Injection Of Endotoxin In Rats Research Articles

Validation of a septic myocardial inhibition model by intraperitoneal injection of endotoxin in rats

To establish septic myocardial inhibition rat model by echocardiography. Twenty adult male Sprague-Dawley (SD) rats were divided into control group and model group according to the random number table method, with 10 rats in each group. The rat model of septic myocardial inhibition was reproduced by intraperitoneal injection of 10 mg/kg lipopolysaccharide, while the control group was given the same volume of saline. The left ventricular end-diastolic diameter (LVDd), left ventricular end-diastolic volume (LVEDV), left ventricular end-systolic diameter (LVDs), left ventricular end-systolic volume (LVESV), left ventricular ejection fraction (LVEF), right ventricular end-diastolic diameter (RVDd), right ventricular end-systolic diameter (RVDs), heart rate (HR), positive pulmonary artery flow rate and aortic flow rate were measured at 8 hours after model establishment by echocardiography. Then the rats were sacrificed to harvest serum and myocardial tissue. The levels of serum tumor necrosis factor-α (TNF-α), nuclear factor-ΚB (NF-ΚB), interleukin-1 (IL-1), cardiac troponin I (cTnI) and B-type brain natriuretic peptide (BNP) were measured by enzyme linked immunosorbent assay (ELISA). The mRNA expressions of TNF-α, IL-1 and NF-ΚB in myocardium were detected by real-time polymerase chain reaction (real-time PCR). The pathological changes of myocardium were observed by hematoxylin-eosin (HE) staining under light microscope. Compared with control group, myocardial inhibition was obviously observed in model group, manifesting as enlargement of overall shape of heart, and prominent increase of HR (bpm: 449.0±21.1 vs. 356.7±23.3, P < 0.01); left ventricular and right ventricular functions were affected, LVDd, LVDs, LVEDV, LVESV were enlarged [LVDd (mm): 10.03±0.95 vs. 7.04±0.71, LVDs (mm): 5.95±0.71 vs. 3.07±0.05, LVEDV (mL): 2.11±0.53 vs. 0.81±0.21, LVESV (mL): 0.51±0.16 vs. 0.07±0.01, all P < 0.05], LVEF was significantly decreased (0.760±0.046 vs. 0.901±0.025, P < 0.01), RVDd was significantly increased (mm: 4.48±0.58 vs. 3.22±0.20, P < 0.05), and positive pulmonary artery velocity was significantly decreased (cm/s: 64.2±9.3 vs. 89.0±0.8, P < 0.05). Compared with control group, the levels of serum NF-ΚB, TNF-α, IL-1, BNP and cTnI in model group were significantly increased [NF-ΚB (ng/L): 103.84±6.55 vs. 57.29±41.34, TNF-α (ng/L): 1 198.32±164.07 vs. 835.45±24.01, IL-1 (ng/L): 1 089.90±221.96 vs. 746.19±165.83, BNP (ng/L): 1 097.36±293.84 vs. 454.71±197.79, cTnI (ng/L): 6 938.59±1 400.21 vs. 3 731.90±1 349.31, all P < 0.01], the mRNA expressions of TNF-α, NF-ΚB and IL-1 in myocardial tissue were significantly increased (2-ΔΔCT: 1.50±0.42 vs. 0.71±0.40, 1.10±0.17 vs. 0.63±0.06, 1.77±0.67 vs. 0.10±0.03, all P < 0.05). It was shown by HE staining that the structure of myocardial tissue in control group was distinct, the arrangement of myocardial fibers was neat, and transverse was clear; the structure of myocardial tissue in model group was loose, blurred, and the cells were swollen, with obvious pathological changes. Cardiac function was assessed by echocardiography, expression of inflammatory factors, myocardial markers and pathological changes. It was verified that intraperitoneal injection of 10 mg/kg endotoxin could successfully prepare a rat model of septic myocardial inhibition.

A single intraperitoneal injection of endotoxin in rats induces long-lasting modifications in behavior and brain protein levels of TNF-α and IL-18

BackgroundSystemic inflammation might cause neuronal damage and sustain neurodegenerative diseases and behavior impairment, with the participation of pro-inflammatory cytokines, like tumor necrosis factor (TNF)-α and interleukin (IL)-18. However, the potential contribution of these cytokines to behavioral impairment in the long-term period has not been fully investigated.MethodsWistar rats were treated with a single intraperitoneal injection of LPS (5 mg/kg) or vehicle. After 7 days and 10 months, the animal behavior was evaluated by testing specific cognitive functions, as mnesic, discriminative, and attentional functions, as well as anxiety levels. Contextually, TNF-α and IL-18 protein levels were measured by ELISA in defined brain regions (that is, frontal cortex, hippocampus, striatum, cerebellum, and hypothalamus).ResultsBehavioral testing demonstrated a specific and persistent cognitive impairment characterized by marked deficits in reacting to environment modifications, possibly linked to reduced motivational or attentional deficits. Concomitantly, LPS induced a TNF-α increase in the hippocampus and frontal cortex (from 7 days onward) and cerebellum (only at 10 months). Interestingly, LPS treatment enhanced IL-18 expression in these same areas only at 10 months after injection.ConclusionsOverall, these results indicate that the chronic neuroinflammatory network elicited by systemic inflammation involves a persistent participation of TNF-α accompanied by a differently regulated contribution of IL-18. This leads to speculation that, though with still unclear mechanisms, both cytokines might take part in long-lasting modifications of brain functions, including behavioral alteration.

Mesenteric Lymph Duct Ligation Attenuates Lung Injury and Neutrophil Activation After Intraperitoneal Injection of Endotoxin in Rats

The release of injurious factors into the mesenteric lymph from the ischemic intestine has been shown to contribute to lung injury and systemic inflammation after shock and trauma. Since endotoxemia is also associated with gut injury, we tested the hypothesis that mesenteric lymph contributes to the lung injury seen in endotoxemia and that the ligation of the mesenteric lymph duct will attenuate this injury. To test this hypothesis, male Sprague-Dawley rats were given intraperitoneal injections (i.p.) of lipopolysaccharide (LPS) (10 mg/kg) with or without mesenteric lymph duct ligation (LDL). At 6 hours after injection of LPS, gut and lung injury, lung permeability, and neutrophil CD11b expression were measured. Lung permeability was quantified by calculating the percentage of Evan's Blue dye and the total protein concentration in the bronchoalveolar lavage fluid (BALF) when compared with the plasma and gut and lung injury were assessed morphologically. LDL attenuated LPS- induced lung injury, lung permeability, and rat PMN CD11b expression but not villous injury. The magnitude of lung permeability as measured by Evan's Blue was approximately twofold greater in the LPS rats when compared with the LPS-treated rats with LDL. The expression of CD11b was greater in the LPS rats when compared with LPS rats with LDL or to sham controls (582 +/- 106 vs. 364 +/- 29 vs. 224 +/- 12 mean fluorescence intensity p < 0.001). Based on the attenuation of lung injury and CD11b expression, these results suggest that LPS-induced lung injury and neutrophil activation is partially mediated through the release of factors from the injured gut into mesenteric lymph.

GH insensitivity induced by endotoxin injection is associated with decreased liver GH receptors.

Sepsis induces a state of growth hormone (GH) resistance associated with a decrease of circulating insulin-like growth factor (IGF) I, a GH-dependent anabolic hormone mainly produced by the liver. To address the mechanisms that might trigger GH insensitivity in sepsis, we investigated the regulation of liver GH receptor (GHR) and its gene expression by endotoxin. Endotoxin injection in rats decreased serum IGF-I and liver GH-binding sites after 10 h. In contrast to liver GHR, circulating GH-binding protein (GHBP) levels were not significantly reduced after endotoxin injection. The parallel decrease in IGF-I and GHR and in their corresponding liver mRNAs suggests that decreased serum IGF-I and liver GHR were likely to result from decreased liver synthesis. Although GH administration in control animals significantly enhanced serum IGF-I, it did fail to prevent the decline in serum IGF-I and liver GH-binding sites in endotoxemic rats. In this study, we showed that endotoxin injection induces a state of GH insensitivity associated with decreased liver GHR. This decline in GHR, which cannot be prevented by exogenous GH, might contribute to the GH insensitivity observed in sepsis.

In vivo antioxidant treatment suppresses nuclear factor-kappa B activation and neutrophilic lung inflammation.

We hypothesized that endotoxin injection in rats would stimulate in vivo nuclear factor-kappa B (NF-kappa B) activation in lung tissue and that antioxidant treatment before endotoxin injection would attenuate endotoxin-induced NF-kappa B activation, chemokine gene expression, and neutrophilic lung inflammation. We studied NF-kappa B activation in rat lung tissue following a single i.p. injection of endotoxin (6 mg/kg). After in vivo endotoxin treatment, lung NF-kappa B activation peaked at 2 h and temporally correlated with the expression of cytokine-induced neutrophil chemoattractant mRNA in lung tissue. Treatment with the antioxidant N-acetylcysteine (NAC) 1 h before endotoxin resulted in decreased lung NF-kappa B activation in a dose-dependent manner (from 200-1000 mg/kg) and diminished cytokine-induced neutrophil chemoattractant mRNA expression in lung tissue. Treatment with NAC significantly suppressed endotoxin-induced neutrophilic alveolitis. The average total lung lavage neutrophil count was 5.5 x 10(6) with endotoxin treatment vs 0.9 x 10(6) with NAC treatment before endotoxin. The NF-kappa B pathway represents an attractive therapeutic target for strategies to control neutrophilic inflammation and lung injury.

Induction mechanism of small intestinal lesions caused by intravenous injection of endotoxin in rats.

The pathogenesis of intestinal damage caused by bolus intravenous injection of endotoxin (ETX; 3 mg/kg) was investigated. Administration of ETX to rats induced reddish discoloration suggestive of bleeding, increased hemoglobin amounts, and leakage of plasma protein in the intestine. However, light microscopic examination of the intestine demonstrated blood congestion of the microvessels. Plasma accumulation was partially inhibited by combined pretreatment with a histamine H1 antagonist and a serotonin (5-HT) antagonist. Neither a 5-lipoxygenase inhibitor, a soybean trypsin inhibitor, nor atropine was observed to inhibit plasma accumulation. Both the intestinal leakage of plasma and the accumulation of hemoglobin were completely inhibited by indomethacin, a selective thromboxane A synthetase inhibitor (OKY 1581), and a stable PGI2 analogue (beraprost). Intravital microscopic observation of the microvessels of the small intestinal villi demonstrated microthrombus formation within several minutes after the injection of ETX, and pretreatment with OKY 1581 attenuated the formation of microthrombus. Platelet counts decreased significantly 10 min after ETX administration, and the decrease was not inhibited by pretreatment with either OKY 1581 or beraprost. Prothrombin time (PT) and activated partial thromboplastin time (APTT) were not prolonged. These observations thus suggest that microcirculatory disturbances by platelet thrombus, which are mediated by thromboxane A2 at least in part, play an important role in ETX-induced intestinal damage.

Thalidomide Inhibits TNF Response and Increases Survival Following Endotoxin Injection in Rats

Sepsis is a leading cause of death following major trauma and complicated abdominal surgery. Tumor necrosis factor-alpha (TNF) is believed to be a central mediator in the inflammatory response syndrome. Numerous methods of blunting the TNF response in sepsis have been attempted with suggestion of increased survival and decreased organ injury. Thalidomide, shown in vitro to selectively inhibit TNF production, has been used clinically in states of chronic TNF elevation with encouraging results. In this study, we examined the effect of thalidomide administration in a rat model of acute septic shock. Femoral artery cannulation was performed and baseline TNF measured. Dose response was determined by giving varying doses of thalidomide by gavage. Rats were injected intraarterially with endotoxin and serial samples drawn. TNF was measured by ELISA. For survival, thalidomide was given by gavage and endotoxin injected intraperitoneally. Serum TNF elevation occurred after endotoxin injection with peak levels at 90 min. Thalidomide treated rats had lower TNF levels at all time points (P = <0.01 at 90 and 120 min), with the inhibition being dose dependent. Survival in treated rats exceeded that of untreated rats (53% vs 19%, P = <0.05) at 48 and 72 hr. In conclusion, we found that thalidomide administration leads to increased survival following acute endotoxemia, which may be due to the observed TNF inhibition.

Endotoxin stimulates the expression of group II PLA2 in rat lung in vivo and in isolated perfused lungs.

Injection of endotoxin in vivo leads to increased phospholipase A2 (PLA2) activity in the lung, but neither the type(s) of PLA2 involved nor the importance of blood components and/or different inflammatory cytokines has been clarified. In the present study, injection of endotoxin in rats caused increased lung levels of group II PLA2, tumor necrosis factor (TNF)-alpha, and interleukin (IL)-1 beta mRNA, while group I PLA2 mRNA levels were unaffected. The augmented group II PLA2 mRNA levels corresponded to a rise in membrane-associated PLA2 enzymatic activity that was inhibited by rutin, an inhibitor of group II PLA2. In blood-free, salt-perfused lungs, addition of endotoxin to the perfusate caused elevated group II PLA2, TNF-alpha, and IL-1 beta mRNA levels and release of PLA2 and TNF-alpha activity into the perfusate, and when instilled intratracheally, endotoxin caused increased PLA2 activity in the lung tissue. It is concluded that 1) endotoxin stimulates group II PLA2, but not group I PLA2, in rat lung cells, 2) this stimulation is accompanied by increased expression of TNF-alpha and IL-1 beta, and 3) endotoxin-induced PLA2 activation and cytokine production in the lung are not dependent on circulating blood components.

Partial hepatectomy reduces the endotoxin-induced peak circulating level of tumor necrosis factor in rats.

The pro-inflammatory cytokine tumor necrosis factor (TNF) is dramatically and transiently elevated in the circulation during endotoxic and septic shock and is a primary mediator in the pathogenesis of the sepsis syndrome. TNF peaks in the circulation 90 min after endotoxin administration with little variation, even among species. The specific cells, tissues, or organs that produce the high circulating level of TNF in septic shock remain unknown. The most likely sources are macrophage-laden tissues such as the liver and the spleen and circulating blood leukocytes. This study evaluated whether the liver is an important source producing the TNF spike 90 min after endotoxin. To test this hypothesis, we measured the peak circulating level of TNF following an endotoxin injection in rats subjected to a two-thirds hepatectomy versus sham-operated controls. Hepatechectomized rats produced 64% less TNF after endotoxin than controls (857 + or - 143 pg/mL plasma vs. 2410 + or - 491, respectively; p < .01). In contrast, splenectomy did not significantly after peak TNF levels versus sham-operated controls following an endotoxin injection (1380 + or - 148 pg/mL plasma vs. 1710 + or - 291). Furthermore, incubation of rat blood with endotoxin for 90 min did not significantly increase TNF above controls. These experiments demonstrate an important role for the liver in producing the high circulating levels of TNF after an endotoxin injection and suggest that hepatic-specific cytokine modulation deserves study for a therapeutic benefit in septic shock.

Thymulin reduces hyperalgesia induced by peripheral endotoxin injection in rats and mice

In a new model of peripheral localized inflammation, induced by intraplantar endotoxin (1.25 micrograms) injection in the hind paw of rats and mice, thymulin, a hormone of the thymus gland involved in immunomodulation, reduced inflammatory pain. High doses of thymulin reduced significantly, and in a dose-dependent manner, mechanical hyperalgesia as assessed by the paw pressure test and thermal hyperalgesia as assessed by the hot plate test and tail immersion test.

Aerosolized and instilled surfactant therapies for acute lung injury caused by intratracheal endotoxin in rats

To compare the effects of surfactant replacement by aerosol inhalation and bolus instillation on acute lung injury caused by the intratracheal injection of endotoxin in rats. Prospective, randomized study. University Laboratory. Male Wistar rats weighing 368 +/- 31 (SD) g. Escherichia coli endotoxin (57 +/- 20 mg/kg) was injected into the tracheas of 36 anesthetized and mechanically ventilated rats (FIO of 1.0). When the PaO2 had decreased to <200 torr (<26.7 kPa), the rats were randomly assigned to one of three groups: a control group (n=12)given no material; a bolus group (n=12) given a modified natural surfactant suspension (100 mg/kg in 2.0 mL/kg saline) by bolus instillation into the trachea; and an aerosol group (n=12) given surfactant aerosolized with an ultra-sonic nebulizer for 60 mins. Bolus instillation transiently decreased the mean blood pressure by approximately 30%. However, aerosol inhalation did not. The PaO2 values of the control group remained <90 torr (<12.0 kPa) until the end of the experiment (180 mins). In contrast, the PaO2 of the bolus group increased to 387 +/- 134 torr (51.6 +/- 17.9 kPa; p<.05 vs. other groups) 15 mins after surfactant replacement, and remained at approximately 400 torr (approximately 53.3 kPa) throughout the experiment. The PaO2 values of the aerosol group increased slowly, peaked at 240 +/- 109 torr (32.0 +/- 14.5 kPa; p<.05 vs. the control group) 60 mins after the start of surfactant replacement, and remained at approximately 200 torr (approximately 26.7 kPa). Bolus instillation was superior to aerosol inhalation concerning maximum efficacy, the rapid onset of therapeutic effects, and the necessary dose of surfactant. However, aerosol that does not cause hypotension may be of use in the treatment of adult respiratory distress syndrome in patients with circulatory instability.

Surfactant replacement reverses respiratory failure induced by intratracheal endotoxin in rats

To evaluate the effect of surfactant replacement on respiratory failure induced by intratracheal injection of endotoxin in rats. Prospective, randomized study. Laboratory at a large university. Male Wistar rats, weighing 353 +/- 50 (SD) g. Escherichia coli endotoxin (53 +/- 19 mg/kg) was injected into the trachea of 32 rats anesthetized with pentobarbital and mechanically ventilated with an FIO2 of 1.0. After PaO2 decreased to < 200 torr (< 26.7 kPa), the rats were assigned to three groups: a) a surfactant group (n = 16), given a modified natural surfactant suspension (100 mg/kg in 2.0 mL/kg of saline) by instillation into the airway; b) a saline control group (n = 8), given 2.0 mL/kg of saline; and c) an air control group (n = 8), given 2.0 mL/kg of air. An additional nine rats were ventilated in the same way but were not given endotoxin. Among the rats receiving endotoxin, the PaO2 of the saline and air control groups remained < 200 torr (< 26.7 kPa), while PaO2 of the surfactant group increased to 390 +/- 116 torr (52.0 +/- 15.5 kPa; p < .05 vs. the preassignment value) 15 mins after the assignment. These high levels were maintained throughout the experiment. Surfactant replacement also led to significant improvements in the PaCO2, the dynamic lung-thorax compliance, the pressure-volume recordings of the lung, and the chest roentgenograms. Histologic examination showed that the alveoli of the surfactant group were better aerated than the alveoli of the control groups. Findings in the rats not given endotoxin were almost normal, indicating that the influences of mechanical ventilation were negligible. Surfactant replacement reversed respiratory failure induced by intratracheal injection of endotoxin in rats.

Fine structure and detoxification kinetics in kupffer cells after injection of endotoxin in rats

The aim of this study was to clarify the role of Kupffer cells in the mechanism of endotoxin-induced liver injury. The study on fine structure of Kupffer cells was performed after the injection of endotoxin. The endotoxin(Escherichia coli lipopolysaccharide 026 : B6. 1.5mg/100 g of body weight) was intraperitoneally injected in Sprague-Dewley rats. Animals were sacrificed at 1/4, 1/2, 1, 2, 4, 8, 16, 24, 72 and 120 hours after the injection of endotoxin. Livers were extirpated and processed to be examined by light and electron microscopy. The results obtained were summerized as follows: Early changes observed in liver after endotoxin injection included the increased number and hypertrophy of Kupffer cells, infiltration of neutrophils and presence of fibrin thrombi within the sinusoids. The continuous increase of the Kupffer cells in number with hypertrophy, congestion and infiltration of inflammatory cells within the sinusoids were observed. Hepatocytes showed fatty change and occasional necrosis. At 72 hours the congestion decreased. At 120 hours the number of Kupffer cells was increased, but the morphology of Kupffer cells became similar to that of the control group. The numbers and sizes of primary and secondary lysosomes and amount of euchromatin of Kupffer cells increased. Swellings and increase in number of mitochondria, Golgi complex, smooth endoplasmic reticulum, rough endoplasmic reticulum were evident. Microthrombi were present within the sinusoids. The swelling of rough endoplasmic reticulum and mitochondria, decrease of glycogen particles, fatty change, hypoxic vacuoles, pyknotic nuclei and occasional necrosis were observed in hepatocytes. At 72 hours the number of secondary lysosomes in Kupffer cells decreased. At 120 hours the morphology of Kupffer cells became similar to that of the control group. According to these results, it was postulated that the endotoxin was initially taken up by pinocytosis into Kupffer cells and degraded in secondary lysosomes of activated Kupffer cells. Kupffer cells may play an important role in the defense mechanism of liver during endotoxemia. The dysfunction of Kupffer cells and ischemia by sinusoidal microthrombi may cause liver injury.

Effect of activated protein C on impaired fibrinolysis in rats with obstructive jaundice.

We studied the effect of activated protein C (APC) on impaired fibrinolysis using a rat model in which disseminated intravascular coagulation (DIC) is induced by the intravenous injection of endotoxin in rats with obstructive jaundice. An intravenous injection of endotoxin in rats with obstructive jaundice resulted in pulmonary hemorrhages and a marked increase in the plasma levels of tissue-type plasminogen activator (t-PA) antigen and plasminogen activator inhibitor activity. Prophylaxis with APC before the injection of endotoxin resulted in a decrease of the number of lung hemorrhages and an accelerated release of t-PA antigen. Thus, DIC in obstructive jaundice may be due to impairment of fibrinolysis and an increased susceptibility of endothelial cells to endotoxin. APC may be effective as a treatment for patients with obstructive jaundice associated with DIC.

P-397 - Multiple responses after intraperitoneal injection of endotoxin in rats.

P-397 - Multiple responses after intraperitoneal injection of endotoxin in rats.

Gold treatment for kala-azar.

We hypothesized that endotoxin injection in rats would stimulate in vivo nuclear factor-kappa B (NF-kappa B) activation in lung tissue and that antioxidant treatment before endotoxin injection would attenuate endotoxin-induced NF-kappa B activation, chemokine gene expression, and neutrophilic lung inflammation. We studied NF-kappa B activation in rat lung tissue following a single i.p. injection of endotoxin (6 mg/kg). After in vivo endotoxin treatment, lung NF-kappa B activation peaked at 2 h and temporally correlated with the expression of cytokine-induced neutrophil chemoattractant mRNA in lung tissue. Treatment with the antioxidant N-acetylcysteine (NAC) 1 h before endotoxin resulted in decreased lung NF-kappa B activation in a dose-dependent manner (from 200-1000 mg/kg) and diminished cytokine-induced neutrophil chemoattractant mRNA expression in lung tissue. Treatment with NAC significantly suppressed endotoxin-induced neutrophilic alveolitis. The average total lung lavage neutrophil count was 5.5 x 10(6) with endotoxin treatment vs 0.9 x 10(6) with NAC treatment before endotoxin. The NF-kappa B pathway represents an attractive therapeutic target for strategies to control neutrophilic inflammation and lung injury.

IRON TRANSPORT AFTER INJECTION OF ENDOTOXIN IN RATS.

The injection of Escherichia coli lipopolysaccharide produces a rapid decline in plasma iron concentration of normal rats. To understand the mechanism of this decrease, combinations of acetylphenylhydrazine and endotoxins were given and measurements of rates of iron transfer from Fe59-labeled damaged red blood cells to the plasma were made. Shortly after giving endotoxin there was an inhibition of the movement of iron from catabolized red blood cells to the plasma transferrin. This inhibition can be partially overcome by increasing the number of erythrocytes which are phagocytized in a given period of time. The major reason for the decrease in plasma iron after an injection of endotoxin was found to be an inhibited reutilization of iron from recently destroyed erythrocytes.