Imaging kidney inflammation using an oxidatively activated MRI probe.

Imaging tools for kidney inflammation could improve care for patients suffering inflammatory kidney diseases by lessening reliance on percutaneous biopsy or biochemical tests alone. During kidney inflammation, infiltration of myeloid immune cells generates a kidney microenvironment that is oxidizing relative to normal kidney. Here, we evaluated whether magnetic resonance imaging (MRI) using the redox-active iron (Fe) complex Fe-PyC3A as an oxidatively activated probe could serve as a marker of kidney inflammation using mouse models of unilateral ischemia-reperfusion injury (IRI) and lupus nephritis (MRL-lpr mice). We imaged unilateral IRI in gp91phox knockout mice, which are deficient in the nicotinamide oxidase II (NOX2) enzyme required for myeloid oxidative burst, as loss of function control, and imaged MRL/MpJ mice as non-kidney involved lupus control. Gadoterate meglumine was used as a non-oxidatively activated control MRI probe. Fe-PyC3A safety was preliminarily examined following a single acute dose. Fe-PyC3A generated significantly greater MRI signal enhancement in the IRI kidney compared to the contralateral kidney in wild-type mice, but the effect was not observed in the NOX2-deficient control. Fe-PyC3A also generated significantly greater kidney enhancement in MRL-lpr mice compared to MRL/MpJ control. Gadoterate meglumine did not differentially enhance the IRI kidney over the contralateral kidney and did not differentially enhance the kidneys of MRL-lpr over MRL/MpJ mice. Fe-PyC3A was well tolerated at the highest dose evaluated, which was a 40-fold greater than required for imaging. Thus, our data indicate that MRI using Fe-PyC3A is specific to an oxidizing kidney environment shaped by activity of myeloid immune cells and support further evaluation of Fe-PyC3A for imaging kidney inflammation.

Numerical Simulation and Experimental Studies of a Ribbon Coil for Trans Spinal Magnetic Stimulation (TSMS) in Rats.

We propose a non-invasive Trans Spinal Magnetic Stimulation (TSMS) coil allowing for focal stimulation. The device is based on a new figure-8 ribbon design, ensuring low R0, and low heating. The two coils were designed and studied using the finite element method (FEM) coupled with NEURON and tested for efficacy on rats. The numerical simulations confirmed the generation of the observed action potentials when the coil was driven with 2.8kA.Clinical Relevance- Chronic neuropathic back and leg pain is one of the main indications for spinal cord stimulation in the United States. Chronic low back pain is one of the most common reasons patients seek medical care, and in 2013 resulted in 87.6 billion dollars in healthcare costs in the USA. Patients would most likely prefer a low-risk, non-invasive procedure, such as TSMS, to surgery with a significant rate of complications.

Gadolinium-based Contrast Agent Biodistribution and Speciation in Rats.

Background Gadolinium retention has been observed in organs of patients with normal renal function; however, the biodistribution and speciation of residual gadolinium is not well understood. Purpose To compare the pharmacokinetics, distribution, and speciation of four gadolinium-based contrast agents (GBCAs) in healthy rats using MRI, mass spectrometry, elemental imaging, and electron paramagnetic resonance (EPR) spectroscopy. Materials and Methods In this prospective animal study performed between November 2021 and September 2022, 32 rats received a dose of gadoterate, gadoteridol, gadobutrol, or gadobenate (2.0 mmol/kg) for 10 consecutive days. GBCA-naive rats were used as controls. Three-dimensional T1-weighted ultrashort echo time images and R2* maps of the kidneys were acquired at 3, 17, 34, and 52 days after injection. At 17 and 52 days after injection, gadolinium concentrations in 23 organ, tissue, and fluid specimens were measured with mass spectrometry; gadolinium distribution in the kidneys was evaluated using elemental imaging; and gadolinium speciation in the kidney cortex was assessed using EPR spectroscopy. Data were assessed with analysis of variance, Kruskal-Wallis test, analysis of response profiles, and Pearson correlation analysis. Results For all GBCAs, the kidney cortex exhibited higher gadolinium retention at 17 days after injection than all other specimens tested (mean range, 350-1720 nmol/g vs 0.40-401 nmol/g; P value range, .001-.70), with gadoteridol showing the lowest level of retention. Renal cortex R2* values correlated with gadolinium concentrations measured ex vivo (r = 0.95; P < .001), whereas no associations were found between T1-weighted signal intensity and ex vivo gadolinium concentration (r = 0.38; P = .10). EPR spectroscopy analysis of rat kidney cortex samples showed that all GBCAs were primarily intact at 52 days after injection. Conclusion Compared with other macrocyclic GBCAs, gadoteridol administration led to the lowest level of retention. The highest concentration of gadolinium was retained in the kidney cortex, but T1-weighted MRI was not sensitive for detecting residual gadolinium in this tissue. © RSNA, 2023 Supplemental material is available for this article. See also the editorial by Tweedle in this issue.

Optimization of an Allysine-Targeted PET Probe for Quantifying Fibrogenesis in a Mouse Model of Pulmonary Fibrosis.

PURPOSE: Idiopathic pulmonary fibrosis (IPF) is a destructive lung disease with a poor prognosis, an unpredictable clinical course, and inadequate therapies. There are currently no measures of disease activity to guide clinicians making treatment decisions. The aim of this study was to develop a PET probe to identify lung fibrogenesis using a pre-clinical model of pulmonary fibrosis, with potential for translation into clinical use to predict disease progression and inform treatment decisions. METHODS: Eight novel allysine-targeting chelators, PIF-1, PIF-2, …, PIF-8, with different aldehyde-reactive moieties were designed, synthesized, and radiolabeled with gallium-68 or copper-64. PET probe performance was assessed in C57BL/6J male mice 2 weeks after intratracheal bleomycin challenge and in naïve mice by dynamic PET/MR imaging and with biodistribution at 90 min post injection. Lung hydroxyproline and allysine were quantified ex vivo and histological staining for fibrosis and aldehyde was performed. RESULTS: In vivo screening of probes identified 68GaPIF-3 and 68GaPIF-7 as probes with high uptake in injured lung, high uptake in injured lung versus normal lung, and high uptake in injured lung versus adjacent liver and heart tissue. A crossover, intra-animal PET/MR imaging study of 68GaPIF-3 and 68GaPIF-7 confirmed 68GaPIF-7 as the superior probe. Specificity for fibrogenesis was confirmed in a crossover, intra-animal PET/MR imaging study with 68GaPIF-7 and a non-binding control compound, 68GaPIF-Ctrl. Substituting copper-64 for gallium-68 did not affect lung uptake or specificity indicating that either isotope could be used. CONCLUSION: A series of allysine-reactive PET probes with variations in the aldehyde-reactive moiety were evaluated in a pre-clinical model of lung fibrosis. The hydrazine-bearing probe, 68GaPIF-7, exhibited the highest uptake in fibrogenic lung, low uptake in surrounding liver or heart tissue, and low lung uptake in healthy mice and should be considered for further clinical translation.

Tailored Chemical Reactivity Probes for Systemic Imaging of Aldehydes in Fibroproliferative Diseases.

During fibroproliferation, protein-associated extracellular aldehydes are formed by the oxidation of lysine residues on extracellular matrix proteins to form the aldehyde allysine. Here we report three Mn(II)-based, small-molecule magnetic resonance probes that contain α-effect nucleophiles to target allysine in vivo and report on tissue fibrogenesis. We used a rational design approach to develop turn-on probes with a 4-fold increase in relaxivity upon targeting. The effects of aldehyde condensation rate and hydrolysis kinetics on the performance of the probes to detect tissue fibrogenesis non-invasively in mouse models were evaluated by a systemic aldehyde tracking approach. We showed that, for highly reversible ligations, off-rate was a stronger predictor of in vivo efficiency, enabling histologically validated, three-dimensional characterization of pulmonary fibrogenesis throughout the entire lung. The exclusive renal elimination of these probes allowed for rapid imaging of liver fibrosis. Reducing the hydrolysis rate by forming an oxime bond with allysine enabled delayed phase imaging of kidney fibrogenesis. The imaging efficacy of these probes, coupled with their rapid and complete elimination from the body, makes them strong candidates for clinical translation.

Tailored chemical reactivity probes for systemic imaging of aldehydes in fibroproliferative diseases.

During fibroproliferation, protein-associated extracellular aldehydes are formed by the oxidation of lysine residues on extracellular matrix proteins to form the aldehyde allysine. Here we report three Mn(II)-based, small molecule magnetic resonance (MR) probes that contain α-effect nucleophiles to target allysine in vivo and report on tissue fibrogenesis. We used a rational design approach to develop turn-on probes with a 4-fold increase in relaxivity upon targeting. The effects of aldehyde condensation rate and hydrolysis kinetics on the performance of the probes to detect tissue fibrogenesis noninvasively in mouse models were evaluated by a systemic aldehyde tracking approach. We showed that for highly reversible ligations, off-rate was a stronger predictor of in vivo efficiency, enabling histologically validated, three-dimensional characterization of pulmonary fibrogenesis throughout the entire lung. The exclusive renal elimination of these probes allowed for rapid imaging of liver fibrosis. Reducing the hydrolysis rate by forming an oxime bond with allysine enabled delayed phase imaging of kidney fibrogenesis. The imaging efficacy of these probes, coupled with their rapid and complete elimination from the body, make them strong candidates for clinical translation.

Molecular MR Imaging of Renal Fibrogenesis in Mice.

BACKGROUND: In most CKDs, lysyl oxidase oxidation of collagen forms allysine side chains, which then form stable crosslinks. We hypothesized that MRI with the allysine-targeted probe Gd-oxyamine (OA) could be used to measure this process and noninvasively detect renal fibrosis. METHODS: Two mouse models were used: hereditary nephritis in Col4a3-deficient mice (Alport model) and a glomerulonephritis model, nephrotoxic nephritis (NTN). MRI measured the difference in kidney relaxation rate, ΔR1, after intravenous Gd-OA administration. Renal tissue was collected for biochemical and histological analysis. RESULTS: ΔR1 was increased in the renal cortex of NTN mice and in both the cortex and the medulla of Alport mice. Ex vivo tissue analyses showed increased collagen and Gd-OA levels in fibrotic renal tissues and a high correlation between tissue collagen and ΔR1. CONCLUSIONS: Magnetic resonance imaging using Gd-OA is potentially a valuable tool for detecting and staging renal fibrogenesis.

Assessment of safety and feasibility of non-invasive vagus nerve stimulation for treatment of acute stroke.

BACKGROUND: Non-invasive vagus nerve stimulation (nVNS) using a hand-held stimulator placed on the neck is an FDA-approved treatment for primary headache disorders. The safety of nVNS is unknown in stroke patients. OBJECTIVE: To assess the safety and feasibility of nVNS for the acute treatment of stroke. METHODS: TR-VENUS (clinicaltrials.gov identifier NCT03733431) was a randomized, sham-controlled, open-label, multicenter trial conducted in patients with acute ischemic stroke (IS) or intracerebral hemorrhage (ICH). Patients were randomly assigned to standard-dose nVNS, high-dose nVNS, or sham stimulation. The primary endpoint was a composite safety outcome defined as bradycardia or reduction in mean arterial blood pressure during treatment or progression of neurological or death within 24 h of treatment. The feasibility endpoints were the proportion of eligible subjects receiving nVNS within 6 h of symptom onset and the proportion completing all pre-specified treatment doses. Efficacy assessments included infarct growth from baseline to 24 h after treatment. RESULTS: Sixty-nine patients (61 IS, 8 ICH) completed the study. The composite safety outcome was achieved in 32.0% in sham and 47.7% in nVNS group (p = 0.203). Treatment was initiated in all but two randomized patients. All dosed subjects received 100% of prespecified stimulations. A non-significant reduction in infarct growth was observed in the high-dose nVNS group (184.2% in sham vs. 63.3% in high-dose nVNS; p = 0.109). CONCLUSIONS: The results of this study suggest that nVNS may be safe and feasible in the setting of acute stroke. These findings support further development of nVNS as a potential treatment for acute ischemic stroke.

Short-pulsed micro-magnetic stimulation of the vagus nerve.

Vagus nerve stimulation (VNS) is commonly used to treat drug-resistant epilepsy and depression. The therapeutic effect of VNS depends on stimulating the afferent vagal fibers. However, the vagus is a mixed nerve containing afferent and efferent fibers, and the stimulation of cardiac efferent fibers during VNS may produce a rare but severe risk of bradyarrhythmia. This side effect is challenging to mitigate since VNS, via electrical stimulation technology used in clinical practice, requires unique electrode design and pulse optimization for selective stimulation of only the afferent fibers. Here we describe a method of VNS using micro-magnetic stimulation (µMS), which may be an alternative technique to induce a focal stimulation, enabling a selective fiber stimulation. Micro-coils were implanted into the cervical vagus nerve in adult male Wistar rats. For comparison, the physiological responses were recorded continuously before, during, and after stimulation with arterial blood pressure (ABP), respiration rate (RR), and heart rate (HR). The electrical VNS caused a decrease in ABP, RR, and HR, whereas µM-VNS only caused a transient reduction in RR. The absence of an HR modulation indicated that µM-VNS might provide an alternative technology to VNS with fewer heart-related side effects, such as bradyarrhythmia. Numerical electromagnetic simulations helped estimate the optimal coil orientation with respect to the nerve to provide information on the electric field's spatial distribution and strength. Furthermore, a transmission emission microscope provided very high-resolution images of the cervical vagus nerve in rats, which identified two different populations of nerve fibers categorized as large and small myelinated fibers.

Imaging High-Risk Atherothrombosis Using a Novel Fibrin-Binding Positron Emission Tomography Probe.

BACKGROUND AND PURPOSE: High-risk atherosclerosis is an underlying cause of cardiovascular events, yet identifying the specific patient population at immediate risk is still challenging. Here, we used a rabbit model of atherosclerotic plaque rupture and human carotid endarterectomy specimens to describe the potential of molecular fibrin imaging as a tool to identify thrombotic plaques. METHODS: Atherosclerotic plaques in rabbits were induced using a high-cholesterol diet and aortic balloon injury (N=13). Pharmacological triggering was used in a group of rabbits (n=9) to induce plaque disruption. Animals were grouped into thrombotic and nonthrombotic plaque groups based on gross pathology (gold standard). All animals were injected with a novel fibrin-specific probe 68Ga-CM246 followed by positron emission tomography (PET)/magnetic resonance imaging 90 minutes later. 68Ga-CM246 was quantified on the PET images using tissue-to-background (back muscle) ratios and standardized uptake value. RESULTS: Both tissue-to-background (back muscle) ratios and standardized uptake value were significantly higher in the thrombotic versus nonthrombotic group (P<0.05). Ex vivo PET and autoradiography of the abdominal aorta correlated positively with in vivo PET measurements. Plaque disruption identified by 68Ga-CM246 PET agreed with gross pathology assessment (85%). In ex vivo surgical specimens obtained from patients undergoing elective carotid endarterectomy (N=12), 68Ga-CM246 showed significantly higher binding to carotid plaques compared to a D-cysteine nonbinding control probe. CONCLUSIONS: We demonstrated that molecular fibrin PET imaging using 68Ga-CM246 could be a useful tool to diagnose experimental and clinical atherothrombosis. Based on our initial results using human carotid plaque specimens, in vivo molecular imaging studies are warranted to test 68Ga-CM246 PET as a tool to stratify risk in atherosclerotic patients.

Positron Emission Tomography-Magnetic Resonance Imaging Pharmacokinetics, In Vivo Biodistribution, and Whole-Body Elimination of Mn-PyC3A.

OBJECTIVES: Mn-PyC3A is an experimental manganese (Mn)-based extracellular fluid magnetic resonance imaging (MRI) contrast agent that is being evaluated as a direct replacement for clinical gadolinium (Gd)-based contrast agents. The goals of this study were to use simultaneous positron emission tomography (PET)-MRI to (1) compare the whole-body pharmacokinetics, biodistribution, and elimination of Mn-PyC3A with the liver-specific contrast agent mangafodipir (Mn-DPDP), (2) determine the pharmacokinetics and fractional excretion of Mn-PyC3A in a rat model of renal impairment, and (3) compare whole-body elimination of Mn-PyC3A to gadoterate (Gd-DOTA) in a rat model of renal impairment. METHODS: Mn-PyC3A and Mn-DPDP were radiolabeled with the positron emitting isotope Mn-52 via Mn2+ exchange with 52MnCl2. Dynamic simultaneous PET-MRI was used to measure whole-body pharmacokinetics and biodistribution of Mn-52 immediately and out to 7 days after an intravenous 0.2 mmol/kg dose of [52Mn]Mn-PyC3A to normal or to 5/6 nephrectomy rats or a 0.01 mmol/kg dose of [52Mn]Mn-DPDP to normal rats. The fractional excretion and 1- and 7-day biodistribution in rats after the injection of 2.0 mmol/kg [52Mn]Mn-PyC3A (n = 11 per time point) or 2.0 mmol/kg Gd-DOTA (n = 8 per time point) were quantified by gamma counting or Gd elemental analysis, respectively. Comparisons of Mn-PyC3A pharmacokinetics and in vivo biodistribution in normal and 5/6 nephrectomy rats and comparisons of ex vivo Mn versus Gd biodistribution data in 5/6 nephrectomy were made with an unpaired t test. RESULTS: Dynamic PET-MRI data demonstrate that both [52Mn]Mn-PyC3A and [52Mn]Mn-DPDP were eliminated by mixed renal and hepatobiliary elimination but that a greater fraction of [52Mn]Mn-PyC3A was eliminated by renal filtration. Whole-body PET images show that Mn-52 from [52Mn]Mn-PyC3A was efficiently eliminated from the body, whereas Mn-52 from [52Mn]Mn-DPDP was retained throughout the body. The blood elimination half-life of [52Mn]Mn-PyC3A in normal and 5/6 nephrectomy rats was 13 ± 3.5 minutes and 23 ± 12 minutes, respectively (P = 0.083). Area under the curve between 0 and 60 minutes postinjection (AUC0-60) in the bladder of normal and 5/6 nephrectomy rats was 2600 ± 1700 %ID/cc*min and 750 ± 180 %ID/cc*min, respectively (P = 0.024), whereas AUC0-60 in the liver of normal and 5/6 nephrectomy rats was 33 ± 13 %ID/cc*min and 71 ± 16 %ID/cc*min, respectively (P = 0.011), indicating increased hepatobiliary elimination in 5/6 nephrectomy rats. The %IDs of Mn from [52Mn]Mn-PyC3A and Gd from Gd-DOTA recovered from 5/6 nephrectomy rats 1 day after injection were 2.0 ± 1.1 and 1.3 ± 0.34, respectively (P = 0.10) and 7 days after injection were 0.14 ± 0.11 and 0.41 ± 0.24, respectively (P = 0.0041). CONCLUSIONS: Mn-PyC3A has different pharmacokinetics and is more efficiently eliminated than Mn-DPDP in normal rats. Mn-PyC3A is efficiently eliminated from both normal and 5/6 nephrectomy rats, with increased fractional hepatobiliary excretion from 5/6 nephrectomy rats. Mn-PyC3A is more completely eliminated than Gd-DOTA from 5/6 nephrectomy rats after 7 days.

Vagus nerve stimulation inhibits cortical spreading depression exclusively through central mechanisms.

Experimental and clinical data strongly support vagus nerve stimulation (VNS) as a novel treatment in migraine. Vagus nerve stimulation acutely suppresses cortical spreading depression (CSD) susceptibility, an experimental model that has been used to screen for migraine therapies. However, mechanisms underlying VNS efficacy on CSD are unknown. Here, we interrogated the central and peripheral mechanisms using VNS delivered either invasively (iVNS) or noninvasively (nVNS) in male Sprague-Dawley rats. Cortical spreading depression susceptibility was evaluated 40 minutes after the stimulation. iVNS elevated the electrical CSD threshold more than 2-fold and decreased KCl-induced CSD frequency by 22% when delivered to intact vagus nerve. Distal vagotomy did not alter iVNS efficacy (2-fold higher threshold and 19% lower frequency in iVNS vs sham). By contrast, proximal vagotomy completely abolished iVNS effect on CSD. Pharmacological blockade of nucleus tractus solitarius, the main relay for vagal afferents, by lidocaine or glutamate receptor antagonist CNQX also prevented CSD suppression by nVNS. Supporting a role for both norepinephrine and serotonin, CSD suppression by nVNS was inhibited by more than 50% after abrogating norepinephrinergic or serotonergic neurotransmission alone using specific neurotoxins; abrogating both completely blocked the nVNS effect. Our results suggest that VNS inhibits CSD through central afferents relaying in nucleus tractus solitarius and projecting to subcortical neuromodulatory centers providing serotonergic and norepinephrinergic innervation to the cortex.

The influence of respiration on brainstem and cardiovagal response to auricular vagus nerve stimulation: A multimodal ultrahigh-field (7T) fMRI study.

BACKGROUND: Brainstem-focused mechanisms supporting transcutaneous auricular VNS (taVNS) effects are not well understood, particularly in humans. We employed ultrahigh field (7T) fMRI and evaluated the influence of respiratory phase for optimal targeting, applying our respiratory-gated auricular vagal afferent nerve stimulation (RAVANS) technique. HYPOTHESIS: We proposed that targeting of nucleus tractus solitarii (NTS) and cardiovagal modulation in response to taVNS stimuli would be enhanced when stimulation is delivered during a more receptive state, i.e. exhalation. METHODS: Brainstem fMRI response to auricular taVNS (cymba conchae) was assessed for stimulation delivered during exhalation (eRAVANS) or inhalation (iRAVANS), while exhalation-gated stimulation over the greater auricular nerve (GANctrl, i.e. earlobe) was included as control. Furthermore, we evaluated cardiovagal response to stimulation by calculating instantaneous HF-HRV from cardiac data recorded during fMRI. RESULTS: Our findings demonstrated that eRAVANS evoked fMRI signal increase in ipsilateral pontomedullary junction in a cluster including purported NTS. Brainstem response to GANctrl localized a partially-overlapping cluster, more ventrolateral, consistent with spinal trigeminal nucleus. A region-of-interest analysis also found eRAVANS activation in monoaminergic source nuclei including locus coeruleus (LC, noradrenergic) and both dorsal and median raphe (serotonergic) nuclei. Response to eRAVANS was significantly greater than iRAVANS for all nuclei, and greater than GANctrl in LC and raphe nuclei. Furthermore, eRAVANS, but not iRAVANS, enhanced cardiovagal modulation, confirming enhanced eRAVANS response on both central and peripheral neurophysiological levels. CONCLUSION: 7T fMRI localized brainstem response to taVNS, linked such response with autonomic outflow, and demonstrated that taVNS applied during exhalation enhanced NTS targeting.

New biomarker for acute ischaemic stroke: plasma glycogen phosphorylase isoenzyme BB.

BACKGROUND: Glycogen phosphorylase is the key enzyme that breaks down glycogen to yield glucose-1-phosphate in order to restore depleted energy stores during cerebral ischaemia. We sought to determine whether plasma levels of glycogen phosphorylase BB (GPBB) isoform increased in patients with acute ischaemic stroke (AIS). METHODS: We studied plasma GPBB levels within 12 hours and again at 48±24 hours of symptom onset in 172 patients with imaging-confirmed AIS and 133 stroke-free individuals. We determined the ability of plasma GPBB to discriminate between cases and controls and examined the predictive value of plasma GPBB for 90-day functional outcome, 90-day survival and acute lesion volumes on neuroimaging. RESULTS: The mean (SD) GPBB levels were higher in cases (46.3±38.6 ng/mL at first measurement and 38.6±36.5 ng/mL at second measurement) than in controls (4.1±7.6 ng/mL, p<0.01 for both). The area under the receiver operating characteristic (ROC) curve for case-control discrimination based on first GPBB measurement was 0.96 (95% CI 0.93 to 0.98). The sensitivity and specificity based on optimal operating point on the ROC curve (7.0 ng/mL) were both 93%. GPBB levels increased in 90% of patients with punctate infarcts (<1.5 mL) and in all patients admitted within the first 4.5 hours of onset. There was no correlation between GPBB concentration and either clinical outcome or acute infarct volume. CONCLUSION: GPBB demonstrates robust response to acute ischaemia and high sensitivity for small infarcts. If confirmed in more diverse populations that also include stroke mimics, GPBB could find utility as a stand-alone marker for acute brain ischaemia.

Transcutaneous Cervical Vagus Nerve Stimulation Ameliorates Acute Ischemic Injury in Rats.

BACKGROUND: Direct stimulation of the vagus nerve in the neck via surgically implanted electrodes is protective in animal models of stroke. We sought to determine the safety and efficacy of a non-invasive cervical VNS (nVNS) method using surface electrodes applied to the skin overlying the vagus nerve in the neck in a model of middle cerebral artery occlusion (MCAO). METHODS: nVNS was initiated variable times after MCAO in rats (n = 33). Control animals received sham stimulation (n = 33). Infarct volume and functional outcome were assessed on day 7. Brains were processed by immunohistochemistry for microglial activation and cytokine levels. The ability of nVNS to activate the nucleus tractus solitarius (NTS) was assessed using c-Fos immunohistochemistry. RESULTS: Infarct volume was 43.15 ± 3.36 percent of the contralateral hemisphere (PCH) in control and 28.75 ± 4.22 PCH in nVNS-treated animals (p < 0.05). The effect of nVNS on infarct size was consistent when stimulation was initiated up to 4 hours after MCAO. There was no difference in heart rate and blood pressure between control and nVNS-treated animals. The number of c-Fos positive cells was 32.4 ± 10.6 and 6.2 ± 6.3 in the ipsilateral NTS (p < 0.05) and 30.4 ± 11.2 and 5.8 ± 4.3 in the contralateral NTS (p < 0.05) in nVNS-treated and control animals, respectively. nVNS reduced the number of Iba-1, CD68, and TNF-α positive cells and increased the number of HMGB1 positive cells. CONCLUSIONS: nVNS inhibits ischemia-induced immune activation and reduces the extent of tissue injury and functional deficit in rats without causing cardiac or hemodynamic adverse effects when initiated up to 4 hours after MCAO.

Vagus nerve stimulation inhibits cortical spreading depression.

Vagus nerve stimulation has recently been reported to improve symptoms of migraine. Cortical spreading depression is the electrophysiological event underlying migraine aura and is a trigger for headache. We tested whether vagus nerve stimulation inhibits cortical spreading depression to explain its antimigraine effect. Unilateral vagus nerve stimulation was delivered either noninvasively through the skin or directly by electrodes placed around the nerve. Systemic physiology was monitored throughout the study. Both noninvasive transcutaneous and invasive direct vagus nerve stimulations significantly suppressed spreading depression susceptibility in the occipital cortex in rats. The electrical stimulation threshold to evoke a spreading depression was elevated by more than 2-fold, the frequency of spreading depressions during continuous topical 1 M KCl was reduced by ∼40%, and propagation speed of spreading depression was reduced by ∼15%. This effect developed within 30 minutes after vagus nerve stimulation and persisted for more than 3 hours. Noninvasive transcutaneous vagus nerve stimulation was as efficacious as direct invasive vagus nerve stimulation, and the efficacy did not differ between the ipsilateral and contralateral hemispheres. Our findings provide a potential mechanism by which vagus nerve stimulation may be efficacious in migraine and suggest that susceptibility to spreading depression is a suitable platform to optimize its efficacy.

A Manganese Alternative to Gadolinium for MRI Contrast.

Contrast-enhanced computed tomography (CT) and magnetic resonance imaging (MRI) are routinely used to diagnose soft tissue and vascular abnormalities. However, safety concerns limit the use of iodinated and gadolinium (Gd)-based CT and MRI contrast media in renally compromised patients. With an estimated 14% of the US population suffering from chronic kidney disease (CKD), contrast media compatible with renal impairment is sorely needed. We present the new manganese(II) complex [Mn(PyC3A)(H2O)](-) as a Gd alternative. [Mn(PyC3A)(H2O)](-) is among the most stable Mn(II) complexes at pH 7.4 (log KML = 11.40). In the presence of 25 mol equiv of Zn at pH 6.0, 37 °C, [Mn(PyC3A)(H2O)](-) is 20-fold more resistant to dissociation than [Gd(DTPA)(H2O)](2-). Relaxivity of [Mn(PyC3A)(H2O)](-) in blood plasma is comparable to commercial Gd contrast agents. Biodistribution analysis confirms that [Mn(PyC3A)(H2O)](-) clears via a mixed renal/hepatobiliary pathway with >99% elimination by 24 h. [Mn(PyC3A)(H2O)](-) was modified to form a bifunctional chelator and 4 chelates were conjugated to a fibrin-specific peptide to give Mn-FBP. Mn-FBP binds the soluble fibrin fragment DD(E) with Kd = 110 nM. Per Mn relaxivity of Mn-FBP is 4-fold greater than [Mn(PyC3A)(H2O)](-) and increases 60% in the presence of fibrin, consistent with binding. Mn-FBP provided equivalent thrombus enhancement to the state of the art Gd analogue, EP-2104R, in a rat model of arterial thrombosis. Mn metabolite analysis reveals no evidence of dechelation and the probe was >99% eliminated after 24 h. [Mn(PyC3A)(H2O)](-) is a lead development candidate for an imaging probe that is compatible with renally compromised patients.

Electrical stimulation of the vagus nerve dermatome in the external ear is protective in rat cerebral ischemia.

BACKGROUND: Although cervical vagus nerve stimulation is effective for reducing infarct volume in rats, it is not feasible for acute human stroke as it requires surgical incision of the neck. We hypothesized that stimulation of the dermatome in the external ear innervated by the vagus nerve (auricular vagus nerve stimulation; aVNS) reduces infarct volume after transient focal ischemia in rats. METHODS: Animals were randomized to active aVNS or sham stimulation. For aVNS, electrical stimulation of the left cavum concha (1 h duration) using percutaneous needles was initiated 30 min after induction of ischemia. Behavioral and tissue outcome were measured 24 h after induction of ischemia. In a separate experimental dataset, c-Fos immunohistochemistry was performed to identify the brain regions activated after the stimulation. RESULTS: Stimulation of the left cavum concha resulted in bilateral c-Fos staining in the nuclei tractus solitarii and the loci coerulei in all animals. There was no c-Fos staining in any part of the brainstem in sham control animals. The mean infarct volume (SD) as calculated by indirect method was 44.20 ± 7.58% in controls and 31.65 ± 9.67% in treated animals (P < 0.0001). The effect of aVNS on tissue outcome was associated with better neurological scores at 24 h after ischemia (P < 0.0001). CONCLUSIONS: Electric stimulation of the vagus nerve dermatome in the external ear activates brainstem afferent vagal nuclei and reduces infarct volume in rats. This finding has potential to facilitate the development of treatments that leverage the brain's endogenous neuroprotective pathways at the setting of acute ischemic stroke.

Effect of Chelate Type and Radioisotope on the Imaging Efficacy of 4 Fibrin-Specific PET Probes.

UNLABELLED: Thrombus formation plays a major role in cardiovascular diseases, but noninvasive thrombus imaging is still challenging. Fibrin is a major component of both arterial and venous thrombi and represents an ideal candidate for imaging of thrombosis. Recently, we showed that (64)Cu-DOTA-labeled PET probes based on fibrin-specific peptides are suitable for thrombus imaging in vivo; however, the metabolic stability of these probes was limited. Here, we describe 4 new probes using either (64)Cu or aluminum fluoride (Al(18)F) chelated to 2 NOTA derivatives. METHODS: Probes were synthesized using a known fibrin-specific peptide conjugated to either NODAGA (FBP8, FBP10) or NOTA-monoamide (FBP9, FBP11) as chelators, followed by labeling with (64)Cu (FBP8 and FBP9) or Al(18)F (FBP10 and FBP11). PET imaging efficacy, pharmacokinetics, biodistribution, and metabolic stability were assessed in a rat model of arterial thrombosis. RESULTS: All probes had similar nanomolar affinity (435-760 nM) for the soluble fibrin fragment DD(E). PET imaging allowed clear visualization of thrombus by all probes, with a 5-fold or higher thrombus-to-background ratio. Compared with the previous DOTA derivative, the new (64)Cu probes FBP8 and FBP9 showed substantially improved metabolic stability (>85% intact in blood at 4 h after injection), resulting in high uptake at the target site (0.5-0.8 percentage injected dose per gram) that persisted over 5 h, producing increasingly greater target-to-background ratios. The thrombus uptake was 5- to 20-fold higher than the uptake in the contralateral artery, blood, muscle, lungs, bone, spleen, large intestine, and heart at 2 h after injection and 10- to 40-fold higher at 5 h. The Al(18)F derivatives FBP10 and FBP11 were less stable, in particular the NODAGA conjugate (FBP10, <30% intact in blood at 4 h after injection), which showed high bone uptake and low thrombus-to-background ratios that decreased over time. The high thrombus-to-contralateral ratios for all probes were confirmed by ex vivo biodistribution and autoradiography. The uptake in the liver (<0.5 percentage injected dose per gram), kidneys, and blood were similar for all tracers, and they all showed predominant renal clearance. CONCLUSION: FBP8, FBP9, and FBP11 showed excellent metabolic stability and high thrombus-to-background ratios and represent promising candidates for imaging of thrombosis in vivo.

In vivo molecular imaging of thrombosis and thrombolysis using a fibrin-binding positron emission tomographic probe.

BACKGROUND: Fibrin is a major component of arterial and venous thrombi and represents an ideal candidate for molecular imaging of thrombosis. Here, we describe imaging properties and target uptake of a new fibrin-specific positron emission tomographic probe for thrombus detection and therapy monitoring in 2 rat thrombosis models. METHODS AND RESULTS: The fibrin-binding probe FBP7 was synthesized by conjugation of a known short cyclic peptide to a cross-bridged chelator (CB-TE2A), followed by labeling with copper-64. Adult male Wistar rats (n=26) underwent either carotid crush injury (mural thrombosis model) or embolic stroke (occlusive thrombosis model) followed by recombinant tissue-type plasminogen activator treatment (10 mg/kg, IV). FBP7 detected thrombus location in both animal models with a high positron emission tomographic target-to-background ratio that increased over time (>5-fold at 30-90 minutes, >15-fold at 240-285 minutes). In the carotid crush injury animals, biodistribution analysis confirmed high probe uptake in the thrombotic artery (≈0.5%ID/g; >5-fold greater than blood and other tissues of the head and thorax). Similar results were obtained from ex vivo autoradiography of the ipsilateral versus contralateral carotid arteries. In embolic stroke animals, positron emission tomographic-computed tomographic imaging localized the clot in the internal carotid/middle cerebral artery segment of all rats. Time-dependent reduction of activity at the level of the thrombus was detected in recombinant tissue-type plasminogen activator-treated rats but not in vehicle-injected animals. Brain autoradiography confirmed clot dissolution in recombinant tissue-type plasminogen activator-treated animals, but enduring high thrombus activity in control rats. CONCLUSIONS: We demonstrated that FBP7 is suitable for molecular imaging of thrombosis and thrombolysis in vivo and represents a promising candidate for bench-to-bedside translation.