Lateral Ventricle Of Adult Mice Research Articles

Blood-brain barrier dysfunction promotes astrocyte senescence through albumin-induced TGFβ signaling activation.

Blood-brain barrier dysfunction (BBBD) and accumulation of senescent astrocytes occur during brain aging and contribute to neuroinflammation and disease. Here, we explored the relationship between these two age-related events, hypothesizing that chronic hippocampal exposure to the blood-borne protein serum albumin could induce stress-induced premature senescence (SIPS) in astrocytes via transforming growth factor beta 1 (TGFβ) signaling. We found that 1 week of albumin exposure significantly increased TGFβ signaling and senescence marker expression in cultured rat hippocampal astrocytes. These changes were preventable by pharmacological inhibition of the type I TGFβ receptor (TGFβR) ALK5. To study these effects in vivo, we utilized an animal model of BBBD in which albumin was continuously infused into the lateral ventricles of adult mice. Consistent with our in vitro results, 1 week of albumin infusion significantly increased TGFβ signaling activation and the burden of senescent astrocytes in hippocampal tissue. Pharmacological inhibition of ALK5 TGFβR or conditional genetic knockdown of astrocytic TGFβR prior to albumin infusion was sufficient to prevent albumin-induced astrocyte senescence. Together, these results establish a link between TGFβ signaling activation and astrocyte senescence and suggest that prolonged exposure to serum albumin due to BBBD can trigger these phenotypic changes.

Role of serotonin in modulating the development and function of adult-born neurons in the olfactory bulb.

Role of serotonin in modulating the development and function of adult-born neurons in the olfactory bulb.

Collection of cerebrospinal fluid from murine lateral ventricles for biomarker determination in mucopolysaccharidosis type IIIA

BackgroundCerebrospinal fluid (CSF) collection is currently the only feasible means to obtain biological fluid for the quantitative determination of biomarkers that may reflect disease activity within the brain. Studies in mouse models of human neurological disease benefit from ascertainment and subsequent analysis of brain tissue that is not afforded in human patients. The CSF provides a translational forum, however, due to the practical constraints presented by the mouse’s small size, CSF is often ignored in experimental mouse models. New methodHere we report a method for the controlled, precise and predictable collection of 10 μL of CSF from the lateral ventricles of adult mice using stereotaxic equipment and a micro-syringe pump. ResultsCollected CSF was clear and manifested the consistency of water and moreover, quantification of a disease-specific biomarker for the neurodegenerative disorder, mucopolysaccharidosis type IIIA (MPS IIIA) was possible in this small volume of CSF. In the naturally occurring mouse model of MPS IIIA, that faithfully recapitulates the human form of the disease, this biomarker was present at concentrations of >100 pmol/mL and undetectable in wild-type mice. Comparison with existing methodAdvantages of this method over the most commonly used cisterna magna collection technique include increased CSF sample volume (10 μL) and reduced blood contamination. ConclusionThe ability to collect CSF from mouse models of neurological disease enables a forum for translating research outcomes in experimental models to the human equivalent in which CSF collection is also possible.

Transplanted Adult Neural Stem Cells Express Sonic Hedgehog In Vivo and Suppress White Matter Neuroinflammation after Experimental Traumatic Brain Injury.

Neural stem cells (NSCs) delivered intraventricularly may be therapeutic for diffuse white matter pathology after traumatic brain injury (TBI). To test this concept, NSCs isolated from adult mouse subventricular zone (SVZ) were transplanted into the lateral ventricle of adult mice at two weeks post-TBI followed by analysis at four weeks post-TBI. We examined sonic hedgehog (Shh) signaling as a candidate mechanism by which transplanted NSCs may regulate neuroregeneration and/or neuroinflammation responses of endogenous cells. Mouse fluorescent reporter lines were generated to enable in vivo genetic labeling of cells actively transcribing Shh or Gli1 after transplantation and/or TBI. Gli1 transcription is an effective readout for canonical Shh signaling. In ShhCreERT2;R26tdTomato mice, Shh was primarily expressed in neurons and was not upregulated in reactive astrocytes or microglia after TBI. Corroborating results in Gli1CreERT2;R26tdTomato mice demonstrated that Shh signaling was not upregulated in the corpus callosum, even after TBI or NSC transplantation. Transplanted NSCs expressed Shh in vivo but did not increase Gli1 labeling of host SVZ cells. Importantly, NSC transplantation significantly reduced reactive astrogliosis and microglial/macrophage activation in the corpus callosum after TBI. Therefore, intraventricular NSC transplantation after TBI significantly attenuated neuroinflammation, but did not activate host Shh signaling via Gli1 transcription.

Purines regulate adult brain subventricular zone cell functions: Contribution of reactive astrocytes

Brain injuries modulate activation of neural stem cells (NSCs) in the adult brain. In pathological conditions, the concentrations of extracellular nucleotides (eNTs) raise several folds, contribute to reactive gliosis, and possibly directly affect subventricular zone (SVZ) cell functioning. Among eNTs and derived metabolites, the P2Y1 receptor agonist ADP strongly promotes astrogliosis and might also influence SVZ progenitor activity. Here, we tested the ability of the stable P2Y1 agonist adenosine 5'-O-(2-thiodiphosphate) (ADPβS) to control adult NSC functions both in vitro and in vivo, with a focus on the possible effects exerted by reactive astrocytes. In the absence of growth factors, ADPβS promoted proliferation and differentiation of SVZ progenitors. Moreover, ADPβS-activated astrocytes markedly changed the pattern of released cytokines and chemokines, and strongly modulated neurosphere-forming capacity of SVZ progenitors. Notably, a significant enhancement in proliferation was observed when SVZ cells, initially grown in the supernatant of astrocytes exposed to ADPβS, were shifted to normal medium. In vivo, ADPβS administration in the lateral ventricle of adult mice by osmotic minipumps caused diffused reactive astrogliosis, and a strong response of SVZ progenitors. Indeed, proliferation of glial fibrillary acidic protein-positive NSCs increased and led to a significant expansion of SVZ transit-amplifying progenitors and neuroblasts. Lineage tracing experiments performed in the GLAST::CreERT2;Rosa-YFP transgenic mice further demonstrated that ADPβS promoted proliferation of glutamate/aspartate transporter-positive progenitors and sustained their progression toward the generation of rapidly dividing progenitors. Altogether, our results show that the purinergic system crucially affects SVZ progenitor activities both directly and through the involvement of reactive astrocytes.

Stimulation of adult neural stem cells with a novel glycolipid biosurfactant

Glycolipids are amphipathic molecules which are highly expressed on cell membranes in skin and brain where they mediate several key cellular processes. Neural stem cells are defined as undifferentiated, proliferative, multipotential cells with extensive self-renewal and are responsive to brain injury. Di-rhamnolipid: α-L-rhamnopyranosyl-(1-2)α-L-rhamnopyranosyl-3-hydroxydecanoyl-3-hydroxydecanoic acid, also referred to as di-rhamnolipid BAC-3, is a glycolipid isolated from the bacteria Pseudomonas aeruginosa. In the previous studies, di-rhamnolipid enhanced dermal tissue healing and regeneration. The present study provides the first assessment of di-rhamnolipid, and glycolipid biosurfactants in general, on the nervous system. Treatment of neural stem cells isolated from the lateral ventricle of adult mice and cultured in defined media containing growth factors at 0.5 and 1 μg/ml of di-rhamnolipid increased the number of neurospheres (2.7- and 2.8-fold, respectively) compared to controls and this effect remained even after passaging in the absence of di-rhamnolipid. In addition, neural stem cells treated with di-rhamnolipid at 50 and 100 μg/ml in defined media supplemented with fetal calf serum and without growth factors exhibited increased cell viability, indicating an interaction between di-rhamnolipid and serum components in the regulation of neural stem cells and neuroprogenitors. Intracerebroventricular administration of di-rhamnolipid at 300 and 120 ng/day increased the number of neurospheres (1.3- and 1.63-fold, respectively) that could be derived from the anterior lateral ventricles of adult mice. These results indicate that di-rhamnolipid stimulates proliferation of neural stem cells and increases their endogenous pools which may have therapeutic potential in managing neurodegenerative or neuropsychiatric disorders and promoting nervous tissue regeneration following injury.

Identification and Mechanism of Action of the Acylguanidine MRT-83, a Novel Potent Smoothened Antagonist

There is a clear need to develop novel pharmacological tools to improve our understanding of Smoothened (Smo) function in normal and pathological states. Here, we report the discovery, the mechanism of action, and the in vivo activity of N-(2-methyl-5-(3-(3,4,5-trimethoxybenzoyl)guanidino)phenyl)biphenyl-4-carboxamide (MRT-83), a novel potent antagonist of Smo that belongs to the acylguanidine family of molecules. MRT-83 fits to a proposed pharmacophoric model for Smo antagonists with three hydrogen bond acceptor groups and three hydrophobic regions. MRT-83 blocks Hedgehog (Hh) signaling in various assays with an IC50 in the nanomolar range, showing greater potency than the reference Smo antagonist cyclopamine. MRT-83 inhibits Bodipy-cyclopamine binding to human and mouse Smo but does not modify Wnt signaling in human embryonic kidney 293 transiently transfected with a Tcf/Lef-dependent Firefly luciferase reporter together with a Renilla reniformis luciferase control reporter. MRT-83 abrogates the agonist-induced trafficking of endogenous mouse or human Smo to the primary cilium of C3H10T1/2 or NT2 cells that derive from a pluripotent testicular carcinoma. Stereotaxic injection into the lateral ventricle of adult mice of MRT-83 but not of a structurally related compound inactive at Smo abolished up-regulation of Patched transcription induced by Sonic Hedgehog in the neighboring subventricular zone. These data demonstrate that MRT-83 efficiently antagonizes Hh signaling in vivo. All together, these molecular, functional and biochemical studies provide evidence that MRT-83 interacts with Smo. Thus, this novel Smo antagonist will be useful for manipulating Hh signaling and may help develop new therapies against Hh-pathway related diseases.

Characterization of novel monoclonal antibodies able to identify neurogenic niches and arrest neurosphere proliferation and differentiation

Two monoclonal antibodies (Nilo1 and Nilo2) were generated after immunization of hamsters with E13.5 olfactory bulb-derived mouse neurospheres. They are highly specific for neural stem and early progenitor cell surface antigens. Nilo positive cells present in the adult mouse subventricular zone (SVZ) were able to initiate primary neural stem cell cultures. Moreover, these antibodies added to neurosphere cultures induced proliferation arrest and interfered with their differentiation. In the lateral ventricles of adult mice, Nilo1 stained a cell subpopulation lining the ventricle and cells located in the SVZ, whereas Nilo2 stained a small population associated with the anterior horn of the SVZ at the beginning of the rostral migratory stream. Co-staining of Nilo1 or Nilo2 and neural markers demonstrated that Nilo1 identifies an early neural precursor subpopulation, whereas Nilo2 detects more differentiated neural progenitors. Thus, these antibodies identify distinct neurogenic populations within the SVZ of the lateral ventricle.

Neurogenic astrocytes transplanted into the adult mouse lateral ventricle contribute to olfactory neurogenesis, and reveal a novel intrinsic subependymal neuron

Spatially and temporally restricted populations of neurogenic astrocytes can generate multipotent neurospheres in vitro. To examine the ability of neurogenic astrocytes to respond to in vivo differentiation cues within a germinal matrix, we provided cultured neonatal cerebellar astrocytes access to the subependymal zone (SEZ) by grafting them directly into the lateral ventricle of adult mice. Here we report three events that follow such transplants. 1) Donor cells attach to periventricular structures, and form “neoplastic-like” spheres that penetrate the ventricular wall. These attached spheres can persist for months, as they give rise to “clones” of cells that infiltrate forebrain parenchyma. 2) Many donor cells enter the rostral migratory stream and migrate into the olfactory bulb where a small percentage differentiates as olfactory interneurons. 3) Finally, within the SEZ, some donor cells formed cell clusters that appear to interact with the SEZ neuronal precursor chains, and some donor cells differentiate into distinctive neurons with extensive, beady projections precisely confined between the ependymal layer and the striatum. Further analysis of normal SEZ anatomy reveals indigenous neurons with identical morphologies—some of which are contacted by 5-HT+ fibers—that we propose represent a heretofore uncharacterized, intrinsic SEZ neuron of unknown function. These results suggest that cultured astrocytes derived from non-SEZ brain regions can respond in different ways to in vivo cues provided by the adult lateral ventricle and SEZ by differentiating into neurons that eventually inhabit both the olfactory bulb and SEZ proper.

Increase of proliferating oligodendroglial progenitors in the adult mouse brain upon Sonic hedgehog delivery in the lateral ventricle

Sonic hedgehog signaling is required for the maintenance of stem cell niches in the postnatal subventricular zone and the proliferation of neural progenitors in the mature hippocampus. We show here that delivery of Sonic hedgehog protein into the lateral ventricle of adult mice increases cell proliferation in the corpus callosum and cerebral cortex. In this latter area, the number of neural progenitors expressing the proteoglycan NG2 is enhanced 2 days after the injection. In both areas, mRNA up-regulation of the transcriptional target gene Patched was observed in cells expressing the oligodendroglial transcription factor Olig1. Twenty-six days following the adenovirus-mediated delivery of Sonic hedgehog into the lateral ventricle, newly generated cells in the cerebral cortex and in the corpus callosum are influenced towards the initial steps of oligodendrogenesis, as indicated by a 50% increase in the number of cells expressing the oligodendroglial marker DM20. Our experiments demonstrate that the number of oligodendrocyte precursor cells in the cerebral cortex and corpus callosum can be increased upon delivery of Sonic hedgehog proteins and highlight the potential capacity of the adult brain to mobilize a pool of premyelinating cells.

Adult Subventricular Zone Neuronal Precursors Continue to Proliferate and Migrate in the Absence of the Olfactory Bulb

Neurons continue to be born in the subventricular zone (SVZ) of the lateral ventricles of adult mice. These cells migrate as a network of chains through the SVZ and the rostral migratory stream (RMS) into the olfactory bulb (OB), where they differentiate into mature neurons. The OB is the only known target for these neuronal precursors. Here, we show that, after elimination of the OB, the SVZ and RMS persist and become dramatically larger. The proportion of dividing [bromodeoxyuridine (BrdU)-labeled] or dying (pyknotic or terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end-labeled) cells in the RMS was not significantly affected at 3 d or 3 weeks after bulbectomy (OBX). However, by 3 months after OBX, the percentage of BrdU-labeled cells in the RMS decreased by half and that of dying cells doubled. Surprisingly, the rostral migration of precursors continued along the RMS after OBX. This was demonstrated by focal microinjections of BrdU and grafts of SVZ cells carrying LacZ under the control of a neuron-specific promoter gene. Results indicate that the OB is not essential for proliferation and the directional migration of SVZ precursors.

Adenovirus-mediated gene delivery into neuronal precursors of the adult mouse brain.

Precursor cells found in the subventricular zone (SVZ) of the adult brain can undergo cell division and migrate long distances before differentiating into mature neurons. We have investigated the possibility of introducing genes stably into this population of cells. Replication-defective adenoviruses were injected into the SVZ of the lateral ventricle of adult mice. The adenoviruses carried a cDNA for the LacZ reporter or the human p75 neurotrophin receptor, for which species-specific antibodies are available. Injection of the viruses into the SVZ led to efficient labeling of neuronal precursors. Two months after viral injection, infected cells were detected in the olfactory bulb, a significant distance from the site of injection. Labeled periglomerular and granular neurons with extensive dendritic arborization were found in the olfactory bulb. These results demonstrate that foreign genes can be efficiently introduced into neuronal precursor cells. Furthermore, adenovirus-directed infection can lead to long-term stable gene expression in progenitor cells found in the adult central nervous system.

Long-Distance Neuronal Migration in the Adult Mammalian Brain

During the development of the mammalian brain, neuronal precursors migrate to their final destination from their site of birth in the ventricular and subventricular zones (VZ and SVZ, respectively). SVZ cells in the walls of the lateral ventricle continue to proliferate in the brain of adult mice and can generate neurons in vitro, but their fate in vivo is unknown. Here SVZ cells from adult mice that carry a neuronal-specific transgene were grafted into the brain of adult recipients. In addition, the fate of endogenous SVZ cells was examined by microinjection of tritiated thymidine or a vital dye that labeled a discrete population of SVZ cells. Grafted and endogenous SVZ cells in the lateral ventricle of adult mice migrate long distances and differentiate into neurons in the olfactory bulb.

Suppression of immunorejection against rat fetal dopaminergic neurons in a mouse brain by 15-deoxyspergualin

In this study, the immunosuppressive effect of 15-deoxyspergualin (DSG) on the survivability of rat embryonic dopaminergic neurons grafted into the lateral ventricle of adult mice was investigated. DSG was administered daily in dose of 5 mg/kg for 2 weeks postgrafting, commencing on the day of transplantation, in the immunosuppressant-treated groups. Animals were then allowed to survive for 2 to 4 weeks after transplantation. Histological analysis revealed that most of transplanted rat fetal tissue was destroyed and scavenged in 2 weeks after transplantation in the non-immunosuppressed group. However, a large number of tyrosine hydroxylase (TH)-positive grafted neurons survived and grew postgrafting in the brain of the host administered with DSG even if they suffered from T lymphocyte infiltrations visualized by cytotoxic T cell immunohistochemistry. The results thus indicated that DSG is an potent immunosuppressant in neural transplantation as well as in transplantation of other organs in animals. It seems to be able to block, at least in part, the ability of mature specific cytotoxic T cells to lyse their target.

The effect of a short-course treatment with FK506 on the survivability of rat-to-mouse cross-species neural graft

We examined the effect of a short-course treatment with a new immunosuppressive agent FK506 (FK) on the survivability of neural xenografts. Pieces of ventral mesencephalic tissues from rat embryos were transplanted into the right lateral ventricle of adult mice. The mice were either assigned to a 4-day FK (10 mg/kg/day) immunosuppressive scheme or were given no immunosuppression. The 4-day course treatment with FK was started on postoperative day 0, 2, 4, 6 or 8. The incidence of graft rejection 28 days after transplantation was 82%, 55%, 55%, 100% and 100% when FK was given on days 0-3, 2-5, 4-7, 6-9 and 8-11, respectively. As a separate group, we examined the cellular infiltration in neural xenografts of the non-immunosuppressed recipients at two different time points (3 days and 7 days) after transplantation. The infiltration of T-lymphocytes was not detected 3 days after transplantation, but had occurred by 7 days after transplantation. We speculate that FK is more effective in preventing the rejection of neural xenografts when it is given just before the initiation of the T-lymphocytic infiltration.

Postmitotic death is the fate of constitutively proliferating cells in the subependymal layer of the adult mouse brain

The early development of the mammalian forebrain involves the massive proliferation of the ventricular zone cells lining the lateral ventricles. A remnant of this highly proliferative region persists into adult life, where it is known as the subependymal layer. We examined the proliferation kinetics and fates of the mitotically active cells in the subependyma of the adult mouse. The medial edge, the lateral edge, and the dorsolateral corner of the subependymal layer of the rostral portion of the lateral ventricle each contained mitotically active cells, but the dorsolateral region had the highest percentage of bromodeoxyuridine (BrdU)-labeled cells per unit area. Repeated injections of BrdU over 14 hr revealed a proliferation curve for the dorsolateral population with a growth fraction of 33%, indicating that 33% of the cells in this subependymal region make up the proliferating population. The total cell cycle time in this population was approximately 12.7 hr, with an S-phase of 4.2 hr. To examine the fate of these proliferating cells, we injected low concentrations of a replication-deficient, recombinant retrovirus directly into the lateral ventricles of adult mice for uptake by mitotically active subependymal cells. Regardless of the survival time postinjection (10 hr, 1 d, 2 d, or 8 d), the number of retrovirally labeled cells per clone remained the same (1 or 2 cells/clone). This suggests that one of the progeny from each cell division dies. Moreover, the clones remained confined to the subependyma and labeled cells were not seen in the surrounding brain tissue. Thus, while 33% of the dorsolateral subependymal cells continue to proliferate in adult life, the fate of the postmitotic progeny is death.

The effect of a new immunosuppressive agent, FK-506, on xenogeneic neural transplantation in rodents

This study examines the effect of a novel immunosuppressive agent FK-506 (FK) on the survivability of embryonic (E14) rat ventral mesencephalic tissue after intracerebral grafting to the lateral ventricle of adult mice. The recipient mice were given FK in doses of 10 mg/kg or 1 mg/kg for 2 weeks postgrafting, at which time they were sacrificed and histologically processed except for one group of animals on the high dose (10 mg/kg). In this group most animals died from side effects of the drug during the following days. Only the mice receiving the high dose of FK displayed healthy grafts without signs of rejection.