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Medical phantoms mimic aspects of procedures like computed tomography (CT), ultrasound (US) imaging, and surgical practices. However, the materials for current commercial phantoms are expensive and the fabrication with these is complex and lacks versatility. Therefore, existing material solutions are not suitable for creating patient-specific phantoms. We present a novel and cost-effective material system (utilizing ubiquitous sodium alginate hydrogel and coconut fat) with independently and accurately tailorable CT, US, and mechanical properties. By varying the concentration of alginate, cross-linker, and coconut fat, the radiological parameters and the elastic modulus were adjusted independently in a wide range. The independence was demonstrated by creating phantoms with features hidden in US, while visible in CT imaging and vice versa. This system is particularly beneficial in resource-scarce areas since the materials are cheap (<$ 1 USD/kg) and easy to obtain, offering realistic and versatile phantoms to practice surgeries and ultimately enhance patient care., Competing Interests: The authors declare the following competing financial interest(s): The authors HQ, JB, RA, and LS have filed a Germany patent under the number DE 10 2022 133 558.5., (© 2024 The Authors. Published by American Chemical Society.)

The human prostate-specific membrane antigen (PSMA) is substantially up-regulated in metastatic prostate cancer (PCa) cells. PSMA can be targeted by 177 Lu conjugated to PSMA-617, a high-affinity ligand for the PSMA. The binding of the radioligand, 177 Lu-PSMA-617, results in its internalisation and delivery of β-radiation into the cancer cells. However, PSMA-617, a component of the final product in the synthesis of the radioligand, may also play a role in the pathophysiology of PCa cells. The present study aimed to clarify the effects of PSMA-617 (10, 50 and 100 nM) on the expression of PSMA in PSMA-positive LNCaP cells, their proliferation, 177 Lu-PSMA-617-induced cell death by WST-1 and lactate dehydrogenase assays, immunohistochemistry, western blotting, immunofluorescence staining and uptake of 177 Lu-PSMA-617. PSMA-617 at 100 nM concentration induced cell-growth arrest, down-regulated cyclin D1 and cyclin E1 (by 43 and 36%, respectively) and up-regulated the cyclin-dependent kinase inhibitor p21 Waf1/Cip1 (by 48%). Immunofluorescence staining demonstrated reduced content of DNA, pointing to a lower rate of cell division. PSMA-617 (up to 100 nM) did not alter the uptake of 177 Lu-PSMA-617 into the LNCaP cells. Interestingly, simultaneous treatment with 177 Lu-PSMA-617 and PSMA-617 for 24 and 48 h substantially potentiated the cell-death promoting effects of the radioligand. In conclusion, the combination of impeding tumour cell proliferation by PSMA-617 and its potentiation of the radiation-induced cell death brought about by 177 Lu-PSMA-617 in PCa cells may considerably improve the outcome of the radiation therapy with 177 Lu-PSMA-617, especially in patients with decreased radiosensitivity of PCa cells to the radioligand., (© 2023. The Author(s).)

Introduction: Performing lymphoscintigraphy in a separate room, frees up the conventional gamma camera, coupled with the desire to directly localize sentinel lymph nodes (SLN) in the operating theatre has led to the development of high-resolution semiconductor-detector based handheld gamma-cameras, CrystalCam., Methods: This work consists of phantom and clinical studies. For the first part, a Jaszczak phantom with hollow spheres of various volumes were filled with the 99m Tc and the camera's sensitivity was measured at various distances to assess the possibilities and limitations of the device. The clinical study evaluates the effectiveness of CrystalCam in localizing SLN in 40 consecutive malignant melanoma patients compared to both conventional planar lymphoscintigraphy and hybrid SPECT/CT. SLNs detected by planar lymphoscintigraphy were marked on the patients' skin using a UV-marker. CrystalCam images were acquired in another room by another examiner and the SLNs were marked with a felt pen. The detected nodes by both camera systems were evaluated using UV-lamp and normal light to visualize the UV- and felt pen marks respectively. The concordance rate of the SLNs and higher-echelon nodes localized by both planar scintigraphy and CrystalCam imaging with respect to the total SLNs and higher-echelon nodes detected by SPECT/CT imaging are compared and statistically analyzed., Results: The results of the phantom study show a good correlation between activity and count-rates for all distancesSPECT/CT, CrystalCamm, and planar lymphoscintigraphy detected 69, 58, and 61 SLNs respectively. The concordance rate of 95.65% by the CrystalCam and planar scintigraphy implies both cameras are statistically coequal in preoperative SLN detection of malignant melanoma. For the higher-echelon nodes, SPECT/CT, planar and CrystalCam imaging systems identified 82, 48, and 13 respectively; thus, CrystalCam was statistically inferior to planar imaging., Conclusion: The handheld CrystalCam is a reliable instrument for localizing SLNs in surgical centers without an on-site nuclear medicine department., (© 2023 The Authors. Journal of Applied Clinical Medical Physics published by Wiley Periodicals, LLC on behalf of The American Association of Physicists in Medicine.)

Radiosynoviorthesis (RSO) is a decades known, effective intra-articular nuclear medicine local therapy, with few rare side-effects, in which inflamed synovial membrane is treated by means of colloidal beta-emitters. There are major variations worldwide in terms of acceptance, frequency of use and approved indications for this procedure. Thus, reliable figures that reflect reality are only available for a few countries. A Europe-wide survey revealed that RSO is carried out most frequently in Germany, where RSO is the most common nuclear medicine therapy with about 70,000 joints treated per year. The main indications include synovitis due to rheumatoid arthritis, hemophilia and pigmented villonodular synovitis (PVNS), and depending on national approvals, osteoarthritis. Despite the many indications, there are very few published scientific studies and therefore, RSO evidence is lacking. Reliable data on the clinical usage of RSO and demographics of RSO specialists are only available in Germany, thus we discuss the future challenges of RSO mainly from a German perspective. In the German healthcare system, RSO is performed primarily on an outpatient basis and plays only a minor role in the university setting. The necessary expertise for RSO is therefore lacking, for the most part, at university training centers. Currently, nearly more than three quarters of the German RSO experts are over fifty years old, illustrating a shortage of young talent. In the future, RSO providers from the non-university or private sector will have to cooperate with universities through networks and will have to intensify their cooperation with referring physicians, such as rheumatologist and orthopedic surgeons, and patients in order to maintain a timely and beneficial exchange of information. In networks of RSO experts, the participants must jointly develop and establish training concepts and facilities for future talents, elaborate on guidelines, if clinically useful expand the range of indications, initiate studies to generate further evidence and finally make the procedure more public. In addition, it is worthwhile to apply this process beyond human medicine to other fields, such as medical physics and veterinary medicine. If these points are implemented, the future of RSO will be bright, if it fails, it looks bleak.

Competing Interests: The authors declare that they have no conflict of interest.

Background: The recent development of quinoline-based radiotracers, which act as fibroblast activation protein inhibitors (FAPIs), has shown promising preclinical and clinical advantages. [ 68 Ga]Ga-FAPI-46 is a new radiotracer for in vivo detection of the fibroblast activation protein by positron emission tomography (PET). Recently, the automated synthesis of [ 68 Ga]Ga-FAPI-46 was reported based on pre-concentration and purification of the generator eluate by using a cation exchange-cartridge. Our aim was to simplify the synthesis and shorten the automated synthesis of [ 68 Ga]Ga-FAPI-46 to make it accessible and thus even more attractive to a broader clinical and scientific community., Results: We developed and evaluated the GMP compliant automatic synthesis of [ 68 Ga]Ga-FAPI-46 using two different 68 Ge/ 68 Ga generators (an Eckert & Ziegler, GalliaPharm generator, 1.85 GBq/50 mCi and an iThemba generator, 1.85 GBq/50 mCi) Somerset West, South Africa) and three different commercial and customized systems: the EasyOne module from Trasis; the GaSy module from Synthra with a customized synthesis template and a customized single use cassette. Additionally, the automatic synthesis of [ 68 Ga]Ga-FAPI-46 was established on a GallElut synthesis module from Scintomics with fixed tubing., Conclusions: Independent of the synthesis modules or the generators employed we were able to complete the synthesis of [ 68 Ga]Ga-FAPI-46 in 12 min including the process of purification and formulation. In all cases, the final products showed more than 99.5% chemical purity and the radiochemical yield reached around 92.5% (decay corrected). All quality control parameters (e.g. sterility, stability and radiochemical purity) were conform to the European Pharmacopoeia., (© 2022. The Author(s).)

Background: Systemic Lutetium-177 prostate-specific membrane antigen-617 radioligand therapy (Lu-177-PSMA-617-RLT) is a novel treatment approach in patients suffering from metastasized castration-resistant prostate cancer. Nonetheless, a therapeutic response may fail to appear in a proportion of patients. This study aims to identify routinely obtainable pre- and intratherapeutic parameters to allow a prediction of overall survival in patients receiving Lu-177-PSMA-617 radioligand therapy., Methods: Between January 2015 and December 2020 52 patients treated with a total of 146 cycles Lu-177-PSMA-617-RLT were retrospectively analysed in a single-center trial. The median overall survival time (OS) was compared to pre-therapeutic serological parameters, the extend of metastatic spread and previously performed therapies using Kaplan-Meier estimators and multivariate Cox-regression. Bonferroni-Holm correction was performed on all statistical tests., Results: The median OS of all patients was 55.6 weeks. Multivariate Cox-regression revealed significant lower survival for decreased pretherapeutic hemoglobin levels (HR 0.698 per g/dl; 95%-CI 0.560-0.872; p = 0.001), increased lactate dehydrogenase (LDH) levels (HR 1.073 per 25 U/l; 95%-CI 1.024-1.125; p = 0.003) and the presence of hepatic metastasis (HR 6.981; 95%-CI 2.583-18.863; p < 0.001). Increased pretherapeutic c-reactive protein (CRP), alkaline phosphatase (ALP) and gamma-glutamyltransferase (GGT) levels were also associated with a shorter survival. A prostate-specific antigen decline after one therapy cycle did not significantly correlate with an increased survival. No significant relations were observed between overall survival time and other serological parameters or previously performed therapies., Conclusion: Pre-therapeutic hemoglobin and LDH levels, as well as the presence of hepatic metastasis are independent predictors of overall survival in patients receiving Lu-177-PSMA-617-RLT. CRP, ALP and GGT levels cloud be utilized as additional decision aids when a Lu-177-PSMA-617-RLT is intended. Trial Registration Not applicable (retrospective observational study)., (© 2022. The Author(s).)

Inflammation is a strong driver of atherosclerotic cardiovascular disease (ASCVD). There is a large unmet need for therapies that prevent or reduce excessive inflammation while avoiding systemic immunosuppression. We showed previously that selective inhibition of pro-inflammatory interleukin-6 (IL-6) trans-signalling by the fusion protein olamkicept (sgp130Fc) prevented and reduced experimental murine atherosclerosis in low-density lipoprotein receptor-deficient ( Ldlr -/- ) mice on a high-fat, high-cholesterol diet independently of low-density lipoprotein (LDL) cholesterol metabolism. Therefore, we allowed compassionate use of olamkicept (600 mg intravenously biweekly for 10 weeks) in a patient with very-high-risk ASCVD. Despite optimal LDL cholesterol under maximum tolerated lipid-lowering treatment, the patient had a remaining very high risk for future cardiovascular events related to significant arterial wall inflammation with lipoprotein (a) [Lp(a)]-cholesterol as the main contributor. 18 Fluorodeoxyglucose positron emission tomography/computed tomography ( 18 FDG PET/CT) measurements were performed before and after the treatment period. Olamkicept reduced arterial wall inflammation in this patient without interfering with lipoprotein metabolism. No clinical or laboratory side effects were observed during or after treatment with olamkicept. Our findings in this patient matched the results from our mechanistic study in Ldlr -/- mice, which were extended by additional analyses on vascular inflammation. Olamkicept may be a promising option for treating ASCVD independently of LDL cholesterol metabolism. A Phase II trial of olamkicept in ASCVD is currently being prepared., Competing Interests: GHW is employed by CONARIS Research Institute AG, a company that owns the intellectual property (IP) rights to olamkicept, and is an inventor of such IP. SR-J and SS are consultants to CONARIS and members of CONARIS’ supervisory board. SR-J is a direct shareholder of CONARIS. CONARIS’ IP regarding olamkicept has been licensed to Ferring Pharmaceuticals (Saint-Prex, CH). None of the other authors have any conflicts of interest, financial or otherwise, to disclose in connection to this work. In particular, the clinicians who decided on the compassionate use of olamkicept and treated the patient, had no conflict of interest. The results of the present work have been protected by a European patent application of Kiel University with the inventors DMS, GHW, ML, and SS., (Copyright © 2022 Schulte, Waetzig, Schuett, Marx, Schulte, Garbers, Lokau, Vlacil, Schulz, Seoudy, Schieffer, Rosenstiel, Seeger, Laudes, Rose-John, Lützen, Grote and Schreiber.)

Background: Magnetic resonance imaging (MRI) is a noninvasive technique that provides excellent contrast for soft tissue organs. However, due to the low density of protons and many air-tissue junctions, its application in the lung is limited. Thus, X‑ray-based methods are often used here (with the well-known disadvantages of ionizing radiation)., Objectives: In this review, we discuss pulmonary MRI with hyperpolarized xenon-129 (Xe-MRI). Xe-MRI provides unique valuable insights into lung microstructure and function, including gas exchange with red blood cells-parameters not accessible by any standard clinical methods., Methods: By magnetic labelling, i.e. hyperpolarization, the signal from xenon-129 is amplified by up to 100,000 times. In this process, electrons from rubidium are first polarized to 100% using laser light and then transferred to xenon by collisions. Then the hyperpolarized gas is brought to the patient in a bag and inhaled shortly before the MRI scan., Results: Using special programming (sequences) of the MRI, the ventilation, microstructure, or gas exchange of the lungs, can be displayed in 3D. This allows, for example, quantitative visualization of ventilation defects, alveolar size, tissue gas uptake and gas transfer to the blood., Conclusions: Xe-MRI provides unique information about the state of the lung-noninvasively, in vivo and in less than a minute., (© 2022. The Author(s), under exclusive licence to Springer Medizin Verlag GmbH, ein Teil von Springer Nature.)

Clinical Issue: Despite being one of the main pillars of modern diagnostics, magnetic resonance imaging (MRI) uses only a tiny fraction of its potential: no more than a millionth of all nuclear spins contribute to the MRI signal. In order to increase this fraction, called polarization, MRI scanners with stronger magnetic fields are being developed. However, even the most modern scanners do not exploit the potential of MRI., Methodological Innovations: To make full use of this potential, hyperpolarized MRI (HP-MRI) is an excellent tool: quantum mechanical tricks can be used to generate contrast agents whose nuclear spins can deliver a MRI signal that is up to a 100,000 times stronger. This signal enhancement allows imaging of in vivo processes that would be otherwise impossible to measure. It is particularly interesting to introduce these magnetically labeled nuclei into metabolic processes so that the metabolism can be investigated non-invasively and in vivo., Performance: Small but diagnostically important changes in metabolism could be found before macroscopic tissue changes were otherwise visible. High-resolution images can be acquired within a few 100 ms, enabling metabolic monitoring in real-time. Heart, brain, and prostate are among the organs that have already been investigated in over 90 clinical trials using this emerging technology., Achievements: So far, displaying tissue in a similar manner was only possible using nuclear medicine, e.g., positron emission tomography (PET) utilizing radionuclides and without resolution of various metabolic steps. A change in tumor metabolism following treatment was shown within hours in HP-MRI. These applications coupled with background information about the technology are the subject of this review., (© 2022. The Author(s), under exclusive licence to Springer Medizin Verlag GmbH, ein Teil von Springer Nature.)

Purpose: According to German law, the [ 131 I]-capsule activity has to be checked in the context of radioiodine therapy (RIT) immediately before application. The measurement leads to significant radiation exposure of the medical personnel, especially of their hands. We aimed to establish a method for estimating [ 131 I]-capsule activity by measuring the dose rate (DR) at contact of the delivered lead closed container carrying the [ 131 I]-capsules and to evaluate radiation exposure in comparison to conventional [ 131 I]-capsule measurement using a dose calibrator., Methods: DR on the surface of the closed lead container was measured at two locations and correlated linearly with the [ 131 I]-capsule activity measured in a dose calibrator to create calibrating curves. The hand and whole body (effective) doses were determined with official dose meters during validation of our method in clinical practice., Results: The determination coefficients (R 2 ) of linear calibration curves were greater than 0.9974. The total relative uncertainty for estimating [ 131 I]-capsule activity with our method was <±7.5% which is lower than the uncertainty of the nominal activity and quite close to the threshold limit for the maximum allowed uncertainty of ± 5% for measuring activity in radioactive drugs. The reduction of the hand dose caused by our method was 97% compared with the conventional measurements of the [ 131 I]-capsules in a dose calibrator., Conclusion: Measuring DR on the surface of the closed lead containers enables the [ 131 I]-capsules activity to be estimated simply, reliably and with sufficient accuracy leading to significant reduction of the radiation exposure for the medical staff., (Copyright © 2022 Associazione Italiana di Fisica Medica e Sanitaria. Published by Elsevier Ltd. All rights reserved.)

Translational photopharmacological applications are limited through irradiation by light showing wavelengths within the bio-optical window. To achieve sufficient tissue penetration, using wavelengths >500 nm is mandatory. Nevertheless, the majority of photopharmacological compounds respond to irradiation with more energetic UV light, which shows only a minor depth of tissue penetration in the µm range. Thus, we became interested in UV light containing Cherenkov radiation (CR) induced as a by-product by clinically employed radionuclides labeling specific tissues. Therefore, CR may be applicable in novel photopharmacological approaches. To provide evidence for the hypothesis, we verified the clinically established radionuclides 68 Ga and 90 Y but not 18 F in clinically used activities to be capable of generating CR in aqueous solutions. We then investigated whether the generated CR was able to photoactivate the caged kinase inhibitor cagedAZD5438 as a photoresponsive model system. Herein, 21% uncaging of the model system cagedAZD5438 occurred by incubation with 90 Y, along with a non-specific compound decomposition for 68 Ga and partly for 90 Y. The findings suggest that the combination of a clinically employed radionuclide with an optimized photoresponsive agent could be beneficial for highly focused photopharmacological therapies.

The present study elucidates the neuroprotective mechanisms of the PPARγ (peroxisome proliferator-activated receptor γ) agonist pioglitazone in survival of ischemic neurons following middle cerebral artery occlusion with reperfusion (MCAO). Intracerebroventricular infusion of pioglitazone over 5 days before and 24 or 48 h after MCAO alleviated neurological impairments, inhibited apoptosis 24 h, and activated the PI3K/Akt pathway along with increased phosphorylation of Akt (ser473) and GSK-3β (ser9) in the peri-infarct cortical areas 48 h after MCAO. In primary cortical neurons, pioglitazone suppressed the glutamate-induced release of lactate dehydrogenase by a PPARγ-dependent mechanism. This protective effect was reversed after co-treatment with PI3K and Akt inhibitors, LY294002 and SH-6, respectively. Pioglitazone enhanced the expression of the antioxidative transcription factor Nrf2 and its target gene protein, heme oxidase-1, in the peri-infarct area. Pioglitazone also increased activation of the antioxidant response element (ARE) in neuronal PC12 cells transfected with the pNQO1-rARE plasmid. We demonstrate in primary cortical neurons from Nrf2 knockout mice that the lack of Nrf2 completely abolished the neuroprotective effects of pioglitazone against oxidative and excitotoxic damage. Our results strongly suggest that the neuroprotective effects of PPARγ in peri-infarct brain tissues comprise the concomitant activation of the PI3K/Akt and Nrf2/ARE pathways. KEY MESSAGES: Pioglitazone inhibits apoptosis in ischemic brain tissue.  Pioglitazone acting on PPARγ activates PI3K/Akt pathway in ischemic brain tissue. Pioglitazone activates via Nrf2 the antioxidant defense pathway in injured neurons. Pioglitazone activates the antioxidant response element in neuronal PC12 cells. Pioglitazone fails to protect primary neurons lacking Nrf2 against oxidative damage. Activation of PPARγ supports the survival of viable neurons in peri-infarct regions., (© 2021. The Author(s).)

Systemic radionuclide therapy with 177 Lu-PSMA-617 is a novel treatment option in patients with metastasized and castration-resistant prostate cancer 4. The molecular target of the 177 Lu-PSMA-617 radioligand therapy is the prostate-specific membrane antigen (PSMA) highly expressed on prostate cancer cells. Beyond the enhanced accumulation of PSMA on prostate cancer cells, PSMA expression is also found on the molecular surface or in the tumor-associated neovasculature of various tumor tissues including sarcomas of the soft tissue 2. Thus, PSMA has theoretically been discussed as a possible future target for systemic radioligand therapy with 177 Lu-PSMA-617 even in non-prostate malignancies 1.Here we report on a female patient with extended metastasized leiomyosarcoma experimentally treated with one application of 177 Lu-PSMA-617 radioligand therapy., Competing Interests: The authors declare that they have no conflict of interest., (© Georg Thieme Verlag KG Stuttgart · New York.)

Objective: Vedolizumab, a monoclonal antibody directed against the integrin heterodimer α4β7, is approved for the treatment of Crohn's disease and ulcerative colitis. The efficacy of vedolizumab has been suggested to result from inhibition of intestinal T cell trafficking although human data to support this conclusion are scarce. We therefore performed a comprehensive analysis of vedolizumab-induced alterations in mucosal and systemic immunity in patients with inflammatory bowel disease (IBD), using anti-inflammatory therapy with the TNFα antibody infliximab as control., Design: Immunophenotyping, immunohistochemistry, T cell receptor profiling and RNA sequencing were performed using blood and colonic biopsies from patients with IBD before and during treatment with vedolizumab (n=18) or, as control, the anti-TNFα antibody infliximab (n=20). Leucocyte trafficking in vivo was assessed using single photon emission computed tomography and endomicroscopy., Results: Vedolizumab was not associated with alterations in the abundance or phenotype of lamina propria T cells and did not affect the mucosal T cell repertoire or leucocyte trafficking in vivo. Surprisingly, however, α4β7 antibody treatment was associated with substantial effects on innate immunity including changes in macrophage populations and pronounced alterations in the expression of molecules involved in microbial sensing, chemoattraction and regulation of the innate effector response. These effects were specific to vedolizumab, not observed in response to the TNFα antibody infliximab, and associated with inhibition of intestinal inflammation., Conclusion: Our findings suggest that modulation of innate immunity contributes to the therapeutic efficacy of vedolizumab in IBD., Trial Registration Number: NCT02694588., Competing Interests: Competing interests: SZ: consulting fees: Biogen, Ferring, Janssen-Cilag, Takeda; Lecture fees: Abbvie, Falk, Ferring, MSD, Roche, Takeda. KA: grant support: Pfizer. AA: consulting fees: Boston Scientific; lecture fees: Boston Scientific. SS: consulting fees: AbbVie, Boehringer, Merck, Janssen, Pfizer, Takeda; lecture fees: AbbVie, Boehringer, Merck, Janssen, Pfizer, Takeda., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2019. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

Mice lacking the substance P (SP) neurokinin-1 (NK1) receptor (NK1R-/-mice) were used to investigate whether SP affects serotonin (5-HT) function in the brain and to assess the effects of acute immobilisation stress on the hypothalamic-pituitary-adrenocortical (HPA) axis and 5-HT turnover in individual brain nuclei. Basal HPA activity and the expression of hypothalamic corticotropin-releasing hormone (CRH) in wild-type (WT)- and NK1R-/- mice were identical. Stress-induced increases in plasma ACTH concentration were considerably higher in NK1R-/- mice than in WT mice while corticosterone concentrations were equally elevated in both mouse lines. Acute stress did not alter the expression of CRH. In the dorsal raphe nucleus (DRN), basal 5-HT turnover was increased in NK1R-/- mice and a 15 min stress further magnified 5-HT utilisation in this region. In the frontoparietal cortex, medial prefrontal cortex, central nucleus of amygdala, and the hippocampal CA1 region, stress increased 5-HT and/or 5-hydroxyindoleacetic acid (5-HIAA) concentrations to a similar extent in WT and NK1R-/- mice. 5-HT turnover in the hypothalamic paraventricular nucleus was not affected by stress, but stress induced similar increases in 5-HT and 5-HIAA in the ventromedial and dorsomedial hypothalamic nuclei in WT and NK1R-/- mice. Our findings indicate that NK1 receptor activation suppresses ACTH release during acute stress but does not exert sustained inhibition of the HPA axis. Genetic deletion of the NK1 receptor accelerates 5-HT turnover in DRN under basal and stress conditions. No differences between the responses of serotonergic system to acute stress in WT and NK1R-/- mice occur in forebrain nuclei linked to the regulation of anxiety and neuroendocrine stress responses.