Autoradiography and inmmunolabeling suggests that lizard blastema contains arginase-positive M2-like macrophages that may support tail regeneration.

BACKGROUND: The regenerating blastema of the tail in the lizard Podarcis muralis contains numerous macrophages among the prevalent mesenchymal cells. Some macrophages are phagocytic but others are devoid of phagosomes suggesting that they have other roles aside phagocytosis. METHODS: The presence of healing macrophages (M2-like) has been tested using autoradiographic, immunohistochemical and ultrastructural studies. RESULTS: Autoradiography shows an uptake of tritiated arginine in sparse cells of the blastema and in the regenerating epidermis. Bioinformatics analysis suggests that epitopes for arginase-1 and -2, recognized by the employed antibody, are present in lizards. Immunofluorescence shows sparse arginase immunopositive macrophages in the blastema and few macrophages also in the apical wound epidermis. The ultrastructural study shows that macrophages contain dense secretory granules, most likely inactive lysosomes, and small cytoplasmic pale vesicles. Some of the small vesicles are arginase-positive while immunolabeling is very diffuse in the macrophage cytoplasm. CONCLUSIONS: The presence of cells incorporating arginine and of arginase 1-positive cells suggests that M2-like macrophages are present among mesenchymal and epidermal cells of the regenerative tail blastema. M2-like macrophages may promote tail regeneration differently from the numerous pro-inflammatory macrophages previously detected in the scarring limb. The presence of M2-like macrophages in addition to hyaluronate, support the hypothesis that the regenerative blastema of the tail in lizards is an immuno-privileged organ where cell proliferation and growth occur without degenerating in a tumorigenic outgrowth.

Cilia-based flow network in the brain ventricles.

Cerebrospinal fluid conveys many physiologically important signaling factors through the ventricular cavities of the brain. We investigated the transport of cerebrospinal fluid in the third ventricle of the mouse brain and discovered a highly organized pattern of cilia modules, which collectively give rise to a network of fluid flows that allows for precise transport within this ventricle. We also discovered a cilia-based switch that reliably and periodically alters the flow pattern so as to create a dynamic subdivision that may control substance distribution in the third ventricle. Complex flow patterns were also present in the third ventricles of rats and pigs. Our work suggests that ciliated epithelia can generate and maintain complex, spatiotemporally regulated flow networks.

Planar Organization of Multiciliated Ependymal (E1) Cells in the Brain Ventricular Epithelium.

Cerebrospinal fluid (CSF) continuously flows through the cerebral ventricles, a process essential for brain homeostasis. Multiciliated ependymal (E1) cells line the walls of the ventricles and contribute importantly to CSF flow through ciliary beating. Key to this function is the rotational and translational planar cell polarity (PCP) of E1 cells. Defects in the PCP of E1 cells can result in abnormal CSF accumulation and hydrocephalus. Here, we integrate recent data on the roles of early CSF flow in the embryonic ventricles, PCP regulators (e.g., Vangl2 and Dishevelled), and cytoskeletal networks in the establishment, refinement, and maintenance of E1 cells' PCP. The planar organization mechanisms of E1 cells could explain how CSF flow contributes to brain function and may help in the diagnosis and prevention of hydrocephalus.

p73 is required for ependymal cell maturation and neurogenic SVZ cytoarchitecture.

The adult subventricular zone (SVZ) is a highly organized microenvironment established during the first postnatal days when radial glia cells begin to transform into type B-cells and ependymal cells, all of which will form regenerative units, pinwheels, along the lateral wall of the lateral ventricle. Here, we identify p73, a p53 homologue, as a critical factor controlling both cell-type specification and structural organization of the developing mouse SVZ. We describe that p73 deficiency halts the transition of the radial glia into ependymal cells, leading to the emergence of immature cells with abnormal identities in the ventricle and resulting in loss of the ventricular integrity. p73-deficient ependymal cells have noticeably impaired ciliogenesis and they fail to organize into pinwheels, disrupting SVZ niche structure and function. Therefore, p73 is essential for appropriate ependymal cell maturation and the establishment of the neurogenic niche architecture. Accordingly, lack of p73 results in impaired neurogenesis. Moreover, p73 is required for translational planar cell polarity establishment, since p73 deficiency results in profound defects in cilia organization in individual cells and in intercellular patch orientation. Thus, our data reveal a completely new function of p73, independent of p53, in the neurogenic architecture of the SVZ of rodent brain and in the establishment of ependymal planar cell polarity with important implications in neurogenesis. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 730-747, 2016.

Myosin Id is required for planar cell polarity in ciliated tracheal and ependymal epithelial cells.

In wild type (WT) tracheal epithelial cells, ciliary basal bodies are oriented such that all cilia on the cell surface beat in the same upward direction. This precise alignment of basal bodies and, as a result, the ciliary axoneme, is termed rotational planar cell polarity (PCP). Rotational PCP in the multi-ciliated epithelial cells of the trachea is perturbed in rats lacking myosin Id (Myo1d). Myo1d is localized in the F-actin and basal body rich subapical cortex of the ciliated tracheal epithelial cell. Scanning and transmission electron microscopy of Myo1d knock out (KO) trachea revealed that the unidirectional bending pattern is disrupted. Instead, cilia splay out in a disordered, often radial pattern. Measurement of the alignment axis of the central pair axonemal microtubules was much more variable in the KO, another indicator that rotational PCP is perturbed. The asymmetric localization of the PCP core protein Vangl1 is lost. Both the velocity and linearity of cilia-driven movement of beads above the tracheal mucosal surface was impaired in the Myo1d KO. Multi-ciliated brain ependymal epithelial cells exhibit a second form of PCP termed translational PCP in which basal bodies and attached cilia are clustered at the anterior side of the cell. The precise asymmetric clustering of cilia is disrupted in the ependymal cells of the Myo1d KO rat. While basal body clustering is maintained, left-right positioning of the clusters is lost.

Ependymal cell differentiation, from monociliated to multiciliated cells.

Primary and motile cilia differ in their structure, composition, and function. In the brain, primary cilia are immotile signalling organelles present on neural stem cells and neurons. Multiple motile cilia are found on the surface of ependymal cells in all brain ventricles, where they contribute to the flow of cerebrospinal fluid. During development, monociliated ependymal progenitor cells differentiate into multiciliated ependymal cells, thus providing a simple system for studying the transition between these two stages. In this chapter, we provide protocols for immunofluorescence staining of developing ependymal cells in vivo, on whole mounts of lateral ventricle walls, and in vitro, on cultured ependymal cells. We also provide a list of markers we currently use to stain both types of cilia, including proteins at the ciliary membrane and tubulin posttranslational modifications of the axoneme.

Targeted gene transfer into ependymal cells through intraventricular injection of AAV1 vector and long-term enzyme replacement via the CSF.

Enzyme replacement via the cerebrospinal fluid (CSF) has been shown to ameliorate neurological symptoms in model animals with neuropathic metabolic disorders. Gene therapy via the CSF offers a means to achieve a long-term sustainable supply of therapeutic proteins within the central nervous system (CNS) by setting up a continuous source of transgenic products. In the present study, a serotype 1 adeno-associated virus (AAV1) vector was injected into a lateral cerebral ventricle in adult mice to transduce the gene encoding human lysosomal enzyme arylsulfatase A (hASA) into the cells of the CNS. Widespread transduction and stable expression of hASA in the choroid plexus and ependymal cells was observed throughout the ventricles for more than 1 year after vector injection. Although humoral immunity to hASA developed after 6 weeks, which diminished the hASA levels detected in CSF from AAV1-injected mice, hASA levels in CSF were maintained for at least 12 weeks when the mice were tolerized to hASA prior of vector injection. Our results suggest that the cells lining the ventricles could potentially serve as a biological reservoir for long-term continuous secretion of lysosomal enzymes into the CSF following intracerebroventricular injection of an AAV1 vector.

Neural stem and progenitor cells in the aged subependyma are activated by the young niche.

Previous studies have demonstrated an age related decline in the size of the neural stem cell (NSC) pool and a decrease in neural progenitor cell proliferation, however, the mechanisms underlying these changes are unclear. In contrast to previous reports, we report that the numbers of NSCs is unchanged in the old age subependyma and the apparent loss is because of reduced proliferative potential in the aged stem cell niche. Transplantation studies reveal that the proliferation kinetics and migratory behavior of neural precursor cells are dependent on the age of the host animal and independent of the age of the donor cells suggesting that young and old age neural precursors are not intrinsically different. Factors from the young stem cell niche rescue the numbers of NSC colonies derived from old age subependyma and enhance progenitor cell proliferation in vivo in old age mice. Finally, we report a loss of Wnt signaling in the old age stem cell niche that underlies the lack of expansion of the NSC pool after stroke.

Ectopic ependymal cells in striatum accompany neurogenesis in a rat model of stroke.

Stroke-induced neurogenesis originates from a neural stem cell (NSC) niche in subventricular zone (SVZ). In mice, NSCs are concentrated in a so-called "neurogenic spot" in the lateral angle area of SVZ. We aimed to identify the "neurogenic spot" in the rat SVZ and to characterize the cellular changes in the ependymal cell compartment in this area at different time points after middle cerebral artery occlusion. The majority of ependymal cells outlining the ventricular wall did not proliferate, and their numbers in the "neurogenic spot" declined at 6 and 16weeks after stroke. Cells with the ultrastructural properties of ependymal cells were detected in the adjacent striatum. The number of these ectopic ependymal cells (EE cells) correlated positively with the magnitude of lateral ventricular enlargement and negatively with the ependymal cell number in the "neurogenic spot". EE cells were found along blood vessels, accumulated in the pericyst regions, and participated in scar formation but did not incorporate BrdU. We provide the first evidence for the occurrence of EE cells in the ischemic striatum following stroke.

Role of estradiol in the dynamic control of tanycyte plasticity mediated by vascular endothelial cells in the median eminence.

In the ever-changing physiological context of the neuroendocrine brain, the mechanisms by which cellular events involving neurons, astroglia, and vascular cells are coordinated to bring forth the appropriate neuronal signaling is not yet known but is amenable to examination. In the median eminence of the hypothalamus, endothelial cells are key players in the plasticity of tanycytes (specialized astroglia) and neuroendocrine synapse efficacy. Here we report that estradiol acts on both purified endothelial cells and isolated tanycytes to trigger endothelial-to-glial communication that leads to a sudden and massive retraction of tanycyte processes. The blockade of endothelial nitric oxide synthase by in vitro adenoviral-mediated gene transfer of a dominant-negative form of endothelial nitric oxide synthase abrogates the estradiol-induced tanycyte plasticity mediated by endothelial cells. In parallel, increases in prostaglandin-E(2) (PGE(2)) due to changes in cyclooxygenase (COX)-1 and COX-2 expression induced by the exposure of tanycytes to estradiol promote acute tanycyte plasticity. We also demonstrate by electron microscopy that the administration of PGE(2) to median eminence explants induces rapid neuroglial plasticity at the neurovascular junction of neurons that release GnRH (the neuropeptide controlling reproduction). Conversely, preventing local PGE(2) synthesis in the median eminence of adult female rats with the COX inhibitor indomethacin impairs the ovarian cycle, a process that requires a pulsatile, coordinated delivery of GnRH into the hypothalamo-hypophyseal portal system. Taken together, our findings show that estradiol controls the dialog between endothelial cells and astroglia to regulate neuroglial plasticity in the neuroendocrine brain.

Lineage analysis of quiescent regenerative stem cells in the adult brain by genetic labelling reveals spatially restricted neurogenic niches in the olfactory bulb.

The subventricular zone (SVZ) of the lateral ventricles is the major neurogenic region in the adult mammalian brain, harbouring neural stem cells within defined niches. The identity of these stem cells and the factors regulating their fate are poorly understood. We have genetically mapped a population of Nestin-expressing cells during postnatal development to study their potential and fate in vivo. Taking advantage of the recombination characteristics of a nestin::CreER(T2) allele, we followed a subpopulation of neural stem cells and traced their fate in a largely unrecombined neurogenic niche. Perinatal nestin::CreER(T2)-expressing cells give rise to multiple glial cell types and neurons, as well as to stem cells of the adult SVZ. In the adult SVZ nestin::CreER(T2)-expressing neural stem cells give rise to several neuronal subtypes in the olfactory bulb (OB). We addressed whether the same population of neural stem cells play a role in SVZ regeneration. Following anti-mitotic treatment to eliminate rapidly dividing progenitors, relatively quiescent nestin::CreER(T2)-targeted cells are spared and contribute to SVZ regeneration, generating new proliferating precursors and neuroblasts. Finally, we have identified neurogenic progenitors clustered in ependymal-like niches within the rostral migratory stream (RMS) of the OB. These OB-RMS progenitors generate neuroblasts that, upon transplantation, graft, migrate and differentiate into granule and glomerular neurons. In summary, using conditional lineage tracing we have identified neonatal cells that are the source of neurogenic and regenerative neural stem cells in the adult SVZ and occupy a novel neurogenic niche in the OB.

Ventricular wall granulations and draining of cerebrospinal fluid in chronic giant hydrocephalus.

BACKGROUND: In rare cases, adults with normal or almost normal cognition may have giant brain ventricles surrounded by a sliver of brain. Because the usual flow of cerebrospinal fluid (CSF) is interrupted in these individuals, they may develop alternative CSF pathways to preserve brain function. OBJECTIVE: To describe novel morphologic autopsy findings in a patient with chronic giant hydrocephalus that suggest the existence of alternative CSF draining pathways. DESIGN: Case report. SETTING: Autopsy study. PATIENT: A 48-year-old man with chronic compensated hydrocephalus associated with a Dandy-Walker malformation. MAIN OUTCOME MEASURE: Autopsy findings. RESULTS: We observed microscopic structures on the ventricular wall that may facilitate CSF resorption. Their histologic appearance, reminiscent of pacchionian granulations, showed the opposite relation in regard to CSF/blood compartments: whereas the core of a pacchionian granulation contains CSF and the granulation is bathed in blood of the venous sinus, the core of the ventricular granulation in our patient contained venules, with the granulation bathed in ventricular CSF. CONCLUSIONS: These previously unreported (to our knowledge) ventricular wall granulations may facilitate draining of CSF into the venous system when CSF outflow from the ventricular system is occluded. The presence of these ventricular structures illustrates biologic adaptation to anomalous conditions and successful compensation.

X-ray exposure induces apoptosis of some proliferative epidermal cells following traumatic spinal cord injury in adult rats.

We investigated whether X-ray radiation induced apoptosis of the proliferative ependymal cells (ECs) in adult rats with spinal cord injury (SCI) and the effect of X-ray radiation on the proliferative activities of ECs. A rat model with SCI was developed and used to determine the proliferation and apoptosis of ECs in the spinal cords after X-ray exposure. TUNEL assay and BrdU incorporation were used to detect apoptosis and proliferation respectively. We found that there were few TUNEL-positive cells in proliferative ependymal zone (EZ) after SCI except at the epicenter, and approximately half of the irradiated ECs became TUNEL-positive. However, these radiated ECs did not lose their proliferative activity until 1 week later and started to decrease rapidly after 1 week. The observation suggested that only part of ECs were sensitive to radiation and the nonsensitive cells continued their mitosis process. These findings indicated that X-ray exposure of the rats with SCI in early stage induced apoptosis of the proliferative ECs and partially inhibited their proliferative activities.

New neurons follow the flow of cerebrospinal fluid in the adult brain.

In the adult brain, neuroblasts born in the subventricular zone migrate from the walls of the lateral ventricles to the olfactory bulb. How do these cells orient over such a long distance and through complex territories? Here we show that neuroblast migration parallels cerebrospinal fluid (CSF) flow. Beating of ependymal cilia is required for normal CSF flow, concentration gradient formation of CSF guidance molecules, and directional migration of neuroblasts. Results suggest that polarized epithelial cells contribute important vectorial information for guidance of young, migrating neurons.

Anatomy and physiology of the leptomeninges and CSF space.

The arachnoid membrane and pia mater are the two membranous layers that comprise the leptomeninges. Cerebrospinal fluid is made within the ventricular system by cells of the choroid plexus and ependyma. This chapter describes in detail the normal anatomic structure and physiologic interactions of the cerebrospinal fluid and leptomeningeal space that are critical to our understanding and treatment of leptomeningeal metastases.

Upregulated and prolonged differentiation potential of the ependymal cells lining the ventriculus terminalis in human fetuses.

The ventriculus terminalis (VT) is a dilated cavity within the conus medullaris of the spinal cord. Although the VT was discovered in the mid-nineteenth century, little is known about its characteristics during development in human fetuses. Ependymal cells lining the cavities within the CNS retain high differentiation potential, and are believed to be responsible for the postnatal neurogenesis. To evaluate the differentiation capacity of the ependymal cells lining the VT during development, we examined glial fibrillary acidic protein (GFAP) and proliferating cell nuclear antigen (PCNA) expression in the spinal cord of 18-24-week-old human fetuses. GFAP is a marker for the degree of ependymal cell differentiation in the human fetus, and PCNA is a well-known marker for cell division. Morphological characteristics of the VT were also examined. At the lower portion of the conus medullaris, the central canal abruptly expands dorsally to become the VT. Then the VT widens bilaterally while its anteroposterior diameter reduces gradually in a caudal direction. Finally, the VT becomes a narrow, transverse slit at the level of the lowermost conus medullaris. Compared with those lining the central canal, more numerous ependymal cells lining the VT showed more intensive GFAP and PCNA expression throughout all gestational ages examined. This suggests that, in the developing human spinal cord, ependymal cells lining the VT retain their differentiation potential, including a higher proliferative capacity, until a later stage of development than those lining the central canal.

Injectable intrathecal delivery system for localized administration of EGF and FGF-2 to the injured rat spinal cord.

The administration of growth factors (GFs) for treatment of experimental spinal cord injury (SCI) has shown limited benefits. One reason may be the mode of delivery to the injury site. We have developed a minimally invasive and safe drug delivery system (DDS) consisting of a highly concentrated collagen solution that can be injected intrathecally at the site of injury providing localized delivery of GFs. Using the injectable DDS, epidermal growth factor (EGF) and basic fibroblast growth factor (FGF-2) were co-delivered in the subarachnoid space of Sprague-Dawley rats. The in vivo distribution of EGF and FGF-2 in both injured and uninjured animals was monitored by immunohistochemistry. Although significant differences in the distribution of EGF and FGF-2 in the spinal cord were evident, localized delivery of the GFs resulted in significantly less cavitation at the lesion epicenter and for at least 720 mum caudal to it compared to control animals without the DDS. There was also significantly more white matter sparing at the lesion epicenter in animals receiving the GFs compared to control animals. Moreover, at 14 days post-injection, delivery of the GFs resulted in significantly greater ependymal cell proliferation in the central canal immediately rostral and caudal to the lesion edge compared to controls. These results demonstrate that the injectable DDS provides a new paradigm for localized delivery of bioactive therapeutic agents to the injured spinal cord.

Neurogenesis in the ependymal layer of the adult rat 3rd ventricle.

Neurogenesis has been described in limited regions of the adult mammalian brain. In this study, we showed that the ependymal layer of the 3rd ventricle is a neurogenic region in the adult rat brain. DiI labeling of the 3rd ventricle revealed that neural progenitor cells were derived from cells at the ependymal layer of the adult 3rd ventricle. The mitosis of these progenitor cells at the ependymal layer was promoted by bFGF administration. Combination of BrdU administration, nestin/GFAP immunohistochemistry, and labeling by GFP-recombinant adenoviral infection (vGFP) indicated that at least some tanycytes might be neural progenitor cells in the ependymal layer of the 3rd ventricle. Tracing by vGFP indicated that neural progenitor cells may have migrated from the 3rd ventricle to the hypothalamic parenchyma, where they were integrated into neural networks by forming synapses. In addition, some BrdU(+) neurons had immunoreactivity for orexin A in the hypothalamus. These results indicate that neural progenitor cells exist in the ependymal layer of the adult rat 3rd ventricle and that they may differentiate into neurons functioning in the hypothalamus.

Vascular endothelial cells promote acute plasticity in ependymoglial cells of the neuroendocrine brain.

Glial and endothelial cells interact throughout the brain to define specific functional domains. Whether endothelial cells convey signals to glia in the mature brain is unknown but is amenable to examination in circumventricular organs. Here we report that purified endothelial cells of one of these organs, the median eminence of the hypothalamus, induce acute actin cytoskeleton remodeling in isolated ependymoglial cells and show that this plasticity is mediated by nitric oxide (NO), a diffusible factor. We found that both soluble guanylyl cyclase and cyclooxygenase products are involved in this endothelial-mediated control of ependymoglia cytoarchitecture. We also demonstrate by electron microscopy that activation of endogenous NO release in the median eminence induces rapid structural changes, allowing a direct access of neurosecretory axons containing gonadotropin-releasing hormone (GnRH) (the neuropeptide controlling reproductive function) to the portal vasculature. Local in vivo inhibition of NO synthesis disrupts reproductive cyclicity, a process that requires a pulsatile, coordinated delivery of GnRH into the hypothalamic-adenohypophyseal portal system. Our results identify a previously unknown function for endothelial cells in inducing neuroglial plasticity and raise the intriguing possibility that endothelial cells throughout the brain may use a similar signaling mechanism to regulate glial-neuronal interactions.

Intracerebroventricular galanin-like peptide induces different brain activation compared with galanin.

Like galanin, the 60-amino-acid peptide, galanin-like peptide (GALP), has orexigenic actions, demonstrated by an acute increase in feeding after central injection in rodents. However, in contrast to galanin, GALP causes a prolonged rise in core body temperature and a reduction in body weight over 24 h. In an attempt to identify potential explanations for the observed differences between GALP and galanin, this study examined which brain areas were activated by these peptides. Intracerebroventricular injection of GALP into conscious rats significantly stimulated feeding over 0-1 h, increased core body temperature, but reduced body weight gain over 24 h. Immunohistochemistry to detect c-fos demonstrated that intracerebroventricular injection of GALP or galanin activated several brain regions in common, including the dorsomedial nucleus of the hypothalamus, lateral hypothalamus, and nucleus tractus solitarius of the brainstem. However, GALP also induced c-fos expression in the periventricular hypothalamic region and supraoptic hypothalamic nucleus. Cell activation induced by GALP in the supraoptic hypothalamic nucleus and nucleus tractus solitarius was dependent on food intake but independent of food consumption in all other brain regions. Double immunohistochemistry indicated that small cells expressing c-fos in the periventricular hypothalamic region after GALP were astrocytes and not microglia.