Neuraminidase (NA) is a sialidase present, among various locations, in the envelope/membrane of some bacteria/viruses (e.g., influenza virus), and is involved in infectiveness and/or dispersion. The ...administration of NA within the brain lateral ventricle represents a model of acute sterile inflammation. The relevance of the Toll-like receptors TLR2 and TLR4 (particularly those in microglial cells) in such process was investigated.
Mouse strains deficient in either TLR2 (TLR2
) or TLR4 (TLR4
) were used. NA was injected in the lateral ventricle, and the inflammatory reaction was studied by immunohistochemistry (IBA1 and IL-1β) and qPCR (cytokine response). Also, microglia was isolated from those strains and in vitro stimulated with NA, or with TLR2/TLR4 agonists as positive controls (P3C and LPS respectively). The relevance of the sialidase activity of NA was investigated by stimulating microglia with heat-inactivated NA, or with native NA in the presence of sialidase inhibitors (oseltamivir phosphate and N-acetyl-2,3-dehydro-2-deoxyneuraminic acid).
In septofimbria and hypothalamus, IBA1-positive and IL-1β-positive cell counts increased after NA injection in wild type (WT) mice. In TLR4
mice, such increases were largely abolished, while were only slightly diminished in TLR2
mice. Similarly, the NA-induced expression of IL-1β, TNFα, and IL-6 was completely blocked in TLR4
mice, and only partially reduced in TLR2
mice. In isolated cultured microglia, NA induced a cytokine response (IL-1β, TNFα, and IL-6) in WT microglia, but was unable to do so in TLR4
microglia; TLR2 deficiency partially affected the NA-induced microglial response. When WT microglia was exposed in vitro to heat-inactivated NA or to native NA along with sialidase inhibitors, the NA-induced microglia activation was almost completely abrogated.
NA is able to directly activate microglial cells, and it does so mostly acting through the TLR4 receptor, while TLR2 has a secondary role. Accordingly, the inflammatory reaction induced by NA in vivo is partially dependent on TLR2, while TLR4 plays a crucial role. Also, the sialidase activity of NA is critical for microglial activation. These results highlight the relevance of microbial NA in the neuroinflammation provoked by NA-bearing pathogens and the possibility of targeting its sialidase activity to ameliorate its impact.
The administration of microbial neuraminidase into the brain ventricular cavities of rodents represents a model of acute aseptic neuroinflammation. Ependymal cell death and hydrocephalus are unique ...features of this model. Here we demonstrate that activated microglia participates in ependymal cell death. Co-cultures of pure microglia with ependymal cells (both obtained from rats) were performed, and neuraminidase or lipopolysaccharide were used to activate microglia. Ependymal cell viability was unaltered in the absence of microglia or inflammatory stimulus (neuraminidase or lipopolysaccharide). The constitutive expression by ependymal cells of receptors for cytokines released by activated microglia, such as IL-1β, was demonstrated by qPCR. Besides, neuraminidase induced the overexpression of both receptors in ventricular wall explants. Finally, ependymal viability was evaluated in the presence of functional blocking antibodies against IL-1β and TNFα. In the co-culture setting, an IL-1β blocking antibody prevented ependymal cell death, while TNFα antibody did not. These results suggest that activated microglia are involved in the ependymal damage that occurs after the administration of neuraminidase in the ventricular cavities, and points to IL-1β as possible mediator of such effect. The relevance of these results lies in the fact that brain infections caused by neuraminidase-bearing pathogens are frequently associated to ependymal death and hydrocephalus.
Soy extracts have been claimed to be neuroprotective against brain insults, an effect related to the estrogenic properties of isoflavones. However, the effects of individual isoflavones on ...obesity-induced disruption of adult neurogenesis have not yet been analyzed. In the present study we explore the effects of pharmacological administration of daidzein, a main soy isoflavone, in cell proliferation, cell apoptosis and gliosis in the adult hippocampus of animals exposed to a very high-fat diet. Rats made obese after 12-week exposure to a standard or high-fat (HFD, 60%) diets were treated with daidzein (50 mg kg(-1)) for 13 days. Then, plasma levels of metabolites and metabolic hormones, cell proliferation in the subgranular zone of the dentate gyrus (SGZ), and immunohistochemical markers of hippocampal cell apoptosis (caspase-3), gliosis (GFAP and Iba-1), food reward factor FosB and estrogen receptor alpha (ERα) were analyzed. Treatment with daidzein reduced food/caloric intake and body weight gain in obese rats. This was associated with glucose tolerance, low levels of HDL-cholesterol, insulin, adiponectin and testosterone, and high levels of leptin and 17β-estradiol. Daidzein increased the number of phospho-histone H3 and 5-bromo-2-deoxyuridine (BrdU)-ir cells detected in the SGZ of standard diet and HFD-fed rats. Daidzein reversed the HFD-associated enhanced immunohistochemical expression of caspase-3, FosB, GFAP, Iba-1 and ERα in the hippocampus, being more prominent in the dentate gyrus. These results suggest that pharmacological treatment with isoflavones regulates metabolic alterations associated with enhancement of cell proliferation and reduction of apoptosis and gliosis in response to high-fat diet.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Innate immune memory explains the plasticity of immune responses after repeated immune stimulation, leading to either enhanced or suppressed immune responses. This process has been extensively ...reported in peripheral immune cells and also, although modestly, in the brain. Here we explored two relevant aspects of brain immune priming: its persistence over time and its dependence on TLR receptors. For this purpose, we used an experimental paradigm consisting in applying two inflammatory stimuli three months apart. Wild type, toll-like receptor (TLR) 4 and TLR2 mutant strains were used. The priming stimulus was the intracerebroventricular injection of neuraminidase (an enzyme that is present in various pathogens able to provoke brain infections), which triggers an acute inflammatory process in the brain. The second stimulus was the intraperitoneal injection of lipopolysaccharide (a TLR4 ligand) or Pam3CSK4 (a TLR2 ligand). One day after the second inflammatory challenge the immune response in the brain was examined. In wild type mice, microglial and astroglial density, as well as the expression of 4 out of 5 pro-inflammatory genes studied (TNFα, IL1β, Gal-3, and NLRP3), were increased in mice that received the double stimulus compared to those exposed only to the second one, which were initially injected with saline instead of neuraminidase. Such enhanced response suggests immune training in the brain, which lasts at least 3 months. On the other hand, TLR2 mutants under the same experimental design displayed an enhanced immune response quite similar to that of wild type mice. However, in TLR4 mutant mice the response after the second immune challenge was largely dampened, indicating the pivotal role of this receptor in the establishment of immune priming. Our results demonstrate that neuraminidase-induced inflammation primes an enhanced immune response in the brain to a subsequent immune challenge, immune training that endures and that is largely dependent on TLR4 receptor.
Endocannabinoid signalling participates in the control of neurogenesis, especially after brain insults. Obesity may explain alterations in physiology affecting neurogenesis, although it is unclear ...whether cannabinoid signalling may modulate neural proliferation in obese animals. Here we analyse the impact of obesity by using two approaches, a high‐fat diet (HFD, 60% fat) and a standard/low‐fat diet (STD, 10% fat), and the response to a subchronic treatment with the cannabinoid receptor type 1 (CB1) inverse agonist AM251 (3 mg/kg) on cell proliferation of two relevant neurogenic regions, namely the subventricular zone in the striatal wall of the lateral ventricle (SVZ) and the subgranular zone of the dentate gyrus (SGZ), and also in the hypothalamus given its role in energy metabolism. We found evidence of an interaction between diet‐induced obesity and CB1 signalling in the regulation of cell proliferation. AM251 reduced caloric intake and body weight in obese rats, as well as corrected plasma levels of cholesterol and triglycerides. AM251 is shown, for the first time, to modulate cell proliferation in HFD‐obese rats only. We observed an increase in the number of 5‐bromo‐2‐deoxyuridine‐labelled (BrdU+) cells in the SGZ, but a decrease in the number of BrdU+ cells in the SVZ and the hypothalamus of AM251‐treated HFD rats. These BrdU+ cells expressed the neuron‐specific βIII‐tubulin. These results suggest that obesity may impact cell proliferation in the brain selectively, and provide support for a role of CB1 signalling regulation of neurogenesis in response to obesity.
There is evidence that viral infections during pre-natal development constitute a risk factor for neuropsychiatric disorders and lead to learning and memory deficits. However, little is known about ...why viral infections during early post-natal development have a different impact on learning and memory depending on the sex of the subject. We previously showed that early post-natal immune activation induces hippocampal-dependent social memory deficits in a male, but not in a female, mouse model of tuberous sclerosis complex (TSC;
mice). Here, we explored the impact of a viral-like immune challenge in object memory. We demonstrate that early post-natal immune activation (during the first 2 weeks of life) leads to object memory deficits in female, but not male, mice that are heterozygous for a gene responsible for tuberous sclerosis complex (
mice), while no effect was observed in wild type (WT) mice. Moreover, we found that the same immune activation in
adult mice was not able to cause object memory deficits in females, which suggests that the early post-natal development stage constitutes a critical window for the effects of immune challenge on adult memory. Also, our results suggest that mTOR plays a critical role in the observed deficit in object memory in female
mice. These results, together with previous results published by our laboratory, showing sex-specific memory deficits due to early post-natal immune activation, reinforce the necessity of using both males and females for research studies. This is especially true for studies related to immune activation, since the higher levels of estrogens in females are known to affect inflammation and to provide neuroprotection.
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Changes in endogenous cannabinoid homeostasis are associated with both ethanol-related neuroinflammation and memory decline. Extensive research is still required to unveil the role of ...endocannabinoid signaling activation on hippocampal microglial cells after ethanol exposure. Either microglial morphology, phenotype and recruitment may become notably altered after chronic alcohol-related neurodegeneration. Here, we evaluated the pharmacological effects of fatty-acid amide-hydrolase (FAAH) inhibitor URB597 (0.3 mg/kg), oleoylethanolamide (OEA, 10 mg/kg), arachidonoylethanolamide (AEA, 10 mg/kg), the CB1 receptor agonist ACEA (3 mg/kg) and the CB2 receptor agonist JWH133 (0.2 mg/kg) administered for 5 days in a rat model of subchronic (2 weeks) ethanol diet (11% v/v) exposure. URB597 turned to be the most effective treatment. URB597 increased microglial (IBA-1+) cell population, and changed morphometric features (cell area and perimeter, roughness, fractal dimension, lacunarity) associated with activated microglia in the hippocampus of ethanol-exposed rats. Regarding innate immune activity, URB597 specifically increased mRNA levels of toll-like receptor 4 (TLR4), glial fibrillary acidic protein (Gfap) and the chemokine stromal cell-derived factor 1 (SDF-1α/CXCL12), and elevated the cell population expressing the chemokine receptors CX3CR1, CCR2 and CCR4 in the ethanol-exposed rat hippocampus. Contrary to ethanol effect, URB597 reduced mRNA levels of Iba-1, Tnfα, IL-6 and the monocyte chemoattractant protein-1 (MCP-1/CCL2), as well as cell population expressing iNOS. URB597 effects on hippocampal immune system were accompanied by changes in short and long-term visual recognition memory. These results suggest that FAAH inhibition may modulates hippocampal microglial recruitment and activation that can be associated with improved hippocampal-dependent memory despite ethanol exposure.
In the rat brain, a single intracerebroventricular injection of neuraminidase from Clostridium perfringens induces ependymal detachment and death. This injury occurs before the infiltration of ...inflammatory blood cells; some reports implicate the complement system as a cause of these injuries. Here, we set out to test the role of complement.
The assembly of the complement membrane attack complex on the ependymal epithelium of rats injected with neuraminidase was analyzed by immunohistochemistry. Complement activation, triggered by neuraminidase, and the participation of different activation pathways were analyzed by Western blot. In vitro studies used primary cultures of ependymal cells and explants of the septal ventricular wall. In these models, ependymal cells were exposed to neuraminidase in the presence or absence of complement, and their viability was assessed by observing beating of cilia or by trypan blue staining. The role of complement in ependymal damage induced by neuraminidase was analyzed in vivo in two rat models of complement blockade: systemic inhibition of C5 by using a function blocking antibody and testing in C6-deficient rats.
The complement membrane attack complex immunolocalized on the ependymal surface in rats injected intracerebroventricularly with neuraminidase. C3 activation fragments were found in serum and cerebrospinal fluid of rats treated with neuraminidase, suggesting that neuraminidase itself activates complement. In ventricular wall explants and isolated ependymal cells, treatment with neuraminidase alone induced ependymal cell death; however, the addition of complement caused increased cell death and disorganization of the ependymal epithelium. In rats treated with anti-C5 and in C6-deficient rats, intracerebroventricular injection of neuraminidase provoked reduced ependymal alterations compared to non-treated or control rats. Immunohistochemistry confirmed the absence of membrane attack complex on the ependymal surfaces of neuraminidase-exposed rats treated with anti-C5 or deficient in C6.
These results demonstrate that the complement system contributes to ependymal damage and death caused by neuraminidase. However, neuraminidase alone can induce moderate ependymal damage without the aid of complement.
is an emerging yeast of worldwide interest due to its antifungal resistance and mortality rates. The aim of this study was to analyse the in vitro and in vivo antifungal activity of a nanoemulsion ...loaded with amphotericin B (NEA) against planktonic cells and biofilm of
clinical isolates belonging to four different clades. In vivo assays were performed using the
model to analyse antifungal activity and histopathological changes. The in vitro results showed that NEA exhibited better antifungal activity than free amphotericin B (AmB) in both planktonic and sessile cells, with >31% inhibition of mature biofilm. In the in vivo assays, NEA demonstrated superior antifungal activity in both haemolymph and tissue. NEA reduced the fungal load in the haemolymph more rapidly and with more activity in the first 24 h after infection. The histological analysis of infected larvae revealed clusters of yeast, immune cells, melanisation, and granulomas. In conclusion, NEA significantly improved the in vitro and in vivo antifungal activity of AmB and could be considered a promising therapy for
infections.
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