Neutrophils and Neutrophil Extracellular Traps Cause Vascular Occlusion and Delayed Cerebral Ischemia After Subarachnoid Hemorrhage in Mice.

BACKGROUND: After subarachnoid hemorrhage (SAH), neutrophils are deleterious and contribute to poor outcomes. Neutrophils can produce neutrophil extracellular traps (NETs) after ischemic stroke. Our hypothesis was that, after SAH, neutrophils contribute to delayed cerebral ischemia (DCI) and worse outcomes via cerebrovascular occlusion by NETs. METHODS: SAH was induced via endovascular perforation, and SAH mice were given either a neutrophil-depleting antibody, a PAD4 (peptidylarginine deiminase 4) inhibitor (to prevent NETosis), DNAse-I (to degrade NETs), or a vehicle control. Mice underwent daily neurological assessment until day 7 and then euthanized for quantification of intravascular brain NETs (iNETs). Subsets of mice were used to quantify neutrophil infiltration, NETosis potential, iNETs, cerebral perfusion, and infarction. In addition, NET markers were assessed in the blood of aneurysmal SAH patients. RESULTS: In mice, SAH led to brain neutrophil infiltration within 24 hours, induced a pro-NETosis phenotype selectively in skull neutrophils, and caused a significant increase in iNETs by day 1, which persisted until at least day 7. Neutrophil depletion significantly reduced iNETs, improving cerebral perfusion, leading to less neurological deficits and less incidence of DCI (16% versus 51.9%). Similarly, PAD4 inhibition reduced iNETs, improved neurological outcome, and reduced incidence of DCI (5% versus 30%), whereas degrading NETs marginally improved outcomes. Patients with aneurysmal SAH who developed DCI had elevated markers of NETs compared with non-DCI patients. CONCLUSIONS: After SAH, skull-derived neutrophils are primed for NETosis, and there are persistent brain iNETs, which correlated with delayed deficits. The findings from this study suggest that, after SAH, neutrophils and NETosis are therapeutic targets, which can prevent vascular occlusion by NETs in the brain, thereby lessening the risk of DCI. Finally, NET markers may be biomarkers, which can predict which patients with aneurysmal SAH are at risk for developing DCI.

Innate immune activation and white matter injury in a rat model of neonatal intraventricular hemorrhage are dependent on developmental stage.

BACKGROUND: Inflammation and white matter injury are consequences of neonatal intraventricular hemorrhage (IVH). Both white matter and the neuroimmune system are developing during the time which IVH occurs and its consequences develop. IVH has been studied in many different animal models; however, the effects of IVH occurring at different developmental time points in the same model have not been examined. Understanding how the timing of IVH affects outcome may provide important insights into both IVH pathophysiology and innate immune development. METHODS: We used intraventricular injection of lysed whole blood to model neonatal IVH in postnatal day (P)2 and P5 rats. Flow cytometry was used to detect innate immune activation. MRI was used to screen animals for the development of increased ventricular size. Immunohistochemistry for myelin basic protein was used to quantify white matter and corpus callosum thickness. RESULTS: P5 animals exhibited significant increases in several measures of classically pro-inflammatory innate immune activation that P2 animals did not. Animals with IVH induced at P5 also developed ventricular enlargement visible on MRI whereas animals with IVH induced at P2 did not. On histological analysis, there were no significant effects of IVH in P2 animals, but IVH in P5 animals reduced white matter labeling and corpus callosum thickness. CONCLUSIONS: IVH induces a strong innate inflammatory response in P5 as well as changes in ventricular size and reduction of white matter. P2 animals do not exhibit significant changes in innate immune activation or white matter structure after IVH. This suggests that white matter pathology from IVH is due in part to innate immune activation; and that the developmental stage of the innate immune system is a key determinant of IVH pathology.

Lactobacillus plantarum isolated from kefir enhances immune responses and survival of white shrimp (Penaeus vannamei) challenged with Vibrio alginolyticus.

Lactobacillus plantarum is known for its probiotics benefit to host, although the effects vary among strains. This study conducted a feeding experiment of three Lactobacillus strains, MRS8, MRS18 and MRS20, which were isolated from kefir and incorporated into the diets of shrimp to evaluate the effects of non-specific immunity, immune-related gene expression, and disease resistance of white shrimp (Penaeus vannamei) against Vibrio alginolyticus. To prepare the experimental feed groups, the basic feed was mixed with different concentrations of L. plantarum strains MRS8, MRS18, and MRS 20, which were incorporated at 0 CFU (control), 1 × 106 CFU (groups 8-6, 18-6, and 20-6), and 1 × 109 CFU (groups 8-9, 18-9, and 20-9) per gram of diet for an in vivo assay. During the rearing period for 28 days of feeding each group, immune responses, namely the total hemocyte count (THC), phagocytic rate (PR), phenoloxidase activity, and respiratory burst were examined on days 0, 1, 4, 7, 14, and 28. The results showed that groups 20-6, 18-9 and 20-9 improved THC, and groups 18-9 and 20-9 improved phenoloxidase activity and respiratory burst as well. The expression of immunity-related genes was also examined. Group 8-9 increased the expression of LGBP, penaeidin 2 (PEN2) and CP, group 18-9 increased the expression of proPO1, ALF, Lysozyme, penaeidin 3 (PEN3) and SOD, and group 20-9 increased the expression of LGBP, ALF, crustin, PEN2, PEN3, penaeidin 4 (PEN4) and CP (p < 0.05). Groups 18-6, 18-9, 2-6, and 20-9 were further used in the challenge test. After feeding for 7 days and 14 days, Vibrio alginolyticus was injected into white shrimp and observed the shrimp survival for 168 h. The results showed that compared to the control, all groups improved the survival rate. Especially, feeding group 18-9 for 14 days improved the survival rate of white shrimp (p < 0.05). After the challenge test for 14 days, the midgut DNA of survival white shrimps was extracted to analyze the colonization of L. plantarum. Among the groups, (6.61 ± 3.58) × 105 CFU/pre shrimp of L. plantarum in feeding group 18-9 and (5.86 ± 2.27) × 105 CFU/pre shrimp in group 20-9 were evaluated by qPCR. Taken together, group 18-9 had the best effects on the non-specific immunity, the immune-related gene expression, and the disease resistance, which might be due to the benefit of the probiotic colonization.

Dietary of Lactobacillus paracasei and Bifidobacterium longum improve nonspecific immune responses, growth performance, and resistance against Vibrio parahaemolyticus in Penaeus vannamei.

This study investigated the effects of two probiotics, namely Lactobacillus paracasei and Bifidobacterium longum, as feed additives on growth performance, nonspecific immunity, immune-related gene expression, and disease resistance against Vibrio parahaemolyticus in Penaeus vannamei. The experimental diets were prepared using L. paracasei and B. longum at concentrations of 105 and 107 CFU/g; these diets were referred to as P5, P7, B5, and B7. After 8 weeks of the diets, regarding growth performance, the B7 group showed the highest weight gain rate (890.34 ± 103.65%), special growth rate (4.08 ± 0.19%), and feed conversion rate (1.52 ± 0.19%) compared with the other groups. Moreover, the total hemocyte counts were significantly increased (p < 0.05) in the P7 groups on day 14 during the 28-day feeding trial. The phagocytosis rate in all experimental groups was increased on day 14 and was persistently significantly activated to day 21, especially in the P7 and B5 group. The phagocytic index of the P7 group showed a significant increase on day 14 and persistent activation to day 21. In the analysis of respiratory burst activity and phenoloxidase activity, the P7 and B5 groups showed a significant increase on day 7 and persistent activation to day 21. The expression level of the immune-related genes of superoxide dismutase, clotting protein, Penaeidin2, Penaeidin3, Penaeidin4, anti-LPS factor, crustin, and lysozyme was significantly increased in the experimental groups, especially in the P7 group. Furthermore, the optimum conditions of feed additives were determined in challenge trials conducted using P7 and B5. Shrimps fed P7 and B5 showed an increased survival rate (72.73% and 66.67%) after the V. parahaemolyticus challenge. In sum, the results revealed that B. longum, as a feed additive at 107 CFU/g, enhanced growth performance. L. paracasei at 107 CFU/g and B. longum at 105 CFU/g can enhance nonspecific immune responses and immune-related gene expression, and 107 CFU/g L. paracasei has the highest resistance ability for V. parahaemolyticus. Thus, dietary supplementation with L. paracasei and B. longum may be a valuable approach in white shrimp aquaculture.

A specific primed immune response in red palm weevil, Rhynchophorus ferrugineus, is mediated by hemocyte differentiation and phagocytosis.

Red palm weevil, Rhynchophorus ferrugineus, is an invasive and destructive pest that causes serious damages to palm trees. Like other invertebrates, red palm weevil relies solely on its innate immune response to fight invading microbes; by definition, innate immunity lacks adaptive characteristics. However, we show here that priming the red palm weevil larvae with heat-killed Bacillus thuringiensis specifically increased survival of the larvae during a secondary lethal infection with live bacteria, and B. thuringiensis primed larvae also showed a higher clearance efficiency for this bacterium, which indicated that the red palm weevil larvae possessed a strong immune priming response. The degree of enhanced immune protection was positively correlated with hemocyte proliferation and the level of phagocytic ability of hemocytes. Moreover, the red palm weevil larvae primed by B. thuringiensis induced the continuous synthesis of serotonin in the hemolymph, which in turn enhanced the phagocytic ability and pathogen clearance ability of the host, representing an important mechanism for the red palm weevil to achieve priming protection. Our findings reveal a specific immune priming of the red palm weevil larvae mediated by the continuous secretion of serotonin, and provide new insights into the mechanisms of invertebrates immune priming.

The Secrets of the Frogs Heart.

The heart of the African clawed frog has a double-inlet and single-outlet ventricle supporting systemic and pulmonary circulations via a truncus, and a lifespan of 25-30 years. We sought to understand the unique cardiac anatomic and physiologic characteristics, with balanced circulation and low metabolic rate, by comparing the basic anatomy structures with focused echocardiography and cardiac magnetic resonance imaging. Twenty-four adult female African clawed frogs were randomly subjected to anatomic dissection (n = 4), echocardiography (n = 10), and cardiac magnetic resonance (n = 10). All anatomical features were confirmed and compared with echocardiography and cardiac magnetic resonance imaging. The main characteristics of the cardiovascular circulation in frogs are the following: Intact interatrial septum, with two separate atrio-ventricular valves, preventing atrial mixing of oxygenated and desaturated blood. Single spongiform ventricular cavity, non-conducive for homogeneous mixing. Single outlet with a valve-like mobile spiral structure, actively streaming into systemic and pulmonary arteries. Intact interatrial septum, spongiform ventricle, and valve-like spiral in the conus arteriosus are likely responsible for balanced systemic and pulmonary circulation in frogs, in spite of double-inlet and single-outlet ventricle.

Selective Endothelial Hyperactivation of Oncogenic KRAS Induces Brain Arteriovenous Malformations in Mice.

OBJECTIVE: Brain arteriovenous malformations (bAVMs) are a leading cause of hemorrhagic stroke and neurological deficits in children and young adults, however, no pharmacological intervention is available to treat these patients. Although more than 95% of bAVMs are sporadic without family history, the pathogenesis of sporadic bAVMs is largely unknown, which may account for the lack of therapeutic options. KRAS mutations are frequently observed in cancer, and a recent unprecedented finding of these mutations in human sporadic bAVMs offers a new direction in the bAVM research. Using a novel adeno-associated virus targeting brain endothelium (AAV-BR1), the current study tested if endothelial KRASG12V mutation induces sporadic bAVMs in mice. METHODS: Five-week-old mice were systemically injected with either AAV-BR1-GFP or -KRASG12V . At 8 weeks after the AAV injection, bAVM formation and characteristics were addressed by histological and molecular analyses. The effect of MEK/ERK inhibition on KRASG12V -induced bAVMs was determined by treatment of trametinib, a US Food and Drug Administration (FDA)-approved MEK/ERK inhibitor. RESULTS: The viral-mediated KRASG12V overexpression induced bAVMs, which were composed of a tangled nidus mirroring the distinctive morphology of human bAVMs. The bAVMs were accompanied by focal angiogenesis, intracerebral hemorrhages, altered vascular constituents, neuroinflammation, and impaired sensory/cognitive/motor functions. Finally, we confirmed that bAVM growth was inhibited by trametinib treatment. INTERPRETATION: Our innovative approach using AAV-BR1 confirms that KRAS mutations promote bAVM development via the MEK/ERK pathway, and provides a novel preclinical mouse model of bAVMs which will be useful to develop a therapeutic strategy for patients with bAVM. ANN NEUROL 2021;89:926-941.

Neuropeptides and G-Protein Coupled Receptors (GPCRs) in the Red Palm Weevil Rhynchophorus ferrugineus Olivier (Coleoptera: Dryophthoridae).

The red palm weevil Rhynchophorus ferrugineus is a devastating, invasive pest that causes serious damages to palm trees, and its invasiveness depends on its strong ability of physiological and behavioral adaptability. Neuropeptides and their receptors regulate physiology and behavior of insects, but these protein partners have not been identified from many insects. Here, we systematically identified neuropeptide precursors and the corresponding receptors in the red palm weevil, and analyzed their tissue expression patterns under control conditions and after pathogen infection. A total of 43 putative neuropeptide precursors were identified, including an extra myosuppressin peptide was identified with amino acid substitutions at two conserved sites. Forty-four putative neuropeptide receptors belonging to three classes were also identified, in which neuropeptide F receptors and insulin receptors were expanded compared to those in other insects. Based on qRT-PCR analyses, genes coding for several neuropeptide precursors and receptors were highly expressed in tissues other than the nervous system, suggesting that these neuropeptides and receptors play other roles in addition to neuro-reception. Some of the neuropeptides and receptors, like the tachykinin-related peptide and receptor, were significantly induced by pathogen infection, especially sensitive to Bacillus thuringiensis and Metarhizium anisopliae. Systemic identification and initial characterization of neuropeptides and their receptors in the red palm weevil provide a framework for further studies to reveal the functions of these ligand- and receptor-couples in regulating physiology, behavior, and immunity in this important insect pest species.

Identification of Novel ARSB Genes Necessary for p-Benzoquinone Biosynthesis in the Larval Oral Secretion Participating in External Immune Defense in the Red Palm Weevil.

External secretions, composed of a variety of chemical components, are among the most important traits that endow insects with the ability to defend themselves against predators, parasites, or other adversities, especially pathogens. Thus, these exudates play a crucial role in external immunity. Red palm weevil larvae are prolific in this regard, producing large quantities of p-benzoquinone, which is present in their oral secretion. Benzoquinone with antimicrobial activity has been proven to be an active ingredient and key factor for external immunity in a previous study. To obtain a better understanding of the genetic and molecular basis of external immune secretions, we identify genes necessary for p-benzoquinone synthesis. Three novel ARSB genes, namely, RfARSB-0311, RfARSB-11581, and RfARSB-14322, are screened, isolated, and molecularly characterized on the basis of transcriptome data. To determine whether these genes are highly and specifically expressed in the secretory gland, we perform tissue/organ-specific expression profile analysis. The functions of these genes are further determined by examining the antimicrobial activity of the secretions and quantification of p-benzoquinone after RNAi. All the results reveal that the ARSB gene family can regulate the secretory volume of p-benzoquinone by participating in the biosynthesis of quinones, thus altering the host's external immune inhibitory efficiency.

Picometer scale vibrometry in the human middle ear using a surgical microscope based optical coherence tomography and vibrometry system.

We have developed a highly phase stable optical coherence tomography and vibrometry system that attaches directly to the accessory area of a surgical microscope common to both the otology clinic and operating room. Careful attention to minimizing sources of phase noise has enabled a system capable of measuring vibrations of the middle ear with a sensitivity of < 5 pm in an awake human patient. The system is shown to be capable of collecting a wide range of information on the morphology and function of the ear in live subjects, including frequency tuning curves below the hearing threshold, maps of tympanic membrane vibrational modes and thickness, and measures of distortion products due to the nonlinearities in the cochlear amplifier.

Rapid and quantitative chemical exchange saturation transfer (CEST) imaging with magnetic resonance fingerprinting (MRF).

PURPOSE: To develop a fast magnetic resonance fingerprinting (MRF) method for quantitative chemical exchange saturation transfer (CEST) imaging. METHODS: We implemented a CEST-MRF method to quantify the chemical exchange rate and volume fraction of the Nα -amine protons of L-arginine (L-Arg) phantoms and the amide and semi-solid exchangeable protons of in vivo rat brain tissue. L-Arg phantoms were made with different concentrations (25-100 mM) and pH (pH 4-6). The MRF acquisition schedule varied the saturation power randomly for 30 iterations (phantom: 0-6 µT; in vivo: 0-4 µT) with a total acquisition time of ≤2 min. The signal trajectories were pattern-matched to a large dictionary of signal trajectories simulated using the Bloch-McConnell equations for different combinations of exchange rate, exchangeable proton volume fraction, and water T1 and T2 relaxation times. RESULTS: The chemical exchange rates of the Nα -amine protons of L-Arg were significantly (P < 0.0001) correlated with the rates measured with the quantitation of exchange using saturation power method. Similarly, the L-Arg concentrations determined using MRF were significantly (P < 0.0001) correlated with the known concentrations. The pH dependence of the exchange rate was well fit (R2 = 0.9186) by a base catalyzed exchange model. The amide proton exchange rate measured in rat brain cortex (34.8 ± 11.7 Hz) was in good agreement with that measured previously with the water exchange spectroscopy method (28.6 ± 7.4 Hz). The semi-solid proton volume fraction was elevated in white (12.2 ± 1.7%) compared to gray (8.1 ± 1.1%) matter brain regions in agreement with previous magnetization transfer studies. CONCLUSION: CEST-MRF provides a method for fast, quantitative CEST imaging.

ATX-MS-1467 Induces Long-Term Tolerance to Myelin Basic Protein in (DR2 × Ob1)F1 Mice by Induction of IL-10-Secreting iTregs.

INTRODUCTION: Antigen-specific immunotherapy could provide a targeted approach for the treatment of multiple sclerosis that removes the need for broad-acting immunomodulatory drugs. ATX-MS-1467 is a mixture of four peptides identified as the main immune-dominant disease-associated T-cell epitopes in myelin basic protein (MBP), an autoimmune target for activated autoreactive T cells in multiple sclerosis. Previous animal studies have shown that ATX-MS-1467 treatment prevented the worsening of signs of disease in experimental autoimmune encephalitis (EAE) in the humanized (DR2 × Ob1)F1 mouse in a dose-dependent fashion. METHODS AND RESULTS: Our study extends these observations to show that subcutaneous treatment with 100 µg of ATX-MS-1467 after induction of EAE in the same mouse model reversed established clinical disability (p < 0.0001) and histological markers of inflammation and demyelination (p < 0.001) compared with vehicle-treated animals; furthermore, in longitudinal magnetic resonance imaging analyses, disruption of blood-brain barrier integrity was reversed, compared with vehicle-treated animals (p < 0.05). Chronic treatment with ATX-MS-1467 was associated with an enduring shift from a pro-inflammatory to a tolerogenic state in the periphery, as shown by an increase in interleukin 10 secretion, relative to interleukin 2, interleukin 17 and interferon γ, a decrease in splenocyte proliferation and an increase in interleukin 10+ Foxp3- T cells in the spleen. CONCLUSION: Our results suggest that ATX-MS-1467 can induce splenic iTregs and long-term tolerance to MBP with the potential to partially reverse the pathology of multiple sclerosis, particularly during the early stages of the disease. FUNDING: EMD Serono, Inc., a business of Merck KGaA.

Microstructural impact of ischemia and bone marrow-derived cell therapy revealed with diffusion tensor magnetic resonance imaging tractography of the heart in vivo.

BACKGROUND: The arrangement of myofibers in the heart is highly complex and must be replicated by injected cells to produce functional myocardium. A novel approach to characterize the microstructural response of the myocardium to ischemia and cell therapy, with the use of serial diffusion tensor magnetic resonance imaging tractography of the heart in vivo, is presented. METHODS AND RESULTS: Validation of the approach was performed in normal (n=6) and infarcted mice (n=6) as well as healthy human volunteers. Mice (n=12) were then injected with bone marrow mononuclear cells 3 weeks after coronary ligation. In half of the mice the donor and recipient strains were identical, and in half the strains were different. A positive response to cell injection was defined by a decrease in mean diffusivity, an increase in fractional anisotropy, and the appearance of new myofiber tracts with the correct orientation. A positive response to bone marrow mononuclear cell injection was seen in 1 mouse. The response of the majority of mice to bone marrow mononuclear cell injection was neutral (9/12) or negative (2/12). The in vivo tractography findings were confirmed with histology. CONCLUSIONS: Diffusion tensor magnetic resonance imaging tractography was able to directly resolve the ability of injected cells to generate new myofiber tracts and provided a fundamental readout of their regenerative capacity. A highly novel and translatable approach to assess the efficacy of cell therapy in the heart is thus presented.

Cerebral blood volume affects blood-brain barrier integrity in an acute transient stroke model.

Insufficient vascular reserve after an ischemic stroke may induce biochemical cascades that subsequently deteriorate the blood-brain barrier (BBB) function. However, the direct relationship between poor cerebral blood volume (CBV) restoration and BBB disruption has not been examined in acute stroke. To quantify BBB integrity at acute stages of transient stroke, in particular for cases in which extravasation of the standard contrast agent (Gd-DTPA) is not observed, we adopted the water exchange index (WEI), a novel magnetic resonance image-derived parameter to estimate the water permeability across the BBB. The apparent diffusion coefficient (ADC) and R2 relaxation rate constant were also measured for outlining the tissue abnormality, while fractional CBV and WEI were quantified for assessing vascular alterations. The significantly decreased ADC and R2 in the ischemic cortices did not correlate with the changes in CBV or WEI. In contrast, a strong negative correlation between the ipsilesional WEI and CBV was found, in which stroke mice were clustered into two groups: (1) high WEI and low CBV and (2) normal WEI and CBV. The low CBV observed for mice with a disrupted BBB, characterized by a high WEI, indicates the importance of CBV restoration for maintaining BBB stability in acute stroke.

Classification of coronary atherosclerotic plaques ex vivo with T1, T2, and ultrashort echo time CMR.

OBJECTIVES: This study sought to determine whether the classification of human coronary atherosclerotic plaques with T1, T2, and ultrashort echo time (UTE) cardiac magnetic resonance (CMR) would correlate well with atherosclerotic plaque classification by histology. BACKGROUND: CMR has been extensively used to classify carotid plaque, but its ability to characterize coronary plaque remains unknown. In addition, the detection of plaque calcification by CMR remains challenging. Here, we used T1, T2, and UTE CMR to evaluate atherosclerotic plaques in fixed post-mortem human coronary arteries. We hypothesized that the combination of T1, T2, and UTE CMR would allow both calcified and lipid-rich coronary plaques to be accurately detected. METHODS: Twenty-eight plaques from human donor hearts with proven coronary artery disease were imaged at 9.4-T with a T1-weighted 3-dimensional fast low-angle shot (FLASH) sequence (250-µm resolution), a T2-weighted rapid acquisition with refocused echoes (RARE) sequence (in-plane resolution 0.156 mm), and an UTE sequence (300-µm resolution). Plaques showing selective hypointensity on T2-weighted CMR were classified as lipid-rich. Areas of hypointensity on the T1-weighted images, but not the UTE images, were classified as calcified. Hyperintensity on the T1-weighted and UTE images was classified as hemorrhage. Following CMR, histological characterization of the plaques was performed with a pentachrome stain and established American Heart Association criteria. RESULTS: CMR showed high sensitivity and specificity for the detection of calcification (100% and 90%, respectively) and lipid-rich necrotic cores (90% and 75%, respectively). Only 2 lipid-rich foci were missed by CMR, both of which were extremely small. Overall, CMR-based classification of plaque was in complete agreement with the histological classification in 22 of 28 cases (weighted κ = 0.6945, p < 0.0001). CONCLUSIONS: The utilization of UTE CMR allows plaque calcification in the coronary arteries to be robustly detected. High-resolution CMR with T1, T2, and UTE contrast enables accurate classification of human coronary atherosclerotic plaque.

Dynamic monitoring of blood-brain barrier integrity using water exchange index (WEI) during mannitol and CO2 challenges in mouse brain.

The integrity of the blood-brain barrier (BBB) is critical to normal brain function. Traditional techniques for the assessment of BBB disruption rely heavily on the spatiotemporal analysis of extravasating contrast agents. However, such methods based on the leakage of relatively large molecules are not suitable for the detection of subtle BBB impairment or for the performance of repeated measurements in a short time frame. Quantification of the water exchange rate constant (WER) across the BBB using strictly intravascular contrast agents could provide a much more sensitive method for the quantification of the BBB integrity. To estimate WER, we have recently devised a powerful new method using a water exchange index (WEI) biomarker and demonstrated BBB disruption in an acute stroke model. Here, we confirm that WEI is sensitive to even very subtle changes in the integrity of the BBB caused by: (i) systemic hypercapnia and (ii) low doses of a hyperosmolar solution. In addition, we have examined the sensitivity and accuracy of WEI as a biomarker of WER using computer simulation. In particular, the dependence of the WEI-WER relation on changes in vascular blood volume, T1 relaxation of cellular magnetization and transcytolemmal water exchange was explored. Simulated WEI was found to vary linearly with WER for typically encountered exchange rate constants (1-4 Hz), regardless of the blood volume. However, for very high WER (>5 Hz), WEI became progressively more insensitive to increasing WER. The incorporation of transcytolemmal water exchange, using a three-compartment tissue model, helped to extend the linear WEI regime to slightly higher WER, but had no significant effect for most physiologically important WERs (WER < 4 Hz). Variation in cellular T1 had no effect on WEI. Using both theoretical and experimental approaches, our study validates the utility of the WEI biomarker for the monitoring of BBB integrity.

Fiber architecture in remodeled myocardium revealed with a quantitative diffusion CMR tractography framework and histological validation.

BACKGROUND: The study of myofiber reorganization in the remote zone after myocardial infarction has been performed in 2D. Microstructural reorganization in remodeled hearts, however, can only be fully appreciated by considering myofibers as continuous 3D entities. The aim of this study was therefore to develop a technique for quantitative 3D diffusion CMR tractography of the heart, and to apply this method to quantify fiber architecture in the remote zone of remodeled hearts. METHODS: Diffusion Tensor CMR of normal human, sheep, and rat hearts, as well as infarcted sheep hearts was performed ex vivo. Fiber tracts were generated with a fourth-order Runge-Kutta integration technique and classified statistically by the median, mean, maximum, or minimum helix angle (HA) along the tract. An index of tract coherence was derived from the relationship between these HA statistics. Histological validation was performed using phase-contrast microscopy. RESULTS: In normal hearts, the subendocardial and subepicardial myofibers had a positive and negative HA, respectively, forming a symmetric distribution around the midmyocardium. However, in the remote zone of the infarcted hearts, a significant positive shift in HA was observed. The ratio between negative and positive HA variance was reduced from 0.96 ± 0.16 in normal hearts to 0.22 ± 0.08 in the remote zone of the remodeled hearts (p < 0.05). This was confirmed histologically by the reduction of HA in the subepicardium from -52.03° ± 2.94° in normal hearts to -37.48° ± 4.05° in the remote zone of the remodeled hearts (p < 0.05). CONCLUSIONS: A significant reorganization of the 3D fiber continuum is observed in the remote zone of remodeled hearts. The positive (rightward) shift in HA in the remote zone is greatest in the subepicardium, but involves all layers of the myocardium. Tractography-based quantification, performed here for the first time in remodeled hearts, may provide a framework for assessing regional changes in the left ventricle following infarction.

Molecular MRI of acute necrosis with a novel DNA-binding gadolinium chelate: kinetics of cell death and clearance in infarcted myocardium.

BACKGROUND: Current techniques to image cell death in the myocardium are largely nonspecific. We report the use of a novel DNA-binding gadolinium chelate (Gd-TO) to specifically detect the exposed DNA in acutely necrotic (ruptured) cells in vivo. METHODS AND RESULTS: In vivo MRI was performed in 20 mice with myocardial infarction (MI). The mice were injected with Gd-TO or Gd-DTPA at varying time points after MI. MRI was performed 2 hours after probe injection, to avoid nonspecific signal from the late gadolinium enhancement effect. Cell rupture (Gd-TO uptake) was present within 2 hours of infarction but peaked 9 to 18 hours after the onset of injury. A significant increase in the longitudinal relaxation rate (R(1)) in the infarct was seen in mice injected with Gd-TO within 48 hours of MI, but not in those injected more than 72 hours after MI (R(1)=1.24±0.08 and 0.92±0.03 s(-1), respectively, P<0.001). Gd-DTPA, unlike Gd-TO, washed completely out of acute infarcts within 2 hours of injection (P<0.001). The binding of Gd-TO to exposed DNA in acute infarcts was confirmed with fluorescence microscopy. CONCLUSIONS: Gd-TO specifically binds to acutely necrotic cells and can be used to image the mechanism and chronicity of cell death in injured myocardium. Cell rupture in acute MI begins early but peaks many hours after the onset of injury. The ruptured cells are efficiently cleared by the immune system and are no longer present in the myocardium 72 hours after injury.

Molecular and Microstructural Imaging of the Myocardium.

The past year has witnessed ongoing progress in the field of molecular MRI of the myocardium. In addition, several novel fluorescent agents have been introduced and used to image remodeling in the injured myocardium. New techniques to image myocardial microstructure, such as diffusion spectrum MRI, have also been introduced and have tremendous potential for integration and synergy with molecular MRI. In the current review we focus on these and other advances in the field that have occurred over the past year.

Manipulation of tissue contrast using contrast agents for enhanced MR microscopy in ex vivo mouse brain.

Detailed 3D mouse brain images may promote better understanding of phenotypical differences between normal and transgenic/mutant mouse models. Previously, a number of magnetic resonance microscopy (MRM) studies have successfully established brain atlases, revealing genotypic traits of several commonly used mouse strains. In such studies, MR contrast agents, mainly gadolinium (Gd) based, were often used to reduce acquisition time and improve signal-to-noise ratio (SNR). In this paper, we intended to extend the utility of contrast agents for MRM applications. Using Gd-DTPA and MnCl(2), we exploited the potential use of MR contrast agents to manipulate image contrast by drawing upon the multiple relaxation mechanisms and tissue-dependent staining properties characteristic of each contrast agent. We quantified r(1) and r(2) of Gd-DTPA and MnCl(2) in both aqueous solution and brain tissue and demonstrated the presence of divergent relaxation mechanisms between solution and tissue for each contrast agent. Further analyses using nuclear magnetic resonance dispersion (NMRD) of Mn(2+) in ex vivo tissue strongly suggested macromolecule binding of Mn(2+), leading to increased T(1) relaxation. Moreover, inductively coupled plasma (ICP) mass spectroscopy revealed that MnCl(2) had higher tissue affinity than Gd-DTPA. As a result, multiple regions of the brain stained by the two agents exhibited different image contrasts. Our results show that differential MRM staining can be achieved using multiple MR contrast agents, revealing detailed cytoarchitecture, and may ultimately offer a window for investigating new techniques by which to understand biophysical MR relaxation mechanisms and perhaps to visualize tissue anomalies even at the molecular level.