Activation of pial and dural macrophages and dendritic cells by cortical spreading depression.

OBJECTIVE: Cortical spreading depression (CSD) has long been implicated in migraine attacks with aura. The process by which CSD, a cortical event that occurs within the blood-brain barrier (BBB), results in nociceptor activation outside the BBB is likely mediated by multiple molecules and cells. The objective of this study was to determine whether CSD activates immune cells inside the BBB (pia), outside the BBB (dura), or in both, and if so, when. METHODS: Investigating cellular events in the meninges shortly after CSD, we used in vivo two-photon imaging to identify changes in macrophages and dendritic cells (DCs) that reside in the pia, arachnoid, and dura and their anatomical relationship to TRPV1 axons. RESULTS: We found that activated meningeal macrophages retract their processes and become circular, and that activated meningeal DCs stop migrating. We found that CSD activates pial macrophages instantaneously, pial, subarachnoid, and dural DCs 6-12 minutes later, and dural macrophages 20 minutes later. Dural macrophages and DCs can appear in close proximity to TRPV1-positive axons. INTERPRETATION: The findings suggest that activation of pial macrophages may be more relevant to cases where aura and migraine begin simultaneously, that activation of dural macrophages may be more relevant to cases where headache begins 20 to 30 minutes after aura, and that activation of dural macrophages may be mediated by activation of migratory DCs in the subarachnoid space and dura. The anatomical relationship between TRPV1-positive meningeal nociceptors, and dural macrophages and DCs supports a role for these immune cells in the modulation of head pain. Ann Neurol 2018;83:508-521.

Molecular components and polarity of radial glial cells during cerebral cortex development.

Originating from ectodermal epithelium, radial glial cells (RGCs) retain apico-basolateral polarity and comprise a pseudostratified epithelial layer in the developing cerebral cortex. The apical endfeet of the RGCs faces the fluid-filled ventricles, while the basal processes extend across the entire cortical span towards the pial surface. RGC functions are largely dependent on this polarized structure and the molecular components that define it. In this review, we will dissect existing molecular evidence on RGC polarity establishment and during cerebral cortex development and provide our perspective on the remaining key questions.

Effect of Transplantation of Mesenchymal Stem Cells on the Density of Pial Microvascular Network in Spontaneously Hypertensive Rats of Different Age.

Using a TV device for studying microcirculation (×40), we analyzed the density of the whole microvascular network and the density of arterioles in the pia mater of the sensorimotor cortex in SHR rats of different ages (3-4 and 12 months) after intracerebral transplantation of human mesenchymal stem cells. We found that the density of pial microvascular network in SHR rats receiving transplantation of human mesenchymal stem cells increased to a level observed in young Wistar-Kyoto rats.

A case of mistaken identity: CD11c-eYFP(+) cells in the normal mouse brain parenchyma and neural retina display the phenotype of microglia, not dendritic cells.

Under steady-state conditions the central nervous system (CNS) is traditionally thought to be devoid of antigen presenting cells; however, putative dendritic cells (DCs) expressing enhanced yellow fluorescent protein (eYFP) are present in the retina and brain parenchyma of CD11c-eYFP mice. We previously showed that these mice carry the Crb1(rd8) mutation, which causes retinal dystrophic lesions; therefore we hypothesized that the presence of CD11c-eYFP(+) cells within the CNS may be due to pathology associated with the Crb1(rd8) mutation. We generated CD11c-eYFP Crb1(wt/wt) mice and compared the distribution and immunophenotype of CD11c-eYFP(+) cells in CD11c-eYFP mice with and without the Crb1(rd8) mutation. The number and distribution of CD11c-eYFP(+) cells in the CNS was similar between CD11c-eYFP Crb1(wt/wt) and CD11c-eYFP Crb1(rd8/rd8) mice. CD11c-eYFP(+) cells were distributed throughout the inner retina, and clustered in brain regions that receive input from the external environment or lack a blood-brain barrier. CD11c-eYFP(+) cells within the retina and cerebral cortex of CD11c-eYFP Crb1(wt/wt) mice expressed CD11b, F4/80, CD115 and Iba-1, but not DC or antigen presentation markers, whereas CD11c-eYFP(+) cells within the choroid plexus and pia mater expressed CD11c, I-A/I-E, CD80, CD86, CD103, DEC205, CD8α and CD135. The immunophenotype of CD11c-eYFP(+) cells and microglia within the CNS was similar between CD11c-eYFP Crb1(wt/wt) and CD11c-eYFP Crb1(rd8/rd8) mice; however, CD11c and I-A/I-E expression was significantly increased in CD11c-eYFP Crb1(rd8/rd8) mice. This study demonstrates that the overwhelming majority of CNS CD11c-eYFP(+) cells do not display the phenotype of DCs or their precursors and are most likely a subpopulation of microglia. GLIA 2016. GLIA 2016;64:1331-1349.

Discovery of a novel fibrous tissue in the spinal pia mater by polarized light microscopy.

Abstract It is very well known that spinal meninges are composed of three layers, dura, arachnoid and pia mater, and that the main components of pia mater are collagen and reticular fibers. However, the distribution of those fibers has not been extensively investigated but just described as a mesh of fibers. In this study, we detected novel structures, which are composed of unidirectionally arranged fibers, in a rat spinal pia mater by using a polarized light microscope. They were seen as three parallel lines, one of which ran along a posterior spinal vein and the rest two of which ran along a pair of posterior spinal arteries. Histological analysis including Masson's trichrome, picrosirius-red staining, Gordon & Sweet's staining and immunohistochemistry with anti-collagen type 1 and 3 antibodies uncovered that they are mainly composed of collagen fibers and some reticular fibers. In addition, a putative primo vessel was detected in the novel fibrous tissue, which was proven out to be different from a blood vessel. In conclusion, we report a newly detected fibrous structure in the spinal pia mater, which may contribute to provide tensile force to the spinal meninges and to harbor the primo vascular system inside.

[Mesenchymal stem sell donor age effect on the cerebral cortex microvascular net density in old age rats-recipients of transplant].

Male Wistar-Kyoto rats aged 22-24 months were intracerebrally transplanted with syngenic bone marrow mesenchymal stem cells (BM MSC) established from the donor aged 3-4 months and 20-22 months, respectively. Using a TV device to study microcirculation in vivo, we have established that transplantation of BM MSC from young donors increased a density of the microvascular network in the pia mater of the sensorimotor cortex in old rats approximately 1.9-fold, comparing to age-matched controls, while a density of the arteriolar compartment increased approximately 2.1-fold. Transplantation of BM MSC from old donors did not lead to the significant increase in the density of the microvascular network in the pia mater, while a density of the arteriolar compartment increased approximately 1.5-fold.

Effect of transplantation of mesenchymal stem cells on the density of pial microvascular network in rats of different age.

Using a TV device for studying microcirculation (×40), we studied the density of the whole microvascular network and arteriolar its compartment in the pia mater of the sensorimotor cortex in rats of different age (2-3, 12, and 24 months) after intracerebral transplantation of mesenchymal stem cells or nutrient medium (control). The density of the microvascular network in the pia mater remained practically unchanged until 1 year, but then decreased by 1.8 times with adding (up to 2 years). MSC transplantation 1.5-1.8-fold increased the density of the pial microvessels in animals of all age groups in comparison with intact and control rats; the density of the arteriolar compartment increased by 2.1-2.4 times. Intracerebral injection of MSC to 1-year-old animals prevented pathological decrease in the density of microvascular network during the next year of life.

Stroke induces disease-specific myeloid cells in the brain parenchyma and pia.

Inflammation triggers secondary brain damage after stroke. The meninges and other CNS border compartments serve as invasion sites for leukocyte influx into the brain thus promoting tissue damage after stroke. However, the post-ischemic immune response of border compartments compared to brain parenchyma remains poorly characterized. Here, we deeply characterize tissue-resident leukocytes in meninges and brain parenchyma and discover that leukocytes respond differently to stroke depending on their site of residence. We thereby discover a unique phenotype of myeloid cells exclusive to the brain after stroke. These stroke-associated myeloid cells partially resemble neurodegenerative disease-associated microglia. They are mainly of resident microglial origin, partially conserved in humans and exhibit a lipid-phagocytosing phenotype. Blocking markers specific for these cells partially ameliorates stroke outcome thus providing a potential therapeutic target. The injury-response of myeloid cells in the CNS is thus compartmentalized, adjusted to the type of injury and may represent a therapeutic target.

The thread-protective cell, a new cell performing multiple tasks.

Our research work, which has led us to discovering the new cerebral cell, has started 30 years ago. An important moment was the year 1986, when we have highlighted it for the first time, during a study upon the clarification of some undiscovered aspects of cerebral atherosclerosis. In 2006 we have initiated the publishing of our results at three congresses (Cape Town - 2006; San Diego - 2009 and Los Angeles - 2011) as well as in three Atlases, form 2006, 2008 and 2010. By means of the electronic microscope we have analyzed to this purpose alone, a number of neurosurgery patients, with 1176 cerebral, vascular, tumoral, cortical, choroid plexus tumor and infectious biopsies. The cell in question was named cordocit-protectocit (thread-protective cell) in order to highlight its morphological aspect of a belt band and its functional one, of protective element of the noble substance of the brain, acting for its defense against various aggressions, especially hemorrhagic. On this occasion we have discovered that the pia mater is made up of such protective cells, which also play a role in preventing the neuroblasts from migrating. When the chemotactants of our cells are not numerous enough, subcortical cell heterotopias will occur, at the level of the corona radiata, double cortex and other neuronal migration disorders which may generate epilepsy. Therefore, the pia mater should be considered from a cytodynamic perspective. The telocyte at the internal organs level (intestine, heart etc.) is nothing else but the interstitial cell of Cajal (ICC), described by Cajal more than 100 years ago. The ICC spontaneously initiate rhythmic electrical activity, much like the peacemaker cells of the heart.

Mechanical Properties of the Cranial Meninges: A Systematic Review.

The meninges are membranous tissues that are pivotal in maintaining homeostasis of the central nervous system. Despite the importance of the cranial meninges in nervous system physiology and in head injury mechanics, our knowledge of the tissues' mechanical behavior and structural composition is limited. This systematic review analyzes the existing literature on the mechanical properties of the meningeal tissues. Publications were identified from a search of Scopus, Academic Search Complete, and Web of Science and screened for eligibility according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. The review details the wide range of testing techniques employed to date and the significant variability in the observed experimental findings. Our findings identify many gaps in the current literature that can serve as a guide for future work for meningeal mechanics investigators. The review identifies no peer-reviewed mechanical data on the falx and tentorium tissues, both of which have been identified as key structures in influencing brain injury mechanics. A dearth of mechanical data for the pia-arachnoid complex also was identified (no experimental mechanics studies on the human pia-arachnoid complex were identified), which is desirable for biofidelic modeling of human head injuries. Finally, this review provides recommendations on how experiments can be conducted to allow for standardization of test methodologies, enabling simplified comparisons and conclusions on meningeal mechanics.

Three-dimensional organization of the perivascular glial limiting membrane and its relationship with the vasculature: a scanning electron microscope study.

To examine the three-dimensional structure of the perivascular glial limiting membrane (Glm) and its relationship with the vasculature in rat/mouse cerebral cortices, serial ion-etched plastic sections were observed under the scanning electron microscope and their images were reconstructed. In the case of arterioles and venules close to the pial surface, cord-like principal processes predominantly formed the endfeet; whereas in the case of capillaries and venules, sheet-like secondary processes chiefly formed Glm. Moreover, it was found that several plate-like structures protruded from the basement membrane surrounding the arterioles to penetrate into the astrocytic somata. The perivascular Glm was formed by monolayers of astrocytic processes and/or somata irrespective of the types of blood vessel. However, the thickness of the perivascular Glm, varied greatly according to the type of blood vessel. The thickness of Glm decreased in the order of arterioles, venules and capillaries. The outer surface of the perivascular Glm was extremely irregular, and sheet-like processes arising from this Glm infiltrated into the surrounding neuropil.

Generation of CSF1-Independent Ramified Microglia-Like Cells from Leptomeninges In Vitro.

Although del Río-Hortega originally reported that leptomeningeal cells are the source of ramified microglia in the developing brain, recent views do not seem to pay much attention to this notion. In this study, in vitro experiments were conducted to determine whether leptomeninges generate ramified microglia. The leptomeninges of neonatal rats containing Iba1+ macrophages were peeled off the brain surface. Leptomeningeal macrophages strongly expressed CD68 and CD163, but microglia in the brain parenchyma did not. Leptomeningeal macrophages expressed epidermal growth factor receptor (EGFR) as revealed by RT-PCR and immunohistochemical staining. Cells obtained from the peeled-off leptomeninges were cultured in a serum-free medium containing EGF, resulting in the formation of large cell aggregates in which many proliferating macrophages were present. In contrast, colony-stimulating factor 1 (CSF1) did not enhance the generation of Iba1+ cells from the leptomeningeal culture. The cell aggregates generated ramified Iba1+ cells in the presence of serum, which express CD68 and CD163 at much lower levels than primary microglia isolated from a mixed glial culture. Therefore, the leptomeningeal-derived cells resembled parenchymal microglia better than primary microglia. This study suggests that microglial progenitors expressing EGFR reside in the leptomeninges and that there is a population of microglia-like cells that grow independently of CSF1.

GPR56 regulates pial basement membrane integrity and cortical lamination.

GPR56 is a member of the family of adhesion G-protein-coupled receptors that have a large extracellular region containing a GPS (G-protein proteolytic site) domain. Loss-of-function mutations in the GPR56 gene cause a specific human brain malformation called bilateral frontoparietal polymicrogyria (BFPP). BFPP is a radiological diagnosis and its histopathology remains unclear. This study demonstrates that loss of the mouse Gpr56 gene leads to neuronal ectopia in the cerebral cortex, a cobblestone-like cortical malformation. There are four crucial events in the development of cobblestone cortex, namely defective pial basement membrane (BM), abnormal anchorage of radial glial endfeet, mislocalized Cajal-Retzius cells, and neuronal overmigration. By detailed time course analysis, we reveal that the leading causal events are likely the breaches in the pial BM. We show further that GPR56 is present in abundance in radial glial endfeet. Furthermore, a putative ligand of GPR56 is localized in the marginal zone or overlying extracellular matrix. These observations provide compelling evidence that GPR56 functions in regulating pial BM integrity during cortical development.

Induced spreading depression evokes cell division of astrocytes in the subpial zone, generating neural precursor-like cells and new immature neurons in the adult cerebral cortex.

BACKGROUND AND PURPOSE: New immature neurons appear out of the germinative zone, in cortical Layers V to VI, after induced spreading depression in the adult rat brain. Because neural progenitors have been isolated in the cortex, we set out to determine whether a subgroup of mature cells in the adult cortex has the potential to divide and generate neural precursors. METHODS: We examined the expression of endogenous markers of mitotic activity, proliferating cell nuclear antigen, and vimentin as a marker for neuronal progenitor cells, if any, in the adult rat cortex after spreading depression stimulation. Immunohistochemical analysis was also performed using antibodies for proliferating cell nuclear antigen, for vimentin, and for nestin. Nestin is a marker for activity dividing neural precursors. RESULTS: At the end of spreading depression (Day 0), glial fibrillary acidic protein-positive cells in the subpial zone and cortical Layer I demonstrated increased mitotic activity, expressing vimentin and nestin. On Day 1, nestin(+) cells were found spreading in deeper cortical layers. On Day 3, vimentin(-)/nestin(+), neural precursor-like cells appeared in cortical Layers V to VI. On Day 6, new immature neurons appeared in cortical Layers V to VI. Induced spreading depression evokes cell division of astrocytes residing in the subpial zone, generating neural precursor-like cells. CONCLUSIONS: Although neural precursor-like cells found in cortical Layers V to VI might have been transferred from the germinative zone rather than the cortical subpial zone, astrocytic cells in the subpial zone may be potent neural progenitors that can help to reconstruct impaired central nervous system tissue. Special caution is required when observing or treating spreading depression waves accompanying pathological conditions in the brain.

Brain mast cell relationship to neurovasculature during development.

Mast cells, derived from the hematopoietic stem cell, are present in the brain from birth. During development, mast cells occur in two locations, namely the pia and the brain parenchyma. The current hypothesis regarding their origin states that brain mast cells (or their precursors) enter the pia and access the thalamus by traveling along the abluminal wall of penetrating blood vessels. The population in the pia reaches a maximum at postnatal (PN) day 11, and declines rapidly thereafter. Chromatin fragmentation suggests that this cell loss is due to apoptosis. In contrast, the thalamic population expands from PN8 to reach adult levels at PN30. Stereological analysis demonstrates that mast cells home to blood vessels. More than 96% of mast cells are inside the blood-brain barrier, with ~90% contacting the blood vessel wall or its extracellular matrix. Mast cells express alpha4 integrins -- a potential mechanism for adhesion to the vascular wall. Despite the steady increase in the volume of microvasculature, at all ages studied, mast cells are preferentially located on large diameter vessels (>16 microm; possibly arteries), and contact only those maturing blood vessels that are ensheathed by astroglial processes. Mast cells not only home to large vessels but also maintain a preferential position at branch points, sites of vessel growth. This observation presents the possibility that mast cells participate in and/or regulate vasculature growth or differentiation. The biochemical and molecular signals that induce mast cell homing in the CNS is an area of active investigation.

Optimizing the transplant dose of a human neuronal cell line graft to treat SCI pain in the rat.

Neuropathic pain is a prevalent and difficult problem in the setting of spinal cord injury (SCI). The use of cellular transplant therapy to treat this pain has been successful with the use of a human neuronal cell line, hNT2.17 [M.J. Eaton, S.Q. Wolfe, M.A. Martinez, M. Hernandez, C. Furst, J. Huang, B.R. Frydel, O. Gomez-Marin, Subarachnoid transplant of a human neuronal cell line attenuates chronic allodynia and hyperalgesia after excitotoxic SCI in the rat, J. Pain 8 (2007) 33-50]. Intrathecal transplant of these cells potently reverses behavioral hypersensitivity after excitotoxic spinal cord injury in the rat model. This study focuses on delineating the optimal dose of these cell grafts in the same model. Two weeks after intraspinal injection of quisqualic acid (QUIS) with subsequent behavioral hypersensitivity, terminally differentiated hNT2.17 cells were transplanted into 300 g Wistar-Furth rats in a logarithmic variation of doses: 10(6), 10(5) and 10(3) cells. Behavioral hypersensitivity testing was performed weekly for 6 weeks following transplant. The dose of 10(6) cells (or approximately 3 million/kg) potently and permanently reversed both cutaneous allodynia (CA) and thermal hyperalgesia (TH). Reduced transplant doses of the hNT2.17 cell line did not permanently reverse behavioral hypersensitivity, suggesting that there is an optimal dose that can be used as a clinical tool to treat SCI-associated neuropathic pain.

[Innervation of the brain: what is innervated by what].

In the pia mater, brain substance and ependyma, peripheral nerves with sensory and effector axons and two types of neurons are located: (1) afferent pseudounipolar and bipolar cells and (2) efferent vegetative Dogiel type I neurons. Together, these nerves and neurons form the intramedullary part of the autonomous nervous system, innervating blood vessels, ependyma and perivascular connective tissue.

Ganglionic axons in motor roots and pia mater.

In addition to motor axons and preganglionic axons, ventral roots contain unmyelinated or thin myelinated sensory axons and postganglionic sympathetic axons. It has been said that ventral roots channel sensory axons to the CNS. However, it now seems that these axons end blindly, shift to the pia or loop and return towards the periphery and that these units reach the CNS via dorsal roots. Sensory ventral root axons project from a variety of somatic or visceral receptors; some of them are third branches of dorsal root afferents and some seem to lack a CNS projection. Many ventral root afferents contain substance P (SP) and/or calcitonin gene-related peptide (CGRP). These fibres are not affected by neonatal capsaicin treatment and they cannot induce radicular or pial extravasation. Some thin ventral root axons are sympathetic and relate to blood vessels. Afferents containing SP and/or CGRP and sympathetic axons also occur in the spinal pia mater. The sensory axons mediate pain. They might also have vasomotor, tissue-regulatory and/or mechanoreceptive functions. The motor roots of cranial nerves IV, VI and XI contain unmyelinated axons arranged like in ventral roots outside the autonomic outflow. However, the motor root of cranial nerve V channels some unmyelinated axons into the CNS. The occurrence of thin axons in ventral roots and pia mater changes during development and ageing. After peripheral nerve injury, ipsilateral ventral roots and pia are invaded by new sensory and postganglionic sympathetic axons.

A critical function of the pial basement membrane in cortical histogenesis.

Mice with a targeted deletion of the nidogen-binding site of laminin gamma1 were used to study the function of the pial basement membrane in cortical histogenesis. The pial basement membrane in the mutant embryos assembled but was unstable and disintegrated at random segments. In segments with a disrupted basement membrane, radial glia cells were retracted from the pial surface, and radially migrating neurons, including Cajal-Retzius cells and cortical plate neurons, passed the meninges or terminated their migration prematurely. By correlating the disruptions in the pial basal lamina with changes in the morphology of radial glia cells, the aberrant migration of Cajal-Retzius cells, and subsequent dysplasia of cortical plate neurons, the present data establish a causal relationship of proper cortical histogenesis with the presence of an intact pial basement membrane.

Ephrin B1 is expressed on neuroepithelial cells in correlation with neocortical neurogenesis.

To identify molecules involved in neurogenesis, we have raised monoclonal antibodies against embryonic day 12.5 mouse telencephalon. One antibody, monoclonal antibody 25H11, stains predominantly the ventricular zone of the anterior and lateral telencephalon. Purification of the 25H11 antigen, a 47 kDa integral membrane protein, from approximately 2500 mouse telencephali reveals its identity with ephrin B1. Ephrin B1 appears at the onset of neocortical neurogenesis, being first expressed in neuron-generating neuroepithelial cells and rapidly thereafter in virtually all neuroepithelial cells. Expression of ephrin B1 persists through the period of neocortical neurogenesis and is downregulated thereafter. Ephrin B1 is present on the ventricular as well as basolateral plasma membrane of neuroepithelial cells and exhibits an ventricular-high to pial-low gradient across the ventricular zone. Expression of ephrin B1 is also detected on radial glial cells, extending all the way to their pial endfeet, and on neurons in the mantle/intermediate zone but not in the cortical plate. Our results suggest that ephrin B1, presumably via ephrin-Eph receptor signaling, has a role in neurogenesis. Given the ventricular-to-pial gradient of ephrin B1 on the neuroepithelial cell surface and its known role in cell migration in other systems mediated by its repulsive properties, we propose that ephrin B1 may be involved in the migration of newborn neurons out from the ventricular zone toward the neocortex.