• Toxicological Research
• /
• v.26 no.3
• /
• pp.203-208
• /
• 2010

Bronchoalveolar lavage (BAL) is a useful tool in researches and in clinical medicine of lung diseases because the BAL fluid contains biochemical and cytological indicators of the cellular response to infection, drugs, or toxicants. However, the variability among laboratories regarding the technique and the processing of the BAL material limits clinical research. The aim of this study was to determine the suction frequency and lavage fraction number necessary to reduce the variability in lavage using male Sprague-Dawley rats. We compared the total cell number and protein level of each lavage fraction and concluded that more cells and protein can be obtained by repetitive lavage with a suction frequency of 2 or 3 than by lavage with a single suction. On the basis of total cell recovery, approximately 70% of cells were obtained from fractions 1~3. The first lavage fraction should be used for evaluation of protein concentration because fractions 2~5 of lavage fluid were diluted in manifolds. These observations were confirmed in bleomycin-induced inflamed lungs of rats. We further compared the BAL data from the whole lobes with data from the right lobes and concluded that BAL data of the right lobes represented data of the whole lobes. However, this conclusion can only be applied to general lung diseases. At the end, this study provides an insight into the technical or analytical problems of lavage study in vivo.

• Toxicological Research
• /
• v.18 no.2
• /
• pp.159-165
• /
• 2002

Recently, we reported (korean J. Biomed. Lab. Sci., 6(4): 245-251, 2000) that cyclohexane (l.56 g/kg of body wt., i.p.) administration led to lung injury in rats. However the detailed mechanism remain to be elucidated. This study was designed to clarify the mechanism of lung damage induced by cyclohexane in rats. First, lung damage was assessed by quantifying bronchoalveolar lavage fluid (BAL) protein content as well us by histopathological examination. Second, activities of serum xanthine oxidase (XO), pulmonary XO and oxygen free radical scavenging enzymes. XO tope conversion (O/D + O, %) ratio and content of reduced glutathione (GSH) were determined. In the histopathological findings, the vasodilation, local edema and hemorrhage were demonstrated in alveoli of lung. And vascular lumens filled with lipid droplets, increased macrophages in luminal margin and increased fibroblast-like interstitial cells in interstitial space were observed in electron micrographs. The introperitoneal treatment of cyclohexane dramatically increased BAL protein by 21-fold compared with control. Cyclohexane administration to rats led to a significant rise of serum and pulmonary XO activities and O/D + O ratio by 47%,30% and 24%, respectively, compared witれ control. Furthermore, activities of pulmonary oxygen free radical scavenging enzymes such as superoxide dismutase, glutathione peroxidase and glutathione S-transferase, and GSH content were not found to be statistically different between control and cyclohexane-treated rats. These results indicate that intraperitoneal injection of cyclohexane to rats may induce the lipid embolism in pulmonary blood vessel and lead to the hypoxia with the ensuing of oxygen free radical generation, and which may be responsible for the pulmonary injury.

• The Journal of Pediatrics of Korean Medicine
• /
• v.20 no.1
• /
• pp.181-194
• /
• 2006

Objective : Author evaluate the effects of Citri Reticulatae Viride Pericarpium for the bronchial asthma using assesment of the vascular endothelial growth factor after Citri Reticulatae Viride Peicarpium was intravenously administered Ovalbumine(OVA)-sensitized and -challenged mice. Material and Methods : Twenty-four female mice, 8-10 weeks old and free of murine specific pathogens, were sensitized by intraperitoneal injection of OVA. Of the sensitized mice, ten mice didn't administrate Citri Reticulatae Viride Pericarpium and the other ten mice administrated Citri Reticulatae Viride Pericarpium. Mice were sensitized on the first and fourteen days introspectional injection of 20 ${\mu}g$ OVA. After$21^{st}\;22^{th}\;and\;23^{rd}$, the initial sensitization, the mice were challenged for 30 minutes with an aerosol of 1% OVA in saline. Citri Reticulatae Viride Pericarpium administered 200mg/kg in the tail of the mouse, one a day, for 7 days and beginning 14 days after first sensitization. Bronchoalveolar lavage was performed 72 hours after the last challenge, and total cell numbers in the BAL fluid were count. Also, lever of VEGF in the BAL fluid was measured by Enzyme immunoassays and Western blot analysis. Results: Total cell numbers in BAL fluid were significantly greater than from 72 hrs after OVA inhalation compared with cell numbers in the control group. However, there was no difference of the total cell numbers between OVA-challenge groups without Citri Reticulatae Viride Pericarpium, and OVA-challenge with Citri Reticulatae Viride Pericarpium. Enzyme immunoassay revealed that VEGF levels in the BAL fluids were significantly increased 72 hrs after OVA inhalation compared with levels in the control group. After administration of the Citri Reticulatae Viride Pericarpiu, the levels of the VEGF in BAL fluids 72 hrs after OVA inhalation reduced dramatically. Western blot analysis revealed that VEGF protein levels were increased in the all mice which were challenge with OVA, without administered Citri Reticulatae Viride Pericarpium, compared the normal mouse. However, in the groups of the administered Citri Reticulatae Viride Pericarpium, the VEGF protein level markedly decreased. Conclusion: Citri Reticulatae Viride Pericarpium might affect the treatment of the bronchial ashma as a inhibition of the VEGF.

• Proceedings of the Korean Society of Potoscience Conference
• /
• spring
• /
• pp.26-27
• /
• 1996

• The Korean Journal of Physiology and Pharmacology
• /
• v.13 no.3
• /
• pp.181-187
• /
• 2009

Intestinal ischemia/reperfusion (I/R) is one of common causes of acute lung injury (ALI). Early and accurate diagnosis of patients who are like to develop serious acute respiratory distress syndrome (ARDS) would give a therapeutic advantage. Ferritin and heme oxygenase-1 (HO-1) are increased by oxidative stress and are potential candidates as a predictive biomarker of ARDS. However, the mechanisms responsible for the increases of ferritin and HO-1, and their relationship to ALI, are unclear. In order to elucidate the interactions between ferritin and HO-1, we studied the changes in ferritin and HO-1 levels in serum and bronchoalveolar lavage (BAL) fluid after intestinal I/R injury in rats. Leukocyte number and protein contents in BAL fluid were elevated following I/R, and the increases were attenuated by mepacrine pretreatment. Both serum ferritin and HO-1 concentrations were progressively elevated throughout the 3 h observation period. Mepacrine pretreatment attenuated the increase of serum and BAL fluid ferritin concentrations, but did not suppress the increase of serum HO-1. Moreover, BAL fluid HO-1 levels did not change after I/R or after mepacrine pretreated I/R compared with sham rats. Unlike ferritin, HO-1 levels are not exactly matched with the ALI. Therefore, there might be a different mechanism between the changes of ferritin and HO-1 in intestinal I/R-induced ALI model.

• Tuberculosis and Respiratory Diseases
• /
• v.44 no.2
• /
• pp.349-359
• /
• 1997

Background : The signal pathways and their precise roles for acute respiratory distress syndrome caused by endotoxin (ETX) has not been established. Since there has been several in vitro experiments suggesting that activation of protein kinase C (PKC) pathway may be responsible for endotoxin-induced inflammatory reaction, we performed in vivo experiments in the rats with the hypothesis that PKC-inhibition can effectively prevent endotoxin-induced acute lung injury. Methods : We studied the role of PKC in ETX-induced ALI using PKC inhibitor (staurosporine, STP) in the rat Specific pathogen free male Sprague-Dawley weighted from 165 to 270g were used for the study. Animals were divided into the normal control (NC)-, vehicle control (VC)-, ETX-, PMA (phorbolmyristateacetate)-, STP+PMA-, and STP+ETX-group. PMA (50mg/kg) or ETX (7mg/kg) was instilled through polyethylen catheter after aseptic tracheostomy with and without STP (0.2mg/kg)-pretreatment STP was injected via tail vein 30min before intratracheal injection (IT) of PMA or ETX. Bronchoalveolar lavage (BAL) was done 3-or 6-hrs after IT of PMA or ETX respectively, to measure protein concentration, total and differential cell counts. Results : The results were as follows. The protein concentrations in BALF in the PMA- and ETX-group were very higher than that of VC-group (p<0.001). When animals were pretreated with STP, the %reduction of the protein concentration in BALF was $64.8{\pm}8.5$ and $30.4{\pm}2.5%$ in the STP+PMA- and STP+ETX-group, respectively (p = 0.028). There was no difference in the total cell counts between the PMA-and VC-group (p = 0.26). However the ETX-group showed markedly increased total cell counts as compared to the VC- (p = 0.003) and PMA-group (p = 0.0027), respectively. The total cell counts in BALF were not changed after pretreatment with STP compared to the PMA- (p = 0.22) and ETX-group (p = 0.46). The percentage of PMN, but not alveolar macrophage, was significantly elevated in the PMA-, and ETX-group. Especially in the ETX-group, the percentage of PMN was 17 times higher than that of PMA (p < 0.001). The differential cell counts was not different between the PMA and STP+PMA On the contrary the STP+ETX-group showed decreased percentage of PMN (p = 0.016). There was no significant relationship between the protein concentration and the total or differential cell counts in each group. Conclusion : Pretreatment with PKC-inhibitor (staurosporine) partially but significantly inhibited ETX-induced ALI.

• The Korean Journal of Physiology and Pharmacology
• /
• v.1 no.1
• /
• pp.71-78
• /
• 1997

In an attempt to investigate the role of phospholipase $A_2$($PLA_2$) in interleukin-l (IL-l) induced acute lung injury, mepacrine was tried to inhibit $PLA_2$ in IL-l induced ARDS rats. For confirmation of acute lung injury by IL-l, and to know the role of neutrophils in this injury, lung leak index, lung myeloperoxidase(MPO), number of neutrophils and protein content in the bronchoalveolar lavage (BAL) and wet lung weight were measured. At the same time lung $PLA_2$ was measured to know the effect of IL-l on $PLA_2$ activity. Pulmonary surfactant was also measured for an investigation of type II alveolar cell function. Neutrophil adhesion assay was performed to know the effect of $PLA_2$ inhibition in vitro with human umbilical vein endothelial cells (HUVEC). For precise location of injury by IL-l, morpholgical study was performed by electron microscopy. Five hours after instillation of IL-l (50 ng/rat), lung leak index, protein content, number of neutrophils, lung MPO and wet lung weight were increased significantly. Five hours after IL-l instillation lung $PLA_2$ activity was increased significantly, and increased surfactant release was observed in IL-l induced ARDS rats' BAL. In contrast, in rats given mepacrine and IL-l, there was decrease of acute lung injury i.e. decrease of lung leak index, wet lung weight, protein content, number of neutrophils in BAL and decreased lung MPO activity. Mepacrine decreased surfactant release also. Interestingly, inhibition of $PLA_2$ decreased adhesion of human neutrophils to HUVEC in vitro. Morphologically, IL-l caused diffuse necrosis of endothelial cells, type I and II epithelial cells and increased the infiltration of neutrophils in the interstitium of the lung but after mepacrine treatment these pathological findings were lessened. On the basis of these experimental results it is suggested that $PLA_2$ has a major role in the pathogenesis of acute lung injury mediated by neutrophil dependent manner in IL-l induced acute lung injury.

• Tuberculosis and Respiratory Diseases
• /
• v.52 no.4
• /
• pp.355-366
• /
• 2002

Background : The heat shock protein (HSP) 70 families are known to protect cells against the irreversible tissue injury induced by stress and to induce the recovery of cell function during stress. Heat pretreatment was reported to decrease the acute lung injury (ALI) of rats induced by lipopolysaccharide (LPS). However, the role of heat shock with LPS co-treatmenton ALI is unclear. The purpose of this study was to investigate the effect of heat treatment, which was given immediately after the beginning of ALI induced by LPS intratracheally administered in rats. Methods : Either saline (saline group) or LPS was intratracheally instilled without heat treatment (LPS group). In addition, heat was conducted 18 hours prior to the instillation of LPS (pre-treatment group) and conducted immediately after instillation of LPS (co-treatment group). Six hours after the LPS or saline treatment, blood, bronchoalveolar lavage (BAL) fluid and lung tissue samples were obtained. The myeloperoxidase (MPO) activity and the heat shock protein expression in the lung tissue, the differential counts of the polymorphonuclear leukocytes (PMN) in the BAL fluids, and the LDH, protein, $IL-1{\beta}$, $TNF-{\alpha}$ and IL-10 levels in BAL fluid and serum were measured. Results : 1) The MPO activity, the differential PMN counts in the BAL fluid, BAL fluid and serum cytokines were higher in the LPS, the heat pre-treatment and co-treatment group than those of the saline group (p value <0.05). 2) The MPO activity and the protein level in the BAL fluid from the heat co-treatment group were similar to those of the LPS group. 3) The serum $TNF-{\alpha}$ level of the heat co-treatment group was significantly higher than that of the LPS group (p=0.01). Conclusion : Heat shock response administered immediately after a LPS instillation did not attenuate the ALI in this model.

• IMMUNE NETWORK
• /
• v.7 no.1
• /
• pp.18-25
• /
• 2007

Background: Although glucocorticoids (GCs) are effective in controlling asthma in the majority of patients, a subset of asthmatics fails to demonstrate a satisfactory response, even to systemic GC therapy. This population is referred to as being "steroid-resistant". The actual mechanism underlying steroid resistance in asthma remains to be elucidated. Methods: We have investigated how dexamethasone (DEX) regulates asthmatic phenotypes in a murine model of asthma, in which mice received i.p. immunization twice, followed by two bronchoprovocations with aerosolized OVA with a one-week interval, which we have recently described. Results: Pretreatment with DEX resulted in an inhibition of NF-${\kappa}B$ activation in asthmatic lungs, and also inhibited bronchoalveolar lavage (BAL) levels of NF-${\kappa}B$-dependent cytokines such as TNF-${\alpha}$ and CC chemokines [eotaxin and monocyte chemotactic protein (MCP)-1]. DEX was effective in suppressing airway hyperresponsiveness (AHR) at 10 h, Th2-dependent asthmatic phenotypes such as airway eosinophilia, BAL levels of Th2 cytokines (IL-5 and IL-13), and mucin production. However, DEX failed to suppress BAL levels of CXC chemokines [macrophage inflammatory protein-2 (MIP-2) and keratinocyte-derived chemokine (KC)] and airway neutrophilia. Conclusion: Airway neutrophilia is among the phenomena observed in patients with severe GC-resistant asthma. This study will provide insight into the molecular basis for airway neutrophila seen in steroid-resistant asthma. Further studies are required to delineate the underlying mechanism of CXC chemokine expression in asthma.

• Tuberculosis and Respiratory Diseases
• /
• v.71 no.2
• /
• pp.97-105
• /
• 2011

Background: It was hypothesized that the immunomodulating effects of moxifloxacin contribute to ameliorate oleic acid (OA)-induced acute lung injury (ALI) by suppression of cytosolic phospholipase A2 (cPLA2). This was based on observations from experiments on rats associated with neutrophilic respiratory burst, cPLA2 activity, and expressions of cPLA2, $TNF{\alpha}$, and COX-II in the lung. Methods: ALI was induced by intravenous injection of OA in male Sprague-Dawley rats. Five hours after OA injection, protein content in bronchoalveolar lavage (BAL), lung myeloperoxidase (MPO) activity, and numbers of BAL neutrophils were measured. As an index of oxidative stress-induced lung injury, the content of malondialdehyde (MDA) in lung tissues was also determined. Lung histology, immunohistochemistry and determination of activity of cPLA2 in lung tissues were carried out. In addition, Western blotting of $TNF{\alpha}$ and COX-II in lung tissues was performed. Results: The accumulation of neutrophils in the lungs was observed after OA injection. BAL protein was increased along with neutrophilic infiltration and migration by OA. Moxifloxacin decreased all of these parameters of ALI and ameliorated ALI histologically. The increased malondialdehyde (MDA) in the lung by OA was also decreased by moxifloxacin. Moxifloxacin not only suppressed cPLA2 expression in the lungs and neutrophils but also decreased cPLA2 activity in lung tissues of rats given OA. The enhanced expressions of $TNF{\alpha}$ and COX-2 in the lung tissues of rats given OA were also suppressed by moxifloxacin. Conclusion: Moxifloxacin inhibited cPLA2 and down-regulated $TNF{\alpha}$ and COX-2 in the lungs of rats given OA, which resulted in the attenuation of inflammatory lung injury.