Variation within the visually evoked neurovascular coupling response of the posterior cerebral artery is not influenced by age or sex.

Neurovascular coupling (NVC) is the temporal and spatial coordination between local neuronal activity and regional cerebral blood flow. The literature is unsettled on whether age and/or sex affect NVC, which may relate to differences in methodology and the quantification of NVC in small sample-sized studies. The aim of this study was to 1) determine the relative and combined contribution of age and sex to the variation observed across several distinct NVC metrics (n = 125, 21-66 yr; 41 males) and 2) present an approach for the comprehensive systematic assessment of the NVC response using transcranial Doppler ultrasound. NVC was measured as the relative change from baseline (absolute and percent change) assessing peak, mean, and total area under the curve (tAUC) of cerebral blood velocity through the posterior cerebral artery (PCAv) during intermittent photic stimulation. In addition, the NVC waveform was compartmentalized into distinct regions, acute (0-9 s), mid (10-19 s), and late (20-30 s), following the onset of photic stimulation. Hierarchical multiple regression modeling was used to determine the extent of variation within each NVC metric attributable to demographic differences in age and sex. After controlling for differences in baseline PCAv, the R2 data suggest that 1.6%, 6.1%, 1.1%, 3.4%, 2.5%, and 4.2% of the variance observed within mean, peak, tAUC, acute, mid, and late response magnitude is attributable to the combination of age and sex. Our study reveals that variability in NVC response magnitude is independent of age and sex in healthy human participants, aged 21-66 yr.NEW & NOTEWORTHY We assessed the variability within the neurovascular coupling response attributable to age and sex (n = 125, 21-66 yr; 41 male). Based on the assessment of posterior cerebral artery responses to visual stimulation, 0%-6% of the variance observed within several metrics of NVC response magnitude are attributable to the combination of age and sex. Therefore, observed differences between age groups and/or sexes are likely a result of other physiological factors.

Recurrent embolisms despite adequate anticoagulant therapy in a patient with prosthetic heart valves and atrial fibrillation: a case report.

BACKGROUND: Antithrombotic management in patients with atrial fibrillation (AF) that have undergone heart valve surgery may be challenging, especially in the context of thromboembolic events during follow-up. The combination of pharmacological therapies with modern transcatheter interventions allows these more complex cases to be overcome. CASE SUMMARY: We present the case of a 66-year-old female with a history of AF and mechanical aortic and mitral valve replacement, which was admitted to the hospital complaining of dizziness and unsteady gait. A computerized tomography scan of the brain confirmed the diagnosis of embolic stroke. Two years later, the patient complained of sudden onset of chest pain, accompanied by electrocardiographic abnormalities and elevated high-sensitivity troponin T. Emergency cardiac catheterization revealed embolic myocardial infarction with distal occlusion of the obtuse marginal artery. Again, 2 years later, the patient suffered a new cerebral embolic event. Given the adequate anticoagulation therapy throughout almost the entire clinical course, percutaneous left atrial appendage closure was proposed as an adjunct to vitamin K antagonist treatment. Notably, intraprocedural transoesophageal echocardiography revealed the presence of a previously undetected left atrial appendage thrombus, thus an embolic protection device was used during the procedure, which was successfully carried out without complications. DISCUSSION: This case report demonstrates the complexity of the antithrombotic management in patients with AF and prosthetic heart valves, and highlights the importance of an individualized approach, integrating new therapeutic strategies to achieve success, in patients that present thromboembolic events despite adequate anticoagulation therapy.

Atrial cholangiocarcinoma diagnosed by cardiac magnetic resonance imaging.

In this rare case of intrahepatic cholangiocarcinoma (ICC) tumor thrombus with right atrial (RA) invasion, we describe its diagnostic workup based on cardiac magnetic resonance imaging (cMRI). An 85-year-old man underwent transthoracic echocardiography due to dyspnea, revealing a RA mass. Comprehensive cMRI evaluation, including cine bright blood, T1- and T2-weighed, fat-suppressed, and contrast-enhanced imaging, was performed and diagnosis of ICC tumor thrombus with RA invasion was made. This first description of cMRI-guided diagnosis of RA invasion by ICC tumor thrombus points out the usefulness of cMRI for the diagnostic approach of RA masses suggestive of tumor thrombus.

Preliminary study of ovariectomy and chronic losartan-induced alterations in brain AT1 receptors.

Women who undergo oophorectomy prior to the age of natural menopause have a higher risk of neurological and psychological impairment. Treatment with the angiotensin receptor blocker (ARB) losartan for 10 weeks following ovariectomy of Long-Evans rats at 3 months of age reduced the ovariectomy-induced cognitive decrements. Following completion of the behavioral experiments, (Campos et al., 2019), the brains were harvested for preliminary receptor autoradiographic studies of AT1 receptor (AT1R) binding in selected brain regions using quantitative densitometric analysis of autoradiograms of 125I-sarcosine1, isoleucine8 angiotensin II binding. Four of the brain regions (amygdala, ventral subiculum, piriform cortex, and cingulate cortex) are associated with cognitive and emotional behavior while one (lateral hypothalamus) is associated with homeostasis. The density of AT1R varied by region: ventral subiculum > amygdala and cingulate cortex, and piriform cortex > cingulate cortex. Losartan treatment decreased AT1R binding in the ventral subiculum of sham and ovariectomized rats by 41.6%, and 46% in the piriform cortex of the sham rats, but tended to increase AT1R binding in the piriform cortex and cingulate cortex 77% and 107%, respectively, in the ovariectomized rats. AT1R binding did not differ significantly between intact male and sham-vehicle female rats among surveyed brain regions. These results suggest that losartan-induced changes in brain AT1R expression may contribute to the reduced anxiety-like behavior and memory impairments seen in ovariectomized rats, but replication of these observations will be needed to determine the extent to which brain AT1R changes mediate the adverse behavioral effects of ovariectomy.

Cardiorespiratory hysteresis during incremental high-altitude ascent-descent quantifies the magnitude of ventilatory acclimatization.

NEW FINDINGS: What is the central question of this study? We assessed the utility of a new metric for quantifying ventilatory acclimatization to high altitude, derived from differential ascent and descent steady-state cardiorespiratory variables (i.e. hysteresis). Furthermore, we aimed to investigate whether the magnitude of cardiorespiratory hysteresis was associated with the development of acute mountain sickness. What is the main finding and its importance? Hysteresis in steady-state cardiorespiratory variables quantifies ventilatory acclimatization to high altitude. The magnitude of cardiorespiratory hysteresis during ascent to and descent from high altitude was significantly related to the development of symptoms of acute mountain sickness. Hysteresis in steady-state chemoreflex drive can provide a simple, non-invasive method of tracking ventilatory acclimatization to high altitude. ABSTRACT: Maintenance of arterial blood gases is achieved through sophisticated regulation of ventilation, mediated by central and peripheral chemoreflexes. Respiratory chemoreflexes are important during exposure to high altitude owing to the competing influence of hypoxia and hypoxic hyperventilation-mediated hypocapnia on steady-state ventilatory drive. Inter-individual variability exists in ventilatory acclimatization to high altitude, potentially affecting the development of acute mountain sickness (AMS). We aimed to quantify ventilatory acclimatization to high altitude by comparing differential ascent and descent values (i.e. hysteresis) in steady-state cardiorespiratory variables. We hypothesized that: (i) the hysteresis area formed by cardiorespiratory variables during ascent and descent would quantify the magnitude of ventilatory acclimatization; and (ii) larger hysteresis areas would be associated with lower AMS symptom scores during ascent. In 25 healthy, acetazolamide-free trekkers ascending to and descending from 5160 m, cardiorespiratory hysteresis was measured in the partial pressure of end-tidal CO2 , peripheral oxygen saturation, minute ventilation, chemoreceptor stimulus index (end-tidal CO2 /peripheral oxygen saturation) and the calculated steady-state chemoreflex drive (SS-CD; minute ventilation/chemoreceptor stimulus index) using portable devices (capnograph, peripheral pulse oximeter and respirometer, respectively). Symptoms of AMS were assessed daily using the Lake Louise questionnaire. We found that: (i) ascent-descent hysteresis was present in all cardiorespiratory variables; (ii) SS-CD is a valid metric for tracking ventilatory acclimatization to high altitude; and (iii) the highest AMS scores during ascent exhibited a significant, moderate and inverse correlation with the magnitude of SS-CD hysteresis (rs  = -0.408, P = 0.043). We propose that ascent-descent hysteresis is a new and feasible way to quantify ventilatory acclimatization in trekkers during high-altitude exposure.

Does sedentary time increase in older adults in the days following participation in intense exercise?

BACKGROUND: Older adults have the highest sedentary time across all age groups, and only a small portion is meeting the minimum recommendations for weekly physical activity. Little research to date has looked at how changes in one of these behaviours influences the other. AIM: To assess changes in 24-h movement behaviours (sedentary time, light intensity physical activity (LPA), moderate-vigorous PA (MVPA) and sleep) over three consecutive days, following acute bouts of exercise of varying intensity in older adults. METHODS: Participants (n = 28, 69.7 ± 6.5 years) completed a maximal exercise test and the following exercise protocols in random order: moderate continuous exercise (MOD), high-intensity interval exercise (HI) and sprint interval exercise (SPRT). A thigh-worn device (ActivPAL™) was used to measure movement behaviours at baseline and the 3 days following each exercise session. RESULTS: Repeated measures analysis of variance indicated that compared to baseline, participants decreased MVPA in the 3 days following all exercise sessions and decreased LPA following HI and SPRT (p < 0.05). Over half of the sample had clinically meaningful increases in sedentary time (30 min/day) in the days following exercise participation. DISCUSSION: Older adults who compensate for exercise participation by reducing physical activity and increasing sedentary time in subsequent days may require behavioural counseling to ensure that incidental and recreational physical activities are not reduced. CONCLUSION: It appears that older adults compensate for acute exercise by decreasing MVPA and LPA, and increasing sedentary time in the days following exercise. Future research is needed to determine whether compensation persists with regular engagement.

Severe food restriction activates the central renin angiotensin system.

We previously showed that 2 weeks of a severe food restricted (sFR) diet (40% of the caloric intake of the control (CT) diet) up-regulated the circulating renin angiotensin (Ang) system (RAS) in female Fischer rats, most likely as a result of the fall in plasma volume. In this study, we investigated the role of the central RAS in the mean arterial pressure (MAP) and heart rate (HR) dysregulation associated with sFR. Although sFR reduced basal mean MAP and HR, the magnitude of the pressor response to intracerebroventricular (icv) microinjection of Ang-[1-8] was not affected; however, HR was 57 ± 13 bpm lower 26 min after Ang-[1-8] microinjection in the sFR rats and a similar response was observed after losartan was microinjected. The major catabolic pathway of Ang-[1-8] in the hypothalamus was via Ang-[1-7]; however, no differences were detected in the rate of Ang-[1-8] synthesis or degradation between CT and sFR animals. While sFR had no effect on the AT1 R binding in the subfornical organ (SFO), the organum vasculosum laminae terminalis (OVLT) and median preoptic nucleus (MnPO) of the paraventricular anteroventral third ventricle, ligand binding increased 1.4-fold in the paraventricular nucleus (PVN) of the hypothalamus. These findings suggest that sFR stimulates the central RAS by increasing AT1 R expression in the PVN as a compensatory response to the reduction in basal MAP and HR. These findings have implications for people experiencing a period of sFR since an activated central RAS could increase their risk of disorders involving over activation of the RAS including renal and cardiovascular diseases.

Novel high molecular weight albumin-conjugated angiotensin II activates ß-arrestin and G-protein pathways.

PURPOSE: To study the ability of a novel bovine serum albumin-angiotensin II (BSA-Ang II) conjugate to effect responses of the AT1 angiotensin II receptor subtype mediated by the G-protein-coupled and the beta-arrestin pathways. METHODS: Angiotensin II (Ang II) was conjugated with bovine serum albumin and compared with Ang II for competition binding to AT1 receptors, to stimulate aldosterone release from adrenocortical cells, to promote beta-arrestin binding to AT1 receptors, to promote calcium mobilization, and stimulate drinking of water and saline by rats. RESULTS: The BSA-Ang II conjugate was less potent competing for AT1R binding, but was equally efficacious at stimulating aldosterone release from H295R adrenocortical cells. Both BSA-Ang II and Ang II stimulated calcium mobilization and beta-arrestin binding to AT1 receptors. BSA-Ang II and Ang II stimulated water appetite equivalently but BSA-Ang II stimulated saline appetite more than Ang II. Both BSA-Ang II and Ang II were considerably more potent at causing calcium mobilization than ß-arrestin binding. CONCLUSIONS: Addition of a high molecular weight molecule to Ang II reduced its AT1 receptor binding affinity, but did not significantly alter stimulation of aldosterone release or water consumption. The BSA-Ang II conjugate caused a greater saline appetite than Ang II suggesting that it may be a more efficacious agonist of this beta-arrestin-mediated response than Ang II. The higher potency calcium signaling response suggests that the G-protein-coupled responses predominate at physiological concentrations of Ang II, while the beta-arrestin response requires pathophysiological or pharmacological concentrations of Ang II to occur.

Peripheral chemoreceptor deactivation attenuates the sympathetic response to glucose ingestion.

Acute increases in blood glucose are associated with heightened muscle sympathetic nerve activity (MSNA). Animal studies have implicated a role for peripheral chemoreceptors in this response, but this has not been examined in humans. Heart rate, cardiac output (CO), mean arterial pressure, total peripheral conductance, and blood glucose concentrations were collected in 11 participants. MSNA was recorded in a subset of 5 participants via microneurography. Participants came to the lab on 2 separate days (i.e., 1 control and 1 experimental day). On both days, participants ingested 75 g of glucose following baseline measurements. On the experimental day, participants breathed 100% oxygen for 3 min at baseline and again at 20, 40, and 60 min after glucose ingestion to deactivate peripheral chemoreceptors. Supplemental oxygen was not given to participants on the control day. There was a main effect of time on blood glucose (P < 0.001), heart rate (P < 0.001), CO (P < 0.001), sympathetic burst frequency (P < 0.001), burst incidence (P = 0.01), and total MSNA (P = 0.001) for both days. Blood glucose concentrations and burst frequency were positively correlated on the control day (r = 0.42; P = 0.03) and experimental day (r = 0.62; P = 0.003). There was a time × condition interaction (i.e., normoxia vs. hyperoxia) on burst frequency, in which hyperoxia significantly blunted burst frequency at 20 and 60 min after glucose ingestion only. Given that hyperoxia blunted burst frequency only during hyperglycemia, our results suggest that the peripheral chemoreceptors are involved in activating MSNA after glucose ingestion.

What Is the Point of the Peak? Assessing Steady-State Respiratory Chemoreflex Drive in High Altitude Field Studies.

Measurements of central and peripheral respiratory chemoreflexes are important in the context of high altitude as indices of ventilatory acclimatization. However, respiratory chemoreflex tests have many caveats in the field, including considerations of safety, portability and consistency. This overview will (a) outline commonly utilized tests of the hypoxic ventilatory response (HVR) in humans, (b) outline the caveats associated with a variety of peak response HVR tests in the laboratory and in high altitude fieldwork contexts, and (c) advance a novel index of steady-state chemoreflex drive (SS-CD) that addresses the many limitations of other chemoreflex tests. The SS-CD takes into account the contribution of central and peripheral respiratory chemoreceptors, and eliminates the need for complex equipment and transient respiratory gas perturbation tests. To quantify the SS-CD, steady-state measurements of the pressure of end-tidal (PET)CO2 (Torr) and peripheral oxygen saturation (SpO2; %) are used to quantify a stimulus index (SI; PETCO2/SpO2). The SS-CD is then calculated by indexing resting ventilation (L/min) against the SI. SS-CD data are subsequently reported from 13 participants during incremental ascent to high altitude (5160 m) in the Nepal Himalaya. The mean SS-CD magnitude increased approximately 96% over 10 days of incremental exposure to hypobaric hypoxia, suggesting that the SS-CD tracks ventilatory acclimatization. This novel SS-CD may have future utility in fieldwork studies assessing ventilatory acclimatization during incremental or prolonged stays at altitude, and may replace the use of complex and potentially confounded transient peak response tests of the HVR in humans.

Discongruence Index　- Simple Indicator to Predict Prosthesis-Patient Mismatch After Transcatheter Aortic Valve Replacement.

BACKGROUND: Prosthesis-patient mismatch (PPM) after transcatheter aortic valve replacement (TAVR) remains an important issue. The aim of this study was to assess the value of a new discongruence index, to predict PPM after TAVR.Methods and Results: A total of 185 patients with severe aortic stenosis who underwent TAVR with the Edwards Sapien prosthesis or CoreValve Revalving system were included (Edwards valve, n=119; Core Valve Revalving system, n=66). Discongruence index was calculated pre-procedurally as the ratio of selected transcatheter valve size (mm) to body surface area (cm2). PPM was defined as effective orifice area (EOA) ≤0.85 cm2/m2 on transthoracic echocardiography before hospital discharge. Mean age was 82±5 years and 72 patients (38.9%) were men. The overall incidence of post-TAVR PPM was 35.1% (n=65). Discongruence index correlated with post-TAVR indexed EOA (y=0.18+0.057x; P<0.001). On multivariate logistic regression analysis, discongruence index was the only independent predictor of post-TAVR PPM (OR, 0.15; 95% CI: 0.03-0.66; P=0.012), and the area under the receiver operating characteristic curve was 0.62 (95% CI: 0.54-0.70, P=0.003), with an optimal cut-off point of 15.02 (sensitivity, 86.2%; specificity, 72.5%; positive predictive value, 74.3%; negative predictive value, 83.4%). CONCLUSIONS: The new discongruence index may be useful tool to predict PPM after TAVR.

Extreme pregnancy: maternal physical activity at Everest Base Camp.

High-altitude natives employ numerous physiological strategies to survive and reproduce. However, the concomitant influence of altitude and physical activity during pregnancy has not been studied above 3,700 m. We report a case of physical activity, sleep behavior, and physiological measurements on a 28-yr-old third-trimester pregnant native highlander (Sherpa) during ascent from 3,440 m to Everest Base Camp (~5,300 m) over 8 days in the Nepal Himalaya and again ~10 mo postpartum during a similar ascent profile. The participant engaged in 250-300 min of moderate to vigorous physical activity per day during ascent to altitude while pregnant, with similar volumes of moderate to vigorous physical activity while postpartum. There were no apparent maternal, fetal, or neonatal complications related to the superimposition of the large volumes of physical activity at altitude. This report demonstrates a rare description of physical activity and ascent to high altitude during pregnancy and points to novel questions regarding the superimposition of pregnancy, altitude, and physical activity in high-altitude natives.

Receptor Autoradiography Protocol for the Localized Visualization of Angiotensin II Receptors.

This protocol describes receptor binding patterns for Angiotensin II (Ang II) in the rat brain using a radioligand specific for Ang II receptors to perform receptor autoradiographic mapping. Tissue specimens are harvested and stored at -80 °C. A cryostat is used to coronally section the tissue (brain) and thaw-mount the sections onto charged slides. The slide-mounted tissue sections are incubated in (125)I-SI-Ang II to radiolabel Ang II receptors. Adjacent slides are separated into two sets: 'non-specific binding' (NSP) in the presence of a receptor saturating concentration of non-radiolabeled Ang II, or an AT1 Ang II receptor subtype (AT1R) selective Ang II receptor antagonist, and 'total binding' with no AT1R antagonist. A saturating concentration of AT2 Ang II receptor subtype (AT2R) antagonist (PD123319, 10 µM) is also present in the incubation buffer to limit (125)I-SI-Ang II binding to the AT1R subtype. During a 30 min pre-incubation at ~22 °C, NSP slides are exposed to 10 µM PD123319 and losartan, while 'total binding' slides are exposed to 10 µM PD123319. Slides are then incubated with (125)I-SI-Ang II in the presence of PD123319 for 'total binding', and PD123319 and losartan for NSP in assay buffer, followed by several 'washes' in buffer, and water to remove salt and non-specifically bound radioligand. The slides are dried using blow-dryers, then exposed to autoradiography film using a specialized film and cassette. The film is developed and the images are scanned into a computer for visual and quantitative densitometry using a proprietary imaging system and a spreadsheet. An additional set of slides are thionin-stained for histological comparisons. The advantage of using receptor autoradiography is the ability to visualize Ang II receptors in situ, within a section of a tissue specimen, and anatomically identify the region of the tissue by comparing it to an adjacent histological reference section.

Structure and vascularization of the ventricular myocardium in Holocephali: their evolutionary significance.

It was generally assumed that the ventricle of the primitive vertebrate heart was composed of trabeculated, or spongy, myocardium, supplied by oxygen-poor luminal blood. In addition, it was presumed that the mixed ventricular myocardium, consisting of a compacta and a spongiosa, and its supply through coronary arteries appeared several times throughout fish evolution. Recent work has suggested, however, that a fully vascularized, mixed myocardium may be the primitive condition in gnathostomes. The present study of the heart ventricles of four holocephalan species aimed to clarify this controversy. Our observations showed that the ventricular myocardium of Chimaera monstrosa and Harriotta raleighana consists of a very thin compacta overlying a widespread spongiosa. The ventricle of Hydrolagus affinis is composed exclusively of trabeculated myocardium. In these three species there is a well-developed coronary artery system. The main coronary artery trunks run along the outflow tract, giving off subepicardial ventricular arteries. The trabeculae of the spongiosa are irrigated by branches of the subepicardial arteries and by penetrating arterial vessels arising directly from the main coronary trunks at the level of the conoventricular junction. The ventricle of Rhinochimaera atlantica has only spongy myocardium supplied by luminal blood. Small coronary arterial vessels are present in the subepicardium, but they do not enter the myocardial trabeculae. The present findings show for the first time that in a wild living vertebrate species, specifically H. affinis, an extensive coronary artery system supplying the whole cardiac ventricle exists in the absence of a well-developed compact ventricular myocardium. This is consistent with the notion derived from experimental work that myocardial cell proliferation and coronary vascular growth rely on distinct developmental programs. Our observations, together with data in the literature on elasmobranchs, support the view that the mixed ventricular myocardium is primitive for chondrichthyans. The reduction or even lack of compacta in holocephali has to be regarded as a derived anatomical trait. Our findings also fit in with the view that the mixed myocardium was the primitive condition in gnathostomes, and that the absence of compact ventricular myocardium in different actinopterygian groups is the result of a repeated loss of such type of cardiac muscle during fish evolution.

Acute repeated intracerebroventricular injections of angiotensin II reduce agonist and antagonist radioligand binding in the paraventricular nucleus of the hypothalamus and median preoptic nucleus in the rat brain.

Angiotensin II (Ang II) stimulates water and saline intakes when injected into the brain of rats. This arises from activation of the AT1 Ang II receptor subtype. Acute repeated injections, however, decrease the water intake response to Ang II without affecting saline intake. Previous studies provide evidence that Ang II-induced water intake is mediated via the classical G protein coupling pathway, whereas the saline intake caused by Ang II is mediated by an ERK 1/2 MAP kinase signaling pathway. Accordingly, the different behavioral response to repeated injections of Ang II may reflect a selective effect on G protein coupling. To test this hypothesis, we examined the binding of a radiolabeled agonist ((125)I-sarcosine(1) Ang II) and a radiolabeled antagonist ((125)I-sarcosine(1), isoleucine(8) Ang II) in brain homogenates and tissue sections prepared from rats given repeated injections of Ang II or vehicle. Although no treatment-related differences were found in hypothalamic homogenates, a focus on specific brain structures using receptor autoradiography, found that the desensitization treatment reduced binding of both radioligands in the paraventricular nucleus of the hypothalamus (PVN) and median preoptic nucleus (MnPO), but not in the subfornical organ (SFO). Because G protein coupling is reported to have a selective effect on agonist binding without affecting antagonist binding, these findings do not support a G protein uncoupling treatment effect. This suggests that receptor number is more critical to the water intake response than the saline intake response, or that pathways downstream from the G protein mediate desensitization of the water intake response.

Distribution of non-AT1, non-AT2 binding of 125I-sarcosine1, isoleucine8 angiotensin II in neurolysin knockout mouse brains.

The recent identification of a novel binding site for angiotensin (Ang) II as the peptidase neurolysin (E.C. 3.4.24.16) has implications for the renin-angiotensin system (RAS). This report describes the distribution of specific binding of 125I-Sarcosine1, Isoleucine8 Ang II (125I-SI Ang II) in neurolysin knockout mouse brains compared to wild-type mouse brains using quantitative receptor autoradiography. In the presence of p-chloromercuribenzoic acid (PCMB), which unmasks the novel binding site, widespread distribution of specific (3 µM Ang II displaceable) 125I-SI Ang II binding in 32 mouse brain regions was observed. Highest levels of binding >700 fmol/g initial wet weight were seen in hypothalamic, thalamic and septal regions, while the lowest level of binding <300 fmol/g initial wet weight was in the mediolateral medulla. 125I-SI Ang II binding was substantially higher by an average of 85% in wild-type mouse brains compared to neurolysin knockout brains, suggesting the presence of an additional non-AT1, non-AT2, non-neurolysin Ang II binding site in the mouse brain. Binding of 125I-SI Ang II to neurolysin in the presence of PCMB was highest in hypothalamic and ventral cortical brain regions, but broadly distributed across all regions surveyed. Non-AT1, non-AT2, non-neurolysin binding was also highest in the hypothalamus but had a different distribution than neurolysin. There was a significant reduction in AT2 receptor binding in the neurolysin knockout brain and a trend towards decreased AT1 receptor binding. In the neurolysin knockout brains, the size of the lateral ventricles was increased by 56% and the size of the mid forebrain (-2.72 to +1.48 relative to Bregma) was increased by 12%. These results confirm the identity of neurolysin as a novel Ang II binding site, suggesting that neurolysin may play a significant role in opposing the pathophysiological actions of the brain RAS and influencing brain morphology.