In vivo imaging of amyloid plaques in the brain

The pathological hallmark of Alzheimer's disease (AD) is the deposition of senile plaques (SPs) and neurofibrillary tangles. The deposition of SPs, which initially occurs in the neocortex, is regarded as the critical event for the pathogenesis of AD. Previous pathological studies indicated that the earliest deposition of SPs is suspected to occur before any detectable cognitive decline. Therefore, noninvasive imaging of brain SPs is considered to be an ideal diagnostic method for presymptomatic detection of AD. For in vivo detection of SPs in the brain, a lipophilic probe that can selectively binds SPs is indispensable to the examination using positron emission tomography (PET) or single photon emission computed tomography (SPECT). For this purpose, many kinds of amyloid binding agents have been developed, such as FDDNP, BTA-1, IMPY. We have also developed a series of promising compounds for in vivo imaging of amyloid deposits. One of these compounds, compound-A, achieves high binding affinity for A beta fibrils. This agent also showed abundant initial brain uptake and short brain clearance half-life in normal mice. Furthermore, to investigate the in vivo binding property of compound-A to amyloid plaques, compound-A was intravenously administered to APP transgenic mice. Brain slices at 120 min post injection demonstrated specific binding of compound-A to amyloid plaques in the brain. These data emphasize the potential usefulness of compound-A as in vivo imaging probe for early diagnosis of AD.     

Copper-induced apoptosis and immediate early gene expression in macrophages.

The death of macrophage-derived foam cells contributes to the formation of the lipid core in atherosclerotic lesions. Although the underlying mechanism is not yet clear, apoptosis has been shown to be responsible, at least in part, for the cell death of lipid-laden macrophages in atherosclerotic plaques. In the present study, we demonstrated that copper, in the presence of 8-hydroxyquinoline, was able to induce apoptosis of murine J774.A1 cells in culture. Ceruloplasmin exerts similar a effect, but not iron or hemin. Further experiments demonstrated that the expression of immediate early genes, including c-jun, c-fos and egr-1, was also induced by copper treatment in these cells, although only egr-1 mRNA was induced in a time- and dose-dependent manner. The antioxidant, N-acetylcysteine, exhibited remarkable inhibitory effect on the copper-induced apoptosis dose-dependently. Time course experiment revealed that prior treatment of cells with N-acetylcysteine is essential for the anti-apoptotic effect of this compound. Results also demonstrated that under the condition; in which N-acetylcysteine inhibited the copper-induced apoptosis, this antioxidant also abolished the gene expression of egr-1. Collectively, these results suggest that egr-1 gene expression is closely associated with the apoptosis induced by copper in macrophages.     

In vivo two-photon imaging reveals monocyte-dependent neutrophil extravasation during pulmonary inflammation

Immune-mediated pulmonary diseases are a significant public health concern. Analysis of leukocyte behavior in the lung is essential for understanding cellular mechanisms that contribute to normal and diseased states. Here, we used two-photon imaging to study neutrophil extravasation from pulmonary vessels and subsequent interstitial migration. We found that the lungs contained a significant pool of tissue-resident neutrophils in the steady state. In response to inflammation produced by bacterial challenge or transplant-mediated, ischemia-reperfusion injury, neutrophils were rapidly recruited from the circulation and patrolled the interstitium and airspaces of the lung. Motile neutrophils often aggregated in dynamic clusters that formed and dispersed over tens of minutes. These clusters were associated with CD115(+) F4/80(+) Ly6C(+) cells that had recently entered the lung. The depletion of blood monocytes with clodronate liposomes reduced neutrophil clustering in the lung, but acted by inhibiting neutrophil transendothelial migration upstream of interstitial migration. Our results suggest that a subset of monocytes serve as key regulators of neutrophil extravasation in the lung and may be an attractive target for the treatment of inflammatory pulmonary diseases.     

In vivo imaging of axonal transport of mitochondria in the diseased and aged mammalian CNS

The lack of intravital imaging of axonal transport of mitochondria in the mammalian CNS precludes characterization of the dynamics of axonal transport of mitochondria in the diseased and aged mammalian CNS. Glaucoma, the most common neurodegenerative eye disease, is characterized by axon degeneration and the death of retinal ganglion cells (RGCs) and by an age-related increase in incidence. RGC death is hypothesized to result from disturbances in axonal transport and in mitochondrial function. Here we report minimally invasive intravital multiphoton imaging of anesthetized mouse RGCs through the sclera that provides sequential time-lapse images of mitochondria transported in a single axon with submicrometer resolution. Unlike findings from explants, we show that the axonal transport of mitochondria is highly dynamic in the mammalian CNS in vivo under physiological conditions. Furthermore, in the early stage of glaucoma modeled in adult (4-mo-old) mice, the number of transported mitochondria decreases before RGC death, although transport does not shorten. However, with increasing age up to 23–25 mo, mitochondrial transport (duration, distance, and duty cycle) shortens. In axons, mitochondria-free regions increase and lengths of transported mitochondria decrease with aging, although totally organized transport patterns are preserved in old (23- to 25-mo-old) mice. Moreover, axonal transport of mitochondria is more vulnerable to glaucomatous insults in old mice than in adult mice. These mitochondrial changes with aging may underlie the age-related increase in glaucoma incidence. Our method is useful for characterizing the dynamics of axonal transport of mitochondria and may be applied to other submicrometer structures in the diseased and aged mammalian CNS in vivo.Globally, longevity is increasing, and the cohort aged 60 y or over is the fastest growing portion of the population. These trends place neurodegenerative diseases among the greatest clinical threats. Glaucoma, the most common progressive neurodegenerative eye disease (1), globally affects an estimated 60.5 million people, of whom 8.4 million are bilaterally blind (2). Similar to other neurodegenerative diseases of the CNS, including Alzheimer’s disease (3) and Parkinson’s disease (4), the incidence of glaucoma (5) increases with aging. Glaucoma is characterized by axon degeneration and the death of retinal ganglion cells (RGCs) (6). Histological studies in glaucoma models have suggested disturbances in the axonal transport of mitochondria in RGCs (7, 8).Axonal transport is essential for delivering the organelles and proteins that are required for axonal function and maintenance. Mitochondria are organelles that must be transported in axons and distributed appropriately to function (9, 10), because mitochondria play a pivotal role in the function and survival of neurons by generating ATP, maintaining Ca²⁺ and reduction-oxidation (redox) homeostasis, and signaling in apoptosis. Disturbances in mitochondrial dynamics are suggested to be involved in neurodegenerative diseases and CNS aging (11–14). In vivo imaging of axonal transport of mitochondria has been reported using explant imaging of the Drosophila nervous system (15) and rat cerebellar slice cultures (16) and intravital imaging (direct in vivo imaging of living animals at subcellular resolution) of the mouse peripheral nervous system (17) and zebrafish nervous system (18). However, intravital imaging of axonal transport of mitochondria has not been achieved in the mammalian CNS. Importantly, the lack of intravital imaging of axonal transport of mitochondria in the mammalian CNS under physiological oxygen levels and metabolism and with intact blood flow has precluded the characterization of the dynamics of axonal transport of mitochondria in the CNS of diseased and aged mammals in vivo.To perform intravital imaging of axonal transport of mitochondria in the mammalian CNS, we developed the technique we call “MIMIR” (for “minimally invasive intravital imaging of mitochondrial axonal transport in RGCs”). MIMIR does not involve thinning or opening of the sclera or produce changes in the intraocular humor. MIMIR directly showed, at submicrometer resolution, that axonal transport of mitochondria is highly dynamic in the mammalian CNS in vivo under physiological conditions. It enabled us to characterize disturbances of mitochondrial transport in a mouse model of glaucoma and age-related changes of mitochondrial transport in old (23- to 25-mo-old) mice.     

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.     

In vivo imaging of gene and cell therapies.

Molecular imaging can be broadly defined as the in vivo characterization and measurement of biological processes at the cellular and molecular level. In contrast to commonly used clinical imaging, it sets forth to probe the molecular abnormalities that are the basis of disease, rather than imaging the end effects of these molecular alterations. Development of new imaging technologies requires a multidisciplinary collaboration between biologists, chemists, physicists, and imaging scientists to create novel agents, signal amplification strategies, and imaging techniques that successfully address these questions. In this article we attempt to present some of the recent developments and show how molecular imaging can be used, at least experimentally, to assess specific molecular targets for gene- and cell-based therapies. In particular, we place emphasis on the development and use of experimental small-animal models, which are particularly inclined toward this approach, primarily in combination with magnetic resonance (MR), radionuclide, and optical imaging. In the future, specific imaging of disease targets will allow earlier detection and characterization of disease, as well as earlier and direct molecular assessment of treatment efficacy.     

Brief visual experience induces immediate early gene expression in the cat visual cortex.

Brief visual experience causes rapid physiological changes in the visual cortex during early postnatal development. A possible mediator of these effects is the immediate early genes whose protein products are involved in the rapid response of neurons to transsynaptic stimulation. Here we report evidence that the levels of immediate early gene mRNAs in the visual cortex can be altered by manipulating the visual environment. Specifically, we find that brief (1 h) visual experience in dark-reared cats causes dramatic transient inductions of egr1, c-fos, and junB mRNAs in the visual cortex but not in the frontal cortex. Levels of c-jun and c-myc mRNAs are unaffected. These results suggest that select combinatorial interactions of immediate early gene proteins are an important step in the cascade of events through which visually elicited activity controls visual cortical development.     

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.     

Homer-1a immediate early gene expression correlates with better cognitive performance in aging

The molecular mechanisms underlying cognitive decline during healthy aging remain largely unknown. Utilizing aged wild-type C57BL/6 mice as a model for normal aging, we tested the hypothesis that cognitive performance, memory, and learning as assessed in established behavioral testing paradigms are correlated with the differential expression of isoforms of the Homer family of synaptic scaffolding proteins. Here we describe a loss of cognitive and motor function that occurs when Homer-1a/Vesl-1S protein levels drop during aging. Our data describe a novel mechanism of age-related synaptic changes contributing to loss of biological function, spatial learning, and memory formation as well as motor coordination, with the dominant negative uncoupler of synaptic protein clustering, Homer-1a/Vesl-1S, as a potential target for the prophylaxis and treatment of age-related cognitive decline.

Electronic supplementary material

The online version of this article (doi:10.1007/s11357-012-9479-6) contains supplementary material, which is available to authorized users.     

Cellular-resolution gene expression profiling in the neonatal marmoset brain reveals dynamic species- and region-specific differences

Precise spatiotemporal control of gene expression in the developing brain is critical for neural circuit formation, and comprehensive expression mapping in the developing primate brain is crucial to understand brain function in health and disease. Here, we developed an unbiased, automated, large-scale, cellular-resolution in situ hybridization (ISH)–based gene expression profiling system (GePS) and companion analysis to reveal gene expression patterns in the neonatal New World marmoset cortex, thalamus, and striatum that are distinct from those in mice. Gene-ontology analysis of marmoset-specific genes revealed associations with catalytic activity in the visual cortex and neuropsychiatric disorders in the thalamus. Cortically expressed genes with clear area boundaries were used in a three-dimensional cortical surface mapping algorithm to delineate higher-order cortical areas not evident in two-dimensional ISH data. GePS provides a powerful platform to elucidate the molecular mechanisms underlying primate neurobiology and developmental psychiatric and neurological disorders.

The mammalian brain contains many functionally distinct regions that are extensively interconnected, enabling rapid and accurate information processing. Each region contains diverse cell types that differ in their molecular, morphological, electrophysiological, and functional characteristics (1, 2). Advanced neuroscience technologies, which are generally optimized and extensively applied in mice, provide complicated approaches to analyze these characteristics and model the genetic basis of this regional- and cell-type diversity. However, clear cognitive differences arise from interspecies differences in these key characteristics, and most contemporary technologies are difficult to apply in species whose study involves extensive ethical, practical, and experimental impediments. The ability to compare gene expression patterns between primates and model species such as mice is crucial for understanding the human brain (3, 4).Recent developments in technologies, such as microarray, single-cell RNA-sequencing (RNA-seq), and Drop-seq, have allowed for high-resolution genome-wide expression analysis in specific regions of the brain or in thousands of individual cells simultaneously (5–10). However, these methods provide limited anatomical information and no morphological observations. Slide-seq solves these problems by transferring RNA from tissue sections onto a surface covered in DNA-barcoded beads, allowing the preservation of positional information (11). However, the unbiased nature of next-generation sequencing technologies may hinder the molecular identification of specific cell types or their direct analysis. In situ hybridization (ISH) databases of humans and nonhuman primates (NHPs) have revealed brain gene expression profiles at cellular resolution, while preserving key morphological and anatomical characteristics, ultimately providing important information about the genetic conservation of analogous brain regions (12). However, these studies are highly resource intensive, requiring substantial time and cost. Consequently, such efforts have been limited, focusing on a few brain regions in limited species (13). Thus, interspecific comparison of brain-cell heterogeneity has not been possible.Here, we describe the development and implementation of an unbiased gene expression profiling system (GePS) for use with the Marmoset Gene Atlas (MGA) (https://gene-atlas.brainminds.riken.jp/) (14), which is adaptable for use with other atlases. The system, developed as a part of “Brain Mapping by Integrated Neurotechnologies for Disease Studies (Brain/MINDS),” a brain mapping program in Japan (15, 16), enables systematic and automatic analysis of gene expression across brain regions. Brain/MINDS aims to develop knowledge-based tools to explore primate-specific brain structure, function, and connectivity in health and disease. Specifically, we analyzed the expression patterns of identified risk genes for psychiatric disorders in the neonatal marmoset brain. There are >2,000 gene expression profiles for the neonatal common-marmoset brain in the MGA. Although each ISH section is complemented by an adjacent neuroanatomically stained section (visualizing the Nissl substance), enabling users to compare gene expression against a brain atlas, mapping gene expression patterns over the entire brain is both time and labor intensive. A large mouse ISH database, the Allen Brain Atlas (ABA; http://developingmouse.brain-map.org/), demonstrated that an anatomic gene expression atlas (AGEA) can characterize local gene expression in the mouse brain without prior knowledge of classical anatomy (17). To enhance the MGA in this manner, we developed GePS to systematically and automatically identify gene expression patterns in specific regions of the neonatal marmoset brain.GePS, including the gene expression comparison system and cortical surface projection mapping function, is publicly available as an open resource on our website (https://gene-atlas.brainminds.riken.jp/). It provides primate-specific whole brain–gene expression patterns and will be a valuable platform in the field of primate neuroscience, helping reveal primate-specific brain functions and connections and providing insights into brain evolution and pathology.     

Aging and in vivo norepinephrine-uptake in mammalian brain.

In order to examine the cellular effect of aging in the central nervous system, the regional distribution and the dynamic aspects of catecholamine metabolism in the brain were investigated. Results indicated that the endogenous norepinephrine (NE) content is lower in hypothalamus and brain stem of older rats (25 mo. n = 6) than younger rats (12 mo. n = 6). We have also observed in these animals that the age pigments were apparently absent in the brain tissue of young rats but become a very distinct feature for the old rats and that the catecholamine strong vesicles appear to be less dense in the nerve terminals of old as compared with the young ones. 3H-NE was administered intracerebrally to mice of different age groups (4, 8, 12, 21 and 24 months) and the NE-uptake activity was examined by measuring the radioactive NE inside the isolated synaptosomes 5 min after 3H-NE injection. The result indicated that the young group (4 mo) has highest uptake activity. The uptake activity decrease with the older mice and reached the lowest in the age group of 21-24 mo. The decrease in NE-uptake activity may be related to the deterioration of synaptosomal membranes.     

In vivo and in vitro gene transfer to mammalian somatic cells by particle bombardment.

Chimeric chloramphenicol acetyltransferase and beta-galactosidase marker genes were coated onto fine gold particles and used to bombard a variety of mammalian tissues and cells. Transient expression of the genes was obtained in liver, skin, and muscle tissues of rat and mouse bombarded in vivo. Similar results were obtained with freshly isolated ductal segments of rat and human mammary glands and primary cultures derived from these explants. Gene transfer and transient expression were also observed in eight human cell culture lines, including cells of epithelial, endothelial, fibroblast, and lymphocyte origin. Using CHO and MCF-7 cell cultures as models, we obtained stable gene transfer at frequencies of 1.7 x 10(-3) and 6 x 10(-4), respectively. The particle bombardment technology thus provides a useful means to transfer foreign genes into a variety of mammalian somatic cell systems. The method is applicable to tissues in vivo as well as to isolated cells in culture and has proven effective with all cell or tissue types tested thus far. This technology may therefore prove to be applicable in various aspects of gene therapy.     

In vivo detection and imaging of phosphatidylserine expression during programmed cell death

One of the earliest events in programmed cell death is the externalization of phosphatidylserine, a membrane phospholipid normally restricted to the inner leaflet of the lipid bilayer. Annexin V, an endogenous human protein with a high affinity for membrane bound phosphatidylserine, can be used in vitro to detect apoptosis before other well described morphologic or nuclear changes associated with programmed cell death. We tested the ability of exogenously administered radiolabeled annexin V to concentrate at sites of apoptotic cell death in vivo. After derivatization with hydrazinonicotinamide, annexin V was radiolabeled with technetium 99m. In vivo localization of technetium 99m hydrazinonicotinamide-annexin V was tested in three models: fuminant hepatic apoptosis induced by anti-Fas antibody injection in BALB/c mice; acute rejection in ACI rats with transplanted heterotopic PVG cardiac allografts; and cyclophosphamide treatment of transplanted 38C13 murine B cell lymphomas. External radionuclide imaging showed a two- to sixfold increase in the uptake of radiolabeled annexin V at sites of apoptosis in all three models. Immunohistochemical staining of cardiac allografts for exogenously administered annexin V revealed intense staining of numerous myocytes at the periphery of mononuclear infiltrates of which only a few demonstrated positive apoptotic nuclei by the terminal deoxynucleotidyltransferase-mediated UTP end labeling method. These results suggest that radiolabeled annexin V can be used in vivo as a noninvasive means to detect and serially image tissues and organs undergoing programmed cell death.     

In vivo laser speckle imaging reveals microvascular remodeling and hemodynamic changes during wound healing angiogenesis

Laser speckle contrast imaging (LSCI) is a high-resolution and high contrast optical imaging technique often used to characterize hemodynamic changes in short-term physiological experiments. In this study, we demonstrate the utility of LSCI for characterizing microvascular remodeling and hemodynamic changes during wound healing angiogenesis in vivo. A 2 mm diameter hole was made in the mouse ear and the periphery of the wound imaged in vivo using LSCI over 12 days. We were able to visualize and quantify the vascular and perfusion changes that accompanied wound healing in the microenvironment proximal to the wound, and validated these changes with histology. We found that consistent with the stages of wound healing, microvessel density increased during the initial inflammatory phase (i.e., day 0–3), stayed elevated through the tissue formation phase (i.e., until day 7) and returned to baseline during the tissue remodeling phase (i.e., by day 12). Concomitant “wide area mapping” of blood flow revealed that tissue perfusion in the wound periphery initially decreased, gradually increased from day 3–7, and subsided as healing completed. Interestingly, some regions exhibited a reestablishment of tissue perfusion approximately 6 days earlier than the ~18 days usually reported for the long term remodeling phase. The results from this study demonstrate that LSCI is an ideal platform for elucidating in vivo changes in microvascular hemodynamics and angiogenesis, and has the potential to offer invaluable insights in a range of disease models involving abnormal hemodynamics, such as diabetes and tumors.     

In vivo imaging of graft-versus-host-disease in mice

We have developed a mouse system by which to track the migration and homing of cells in a setting of bone marrow transplantation (BMT)-induced graft-versus-host disease (GVHD) after systemic infusion using enhanced green fluorescence protein (eGFP) transgenic (Tg) cells and a simple application of a fluorescence stereomicroscope outfitted with a color charge-coupled device (CCD) camera. Whole body images of anesthetized mice taken at various time points after cell infusion revealed the early migration of allogeneic cells to peripheral lymphoid organs, with later infiltration of GVHD target organs. Localization of eGFP Tg cells could be seen through the skin of shaved mice, and internal organs were easily discernible. After allogeneic or syngeneic eGFP Tg cell infusion, representative mice were dissected to better visualize deeper internal organs and tissues. Infusion of different cell populations revealed distinct homing patterns, and this method also provided a simple way to identify the critical time points for expansion of the transplanted cells in various organs. This simple application of the fluorescence stereomicroscope will be valuable for GVHD and graft-versus-tumor studies in which visualization of cellular migration, expansion, and cell-cell interactions will be more informative when analyzed by such an intravital method.     

In vivo imaging of alphaherpesvirus infection reveals synchronized activity dependent on axonal sorting of viral proteins

A clinical hallmark of human alphaherpesvirus infections is peripheral pain or itching. Pseudorabies virus (PRV), a broad host range alphaherpesvirus, causes violent pruritus in many different animals, but the mechanism is unknown. Previous in vitro studies have shown that infected, cultured peripheral nervous system (PNS) neurons exhibited aberrant electrical activity after PRV infection due to the action of viral membrane fusion proteins, yet it is unclear if such activity occurs in infected PNS ganglia in living animals and if it correlates with disease symptoms. Using two-photon microscopy, we imaged autonomic ganglia in living mice infected with PRV strains expressing GCaMP3, a genetically encoded calcium indicator, and used the changes in calcium flux to monitor the activity of many neurons simultaneously with single-cell resolution. Infection with virulent PRV caused these PNS neurons to fire synchronously and cyclically in highly correlated patterns among infected neurons. This activity persisted even when we severed the presynaptic axons, showing that infection-induced firing is independent of input from presynaptic brainstem neurons. This activity was not observed after infections with an attenuated PRV recombinant used for circuit tracing or with PRV mutants lacking either viral glycoprotein B, required for membrane fusion, or viral membrane protein Us9, required for sorting virions and viral glycoproteins into axons. We propose that the viral fusion proteins produced by virulent PRV infection induce electrical coupling in unmyelinated axons in vivo. This action would then give rise to the synchronous and cyclical activity in the ganglia and contribute to the characteristic peripheral neuropathy.Alphaherpesviruses are pathogens of the mammalian nervous system that often evoke manifestations of peripheral pain or itching after infection. The severity and extent of symptoms vary depending on the herpesvirus strain and the host. Herpes simplex virus (HSV) and varicella-zoster virus (VZV) infections in humans characteristically cause lesions that are preceded by sensations of localized pain, which also sometimes lasts long after lesions have been cleared (1, 2). Pseudorabies virus (PRV) is a swine alphaherpesvirus that is related to HSV and VZV. Whereas in its natural porcine host PRV establishes latency in the peripheral sensory ganglia, in nonnative animal hosts, including laboratory mice, PRV causes a severe acute peripheral neuropathy called the “mad itch” (3). This neuropathy is characterized by a strong impulse to bite and scratch the skin of the infected dermatome feverishly and incessantly, which results in severe self-inflicted wounds (4). PRV has proved useful for elucidating pathways involved in the manifestation of these clinical symptoms. Specific PRV gene products have been identified as virulence factors and these research efforts have led to successful live vaccines, such as the strain PRV Bartha and its derivatives (see ref. 5 for review). Despite this effort, the molecular basis for the pronounced peripheral neuropathies produced by PRV and the other alpha herpesviruses remain an active area of research.In the 1950s, Dempsher and colleagues recorded spontaneous bursts of neuronal activity in rat sympathetic superior cervical ganglia (SCG) infected with virulent PRV and the onset correlated with the mad itch symptomology (6). Other in vivo studies showed that the activity induced by PRV infection was reversibly blocked by curare and was cholinergic in origin (7, 8). Denervation of the preganglionic nerve before infection did not affect mad itch symptoms, yet no spontaneous electrical discharges were recorded in the ganglia (8). These studies suggested that increased infection-induced electrical activity originated in the presynaptic terminals within the ganglia (8, 9). A recent publication examined the electrical activity of homogenous cultures of peripheral nervous system (PNS) neurons from the SCG infected with virulent or attenuated PRV strains in vitro using patch clamp recordings (10). They found that neurons infected with a virulent PRV strain had increased and sustained rates of action potential firing late in infection that were synchronized among the neuronal population. Infection by the attenuated PRV strain Bartha exhibited a marked delay in the onset of abnormal firing. Importantly, these studies showed that the PRV membrane fusion protein glycoprotein B (gB) was required for elevated firing rates and fusion of infected neuron cell bodies. The authors concluded that fusion pores were formed during PRV infection, which enabled ions to flow between neurons to cause direct electrical coupling (10). As the pores increased in size, spontaneous action potentials spread into coupled neurons and throughout the network, causing firing events to synchronize and resonate in the culture. This synchronous firing phenomenon also manifests itself as a continuous and rapid calcium flux in infected cultured neurons that can lead to other dramatic changes, including changes in mitochondrial shape and dynamics (11). Therefore, the formations of viral-induced excitatory synaptic connections that allow for continuous propagation of action potentials do not reflect normal chemical transmission at conventional synapses, but rather an abnormal coupling that requires viral DNA replication and that can be strain dependent (12, 13).Whereas the results with cultured neurons suggested a mechanism by which PRV may induce neuropathy, the relatively simplified in vitro system still leaves unanswered questions about what happens during infection of animals. In particular, studies using the mouse flank scarification model showed that virulent and attenuated PRV strains, such as PRV Bartha, have two different mechanisms of neuroinvasion and lethality in mice (4). Specifically, the virulent strain induces the classic symptomology of violent pruritus with rapid onset of death, little viral antigen in the brain, and no signs of CNS pathology. In contrast, mice infected with PRV Bartha survive much longer, do not exhibit any peripheral neuropathy, have abundant infectious virus in the brain, but do present CNS abnormalities at very late times after infection and still succumb (4). Therefore, it appears that during infection with virulent PRV strains, but not attenuated PRV Bartha strains, the infected peripheral neural circuits are the primary players in determining the severe clinical manifestations. Our previous study in living mice showed that a PRV Bartha recombinant, expressing a genetically encoded calcium indicator, did not induce synchronous activity among neurons in an autonomic ganglia (14), However, we did not examine neuronal activity after virulent PRV infection. The differences between in vitro and in vivo infections are obvious, such as the organization of innervated tissues, structure of ganglia, and the involvement of glial cells, but their impact on infection and symptomology after infection with virulent or attenuated PRV strains is not clear.In this report, we examined neuronal activity during a virulent PRV infection of anesthetized mice by imaging infected PNS ganglia directly in vivo. To do so, we constructed virulent and mutant PRV recombinants that expressed the genetically encoded fluorescent calcium indicator protein GCaMP3 (15). This calcium sensor fluoresces in the presence of calcium transients and is therefore a useful correlate of neuronal activity (16). Calcium imaging enabled us to monitor the activity in vivo of multiple infected neurons simultaneously with single-cell resolution using two-photon microscopy. Interestingly, 2 d after infection of the salivary glands by virulent PRV, infected neurons in the submandibular ganglia (SMG) flashed synchronously and cyclically. There were no qualitative signs of fusion between soma in the ganglia. When we severed the axons between the brainstem and the ganglia in vivo and continued imaging, the flashing phenotype continued unabated, suggesting that the impetus to fire did not arise in the presynaptic brainstem neurons. In contrast, infection by a replicating, but attenuated PRV-Bartha–derived recombinant that does not cause violent pruritus showed no signs of synchronous or cyclical firing even at later times after infection. In addition, mutants that do not express either viral gB involved in membrane fusion or viral membrane protein Us9 required for sorting and transport of virions and viral glycoproteins such as gB in axons, showed no signs of synchronous or cyclical firing. These data validate in vivo the in vitro observation that virulent PRV infection induces electrical coupling via fusion events. Importantly, we further propose that in vivo, this electrical coupling may occur in the infected, unmyelinated axons that bundle together as they project to the salivary gland, and therefore the spatial organization of natural synaptic partners in vivo would contribute to the specific phenotype, which cannot be distinguished in vitro. Our in vivo evidence supports the hypothesis that the peripheral neuropathy observed during a virulent PRV infection in living mice results from sustained, aberrant firing of infected PNS neurons.