In vivo real-time imaging reveals megalin as the aminoglycoside gentamicin transporter into cochlea whose inhibition is otoprotective

Aminoglycosides (AGs) are commonly used antibiotics that cause deafness through the irreversible loss of cochlear sensory hair cells (HCs). How AGs enter the cochlea and then target HCs remains unresolved. Here, we performed time-lapse multicellular imaging of cochlea in live adult hearing mice via a chemo-mechanical cochleostomy. The in vivo tracking revealed that systemically administered Texas Red–labeled gentamicin (GTTR) enters the cochlea via the stria vascularis and then HCs selectively. GTTR uptake into HCs was completely abolished in transmembrane channel-like protein 1 (TMC1) knockout mice, indicating mechanotransducer channel-dependent AG uptake. Blockage of megalin, the candidate AG transporter in the stria vascularis, by binding competitor cilastatin prevented GTTR accumulation in HCs. Furthermore, cilastatin treatment markedly reduced AG-induced HC degeneration and hearing loss in vivo. Together, our in vivo real-time tracking of megalin-dependent AG transport across the blood–labyrinth barrier identifies new therapeutic targets for preventing AG-induced ototoxicity.

Drug-induced ototoxicity is a major side effect of several pharmacological treatments for life-threatening diseases (1). Aminoglycosides (AGs) which are widely used antibiotics, are one of the most common ototoxic drugs in clinical use (2). To treat severe Gram-negative infections, sepsis and, tuberculosis (3), over 10 million annual doses of AG are used in the United States (4). In some developing countries, AGs are used as a first line treatment owing to their broad-spectrum efficacy, a low incidence of allergic reactions, and limited antibiotic resistance (3, 4). Nevertheless, AGs can cause irreversible hearing loss by inducing selective degeneration of cochlear sensory hair cells (HCs) (5).The cochlea is composed of three fluid-filled chambers, the scala vestibuli (SV), the scala media (SM), and the scala tympani (ST) (Fig. 1A) (6). SM is comprised of a high K⁺/low Ca²⁺ endolymph solution, while SV and ST are filled with a perilymph solution having a similar ionic composition as extracellular fluid (7, 8). The unique endolymph ionic composition is maintained by the stria vascularis, where the blood–labyrinth barrier is presumably housed. Dense blood vessels and multiple transporters in the cells of the stria vascularis selectively regulate ion transport to the endolymph (9, 10). HCs that are in the organ of Corti (OoC) lie on the basilar membrane separating the SM and ST. More specifically, HC stereocilia which include mechanotransducer (MET) channels are exposed to the endolymph in SM, while the cell body is exposed to the perilymph (11–13).Open in a separate windowFig. 1.Surgical approach for generating an IW for in vivo imaging of cochlear cells with preserved hearing function. (A) Illustrations of cochlea show three fluid-filled chambers (SV, SM, and ST), OoC, and stria vascularis. Eye ball indicates the direction of imaging path. The range of two z-scans are demonstrated by green (z-scan1, stria vascularis region, 100 µm depth) and yellow (z-scan 2, OoC region, 60 µm depth) boxes. (B) Illustrations of cochlea within a mouse are shown to provide orientation; Left, side view; Right, transverse view. In the transverse view, the mouse head was divided to display pre- and post-surgical conditions. Post-surgery image shows the bulla opening with a preserved tympanic membrane and an IW (white circle). Schematics of (C) the CMC for preventing intracochlear fluid leakage during IW formation and (D) the position of the IW. Note that the cochlear endosteum (dark blue) remains intact after IW formation, thus avoiding intracochlear fluid leaking. Cochlear apex images (E) before and (F) after IW formation (red box and black arrowhead) with a diameter of ∼200–300 µm. Yellow circle indicates cochlea. The IW is near the 8–10 kHz region (43). (G) ABR thresholds were obtained after each surgical step and over time after IW formation with CMC (n = 5). ANOVA shows that no statistically significant differences were seen between comparisons (P > 0.05). (H) Representative two-photon in vivo image of the cochlear apex through the IW. AM1-43 dye (1 µL of 2.5 mM solution) was injected through the round window after intracochlear pressure release (by formation of a SCC hole) to visualize cells in the OoC. Note that the mouse lost its hearing after the dye injection.Several possible mechanisms of AG transport into the cochlea and HCs have been proposed. AG entry into endolymph via the stria vascularis is reported based on localization of gentamicin-conjugated Texas Red (GTTR) (14, 15) or AG immunolabeling (16). Reissner’s and basilar membranes have also been considered as possible entry routes of AG into the endolymph (17), but trafficking across the strial blood–endolymph barrier predominates compared to the uptake from perilymph (18). Despite the convergence of information supporting AG transport across the stria vascularis into endolymph, the molecular mechanism for transport remains unknown. In vitro assays using neonatal cochlea tissue demonstrated that MET channels are the main mediator of AG entry into HCs from the endolymph (19–22). Whether this remains true in adult in vivo conditions needs to be determined (21, 23). Modified AGs that show less permeation through the MET are also less toxic in vivo (24). Other routes like transient receptor potential (TRP) channels (25, 26), endocytosis (27), and purinergic receptors (28) may also contribute to HC uptake. However, the state of these routes under in vivo conditions and whether their participation occurs under normal or pathophysiological states is unknown. Regardless, these channels have a large pore, MET (1.3 nm) (20, 29), TRPA1 (∼1.1–1.4 nm) (30), and TRPV1 (∼1 nm) (31) in common, which supports an argument for direct permeation of AG (0.8–0.9 nm) (20, 32) into the cells. Possible therapeutic targets for preventing AG ototoxicity also have been explored by blocking one of the AG transport pathways or intracellular mechanisms (32, 33). For example, MET channel blockers (34–36) as well as antioxidants and anti-apoptotic agents were proposed with some limited efficacy (37–39).Previous efforts to identify uptake pathways have primarily relied on in vitro assays using the immature, neonatal cochlea or fixed tissues from the mature cochlea, which have led to some conflicting results (14, 16). To overcome these limitations, we developed an in vivo multicellular cochlear imaging method in live, hearing adult animals. In vivo imaging of the cochlea at the cellular level is especially challenging (Fig. 1B) because: (i) the deep location within the temporal bone makes access difficult, (ii) the fluid-filled bony structure prevents imaging cells, and (iii) existing surgical approaches for in vivo studies cause hearing loss. We have overcome these obstacles by creating an imaging window (IW) on the otic capsule using a chemo-mechanical cochleostomy (CMC) that prevented intracochlear fluid leakage and hearing loss.We used this surgical approach to track AG transport into cochlea and HCs in adult hearing animals in vivo in real-time. Systemically administered GTTR first appeared in the stria vascularis and later HCs in the OoC. We revealed that the major route of AG entry into HCs is via MET channels. We further demonstrated that entry into the endolymph is regulated by the endocytic transporter, megalin. Cilastatin, which inhibits the binding between megalin and AGs, significantly reduced HC uptake of GTTR and reduced AG-induced HC degeneration and hearing loss in vivo.     

In vitro reconstitution of calcium-dependent recruitment of the human ESCRT machinery in lysosomal membrane repair

The endosomal sorting complex required for transport (ESCRT) machinery is centrally involved in the repair of damage to both the plasma and lysosome membranes. ESCRT recruitment to sites of damage occurs on a fast time scale, and Ca²⁺ has been proposed to play a key signaling role in the process. Here, we show that the Ca²⁺-binding regulatory protein ALG-2 binds directly to negatively charged membranes in a Ca²⁺-dependent manner. Next, by monitoring the colocalization of ALIX with ALG-2 on negatively charged membranes, we show that ALG-2 recruits ALIX to the membrane. Furthermore, we show that ALIX recruitment to the membrane orchestrates the downstream assembly of late-acting CHMP4B, CHMP3, and CHMP2A subunits along with the AAA⁺ ATPase VPS4B. Finally, we show that ALG-2 can also recruit the ESCRT-III machinery to the membrane via the canonical ESCRT-I/II pathway. Our reconstitution experiments delineate the minimal sets of components needed to assemble the entire membrane repair machinery and open an avenue for the mechanistic understanding of endolysosomal membrane repair.

The endolysosomal system is susceptible to damage by a number of factors, including protein aggregates, lysosomotropic compounds, reactive oxygen species, and lipid metabolites (1). Endosomal escape mediates the entry into the cytosol of many viruses (2), therapeutic agents (3, 4), and the transmission of prion-like molecular aggregates (5). Escape is counteracted by the protective effects of lysosomal membrane repair (6), which is carried out by the endosomal sorting complex required for transport (ESCRT) membrane sealing machinery. The ESCRTs are a conserved membrane scission and sealing machinery consisting of about 30 proteins in humans (7, 8). In particular, ALIX, ESCRT-I, and the ESCRT-III subunits CHMP6, CHMP2A, and CHMP2B have been implicated in cytoprotective lysosomal membrane repair events (6, 9, 10). ESCRTs are also involved in the repair of the plasma membrane (11), where extracellular Ca²⁺ influx has been shown to trigger the subsequent recruitment of ESCRT-III machinery to the damaged plasma membrane site (12). The eventual closure of damage-induced holes in the plasma membrane is achieved by membrane budding and shedding vesicles toward the extracellular space by ESCRT-III machinery (11).Lysosomes have nearly a 5,000-fold higher concentration of Ca²⁺ (∼0.5 mM) compared to that in cytosol (∼100 nM) (13, 14). Therefore, damage to the endolysosomal membrane locally increases Ca²⁺ concentration near the site of damage. Increased local Ca²⁺ efflux into the cytosol has been proposed to be a trigger for the recruitment of endolysosomal membrane repair machinery (9). The Ca²⁺ binding protein ALG-2 coaccumulates with ALIX upon damage and has been proposed to have an upstream role in the endolysosomal membrane repair sequence (9). ALG-2 contains five serially repetitive EF-hand structures and is the most conserved protein among the penta-EF-hand (PEF) family (15). Upon binding to Ca²⁺, ALG-2 undergoes conformational changes, rendering it amenable to bind to proline-rich proteins such as ALIX (16). Therefore, ALG-2 is a prominent candidate to trigger the recruitment of ESCRT-III machinery, and therefore endolysosomal membrane repair, in response to increased local Ca²⁺ concentration around a damaged endolysosome. However, whether ALG-2 is sufficient to trigger the membrane recruitment of repair machinery at the site of damage is unclear (17).Here, we investigated the mechanism directly through in vitro reconstitution in a completely defined system of purified proteins and synthetic lipids. In vitro reconstitution is a powerful tool to determine the sufficiency of a biochemical factor, which we applied here to probe whether ALG-2 is sufficient to recruit downstream components of the endolysosomal membrane repair pathway. We used a giant unilamellar vesicle (GUV) reconstitution system to take advantage of fluorescence microscopy for our reconstitution experiments. We first showed that ALG-2 can be recruited to the negatively charged membranes without the need for an upstream adaptor protein. By using a Ca²⁺-binding–deficient mutant of ALG-2, we confirmed that ALG-2 membrane recruitment is mediated by Ca²⁺. By monitoring the colocalization of ALIX with ALG-2 on the negatively charged membrane, we confirmed the upstream role of ALG-2 in bringing ALIX to the membrane. We showed the complete downstream recruitment of human ESCRT-III machinery vis-à-vis CHMP4B, CHMP2A, and CHMP3 along with the AAA⁺ ATPase VPS4B to the negatively charged membranes in an ALG-2– and Ca²⁺-dependent manner. We demonstrated that ALG-2 also recruits the ESCRT-III machinery via the canonical ESCRT-I/II pathway. Finally, we validated that endolysosomal membrane damage leads to colocalization of ALG-2, ALIX, and ESCRT-I in vivo.     

Severe brain injury after cardiac surgery in children: consequences for the family and the need for assistance

Objective—To identify the short and longer term needs of parents whose children sustain severe brain injury after cardiac surgery and to determine what further measures could be of use to the family after such a catastrophe.
Design—Qualitative analysis of data generated by semistructured interviews and a series of self report questionnaires.
Setting—Tertiary cardiothoracic referral centre.
Subjects—Group 1: four sets of parents (eight individuals) whose children had suffered severe brain injury after heart surgery; group 2: four sets of parents (seven individuals) caring for children with acute brain injury from other causes.
Results—The data provide evidence of social, emotional, physical, practical, and financial difficulties. After the children suffered brain injury following cardiac surgery their parents did not receive information, support, and practical assistance as early as they needed it.
Conclusions—Although a small population was studied, it would seem that a structured, planned health care service response to this devastating event is not established; therefore, the needs of these parents are not well met. The parents and our inquiries suggest that a coordinator who is not attached to the hospital where brain injury occurred might optimally fulfil this role.

Keywords: brain damage;  cardiac surgery;  parental support     

In vivo imaging reveals an essential role of vasoconstriction in rupture of the ovarian follicle at ovulation

Rupture of the ovarian follicle releases the oocyte at ovulation, a timed event that is critical for fertilization. It is not understood how the protease activity required for rupture is directed with precise timing and localization to the outer surface, or apex, of the follicle. We hypothesized that vasoconstriction at the apex is essential for rupture. The diameter and blood flow of individual vessels and the thickness of the apical follicle wall were examined over time to expected ovulation using intravital multiphoton microscopy. Vasoconstriction of apical vessels occurred within hours preceding follicle rupture in wild-type mice, but vasoconstriction and rupture were absent in Amhr2^cre/+SmoM2 mice in which follicle vessels lack the normal association with vascular smooth muscle. Vasoconstriction is not simply a response to reduced thickness of the follicle wall; vasoconstriction persisted in wild-type mice when thinning of the follicle wall was prevented by infusion of protease inhibitors into the ovarian bursa. Ovulation was inhibited by preventing the periovulatory rise in the expression of the vasoconstrictor endothelin 2 by follicle cells of wild-type mice. In these mice, infusion of vasoconstrictors (either endothelin 2 or angiotensin 2) into the bursa restored the vasoconstriction of apical vessels and ovulation. Additionally, infusion of endothelin receptor antagonists into the bursa of wild-type mice prevented vasoconstriction and follicle rupture. Processing tissue to allow imaging at increased depth through the follicle and transabdominal ultrasonography in vivo showed that decreased blood flow is restricted to the apex. These results demonstrate that vasoconstriction at the apex of the follicle is essential for ovulation.During ovulation in typically mono-ovulatory species such as humans, as well as in poly-ovulatory species such as rodents, the oocyte is released from the preovulatory follicle by extrusion through a rupture site on the outer surface, or apex, of the follicle, which protrudes from the surface of the ovary (1). Precise timing and accurate spatial localization of rupture at the apex are essential to allow capture of the oocyte by a hormonally primed oviduct where fertilization occurs, but the mechanisms involved are not yet understood. The rupture site breaches multiple layers of cells and their associated extracellular matrix and basement membranes (2). These include the single layer of epithelial cells that covers the surface of the ovary, the basement membrane that supports it, and the multiple cell layers comprising the wall of the preovulatory follicle. The outer wall of the ovarian follicle contains androgen-secreting theca cells and extensive vasculature. This vasculature consists of an inner and an outer plexus of capillaries with associated arterioles and venules that supply nutrients to the entire follicle (3–5). Underlying the theca and separated from it by a basement membrane is the avascular granulosa cell layer that serves as the major source of estrogen. The oocyte resides in the center of the follicle surrounded by multiple layers of specialized granulosa cells known as “cumulus cells.” In a mature preovulatory follicle, formation of a fluid-filled antral cavity separates the oocyte–cumulus complex from the mural granulosa cells that form the wall of the follicle except at a region known as the “stalk,” which connects the oocyte–cumulus complex to the antral granulosa cells of the follicle wall. At ovulation the oocyte is released from the follicle in association with attached cumulus cells.The preovulatory release of surge levels of luteinizing hormone (LH) from the anterior pituitary acts on receptors in the follicle to trigger events critical for the rupture and remodeling of the follicle and differentiation of granulosa and theca cells into progesterone-producing cells of the corpus luteum. The cumulus cells are induced to secrete a mucoelastic extracellular matrix which causes loosening of contacts between granulosa cells and between granulosa cells and the oocyte, a process known as “cumulus expansion,” which is essential for ovulation (1). Expression of proteases belonging to several major families, including the matrix metalloproteinase, plasminogen activator/plasmin, and ADAMTS (a disintegrin and metalloproteinase with thrombospondin-like motifs) families, increases. Simultaneously, follicle cells express protease inhibitors such as tissue inhibitors of metalloproteinases (TIMPs 1–4) and plasminogen activator inhibitors (PAI 1–3) (6, 7). The increase in protease activity is essential for rupture of the follicle and for the breakdown of the basement membrane separating theca and granulosa cells to allow the ingrowth of blood vessels to establish the corpus luteum. The mechanisms that regulate the balance of protease and protease inhibitor activity in the follicle to allow precise rupture at the apex while protecting most of the follicle structure from protease activity are not understood (1, 6, 7).We postulated that vasoconstriction of vessels within the theca at the apex of the follicle is required to promote follicle rupture. Our first approach was to examine mice with conditional expression of a dominant active allele of smoothened (SMO), the transmembrane protein that relays signaling by the hedgehog (HH) pathway. In these Amhr2^cre/+SmoM2 mice, preovulatory follicles develop normally in many respects, including changes in the expression of critical genes in response to the preovulatory LH surge (8, 9). However, follicles fail to rupture, and oocytes remain trapped as the follicles luteinize. The major ovarian phenotype in these mice is a pronounced deficiency of vascular smooth muscle (VSM) surrounding vessels in the theca cell layer, whereas other vessels that are present throughout the stroma of the ovary have normal maturation with VSM. Because VSM is required for vasoconstriction, the mice provided a model to test whether failure of vasoconstriction contributes to anovulation. In additional experiments with wild-type mice, we blocked the increase in the expression of endothelin 2 (Edn2) by granulosa cells that normally occurs within hours before follicle rupture (10, 11). Because EDN2 is a potent vasoconstrictor, this approach allowed us to test the effect on follicle rupture of inhibiting vasoconstriction versus treatment with exogenous compounds to restore vasoconstriction. In addition, treatment of wild-type mice with EDN2 receptor antagonists was used to test the role of EDN2 in vasoconstriction and rupture. Vasoconstriction and changes in the follicle wall were monitored repeatedly relative to the time of ovulation using intravital multiphoton microscopy.     

Unruptured aneurysm of the left sinus of Valsalva extending into the left ventricular outflow tract: presentation and imaging

The symptomatic presentation of an unruptured sinus of Valsalva aneurysm is rare. A 48 year old man with a history of treated hypothyroidism, and a five year history of ileocolonic Crohn's disease of chronic low grade activity presented with a profound left hemiplegia. He was in sinus rhythm and normotensive. Cardiac auscultation was repeatedly normal. Computed tomography of the head performed early in the course of the illness was reported as normal. Duplex Doppler examination of the carotid arteries performed six months later revealed no significant atheroma. There was complete resolution of the neurological deficit over a period of months. A year later he presented with chest pain suggestive of myocardial ischaemia. Computed tomography, magnetic resonance imaging, transthoracic and transoesophageal echocardiography, and cardiac catheterisation pointed to a sinus of Valsalva aneurysm protruding into the left ventricular outflow tract. In view of the previous neurological event and ongoing chest pain suggestive of myocardial ischaemia, the lesion was resected. The patient made a good recovery and postoperative transoesophageal echocardiography showed normal aortic valve function with no residual regurgitation. This is the first reported case of pure left ventricular outflow tract extension of an unruptured left sinus aneurysm. The presentation with ischaemic cardiac pain does not seem to be explained by conventional mechanisms.

Keywords: sinus of Valsalva aneurysm;  non-invasive imaging;  transoesophageal echocardiography     

SD + SV4 diagnosis of left ventricular hypertrophy,a revaluation of ECG criterion by cardiac magnetic resonance imaging

BackgroudPresent electrocardiogram (ECG) criteria for diagnosing left ventricular hypertrophy (LVH) usually have low sensitivity, while the newly proposed SD + SV4 criterion, namely the deepest S‐wave amplitude in any lead (SD) plus SV4 amplitude, has been reported to have higher sensitivity and accuracy compared with other existing criteria. We aimed to further evaluate the diagnostic value of the SD + SV4 criterion in reference to the gold standard cardiac magnetic resonance imaging (CMR) in LVH diagnosis.MethodsThis retrospective study enrolled 138 patients who received CMR examination—60 patients with reduced ejection fraction (EF) and 78 patients with preserved EF. The left ventricular mass index (LVMI) measured by CMR was used as the gold standard for diagnosing LVH.ResultThe diagnostic value of the SD + SV4 criterion was compared with other 4 commonly used criteria. By CMR, 29 out of 138 people (21%) were diagnosed with LVH in reference to CMR. The SD + SV4 criterion had markedly higher sensitivity in diagnosing LVH compared with other criteria, but no higher specificity. There was no significant difference in area under receiver operating characteristic (ROC) curve among these criteria. The SD + SV4 criterion was not markedly consistent with CMR in diagnosing LVH. Compared to the other criteria, the SD + SV4 criterion had the highest sensitivity in patients with reduced ejection fraction; however, the area under the curve (AUC) of the SD + SV4 criterion in patients with reduced EF was significantly lower than in patients with preserved EF.ConclusionThe newly proposed SD + SV4 criterion did not have a better diagnostic value compared with other existing criteria, and the statistical power of the SD + SV4 criterion was influenced by EF.     

High-resolution photoacoustic tomography of resting-state functional connectivity in the mouse brain

The increasing use of mouse models for human brain disease studies presents an emerging need for a new functional imaging modality. Using optical excitation and acoustic detection, we developed a functional connectivity photoacoustic tomography system, which allows noninvasive imaging of resting-state functional connectivity in the mouse brain, with a large field of view and a high spatial resolution. Bilateral correlations were observed in eight functional regions, including the olfactory bulb, limbic, parietal, somatosensory, retrosplenial, visual, motor, and temporal regions, as well as in several subregions. The borders and locations of these regions agreed well with the Paxinos mouse brain atlas. By subjecting the mouse to alternating hyperoxic and hypoxic conditions, strong and weak functional connectivities were observed, respectively. In addition to connectivity images, vascular images were simultaneously acquired. These studies show that functional connectivity photoacoustic tomography is a promising, noninvasive technique for functional imaging of the mouse brain.Resting-state functional connectivity (RSFC) is an emerging neuroimaging approach that aims to identify low-frequency, spontaneous cerebral hemodynamic fluctuations and their associated functional connections (1, 2). Recent research suggests that these fluctuations are highly correlated with local neuronal activity (3, 4). The spontaneous fluctuations relate to activity that is intrinsically generated by the brain, instead of activity attributable to specific tasks or stimuli (2). A hallmark of functional organization in the cortex is the striking bilateral symmetry of corresponding functional regions in the left and right hemispheres (5). This symmetry also exists in spontaneous resting-state hemodynamics, where strong correlations are found interhemispherically between bilaterally homologous regions as well as intrahemispherically within the same functional regions (3). Clinical studies have demonstrated that RSFC is altered in brain disorders such as stroke, Alzheimer’s disease, schizophrenia, multiple sclerosis, autism, and epilepsy (6–12). These diseases disrupt the healthy functional network patterns, most often reducing correlations between functional regions. Due to its task-free nature, RSFC imaging requires neither stimulation of the subject nor performance of a task during imaging (13). Thus, it can be performed on patients under anesthesia (14), on patients unable to perform cognitive tasks (15, 16), and even on patients with brain injury (17, 18).RSFC imaging is also an appealing technique for studying brain diseases in animal models, in particular the mouse, a species that holds the largest variety of neurological disease models (3, 13, 19, 20). Compared with clinical studies, imaging genetically modified mice allows exploration of molecular pathways underlying the pathogenesis of neurological disorders (21). The connection between RSFC maps and neurological disorders permits testing and validation of new therapeutic approaches. However, conventional neuroimaging modalities cannot easily be applied to mice. For instance, in functional connectivity magnetic resonance imaging (fcMRI) (22), the resting-state brain activity is determined via the blood-oxygen-level–dependent (BOLD) signal contrast, which originates mainly from deoxy-hemoglobin (23). The correlation analysis central to functional connectivity requires a high signal-to-noise ratio (SNR). However, achieving a sufficient SNR is made challenging by the high magnetic fields and small voxel size needed for imaging the mouse brain, as well as the complexity of compensating for field inhomogeneities caused by tissue–bone or tissue–air boundaries (24). Functional connectivity mapping with optical intrinsic signal imaging (fcOIS) was recently introduced as an alternative method to image functional connectivity in mice (3, 20). In fcOIS, changes in hemoglobin concentrations are determined based on changes in the reflected light intensity from the surface of the brain (3, 25). Therefore, neuronal activity can be measured through the neurovascular response, similar to the method used in fcMRI. However, due to the diffusion of light in tissue, the spatial resolution of fcOIS is limited, and experiments have thus far been performed using an exposed skull preparation, which increases the complexity for longitudinal imaging.Photoacoustic imaging of the brain is based on the acoustic detection of optical absorption from tissue chromophores, such as oxy-hemoglobin (HbO₂) and deoxy-hemoglobin (Hb) (26, 27). This imaging modality can simultaneously provide high-resolution images of the brain vasculature and hemodynamics with intact scalp (28, 29). In this article, we perform functional connectivity photoacoustic tomography (fcPAT) to study RSFC in live mice under either hyperoxic or hypoxic conditions, as well as in dead mice. Our experiments show that fcPAT is able to detect connectivities between different functional regions and even between subregions, promising a powerful functional imaging modality for future brain research.     

Prognostic significance of electrical alternans versus signal averaged electrocardiography in predicting the outcome of electrophysiological testing and arrhythmia-free survival

Objective—To investigate the accuracy of signal averaged electrocardiography (SAECG) and measurement of microvolt level T wave alternans as predictors of susceptibility to ventricular arrhythmias.
Design—Analysis of new data from a previously published prospective investigation.
Setting—Electrophysiology laboratory of a major referral hospital.
Patients and interventions—43 patients, not on class I or class III antiarrhythmic drug treatment, undergoing invasive electrophysiological testing had SAECG and T wave alternans measurements. The SAECG was considered positive in the presence of one (SAECG-I) or two (SAECG-II) of three standard criteria. T wave alternans was considered positive if the alternans ratio exceeded 3.0.
Main outcome measures—Inducibility of sustained ventricular tachycardia or fibrillation during electrophysiological testing, and 20 month arrhythmia-free survival.
Results—The accuracy of T wave alternans in predicting the outcome of electrophysiological testing was 84% (p < 0.0001). Neither SAECG-I (accuracy 60%; p < 0.29) nor SAECG-II (accuracy 71%; p < 0.10) was a statistically significant predictor of electrophysiological testing. SAECG, T wave alternans, electrophysiological testing, and follow up data were available in 36 patients while not on class I or III antiarrhythmic agents. The accuracy of T wave alternans in predicting the outcome of arrhythmia-free survival was 86% (p < 0.030). Neither SAECG-I (accuracy 65%; p < 0.21) nor SAECG-II (accuracy 71%; p < 0.48) was a statistically significant predictor of arrhythmia-free survival.
Conclusions—T wave alternans was a highly significant predictor of the outcome of electrophysiological testing and arrhythmia-free survival, while SAECG was not a statistically significant predictor. Although these results need to be confirmed in prospective clinical studies, they suggest that T wave alternans may serve as a non-invasive probe for screening high risk populations for malignant ventricular arrhythmias.

     

Generation of a highly inducible Gal4-->Fluc universal reporter mouse for in vivo bioluminescence imaging

Full understanding of the functional complexity of the protein interactome requires mapping of biomolecular complexes within the cellular environment over biologically relevant time scales. New approaches to imaging interacting protein partners in vivo will allow the study of functional proteomics of human biology and disease within the context of living animals. Herein, we describe a universal transgenic reporter mouse strain that expresses firefly luciferase (Fluc) under the regulatory control of a concatenated Gal4 promoter (Tg^G4F(+/−)). Using an adenovirus to deliver a fused binding-domain-activator chimera (Gal4BD-VP16), induction of bioluminescence in Tg^G4F(+/−) tissues of up to 4 orders of magnitude was observed in fibroblasts, liver, respiratory epithelia, muscle, and brain. The Tg^G4F(+/−) reporter strain allowed noninvasive detection of viral infectivity, duration of the infection as well as viral clearance in various tissues in vivo. To demonstrate protein–protein interactions in live mice, the well characterized interaction between tumor suppressor p53 (fused to Gal4BD) and large T antigen (TAg) (fused to VP16) was visualized in vivo by using a two-hybrid strategy. Hepatocytes of Tg^G4F(+/−) mice transfected with p53/TAg demonstrated 48-fold greater induction of Fluc expression in vivo than noninteracting pairs. Furthermore, to demonstrate the feasibility of monitoring experimental therapy with siRNA in vivo, targeted knockdown of p53 resulted in markedly reduced light output, whereas use of a control siRNA had no effect on protein interaction-dependent induction of Fluc. Thus, this highly inducible Gal4→Fluc conditional reporter strain should facilitate imaging studies of protein interactions, signaling cascades, viral dissemination, and therapy within the physiological context of the whole animal.     

Nanoparticles enhance brain delivery of blood-brain barrier-impermeable probes for in vivo optical and magnetic resonance imaging

Several imaging modalities are suitable for in vivo molecular neuroimaging, but the blood-brain barrier (BBB) limits their utility by preventing brain delivery of most targeted molecular probes. We prepared biodegradable nanocarrier systems made up of poly(n-butyl cyanoacrylate) dextran polymers coated with polysorbate 80 (PBCA nanoparticles) to deliver BBB-impermeable molecular imaging probes into the brain for targeted molecular neuroimaging. We demonstrate that PBCA nanoparticles allow in vivo targeting of BBB-impermeable contrast agents and staining reagents for electron microscopy, optical imaging (multiphoton), and whole brain magnetic resonance imaging (MRI), facilitating molecular studies ranging from individual synapses to the entire brain. PBCA nanoparticles can deliver BBB-impermeable targeted fluorophores of a wide range of sizes: from 500-Da targeted polar molecules to 150,000-Da tagged immunoglobulins into the brain of living mice. The utility of this approach is demonstrated by (i) development of a "Nissl stain" contrast agent for cellular imaging, (ii) visualization of amyloid plaques in vivo in a mouse model of Alzheimer's disease using (traditionally) non-BBB-permeable reagents that detect plaques, and (iii) delivery of gadolinium-based contrast agents into the brain of mice for in vivo whole brain MRI. Four-dimensional real-time two-photon and MR imaging reveal that brain penetration of PBCA nanoparticles occurs rapidly with a time constant of ～18 min. PBCA nanoparticles do not induce nonspecific BBB disruption, but collaborate with plasma apolipoprotein E to facilitate BBB crossing. Collectively, these findings highlight the potential of using biodegradable nanocarrier systems to deliver BBB-impermeable targeted molecular probes into the brain for diagnostic neuroimaging.     

Imaging T-cell movement in the brain during experimental cerebral malaria

T-cells are known to play a role in the pathology associated with experimental cerebral malaria, although it has not previously been possible to examine their behaviour in brain. Using multiphoton laser scanning microscopy, we have examined the migration and movement of these cells in brain tissue. We believe that this approach will help define host–parasite interactions and examine how intervening in these relationships affects the development of cerebral pathology.     

Near-infrared fluorescence imaging with indocyanine green-lactosomes in a mouse model of rheumatoid arthritis

Objectives: The early diagnosis and treatment of rheumatoid arthritis (RA) is important to reduce joint destruction. Many of the current imaging techniques have disadvantages, such as the need for contrast agents and interpretation by specialists. Fluorescence imaging is an emerging technique that overcomes some of these problems. The aim of this study was to determine whether near-infrared (NIR) fluorescence imaging of indocyanine green (ICG)-lactosomes can detect joint inflammation in a mouse model of RA.

Methods: Control and arthritic SKG/Jcl mice were injected with ICG alone or ICG-lactosomes and examined by NIR fluorescence imaging. Arthritis severity was assessed macroscopically and histopathologically.

Results: ICG fluorescence was detected in the liver soon after injection and then decreased over the next several hours. ICG was not detected in the joints of control or arthritic mice. In contrast, ICG-lactosomes remained in mice for at least 48?h and accumulated specifically at inflamed joints. ICG-lactosome fluorescence was higher in arthritic versus normal joints at all times examined and was maximal at 24?h after injection.

Conclusions: NIR fluorescence imaging of ICG-lactosomes detects arthritic joints in a mouse model of RA. ICG-lactosomes may preferentially localize to inflamed joints via enhanced permeability and retention.     

Relation between the insulin lowering rate and changes in bone mineral density: Analysis among subtypes of type 1 diabetes mellitus

Aims/IntroductionThe bone mineral density in patients with type 1 diabetes mellitus is reduced due to impaired insulin secretion. However, it is unclear whether the rate of bone mineral density reduction is affected by the type 1 diabetes mellitus subtype. This study aimed to clarify the difference in bone mineral density across type 1 diabetes mellitus subtypes: slowly progressive (SP), acute‐onset (AO), and fulminant (F).MethodsThis was a retrospective, single‐center, cross‐sectional study conducted on 98 adult type 1 diabetes mellitus patients. The main outcome included the bone mineral density Z‐score (BMD‐Z) measured at the lumbar spine and femoral neck.ResultsThe lumbar spine BMD‐Z was lower in the acute‐onset than in the slowly progressive subtype (P = 0.03). No differences were observed when compared with the fulminant subtype. The femoral neck BMD‐Z tended to be higher in the slowly progressive than in the acute‐onset and fulminant subtypes. Multiple regression analyses showed that the lumbar spine BMD‐Z was associated with subtypes (AO vs SP) (P = 0.01), but not subtypes (F vs SP), adjusted for sex, duration, retinopathy, and C‐peptide immunoreactivity (CPR). When the patients were divided into disease duration tertiles, in the first and second tertiles, the CPR levels were lower in the acute‐onset or fulminant than in the slowly progressive subtype. In contrast, the lumbar spine and femoral neck BMD‐Z differed between the acute‐onset and slowly progressive only in the second tertiles (both P < 0.01), with a similar tendency between the fulminant and slowly progressive subtypes.ConclusionsAmong the type 1 diabetes mellitus subtypes, bone mineral density undergoes time‐dependent changes, which reveals that the bone mineral density decline follows the impaired insulin secretion. These results provide novel insights into the association between the low insulin exposure duration and bone mineral density.     

Impact of glucagon response on early postprandial glucose excursions irrespective of residual β‐cell function in type 1 diabetes: A cross‐sectional study using a mixed meal tolerance test

Aims/IntroductionControlling postprandial glucose levels in patients with type 1 diabetes is challenging even under the adequate treatment of insulin injection. Recent studies showed that dysregulated glucagon secretion exacerbates hyperglycemia in type 2 diabetes patients, but little is known in type 1 diabetes patients. We investigated whether the glucagon response to a meal ingestion could influence the postprandial glucose excursion in patients with type 1 diabetes.Materials and MethodsWe enrolled 34 patients with type 1 diabetes and 23 patients with type 2 diabetes as controls. All patients underwent a liquid mixed meal tolerance test. We measured levels of plasma glucose, C‐peptide and glucagon at fasting (0 min), and 30, 60 and 120 min after meal ingestion. All type 1 diabetes patients received their usual basal insulin and two‐thirds of the necessary dose of the premeal bolus insulin.ResultsThe levels of plasma glucagon were elevated and peaked 30 min after the mixed meal ingestion in both type 1 diabetes and type 2 diabetes patients. The glucagon increments from fasting to each time point (30, 60 and 120 min) in type 1 diabetes patients were comparable to those in type 2 diabetes patients. Among the type 1 diabetes patients, the glucagon response showed no differences between the subgroups based on diabetes duration (<5 vs ≥5 years) and fasting C‐peptide levels (<0.10 vs ≥0.10 nmol/L). The changes in plasma glucose from fasting to 30 min were positively correlated with those in glucagon, but not C‐peptide, irrespective of diabetes duration and fasting C‐peptide levels in patients with type 1 diabetes.ConclusionsThe dysregulated glucagon likely contributes to postprandial hyperglycemia independent of the residual β‐cell functions during the progression of type 1 diabetes.     

Microbubbles Remove Listeria monocytogenes from the Surface of Stainless Steel,Cucumber, and Avocado

Fresh produce may be contaminated by bacterial pathogens, including Listeria monocytogenes, during harvesting, packaging, or transporting. A low-intensity cavitation process with air being injected into water was studied to determine the microbubbles’ efficiency when detaching L. monocytogenes from stainless steel and the surface of fresh cucumber and avocado. Stainless steel coupons (1″ × 2″), cucumber, and avocado surfaces were inoculated with L. monocytogenes (LCDC strain). After 1, 24 or 48 h, loosely attached cells were washed off, and inoculated areas were targeted by microbubbles (~0.1–0.5 mm dia.) through a bubble diffuser (1.0 L air/min) for 1, 2, 5, or 10 min. For steel, L. monocytogenes (48 h drying) detachment peaked at 2.95 mean log reduction after 10 min of microbubbles when compared to a no-bubble treatment. After 48 h pathogen drying, cucumbers treated for 10 min showed a 1.78 mean log reduction of L. monocytogenes. For avocados, L. monocytogenes (24 h drying) detachment peaked at 1.65 log reduction after 10 min of microbubbles. Microbubble applications may be an effective, economical, and environmentally friendly way to remove L. monocytogenes, and possibly other bacterial pathogens, from food contact surfaces and the surfaces of whole, intact fresh produce.     

In vivo optical imaging of revascularization after brain trauma in mice

Revascularization following brain trauma is crucial to the repair process. We used optical micro-angiography (OMAG) to study endogenous revascularization in living mice following brain injury. OMAG is a volumetric optical imaging method capable of in vivo mapping of localized blood perfusion within the scanned tissue beds down to capillary level imaging resolution. We demonstrated that OMAG can differentiate revascularization progression between traumatized mice with and without soluble epoxide hydrolase (sEH) gene deletion. The time course of revascularization was determined from serial imaging of the traumatic region in the same mice over a one-month period of rehabilitation. Restoration of blood volume at the lesion site was more pronounced in sEH knockout mice than in wild-type mice as determined by OMAG. These OMAG measurements were confirmed by histology and showed that the sEH knockout effect may be involved in enhancing revascularization. The correlation of OMAG with histology also suggests that OMAG is a useful imaging tool for real-time in vivo monitoring of post-traumatic revascularization and for evaluating agents that inhibit or promote endogenous revascularization during the recovery process in small rodents.     

Quantitative analysis by in vivo imaging of the dynamics of vascular and axonal networks in injured mouse spinal cord

Understanding the endogenous repair capacity of spinal cord is pivotal to develop strategies to improve it. Here we design a paradigm of spinal cord lesion in the dorsal column using a 2-photon microscopy technique to dynamically and chronically monitor simultaneous changes of vascular and axonal networks in living mice up to 4 months postinjury. High-resolution images showed that early explorative sprouting of surviving injured axons resulted in extensive regrowth until and past the lesion site within 2 months. Blood vessel density was transiently up-regulated and most neurovascular interactions occurred within 2 weeks. Time-lapse analysis showed that neovessels exerted a potent growth stimulating action, but no guidance effect on neighboring sprouts, possibly because of their geometry and plasticity. Nevertheless, if reconnection depends on axon sprout density, stimulation of angiogenesis would probably be beneficial to repair. More generally, this imaging approach is showing promise to aid in monitoring brain diseases and the efficacy of potential treatments.