Effects of aging and sensory loss on glial cells in mouse visual and auditory cortices.

Normal aging is often accompanied by a progressive loss of receptor sensitivity in hearing and vision, whose consequences on cellular function in cortical sensory areas have remained largely unknown. By examining the primary auditory (A1) and visual (V1) cortices in two inbred strains of mice undergoing either age-related loss of audition (C57BL/6J) or vision (CBA/CaJ), we were able to describe cellular and subcellular changes that were associated with normal aging (occurring in A1 and V1 of both strains) or specifically with age-related sensory loss (only in A1 of C57BL/6J or V1 of CBA/CaJ), using immunocytochemical electron microscopy and light microscopy. While the changes were subtle in neurons, glial cells and especially microglia were transformed in aged animals. Microglia became more numerous and irregularly distributed, displayed more variable cell body and process morphologies, occupied smaller territories, and accumulated phagocytic inclusions that often displayed ultrastructural features of synaptic elements. Additionally, evidence of myelination defects were observed, and aged oligodendrocytes became more numerous and were more often encountered in contiguous pairs. Most of these effects were profoundly exacerbated by age-related sensory loss. Together, our results suggest that the age-related alteration of glial cells in sensory cortical areas can be accelerated by activity-driven central mechanisms that result from an age-related loss of peripheral sensitivity. In light of our observations, these age-related changes in sensory function should be considered when investigating cellular, cortical, and behavioral functions throughout the lifespan in these commonly used C57BL/6J and CBA/CaJ mouse models.

Second harmonic properties of tumor collagen: determining the structural relationship between reactive stroma and healthy stroma.

We utilize the polarization and directionality of light emitted by fibrillar collagen via second harmonic generation to determine structural relationships between collagen in mouse mammary tumor models and the healthy mammary fat pad. In spite of the aberrations in collagen production and degradation that are the hallmarks of tumor stroma, we find that the characteristic angle of SHG scatterers within collagen fibrils, and the spatial extent over which they are appropriately ordered for SHG production, are the same in tumor and healthy collagen. This suggests that the SHGproducing subpopulation of collagen is unaffected by the altered collagen synthesis of the tumor stroma, and protected from its aberrant degradative environment.

Age-related decline in Kv3.1b expression in the mouse auditory brainstem correlates with functional deficits in the medial olivocochlear efferent system.

Kv3.1b channel protein is widely distributed in the mammalian auditory brainstem, but studies have focused mainly on regions critical for temporal processing, including the medial nucleus of the trapezoid body (MNTB) and anteroventral cochlear nucleus (AVCN). Because temporal processing declines with age, this study was undertaken to determine if the expression of Kv3.1b likewise declines, and if changes are specific to these nuclei. Immunocytochemistry using an anti-Kv3.1b antibody was performed, and the relative optical density of cells and neuropil was determined from CBA/CaJ mice of four age groups. Declines in expression in AVCN, MNTB, and lateral superior olive (35, 26, and 23%) were found, but changes were limited to neuropil. Interestingly, cellular optical density declines were found in superior paraolivary nucleus, ventral nucleus of the trapezoid body, and lateral nucleus of the trapezoid body (24, 29, and 26%), which comprise the medial olivocochlear (MOC) feedback system. All declines occurred by middle age (15 months old). No age-related changes were found in the remaining regions of cochlear nucleus or in the inferior colliculus. Contralateral suppression of distortion-product otoacoustic emission amplitudes of age-matched littermates also declined by middle age, suggesting a correlation between Kv3.1 expression and MOC function. In search of more direct evidence for such a correlation, Kv3.1b knockout mice were examined. Knockouts show poor MOC function as compared to +/+ and +/- genotypes. Thus, Kv3.1b expression declines in MOC neurons by middle age, and these changes appear to correlate with functional declines in efferent activity in both middle-aged CBA mice and Kv3.1b knockout mice.

Glutamate-related gene expression changes with age in the mouse auditory midbrain.

Glutamate is the main excitatory neurotransmitter in both the peripheral and central auditory systems. Changes of glutamate and glutamate-related genes with age may be an important factor in the pathogenesis of age-related hearing loss-presbycusis. In this study, changes in glutamate-related mRNA gene expression in the CBA mouse inferior colliculus with age and hearing loss were examined and correlations were sought between these changes and functional hearing measures, such as the auditory brainstem response (ABR) and distortion product otoacoustic emissions (DPOAEs). Gene expression of 68 glutamate-related genes was investigated using both genechip microarray and real-time PCR (qPCR) molecular techniques for four different age/hearing loss CBA mouse subject groups. Two genes showed consistent differences between groups for both the genechip and qPCR. Pyrroline-5-carboxylate synthetase enzyme (Pycs) showed down-regulation with age and a high-affinity glutamate transporter (Slc1a3) showed up-regulation with age and hearing loss. Since Pycs plays a role in converting glutamate to proline, its deficiency in old age may lead to both glutamate increases and proline deficiencies in the auditory midbrain, playing a role in the subsequent inducement of glutamate toxicity and loss of proline neuroprotective effects. The up-regulation of Slc1a3 gene expression may reflect a cellular compensatory mechanism to protect against age-related glutamate or calcium excitoxicity.

The effects of early bilateral deafening on calretinin expression in the dorsal cochlear nucleus of aged CBA/CaJ mice.

The aim of this study was to test the hypothesis that calretinin (CR) levels in the aged mouse auditory brainstem depend upon hearing ability. Old animals with good hearing, and thus higher sound-evoked activity levels, were predicted to have higher levels of CR immunoreactivity than old animals with hearing loss. CR immunoreactivity was analyzed in the deep layer (layer III) of the dorsal cochlear nucleus (DCN) in CBA/CaJ mice that were bilaterally deafened at 3 months of age with kanamycin, and then aged until 24 months. This manipulation partially mimics the lack of sound-evoked auditory activity experienced by old C57BL/6J mice, who are deaf at 24 months of age (but show residual hearing at 15 months) and have lower levels of CR immunoreactivity than old CBA mice with normal hearing [Hear. Res. 158 (2001) 131]. Cell counts revealed that the density of CR+ cells in DCN layer III of the deafened CBA mice was statistically different from old intact CBA mice raised under identical conditions. Old deafened CBAs showed a decline of 47% in the mean density of CR+ cells compared to old hearing CBAs, thus supporting the hypothesis. Interestingly, while there tended to be fewer CR+ cells in the old deaf C57s as compared to young C57s and young and old CBAs with normal hearing, the difference was not statistically significant. It is possible that the residual hearing of C57 mice at 15 months may provide sufficient auditory input to maintain CR at levels higher than CBA mice that are deafened completely at 3 months of age, and are profoundly deaf for a much longer time (21 months).

Tumor-associated macrophages and stromal TNF-α regulate collagen structure in a breast tumor model as visualized by second harmonic generation.

Collagen fibers can be imaged with second harmonic generation (SHG) and are associated with efficient tumor cell locomotion. Preferential locomotion along these fibers correlates with a more aggressively metastatic phenotype, and changes in SHG emission properties accompany changes in metastatic outcome. We therefore attempted to elucidate the cellular and molecular machinery that influences SHG in order to understand how the microstructure of tumor collagen fibers is regulated. By quantifying SHG and immunofluorescence (IF) from tumors grown in mice with and without stromal tumor necrosis factor (TNF)-α and in the presence or absence of tumor-associated macrophages (TAMs), we determined that depletion of TAMs alters tumor collagen fibrillar microstructure as quantified by SHG and IF. Furthermore, we determined that abrogation of TNF-α expression by tumor stromal cells also alters fibrillar microstructure and that subsequent depletion of TAMs has no further effect. In each case, metastatic burden correlated with optical readouts of collagen microstructure. Our results implicate TAMs and stromal TNF-α as regulators of breast tumor collagen microstructure and suggest that this regulation plays a role in tumor metastasis. Furthermore, these results indicate that quantification of SHG represents a useful strategy for evaluating the cells and molecular pathways responsible for manipulating fibrillar collagen in breast tumor models.

Brain tumor imaging: live imaging of glioma by two-photon microscopy.

Glioblastoma is a highly invasive and aggressive brain tumor that is very difficult to treat. The rat glioma cell line CNS-1 is a widely used, well-characterized model of infiltrative glioma. We have used CNS-1 to study tumors that initiate within the brain parenchyma. The CNS-1 cells were stably transfected with the yellow fluorescent protein (YFP) variant Venus to enable standard one-photon and two-photon imaging. In this protocol, we describe how to prepare a cranial window and how to inject the tumor cells into the cerebral cortex at a depth suitable for two-photon imaging. Imaging can begin 24 h after implantation of the cells. Two-photon imaging uses a long excitation wavelength (920 nm); the emission spectrum is the same for the fluorophore as for standard one-photon imaging (emission maximum = 528 nm). Two-photon microscopy permits tissue imaging to depths of 500 µm with three-dimensional (3D) high resolution and minimal photodamage to surrounding tissues. Multiple imaging sessions can be conducted over weeks in the same animal, depending on how long the cranial window remains clear and how quickly the tumor grows. Using this technique, the kinetics of tumor growth and invasion into the surrounding brain parenchyma can be measured in the same animal. This model can be used for determining the molecular and cellular players in brain tumor growth and invasion and for testing potential drug therapies to prevent brain metastasis.

Brain tumor imaging: imaging brain metastasis using a brain-metastasizing breast adenocarcinoma.

Brain metastases from primary or secondary breast tumors are difficult to model in the mouse. When metastatic breast cancer cell lines are injected directly into the arterial circulation, only a small fraction of cells enter the brain to form metastatic foci. To study the molecular and cellular mechanisms of brain metastasis, we have transfected MB-231BR, a brain-homing derivative of a human breast adenocarcinoma line MDA-MB-231, with the yellow fluorescent protein (YFP) variant Venus. MB-231BR selectively enters the brain after intracardiac injection into the arterial circulation, resulting in accumulation of fluorescent foci of cells in the brain that can be viewed by standard fluorescence imaging procedures. We describe how to perform the intracardiac injection and the parameters used to quantify brain metastasis in brain sections by standard one-photon fluorescence imaging. The disadvantage of this model is that the kinetics of growth over time cannot be determined in the same animal. In addition, the injection technique does not permit precise placement of tumor cells within the brain. This model is useful for determining the molecular determinants of brain tumor metastasis.

Serotonin 2B receptor: upregulated with age and hearing loss in mouse auditory system.

Serotonin (5-HT) is a monoamine neurotransmitter. Serotonin may modulate afferent fiber discharges in the cochlea, inferior colliculus (IC) and auditory cortex. Specific functions of serotonin are exerted upon its interaction with specific receptors; one of those receptors is the serotonin 2B receptor. The aim of this study was to investigate the differences in gene expression of serotonin 2B receptors with age in cochlea and IC, and the possible correlation between gene expression and functional hearing measurements in CBA/CaJ mice. Immunohistochemical examinations of protein expression of IC in mice of different age groups were also performed. Gene expression results showed that serotonin 2B receptor gene was upregulated with age in both cochlea and IC. A significant correlation between gene expression and functional hearing results was established. Immunohistochemical protein expression studies of IC showed more serotonin 2B receptor cells in old mice relative to young adult mice, particularly in the external nucleus. We conclude that serotonin 2B receptors may play a role in the pathogenesis of age-related hearing loss.