Injection Of Low-dose Lipopolysaccharide Research Articles

Plasminogen Activator Inhibitor-1 Mitigates Brain Injury in a Rat Model of Infection-Sensitized Neonatal Hypoxia-Ischemia

Intrauterine infection exacerbates neonatal hypoxic-ischemic (HI) brain injury and impairs the development of cerebral cortex. Here we used low-dose lipopolysaccharide (LPS) pre-exposure followed by unilateral cerebral HI insult in 7-day-old rats to study the pathogenic mechanisms. We found that LPS pre-exposure blocked the HI-induced proteolytic activity of tissue-type plasminogen activator (tPA), but significantly enhanced NF-κB signaling, microglia activation, and the production of pro-inflammatory cytokines in newborn brains. Remarkably, these pathogenic responses were all blocked by intracerebroventricular injection of a stable-mutant form of plasminogen activator protein-1 called CPAI. Similarly, LPS pre-exposure amplified, while CPAI therapy mitigated HI-induced blood-brain-barrier damage and the brain tissue loss with a therapeutic window at 4 h after the LPS/HI insult. The CPAI also blocks microglia activation following a brain injection of LPS, which requires the contribution by tPA, but not the urinary-type plasminogen activator (uPA), as shown by experiments in tPA-null and uPA-null mice. These results implicate the nonproteolytic tPA activity in LPS/HI-induced brain damage and microglia activation. Finally, the CPAI treatment protects near-normal motor and white matter development despite neonatal LPS/HI insult. Together, because CPAI blocks both proteolytic and nonproteolytic tPA neurotoxicity, it is a promising therapeutics of neonatal HI injury either with or without infection.

Long-term effect of intraventricular injection of low-dose lipopolysaccharide on behavior, microglias and dopaminergic neurons in the substantia nigra of rats

Objective To investigate the long-term effect of inflammation on behavior,microglias and dopaminergic （DA） neurons in the substantia nigra of intracephalic inflammation rat models induced by intracerebroventricular injection of low-dose（10μg） lipopolysaccharide （LPS）. To analyze the relationship between activa- tion of microglias and DA neurons degeneration in order to explore the mechanism of inflammation in the progres- sive process of Parkinson＇s disease （PD）. Methods 50 healthy male SD rats were randomly assigned into sa- line-injected control group and 10μg LPS-injected group. All injections were made intracerebroventricularly on right side of rats with saline or LPS. Moving speed was measured at different time points. At 24 weeks and 40 weeks after saline or LPS injection, specific antibodies of OX-42 and OX-6 were used separately to detect the changes of microglia in the substantia nigra of rat. The changes in morphology and numbers of substantia nigra DA neurons were observed by tyrosine hydroxylase（TH） immunohistochemical staining. The expression and distribution of the degenerated neurons in substantia nigra were detected by using Fluoro-Jade B （FJB）. Results （1）Analysis of moving speed showed that the moving speed of 10μg LPS-injected group rats and saline-injected group rats was similar from 4 weeks to 36 weeks after injection. At 40 weeks post injection, moving speed of 10μg LPS-injected group rats decreased by 24.6% compared with that of saline-injected group rats （P〉 0.05 ）. （2）At 24 weeks and 40 weeks after injection,there were many activated 0X-42 positive microglias in the substantia nigra of 10μg LPS-in- jected group rats, but there was almost no significant activated OX-42 positive microglia in saline-injected group. OX-6 positive microglias were not found in the substantia nigra of both of two groups. （1）At 24 weeks and 40 weeks post injection, the number of TH-positive neurons in the substantia nigra of 10μg LPS-iniected rout3 rats decreasedby 24.2% （ t=4.803, P〈0.01 ） and 27.6% （ t= 3. 212, P〈0.01 ） respectively compared with those of salineinjected group. （4） There was no FJB positive neurons in the suhstantia nigra of the two group rats. Conclusion Intraventricular injection of low-dose LPS （10μg） in rats may induce long-term activation of microglias and chronic degeneration of DA neurons in the substantia nigra of rats although the necrosis are not occurs to DA neurons till 40 weeks post LPS injection. Intravenlricular injection of low-dose LPS in rats could he ideal model to study the mechanism of chronic degeneration of DA neurons in PD. Key words: Parkinson＇ s disease ; Inflammation ; Behaviour ; Microglia ; Dopaminergic neurons

Proinflammatory cytokines cause down-regulation of renal chloride entry pathways during sepsis*

Sepsis is the most important trigger for acute renal failure, with tubular dysfunction and collapse in urine concentration. As chloride plays a major role in the urinary concentrating mechanisms, we aimed to investigate the regulation of renal chloride entry pathways, such as kidney-specific chloride channel 1, kidney-specific chloride channel 2, Barttin, thiazide-sensitive Na+-Cl- cotransporter, renal outer medullary potassium channel, and Na+/K+-adenosine triphosphatase during sepsis. Prospective animal trial. Laboratory of the Department of Anesthesiology. Male C57/BL6 and B6129SF2/J mice and mice deficient for tumor necrosis factor-alpha, interleukin-1-receptor-1, interferon-gamma, or interleukin-6. Mice were injected with lipopolysaccharide (LPS) or proinflammatory cytokines. Hemodynamic and renal variables, cytokine concentrations, and expression of renal chloride-reabsorbing systems were investigated. Experiments with cytokine knockout mice, renal artery-clipped mice, and mice treated with glucocorticoids, low-dose LPS, and sodium nitroprusside were performed. LPS-injected mice presented with decreased blood pressure and glomerular filtration rate, increased fractional chloride excretion, and depressed expression of renal chloride transporters/channels. Similar alterations were observed after application of tumor necrosis factor-alpha, interleukin-1beta, interferon-gamma, or interleukin-6. LPS-induced down-regulation of chloride transporters/channels was not affected in cytokine knockout mice. Glucocorticoid treatment inhibited LPS-induced increase of cytokine concentrations, diminished LPS-induced renal dysfunction, and attenuated the down-regulation of renal chloride transporters/channels. Injection of low-dose LPS increased renal tissue cytokines and down-regulated chloride entry pathways without arterial hypotension, indicating that renal ischemia due to systemic hypotension does not mediate down-regulation of renal chloride transporters/channels. In addition, renal ischemia induced by renal artery clipping or sodium nitroprusside administration did not influence chloride transporter/channel expression. Our results demonstrate down-regulation of renal chloride transporters/channels during sepsis, which is probably mediated by proinflammatory cytokines and accounts for the development of LPS-induced tubular dysfunction. Our findings contribute to the understanding, on one hand, the failure of single-anticytokine strategies and, on the other hand, the beneficial effects of glucocorticoids in the therapy of septic patients.

Pro-inflammatory cytokine and acute phase protein responses to low-dose lipopolysaccharide (LPS) challenge in pigs

AbstractThe objective of this study was to establish the pro-inflammatory cytokine and acute phase protein responses to low-dose lipopolysaccharide (LPS) challenge in pigs and to determine whether these immune parameters could also be measured in saliva. Possible gender differences in the acute phase reaction were also assessed. At 6 weeks of age, 24 male and 24 female pigs were injected intraperitoneally with a single dose of 0 or 5 μg/kg live weight (LW) of LPS fromEscherichia coli(treatment). Matched saliva and blood samples were taken at 0, 2, 4, 8, 12 or 24 h after treatment administration. Samples were analysed for concentrations of the pro-inflammatory cytokines tumor necrosis factor-alpha (TNF-α) and interleukin-1beta (IL-1β), the acute phase proteins C-reactive protein (CRP), serum amyloid A (SAA), haptoglobin (Hp), and cortisol. Low-dose LPS administration increased plasma levels of TNF-α (P<0·001), CRP (P<0·05) and SAA (P<0·05) but did not affect plasma concentrations of IL-1β or Hp (P>0·1). Treatment by time interactions showed that plasma levels of TNF-α and CRP in LPS-treated pigs peaked at 2 h (P<0·001) and 12 h (P<0·01), respectively. Low-dose LPS injection tended to increase plasma concentrations of cortisol (P=0·056) and the response to LPS differed between genders (P<0·05), with females showing higher cortisol responsiveness to the challenge (P<0·01). Males showed higher levels of both cytokines regardless of the treatment (P<0·05), probably due to the inhibition of cytokine synthesis by cortisol. Concentrations of both pro-inflammatory cytokines were consistently detectable in saliva and were present in higher concentrations than in plasma (P<0·001). Hence, plasma TNF-α, CRP and SAA are useful indicators of sub-acute inflammation/infection in pigs as simulated by a low-dose LPS challenge and gender differences exist in the pro-inflammatory cytokine response after a low dose of LPS.

Multiple bioimaging modalities in evaluation of an experimental osteonecrosis induced by a combination of lipopolysaccharide and methylprednisolone

Aims: The present study employed both static and dynamic imaging modalities to study both intra- and extravascular events attributing to steroid-associated osteonecrosis (ON) using an experimental protocol with a single low-dose lipopolysaccharide (LPS) injection and subsequently three injections of high-dose methylprednisolone (MPS). Methods: Fourteen 28-week-old male New Zealand white rabbits received one intravenous injection of LPS (10 μg/kg). 24 h later, three injections of 20 mg/kg of MPS were given intramuscularly at a time interval of 24 h. Additional 6 rabbits were used as controls. Dynamic MRI was performed on bilateral femora for local intraosseous perfusion before and after LPS injection. Blood samples were collected for hematological examinations before and after LPS injection. Bilateral femora were dissected and decalcified for microCT-based microangiography. ON lesion, intravascular thrombus and extravascular marrow fat cell size were examined histopathologically. Results: Intravascular thrombus was observed in all ON rabbits. Extravascular marrow fat cell size was significantly increased in ON rabbits than that of the controls ( P < 0.05). Compared to baseline, a significant decrease in ratio of tissue-type plasminogen activator/plasminogen activator inhibitor 1, activated partial thromboplatin time and a significant increase in ratio of low-density lipoprotein/high-density lipoprotein were only found in ON rabbits ( P < 0.05). Dynamic MRI showed a significant decrease in the perfusion index ‘maximum enhancement’ in the ON rabbits ( P < 0.05), and microCT-based microangiography showed blocked stem vessels in ON samples. Overall, 93% of the rabbits (13/14) developed ON, and no rabbits died throughout the experiment period. Conclusion: Both intra- and extravascular events were found attributing to the steroid-associated ON based on our experimental protocol with a single low-dose LPS injection and subsequent three injections of high-dose MPS. Both high ON incidence and no mortality in rabbits treated with this inductive protocol suggested its effectiveness for future studies on evaluation of therapeutic efficacy of interventions developed for prevention of steroid-associated ON.

Improved survival following induction of GVHD following lipopolysaccharide immunization

Graft-vs-host disease (GVHD) is still the primary limitation to the wider application of allogeneic bone marrow transplantation (BMT). On the one hand, it predisposes transplant recipients to risk of bacterial, fungal, and viral infections, on the other, lipopolysaccharide (LPS), an endotoxin found in the cell walls of gram-negative bacteria, has been shown to play a significant role in the development and severity of GVHD following allogeneic myeloablative BMT. Our study focused on immunization of recipient and donor mice with endotoxin prior to transplantation, in an attempt to reduce mortality caused by gram-negative bacterial infections posttransplantation. In one experiment, recipient mice were immunized with LPS prior to BMT, whereas in another experiment, donor mice were immunized prior to BMT. The mice were evaluated for development of GVHD and for survival. Our results showed that injection of low-dose LPS to mice prior to induction of GVHD with allogeneic spleen cells saved more than 40% of the recipients, whereas all mice in the untreated control group died. The survival of recipients of spleen cells from immunized donors rose to 54% and clinical signs of GVHD were attenuated as compared to control mice inoculated with spleen cells obtained from unimmunized donors. This immunization protocol suggests that immunization of the donor or the recipient against LPS prior to transplantation may be protective against gram-negative bacteria.

Osteonecrosis induced by a single administration of low-dose lipopolysaccharide in rabbits

We succeeded in developing a novel rabbit model of nonsteroid and nontraumatic osteonecrosis (ON) by use of a single- and low-dose lipopolysaccharide (LPS) injection. This model is simple and highly reproducible for the frequent development of multifocal and widespread ON lesions. Male adult Japanese white rabbits intravenously injected with a single injection of 10 μg/kg body weight of LPS were histopathologically examined in the early phase (3 [n = 3], 5 [n = 3], and 24 h [n = 3]) and at 4 weeks (n = 22). Seventy-seven percent of the rabbits developed multifocal ON 4 weeks after LPS injection. ON was also observed in the femoral and humeral condyle. The average percentage of necrotic area/total area examined was 86.7 ± 29.1% and 78.8 ± 16.7% in the proximal one third of both the femoral and humeral bones, respectively. Organized thrombi in the intraosseous small-sized arteries and arterioles were frequently seen in and around the necrotic tissues. In the early phase, LPS treatment prominently induced thrombocytopenia, hyperlipidemia, and increased plasma levels of plasminogen activator inhibitor-1 (PAI-1). The plasma level of PAI-1 was significantly higher in the rabbits with ON than in those without ON ( p < 0.01). The immunohistochemical expression of tissue factor was exaggerated in monocytes/macrophages and adipocytes in both the femoral and humeral bones of the LPS-treated rabbits. Histologically, marrow necrosis and fibrin thrombi could be observed at 24 h. In addition, pretreatment with an anticoagulant, warfarin potassium, significantly decreased the incidence of LPS-induced ON (33%, n = 9, p < 0.05) associated with elongation of prothrombin time. The results of our study show that a single administration of low-dose lipopolysaccharide induces multifocal and widespread ON characterized by the pathophysiological participation of hypercoagulability in ON development. Therefore, this model would be useful for elucidating the pathogenesis of nonsteroid ON in humans especially inflammatory hypercoagulability-induced as well as for developing preventive and therapeutic strategies.

Electron paramagnetic resonance investigations of nitrosyl complex formation during endotoxin tolerance

The prior administration of low dose endotoxin induces a state of hyporesponsiveness or tolerance to the lethal effects of endotoxin. It is generally accepted that macrophages are main cellular components in the development of tolerance, hence, nitric oxide ( ∗NO) as one of the macrophage mediators may play a role in host defense mechanisms during tolerance. In this study, we utilized EPR spectroscopy to directly detect nitrosyl complexes as products of ∗NO in whole blood, livers and intestines of lipopolysaccharide (LPS)-tolerant rats. Male Sprague-Dawley rats were injected with a “low dose” LPS (0.5 mg/kg) 12–168 h prior to a “high dose” LPS (3 mg/kg), then sacrificed 6 h later. EPR signals of nitrosyl hemoprotein complexes were detected in specimens after high dose LPS. The post-LPS EPR signals of nitrosyl complexes from all samples were attenuated by a prior injection of low dose LPS. The signals of dinitrosyl-iron-dithiolate became apparent in samples from tolerant rats as signals of nitrosyl hemoprotein decreased. The maximal tolerance in terms of diminished ∗NO production was observed when low dose LPS was given 48–96 h prior to high dose LPS. Hemoglobin concentrations in the intestine used as biomarkers of hemorrhagic damage, were concomitantly attenuated in the jejunum of tolerant rats. These results together with our previous studies indicate that suppression of ∗NO production may contribute to the amelioration of hepatic and intestinal injury during endotoxin tolerance.

Acute endotoxin tolerance downregulates superoxide anion release by the perfused liver and isolated hepatic nonparenchymal cells

This work is based on the hypothesis that low-dose lipopolysaccharide (LPS) suppresses the stimulatory and priming effects of a subsequent high-dose endotoxin on the formation of toxic oxygen-derived radicals by the perfused liver and isolated hepatic nonparenchymal cells. Such effects may in turn contribute to hyposensitivity to the lethal effect of large doses of endotoxin. Male Sprague-Dawley rats received a nonlethal ("low-dose") intravenous injection of Escherichia coli LPS (0.5 mg/kg body weight) 12 to 120 hours before they were challenged by a "large dose" of endotoxin (10 mg/kg). Three hours after LPS challenge, the livers were perfused, and superoxide release was determined. Nonparenchymal cells were also isolated for the determination of superoxide anion formation in vitro. There was a low rate (0.14 +/- 0.1 nmol/min/g liver weight) of superoxide generated by the perfused livers from rats that received the low-dose LPS 1 to 5 days previously. Control livers generated less than 0.08 nmol superoxide. A high rate (1.3 +/- 0.1 nmol/min/g) of superoxide release was measured in the perfused liver 4 hours after treatment of previously untreated control rats with large-dose LPS. This was attenuated to 0.7 +/- 0.04 nmol/min/g by an injection of low-dose LPS before challenge. This attenuation was time dependent; it failed to manifest at 12, 24, or 120 hours after low-dose LPS. Isolated endothelial cells, Kupffer cells, and sequestered hepatic neutrophils from rats given a high-dose LPS also generated significant amounts of superoxide both in the presence or absence of agonists, i.e., phorbol myristate acetate or opsonized zymosan.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute endotoxin tolerance downregulates superoxide anion release by the perfused liver and isolated hepatic nonparenchymal cells

This work is based on the hypothesis that low-dose lipopolysaccharide (LPS) suppresses the stimulatory and priming effects of a subsequent high-dose endotoxin on the formation of toxic oxygen-derived radicals by the perfused liver and isolated hepatic nonparenchymal cells. Such effects may in turn contribute to hyposensitivity to the lethal effect of large doses of endotoxin. Male Sprague-Dawley rats received a nonlethal (“low-dose”) intravenous injection of Escherichia coli LPS (0.5 mg/kg body weight) 12 to 120 hours before they were challenged by a “large dose” of endotoxin (10 mg/kg). Three hours after LPS challenge, the livers were perfused, and superoxide release was determined. Nonparenchymal cells were also isolated for the determination of superoxide anion formation in vitro. There was a low rate (0.14 ± 0.1 nmol/min/g liver weight) of superoxide generated by the perfused livers from rats that received the low-dose LPS 1 to 5 days previously. Control livers generated less than 0.08 nmol superoxide. A high rate (1.3 ± 0.1 nmol/min/g) of superoxide release was measured in the perfused liver 4 hours after treatment of previously untreated control rats with large-dose LPS. This was attenuated to 0.7 ± 0.04 nmol/min/g by an injection of low-dose LPS before challenge. This attenuation was time dependent; it failed to manifest at 12, 24, or 120 hours after low-dose LPS. Isolated endothelial cells, Kupffer cells, and sequestered hepatic neutrophils from rats given a high-dose LPS also generated significant amounts of superoxide both in the presence or absence of agonists, i.e., phorbol myristate acetate or opsonized zymosan. Pretreatment of rats with the low dose of LPS 48 hours before the large dose attenuated the spontaneous and primed release of superoxide by isolated nonparenchymal cells. Serum transaminase activities were also reduced in these LPS-tolerant rats. The suppressed oxygen-derived radical formation by the liver and nonparenchymal cells during acute LPS tolerance was associated with down-regulation of tumor necrosis factor (TNF) release in vivo. TNF has been shown to stimulate and prime neutrophils and Kupffer cells for increased respiratory burst. These results suggest that suppression of free radical formation may contribute to the amelioration of hepatic injury during acute endotoxin tolerance.