OBJECTIVE

To describe and compare characteristics of premacular membranes in idiopathic macular pucker (iMP) and proliferative vitreoretinopathy (PVR) using immunofluorescence and transmission electron microscopy.

METHODS

For immunocytochemical and ultrastructural analyses, premacular membranes were harvested during vitrectomy from 16 eyes with iMP and 12 eyes with PVR. All specimens were processed as flat mounts for phase-contrast and fluorescence microscopy. We used 19 different primary antibodies such as anti-α-smooth muscle actin (α-SMA), anti-integrin-αv, anti-galectin, anti-IBA-1, anti-EMMPRIN (CD147), anti-ricinus (RCS) and anti-collagen-type I. Eight of 28 eyes were also prepared for transmission electron microscopy.

RESULTS

In all eyes with iMP and PVR, positive immunoreactivity of integrins, especially αvβ3 was found. There was also a strong staining of anti-α-SMA, anti-galectin, anti-EMMPRIN, anti-RCS, anti-IBA1 and anti-collagen-type I. Transmission electron microscopy showed that premacular membrane of iMP composed of myofibroblasts, glial cells and fibroblasts. In eyes with PVR, retinal pigment epithelial cells and myofibroblasts were seen as predominant cell types.

CONCLUSIONS

Premacular membranes of iMP and PVR presented with similarities in cell distribution and immunoreactivity, but showed differences in cell composition. Herein, we demonstrate immunocytochemical characteristics involved in fibrotic processes. Cell transdifferentiation into myofibroblasts represents an important process in pathogenesis of both entities. In order to address future anti-fibrotic treatment strategies, we emphasize that both fibrotic diseases share distinct immunocytochemical and ultrastructural features.

BACKGROUND

Chemokine signaling has been implicated in the pathogenesis of diabetic neuropathy; however, the involvement of the chemokine CC motif ligand 1 (CCL1)-chemokine CC motif receptor 8 (CCR8) interaction remains unknown. The goal of this study was to examine the role of CCL1-CCR8 signaling interplay in the development of hypersensitivity and in opioid effectiveness in diabetic neuropathy.

METHODS

Primary glial cell cultures and a streptozotocin (STZ; 200mg/kg, intraperitoneal)-induced mouse model of diabetic neuropathy were used. Analysis of mRNA/protein expression of glial markers and CCL1/CCR8 was performed by qRT-PCR, Western blotting and/or protein arrays. The co-localization of CCL1/CCR8 with neural/glial cells was visualized by immunofluorescence. The pharmacological tools were injected intrathecally, and pain behavior was evaluated by von Frey/cold plate tests.

RESULTS

Single STZ injection increased blood glucose levels and induced the development of hypersensitivity as measured on days 7-21. On day 7 after STZ, the protein levels of CCL1 and IBA1 but not of CCR8 or GFAP were elevated. Immunofluorescent staining revealed that CCR8 was predominantly localized in neurons, which are also the main source of spinal CCL1. Lipopolysaccharide stimulation of primary microglial cultures resulted in decreases in the levels of CCL1 and CCR8. Single intrathecal injection of CCL1 (10-500ng) induced the development of hypersensitivity, whereas on day 7 after STZ, a CCL1-neutralizing antibody dose-dependently (2-8μg) delayed pain behavior. Repeated administration of the CCL1-neutralizing antibody (4μg) also enhanced the effectiveness of morphine and buprenorphine (1μg).

CONCLUSIONS

These results reveal that CCL1/CCR8 neuronal signaling plays an important role in the development of diabetic neuropathy and the effectiveness of opioids.

The present study was designed to follow neuroinflammation after ischemic brain injury in the long-term survival rat model. Immunohistochemistry was performed 2 years after 10 min global brain ischemia due to cardiac arrest. For the visualization of the cellular inflammatory reaction microglial marker Iba1 and astrocyte marker GFAP were used. In post-ischemic animals our study revealed significant activation of astrocytes in all tested brain regions (hippocampal CA1 and CA3 areas and dentate gyrus, motor and somatosensory cortex, striatum and thalamus), while microglial activation was only found in CA1 and CA3 areas, and the motor cortex. In the specifically sensitive brain areas microglia and astrocytes showed simultaneously significant activation, while in the resistant brain areas only astrocytes were activated. Thus, there was clear evidence of less intensive neuroinflammation in brain areas resistant to ischemia. Such neuroinflammatory processes are backed by microglia and astrocytes activity even up to 2 years after ischemia-reperfusion brain injury. Our study thus revealed a chronic effect of global cerebral ischemia on the neuroinflammatory reaction in the rat brain even 2 years after the insult.

Keywords: Alzheimer’s disease; aging; brain ischemia; glia; neuroinflammation.

Background: Like the brain, the human inner ear was long thought to be devoid of immune activity. Only the endolymphatic sac (ES) was known to be endowed with white blood cells that could process antigens and serve as an immunologic defense organ for the entire inner ear. Unexpectedly, the cochlear and vestibular organs, including the eighth cranial nerve, were recently shown to contain macrophages whose functions and implication in ear disease are somewhat undefined. Here, we review recent inner ear findings in man and extend the analyses to the vestibular nerve using super-resolution structured illumination microscopy (SR-SIM). Materials and Methods: Human ESs and cochleae were collected during surgery to treat patients with vestibular schwannoma and life-threatening petro-clival meningioma compressing the brainstem. The ESs and cochleae were placed in fixative, decalcified, and rapidly frozen and cryostat sectioned. Antibodies against ionized calcium-binding adaptor molecule 1-expressing cells (IBA1 cells), laminin β2 and type IV collagen TUJ1, cytokine fractalkine (CX3CL1), toll-like receptor 4 (TLR4), CD68, CD11b, CD4, CD8, the major histocompatibility complex type II (MHCII), and the microglial marker TEME119 were used. Results: IBA1-positive cells were present in the ESs, the cochlea, central and peripheral axons of the cochlear nerve, and the vestibular nerve trunk. IBA1 cells were found in the cochlear lateral wall, spiral limbus, and spiral ganglion. Notable variants of IBA1 cells adhered to neurons with "synapse-like" specializations and cytoplasmic projections. Slender IBA1 cells occasionally protracted into the basal lamina of the Schwann cells and had intimate contact with surrounding axons. Discussion: The human eighth nerve may be under the control of a well-developed macrophage cell system. A small number of CD4+ and CD8+ cells were found in the ES and occasionally in the cochlea, mostly located in the peripheral region of Rosenthal's canal. A neuro-immunologic axis may exist in the human inner ear that could play a role in the protection of the auditory nerve. The implication of the macrophage system during disease, surgical interventions, and cell-based transplantation should be further explored.

The use of small non-coding nucleic acids, such as siRNA and miRNA, has allowed for a deeper understanding of gene functions, as well as for development of gene therapies for complex neurodegenerative diseases, including retinal degeneration. For effective delivery into the eye and transfection of the retina, suitable transfection methods are required. We investigated the use of a lipid-based transfection agent, Invivofectamine^® 3.0 (Thermo Fisher Scientific), as a potential method for delivery of nucleic acids to the retina.

Rodents were injected intravitreally with formulations of Invivofectamine 3.0 containing scrambled, Gapdh, Il-1β, and C3 siRNAs, or sterile PBS (control) using a modified protocol for encapsulation of nucleic acids. TdT-mediated dUTP nick-end labeling (TUNEL) and IBA1 immunohistochemistry was used to determine histological cell death and inflammation. qPCR were used to determine the stress and inflammatory profile of the retina. Electroretinography (ERG) and optical coherence tomography (OCT) were employed as clinical indicators of retinal health.

We showed that macrophage recruitment, retinal stress, and photoreceptor cell death in animals receiving Invivofectamine 3.0 were comparable to those in negative controls. Following delivery of Invivofectamine 3.0 alone, no statistically significant changes in expression were found in a suite of inflammatory and stress genes, and ERG and OCT analyses revealed no changes in retinal function or morphology. Injections with siRNAs for proinflammatory genes (C3 and Il-1β) and Gapdh, in combination with Invivofectamine 3.0, resulted in statistically significant targeted gene knockdown in the retina for up to 4 days following injection. Using a fluorescent Block-It siRNA, transfection was visualized throughout the neural retina with evidence of transfection observed in cells of the ganglion cell layer, inner nuclear layer, and outer nuclear layer.

This work supports the use of Invivofectamine 3.0 as a transfection agent for effective delivery of nucleic acids to the retina for gene function studies and as potential therapeutics.

An increased risk of narcolepsy following administration of an AS03-adjuvanted A(H1N1)pdm09 pandemic influenza vaccine (Pandemrix™) was described in children and adolescents in certain European countries. We investigated the potential effects of administration of the AS03-adjuvanted vaccine, non-adjuvanted vaccine antigen and AS03 Adjuvant System alone, on the central nervous system (CNS) in one-month-old cotton rats. Naïve or A(H1N1)pdm09 virus-primed animals received 2 or 3 intramuscular injections, respectively, of test article or saline at 2-week intervals. Parameters related to systemic inflammation (hematology, serum IL-6/IFN-γ/TNF-α) were assessed. Potential effects on the CNS were investigated by histopathological evaluation of brain sections stained with hematoxylin-and-eosin, or by immunohistochemical staining of microglia, using Iba1 and CD68 as markers for microglia identification/activation, albumin as indicator of vascular leakage, and hypocretin. We also determined cerebrospinal fluid (CSF) hypocretin levels and hemagglutination-inhibiting antibody titers. Immunogenicity of the AS03-adjuvanted A(H1N1)pdm09 pandemic influenza vaccine was confirmed by the induction of hemagglutination-inhibiting antibodies. Both AS03-adjuvanted vaccine and AS03 alone activated transient innate (neutrophils/eosinophils) immune responses. No serum cytokines were detected. CNS analyses revealed neither microglia activation nor inflammatory cellular infiltrates in the brain. No differences between treatment groups were detected for albumin extravascular leakage, CSF hypocretin levels, numbers of hypocretin-positive neuronal bodies or distributions of hypocretin-positive axonal/dendritic projections. Consequently, there was no evidence that intramuscular administration of the test articles promoted inflammation or damage in the CNS, or blood-brain barrier disruption, in this model.

There is a vital need to develop in vitro models of the developing human brain to recapitulate the biological effects that toxic compounds have on the brain. To model perineural vascular plexus (PNVP) in vitro, which is a key stage in embryonic development, human embryonic stem cells (hESC)-derived endothelial cells (ECs), neural progenitor cells, and microglia (MG) with primary pericytes (PCs) in synthetic hydrogels in a custom-designed microfluidics device are cocultured. The formation of a vascular plexus that includes networks of ECs (CD31+, VE-cadherin+), MG (IBA1+), and PCs (PDGFRβ+), and an overlying neuronal layer that includes differentiated neuronal cells (βIII Tubulin+, GFAP+) and radial glia (Nestin+, Notch2NL+), are characterized. Increased brain-derived neurotrophic factor secretion and differential metabolite secretion by the vascular plexus and the neuronal cells over time are consistent with PNVP functionality. Multiple concentrations of developmental toxicants (teratogens, microglial disruptor, and vascular network disruptors) significantly reduce the migration of ECs and MG toward the neuronal layer, inhibit formation of the vascular network, and decrease vascular endothelial growth factor A (VEGFA) secretion. By quantifying 3D cell migration, metabolic activity, vascular network disruption, and cytotoxicity, the PNVP model may be a useful tool to make physiologically relevant predictions of developmental toxicity.

Keywords: developmental toxicity; engineered organoids; human microphysiological systems; microfluidics devices; synthetic hydrogels.

Neuroinflammation is an integral part of the neurodegeneration process inherent to several aging dysfunctions. Within the central nervous system, microglia are the effective immune cells, responsible for neuroinflammatory responses. In this study, raspberries were subjected to in vitro digestion simulation to obtain the components that result from the gastrointestinal (GI) conditions, which would be bioaccessible and available for blood uptake. Both the original raspberry extract and the gastrointestinal bioaccessible (GIB) fraction protected neuronal and microglia cells against H2O2-induced oxidative stress and lipopolysaccharide (LPS)-induced inflammation, at low concentrations. Furthermore, this neuroprotective capacity was independent of intracellular ROS scavenging mechanisms. We show for the first time that raspberry metabolites present in the GIB fraction significantly inhibited microglial pro-inflammatory activation by LPS, through the inhibition of Iba1 expression, TNF-α release and NO production. Altogether, this study reveals that raspberry polyphenols may present a dietary route to the retardation or amelioration of neurodegenerative-related dysfunctions.

After the onset of stroke, a series of progressive and degenerative reactions, including inflammation, is activated, which leads to cell death. We recently reported that human placenta-derived multipotent stem cells (hPDMCs) process potent anti-inflammatory effects. In this study, we examined the protective effect of hPDMC transplants in a rodent model of stroke. Adult male Sprague-Dawley rats were anesthetized. hPDMCs labeled with a vital dye of fluorescing microparticles, DiI, or vehicle were transplanted into three cortical areas adjacent to the right middle cerebral artery (MCA). Five minutes after grafting, the right MCA was transiently occluded for 60 min. Stroke animals receiving hPDMCs showed a significant behavioral improvement and reduction in lesion volume examined by T2-weighted images 4 days poststroke. Brain tissues were collected 1 day later. Human-specific marker HuNu immunoreactivity and DiI fluorescence were found at the hPDMC graft sites, suggesting the survival of hPDMCs in host brain. Grafting of hPDMCs suppressed IBA1 immunoreactivity and deramification of IBA1(+) cells in the perilesioned area, suggesting activation of microglia was attenuated by the transplants. Taken together, our data indicate that hPDMC transplantation reduced cortical lesions and behavioral deficits in adult stroke rats, and these cells could serve as a unique anti-inflammatory reservoir for the treatment of ischemic brain injury.

Obesity-induced fatty liver disease is associated with increased hypothalamic inflammation. Previous reports have demonstrated that the deletion of SIRT1 in hepatocytes increases hepatic steatosis and inflammation. Using myeloid cell-specific SIRT1 knockout (KO) mice, we investigated whether ablation of SIRT1 in macrophages plays a role in regulating hepatic steatosis and hypothalamic inflammation. When challenged with a high-fat diet (HFD) for 24 weeks, hyperleptinemia, hyperinsulinemia, hepatic steatosis and macrophage infiltrations in HFD-fed KO mice were increased compared with HFD-fed WT mice. Hypothalamic expression levels of iba1 were increased in HFD-fed KO mice compared with HFD-fed WT mice. In particular, the expression levels of choline acetyltransferase were decreased in the hypothalamus of HFD-fed KO mice compared with HFD-fed WT mice. Thus, our findings suggest that SIRT1 plays a key role for hepatic steatosis and hypothalamic inflammation and that anti-inflammatory effect of SIRT1 may be important for the prevention of obesity-induced metabolic syndromes.

The present study investigates the potential protective effects of granulocyte colony-stimulating factor (G-CSF) and underlying mechanisms in a gerbil model of global cerebral ischemia. We examined neuronal death, inflammatory reaction and neurogenesis in hippocampus 72 h after transient forebrain ischemia and investigated functional deficits. G-CSF was administered intraperitoneally 24 h before ischemia and then daily. Treatment with G-CSF at 25-50 μg/kg significantly reduced neuronal loss in the hippocampus CA1 area but not at 10 ug/kg. G-CSF at 50 μg/kg significantly decreased the level of TNF-α, the number of Iba1 (microglia marker) positive cells and reduced locomotor activity 72 h after transient forebrain ischemia. Furthermore, the number of DCX-positive cells in the hippocampal dentate gyrus increased in with G-CSF treatment. Our findings indicate that G-CSF reduces hippocampal neuronal cell death dose-dependently and attenuates sensorimotor deficits after transient forebrain ischemia. These neuroprotective effects of G-CSF may be linked to inhibition of inflammation and possibly increased neurogenesis in the hippocampus.

Increasing evidence has suggested that human umbilical cord blood cells (hUCBC) have a favorable effect on hypoxic-ischemic (HI) brain injury. However, the efficacy of using hUCBCs to treat this injury has been variable and the underlying mechanism remains elusive. Here, we investigated its effectiveness using stereological analysis in an allogeneic system to examine whether intraperitoneal injection of cells derived from UCBCs of green fluorescent protein (GFP)-transgenic rats could ameliorate brain injury in neonatal rats. Three weeks after the HI event, the estimated residual brain volume was larger and motor function improved more in the cell-injected rats than in the control (PBS-treated) rats. The GFP-positive cells were hardly detectable in the brain (0.0057% of injected cells) 9 days after injection. Although 60% of GFP-positive cells in the brain were Iba1-positive, none of these were positive for NeuroD or DCX. While the number of proliferating cells increased in the hippocampus, that of activated microglia/macrophages decreased and a proportion of M2 microglia/macrophages increased in the ipsilateral hemisphere of cell-injected rats. These results suggest that intraperitoneal injection of cells derived from UCBCs could ameliorate HI injury, possibly through an endogenous response and not by supplying differentiated neurons derived from the injected stem cells.

Systemic inflammation induces cognitive impairment, yet the mechanism involved in this process is unclear. Neurotrophin receptor p75 (p75NTR) signaling is a key pathological factor contributing to neurobehavioral abnormalities in many neurodegenerative diseases. However, the role of p75NTR signaling in the regulation of sepsis-induced cognitive impairment remains largely to be elucidated. In this study, systemic inflammation was induced by cecal ligation and puncture (CLP). Neurobehavioral performances were evaluated by open field, novel object recognition, and fear conditioning tests. The expressions of proinflammatory cytokines (tumor necrosis factor (TNF-α), interleukin-1β (IL-1β), IL-6, IL-10), apoptosis marker cleaved caspase-3, ionized calcium binding adaptor molecule 1 (IBA1), proBDNF, p75NTR, c-Jun N-terminal kinase (JNK), and pJNK in the hippocampus were determined by enzyme-linked immunosorbent assay, western blot analysis, and immunofluorescence. The synaptic marker in the CA1 region of the hippocampus was assessed by Golgi staining. In the present study, we showed that systemic inflammation induced cognitive impairment, which was accompanied by increased expressions of hippocampcal proBDNF and p75NTR. Of note, we found that LM11A-31, an orally available, blood-brain barrier-permeant small-molecule p75NTR signaling modulator significantly reversed the sepsis-induced cognitive impairment and restored most of the abnormal biochemical parameters. Taken together, our study suggests that proBDNF/p75NTR signaling pathway might play a key role in the development of sepsis-induced cognitive impairment, whereas specific p75NTR inhibitor may provide a novel therapeutic approach for this disorder and possible other neurodegenerative diseases.

Key questions remain regarding the processes governing gliogenesis following central nervous system injury that are critical to understanding both beneficial brain repair mechanisms and any long-term detrimental effects, including increased risk of seizures. We have used cortical injury produced by intracranial electrodes (ICEs) to study the time-course and localization of gliosis and gliogenesis in surgically resected human brain tissue. Seventeen cases with ICE injuries of 4-301 days age were selected. Double-labelled immunolabelling using a proliferative cell marker (MCM2), markers of fate-specific transcriptional factors (PAX6, SOX2), a microglial marker (IBA1) and glial markers (nestin, GFAP) was quantified in three regions: zone 1 (immediate vicinity: 0-350 μm), zone 2 (350-700 μm) and zone 3 (remote ≥2000 μm) in relation to the ICE injury site. Microglial/macrophage cell densities peaked at 28-30 days post-injury (dpi) with a significant decline in proliferating microglia with dpi in all zones. Nestin-expressing cells (NECs) were concentrated in zones 1 and 2, showed the highest regenerative capacity (MCM2 and PAX6 co-expression) and were intimately associated with capillaries within the organizing injury cavity. There was a significant decline in nestin/MCM2 co-expressing cells with dpi in zones 1 and 2. Nestin-positive fibres remained in the chronic scar, and NECs with neuronal morphology were noted in older injuries. GFAP-expressing glia were more evenly distributed between zones, with no significant decline in density or proliferative capacity with dpi. Colocalization between nestin and GFAP in zone 1 glial cells decreased with increasing dpi. In conclusion, NECs at acute injury sites are a proliferative, transient cell population with capacity for maturation into astrocytes with possible neuronal differentiation observed in older injuries.

BACKGROUND

Altered hypothalamo-pituitary-adrenal (HPA) axis activity may be accompanied by a modulation of pain sensitivity. In a model of neuropathic pain (chronic constriction injury, CCI) we investigated the onset and maintenance of mechanical allodynia/hyperalgesia and the expression of biochemical mediators potentially involved in spinal cell modulation in two rat strains displaying either hypo- (Lewis-LEW) or hyper- (Fischer 344-FIS) reactivity of the HPA axis.

RESULTS

Mechanical pain thresholds and plasmatic corticosterone levels were assessed before and during periods of 4 or 21 days following CCI surgery. At the end of the respective protocols, the mRNA expression of glial cell markers (GFAP and Iba1) and glutamate transporters (EAAT3 and EAAT2) were examined. We observed a correlation between the HPA axis reactivity and the pain behavior but not as commonly described in the literature; LEW rats seemed to be less sensitive than FIS from 4 to 14 days after the CCI surgery when looking at the mechanical allodynia/hyperalgesia. However, the biochemical spinal markers expression we observed is conflicting.

CONCLUSIONS

We did not find a specific causal relation between the pain behavior and the glial cell activation or the expression of the glutamate transporters, suggesting that the interaction between the HPA axis and the spinal activation pattern is more complex in a context of neuropathic pain.

BackgroundTherapeutic hypothermia (TH) is the standard of care for neonates with hypoxic-ischemic encephalopathy, but it is not fully protective in the clinical setting. Hypoxia-ischemia (HI) may cause white matter injury (WMI), leading to neurological and cognitive dysfunction.MethodsP9 mice were subjected to HI as previously described. Pups underwent 3.5 h of systemic hypothermia or normothermia. Cresyl violet and Perl's iron staining for histopathological scoring of brain sections was completed blindly on all brains. Immunocytochemical (ICC) staining for myelin basic protein (MBP), microglia (Iba1), and astrocytes (glia fibrillary acidic protein (GFAP)) was performed on adjacent sections. Volumetric measurements of MBP coverage were used for quantitative analysis of white matter.ResultsTH provided neuroprotection by injury scoring for the entire group (n=44; P<0.0002). ICC analysis of a subset of brains showed that the lateral caudate was protected from WMI (P<0.05). Analysis revealed decreased GFAP and Iba1 staining in hippocampal regions, mostly CA2/CA3. GFAP and Iba1 directly correlated with injury scores of normothermic brains.ConclusionTH reduced injury, and qualitative data suggest that hippocampus and lateral caudate are protected from HI. Mildly injured brains may better show the benefits of TH. Overall, these data indicate regional differences in WMI susceptibility and inflammation in a P9 murine HI model.

Mounting evidence shows that chronic stress can affect both the structure and function of the brain resulting in decreased synaptic plasticity and cognitive dysfunction. Although several studies have indicated that aged brains are more vulnerable to chronic stress, it remains unknown how to prevent stress-induced memory deficits in aged animals. Neuroinflammation plays an important role in the pathogenesis of chronic stress-related brain dysfunction. Receptor-interacting protein 1 (RIP1) is a key molecule that can modulate inflammation, apoptosis, and necroptosis. Here, we investigated whether inhibiting RIP1 using necrostatin-1 during chronic stress could improve chronic stress-related brain dysfunction in D-galactose-induced aging mice. The stressed mice underwent restraint stress for 14 days. Necrostatin-1 (6.25 mg/kg) or vehicle was administered intraperitoneally once every 3 days during the stress period. Locomotor activity was tested using the open field test and cognitive function was assessed using the novel object recognition and Barnes maze tests. The hippocampus was collected to assess neuroinflammation (Iba1, IL-1α, IL-1β, TNF-α, and C1q), necroptosis [RIP1, RIP3, mixed lineage kinase domain-like (MLKL), and NF-κB], neuroplasticity (doublecortin, NR1, NR2A, NR2B, GluA1, and GluA2), and the expression of glucocorticoid and mineralocorticoid receptors. Blood samples were collected to quantify the levels of corticosterone. We found that chronic stress induced obvious memory impairment and neuroinflammation, decreased neurogenesis and GluA2 expression, and increased the expression of RIP1 and NF-κB. Inhibiting RIP1 by necrostatin-1 during chronic stress rescued the memory impairment and alleviated the pathological changes induced by stress. These suggest that inhibiting RIP1 using necrostatin-1 improves chronic stress-related brain dysfunction in D-galactose-induced aging mice. The potential mechanisms include limitation of neuroinflammation and the rescue of neurogenesis and GluA2 expression.

Histamine is a major peripheral inflammatory mediator and a neurotransmitter in the central nervous system. We have reported that histamine induces microglia activation and releases proinflammatory factors in primary cultured microglia. Whether histamine has similar effects in vivo is unknown. In the present study, we aimed to investigate the role of histamine and its receptors in the release of inflammatory mediators and activation of microglia in rat brain. We site-directed injected histamine, histamine receptor agonists or histamine receptor antagonists in the rat lateral ventricle using stereotaxic techniques. Flow cytometry was employed to determine histamine receptor expression in rat microglia. Microglia activation was assessed by Iba1 immunohistochemistry. The levels of tumour necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1β) and interleukin-10 (IL-10) were measured with commercial enzyme-linked immunosorbent assay (ELISA) kits, TNF-α, IL-1β and IL-10 mRNA expressions were determined with Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR). We found that all four types of histamine receptors were expressed in rat brain microglia. Histamine was able to induce microglia activation and subsequent production of the inflammatory factors TNF-α, IL-1β and IL-10, and these effects were partially abolished by H₁R and H₄R antagonists. However, H₂R and H₃R antagonists significantly increased production of TNF-α and IL-1β, and decreased IL-10 levels. The H₁R or H₄R agonists stimulated the production of TNF-α and IL-1β, while the H₂R or H₃R agonists increased IL-10 release. Our results demonstrate that histamine induces microglia activation and the release of both proinflammatory and anti-inflammatory factors in rat brain, thus contributing to the development of inflammation in the brain. Graphical Abstract Histamine induces microglia activation and the release of both proinflammatory (TNF-α and IL-1β) and anti-inflammatory factors (IL-10) in rat brain, thus contributing to the development of inflammation in the brain.

OBJECTIVE

To evaluate in adult rats, previously suckled under favorable and unfavorable conditions, the brain electrophysiological and microglial effects of the treatment early in life with the lectin (ConA) from Canavalia ensiformis.

METHODS

Male Wistar newborn rats (n = 89) were suckled under favorable or unfavorable conditions, represented by litters with 6-7 pups or 12-14 pups (groups N6 and N12, respectively). From postnatal days 5-24, they were treated intraperitoneally with 1 or 10 mg/kg ConA (groups L1 and L10, respectively), or with saline solution (group Sal), or no treatment (group Naïve). At 90-120 days of age, cortical spreading depression (CSD) was recorded at two parietal points for 4 hours, and CSD parameters (velocity of propagation and amplitude and duration of the DC slow potential change) were measured. Fixative-perfused brain sections were reacted with anti-Iba1 antibodies to quantify immunolabeled microglia.

RESULTS

Compared with the control groups, ConA-treated animals dose-dependently presented with reduced CSD propagation velocities and increased amplitude and duration of the CSD slow potential change. Microglia Iba-1 immunoreactivity was lower in both nutritional groups treated with ConA, in comparison with the control groups. The CSD hemisphere presented with higher immunoreactivity compared with the CSD-free hemisphere.

CONCLUSIONS

Attenuation in CSD propagation and microglia reaction was associated in adulthood with ConA treatment during brain development, indicating that the lectin can affect the electrophysiological and microglial development, and suggesting long-lasting protective action of the lectin on the rat brain, which is not impeded by the unfavorable suckling condition.

Lysophosphatidic acid receptor 1 (LPA₁) contributes to brain injury following transient focal cerebral ischemia. However, the mechanism remains unclear. Here, we investigated whether nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome activation might be an underlying mechanism involved in the pathogenesis of brain injury associated with LPA₁ following ischemic challenge with transient middle cerebral artery occlusion (tMCAO). Suppressing LPA₁ activity by its antagonist attenuated NLRP3 upregulation in the penumbra and ischemic core regions, particularly in ionized calcium-binding adapter molecule 1 (Iba1)-expressing cells like macrophages of mouse after tMCAO challenge. It also suppressed NLRP3 inflammasome activation, such as caspase-1 activation, interleukin 1β (IL-1β) maturation, and apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) speck formation, in a post-ischemic brain. The role of LPA₁ in NLRP3 inflammasome activation was confirmed in vitro using lipopolysaccharide-primed bone marrow-derived macrophages, followed by LPA exposure. Suppressing LPA₁ activity by either pharmacological antagonism or genetic knockdown attenuated NLRP3 upregulation, caspase-1 activation, IL-1β maturation, and IL-1β secretion in these cells. Furthermore, nuclear factor-κB (NF-κB), extracellular signal-regulated kinase 1/2 (ERK1/2), and p38 were found to be LPA₁-dependent effector pathways in these cells. Collectively, results of the current study first demonstrate that LPA₁ could contribute to ischemic brain injury by activating NLRP3 inflammasome with underlying effector mechanisms.

Keywords: LPA; LPA1; NLRP3 inflammasome; bone marrow-derived macrophage; lipopolysaccharide; transient middle cerebral artery occlusion.

Background: Postoperative cognitive dysfunction (POCD) is a very common complication that might increase the morbidity and mortality of elderly patients after surgery. However, the mechanism of POCD remains largely unknown. The NAD-dependent deacetylase protein Sirtuin 3 (SIRT3) is located in the mitochondria and regulates mitochondrial function. SIRT3 is the only sirtuin that specifically plays a role in extending lifespan in humans and is associated with neurodegenerative diseases. Therefore, the aim of this study was to evaluate the effect of SIRT3 on anesthesia/surgery-induced cognitive impairment in aged mice.

Methods: SIRT3 expression levels were decreased after surgery. For the interventional study, an adeno-associated virus (AAV)-SIRT3 vector or an empty vector was microinjected into hippocampal CA1 region before anesthesia/surgery. Western blotting, immunofluorescence staining, and enzyme-linked immune-sorbent assay (ELISA) were used to measure the oxidative stress response and downstream microglial activation and proinflammatory cytokines, and Golgi staining and long-term potentiation (LTP) recording were applied to evaluate synaptic plasticity.

Results: Overexpression of SIRT3 in the CA1 region attenuated anesthesia/surgery-induced learning and memory dysfunction as well as synaptic plasticity dysfunction and the oxidative stress response (superoxide dismutase [SOD] and malondialdehyde [MDA]) in aged mice with POCD. In addition, microglia activation (ionized calcium binding adapter molecule 1 [Iba1]) and neuroinflammatory cytokine levels (tumor necrosis factor-alpha [TNF-α], interleukin [IL]-1β and IL-6) were regulated after anesthesia/surgery in a SIRT3-dependent manner.

Conclusion: The results of the current study demonstrate that SIRT3 has a critical effect in the mechanism of POCD in aged mice by suppressing hippocampal neuroinflammation and reveal that SIRT3 may be a promising therapeutic and diagnostic target for POCD.

Keywords: Microglia; Mitochondrial oxidative stress; Neuroinflammation; Postoperative cognitive dysfunction; SIRT3; Synaptic plasticity.

Cholesterol excess in the brain is mainly disposed via cholesterol 24-hydroxylation catalysed by cytochrome P450 46A1, a CNS-specific enzyme. Cytochrome P450 46A1 is emerging as a promising therapeutic target for various brain diseases with both enzyme activation and inhibition having therapeutic potential. The rate of cholesterol 24-hydroxylation determines the rate of brain cholesterol turnover and the rate of sterol flux through the plasma membranes. The latter was shown to affect membrane properties and thereby membrane proteins and membrane-dependent processes. Previously we found that treatment of 5XFAD mice, an Alzheimer's disease model, with a small dose of anti-HIV drug efavirenz allosterically activated cytochrome P450 46A1 in the brain and mitigated several disease manifestations. Herein, we generated Cyp46a1^-/- 5XFAD mice and treated them, along with 5XFAD animals, with efavirenz to ascertain cytochrome P450 46A1-dependent and independent drug effects. Efavirenz-treated versus control Cyp46a1^-/- 5XFAD and 5XFAD mice were compared for the brain sterol and steroid hormone content, amyloid β burden, protein and mRNA expression as well as synaptic ultrastructure. We found that the cytochrome P450 46A1-dependent efavirenz effects included changes in the levels of brain sterols, steroid hormones, and such proteins as glial fibrillary acidic protein, Iba1, Munc13-1, post-synaptic density-95, gephyrin, synaptophysin and synapsin-1. Changes in the expression of genes involved in neuroprotection, neurogenesis, synaptic function, inflammation, oxidative stress and apoptosis were also cytochrome P450 46A1-dependent. The total amyloid β load was the same in all groups of animals, except lack of cytochrome P450 46A1 decreased the production of the amyloid β40 species independent of treatment. In contrast, altered transcription of genes from cholinergic, monoaminergic, and peptidergic neurotransmission, steroid sulfation and production as well as vitamin D₃ activation was the main CYP46A1-independent efavirenz effect. Collectively, the data obtained reveal that CYP46A1 controls cholesterol availability for the production of steroid hormones in the brain and the levels of biologically active neurosteroids. In addition, cytochrome P450 46A1 activity also seems to affect the levels of post-synaptic density-95, the main postsynaptic density protein, possibly by altering the calcium/calmodulin-dependent protein kinase II inhibitor 1 expression and activity of glycogen synthase kinase 3β. Even at a small dose, efavirenz likely acts as a transcriptional regulator, yet this regulation may not necessarily lead to functional effects. This study further confirmed that cytochrome P450 46A1 is a key enzyme for cholesterol homeostasis in the brain and that the therapeutic efavirenz effects on 5XFAD mice are likely realized via cytochrome P450 46A1 activation.

Keywords: Alzheimer’s disease; CYP46A1; brain; cholesterol; membrane properties.

White matter lesions (WMLs) are a common manifestation of small vessel disease (SVD) in the elderly population. They are associated with an enhanced risk of developing gait abnormalities, poor executive function, dementia, and stroke with high mortality. Hypoperfusion and the resulting endothelial damage are thought to contribute to the development of WMLs. The focus of the present study was the analysis of the microvascular bed in SVD patients with deep WMLs (DWMLs) by using double- and triple-label immunohistochemistry and immunofluorescence. Simultaneous visualization of collagen IV (COLL4)-positive membranes and the endothelial glycocalyx in thick sections allowed us to identify endothelial recession in different types of string vessels, and two new forms of small vessel/capillary pathology, which we called vascular bagging and ghost string vessels. Vascular bags were pouches and tubes that were attached to vessel walls and were formed by multiple layers of COLL4-positive membranes. Vascular bagging was most severe in the DWMLs of cases with pure SVD (no additional vascular brain injury, VBI). Quantification of vascular bagging, string vessels, and the density/size of CD68-positive cells further showed widespread pathological changes in the frontoparietal and/or temporal white matter in SVD, including pure SVD and SVD with VBI, as well as a significant effect of the covariate age. Plasma protein leakage into vascular bags and the white matter parenchyma pointed to endothelial damage and basement membrane permeability. Hypertrophic IBA1-positive microglial cells and CD68-positive macrophages were found in white matter areas covered with networks of ghost vessels in SVD, suggesting phagocytosis of remnants of string vessels. However, the overall vessel density was not altered in our SVD cohort, which might result from continuous replacement of vessels. Our findings support the view that SVD is a progressive and generalized disease process, in which endothelial damage and vascular bagging drive remodeling of the microvasculature.