Therapeutic strategies to recover ependymal barrier after inflammatory damage: relevance for recovering neurogenesis during development

Abstract

The epithelium covering the surfaces of the cerebral ventricular system is known as the ependyma, and is essential for maintaining the physical and functional integrity of the central nervous system. Additionally, the ependyma plays an essential role in neurogenesis, neuroinflammatory modulation and neurodegenerative diseases. Ependyma barrier is severely affected by perinatal hemorrhages and infections that cross the blood brain barrier. The recovery and regeneration of ependyma after damage are key to stabilizing neuroinflammatory and neurodegenerative processes that are critical during early postnatal ages. Unfortunately, there are no effective therapies to regenerate this tissue in human patients. Here, the roles of the ependymal barrier in the context of neurogenesis and homeostasis are reviewed, and future research lines for development of actual therapeutic strategies are discussed.

Keywords: FoxJ1; cell therapy; ependyma; germinal matrix hemorrhage; neural stem cell; neurogenesis; neurogenic niche; posthemorrhagic hydrocephalus.

Figure 1

Representative scheme illustrating the transition of the ventricular wall epithelium during human brain development. The ventricular wall epithelium is constituted by different cell types during embryonic development. The first components are the neuroepithelial cells, polarized cells, highly proliferative, with an apical single cilium and a long basal prolongation that extends and contacts the marginal zone. The neuroepithelial cells give rise to radial glial cells with a morphology similar to neuroepithelial cells but with long basal processes that contact the meninges or blood vessels, and their cell bodies are in the ventricular zone. Radial glial cells differentiate mostly into multiciliated ependymal cells. This occurs at around 25 weeks of gestation in humans. Finally, a small subpopulation of radial glial cells differentiates into postnatal stem cells that give rise to neurons and oligodendrocytes. The germinal matrix is situated under and along the ventricular wall epithelium and hosts progenitor and intermediate progenitor cells with high mitotic activity. Neurogenesis and gliogenesis takes place in this region during the embryonic neural development until the 32nd -34 th gestational week in humans, when a decrease in mitotic activity becomes evident. Germinal matrix thickness begins to decrease after the 24 th week of gestation and is practically absent at 36 and 37 weeks of gestation. After the 34 th week, the brain region that is equivalent of the germinal matrix is commonly referred to as germinal ventricular zone or subventricular zone (SVZ) or subependymal zone (SEZ).

Figure 2

Representative scheme illustrating the factors that can influence the IVH in the germinal matrix. There are two main group of risk factors that increase the probability of suffering IVH in the germinal matrix, one related to fragility of the blood vessels, and another related to increased demand for blood flow. Hypoxia, seizures, coagulation disorders or problems during the vaginal delivery are also risk factors that may trigger IVH.

Figure 3

Main characteristics that define of GMH/IVH in the Papile System and Volpe System. For each grade, the extent of the hemorrhage and the percentage of blood detected into the ventricle cavities that allows the classification is indicated.

Figure 4

Representative drawings illustrating how the germinal matrix IVH affects the surrounding structures depending on the grade of severity of the IVH. The IVH has been graded under the Papile system. In grade I, hemorrhage affects only to the germinal matrix. In this case, IVH will affect to the ependyma if the hemorrhage occurs close to the ventricular border. The damage is usually minimal. In grade II, the hemorrhage occurs in the germinal matrix but progresses into the ventricles, however, IVH does not cause ventricular dilatation. Ependyma is damaged and blood products spread into the CSF affecting more ependymal areas. In grade III the hemorrhage that occurs in the germinal matrix progresses into the ventricles and, additionally, causes ventricular dilatation. It will be also considered grade III if the IVH occupies more than 50% of the ventricular cavity, even if no ventricle dilation is detected. The damage in the ependyma in this grade is severe, the blood products reach more areas of the ventricle cavities and blood by-products induce a more generalized inflammatory response. In grade IV the hemorrhage has spread to the brain parenchyma and the ependyma disruption and inflammatory response will be severe. CC, Corpus Callosum; Ep, Ependyma; GMH, Germinal Matrix Hemorrhage; H, Hemorrhage: IVH, Intraventricular Hemorrhage; LV, Lateral Ventricle; St, Striatum.

Figure 5

The different roles of ependyma barrier to control the brain homeostasis and neurogenesis. Center, Schematic representation of ependyma epithelium showing the position of the stem cells and the astrocytes located in the subventricular zone. Ependyma provides metabolic support for the astrocytes and stem cells that are in close proximity and will create the proper environment for stem cell function by the various mechanisms represented in this figure. Ep, Ependyma; SC, stem cells; A, astrocytes. Top left, Ependyma actively controls water transport and ion homeostasis by the presence of aquaporins, channels to regulates water flow and osmotic pressure, and different types of cotransporters such as KCC or NKCC1. Top Center, Ependyma plays an active role in preventing infection by expressing receptors in its surface that can activate an immune response against viruses or bacteria. Some representative receptors for this purpose are CD55, or CD59. Additionally, ependyma present components of the complement pathway. The most representative is TLR4. Ependyma will sense and react to infections and will active the nearby microglia. Bottom Left, To control without completely blocking paracellular transport, ependymal cells presents adherent junctions generated by the interactions of cadherins and catenins in a homotypic fashion among apical-lateral surfaces of adjacent ependymal cells; Ependymal cells lack complete tight junctions. These associations are crucial to maintain the differentiation of the CSF and brain parenchyma environments. Bottom Right, Ependyma controls the concentration of surrounding regulatory peptides that are present in the CSF by expressing enzymes that break down the molecules that may affects the ependyma itself or the germinal matrix/subventricular zone. Therefore, ependyma controls growth factors, chemokines, hormones, and neuropeptides on itself and in brain parenchyma. Top Right, Ependyma prevents the diffusion of damaging molecules in the ventricular zone or in brain parenchyma by the presence of receptors located in their membrane. Ependyma presents receptors for drugs, pathological proteins, or enzymatic degradation products. If these products persist, the ependyma function is compromised.

Figure 6

Representative scheme illustrating and summarizing the direct interaction between the multiciliated ependyma and stem cells key for normal neurogenesis. Top left, Cross-section of ependyma epithelium showing the position of the multiciliated ependymal cells and the stem cells. Multiciliated ependymal cells are cuboidal in shape and are distributed throughout the ventricular surface. However, the stem cells have an astrocytic-like shape, and appear in clusters, with the cell body bellow the multiciliated ependymal cells. Only a thin cytoplasmatic extension projects to the ventricular surface. In the apical membrane contacting the CSF, the stem cells present only a single cilium. Top right, Schematic representation of a top-view of ependyma epithelium showing the position of the multiciliated ependymal cells and the stem cells organized in a pinwheel-like structure. The center of each pinwheel-like structure consists of a cluster of stem cells surrounded by multiciliated ependymal cells. This structure ensures a direct and permanent interaction between multiciliated ependymal cells and stem cells that allows to the ependymal cells to exert a decisive control of the stem cell function. Bottom, Summary of the principal mechanisms used by multiciliated ependymal cells to control stem cell function. Both, direct physical interactions through basolateral adhesion molecules (N-Cadherin, and fractone Bulbs), or paracrine signaling (Noggin, PRL2, PEDF, CCN1, MMP12, TSK) and their effects are represented.

Figure 7

Inflammatory response that takes place after a GMH/IVH in the germinal matrix and ependyma. After the GMH/IVH, erythrocytes are released into the ventricular system. Lysed erythrocytes will release their potentially neurotoxic components (PRX2, hemoglobin, and iron), that together with thrombin released into the CSF after the hemorrhage will induce and the potentiate primary and secondary lesion. The primary and secondary lesions are also initiated and potentiated by the microglia activation. Activation of microglia occurs after the GMH/IVH by the Toll-like receptors, the NOD-like receptors, receptors for nucleic acids, and C-type lectin receptors. The release of cytokines will strongly increase neuroinflammation, edema and oxidative stress. Neuroinflammation will also increase edema and mechanical pressure over time.

Figure 8

Molecular mechanisms behind the de-differentiation of ependymal cells during neuroinflammation. In normal ependyma, phosphorylation of Foxj1 transcription factor by IKK2 complex (the kinase complex subunit 2) in a non-canonical manner (IKKγ/NF-KB-independent manner) prevents its degradation by ubiquitin proteosome system and allows the Foxj1 transcription factor to reach the nucleus to maintain mature ependyma status. Under pathological conditions/inflammatory conditions, the TLR4 pathway is activated, IKKγ/NF-KB canonical activity triggered and phosphorylation of Foxj1 transcription factor is reduced, Foxj1 transcription factor is degradated by the ubiquitin proteosome system and loss of mature status occurs.

Figure 9

The role of ependyma on neurogenesis in normal and neuroinflammation induced by GMH/IVH conditions. In healthy ependyma, phosphorylation of Foxj1 transcription factor through non-canonical IKK2 signaling (IKKγ/NF-KB-independent manner) activates Ank3 pathway to maintain the mature multiciliated ependyma. In this status, ependymal cells exert direct control on neurogenesis by maintaining the pinwheel-like structure, by cell–cell contacts through basolateral adhesion molecules (N-Cadherin, and fractone Bulbs), and by paracrine signaling (Noggin, PRL2, PEDF, CCN1, MMP12, TSK). Additionally, ependymal cells maintain the appropriated microenvironment for stem cell function through its protective and homeostatic barrier function. In this conditions, neural stem cells produce the needed progeny. After GMH/IVH, TLR4 pathway is activated, IKKγ/NF-KB canonical activity triggered, Foxj1 transcription factor is reduced and its degradation by ubiquitin proteosome system is potentiated, initiating loss of mature status. Pinwheel-like structure disappears, control of stem cell function by cell–cell contacts through basolateral adhesion molecules is altered and the paracrine control of stem cell function becomes defective. The microenvironment that ependymal cells generate for proper stem cell function is disrupted and homeostatic barrier function of ependyma is lost. Additionally, IKKγ/NF-KB canonical activity induces SPAK activation that leads to NKCC1 upregulation and hypersecretion of CSF, contributing to edema and hydrocephalus, that will further alter the microenvironment. Neural stem cells produce mostly astrocytes under these pathological conditions.

Figure 10

Risks of untreated ependymal damage. In healthy conditions, normal ependyma maintains a mature status capable of regulating brain homeostasis and sustaining neurogenesis. If GMH/IVH occurs, neuroinflammatory conditions induce the de-differentiation of mature ependyma, neurogenesis defects and edema/hydrocephalus. Additionally, mature ependyma discontinuities in the ependymal lining, which then induces short and mid-term proliferation of periventricular astrocytes, preventing proper function of remaining ependyma and, therefore, to the normal stem cell function. Astrocytes additionally increase expression of AQP4 that contributes to abnormal CSF flux and circulation, which aggravates edema and hydrocephalus. These two phenomena will increase cytotoxicity, mechanical pressure, and oxidative stresses that will potentiate inflammatory reactions, and will increase ependyma damage and periventricular astrocytic reaction.

Figure 11

Current treatments used to treat GMH/IVH and PHH. Surgical interventions to treat GMH/IVM/ and PHH aim to alleviate ventricular pressure by drainage of CSF and blood to reduce the risk of hydrocephalus. Those treatments (green arrows) are efficient for their specific purpose, but neglect to treat or recover the underlying ependymal damage that persists. In the same way, these treatments are not directed to treat or recover the lost neurogenesis aspect, critical for proper neonatal development. Stem cell-based treatments with MSCs are efficient in reducing inflammatory conditions after bleeding (green arrows), including a marked reduction of astroglia reactions (green arrow). However, attempts using MSCs to recover the ependyma or restore neurogenesis have not yet been successful (dashed line). While introduced NSCs do integrate into the ventricular wall, no efficient treatment based on NSCs has been successful in restoring neurogenesis (dashed line). Therefore, loss of mature ependyma status and the consequent alteration in neurogenesis is still lacking treatment and is the main gap remaining to be addressed.