Deletion of the Pedf gene leads to inflammation, photoreceptor loss and vascular disturbances in the retina.

Retinal diseases are often accompanied by inflammation, vascular abnormalities, and neurodegeneration that decrease vision. Treatment with exogenous PEDF is widely shown to alleviate these conditions leading us to hypothesize that loss of function of the PEDF gene disrupts these pathways and leads to visual loss. Measurements were carried out by detailed phenotyping of PEDF null mice to assess expression of immunomodulators, glia activation, systemic inflammation, vascular disturbances, and visual sensitivity often associated with retinal pathologies. With a deletion of the Pedf gene, there was increased expression of several immune modulators in Pedf-/- retinas and serum with IL-2 and GM-CSF upregulated in both. Increases in retina glia activation and macrophage infiltration, levels of serum c-reactive protein (CRP), numbers of white and red blood cells and platelets and decreased blood glucose levels were all features associated with PEDF null mice. With PEDF gene deletion, there was also a notable increase in apoptosis in early developing retinas (PN3), reduced thickness of the photoreceptor layer, swelling of the inner plexiform layer, reduced retinal sensitivity and steady-state reduced activation of Erk and Akt, two signaling pathways used by PEDF. There is a substantial body of animal data emphasizing utility of PEDF treatment in homeostatic regulation of retinal diseases, including diabetic retinopathy and age-related macular degeneration but there is little agreement or evidence on the role of endogenous PEDF in retinal diseases. Our findings strongly support the concept that a deletion of the PEDF gene makes the retina vulnerable to diseases, and argue that endogenous PEDF plays a critical role in limiting pathological events in the retina.

Lupeol Treatment Attenuates Activation of Glial Cells and Oxidative-Stress-Mediated Neuropathology in Mouse Model of Traumatic Brain Injury.

Traumatic brain injury (TBI) signifies a major cause of death and disability. TBI causes central nervous system (CNS) damage under a variety of mechanisms, including protein aggregation, mitochondrial dysfunction, oxidative stress, and neuroinflammation. Astrocytes and microglia, cells of the CNS, are considered the key players in initiating an inflammatory response after injury. Several evidence suggests that activation of astrocytes/microglia and ROS/LPO have the potential to cause more harmful effects in the pathological processes following traumatic brain injury (TBI). Previous studies have established that lupeol provides neuroprotection through modulation of inflammation, oxidative stress, and apoptosis in Aß and LPS model and neurodegenerative disease. However, the effects of lupeol on apoptosis caused by inflammation and oxidative stress in TBI have not yet been investigated. Therefore, we explored the role of Lupeol on antiapoptosis, anti-inflammatory, and antioxidative stress and its potential mechanism following TBI. In these experiments, adult male mice were randomly divided into four groups: control, TBI, TBI+ Lupeol, and Sham group. Western blotting, immunofluorescence staining, and ROS/LPO assays were performed to investigate the role of lupeol against neuroinflammation, oxidative stress, and apoptosis. Lupeol treatment reversed TBI-induced behavioral and memory disturbances. Lupeol attenuated TBI-induced generation of reactive oxygen species/lipid per oxidation (ROS/LPO) and improved the antioxidant protein level, such as nuclear factor erythroid 2-related factor 2 (Nrf2) and heme-oxygenase 1 (HO-1) in the mouse brain. Similarly, our results indicated that lupeol treatment inhibited glial cell activation, p-NF-κB, and downstream signaling molecules, such as TNF-α, COX-2, and IL-1ß, in the mouse cortex and hippocampus. Moreover, lupeol treatment also inhibited mitochondrial apoptotic signaling molecules, such as caspase-3, Bax, cytochrome-C, and reversed deregulated Bcl2 in TBI-treated mice. Overall, our study demonstrated that lupeol inhibits the activation of astrocytes/microglia and ROS/LPO that lead to oxidative stress, neuroinflammation, and apoptosis followed by TBI.

Resveratrol suppresses glial activation and alleviates trigeminal neuralgia via activation of AMPK.

BACKGROUND: Glial activation and neuroinflammation in the spinal trigeminal nucleus (STN) play a pivotal role in the genesis and maintenance of trigeminal neuralgia (TN). Resveratrol, a natural compound from grape and red wine, has a potential anti-inflammatory effect. We hypothesized that resveratrol could significantly suppress neuroinflammation in the STN mediated by glial activation and further relieve TN. In this study, we evaluated whether resveratrol could alleviate trigeminal allodynia and explore the mechanism underlying the antinociceptive effect of resveratrol. METHODS: Animals were orally injected with resveratrol after chronic constriction injury (CCI) of the infraorbital nerve. Mechanical thresholds of the affected whisker pad were measured to assess nociceptive behaviors. The STN was harvested to quantify the changing levels of p-NR1, p-PKC, TNF-α, and IL1-ß by western blotting and detect the expression of calcitonin gene-related peptide (CGRP) and c-Fos by immunofluorescence. Glial activation was observed by immunofluorescence and western blotting. Mitogen-activated protein kinase (MAPK) phosphorylation in vivo and in vitro was examined by western blotting. RESULTS: We found that resveratrol significantly attenuated trigeminal allodynia dose-dependently and decreased the increased expression of CGRP and c-Fos in the STN. Additionally, resveratrol showed an inhibitory effect on CCI-evoked astrocyte and microglia activation and reduced production of pro-inflammatory cytokines in the STN. Furthermore, the antinociceptive effect of resveratrol was partially mediated by reduced phosphorylation of MAP kinases via adenosine monophosphate-activated protein kinase (AMPK) activation. CONCLUSIONS: AMPK activation in the STN glia via resveratrol has utility in the treatment of CCI-induced neuroinflammation and further implicates AMPK as a novel target for the attenuation of trigeminal neuralgia.

Metformin reduces morphine tolerance by inhibiting microglial-mediated neuroinflammation.

BACKGROUND: Tolerance seriously impedes the application of morphine in clinical medicine. Thus, it is necessary to investigate the exact mechanisms and efficient treatment. Microglial activation and neuroinflammation in the spinal cord are thought to play pivotal roles on the genesis and maintaining of morphine tolerance. Activation of adenosine monophosphate-activated kinase (AMPK) has been associated with the inhibition of inflammatory nociception. Metformin, a biguanide class of antidiabetic drugs and activator of AMPK, has a potential anti-inflammatory effect. The present study evaluated the effects and potential mechanisms of metformin in inhibiting microglial activation and alleviating the antinociceptive tolerance of morphine. METHODS: The microglial cell line BV-2 cells and mouse brain-derived endothelial cell line bEnd3 cells were used. Cytokine expression was measured using quantitative polymerase chain reaction. Cell signaling was assayed by western blot and immunohistochemistry. The antinociception and morphine tolerance were assessed in CD-1 mice using tail-flick tests. RESULTS: We found that morphine-activated BV-2 cells, including the upregulation of p38 mitogen-activated protein kinase (p38 MAPK) phosphorylation, pro-inflammatory cytokines, and Toll-like receptor-4 (TLR-4) mRNA expression, which was inhibited by metformin. Metformin suppressed morphine-induced BV-2 cells activation through increasing AMPK phosphorylation, which was reversed by the AMPK inhibitor compound C. Additionally, in BV-2 cells, morphine did not affect the cell viability and the mRNA expression of anti-inflammatory cytokines. In bEnd3 cells, morphine did not affect the mRNA expression of interleukin-1ß (IL-1ß), but increased IL-6 and tumor necrosis factor-α (TNF-α) mRNA expression; the effect was inhibited by metformin. Morphine also did not affect the mRNA expression of TLR-4 and chemokine ligand 2 (CCL2). Furthermore, systemic administration of metformin significantly blocked morphine-induced microglial activation in the spinal cord and then attenuated the development of chronic morphine tolerance in mice. CONCLUSIONS: Metformin significantly attenuated morphine antinociceptive tolerance by suppressing morphine-induced microglial activation through increasing AMPK phosphorylation.

ω-3 PUFAs Ameliorated the Maternal High-Fructose Diet-Induced Early-Onset Retinopathy in Offspring via Inhibiting NLRP3-Associated Neuroinflammation.

SCOPE: Maternal high fructose diet (HFD) during pregnancy and lactation can initiate retinal dysfunction. However, the underlying mechanism remains largely unknown. METHODS AND RESULTS: By using the rodent model of maternal HFD in this study, the results from electroretinography (ERG) indicate that b-wave amplitude, an index of inner retinal function, is significantly reduced as early as 3 months old and the deteriorated effect can be detected at 15 months old. Further, the protein expressions of CD11b (a marker of active microglia), p40phox subunit of NADPH oxidase, GFAP (a marker of active astrocytes), and NLPR3 examined by western blot and immunofluorescence are significantly increased in the retina of the male HFD offspring at 3 months old. Treatment with omega-3 polyunsaturated fatty acids (ω-3 PUFAs) for 2 weeks (from 2.5 to 3 months old) effectively reverses the aforementioned changes. CONCLUSION: Together, these results indicate that the early onset and extensive retinal dysfunction may be a result of glial activation which is induced by maternal HFD to initiate an inflammatory microenvironment leading to a long-term progression of retinopathy. Short-term administration of ω-3 PUFA at a young age may be a feasible strategy to intervene in the maternal HFD-programmed retinal impairment in male offspring.

Glial cell transcriptome analyses in 3xTg-AD mice: Effects of aging, disease progression, and anti-Aß immunotherapy.

Background: To investigate how changes in expression of glial genes relate to a progression of Alzheimer's disease (AD) pathology, and how anti-Aß immunotherapy impact these changes, we conducted a transcriptomic analysis for brains from cohorts of 2-, 10-, and 20 month old 3xTg-AD mice, and a cross-sectional study in groups of 20 month-old mice treated with active DNA Aß42 immunization, passive immunotherapy, untreated, and wild-type (wt) controls. Methods: Twenty-four Formalin-Fixed Paraffin-Embedded (FFPE) mouse brain sections were used for the gene expression analyses (nanostring). Adjacent sections from these and additional mouse brains were stained for microglia using antibodies detecting IbaI and Gal3. For a semi-quantitative analysis of increased tau and amyloid pathology with aging and disease progression, a comparison of ELISA results from brains of 12 and 20 months old 3xTg-AD mice were shown. Results: Based on the different comparisons of transcript numbers found the 3xTg-AD age groups with the senescent 20 months old wt control mouse brains, and the 20 months old 3xTg-AD mouse brains with the 20 months old wt control mouse brains, genes were assigned as upregulated due to aging, or due to disease progression, or due to both. The immunohistochemistry of microglia markers revealed that Gal3 might be an important marker for phagocytosing microglia around amyloid plaques. The comparison of the two anti-Aß immunotherapy approaches showed a differential downregulation of inflammatory glial genes. Conclusion: These results are relevant for future clinical trials using active anti-amyloid immunotherapy.

Potential Role of Cannabinoid Type 2 Receptors in Neuropsychiatric and Neurodegenerative Disorders.

The endocannabinoid system (ECS) is composed of the two canonical receptor subtypes; type-1 cannabinoid (CB1R) and type 2 receptor (CB2R), endocannabinoids (eCBs) and enzymes responsible for the synthesis and degradation of eCBs. Recently, with the identification of additional lipid mediators, enzymes and receptors, the expanded ECS called the endocannabinoidome (eCBome) has been identified and recognized. Activation of CB1R is associated with a plethora of physiological effects and some central nervous system (CNS) side effects, whereas, CB2R activation is devoid of such effects and hence CB2Rs might be utilized as potential new targets for the treatment of different disorders including neuropsychiatric disorders. Previous studies suggested that CB2Rs were absent in the brain and they were considered as peripheral receptors, however, recent studies confirmed the presence of CB2Rs in different brain regions. Several studies have now focused on the characterization of its physiological and pathological roles. Studies done on the role of CB2Rs as a therapeutic target for treating different disorders revealed important putative role of CB2R in neuropsychiatric disorders that requires further clinical validation. Here we provide current insights and knowledge on the potential role of targeting CB2Rs in neuropsychiatric and neurodegenerative disorders. Its non-psychoactive effect makes the CB2R a potential target for treating CNS disorders; however, a better understanding of the fundamental pharmacology of CB2R activation is essential for the design of novel therapeutic strategies.

Glaucoma and neuroinflammation: An overview.

Glaucoma is an optic neuropathy characterized by well-defined optic disc morphological changes (i.e., cup enlargement, neuroretinal border thinning, and notching, papillary vessel modifications) consequent to retinal ganglion cell loss, axonal degeneration, and lamina cribrosa remodeling. These modifications tend to be progressive and are the main cause of functional damage in glaucoma. Despite the latest findings about the pathophysiology of the disease, the exact trigger mechanisms and the mechanism of degeneration of retinal ganglion cells and their axons have not been completely elucidated. Neuroinflammation may play a role in both the development and the progression of the disease as a result of its effects on retinal environment and retinal ganglion cells. We summarize the latest findings about neuroinflammation in glaucoma and examine the connection between risk factors, neuroinflammation, and retinal ganglion cell degeneration.

NIR Laser Photobiomodulation Induces Neuroprotection in an In Vitro Model of Cerebral Hypoxia/Ischemia.

Brain photobiomodulation (PBM) is an innovative treatment for a variety of neurological conditions, including cerebral ischemia. However, the capability of PBM for ischemic stroke needs to be further explored and its mechanisms of action remain currently unclear. The aim of the present research was to identify a treatment protocol capable of inducing neuroprotection and to investigate the molecular mechanisms activated by a dual-wavelength near infrared (NIR) laser source in an organotypic hippocampal slice model of hypoxia/ischemia. Hippocampal slices were exposed to oxygen and glucose deprivation (OGD) for 30 min followed by NIR laser light (fluence 3.71, 7.42, or 14.84 J/cm2; wavelengths 808 nm and 905 nm) delivered immediately or 30 min or 60 min after OGD, in order to establish a therapeutic window. Neuronal injury was assessed by propidium iodide fluorescence 24 h later. Our results show that NIR laser irradiation attenuates OGD neurotoxicity once applied immediately or 30 min after OGD. Western blot analysis of proteins involved in neuroinflammation (iNOS, COX-2, NFkB subunit p65, and Bcl-2) and in glutamatergic-mediated synaptic activity (vGluT1, EAAT2, GluN1, and PSD95) showed that the protein modifications induced by OGD were reverted by NIR laser application. Moreover, CA1 confocal microscopy revealed that the profound morphological changes induced by OGD were reverted by NIR laser radiation. In conclusion, NIR laser radiation attenuates OGD neurotoxicity in organotypic hippocampal slices through attenuation of inflammatory mechanisms. These findings shed light on molecular definition of NIR neuroprotective mechanisms, thus underlining the potential benefit of this technique for the treatment of cerebral ischemia.

Molecular mechanisms of neurodegeneration in the entorhinal cortex that underlie its selective vulnerability during the pathogenesis of Alzheimer's disease.

The entorhinal cortex (EC) is a vital component of the medial temporal lobe, and its contributions to cognitive processes and memory formation are supported through its extensive interconnections with the hippocampal formation. During the pathogenesis of Alzheimer's disease (AD), many of the earliest degenerative changes are seen within the EC. Neurodegeneration in the EC and hippocampus during AD has been clearly linked to impairments in memory and cognitive function, and a growing body of evidence indicates that molecular and functional neurodegeneration within the EC may play a primary role in cognitive decline in the early phases of AD. Defining the mechanisms underlying molecular neurodegeneration in the EC is crucial to determining its contributions to the pathogenesis of AD. Surprisingly few studies have focused on understanding the mechanisms of molecular neurodegeneration and selective vulnerability within the EC. However, there have been advancements indicating that early dysregulation of cellular and molecular signaling pathways in the EC involve neurodegenerative cascades including oxidative stress, neuroinflammation, glia activation, stress kinases activation, and neuronal loss. Dysfunction within the EC can impact the function of the hippocampus, which relies on entorhinal inputs, and further degeneration within the hippocampus can compound this effect, leading to severe cognitive disruption. This review assesses the molecular and cellular mechanisms underlying early degeneration in the EC during AD. These mechanisms may underlie the selective vulnerability of neuronal subpopulations in this brain region to the disease development and contribute both directly and indirectly to cognitive loss.This paper has an associated Future Leader to Watch interview with the first author of the article.

The role of resveratrol as a natural modulator in glia activation in experimental models of stroke.

OBJECTIVE: Stroke is one of the most important causes of death and disability in modern and developing societies. In a stroke, both the glial cells and neurons develop apoptosis due to decreased cellular access to glucose and oxygen. Resveratrol (3, 5, 4'-trihydroxy-trans-stilbene) as a herbal compound shows neuroprotective and glioprotective effects. This article reviews how resveratrol can alleviate symptoms after stroke to help neurons to survive by modulating some signaling pathways in glia. MATERIALS AND METHODS: Various databases such as ISI Web of Knowledge, Scopus, Medline, PubMed, and Google Scholar, were searched from 2000 to February 2020 to gather the required articles using appropriate keywords. RESULTS: Resveratrol enhances anti-inflammatory and decreases inflammatory cytokines by affecting the signaling pathways in microglia such as AMP-activated protein kinase (5' adenosine monophosphate-activated protein kinase, AMPK), SIRT1 (sirtuin 1) and SOCS1 (suppressor of cytokine signaling 1). Furthermore, through miR-155 overexpressing in microglia, resveratrol promotes M2 phenotype polarization. Resveratrol also increases AMPK and inhibits GSK-3ß (glycogen synthase kinase 3 beta) activity in astrocytes, which release energy, makes ATP available to neurons and reduces reactive oxygen species (ROS). Besides, resveratrol increases oligodendrocyte survival, which can lead to maintaining post-stroke brain homeostasis. CONCLUSION: These results suggest that resveratrol can be considered a novel therapeutic agent for the reduction of stroke symptoms that can not only affect neuronal function but also play an important role in reducing neurotoxicity by altering glial activity and signaling.

Prolactin mitigates deficiencies of retinal function associated with aging.

Aging causes the progressive degeneration of retinal cells leading to the eventual loss of vision. The hormone prolactin (PRL) is a neurotrophic factor able to compensate for photoreceptor cell death and electroretinogram deficits induced by light retinal damage. Here, we used adult 4-month old and aged 20-month old pigmented mice, null or not for the PRL receptor to explore whether PRL provides trophic support against age-related retinal dysfunction. Retinal functionality, apoptosis, glia activation, and neurotrophin expression were assessed by electroretinogram, TUNEL, glial fibrillary acidic protein and ionized calcium binding adaptor molecule 1 immunohistochemistry, and real-time PCR, respectively. Lack of PRL signaling in aged mice, but not in adult mice, correlated with photosensitive retinal dysfunction, increased photoreceptor apoptosis, differential expression of proapoptotic mediators, and microglia activation. We conclude that PRL is required for maintaining retinal functionality in both female and male mice during aging and has potential therapeutic value against age-related retinal disorders.

Cognitive improvements and reduction in amyloid plaque deposition by saikosaponin D treatment in a murine model of Alzheimer's disease.

Alzheimer's disease (AD), is a severe neurodegenerative disease that currently lacks an optimally effective therapeutic agent for its management. Saikosaponin D (SSD) is a component extracted from the herb Bupleurum falcatum that is commonly used in Chinese medicine. Although SSD has been reported to exert neuroprotective effects, its pharmacological role in AD has not been previously elucidated. Therefore, the aim of the present study was to investigate whether SSD treatment improves the cognitive function and pathological features of 3xTg mice, a triple-transgenic mouse model of AD that displays classical pathological features of AD. The effects of SSD treatment on the behavioral, histological and physiological features of the animal were quantified. Results from the behavioral experiments on the SSD-treated 3xTg mice identified a significant reduction in memory impairment. In addition, histological staining results indicated that SSD application could preserve the morphology of neurons, reduce apoptosis and significantly inhibit amyloid-ß deposition in the hippocampus of 3xTg mice. SSD treatment also decelerated the activation of microglia and astrocytes in the hippocampus of 3xTg mice, possibly via the inhibition of the NF-κB signal transduction pathway. Therefore, the present study demonstrated the protective effects of SSD against progressive neurodegeneration and identified the potential underlying pharmacological mechanism. It was speculated that SSD may serve as a possible therapeutic agent in AD treatment in the future.

Analysis of the behavioral, cellular and molecular characteristics of pain in severe rodent spinal cord injury.

Human SCI is frequently associated with chronic pain that is severe and refractory to medical therapy. Most rodent models used to assess pain outcomes in SCI apply moderate injuries to lower thoracic spinal levels, whereas the majority of human lesions are severe in degree and occur at cervical or upper thoracic levels. To better model and understand mechanisms associated with chronic pain after SCI, we subjected adult rats to T3 severe compression or complete transection lesions, and examined pain-related behaviors for three months. Within one week after injury, rats developed consistent forepaw pain-related behaviors including increased spontaneous lifts, tactile allodynia and cold sensitivity that persisted for three months. Place escape avoidance testing confirmed that withdrawal of the forepaws from a von Frey stimulus represented active pain-related aversion. Spontaneous and evoked pain-related measures were attenuated by gabapentin, further indicating that these behaviors reflect development of pain. Spinal level of injury was relevant: rats with T11 severe SCI did not exhibit forepaw pain-related behaviors. Immunoblotting and immunofluorescence of C6-C8 spinal dorsal horn, reflecting sensory innervation of the forepaw, revealed: 1) expansion of CGRP immunoreactivity in lamina I/II; 2) increased GAP-43 expression; and 3) increased IBA1, GFAP and connexin-43 expression. These findings indicate that aberrant pain fiber sprouting and gliopathy occur after severe SCI. Notably, satellite glial cells (SGCs) in C6-C8 DRGs exhibited increases in GFAP and connexin-43, suggesting ongoing peripheral sensitization. Carbenoxolone, a gap junction inhibitor, and specific peptide inhibitors of connexin-43, ameliorated established tactile allodynia after severe SCI. Collectively, severe T3 SCI successfully models persistent pain states and could constitute a useful model system for examining candidate translational pain therapies after SCI.

Protective effect of pioglitazone on retinal ischemia/reperfusion injury in rats.

PURPOSE: To investigate the protective effect of pioglitazone on the rat retina after ischemia/reperfusion (I/R) injury and to explore its possible mechanisms. METHODS: Retinal ischemia was induced by increasing the intraocular pressure to 110 mm Hg for 60 minutes, and pioglitazone was delivered 3 hours before the I/R. Retinal damage was quantified by measuring the thickness of the retina, the functional changes of visual evoked potential (VEP) and electroretinography (ERG), and the number of retinal ganglion cells (RGCs) at 7 days after I/R injury. Real-time PCR and Western blot analysis were performed to measure the glial fibrillary acidic protein (GFAP) expression. Retinal cell apoptosis was detected by TUNEL assay at 24 hours after reperfusion. Nuclear factor-κB (NF-κB), Bax, and Bcl-2 in the retina were determined by Western blot analysis. RESULTS: The I/R produced a degenerative effect primarily in the ganglion cell layer, inner plexiform layer, and inner nuclear layer. Pioglitazone maintained the retinal thickness, promoted the survival of RGCs, and attenuated the destruction of ERG and VEP caused by I/R. Pioglitazone pretreatment also suppressed NF-κB activation and altered GFAP overexpression. The number of TUNEL-labeled cells significantly decreased in the retinas pretreated with pioglitazone, and the Bax-Bcl-2 ratio was much lower in the retinas pretreated with pioglitazone than in the I/R group. CONCLUSIONS: Pioglitazone could inhibit activation of the glia cells, prevent cell apoptosis, and protect the retina from subsequent cellular damage caused by the retinal I/R. The possible mechanism might involve the NF-κB pathway.