Binding Adaptor Research Articles

Mouse maternal odontogenic infection with Porphyromonas gingivalis induces cognitive decline in offspring.

Porphyromonas gingivalis (P. gingivalis), a major periodontal pathogen, causes intrauterine infection/inflammation. Offspring exposed to intrauterine infection/inflammation have an increased risk of neurological disorders, regardless of gestational age. However, the relationship between maternal periodontitis and offspring functional/histological changes in the brain has not yet been elucidated. In this study, we used a gestational mouse model to investigate the effects of maternal odontogenic infection of P. gingivalis on offspring behavior and brain tissue. The step-through passive avoidance test showed that the latency of the acquisition trial was significantly shorter in the P. gingivalis group (p < 0.05), but no difference in spontaneous motor/exploratory parameters by open-field test. P. gingivalis was diffusely distributed throughout the brain, especially in the hippocampus. In the hippocampus and amygdala, the numbers of neuron cells and cyclic adenosine monophosphate response element binding protein-positive cells were significantly reduced (p < 0.05), whereas the number of ionized calcium binding adapter protein 1-positive microglia was significantly increased (p < 0.05). In the hippocampus, the number of glial fibrillary acidic protein-positive astrocytes was also significantly increased (p < 0.05). The offspring of P. gingivalis-infected mothers have reduced cognitive function. Neurodegeneration/neuroinflammation in the hippocampus and amygdala may be caused by P. gingivalis infection, which is maternally transmitted. The importance of eliminating maternal P. gingivalis-odontogenic infection before or during gestation in maintenance healthy brain function in offspring should be addressed in near future.

Dietary wheat gluten induces astro- and microgliosis in the hypothalamus of male mice.

Gluten, which is found in cereals such as wheat, rye and barley, makes up a major dietary component in most western nations, and has been shown to promote body mass gain and peripheral inflammation in mice. In the current study, we investigated the impact of gluten on central inflammation that is typically associated with diet-induced obesity. While we found no effect of gluten when added to a low-fat diet (LFD), male mice fed high fat diet (HFD) enriched with gluten increased body mass and adiposity compared with mice fed HFD without gluten. We furthermore found that gluten, when added to the LFD, increases circulating C-reactive protein levels. Gluten regardless of whether it was added to LFD or HFD led to a profound increase in the number of microglia and astrocytes in the arcuate nucleus of the hypothalamus, as detected by immunohistochemistry for ionised calcium binding adaptor molecule 1 (Iba-1) and glial fibrillary acidic protein (GFAP), respectively. In mice fed LFD, gluten mimicked the immunogenic effects of HFD exposure and when added to HFD led to a further increase in the number of immunoreactive cells. Taken together, our results confirm a moderate obesogenic effect of gluten when fed to mice exposed to HFD and for the first-time report gluten-induced astro- and microgliosis suggesting the development of hypothalamic injury in rodents.

Granulomatous mural folliculitis in 16 domestic goats: Infection with malignant catarrhal fever viruses and colocalization with ovine herpesvirus-2 using in situ hybridization.

Granulomatous mural folliculitis (GMF) is an uncommon reaction pattern occasionally observed in nonadapted ruminant hosts infected with malignant catarrhal fever viruses. This report characterizes GMF and concurrent cutaneous lesions in 16 goats with crusting dermatitis using histochemistry including hematoxylin and eosin, periodic acid-Schiff, and Grocott's methenamine silver, and immunohistochemistry for CD3, CD20, ionized calcium binding adaptor molecule 1, and cytokeratin AE1/3. Infiltrates in all 16 GMF cases consisted of macrophages and fewer T lymphocytes, and variably included eosinophils, multinucleated histiocytic giant cells, and/or neutrophils. Formalin-fixed paraffin-embedded skin and fresh skin samples from caprine GMF cases were tested using pan-herpesvirus nested conventional polymerase chain reaction (PCR) and partial sequencing, ovine herpesvirus-2 (OvHV-2) real-time PCR, and OvHV-2 colorimetric in situ hybridization (ISH). Five of 16 goats with GMF (31%) were PCR positive for malignant catarrhal fever viruses, including caprine herpesvirus 3 in 1 goat and OvHV-2 in 4 goats. Three goats also had positive intranuclear OvHV-2 hybridization signal in follicular keratinocytes, among other cell types, localized to areas of GMF. Herpesviruses were not detected in the formalin-fixed paraffin-embedded skin of 9 goats without GMF. This case series describes relatively frequent detections of malignant catarrhal fever viruses in the skin of goats with GMF, including the first report of caprine herpesvirus 3, and localizes OvHV-2 infected follicular keratinocytes within areas of GMF.

The role of gut-brain axis in a rotenone-induced rat model of Parkinson's disease

Parkinson's disease (PD) is a widespread neurodegenerative condition affecting millions globally. This investigation centered on the gut-brain axis in a rotenone-induced PD rat model. Researchers monitored behavioral shifts, histological modifications, neurodegeneration, and inflammation markers throughout the rats' bodies. Results revealed that rotenone-treated rats displayed reduced exploration (p = 0.004) and motor coordination (p < 0.001), accompanied by decreased Nissl staining and increased alpha-synuclein immunoreactivity in the striatum (p = 0.009). Additionally, these rats exhibited weight loss (T3, mean = 291.9 ± 23.67; T19, mean = 317.5 ± 17.53; p < 0.05) and substantial intestinal histological alterations, such as shortened villi, crypt architecture loss, and inflammation. In various regions, researchers noted elevated immunoreactivity to ionized binding adapter molecule (IBA)-1 (p < 0.05) and reduced immunoreactivity to glial fibrillary acidic protein (p < 0.05) and S100B (p < 0.001), indicating altered glial cell activity. Overall, these findings imply that PD is influenced by gut-brain axis changes and may originate in the intestine, impacting bidirectional gut-brain communication.

Metformin restores cognitive dysfunction and histopathological deficits in an animal model of sporadic Alzheimer's disease

BackgroundMetformin has been introduced as a neuroprotective agent in recent years. Here we evaluate the therapeutic effects of metformin in sporadic mouse model of Alzheimer's disease (SAD). MethodsAD was induced by streptozotocin (STZ, 0.5 mg/kg) on days 1 and 3. Metformin (MET, 200 mg/kg per day) was used for two weeks. Novel objective recognition (NOR) and Barnes Maze test were used to test the learning and memory. Nissl staining was used as s histological method for counting the dying neurons in different regions of hippocampus. Immunofluorescence staining against glial fibrillary acidic protein (GFAP), ionized calcium binding adaptor molecule 1 (Iba1) and NeuN were used to visualize reactive astrocytes, microglia and neurons, respectively. ResultsIn NOR test, the discrimination indices in the STZ group were significantly lower than the control and treatment groups. Goal sector/non-goal sector (GS/NGS) ratio index in Barnes maze was increased in metformin group compared to other groups. The number of dying neurons was increased by SAD and metformin reduced it. GFAP level was increased in CA1, CA3 and cortex of STZ group and reversed following the treatment. Iba1 level was significantly higher in STZ group in CA3 and cortex regions compared to Control and decreased by metformin in CA3 and cortex. Counting NeuN+ cells demonstrated significant reduction of neurons in DG+CA1 and CA3 after SAD induction. SignificanceMetformin decreased inflammatory cells and reactive astrocytes as well as the dying neurons in the hippocampus region and the cortex in SAD, and improved the cognitive performance.

Construction of a Brain-specific SLC23A2 Gene Knockout Mice Model

Vitamin C (VC) is a key antioxidant of the Central Nervous System (CNS) and SLC23A2 (SVCT2) is the only transporter that actively transports VC into the brain. While the existing animal models of VC deficiency are in the whole body, the essential role of VC in brain development remains elusive. In our study presented here, the CRISPR/Cas9 technology was applied for the construction of a C57BL/6J-SLC23A2 em1(flox)Smoc mouse model, which was crossed with the Glial fibrillary acidic protein-driven Cre Recombinase (GFAP-Cre) genotype mice to generate a conditional knockout model of SLC23A2(SVCT2) gene in mice brain (GFAP-Cre;SLC23A2 flox/flox) after generations of crossbreeding. Our results showed that the expression of SVCT2 in GFAP-Cre;SLC23A2 flox/flox (Cre;svct2 f/f) mice brain was significantly decreased, and consistently, the expression of Neuronal nuclei antigen (NeuN), Glial fibrillary acidic protein (GFAP), calbindin-28k, brain-derived neurotrophic factor (BDNF) was down-regulated but Ionized calcium binding adapter molecule 1 (Iba-1) was up-regulated in Cre;svct2 f/f mice brain tissues. On the other hand, the levels of Glutathione, Reduced (GSH), myeloperoxidase (MDA), 8-isoprostane, tumor necrosis factor-α (TNF-α) and interleukin-6(IL-6) were significantly increased, but the levels of VC in brain tissue of the model group were decreased in Cre;svct2 f/f mice brain tissues, indicating the protective effect of VC against oxidative stress and inflammation during pregnancy. Thus, the conditional knockout of the SLC23A2 gene in the brain of mouse was successfully established by the CRISPR/Cas9 technology in our study, providing an effective animal model for studying the role of VC in fetal brain development.

Ceftriaxone ameliorates hippocampal synapse loss by inhibiting microglial/macrophages activation in glial glutamate transporter-1 dependent manner in the APP/PS1 mouse model of Alzheimer’s disease

Synapse loss is a major contributor to cognitive dysfunction in Alzheimer’s disease (AD). Impairments in the expression and/or glutamate uptake activity of glia glutamate transporter-1 (GLT-1) contribute to synapse loss in AD. Hence, targeting the restoration of GLT-1 activity may have potential for alleviating synapse loss in AD. Ceftriaxone (Cef) can upregulate the expression and glutamate uptake activity of GLT-1 in many disease models, including those for AD. The present study investigated the effects of Cef on synapse loss and the role of GLT-1 using APP/PS1 transgenic and GLT-1 knockdown APP/PS1 AD mice. Furthermore, the involvement of microglia in the process was investigated due to its important role in synapse loss in AD. We found that Cef treatment significantly ameliorated synapse loss and dendritic degeneration in APP/PS1 AD mice, evidenced by an increased dendritic spine density, decreased dendritic beading density, and upregulated levels of postsynaptic density protein 95 (PSD95) and synaptophysin. The effects of Cef were suppressed by GLT-1 knockdown in GLT-1+/-/APP/PS1 AD mice. Simultaneously, Cef treatment inhibited ionized calcium binding adapter molecule 1 (Iba1) expression, decreased the proportion of CD11b+CD45hi cells, declined interleukin-6 (IL-6) content, and reduced the co-expression of Iba1 with PSD95 or synaptophysin in APP/PS1 AD mice. In conclusion, Cef treatment ameliorated synapse loss and dendritic degeneration in APP/PS1 AD mice in a GLT-1-dependent manner, and the inhibitory effect of Cef on the activation of microglia/macrophages and their phagocytosis for synaptic elements contributed to the mechanism.

Metformin Exerted Neuroprotective Effects on Cognition in Rats with Trastuzumap‐Induced Brain Toxicity

AbstractBackgroundTrastuzumap (Trz) is effectively used for breast cancer patients to improve health status and reduce mortality rate. Nevertheless, several studies reported its adverse effect on brain toxicity. To date, the mechanisms of Trz‐induced brain toxicity are not fully understood. Metformin is a generic prescribed drug for type 2 diabetes mellitus, while in the past decade it also used for attenuating the adverse effects of chemotherapy. However, the effects of metformin on brain mitochondrial function, hippocampal microglial function, hippocampal synaptic plasticity, and cognitive function in Trz‐induced brain toxicity have never been investigated.MethodEighteen male Wistar rats were randomly divided into two groups; control group (0.9% NSS, intraperitoneal (i.p.) injection, n = 8) and Trz‐treated group (4 mg/kg for 7 days, i.p. injection, n = 16). Then, Trz‐treated rats were divided into two subgroups (n = 6/ subgroup) to receive either vehicle (0.9% NSS, daily via oral gavage) or metformin (250 mg/kg/day for 7 days, daily via oral gavage). At the end of experiment protocol, cognitive function was measured, and brains were obtained for further molecular investigation.ResultTrz‐treated rats demonstrated an increase of mitochondrial ROS production, increased mean fluorescence intensity of ionized calcium binding adaptor molecule 1 (Iba1), decreased hippocampal dendritic spine density, decreased preferent index in novel object location (NOL) and novel object recognition (NOR) test (p&lt;0.05, Figure 1A‐F). Metformin‐treated in Trz‐rats reversed the adverse effect of Trz by attenuated mitochondrial ROS production, decreased mean fluorescence intensity of Iba1, increased hippocampal dendritic spine density, as well as increased preferent index in NOL and NOR test (p&lt;0.05, Figure 1A‐F).ConclusionTrz‐induced brain toxicity as indicated by brain mitochondrial dysfunction, decreased hippocampal microglial function, impaired hippocampal synaptic plasticity, and cognitive dysfunction, while metformin alleviated those deleterious effects. The findings in this study emphasize the roles of metformin against Trz‐induced brain toxicity and strengthen

Safety profile of lycopene, ellagic acid and hesperetin combination regimen in the brain of balb c mice

AbstractBackgroundThe pathophysiology of neurodegenerative diseases, particularly Alzheimer’s disease is multifactorial. However, with scientific evidences suggesting that various antioxidants possess different protective mechanisms, thus hypothetically/theoretically no single natural compound can adequately abrogate these plethora of neurodegenerative cascades. Hence, there is a need for a paradigm shift from the usual monotherapy directed management of neurodegeneration. Therefore, this study investigated the effects of combined administration of three antioxidants namely; hesperetin, lycopene and ellagic acid in the brain of balb c mice.MethodThirty (30) male balb/c mice were grouped and administered orally with respect to each of their weights for Twenty eight (28) days as follows: Group I (Control)‐ Dimethyl sulfoxide (DMSO) in olive oil, Group II‐ Lycopene (5mg/kg body wt), Ellagic acid (15mg/kg body wt), Hesperetin (20mg/kg body wt) dissolved in DMSO and olive oil, Group III‐lycopene(10mg/kg body wt), Ellagic acid (30mg/kg body wt), Hesperetin(40mg/kg body wt) dissolved in DMSO and olive oil.ResultThe combination regimen at both doses show no significant difference in the concentration of nitric oxide (NO), protein carbonyl (PC), reduced glutathione (GSH), hydrogen peroxide (H2O2), malondialdehyde (MDA) level, activity of catalase (CAT) in the brain. Furthermore, the numbers of spared pyramidal and purkinje neurons of the cortex (sensorimotor cortex, hippocampus) and cerebellum at both doses were increased respectively but not significant when compared with their age‐matched control. Also, neuronal nuclei (NeuN) immunohistochemical staining revealed a slightly elevated matured neuronal population, as well as decreased ionized calcium binding adaptor molecule 1 (Iba1) expressions.ConclusionIn conclusion, this study suggests that the combined regimen is not only safe, but also enhanced cell viability in normal condition and could be promising in the management of neurodegenerative diseases.

Effect of electroacupuncture on the expression of Iba-1 in spinal dorsal horn and hippocampus of chronic pain rats with knee osteoarthritis

To observe the effect of electroacupuncture(EA) on pain-ralated behaviors, morphology of hippocampus, concentrations of inflammatory cytokines and expression of ionized calcium binding adapter molecule 1(Iba-1) in dorsal horn of the spinal cord and the hippocampus, and brain-derived neurotrophic factor (BDNF) in hippocampus of rats with knee osteoarthritis (KOA), so as to explore the mechanism of EA in improving chronic pain of KOA. Forty SD rats were randomly divided into blank group, saline group, model group and EA group, with 10 rats in each group. Monosodium iodoacetate(MIA, 80 mg/mL, 50 μL) was injected into the left knee joint cavity of rats in the model group and EA group to establish the chronic pain model of KOA, while the same volume of normal saline was injected into the left knee joint cavity of rats in the saline group. Rats in the EA group received EA stimulation(2 Hz/100 Hz, 1-2 mA) at left "Yanglingquan"(GB34) and "Neixiyan"(EX-LE4) for 15 min, 14 d after MIA injection. The treatment was given once daily, 5 d as 1 session and 2 sessions of treatment were required. Methanical withdrawl threshold(MWT) and weight-bearing capacity tests on left hind limbs were carried out 1 d before, 7 d，14 d, 20 d and 26 d after MIA injection. At the 27th day, rats were sacrificed and HE staining was used to observe the morphology of hippocampal CA1 area. Concentrations of interleukin(IL)-1β and tumor necrosis factor(TNF)-α in the left L3-L5 spinal dorsal horn and hippocampal CA1 area were detected by ELISA, the expressions of Iba-1 in the spinal dorsal horn and hippo-campal CA1 area were detected by immunofluorescence, and the expression of BDNF in left hippocampal CA1 area was detected by Western blot. The HE staining results of the hippocampal CA1 area showed reduced number of neurons, unclear cell contour and boundary between nucleus and cytoplasm, and nuclear pyknosis in the model group, which was relatively milder in the EA group. Compared with the blank group, MWT and weight-bearing capacity of rats' left hind limbs, and expression of BDNF protein in hippocampal CA1 area were significantly decreased (P<0.01), while the contents of IL-1β and TNF-α, the expression of Iba-1 in spinal dorsal horn and hippocampal CA1 area were significantly increased (P<0.01) in the model group. In comparison with the model group, MWT and weight-bearing capacity of rats' left hind limbs, and protein expression of BDNF in hippocampal CA1 area were significantly increased (P<0.01), while the contents of IL-1β and TNF-α, and the expression of Iba-1 protein in spinal dorsal horn and hippocampal CA1 area were significantly decreased after EA intervention(P<0.01). EA at GB34 and EX-LE4 can alleviate the pain-related behaviors of KOA rats. The mechanism might be related to the inhibition of inflammatory reaction mediated by microglia in spinal dorsal horn and hippocampus, and the up-regulation of BDNF expression in hippocampus.

Adiponectin Attenuates Splenectomy-Induced Cognitive Deficits by Neuroinflammation and Oxidative Stress via TLR4/MyD88/NF-κb Signaling Pathway in Aged Rats.

Perioperative neurocognitive disorder (PND) is a common adverse event after surgical trauma in elderly patients. The pathogenesis of PND is still unclear. Adiponectin (APN) is a plasma protein secreted by adipose tissue. We have reported that a decreased APN expression is associated with PND patients. APN may be a promising therapeutic agent for PND. However, the neuroprotective mechanism of APN in PND is still unclear. In this study, 18 month old male Sprague-Dawley rats were assigned to six groups: the sham, sham + APN (intragastric (i.g.) administration of 10 μg/kg/day for 20 days before splenectomy), PND (splenectomy), PND + APN, PND + TAK-242 (intraperitoneal (i.p.) administration of 3 mg/kg TAK-242), and PND + APN + lipopolysaccharide (LPS) (i.p. administration of 2 mg/kg LPS). We first found that APN gastric infusion significantly improved learning and cognitive function in the Morris water maze (MWM) test after surgical trauma. Further experiments indicated that APN could inhibit the Toll-like receptor 4 (TLR4)/myeloid differentiation factor 88 (MyD88)/nuclear factor kappa B (NF-κb) p65 pathway to decrease the degree of oxidative damage (malondialdehyde (MDA) and superoxide dismutase (SOD)), microglia-mediated neuroinflammation (ionized calcium binding adapter molecule 1 (IBA1), caspase-1, tumor necrosis factor (TNF)-α, interleukin-1β (IL-1β), and interleukin-6 (IL-6)), and apoptosis (p53, Bcl2, Bax, and caspase 3) in hippocampus. By using LPS-specific agonist and TAK-242-specific inhibitor, the involvement of TLR4 engagement was confirmed. APN intragastric administration exerts a neuroprotective effect against cognitive deficits induced by peripheral trauma, and the possible mechanisms include the inhibition of neuroinflammation, oxidative stress, and apoptosis, mediated by the suppression of the TLR4/MyD88/NF-κb signaling pathway. We propose that oral APN may be a promising candidate for PND treatment.

Effects of intranasal administration of tripterygium glycoside-bearing liposomes on behavioral cognitive impairment of mice induced by central nervous system inflammation

Tripterygium glycosides liposome(TPGL) were prepared by thin film-dispersion method, which were optimized accor-ding to their morphological structures, average particle size and encapsulation rate. The measured particle size was(137.39±2.28) nm, and the encapsulation rate was 88.33%±1.82%. The mouse model of central nervous system inflammation was established by stereotaxic injection of lipopolysaccharide(LPS). TPGL and tripterygium glycosides(TPG) were administered intranasally for 21 days. The effects of intranasal administration of TPG and TPGL on behavioral cognitive impairment of mice due to LPS-induced central ner-vous system inflammation were estimated by animal behavioral tests, hematoxylin-eosin(HE) staining of hippocampus, real-time quantitative polymerase chain reaction(RT-qPCR) and immunofluorescence. Compared with TPG, TPGL caused less damage to the nasal mucosa, olfactory bulb, liver and kidney of mice administered intranasally. The behavioral performance of treated mice was significantly improved in water maze, Y maze and nesting experiment. Neuronal cell damage was reduced, and the expression levels of inflammation and apoptosis related genes [tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β), BCL2-associated X(Bax), etc.] and glial activation markers [ionized calcium binding adaptor molecule 1(IBA1) and glial fibrillary acidic protein(GFAP)] were decreased. These results indicated that liposome technique combined with nasal delivery alleviated the toxic side effects of TPG, and also significantly ameliorated the cognitive impairment of mice induced by central nervous system inflammation.

A Lipopolysaccharide Mouse Model Mirrors Neuroinflammatory Transcriptional Signatures of Human AD, and the Glucagon-Like Peptide-1 Receptor Agonist Semaglutide Attenuates Neuroinflammation in this Model (P1-6.007)

<h3>Objective:</h3> We aimed to investigate the molecular effects of semaglutide on lipopolysaccharide (LPS) induced hippocampal neuroinflammation in mice and further to compare human gene expression signatures in Alzheimer’s disease (AD) brain tissue with those induced by LPS. <h3>Background:</h3> Neuroinflammation is part of the pathophysiology in AD. AD neuroimaging studies highlight increased inflammation, while genome wide association studies indicate that many AD-associated genes are expressed in glial cells. The glucagon-like peptide-1 receptor agonist semaglutide is currently being investigated in two phase 3a randomized, placebo-controlled trials in people with early Alzheimer’s disease (evoke/evoke+). <h3>Design/Methods:</h3> Mice were treated daily with subcutaneous semaglutide (30nmol/kg) or vehicle for 28 days, and LPS or vehicle was administered on days 15–17. RNA-sequencing and immunohistochemistry were performed to assess transcriptional and morphological changes in the hippocampus at Day 19 and Day 28 (Day 2 and 11 post-LPS, respectively). RNA-sequencing analyses included differentially expressed gene analysis, weighted gene co-regulated network analysis, pathway analysis and gene set enrichment analysis on published human AD datasets. <h3>Results:</h3> Semaglutide treatment significantly decreased the area of ionized calcium binding adaptor molecule 1 (Iba1) positive microglia (quantitative assessment) and attenuated co-expressed inflammatory genes vs vehicle in LPS-treated mice (p&lt;0.01) on Day 28. Pathway analysis was consistent with an overall dampening of inflammatory processes in mice treated with LPS and semaglutide. The mouse LPS model mirrored the transcriptional signatures identified in people with AD in data of human hippocampal subtype transcriptional signatures, suggesting that semaglutide could potentially alleviate homologous inflammatory molecular processes in humans. <h3>Conclusions:</h3> In an LPS-induced neuroinflammation mouse model, semaglutide reduced hippocampal neuroinflammation as measured by transcriptional changes and microglial area (Iba1). This could represent a novel mechanism whereby semaglutide may affect neuronal integrity and function, and thereby AD pathophysiology. <b>Disclosure:</b> Dr. Ludwig has nothing to disclose. Dr. Rausch has nothing to disclose. Dr. Secher has received personal compensation for serving as an employee of Novo Nordisk. An immediate family member of Dr. Secher has received personal compensation for serving as an employee of Novo Nordisk. Dr. Secher has stock in Novo Nordisk. Dr. Bentsen has received personal compensation for serving as an employee of Novo Nordisk. An immediate family member of Dr. Bentsen has stock in Novo Nordisk. Dr. Hansen has received personal compensation for serving as an employee of Neumirna Therapeutics. Dr. Hansen has received intellectual property interests from a discovery or technology relating to health care. Dr. Bjerregaard has received personal compensation for serving as an employee of Novo Nordisk A/S. Dr. Niss has nothing to disclose. Kristoffer L Egerod has nothing to disclose. Dr. Hansen has received personal compensation for serving as an employee of Novo Nordisk A/S. Dr. Hansen has stock in Novo Nordisk A/S. Dr. Dalgaard has received personal compensation for serving as an employee of Novo Nordisk. Dr. Merkestein has received personal compensation for serving as an employee of Novo Nordisk A/S. Dorte Holst has nothing to disclose. Charles Pyke has nothing to disclose. Mrs. Wichern has received personal compensation for serving as an employee of Gubra ApS. Dr. Polex-Wolf has received personal compensation for serving as an employee of Novo Nordisk A/S. Tune H Pers has nothing to disclose. Lotte B Knudsen has nothing to disclose.

Early electroacupuncture intervention delays progression of disease in mice with amyotrophic lateral sclerosis by down-regulating TLR4/NF-κB signaling

To observe the effect of early electroacupuncture (EA) intervention on Toll-like receptor 4 (TLR4)/nuclear factor-κB (NF-κB) signaling pathway in mice with amyotrophic lateral sclerosis (ALS), so as to explore its mechanisms underlying alleviation of ALS. Fifty-four ALS (ALS-SOD1G93A) mice with SOD1G93A gene mutation identified by PCR were randomly divided into model group, 60 day(d) EA group and 90 d EA group(n=18 mice in each group), and other 18 ALS-SOD1G93A negative mice were used as the control group. At the age of 60 and 90 days, mice of the two EA groups received EA (2 Hz, 1 mA) stimulation of bilateral "Jiaji" (EX-B2) of L1-L2 and L5-L6 for 20 min, twice a week for 4 weeks,respectively. When being 60 days old, the mice in the model and control groups were subjected to the same binding as that in the two EA groups but without EA intervention. The tail suspension test was used to judge the onset time of disease and the survival period, and rotary rod fatigue test was used to evaluate the hind limb motor function. Nissl staining method was used to observe the content of Nissl bodies in the anterior horn of the lumbar spinal cord. Immunohistochemical staining was used to observe the expression of ionized calcium binding adaptor molecule-1 (Iba-1) in the anterior horn of the lumbar spinal cord, and Western blot was used to detect the relative expression of TLR4, NF-κB and tumor necrosis factor-α (TNF-α) in the lumbar spinal cord. The disease onset time apparently delayed in the 60 d EA group than in the model group (P<0.01). The survival time was apparently shorter in the model group than in the control group (P<0.01), and obviously prolonged in the 60 d EA and 90 d EA groups than in the model group (P<0.01). The rotatory rod time was obviously shorter in the model group than in the control group (P<0.05), and apparently longer in the 60 d EA group than in the model group and 90 d EA group (P<0.05). Compared with the control group, the model group had a decrease in the number of Nissl bodies in the anterior horn of the lumbar spinal cord (P<0.01), and an increase in the expression levels of Iba-1, TLR4, NF-κB and TNF-α in the lumbar spinal cord (P<0.01). In contrast to the model group, both 60 d EA and 90 d EA groups had an apparent increase in the number of Nissl bodies and a marked decrease in the expression levels of Iba-1, TLR4, NF-κB and TNF-α in the lumbar spinal cord (P<0.05, P<0.01). The therapeutic effects of 60 d EA group were evidently superior to those of 90 d EA group in delaying the onset time of disease, prolonging the survival time and rotatory rod time, increasing the number of Nissl bodies, and in down-regulating the expression of Iba-1, TLR4, NF-κB and TNF-α (P<0.05, P<0.01). The early intervention of EX-B2 EA is more effective in delaying the progression of ALS than post-onset intervention in ALS-SOD1G93A mice, which may be related to its functions in inhibiting the excessive activation of microglia, and down-regulating TLR4/NF-κB signaling.

Altered endocannabinoid metabolism compromises the brain-CSF barrier and exacerbates chronic deficits after traumatic brain injury in mice.

Endocannabinoids [2-arachidonoylglycerol (2-AG) and N-arachidonoylethanolamine (AEA)], endogenously produced arachidonate-based lipids, are anti-inflammatory physiological ligands for two known cannabinoid receptors, CB1 and CB2, yet the molecular and cellular mechanisms underlying their effects after brain injury are poorly defined. In the present study, we hypothesize that traumatic brain injury (TBI)-induced loss of endocannabinoids exaggerates neurovascular injury, compromises brain-cerebrospinal fluid (CSF) barriers (BCB) and causes behavioral dysfunction. Preliminary analysis in human CSF and plasma indicates changes in endocannabinoid levels. This encouraged us to investigate the levels of endocannabinoid-metabolizing enzymes in a mouse model of controlled cortical impact (CCI). Reductions in endocannabinoid (2-AG and AEA) levels in plasma were supported by higher expression of their respective metabolizing enzymes, monoacylglycerol lipase (MAGL), fatty acid amide hydrolase (FAAH), and cyclooxygenase 2 (Cox-2) in the post-TBI mouse brain. Following increased metabolism of endocannabinoids post-TBI, we observed increased expression of CB2, non-cannabinoid receptor Transient receptor potential vanilloid-1 (TRPV1), aquaporin 4 (AQP4), ionized calcium binding adaptor molecule 1 (IBA1), glial fibrillary acidic protein (GFAP), and acute reduction in cerebral blood flow (CBF). The BCB and pericontusional cortex showed altered endocannabinoid expressions and reduction in ventricular volume. Finally, loss of motor functions and induced anxiety behaviors were observed in these TBI mice. Taken together, our findings suggest endocannabinoids and their metabolizing enzymes play an important role in the brain and BCB integrity and highlight the need for more extensive studies on these mechanisms.

Inhibition of Microglial Activation by Delayed Mild Hypothermia Reduced Preoligodendrocyte Injury in a Neonatal Rat Brain Slice Model.

Periventricular leukomalacia (PVL), characterized by distinctive form of white matter injury, often arises after neonatal cardiac surgery. Proven therapies for PVL are absent. In this study, we designed to quest therapeutic effects of delayed mild hypothermia on PVL and its mechanism in a neonatal rat brain slice model. With the increase of delayed mild hypothermia-treating time, the reduced expression of myelin basic protein and loss of preoligodendrocytes were significantly attenuated after oxygen-glucose deprivation. In addition, the proportion of ionized calcium binding adapter molecule 1 (Iba-1)-positive cells and the expression of Iba-1 were apparently reduced with the increased duration of mild hypothermia treatment. Furthermore, the levels of tumor necrosis factor alpha and interleukin-6 reduced after the mild hypothermia treatment relative to the control. Inhibition of microglial activation with prolonged mild hypothermia may be a potential strategy for white matter protection during cardiopulmonary bypass and hypothermic circulatory arrest.

Protective effects of three kinds of borneol on different brain regions in acute cerebral ischemia/reperfusion model rats

This study compared the ameliorating effects of L-borneol, natural borneol, and synthetic borneol on the injury of different brain regions in the rat model of acute phase of cerebral ischemia/reperfusion(I/R) for the first time, which provides a reference for guiding the rational application of borneol in the early treatment of ischemic stroke and has important academic and application values. Healthy specific pathogen-free(SPF)-grade SD male rats were randomly assigned into 13 groups: a sham-operation group, a model group, a Tween model group, a positive drug(nimodipine) group, and high-, medium-, and low-dose(0.2, 0.1, and 0.05 g·kg~(-1), respectively) groups of L-borneol, natural borneol, and synthetic borneol according to body weight. After 3 days of pre-administration, the rat model of I/R was established by suture-occluded method and confirmed by laser speckle imaging. The corresponding agents in different groups were then administered for 1 day. The body temperature was monitored regularly before pre-administration, days 1, 2, and 3 of pre-administration, 2 h after model awakening, and 1 d after model establishment. Neurological function was evaluated based on Zea-Longa score and modified neurological severity score(mNSS) 2 h and next day after awakening. The rats were anesthetized 30 min after the last administration, and blood was collected from the abdominal aorta. Enzyme-linked immunoassay assay(ELISA) was employed to determine the serum levels of tumor necrosis factor-alpha(TNF-α), interleukin-6(IL-6), IL-4, and transforming growth factor-beta1(TGF-β1). The brain tissues were stained with triphenyltetrazolium chloride(TTC) for the calculation of cerebral infarction rate, and hematoxylin-eosin(HE) staining was used for observing and semi-quantitatively evaluating the pathological damage in different brain regions. Immunohistochemistry was employed to detect the expression of ionized calcium binding adapter molecule 1(IBA1) in microglia. q-PCR was carried out to determine the mRNA levels of iNOS and arginase 1(Arg1), markers of polarization phenotype M1 and M2 in microglia. Compared with the sham-operation group, the model group and the Tween model group showed significantly elevated body temperature, Zea-Longa score, mNSS, and cerebral infarction rate, severely damaged cortex, hippocampus, and striatum, increased serum levels of IL-6 and TNF-α, and decreased serum levels of IL-4 and TGF-β1. The three borneol products had a tendency to reduce the body temperature of rats 1 day after modeling. Synthetic borneol at the doses of 0.2 and 0.05 g·kg~(-1), as well as L-borneol of 0.1 g·kg~(-1), significantly reduced Zea-Longa score and mNSS. The three borneol products at the dose of 0.2 g·kg~(-1) significantly reduced the cerebral infarction rate. L-borneol at the doses of 0.2 and 0.1 g·kg~(-1) and natural borneol at the dose of 0.1 g·kg~(-1) significantly reduced the pathological damage of the cortex. L-borneol and natural borneol at the dose of 0.1 g·kg~(-1) attenuated the pathological damage of hippocampus, and 0.2 g·kg~(-1) L-borneol attenuated the damage of striatum. The 0.2 g·kg~(-1) L-borneol and the three doses of natural borneol and synthetic borneol significantly reduced the serum level of TNF-α, and the 0.1 g·kg~(-1) synthetic borneol reduced the level of IL-6. L-borneol and synthetic borneol at the dose of 0.2 g·kg~(-1) significantly inhibited the activation of cortical microglia, and 0.2 g·kg~(-1) L-borneol up-regulated the expression of Arg1 and down-regulated the expression level of iNOS. In conclusion, the three borneol products may alleviate inflammation to ameliorate the pathological damage of brain regions of rats in the acute phase of I/R by inhibiting the activation of microglia and promoting the polarization of microglia from M1 type to M2 type. The protective effect on brain followed a trend of L-borneol &gt; synthetic borneol &gt; natural borneol. We suggest L-borneol the first choice for the treatment of I/R in the acute phase.

Combining SOS1 and MEK Inhibitors in a Murine Model of Plexiform Neurofibroma Results in Tumor Shrinkage

Individuals with neurofibromatosis type 1 develop rat sarcoma virus (RAS)-mitogen-activated protein kinase-mitogen-activated and extracellular signal-regulated kinase (RAS-MAPK-MEK)-driven nerve tumors called neurofibromas. Although MEK inhibitors transiently reduce volumes of most plexiform neurofibromas in mouse models and in neurofibromatosis type 1 (NF1) patients, therapies that increase the efficacy of MEK inhibitors are needed. BI-3406 is a small molecule that prevents Son of Sevenless (SOS)1 interaction with Kirsten rat sarcoma viral oncoprotein (KRAS)-GDP, interfering with the RAS-MAPK cascade upstream of MEK. Single agent SOS1 inhibition had no significant effect in the DhhCre;Nf1 fl/fl mouse model of plexiform neurofibroma, but pharmacokinetics (PK)-driven combination of selumetinib with BI-3406 significantly improved tumor parameters. Tumor volumes and neurofibroma cell proliferation, reduced by MEK inhibition, were further reduced by the combination. Neurofibromas are rich in ionized calcium binding adaptor molecule 1 (Iba1)+ macrophages; combination treatment resulted in small and round macrophages, with altered cytokine expression indicative of altered activation. The significant effects of MEK inhibitor plus SOS1 inhibition in this preclinical study suggest potential clinical benefit of dual targeting of the RAS-MAPK pathway in neurofibromas. SIGNIFICANCE STATEMENT: Interfering with the RAS-mitogen-activated protein kinase (RAS-MAPK) cascade upstream of mitogen activated protein kinase kinase (MEK), together with MEK inhibition, augment effects of MEK inhibition on neurofibroma volume and tumor macrophages in a preclinical model system. This study emphasizes the critical role of the RAS-MAPK pathway in controlling tumor cell proliferation and the tumor microenvironment in benign neurofibromas.

Electroacupuncture inhibits neuron injury of SAMP8 mice by reducing inflammatory response

To observe the effect of electroacupuncture (EA) at "Baihui"(GV20) and "Shenshu" (BL23) on the pathological injury of neurons in SAMP8 mice and the anti-inflammatory effect on neuron repair, providing a new experimental basis for EA prevention and treatment of Alzheimer's disease. Twelve 7-month-old SAMP8 mice were randomly divided into model and EA groups, and 6 SAMR1 mice of the same age and genetic background were used as normal group. Mice in the EA group were needled at GV20 and bilateral BL23, and EA (1 mA, 2 Hz) was applied to bilateral BL23 for 15 min, once daily, 10 d as a course for a total of 4 courses, with an interval of 1 d. Mice in the normal and model groups were captured and fixed in the same way as the EA group. The spatial learning and memory ability was detected by Morris water maze test. Neuronal nuclear antigen (NeuN) positive expression and the number of NeuN-positive cells in dentate gyrus (DG) were detected by immunofluorescence staining. The protein expression levels of ionized calcium binding adapter molecule 1(Iba-1), tumor necrosis factor-α(TNF-α), interleukin (IL)-6 and IL-1β in hippocampus were detected by Western blot. The ultrastructure of nerve cells in DG was observed by transmission electron microscopy. Compared with the normal group, the average escape latency was prolonged(P<0.01), the number of platform crossing was significantly reduced (P<0.01), the average fluorescence intensity of NeuN and the number of NeuN-positive cells in hippocampus DG region decreased (P<0.05), the expression levels of Iba-1, TNF-α, IL-6 and IL-1β in hippocampus were increased (P<0.05) in the model group.Compared with the model group, the ave-rage escape latency was shortened (P<0.01), the number of platform crossing times was significantly increased (P<0.01), the average fluorescence intensity of NeuN and the number of NeuN-positive cells in hippocampus DG region increased (P<0.05), the expression levels of Iba-1, TNF-α, IL-6 and IL-1β in hippocampus were decreased (P<0.05) in the EA group. The morphology of nerve cells in the hippocampus DG region was normal， and the organelles in the cytoplasm were clear, complete and regularly distributed in the normal group. However, the morphology of nerve cells in the model group was seriously irregular, which was also irregular in EA group but somewhat relieved compared with model group. EA at GV20 and BL23 can improve the learning and memory ability of SAMP8 mice, which may be related to inhibiting the neuroinflammatory response, increasing the number of neurons and improving the ultrastructure of the DG region of the hippocampus to play the role of neuron protection.

Treatment with Granulocyte-Macrophage Colony-Stimulating Factor Reduces Viral Titers in the Brains of West Nile Virus-Infected Mice and Improves Survival.

West Nile virus (WNV) is the leading cause of epidemic arboviral encephalitis in the United States. As there are currently no proven antiviral therapies or licensed human vaccines, understanding the neuropathogenesis of WNV is critical for rational therapeutic design. In WNV-infected mice, the depletion of microglia leads to enhanced viral replication, increased central nervous system (CNS) tissue injury, and increased mortality, suggesting that microglia play a critical role in protection against WNV neuroinvasive disease. To determine if augmenting microglial activation would provide a potential therapeutic strategy, we administered granulocyte-macrophage colony-stimulating factor (GM-CSF) to WNV-infected mice. Recombinant human GM-CSF (rHuGMCSF) (sargramostim [Leukine]) is an FDA-approved drug used to increase white blood cells following leukopenia-inducing chemotherapy or bone marrow transplantation. Daily treatment of both uninfected and WNV-infected mice with subcutaneous injections of GM-CSF resulted in microglial proliferation and activation as indicated by the enhanced expression of the microglia activation marker ionized calcium binding adaptor molecule 1 (Iba1) and several microglia-associated inflammatory cytokines, including CCL2 (C-C motif chemokine ligand 2), interleukin 6 (IL-6), and IL-10. In addition, more microglia adopted an activated morphology as demonstrated by increased sizes and more pronounced processes. GM-CSF-induced microglial activation in WNV-infected mice was associated with reduced viral titers and apoptotic activity (caspase 3) in the brains of WNV-infected mice and significantly increased survival. WNV-infected ex vivo brain slice cultures (BSCs) treated with GM-CSF also showed reduced viral titers and caspase 3 apoptotic cell death, indicating that GM-CSF specifically targets the CNS and that its actions are not dependent on peripheral immune activity. Our studies suggest that stimulation of microglial activation may be a viable therapeutic approach for the treatment of WNV neuroinvasive disease. IMPORTANCE Although rare, WNV encephalitis poses a devastating health concern, with few treatment options and frequent long-term neurological sequelae. Currently, there are no human vaccines or specific antivirals against WNV infections, so further research into potential new therapeutic agents is critical. This study presents a novel treatment option for WNV infections using GM-CSF and lays the foundation for further studies into the use of GM-CSF as a treatment for WNV encephalitis as well as a potential treatment for other viral infections.