskinesia had GPi implantation.Receiving a DBS implant represents a milestone in the clinical course of PD, which could also be seen as a marker of a particular endophenotype. The predisposition to developing this endophenotype is likely shaped by genetics, implying that relevant genetic variants should be enriched in DBS-treated cohorts. We found three studies comparing the fraction of mutation carriers in a large group of PD patients to a non-DBS group.LRRK2 and GBA mutations in young onset PD patients, including 99 receiving DBS treatment and 684 without DBS.21 When analyzed together, mutation carriers were significantly more common in the DBS group compared to the non-DBS group . This enrichment did not reach statistical significance for any individual gene, although a slightly higher rate of carriers was observed for all three genes investigated.Pal et al. screened for parkin, LRRK2 or GBA mutations in 29%.20 No mutations were found in SNCA, PINK1 or DJ-1. No non-DBS control group was included in the study, but comparing with published reports the authors state that the carrier frequency of 29% was much greater than in population-representative cohorts of PD. Interestingly, both these studies highlighted that parkin mutation carriers had earlier disease onset, yet longer disease duration at the time of DBS. In contrast, GBA carriers, who are known to progress faster, had DBS earlier in the disease course.Performing genetic screening in a cohort of 94 DBS-treated PD patients, Angeli et al. identified parkin, 19 All patients were screened for LRRK2 and SNCA mutations, whereas investigation of parkin, PINK1, GBA and other genes was performed depending on age at onset and family history. PD patients carrying mutations in LRRK2 or parkin were significantly overrepresented in the group who underwent STN-stimulation. Interestingly, this study also included 21 patients receiving VIM stimulation for levodopa-refractory tremor. No mutation was identified in any of these patients.A study by Johansen et al. reported on genetic screening in 60 DBS-treated and 570 non-DBS PD patients consecutively enrolled in a movement disorder centre.20 Findings relating to specific genes are summarized in the following sections.The two latter studies also compared the postoperative clinical outcomes of mutation-positive patients to that of non-carriers, concluding that no significant differences can be detected when mutations in all investigated genes are lumped together.LRRK2, encoding leucine-rich repeat kinase-223, of which the G2019S mutation is the most common.24 The normal function of the lrrk2 protein is incompletely understood, but current evidence indicates a role in pathways relating to vesicular transport and lysosomal degradation. The phenotype is similar to idiopathic PD with evidence indicating a slightly more benign course of disease with good response to levodopa, relatively slow decline in motor and cognitive functions and a low burden of non-motor symptoms.27 We found 11 studies assessing the efficacy of DBS in LRRK2 mutation carriers. Five of these had sufficient sample size to perform group-wise comparisons of the motor improvement in LRRK2 PD versus idiopathic PD, while the rest were descriptive studies of smaller cohorts or case reports.The most frequent form of monogenic PD is caused by autosomal dominant mutations in LRRK2 carriers and idiopathic PD. A study in Ashkenazi Jewish patients followed 13 G2019S positive PD patients and 26 matched non-carriers for three years postoperatively and assessed the impact of the mutation on treatment outcome in a linear mixed model.28 No significant difference was observed in UPDRS off medication on stimulation or LEDD reduction across the two groups. Similarly, relative improvement of UPDRS II-IV and LEDD reduction were not significantly different after 6–12 months in a French study comparing nine LRRK2-PD patients, mainly G2019S carriers, to 60 idiopathic PD patients using a t-test.29 The UK screening study mentioned above also reported that differences in outcome were statistically non-significant, yet this study included only five LRRK2 patients.20Two studies with similarly sized mutation carrier groups have used statistical tests to compare measures of DBS efficacy, both reporting no significant differences between 30 UPDRS III improvement the medication off-state was reported at 51.1% in mutation-positive patients versus 25.5% in non-carriers, and similar differences were seen for Hoehn & Yahr and Schwab & England scales, yet without formal statistical testing across groups. Conversely, a study including four LRRK2 R1441G PD patients in the Basque Country of Spain stated that these had a limited DBS response on motor function, daily life activities and quality of life, an inferior outcome compared to 41 LRRK2 mutation-negative DBS-treated control patients, yet not supported by statistical hypothesis testing.31A claim that LRRK2 G2019S patients have greater improvement following surgery for STN-DBS than idiopathic patients was made in an Algerian comparative study of 15 mutation carriers and 12 non-carriers with two years’ follow-up.LRRK2 mutations  and seven had one identified mutation.43 In particular, there are reports of DBS efficacy after extremely long disease durations (up to 45 years)45, and a sustained response many years postoperatively.13 A few reported parkin patients had electrodes implanted in GPi mainly to control dyskinesias, one even as a second target after several years of STN-DBS.14 A publication reporting long-term follow-up in Arabic parkin kindreds emphasize that only modest improvement was seen after DBS in three patients where axial symptoms were prominent.46We identified seven further reports of one or a few parkin PD patients receiving DBS treatment, most of which highlight a good outcome.47 and genomic multiplications48 involving SNCA are rare causes of autosomal dominant PD. Triplication is associated with a severe form of PD with early-onset, rapid progression and dementia whereas patients with duplications may resemble idiopathic PD.49 We identified two case reports, each describing a patient with SNCA duplication treated with STN-DBS. Both reported a good response with substantial improvement of UPDRS-III scores and reduction of LEDD postoperatively, and only slight to moderate decline in cognitive function after one and 3 years, respectively, in line with overall STN-DBS results in PD.51 A third case report describe a patient with a mosaicism of SNCA duplication show an overall good outcome of DBS, where GPi was chosen as target due to prominent dyskinesias and mild cognitive impairment.15Oligomerization and aggregation of alpha-synuclein plays a pivotal role in PD pathogenesis, and the protein forms the main component of the neuropathological hallmark, Lewy bodies. Point mutationsVPS35 cause autosomal PD with a phenotype that is clinically indistinguishable from idiopathic PD.53 We identified a total of five articles, all describing PD-patients with the D620N VPS35 mutation treated with DBS. As part of screening efforts following the identification of the gene, two mutation carriers treated with DBS were reported, one with a good motor outcome54, the other implanted at high age with a small benefit, yet complicated by dysarthria.55 Two publications have followed-up kindreds included in one of the studies that originally linked the gene to PD, reporting DBS treatment in one US56 and two Swiss patients57 with a good response, sustained for up to eight years postoperatively. Similarly, an excellent long-term motor response to DBS was described in a Taiwanese patient carrying the VPS35 D620N mutation.58Mutations in PINK1 encodes PTEN-induced putative kinase 1, which has a role in mitochondrial quality control forming protein-protein interactions with parkin. Similar to parkin, PINK1 mutations cause autosomal recessive PD that is clinically characterized by early onset, slow progression and a good response to dopaminergic treatment. One patient with homozygous PINK1 mutation was reported together with parkin mutations in a German-Canadian study mentioned above.41 Apart from this publication, we identified only one article showing the DBS outcome of a homozygous PINK1 mutation carrier, highlighting successful treatment with GPi-DBS in a patient with prominent dystonia and dyskinesias.1659 Different GBA mutations show a spectrum of severity with respect to impact on enzyme activity and effect on PD susceptibility, ranging from around tenfold increased risk for carriers of L444P60 to less than twofold in the low-frequency variant E326K,62 which does not cause Gaucher’s disease in the homozygous state. It is now well established that GBA mutations are associated with a more severe PD phenotype characterized by early onset, rapid motor progression, more prominent cognitive decline and a high burden of other non-motor symptoms, with worse outcomes for the most severe mutations.65 We found five articles reporting on the outcome of DBS treatment in patients with GBA mutations.This gene encodes the lysosomal enzyme alpha-glucocerebrosidase. Homozygous mutations in this gene cause Gaucher’s disease, an autosomal recessive lysosomal storage disorder where Parkinsonism occurs in a subset of patients. In the heterozygous state, however, the same pathogenic variants are strong risk factors for PD, yet without sufficient penetrance to cause a Mendelian inheritance pattern.17 Motor symptoms, LEDD and stimulation settings did not differ significantly between groups. On long-term follow-up, cognitive decline was more prevalent and more severe in GBA mutation carriers, and outcomes were worse for non-motor symptoms and quality of life. The reported results were likely underestimates, as five GBA positive patients were lost to follow-up due to severe disability or death. Similar results were also highlighted in an earlier publication from the same group, where cognitive and axial impairment were significantly more pronounced in the group of 15 GBA mutation carriers on longitudinal follow-up after GPi or STN-DBS.20 A German study identified four DBS-treated PD patients carrying a GBA mutation through screening, matched each GBA case to two non-carriers and compared clinical data from 4–10 years of follow-up.66 Both mutation carriers and non-carriers reduced their LEDD and had well controlled motor fluctuations and dyskinesias postoperatively. However, on long-term follow-up therapy-resistant axial symptoms and cognitive decline were markedly more pronounced in the GBA group. A large-scale French GBA screening study reported two GBA carriers treated successfully with DBS, one of these being homozygote for the N370S mutation.67 A retrospective study from a Gaucher’s disease clinic in Israel identified two Gaucher’s disease patients also diagnosed with PD who were treated with DBS with a dramatic and sustained symptomatic improvement.68A study from the UK matched 17 patients with GBA mutations to 17 non-carriers and assessed DBS outcome with a mean postoperative follow-up of 7.5 years.18We found one article describing the 22q11.2 microdeletion, known to increase the risk of PD, in which three of the patients who underwent DBS had a satisfactory improvement of UPDRS-III score with 30–70%.69 Individually, each of these common variants has a small effect on disease susceptibility, and in general, large samples are required for adequate statistical power in genetic association studies of common variants. There is currently increasing evidence showing that common variant risk profile plays a role in shaping PD phenotype, although sample size remains a challenge for association studies of clinical outcomes.70Large-scale meta-analyses of GWAS in PD have identified an increasing number of common genetic risk variants.71 Eighty-five patients who underwent STN-DBS were genotyped for single-nucleotide polymorphisms in the SNCA (rs356220) and LRRK2 (rs1491923) loci. Treatment response was measured as difference in UPDRS III in the stimulation on/medication off-state two years postoperatively versus medication off before surgery. The risk-allele of the SNCA variant rs356220 was weakly associated with UPDRS III improvement at p = 0.03, but not with baseline scores, indicating that the variant could be a prognostic marker for DBS outcome. No association was seen for the LRRK2 variant.We identified one study assessing the association between DBS outcome and common low-risk variants from GWAS.Surgical therapy with DBS can alleviate symptoms and improve quality of life in a subset of patients with advanced PD. Where current patient and target selection relies on assessing the individual risk-benefit profile through careful clinical screening, future DBS considerations could potentially incorporate genetic information, in line with the principle of personalized medicine. To summarize the current evidence on genetic variability and DBS treatment and outcomes in PD, we performed a systematic review of the literature, identifying 39 publications of interest. Overall, the numbers of included patients are small and the methodology is heterogeneous across studies, providing scarce evidence for any firm conclusions.LRRK2 and parkin) and carriers of the strong GBA risk alleles may be overrepresented in the DBS group. This is in line with the observation that these genes predispose to particular phenotypic profiles that to some extent overlap with clinical criteria for DBS eligibility. Interestingly, no enrichment of genetic forms of PD was reported in patients receiving VIM-DBS for treatment-refractory tremor, indicating that genetics may be less clinically relevant for this group, compared to DBS candidates with motor fluctuations.19Studies based on genetic screening of patient cohorts comparing DBS-treated PD to patients not receiving DBS indicate that both certain forms of monogenic PD (prognosis. DBS prognosis, in turn, depends largely on whether fluctuations of dopa-responsive motor symptoms remain predominant, or are outweighed by non-motor and axial symptoms. For the three most studied genes, parkin, LRRK2 and GBA, the current literature gives some indication about DBS outcomes on a group level.When DBS electrodes are implanted and the stimulation parameters established, we expect a therapeutic response on a subset of motor symptoms. Overall, the evidence gives no reason to believe that this immediate postoperative effect should be any different in patients with a specific genotype. Consequently, the potential clinical relevance of genetic information would relate to its impact on The PD phenotype caused by parkin mutations corresponds well with the prototypic DBS patient, with early onset of a largely pure motor syndrome and pronounced motor fluctuations, yet minimal cognitive decline, even after many years of disease. Several reports document good response to DBS despite long disease duration both before and after surgery in parkin patients. It could be argued that a status with biallelic parkin mutations would weigh somewhat in favor of surgery, probably sooner rather than later, in a case where DBS treatment is considered.LRRK2 mutations generally have a phenotype similar to idiopathic PD, possibly with a slightly more benign disease course. In line with this, the largest studies of DBS in LRRK2 patients show no significant differences in outcome compared to non-carriers.29 Some conflicting evidence is presented in small studies and case reports, but taken together we see no current rationale for LRRK2 status to impact clinical considerations concerning DBS treatment in PD.Carriers of GBA mutations are associated with a severe phenotype and fast progression of both motor and non-motor symptoms. Several studies show that these patients tend to require DBS early in the disease course because of rapid motor progression.21 Furthermore, current evidence suggests that GBA carriers show faster cognitive decline and development of axial symptoms following surgery.66 The authors of the largest study on GBA and DBS conclude that GBA status could be an important consideration when weighing the benefits and risks associated with surgery.17 It should be emphasized, that even though mutation carriers may have less clinical benefit than non-carriers, this does not imply that there is no relevant benefit. A critical question concerns cognitive decline, where some changes are normally seen following DBS, yet no general reduction in overall cognition.72 Whether GBA carriers are particularly vulnerable to cognitive side effects of DBS, or if worse postoperative outcomes merely represent disease progression in itself, remains to be determined. Some authors argue that GBA mutation carrier status could weigh in favor of targeting GPi, which is known to have less impact on cognition than STN-DBS.17Heterozygous It is worth noting that a positive association between genotype and DBS outcome, as reported in a number of publications reviewed here, does not necessarily imply that genetic testing is clinically helpful. To definitely prove its independent utility, studies would have to show that genetic testing gives an added prognostic value, over and above what can already be obtained through careful clinical assessment when the patient is evaluated for DBS. Large prospective cohorts combining genetic profiling with deep phenotyping may provide suitable data to address this question.71 Polygenic scores capturing the cumulative effect of multiple common variants could ultimately make the genetic background clinically relevant also for the many PD patients falling into the “mutation-negative” category in the majority of studies reviewed here.Careful assessment of clinical symptoms remains the dominant basis for clinical decisions associated with DBS surgery in PD. We believe however, that weighing in genetic information could probably be justified in very special cases. In the future, we anticipate that our ability to predict PD subtype based on genetic profiling will increase substantially, and that genetic information will be one important resource among many for clinicians tailoring individualized medical or surgical therapy to PD patients. Although this review has emphasized coding variants in single genes, one small article points towards the possibility of genetic profiling based also on common risk variants.We defined publications of interest as articles reporting patient series or cases characterized by both genetic status and DBS outcome, meaning either DBS treatment versus no DBS as a clinical outcome in itself, or any measure of DBS efficacy or safety. The strategy of the systematic search is shown in Fig.


Adult opioid use and neonatal abstinence syndrome (NAS) are growing public health problems in the United States (U.S.). Our objective was to determine how opioid use disorder treatment access impacts the relationship between adult opioid use and NAS.We conducted a cross-sectional state-level ecologic study using 36 states with available Healthcare Cost and Utilization Project State Inpatient Databases in 2014. Opioid use disorder treatment access was determined by the: 1) proportion of people needing but not receiving substance use treatment, 2) density of buprenorphine-waivered physicians, and 3) proportion of individuals in outpatient treatment programs (OTPs). The incidence of NAS was defined as ICD-9 code 779.5  from any discharge diagnosis field per 1000 live births in that state.Unmet need for substance use disorder treatment correlated with NAS . The correlation between adult illicit drug use/dependence and NAS was higher in states with a lower density of buprenorphine-waivered physicians and individuals in OTPs.Measures of opioid use disorder treatment access dampened the correlation between illicit drug use/dependence and NAS. Future studies using community- or individual-level data may be better poised to answer the question of whether or not opioid use disorder treatment access improves NAS relative to adult opioid use. Neonatal abstinence syndrome (NAS) is a drug withdrawal syndrome experienced by newborns whose mothers have taken opioids or other substances during pregnancy. Opioids that can cause NAS include illicit substances  as well as legally prescribed medications . It is estimated that 60–80% of infants exposed to opioids develop NAS . Clinicatreating opioid use disorder in pregnant women (e.g. methadone and buprenorphine) can also result in NAS, [Concurrent with the surge in adult opioid use across the United States , the inc in NAS, , 8 the rIn addition to opioid replacement therapy, opioid use disorder treatment programs may also include prenatal care, case management, behavioral and mental health interventions and social service support. We hypothesized that there may be non-pharmacologic ways in which opioid use disorder treatment programs might lower rates of NAS such as improved mother-infant bonding, increased breastfeeding, and decreased use of non-opioid drugs . This stWe conducted a cross-sectional state-level ecologic analysis of pediatric discharge data from the Healthcare Cost and Utilization Project’s State Inpatient Databases (HCUP SID) and state-level variables from other publicly-available databases.The percentage of patients needing but not receiving treatment was obtained from open publicly available databases including the National Survey on Drug Use and Health (NSDUH), which provides state-level data on illicit drug dependence or abuse in the past year and individuals who needed but did not receive treatment for substance use . The extWe identified cases of NAS by abstracting ICD-9 code 779.5   from anyn = 36) prevented an analysis of opioid use disorder treatment access measures as classical effect modifiers (i.e. interaction terms). Instead, states were stratified as having low or high density of buprenorphine-waivered physicians and individuals in OTPs, based on whether they were above or below the national median. The MEANS, CORR and GLIMMIX procedures in the SAS Statistical Software platform  were used for all summaries and analyses. NAS frequencies were mapped using EsriPress ArcGIS 10.2.Associations between continuous measurements and NAS were measured using Pearson linear correlation coefficients, while NAS was compared between levels of binary classifications using an equal variance two-sample t-test and was compared between levels of polytomous classifications using the Kruskal-Wallis test . For any prevalence measures that had significant associations with NAS, analysis of covariance without interaction was used to reassess that association for significance (and sign: positive or negative) in the presence of access and demographic measurements. Due to small sample size, each access/demographic measurement was included separately, and no interaction effects were modeled. The study’s relatively small sample size (Although the median density of buprenorphine-waivered physicians was 8 IQR: 5–14) per 100,000 population . The correlation between NAS and illicit drug use/dependence was stronger in states with a As expected, we found that higher state averages for adult opioid prescribing and illicit drug use correlated with higher rates of NAS. Adult opioid use and NAS have both dramatically increased across the U.S. over the past 10 years , 5. The Between states, there was wide variation in access to opioid use disorder treatment. The states with the highest levels of access tended to be in the Northeast and the Pacific Northwest. New Mexico also had high levels of opioid use disorder treatment access Fig. . The AppUnmet need for substance use disorder treatment was positively and significantly correlated with NAS. This finding is in concordance with a county-level study that found an association between shortage of mental health providers and NAS . Howevergreater density of buprenorphine-waivered physicians. One explanation for this phenomenon is that buprenorphine itself can result in NAS. There may also be a diversion of prescribed buprenorphine to unintended recipients. In addition, some states have graduate medical education that includes buprenorphine-waivers for residents, but some of these physicians may not actually use the waivers for opioid use disorder treatment.In contrast to illicit drug use/dependence, the correlation between NAS and opioid prescribing was actually stronger in states with a There are several potential mechanisms for how access to opioid use disorder treatment might reduce the likelihood of NAS. First, long-acting opioids used in opioid use disorder treatment may pose a lower risk for NAS than do short-acting drugs such as heroin or oxycodone  due to sOur study has several limitations. The first limitation is missing data: we lacked NAS data for 14 (28%) of the 50 states. Lacking a national database for NAS, we relied on data from individual states, some of which do not participate in centralized HCUP distribution. We do not know if exclusion of these 14 states biased our results and what direction that bias may have taken. Second, the study relied on ICD-9 codes for diagnoses of NAS. ICD-9 codes have been used widely by groups such as the Centers for Disease Control and Prevention to characterize NAS trends. Nevertheless, ICD-9 codes have several limitations. ICD-9 codes can be generated by non-medically trained personnel and may not always accurately reflect patients’ medical conditions. Some studies have found that ICD-9 codes tend to underestimate cases of NAS , 32. We Our small sample size and inability to control for other state-level factors did not allow us to identify other characteristics that could have moderated the effect of adult opioid use on NAS. We did not have data on how many pregnant women had opioid use disorder in various states. The proportion of buprenorphine-waivered physicians and individuals in OTPs are indirect measures of treatment access. Certain clinics may have improved access for pregnant women relative to men and non-pregnant women. Other factors that could conceivably influence the relationship between adult opioid use and NAS include 1) the fertility rate amongst opioid-addicted mothers, 2) the proportion of pregnant women using non-opioid drugs such as tobacco, anti-depressants or benzodiazepines  3) the comprehensiveness or content of opioid use disorder-treatment programs in each state, 4) the promotion of abstinence vs. opioid replacement within opioid use disorder treatment programs, 5) the ratio of public to private treatment programs 6) the willingness of licensed providers to treat pregnant women and 7) variability in the legal measures taken against substance-abusing mothers . Some opIn addition, our findings—which rely on state-level statistics—are vulnerable to the “ecologic fallacy,” in which inferences drawn from a population do not apply to individual members. For example, just because a state has a certain proportion of physicians who can prescribe buprenorphine, does not mean that these physicians are equally distributed throughout the population or accessible to those in need. Furthermore, there is a wide variability in how many patients each buprenorphine-waivered physician treats . Future In summary, unmet need for substance use disorder treatment was positively correlated with NAS. The correlation between adult illicit drug use/dependence and NAS was dampened in states with higher levels of opioid use disorder treatment access. Future studies utilizing community or individual level data may be better able to address whether adding opioid use disorder treatment programs or buprenorphine-waivered physicians can reduce incidence of NAS. Since NAS results in substantial infant morbidity and healthcare costs, the impact of NAS should be taken into account when considering public health measures that would improve access to opioid use disorder treatment.