Chronic Hyperhomocysteinemia Increases Inflammatory Markers in Hippocampus and Serum of Rats

This study investigated the effects of chronic homocysteine administration on some parameters of inflammation, such as cytokines (TNF-α, IL-1β and IL-6), chemokine CCL(2) (MCP-1), nitrite and prostaglandin E(2) levels, as well as on immunocontent of NF-κB/p65 subunit in hippocampus and/or serum of rats. Since acetylcholinesterase has been associated with inflammation, we also evaluated the effect of homocysteine on this enzyme activity in hippocampus of rats. Wistar rats received daily subcutaneous injections of homocysteine (0.3-0.6 μmol/g body weight) or saline (control) from the 6th to the 28th days-of-age. One or 12 h after the last injection, rats were euthanized and hippocampus and serum were used. Results showed that chronic hyperhomocysteinemia significantly increased pro-inflammatory cytokines (TNF-α, IL-1β and IL-6), chemokine CCL(2) (MCP-1) and prostaglandin E(2) in hippocampus and serum of rats at 1 and 12 h after the last injection of homocysteine. Nitrite levels increased in hippocampus, but decreased in serum at 1 h after chronic hyperhomocysteinemia. Acetylcholinesterase activity and immunocontent of citoplasmic and nuclear NF-κB/p65 subunit were increased in hippocampus of rats subjected to hyperhomocysteinemia at 1 h, but did not alter at 12 h after the last injection of homocysteine. According to our results, chronic hyperhomocysteinemia increases inflammatory parameters, suggesting that this process might be associated, at least in part, with the cerebrovascular and vascular dysfunctions characteristic of some homocystinuric patients.     

Mitochondrial respiratory chain and creatine kinase activities in rat brain after sepsis induced by cecal ligation and perforation

The mechanisms responsible to the development of brain dysfunction during sepsis are not well understood. The objective of this study is to evaluate mitochondrial respiratory chain and creatine kinase activities in the brain after cecal ligation and perforation (CLP) in rats. We performed a prospective, controlled experiment in male Wistar rats. Rats were subjected to CLP (sepsis group) with saline resuscitation (at 50mL/kg immediately and 12h after cecal ligation and perforation) or sham operation (control group). Several times (0, 6, 12, 24, 48 and 96h) after CLP six rats were killed by decapitation, and brain structures (cerebellum, hippocampus, striatum and cortex) were isolated. Mitochondrial respiratory chain and creatine kinase activity were then measured. It was observed that animals submitted to CLP presented decreased mitochondrial respiratory chain activity in complex I, but not in complex II, III and IV, 24, 48 and 96h in all analyzed structures. Activity of succinate dehydrogenase was decreased in 48 and 96h in all analyzed structures. Creatine kinase activity increased after CLP in cerebellum, hippocampus and cortex (after 0h) and striatum (after 6h). Sepsis associated brain injury may include dysfunction in the mitochondrial respiratory chain activity.     

Il1-β Involvement in Cognitive Impairment after Sepsis

Sepsis is defined as the host's reaction to infection and characterised by a systemic inflammatory response with important clinical implications. Central nervous system dysfunction secondary to sepsis is associated with local generation of pro- and anti-inflammatory cytokines, impaired cerebral microcirculation, an imbalance of neurotransmitters, apoptosis and cognitive impairment. It's known that the IL-1β is one of the first cytokines to be altered. Thus, the objective of this study was to evaluate the role of IL-1β in cognitive parameters in brain tissue through the use of an IL-1β (IL-1ra) receptor antagonist up to 10 days and to assess blood–brain barrier permeability, cytokine levels, oxidative parameters and energetic metabolism up to 24 h, after sepsis induction. To this aim, we used sham-operated Wistar rats or submitted to the cecal ligation and perforation (CLP) procedure. Immediately after, the animals received one dose of 10 μg of IL-1ra. After 24 h, the rats were killed and were evaluated for biochemical parameters in the pre-frontal cortex, hippocampus and striatum. After 10 days, the animals were submitted to the habituation to the open field and step-down inhibitory avoidance task. We observed that the use of IL-1ra reverted the increase of blood–brain barrier permeability in the pre-frontal cortex, hippocampus and striatum; the increase of IL-1β, IL1-6 and TNF-α levels in the pre-frontal cortex and striatum; the decrease of complex I activity in the pre-frontal, hippocampus and striatum; the increase of oxidative parameters in pre-frontal cortex, hippocampus and striatum; and cognitive impairment. In conclusion, the results observed in this study reinforce the role of acute brain inflammatory response, in particular, the IL1β response, in the cognitive impairment associated with sepsis.     

The time course of inflammatory cytokine secretion in a rat model of postoperative pain does not coincide with the onset of mechanical hyperalgesia

We characterized the time course of inflammatory cytokine release at the site of injury and in plasma after surgery on the rat tail. Anesthetized Sprague-Dawley rats had a 20 mm long incision made through the skin and fascia of their tails. Control rats were anesthetized, but no incision was made. Blood and tissue samples were taken 2 h and 1, 2, 4, and 8 days after surgery and analysed by ELISA for interleukin-1beta (IL-1beta), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), and cytokine-induced neutrophil chemoattractant-1 (CINC-1). In another group of rats, daily behavioral measurements were made of the rats' responses to a blunt noxious mechanical stimulus (4 Newtons) applied to their tails. Primary hyperalgesia developed within 2 h of surgery and lasted for 6 days. The tissue concentrations of IL-1beta, IL-6, and CINC-1 increased within 24 h of surgery, and TNF-alpha concentration increased within 48 h of surgery. Thereafter, cytokine concentrations remained elevated for 4 (IL-1beta and IL-6) to 8 days (CINC-1, TNF-alpha) after surgery. Control animals did not develop hyperalgesia and no changes in cytokines concentrations were detected. Thus, in our model of postoperative pain, secretion of inflammatory cytokines IL-1beta, IL-6, TNF-alpha, and CINC-1 was not essential for the initiation of postoperative hyperalgesia.     

Blood-brain barrier tight junctions are altered during a 72-h exposure to lambda-carrageenan-induced inflammatory pain

In this study, we examined the effect of lambda-carrageenan-induced inflammatory pain on the functional and structural properties of the rat blood-brain barrier (BBB) over a 72-h time period. Systemic inflammation was induced by an intraplantar injection of 3% lambda-carrageenan into the right hind paw of female Sprague-Dawley rats. In situ brain perfusion and Western blot analyses were performed at 1, 3, 6, 12, 24, 48, and 72 h. In situ brain perfusion showed lambda-carrageenan significantly increased brain uptake of [(14)C]sucrose at 1, 3, 6, and 48 h (139 +/- 9%, 166 +/- 19%, 138 +/- 13%, and 146 +/- 7% compared with control, respectively). Capillary depletion analysis insured the increased brain uptake was due to increased BBB permeability and not vascular trapping. Western blot analyses for zonula occludens-1 (ZO-1) and occludin were performed on isolated cerebral microvessels. ZO-1 expression was significantly increased at 1, 3, and 6 h and returned to control expression levels by 12 h. Total occludin expression was significantly reduced at 1, 3, 6, 12, and 48 h. This investigation demonstrated that lambda-carrageenan-induced inflammatory pain elicits a biphasic increase in BBB permeability with the first phase occurring from 1-6 h and the second phase occuring at 48 h. Furthermore, changes in BBB function are correlated with altered tight junctional protein expression of occludin and ZO-1. Changes in the structure of tight junctions may have important clinical ramifications concerning central nervous system homeostasis and therapeutic drug delivery.     

Acetylcholinesterase activity in the rat brain after pneumococcal meningitis

Pneumococcal meningitis is a life-threatening disease characterized by acute purulent infection of the meninges causing neuronal injury, cortical necrosis and hippocampal apoptosis. Cholinergic neurons and their projections are extensively distributed throughout the central nervous system. The aim of this study was to assess acetylcholinesterase activity in the rat brain after pneumococcal meningitis. In the hippocampus, frontal cortex and cerebrospinal fluid, acetylcholinesterase activity was found to be increased at 6, 12, 24, 48 and 96 hr without antibiotic treatment, and at 48 and 96 hr with antibiotic treatment. Our data suggest that acetylcholinesterase activity could be related to neuronal damage induced by pneumococcal meningitis.     

Altered chemokine expression in the spinal cord and brain contributes to differential interleukin-1beta-induced neutrophil recruitment

The pattern of neutrophil recruitment that accompanies inflammation in the CNS depends on the site of injury and the stage of development. The adult brain parenchyma is refractory to neutrophil recruitment and associated damage as compared to the spinal cord or juvenile brain. Using quantitative Taqman RT-PCR and enzyme-liked immunosorbent assay (ELISA), we compared mRNA and protein expression of the rat neutrophil chemoattractant chemokines (CINC) in spinal cord and brain of adult and juvenile rats to identify possible association with the observed differences in neutrophil recruitment. Interleukin-1beta (IL-1beta) injection resulted in up-regulated chemokine expression in both brain and spinal cord. CINC-3 mRNA was elevated above CINC-1 and CINC-2alpha, with expression levels for each higher in spinal cord than in brain. By ELISA, IL-1beta induced greater CINC-1 and CINC-2alpha expression compared to CINC-3, with higher protein levels in spinal cord than in brain. In the juvenile brain, significantly higher levels of CINC-2alpha protein were observed in response to IL-1beta injection than in the adult brain following an equivalent challenge. Correspondingly, neutrophil recruitment was observed in the juvenile brain and adult spinal cord, but not in the adult brain. No expression of CINC-2beta mRNA was detected. Thus differential chemokine induction may contribute to variations in neutrophil recruitment in during development and between the different CNS compartments.     

Marker enzyme assessment in the liver of cyprinus carpio (L.) exposed to 2,4-D and azinphosmethyl

The potential utility of antioxidant enzymes and lipid peroxidation as indicators of exposure to 2,4-D and azinphosmethyl together with the toxic effects of these compounds in freshwater fish Cyprinus carpio were evaluated. Biochemical parameters were recorded spectrophotometrically in fish liver, which were exposed to a single dose of 2,4-D and azinphosmehtyl (1/3 LC(50)), and their mixture at 1:1 ratio for 24, 48, 72, and 96 h. The most sensitive parameter was glutathione S-transferase (GST) activity, which significantly increased with experimental exposures. Glucose 6-phosphate dehydrogenase activity did not change after 24 and 48 h while there was an elevation after 72 h in all exposure groups. The activity decreased only when these were applied in combination at 96 h. Superoxide dismutase activity increased after azinphosmethyl exposure for 48 and 96 h. 2,4-D decreased the activity after 24 h while the activity remained at the same level with control after 48 h. An elevation was found between 72 and 96 h. Mixture treatment did not changed the activity. Glutathione reductase and catalase enzyme activities, and malondialdehyde levels remained constant in all the treatment groups compared with controls. These results suggest that induction of GST activity may be used as biomarker for the assessment of water pollution in C. carpio.     

Blood-brain barrier dysfunctions following systemic injection of kainic acid in the rat.

Changes in blood-brain barrier (BBB) permeability and cerebral metabolic activity following intravenous injection of kainic acid (KA; 6, 12 mg/Kg) in rats were assessed by calculating respectively a blood-to-brain transfer constant (Ki) for [14C]alpha-aminoisobutyric acid and local cerebral glucose utilization (LCGU) values, at different times (1 h, or acute seizures phase, and 48 h, or chronic pathology phase) after the induction of seizures. A significant increase in the local permeability of the BBB was observed 1 h after the injection of KA 6 mg/Kg (eliciting no significant changes in cerebral metabolic activity, except within the frontal cortex and the hippocampus) and 12 mg/Kg (which induced a marked and widespread enhancement of LCGU). On the contrary, during the pathology phase, persistent regional increases in Ki values were evidenced in rats treated with the lowest dose of the convulsant, but not in rats injected with KA 12 mg/Kg (a dose able to cause extensive neuronal damage). Thus one can speculate that: 1) KA-induced regional changes in the permeability of the BBB are not correlated with changes in neuronal activity; 2) opening of the BBB is not reliably associated with neuronal injury.     

连续观察兔油酸ARDS模型稳定性及血L-1、IL-8近期变化

目的：探讨油酸（OA）构建兔急性呼吸窘迫综合征（ARDS）模型3天内的稳定性及血浆炎症因子IL-1、IL-8含量变化的意义。方法：健康新西兰大耳白兔30只随机分为5组,每组6只,实验组（n=24）耳缘静脉注射油酸（0.1mL/kg）建立ARDS模型,对照组（n=6）注射等量生理盐水。分别检测对照组6h和实验组6h、24h、48h、72h（n=6）动脉血PH值、PaO2、PaO2/FiO2、PaCO2、肺湿/干重比值（W/D）,ELISA法检测血浆细胞因子IL-1、IL-8含量,HE染色观察肺组织病理学改变。结果：和对照组比较,实验组血中细胞因子IL-1在6h、24h组升高;细胞因子IL-8在6h组、24h组、48h组升高。6h组、24h组、48h组氧合指数〈200mmHg。结论：兔油酸ARDS模型48h内稳定,炎性细胞和IL-1、IL-8可能是导致ARDS的发生和发展主要原因之一。     

PA-MSHA引起注射局部急性炎症及白细胞介素17、肿瘤坏死因子α和Toll样受体4的表达改变

为研究皮下注射铜绿假单胞菌甘露糖敏感血凝菌毛株(Pseudomonas aeruginosa mannose sensitive hemagglutinin,PA-MSHA)注射液后局部组织的病理学改变及白细胞介素17(interleukin 17,IL-17)、肿瘤坏死因子α(tumor necrosis factorα,TNF-α)和Toll样受体4(Toll-like receptor 4,TLR4)表达情况,将30只小鼠随机分为两组,分别皮下注射PA-MSHA和生理盐水,在注射后6、12、24、48及72h取注射局部组织,苏木精-伊红(hematoxylin-eosin,HE)染色后观察病理形态改变,并用免疫组化染色和图像分析检测IL-17、TNF-α和TLR4表达水平。结果显示,小鼠皮下注射PA-MSHA后,在给药部位引起以中性粒细胞浸润为主的急性炎症;局部炎症灶中IL-17、TNF-α和TLR4表达在注射后6h升高,24h达到高峰,然后逐渐减低,至72h实验组与对照组无差异;3种炎症因子的表达与炎症反应程度相平行。结果表明,皮下注射PA-MSHA能引起局部组织急性炎症反应,且能改变局部免疫状态。     

N-Acetylcysteine and Selenium Modulate Oxidative Stress,Antioxidant Vitamin and Cytokine Values in Traumatic Brain Injury-Induced Rats

It has been suggested that oxidative stress plays an important role in the pathophysiology of traumatic brain injury (TBI). N-acetylcysteine (NAC) and selenium (Se) display neuroprotective activities mediated at least in part by their antioxidant and anti-inflammatory properties although there is no report on oxidative stress, antioxidant vitamin, interleukin-1 beta (IL)-1β and IL-4 levels in brain and blood of TBI-induced rats. We investigated effects of NAC and Se administration on physical injury-induced brain toxicity in rats. Thirty-six male Sprague–Dawley rats were equally divided into four groups. First and second groups were used as control and TBI groups, respectively. NAC and Se were administrated to rats constituting third and forth groups at 1, 24, 48 and 72 h after TBI induction, respectively. At the end of 72 h, plasma, erythrocytes and brain cortex samples were taken. TBI resulted in significant increase in brain cortex, erythrocytes and plasma lipid peroxidation, total oxidant status (TOS) in brain cortex, and plasma IL-1β values although brain cortex vitamin A, β-carotene, vitamin C, vitamin E, reduced glutathione (GSH) and total antioxidant status (TAS) values, and plasma vitamin E concentrations, plasma IL-4 level and brain cortex and erythrocyte glutathione peroxidase (GSH-Px) activities decreased by TBI. The lipid peroxidation and IL-1β values were decreased by NAC and Se treatments. Plasma IL-4, brain cortex GSH, TAS, vitamin C and vitamin E values were increased by NAC and Se treatments although the brain cortex vitamin A and erythrocyte GSH-Px values were increased through NAC only. In conclusion, NAC and Se caused protective effects on the TBI-induced oxidative brain injury and interleukin production by inhibiting free radical production, regulation of cytokine-dependent processes and supporting antioxidant redox system.     

The influence of interleukin-1beta on gamma-glutamyl transpepidase activity in rat hippocampus

Brain infections as well as peripheral challenges to the immune system lead to an increased production of interleukin-1beta (IL-1beta), a cytokine involved in leukocyte-mediated breakdown of the blood-brain barrier. The effects of IL-1beta have been reported to depend on whether the route of administration is systemic or intracerebral. Using 50-day-old male rats, we compared the effects of IL-1beta on brain gamma-glutamyl transpeptidase (GGT; an enzymatic marker of brain capillary endothelium) at 2, 24 and 96 h after either an intravenous (i.v.) injection of 5 microg IL-1beta or an intracerebroventricular (i.c.v. - lateral ventricle) infusion of 50 ng IL-1beta. When the i.v. route was used, the GGT activity underwent small but significant changes; decreasing in the hippocampus 2 h after the i.v. injection, increasing 24 h later and returning to control levels at 96 h. No significant changes in the hippocampal GGT activity were observed at 2 and 24 h following the i.c.v. infusion. The GGT activity in the hypothalamus remained unchanged regardless of the route of IL-1beta administrations. Similar changes in GGT activity were revealed histochemically. The labeling was found mainly in the capillary bed, the changes being most evident in the hippocampal stratum radiatum and stratum lacunosum-moleculare. A transient increase in GGT activity at 24 h, together with a less sharp delineation of GGT-stained vessels, may reflect IL-1beta induced increased turnover of glutathione and/or oxidative stress, that may in turn, be related to altered permeability of the blood-brain barrier in some neurological and mental disorders, including schizophrenia.     

Time-course of changes in inflammatory response after whole-body cryotherapy multi exposures following severe exercise

The objectives of the present investigation was to analyze the effect of two different recovery modalities on classical markers of exercise-induced muscle damage (EIMD) and inflammation obtained after a simulated trail running race. Endurance trained males (n = 11) completed two experimental trials separated by 1 month in a randomized crossover design; one trial involved passive recovery (PAS), the other a specific whole body cryotherapy (WBC) for 96 h post-exercise (repeated each day). For each trial, subjects performed a 48 min running treadmill exercise followed by PAS or WBC. The Interleukin (IL) -1 (IL-1), IL-6, IL-10, tumor necrosis factor alpha (TNF-α), protein C-reactive (CRP) and white blood cells count were measured at rest, immediately post-exercise, and at 24, 48, 72, 96 h in post-exercise recovery. A significant time effect was observed to characterize an inflammatory state (Pre vs. Post) following the exercise bout in all conditions (p<0.05). Indeed, IL-1β (Post 1 h) and CRP (Post 24 h) levels decreased and IL-1ra (Post 1 h) increased following WBC when compared to PAS. In WBC condition (p<0.05), TNF-α, IL-10 and IL-6 remain unchanged compared to PAS condition. Overall, the results indicated that the WBC was effective in reducing the inflammatory process. These results may be explained by vasoconstriction at muscular level, and both the decrease in cytokines activity pro-inflammatory, and increase in cytokines anti-inflammatory.     

Cellular Localization and Temporal Elevation of Tumor Necrosis Factor-α, Interleukin-1α, and Transforming Growth Factor-β1 mRNA in Hippocampal Injury Response Induced by Trimethyltin

Abstract: In certain pathologic states, cytokine production may become spatially and temporally dysregulated, leading to their inappropriate production and potentially detrimental consequences. Tumor necrosis factor-α (TNF-α), interleukin (IL)-1, IL-6, and transforming growth factor-β (TGF-β) mediate a range of host responses affecting multiple cell types. To study the role of cytokines in the early stages of brain injury, we examined alterations in the 17-day-old mouse hippocampus during trimethyltin-induced neurodegeneration characterized by neuronal necrosis, microglia activation in the dentate, and astrocyte reactivity throughout the hippocampus. By 24 h after dosing, elevations in mRNA levels for TNF-α, IL-1α, IL-1β, and IL-6 mRNA were seen. TGF-β1 mRNA was elevated at 72 h. In situ hybridization showed that TNF-α and IL-1α were localized to the microglia, whereas TGF-β1 was expressed predominantly in hippocampal pyramidal cells. Intercellular adhesion molecule-1, EB-22, Mac-1, and glial fibrillary acidic protein mRNA levels were elevated within the first 3 days of exposure in the absence of increased inducible nitric oxide synthetase and interferon-γ mRNA. These data suggest that pro-inflammatory cytokines contribute to the progression and pattern of neuronal degeneration in the hippocampus.     

Longitudinal changes in zinc transport kinetics, metallothionein and zinc transporter expression in a blood-brain barrier model in response to a moderately excessive zinc environment

A blood-brain barrier (BBB) model composed of porcine brain capillary endothelial cells (BCEC) was exposed to a moderately excessive zinc environment (50 micromol/L Zn) in cell culture, and longitudinal measurements were made of zinc transport kinetics, ZnT-1 (SLC30A1) expression and changes in the protein concentration of metallothionein (MT), ZnT-1, ZnT-2 (SLC30A2) and Zip1 (SLC39A1). Zinc release by cells of the BBB model significantly increased after 12-24 h of exposure, but decreased back to control levels after 48-96 h, as indicated by transport across the BBB from both the ablumenal (brain) and the lumenal (blood) directions. Expression of ZnT-1, the zinc export protein, increased by 169% within 12 h, but was no longer different from controls after 24 h. Likewise, ZnT-1 protein content increased transiently after 12 h of exposure, but returned to control levels by 24 h. Capacity for zinc uptake and retention increased from both the lumenal and the ablumenal directions within 12-24 h of exposure and remained elevated. MT and ZnT-2 were elevated within 12 h and remained elevated throughout the study. Zip1 was unchanged by the treatment. The BBB's response to a moderately high zinc environment was dynamic and involved multiple mechanisms. The initial response was to increase the cells' capacity to sequester zinc with additional MT and to increase zinc export with the ZnT-1 protein. But the longer-term strategy involved increasing ZnT-2 transporters, presumably to sequester zinc into intracellular vesicles as a mechanism to protect the brain and to maintain brain zinc homeostasis.