Establishing co-culture blood–brain barrier models for different neurodegeneration conditions to understand its effect on BBB integrity

The blood–brain barrier (BBB) is a functional interface that provides selective permeability, protection from toxic substances, transport of nutrients, and clearance of brain metabolites. Additionally, BBB disruption has been shown to play a role in many neurodegenerative conditions and diseases. Therefore, the aim of this study was to establish a functional, convenient, and efficient in vitro co-cultured BBB model that can be used for several physiological conditions related to BBB disruption. Mouse brain-derived endothelial (bEnd.3) and astrocyte (C8-D1A) cells were co-cultured on transwell membranes to establish an intact and functional in vitro model. The co-cultured model and its effects on different neurological diseases and stress conditions, including Alzheimer’s disease (AD), neuroinflammation, and obesity, have been examined by transendothelial electrical resistance (TEER), fluorescein isothiocyanate (FITC) dextran, and tight junction protein analyses. Scanning electron microscope images showed evidence of astrocyte end-feet processes passing through the membrane of the transwell. Moreover, the co-cultured model showed effective barrier properties in the TEER, FITC, and solvent persistence and leakage tests when compared to the mono-cultured model. Additionally, the immunoblot results showed that the expression of tight junction proteins such as zonula occludens-1 (ZO-1), claudin-5, and occludin-1 was enhanced in the co-culture. Lastly, under disease conditions, the BBB structural and functional integrity was decreased. The present study demonstrated that the co-cultured in vitro model mimicked the BBB’s structural and functional integrity and, under disease conditions, the co-cultured model showed similar BBB damages. Therefore, the present in vitro BBB model can be used as a convenient and efficient experimental tool to investigate a wide range of BBB-related pathological and physiological studies

Deciphering the Potential Neuroprotective Effects of Luteolin against Aβ1–42-Induced Alzheimer’s Disease

The current study was undertaken to unveil the protective effects of Luteolin, a natural flavonoid, against amyloid-beta (Aβ1–42)-induced neuroinflammation, amyloidogenesis, and synaptic dysfunction in mice. For the development of an AD mouse model, amyloid-beta (Aβ1–42, 5 μL/5 min/mouse) oligomers were injected intracerebroventricularly (i.c.v.) into mice’s brain by using a stereotaxic frame. After that, the mice were treated with Luteolin for two weeks at a dose of 80 mg/kg/day. To monitor the biochemical changes, we conducted western blotting and immunofluorescence analysis. According to our findings, the infusion of amyloid-beta activated c-Jun N-terminal kinases (p-JNK), p38 mitogen-activated protein kinases, glial fibrillary acidic protein (GFAP), and ionized calcium adaptor molecule 1 (Iba-1) in the cortex and hippocampus of the experimental mice; these changes were significantly inhibited in Aβ1–42 + Luteolin-treated mice. Likewise, we also checked the expression of inflammatory markers, such as p-nuclear factor-kB p65 (p-NF-kB p65 (Ser536), tissue necrosis factor (TNF-α), and Interleukin1-β (IL-1β), in Aβ1–42-injected mice brain, which was attenuated in Aβ1–42 + Luteolin-treated mice brains. Further, we investigated the expression of pro- and anti-apoptotic cell death markers such as Bax, Bcl-2, Caspase-3, and Cox-2, which was significantly reduced in Aβ1–42 + Lut-treated mice brains compared to the brains of the Aβ-injected group. The results also indicated that with the administration of Aβ1–42, the expression levels of β-site amyloid precursor protein cleaving enzyme (BACE-1) and amyloid-beta (Aβ1–42) were significantly enhanced, while they were reduced in Aβ1–42 + Luteolin-treated mice. We also checked the expression of synaptic markers such as PSD-95 and SNAP-25, which was significantly enhanced in Aβ1–42 + Lut-treated mice. To unveil the underlying factors responsible for the protective effects of Luteolin against AD, we used a specific JNK inhibitor, which suggested that Luteolin reduced Aβ-associated neuroinflammation and neurodegeneration via inhibition of JNK. Collectively, our results indicate that Luteolin could serve as a novel therapeutic agent against AD-like pathological changes in mice

Oral Administration of Gintonin Protects the Brains of Mice against Aβ-Induced Alzheimer Disease Pathology: Antioxidant and Anti-Inflammatory Effects

The study was aimed at analyzing the protective effects of gintonin in an amyloid beta- (Aβ-) induced Alzheimer’s disease (AD) mouse model. For the development of the Aβ-induced AD mouse model, the amyloid-β (Aβ1-42) peptide was stereotaxically injected into the brains of mice. Subsequently, gintonin was administered at a dose of 100 mg/kg/day/per oral (p.o) for four weeks daily, and its effects were evaluated by using western blotting, fluorescence analysis of brain sections, biochemical tests, and memory-related behavioral evaluations. To elucidate the effects of gintonin at the mechanistic level, the activation of endogenous antioxidant mechanisms, as well as the activation of astrocytes, microglia, and proinflammatory mediators such as nuclear factor erythroid 2-related factor 2 (NRF-2) and heme oxygenase-1 (HO-1), was evaluated. In addition, microglial cells (BV-2 cells) were used to analyze the effects of gintonin on microglial activation and signaling mechanisms. Collectively, the results suggested that gintonin reduced elevated oxidative stress by improving the expression of NRF-2 and HO-1 and thereby reducing the generation of reactive oxygen species (ROS) and lipid peroxidation (LPO). Moreover, gintonin significantly suppressed activated microglial cells and inflammatory mediators in the brains of Aβ-injected mice. Our findings also indicated improved synaptic and memory functions in the brains of Aβ-injected mice after treatment with gintonin. These results suggest that gintonin may be effective for relieving AD symptoms by regulating oxidative stress and inflammatory processes in a mouse model of AD. Collectively, the findings of this preclinical study highlight and endorse the potential, multitargeted protective effects of gintonin against AD-associated oxidative damage, neuroinflammation, cognitive impairment, and neurodegeneration

Cycloastragenol, a Triterpenoid Saponin, Regulates Oxidative Stress, Neurotrophic Dysfunctions, Neuroinflammation and Apoptotic Cell Death in Neurodegenerative Conditions

Here, we have unveiled the effects of cycloastragenol against Aβ (Amyloid-beta)-induced oxidative stress, neurogenic dysfunction, activated mitogen-activated protein (MAP) kinases, and mitochondrial apoptosis in an Aβ-induced mouse model of Alzheimer’s disease (AD). The Aβ-induced mouse model was developed by the stereotaxic injection of amyloid-beta (5 μg/mouse/intracerebroventricular), and cycloastragenol was given at a dose of 20 mg/kg/day/p.o for 6 weeks daily. For the biochemical analysis, we used immunofluorescence and Western blotting. Our findings showed that the injection of Aβ elevated oxidative stress and reduced the expression of neurogenic markers, as shown by the reduced expression of brain-derived neurotrophic factor (BDNF) and the phosphorylation of its specific receptor tropomyosin receptor kinase B (p-TrKB). In addition, there was a marked reduction in the expression of NeuN (neuronal nuclear protein) in the Aβ-injected mice brains (cortex and hippocampus). Interestingly, the expression of Nrf2 (nuclear factor erythroid 2–related factor 2), HO-1 (heme oxygenase-1), p-TrKB, BDNF, and NeuN was markedly enhanced in the Aβ + Cycloastragenol co-treated mice brains. We have also evaluated the expressions of MAP kinases such as phospho c-Jun-N-terminal kinase (p-JNK), p-38, and phospho-extracellular signal-related kinase (ERK1/2) in the experimental groups, which suggested that the expression of p-JNK, p-P-38, and p-Erk were significantly upregulated in the Aβ-injected mice brains; interestingly, these markers were downregulated in the Aβ + Cycloastragenol co-treated mice brains. We also checked the expression of activated microglia and inflammatory cytokines, which showed that cycloastragenol reduced the activated microglia and inflammatory cytokines. Moreover, we evaluated the effects of cycloastragenol against mitochondrial apoptosis and memory dysfunctions in the experimental groups. The findings showed significant regulatory effects against apoptosis and memory dysfunction as revealed by the Morris water maze (MWM) test. Collectively, the findings suggested that cycloastragenol regulates oxidative stress, neurotrophic processes, neuroinflammation, apoptotic cell death, and memory impairment in the mouse model of AD

LDHB Deficiency Promotes Mitochondrial Dysfunction Mediated Oxidative Stress and Neurodegeneration in Adult Mouse Brain

Age-related decline in mitochondrial function and oxidative stress plays a critical role in neurodegeneration. Lactate dehydrogenase-B (LDHB) is a glycolytic enzyme that catalyzes the conversion of lactate, an important brain energy substrate, into pyruvate. It has been reported that the LDHB pattern changes in the brain during ageing. Yet very little is known about the effect of LDHB deficiency on brain pathology. Here, we have used Ldhb knockout (Ldhb−/−) mice to test the hypothesis that LDHB deficiency plays an important role in oxidative stress-mediated neuroinflammation and neurodegeneration. LDHB knockout (Ldhb−/−) mice were generated by the ablation of the Ldhb gene using the Cre/loxP-recombination system in the C57BL/6 genetic background. The Ldhb−/− mice were treated with either osmotin (15 μg/g of the body; intraperitoneally) or vehicle twice a week for 5-weeks. After behavior assessments, the mice were sacrificed, and the cortical and hippocampal brain regions were analyzed through biochemical and morphological analysis. Ldhb−/− mice displayed enhanced reactive oxygen species (ROS) and lipid peroxidation (LPO) production, and they revealed depleted stores of cellular ATP, GSH:GSSG enzyme ratio, and downregulated expression of Nrf2 and HO-1 proteins, when compared to WT littermates. Importantly, the Ldhb−/− mice showed upregulated expression of apoptosis mediators (Bax, Cytochrome C, and caspase-3), and revealed impaired p-AMPK/SIRT1/PGC-1alpha signaling. Moreover, LDHB deficiency-induced gliosis increased the production of inflammatory mediators (TNF-α, Nf-ĸB, and NOS2), and revealed cognitive deficits. Treatment with osmotin, an adipoR1 natural agonist, significantly increased cellular ATP production by increasing mitochondrial function and attenuated oxidative stress, neuroinflammation, and neuronal apoptosis, probably, by upregulating p-AMPK/SIRT1/PGC-1alpha signaling in Ldhb−/− mice. In brief, LDHB deficiency may lead to brain oxidative stress-mediated progression of neurodegeneration via regulating p-AMPK/SIRT1/PGC-1alpha signaling, while osmotin could improve mitochondrial functions, abrogate oxidative stress and alleviate neuroinflammation and neurodegeneration in adult Ldhb−/− mice

Quinovic Acid Impedes Cholesterol Dyshomeostasis, Oxidative Stress, and Neurodegeneration in an Amyloid-β-Induced Mouse Model

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder typified by several neuropathological features including amyloid-beta (Aβ) plaque and neurofibrillary tangles (NFTs). Cholesterol retention and oxidative stress (OS) are the major contributors of elevated β- and γ-secretase activities, leading to excessive Aβ deposition, signifying the importance of altered cholesterol homeostasis and OS in the progression of Aβ-mediated neurodegeneration and cognitive deficit. However, the effect of Aβ on cholesterol metabolism is lesser-known. In this study, we evaluated the effect of quinovic acid (QA; 50 mg/kg body weight, i.p.) against the intracerebroventricular (i.c.v.) injection of Aβ (1-42)-induced cholesterol dyshomeostasis, oxidative stress, and neurodegeneration in the cortex and hippocampal brain regions of wild-type male C57BL/6J mice. Our results indicated that Aβ (1-42)-treated mice have increased Aβ oligomer formation along with increased β-secretase expression. The enhanced amyloidogenic pathway in Aβ (1-42)-treated mice intensified brain cholesterol accumulation due to increased expressions of p53 and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) enzyme. Importantly, we further confirmed the p53-mediated HMGCR axis activation by using pifithrin-α (PFT) in SH-SY5Y cells. Furthermore, the augmented brain cholesterol levels were also associated with increased OS. However, the QA administration to Aβ (1-42)-injected mice significantly ameliorated the Aβ burden, p53 expression, and cholesterol accumulation by deterring the oxidative stress through upregulating the Nrf2/HO-1 pathway. Moreover, the QA downregulated gliosis, neuroinflammatory mediators (p-NF-κB and IL-1β), and the expression of mitochondrial apoptotic markers (Bax, cleaved caspase-3, and cytochrome c). QA treatment also reversed the deregulated synaptic markers (PSD-95 and synaptophysin) and improved spatial learning and memory behaviors in the Aβ-treated mouse brains. These results suggest that Aβ (1-42) induces its acute detrimental effects on cognitive functions probably by increasing brain cholesterol levels through a possible activation of the p53/HMGCR axis. However, QA treatment reduces the cholesterol-induced oxidative stress, neuroinflammation, and neurodegeneration, leading to the restoration of cognitive deficit after Aβ (1-42) i.c.v. injection in mice

17β-Estradiol Modulates SIRT1 and Halts Oxidative Stress-Mediated Cognitive Impairment in a Male Aging Mouse Model

Oxidative stress has been considered the main mediator in neurodegenerative disease and in normal aging processes. Several studies have reported that the accumulation of reactive oxygen species (ROS), elevated oxidative stress, and neuroinflammation result in cellular malfunction. These conditions lead to neuronal cell death in aging-related neurodegenerative disorders such as Alzheimer's disease (AD) and Parkinson's disease. Chronic administration of d-galactose (d-gal) for a period of 10 weeks causes ROS generation and neuroinflammation, ultimately leading to cognitive impairment. In this study, we evaluated the estrogen receptor alpha (ER alpha)/silent mating type information regulation 2 homolog 1 (SIRT1)-dependent antioxidant efficacy of 17 beta-estradiol against d-gal-induced oxidative damage-mediated cognitive dysfunction in a male mouse model. The results indicate that 17 beta-estradiol, by stimulating ER alpha/SIRT1, halts d-gal-induced oxidative stress-mediated JNK/NF-?B overexpression, neuroinflammation and neuronal apoptosis. Moreover, 17 beta-estradiol ameliorated d-gal-induced AD-like pathophysiology, synaptic dysfunction and memory impairment in adult mouse brains. Interestingly, inhibition of SIRT1 with Ex527 (a potent and selective SIRT1 inhibitor) further enhanced d-gal-induced toxicity and abolished the beneficial effect of 17 beta-estradiol. Most importantly, for the first time, our molecular docking study reveals that 17 beta-estradiol allosterically increases the expression of SIRT1 and abolishes the inhibitory potential of d-ga. In summary, we can conclude that 17 beta-estradiol, in an ER alpha/SIRT1-dependent manner, abrogates d-gal-induced oxidative stress-mediated memory impairment, neuroinflammation, and neurodegeneration in adult mice