Neuroprotective Effects of a Combined Therapy With Memantine, Donepezil and Vitamin D in Ovariectomized Female Mice Subjected to Dementia Model

The postmenopausal period is characterized by a decrease in the hormonal supply which is associated with Alzheimer's Disease (AD). Vitamin D is neuroprotective and can be used in combination with pre-existing medications to improve its effects. The objective was to evaluate the effect of vitamin D associated with memantine and donepezil in female mice submitted to ovariectomy (OVX) for 5 months and subjected to an AD-induced dementia model. Animals were divided into 5 groups who received 17 days of treatment and were subjected to behavioral tests. The animals underwent euthanasia at 18th day. OVX groups exhibit reduced levels of E2 and triple treatment group had high levels of vitamin D. The induction of dementia with OVX induced short- and long-term spatial and habituation memories damage. Also, induced reduction of BDNF and IL-4 levels in hippocampus, and increasing levels of TNFα in hippocampus and of IL-1β in hippocampus and frontal cortex of animals, as well as a significant increase on GFAP immunoreactivity. Triple-association treatment reversed the effects of long-term spatial and habituation memories damage, as well as reversed changes in TNFα, IL-1β, IL-4 and GFAP immunoreactivity levels in hippocampus of treated animals. Therapeutic association has beneficial effects on memory and inflammation parameters in female mice subjected to OVX and the AD animal model of dementia.

Astrocytes in the dorsal vagal complex are not activated by systemic glucoprivation and their chemogenetic activation does not elicit homeostatic glucoregulatory responses in mice

The dorsal vagal complex (DVC) is a brainstem site regulating diverse aspects of physiology including food intake and blood glucose homeostasis. Astrocytes are purported to play an active role in regulating DVC function and, by extension, physiological parameters. Previous work has demonstrated that DVC astrocytes directly sense hormones that regulate food intake and blood glucose and are critical for their effect. In addition, DVC astrocytes in ex vivo slices respond to low tissue glucose. The response of neurons, including catecholaminergic neurons, to low glucose is conditional on intact astrocyte signalling in slice preparations suggesting astrocytes are possibly the primary sensors of glucose deprivation (glucoprivation). Based on these findings we hypothesised that if DVC astrocytes act as glucoprivation sensors in vivo they would both show a response to systemic glucoprivation and drive physiological responses to restore blood glucose. We found that 2 hours of systemic glucoprivation induced neither FOS nor glial fibrillary acidic protein (GFAP)-immunoreactivity in DVC astrocytes, specifically those in the nucleus of the solitary tract (NTS). Furthermore, we found that while chemogenetic activation of DVC astrocytes suppressed food intake by reducing both meal size and meal number, this manipulation also suppressed food intake under conditions of glucoprivation. Chemogenetic activation of DVC astrocytes did not increase basal blood glucose nor protect against insulin-induced hypoglycaemia. In male mice chemogenetic DVC astrocyte activation did not alter glucose tolerance, in female mice the initial glucose excursion was reduced, suggesting enhanced glucose absorption. Taken together this suggests that as a whole-population DVC astrocytes do not function as glucoprivation sensors in vivo in mice. Instead, we propose that DVC astrocytes play an indispensable, homeostatic role to maintain the function of glucoregulatory neuronal circuitry.

Increases in GFAP immunoreactive astrocytes in the cerebellar molecular layer of young adult CBA/J mice

Background CBA/J mice are standard experimental animals in auditory studies, and age-related changes in auditory pathways are well documented. However, changes in locomotion-related brain regions have not been systematically explored. Results We showed an increase in immunoreactivity for glial fibrillary acidic protein (GFAP) in the cerebellar molecular layer associated with Purkinje cells in mice at 24 weeks of age but not in the younger mice. Increased GFAP immunoreactivity appeared in the form of clusters and distributed multifocally consistent with hyperplasia of astrocytes that were occasionally associated with Purkinje cell degeneration. Three out of 12 animals at 16 and 24 weeks of age exhibited pre-convulsive clinical signs. Two of these 3 animals also showed increased GFAP immunoreactivity in the cerebellum. Rotarod behavioral assessments indicated decreased performance at 24 weeks of age. Conclusions These results suggest minimal to mild reactive astrocytosis likely associated with Purkinje cell degeneration in the cerebellum at 24 weeks of age in CBA/J mice. These findings should be taken into consideration prior to using this mouse strain for studying neuroinflammation or aging.

Differential Response of Müller Cells and Microglia in a Mouse Retinal Detachment Model and Its Implications in Detached and Non-Detached Regions

Retinal detachment (RD) is a sight-threatening condition, leading to photoreceptor cell death; however, only a few studies provide insight into its effects on the entire retinal region. We examined the spatiotemporal changes in glial responses in a mouse RD model. In electroretinography, a- and b-waves were reduced in a time-dependent manner. Hematoxylin and eosin staining revealed a gradual decrease in the outer nuclear layer throughout the retinal region. Terminal deoxynucleotidyltransferase dUTP nick end labeling (TUNEL) assay showed that TUNEL-positive photoreceptors increased 5 days after RD and decreased by 14 days. Glial response was evaluated by immunohistochemistry using antibodies against glial fibrillary acidic protein (GFAP, Müller glial marker) and Iba-1 (microglial marker) and osteopontin (OPN, activated microglial marker). GFAP immunoreactivity increased after 7 days in complete RD, and was retained for 14 days. OPN expression increased in microglial cells 3–7 days after RD, and decreased by 14 days in the detached and border regions. Although OPN was not expressed in the intact region, morphologically activated microglial cells were observed. These retinal glial cell responses and photoreceptor degeneration in the border and intact regions suggest that the effects of RD in the border and intact retinal regions need to be understood further.

Protective Effect of Memantine on Bergmann Glia and Purkinje Cells Morphology in Optogenetic Model of Neurodegeneration in Mice

Spinocerebellar ataxias are a family of fatal inherited diseases affecting the brain. Although specific mutated proteins are different, they may have a common pathogenetic mechanism, such as insufficient glutamate clearance. This function fails in reactive glia, leading to excitotoxicity and overactivation of NMDA receptors. Therefore, NMDA receptor blockers could be considered for the management of excitotoxicity. One such drug, memantine, currently used for the treatment of Alzheimer’s disease, could potentially be used for the treatment of other forms of neurodegeneration, for example, spinocerebellar ataxias (SCA). We previously demonstrated close parallels between optogenetically induced cerebellar degeneration and SCA1. Here we induced reactive transformation of cerebellar Bergmann glia (BG) using this novel optogenetic approach and tested whether memantine could counteract changes in BG and Purkinje cell (PC) morphology and expression of the main glial glutamate transporter—excitatory amino acid transporter 1 (EAAT1). Reactive BG induced by chronic optogenetic stimulation presented increased GFAP immunoreactivity, increased thickness and decreased length of its processes. Oral memantine (~90 mg/kg/day for 4 days) prevented thickening of the processes (1.57 to 1.81 vs. 1.62 μm) and strongly antagonized light-induced reduction in their average length (186.0 to 150.8 vs. 171.9 μm). Memantine also prevented the loss of the key glial glutamate transporter EAAT1 on BG. Finally, memantine reduced the loss of PC (4.2 ± 0.2 to 3.2 ± 0.2 vs. 4.1 ± 0.3 cells per 100 μm of the PC layer). These results identify memantine as potential neuroprotective therapeutics for cerebellar ataxias.

GFAP-Negative Subcutaneous Sacrococcygeal Extraspinal Ependymoma

Ependymomas are slowly growing glial tumors derived from the ependymal cells and usually occur in the central nervous system (CNS). Ependymomas rarely occur outside of the CNS and they are called extraspinal ependymomas. In spite of their metastatic potential, extraspinal ependymomas can be misdiagnosed for other benign mass like pilonidal cysts. The diagnosis is confirmed by histopathology and most of the cases are known to show glial fibrillary acidic protein (GFAP), S-100 protein, and keratin (AE1AE3) immunoreactivity. Herein, we present a case of GFAP-negative ependymoma, which presented as asymptomatic subcutaneous tumor of the left buttock and was clinically misdiagnosed as epidermal cyst. Our case indicates that ependymomas cannot be ruled out by lack of GFAP immunoreactivity and an asymptomatic subcutaneous mass could be a malignant tumor like ependymomas, which requires careful examinations.

Loss of TDP-43 in astrocytes leads to motor deficits by triggering A1-like reactive phenotype and triglial dysfunction

Patients with amyotrophic lateral sclerosis (ALS) can have abnormal TDP-43 aggregates in the nucleus and cytosol of their surviving neurons and glia. Although accumulating evidence indicates that astroglial dysfunction contributes to motor neuron degeneration in ALS, the normal function of TDP-43 in astrocytes are largely unknown, and the role of astroglial TDP-43 loss to ALS pathobiology remains to be clarified. Herein, we show that TDP-43–deleted astrocytes exhibit a cell-autonomous increase in GFAP immunoreactivity without affecting astrocyte or microglia proliferation. At the transcriptomic level, TDP-43–deleted astrocytes resemble A1-reactive astrocytes and induce microglia to increase C1q expression. These astrocytic changes do not cause loss of motor neurons in the spinal cord or denervation at the neuromuscular junction. In contrast, there is a selective reduction of mature oligodendrocytes, but not oligodendrocyte precursor cells, suggesting triglial dysfunction mediated by TDP-43 loss in astrocytes. Moreover, mice with astroglial TDP-43 deletion develop motor, but not sensory, deficits. Taken together, our results demonstrate that TDP-43 is required to maintain the protective functions of astrocytes relevant to the development of motor deficits in mice.

Bone Marrow-Derived Mononuclear Cell Transplants Decrease Retinal Gliosis in Two Animal Models of Inherited Photoreceptor Degeneration

Inherited photoreceptor degenerations are not treatable diseases and a frequent cause of blindness in working ages. In this study we investigate the safety, integration and possible rescue effects of intravitreal and subretinal transplantation of adult human bone-marrow-derived mononuclear stem cells (hBM-MSCs) in two animal models of inherited photoreceptor degeneration, the P23H-1 and the Royal College of Surgeons (RCS) rat. Immunosuppression was started one day before the injection and continued through the study. The hBM-MSCs were injected in the left eyes and the animals were processed 7, 15, 30 or 60 days later. The retinas were cross-sectioned, and L- and S- cones, microglia, astrocytes and Müller cells were immunodetected. Transplantations had no local adverse effects and the CD45+ cells remained for up to 15 days forming clusters in the vitreous and/or a 2–3-cells-thick layer in the subretinal space after intravitreal or subretinal injections, respectively. We did not observe increased photoreceptor survival nor decreased microglial cell numbers in the injected left eyes. However, the injected eyes showed decreased GFAP immunoreactivity. We conclude that intravitreal or subretinal injection of hBM-MSCs in dystrophic P23H-1 and RCS rats causes a decrease in retinal gliosis but does not have photoreceptor neuroprotective effects, at least in the short term. However, this treatment may have a potential therapeutic effect that merits further investigation.

Lack of Overt Retinal Degeneration in a K42E Dhdds Knock-In Mouse Model of RP59

Dehydrodolichyl diphosphate synthase (DHDDS) is required for protein N-glycosylation in eukaryotic cells. A K42E point mutation in the DHDDS gene causes an autosomal recessive form of retinitis pigmentosa (RP59), which has been classified as a congenital disease of glycosylation (CDG). We generated K42E Dhdds knock-in mice as a potential model for RP59. Mice heterozygous for the Dhdds K42E mutation were generated using CRISPR/Cas9 technology and crossed to generate DhddsK42E/K42E homozygous mice. Spectral domain-optical coherence tomography (SD-OCT) was performed to assess retinal structure, relative to age-matched wild type (WT) controls. Immunohistochemistry against glial fibrillary acidic protein (GFAP) and opsin (1D4 epitope) was performed on retinal frozen sections to monitor gliosis and opsin localization, respectively, while lectin cytochemistry, plus and minus PNGase-F treatment, was performed to assess protein glycosylation status. Retinas of DhddsK42E/K42E mice exhibited grossly normal histological organization from 1 to 12 months of age. Anti-GFAP immunoreactivity was markedly increased in DhddsK42E/K42E mice, relative to controls. However, opsin immunolocalization, ConA labeling and PNGase-F sensitivity were comparable in mutant and control retinas. Hence, retinas of DhddsK42E/K42E mice exhibited no overt signs of degeneration, yet were markedly gliotic, but without evidence of compromised protein N-glycosylation. These results challenge the notion of RP59 as a DHDDS loss-of-function CDG and highlight the need to investigate unexplored RP59 disease mechanisms.

Involvement of Enteric Glia in Small Intestine Neuromuscular Dysfunction of Toll-Like Receptor 4-Deficient Mice

Enteric glial cells (EGCs) influence nitric oxide (NO)− and adenosine diphosphate (ADP)− mediated signaling in the enteric nervous system (ENS). Since Toll-like receptor 4 (TLR4) participates to EGC homoeostasis, this study aimed to evaluate the possible involvement of EGCs in the alterations of the inhibitory neurotransmission in TLR4−/− mice. Ileal segments from male TLR4−/− and wild-type (WT) C57BL/6J mice were incubated with the gliotoxin fluoroacetate (FA). Alterations in ENS morphology and neurochemical coding were investigated by immunohistochemistry whereas neuromuscular responses were determined by recording non-adrenergic non-cholinergic (NANC) relaxations in isometrically suspended isolated ileal preparations. TLR4−/− ileal segments showed increased iNOS immunoreactivity associated with enhanced NANC relaxation, mediated by iNOS-derived NO and sensitive to P2Y1 inhibition. Treatment with FA diminished iNOS immunoreactivity and partially abolished NO− and ADP− mediated relaxation in the TLR4−/− mouse ileum, with no changes of P2Y1 and connexin-43 immunofluorescence distribution in the ENS. After FA treatment, S100β and GFAP immunoreactivity in TLR4−/− myenteric plexus was reduced to levels comparable to those observed in WT. Our findings show the involvement of EGCs in the alterations of ENS architecture and in the increased purinergic and nitrergic-mediated relaxation, determining gut dysmotility in TLR4−/− mice.