静脉注射和气管内滴入博莱霉素诱导小鼠肺纤维化的差异

目的 探讨静脉注射和气管内滴入博莱霉素(BLM)对小鼠肺纤维化形成的差异.方法 8周龄雌性C57BL/6小鼠40只,随机分为静脉注射组(V组)20只、气管内滴入组(I组)20只,分别经尾静脉一次性注射BLM 150 mg/kg和气管内滴入BLM 5 mg/kg.观察每组小鼠生存率、肺组织病理改变及肺组织羟脯胺酸的含量.结果 ① V组和I组小鼠的生存率分别为50%和75%,两者间统计学无显著性差异(P>0.05).②注射处置后28 d,两组小鼠均形成广泛、稳定的间质纤维化病理改变,但I组主要分布在肺门和支气管周围,而V组主要分布在胸膜下及血管周围.肺纤维化病理评分I组与V组之间无显著的统计学差异(P>0.05).③小鼠注射处置后28 d,V组与I组肺羟脯氨酸含量分别为634.4±67.1 μg/g和696.6±41.2 μg/g,两组间无显著的统计学差异(P>0.05).结论 利用BLM静脉注射和气管内滴入制备肺间质纤维化动物模型,纤维化形成的部位存在着一定的差异,而肺间质形成纤维化的程度和病理改变大致相同.     

Intratracheal Instillation of High Dose Adenoviral Vectors Is Sufficient to Induce Lung Injury and Fibrosis in Mice

Rationale

Replication deficient adenoviruses (Ad) vectors are common tools in gene therapy. Since Ad vectors are known to activate innate and adaptive immunity, we investigated whether intratracheal administration of Ad vectors alone is sufficient to induce lung injury and pulmonary fibrosis.

Methods

We instilled Ad viruses ranging from 10⁷ to 1.625×10⁹ ifu/mouse as well as the same volume of PBS and bleomycin. 14 and 21 days after administration, we collected bronchoalveolar lavage fluid (BALF) and mouse lung tissues. We measured the protein concentration, total and differential cell counts, and TGF-β1 production, performed Trichrome staining and Sircol assay, determined gene and protein levels of profibrotic cytokines, MMPs, and Wnt signaling proteins, and conducted TUNEL staining and co-immunofluorescence for GFP and α-SMA staining.

Results

Instillation of high dose Ad vectors (1.625×10⁹ ifu/mouse) into mouse lungs induced high levels of protein content, inflammatory cells, and TGF-β1 in BALF, comparable to those in bleomycin-instilled lungs. The collagen content and mRNA levels of Col1a1, Col1a2, PCNA, and α-SMA were also increased in the lungs. Instillation of both bleomycin and Ad vectors increased expression levels of TNFα and IL-1β but not IL-10. Instillation of bleomycin but not Ad increased the expression of IL-1α, IL-13 and IL-16. Treatment with bleomycin or Ad vectors increased expression levels of integrin α1, α5, and αv, MMP9, whereas treatment with bleomycin but not Ad vectors induced MMP2 expression levels. Both bleomycin and Ad vectors induced mRNA levels of Wnt2, 2b, 5b, and Lrp6. Intratracheal instillation of Ad viruses also induced DNA damages and Ad viral infection-mediated fibrosis is not limited to the infection sites.

Conclusions

Our results suggest that administration of Ad vectors induces an inflammatory response, lung injury, and pulmonary fibrosis in a dose dependent manner.     

Non-surgical Intratracheal Instillation of Mice with Analysis of Lungs and Lung Draining Lymph Nodes by Flow Cytometry

Phagocytic cells such as alveolar macrophages and lung dendritic cells (LDCs) continuously sample antigens from the alveolar spaces in the lungs. LDCs, in particular, are known to migrate to the lung draining lymph nodes (LDLNs) where they present inhaled antigens to T cells initiating an appropriate immune response to a variety of immunogens^1,2. To model interactions between the lungs and airborne antigens in mice, antigens can be administered intranasally^1,3,4, intratracheally⁵ or as aerosols⁶. Delivery by each route involves distinct technical skills and limitations that need to be considered before designing an experiment. For example, intranasal and aerosolized exposure delivers antigens to both the lungs and the upper respiratory tract. Hence antigens can access the nasal associated lymphoid tissue (NALT)⁷, potentially complicating interpretation of the results. In addition, swallowing, sneezing and the breathing rate of the mouse may also lead to inconsistencies in the doses delivered. Although the involvement of the upper respiratory tract may be preferred for some studies, it can complicate experiments focusing on events specifically initiated in the lungs. In this setting, the intratracheal (i.t) route is preferable as it delivers test materials directly into the lungs and bypasses the NALT. Many i.t injection protocols involve either blind intubation of the trachea through the oral cavity or surgical exposure of the trachea to access the lungs. Herein, we describe a simple, consistent, non-surgical method for i.t instillation. The opening of the trachea is visualized using a laryngoscope and a bent gavage needle is then inserted directly into the trachea to deliver the innoculum. We also describe procedures for harvesting and processing of LDLNs and lungs for analysis of antigen trafficking by flow cytometry.Download video file.^{(48M, mov)}     

Instillation and Fixation Methods Useful in Mouse Lung Cancer Research

The ability to instill live agents, cells, or chemicals directly into the lung without injuring or killing the mice is an important tool in lung cancer research. Although there are a number of methods that have been published showing how to intubate mice for pulmonary function measurements, none are without potential problems for rapid tracheal instillation in large cohorts of mice. In the present paper, a simple and quick method is described that enables an investigator to carry out such instillations in an efficient manner. The method does not require any special tools or lighting and can be learned with very little practice. It involves anesthetizing a mouse, making a small incision in the neck to visualize the trachea, and then inserting an intravenous catheter directly. The small incision is quickly closed with tissue adhesive, and the mice are allowed to recover. A skilled student or technician can do instillations at an average rate of 2 min/mouse. Once the cancer is established, there is frequently a need for quantitative histologic analysis of the lungs. Traditionally pathologists usually do not bother to standardize lung inflation during fixation, and analyses are often based on a scoring system that can be quite subjective. While this may sometime be sufficiently adequate for gross estimates of the size of a lung tumor, any proper stereological quantification of lung structure or cells requires a reproducible fixation procedure and subsequent lung volume measurement. Here we describe simple reliable procedures for both fixing the lungs under pressure and then accurately measuring the fixed lung volume. The only requirement is a laboratory balance that is accurate over a range of 1 mg–300 g. The procedures presented here thus could greatly improve the ability to create, treat, and analyze lung cancers in mice.     

Lead Induces Apoptosis and Histone Hyperacetylation in Rat Cardiovascular Tissues

Acute and chronic lead (Pb) exposure might cause hypertension and cardiovascular diseases. The purpose of this study was to evaluate the effects of early acute exposure to Pb on the cellular morphology, apoptosis, and proliferation in rats and to elucidate the early mechanisms involved in the development of Pb-induced hypertension. Very young Sprague-Dawley rats were allowed to drink 1% Pb acetate for 12 and 40 days. Western blot analysis indicated that the expression of proliferating cell nuclear antigen (PCNA) decreased in the tissues of the abdominal and thoracic aortas and increased in the cardiac tissue after 12 and 40 days of Pb exposure, respectively. Bax was upregulated and Bcl-2 was downregulated in vascular and cardiac tissues after 40 days of Pb exposure. In addition, an increase in caspase-3 activity was observed after 40 days of exposure to Pb. In terms of morphology, we found that the internal elastic lamina (IEL) of aorta lost the original curve and the diameter of cardiac cell was enlarged after 40 days. Furthermore, the exposure led to a marked increase in acetylated histone H3 levels in the aortas and cardiac tissue after 12 and 40 days, than that in the control group. These findings indicate that Pb might increase the level of histone acetylation and induce apoptosis in vascular and cardiac tissues. However, the mechanism involved need to be further investigated.     

Lipid and Protein Arrangement in Bacteriophage PM2

X-ray structure analysis shows that the lipid of bacteriophage PM2 is organized in a bilayer. These results provide a basis for interpreting electron micrographs of the virus in molecular terms.     

氧化苦参碱对哮喘大鼠肺组织IL-10 表达的影响

目的:观察不同浓度氧化苦参碱(Oxymatrinem,Oxy)对哮喘大鼠肺组织IL~(-1)0表达的影响,并探讨其作用机制。方法:构建哮喘大鼠模型,将40只清洁级健康雌性SD大鼠随机分成5组,每组8只:A:哮喘组(仅卵蛋白(Ovalbumin,OVA)致敏)、B:低浓度组(Oxy 50 mg/kg)、C:中浓度组(Oxy 100 mg/kg)、D:高浓度组(Oxy 150 mg/kg)、E:对照组(生理盐水),末次激发24 h后处死全部大鼠,取大鼠肺脏,HE染色观察肺组织病理改变,采用RT-PCR、Western Blot测定各组肺组织中IL~(-1)0基因及蛋白水平的表达。结果:HE结果显示,哮喘组可见大量炎症细胞浸润,气管平滑肌明显增厚。对照组肺泡壁薄且光滑,未见明显炎性细胞的浸润,不同浓度氧化苦参碱药物干预组其肺组织炎症细胞浸润及气管平滑肌病变程度随着用药浓度的增高呈逐渐减轻趋势。RT-PCR以及Western blot检测IL~(-1)0发现,哮喘组、氧化苦参碱低浓度组、氧化苦参碱中浓度组与对照组相比IL~(-1)0的表达均有所减低(P0.05),而氧化苦参碱高浓度组与对照组比较,IL~(-1)0的表达无统计学意义(P0.05);氧化苦参碱中浓度组、氧化苦参碱高浓度组与哮喘组相比IL~(-1)0的表达均有所增高(P0.05),氧化苦参碱低浓度组与哮喘组相比IL~(-1)0的表达无统计学意义(P0.05)。结论:氧化苦参碱抑制、控制哮喘发作可能与促进肺组织中IL~(-1)0基因、蛋白的表达相关,且促进程度在一定范围内与浓度呈正比。     

Noninvasive Intratracheal Intubation to Study the Pathology and Physiology of Mouse Lung

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular disease and for developing therapeutic intervention. With the increasing availability of knockout and transgenic derivatives, together with a vast amount of genetic information, mice provide one of the best models to study the molecular mechanisms underlying the pathology and physiology of lung diseases. Inhalation, intranasal instillation, intratracheal instillation, and intratracheal intubation are the most widely used techniques by a number of investigators to administer materials of interest to mouse lungs. There are pros and cons for each technique depending on the goals of a study. Here a noninvasive intratracheal intubation method that can directly deliver exogenous materials to mouse lungs is presented. This technique was applied to administer bleomycin to mouse lungs as a model to study pulmonary fibrosis.     

Effects of Physical Properties of the Lung Parenchyma and Airways on Cough Efficiency

The effects of the physical parameters of the parenchyma and airways on the temporal relationships between the volume flow rate and the linear velocity of airflow in the trachea during coughing were studied using a mathematical model. It was shown that the relationship between the volume flow rate and the duration of the act of conghing can be used for assessment of inertial and viscous properties of the posterior wall of the trachea and for assessment of the parameter characterizing the nonlinearity of the intrapulmonary airway resistance. Computations indicated that the linear air velocity in the trachea and, therefore, the cough efficiency depend weakly on the physical properties of the posterior wall of the trachea and lung parenchyma.     

Chlamydia pneumoniae Infection in Mice Induces Chronic Lung Inflammation,iBALT Formation,and Fibrosis

Chlamydia pneumoniae (CP) lung infection can induce chronic lung inflammation and is associated with not only acute asthma but also COPD exacerbations. However, in mouse models of CP infection, most studies have investigated specifically the acute phase of the infection and not the longer-term chronic changes in the lungs. We infected C57BL/6 mice with 5×10⁵ CP intratracheally and monitored inflammation, cellular infiltrates and cytokine levels over time to investigate the chronic inflammatory lung changes. While bacteria numbers declined by day 28, macrophage numbers remained high through day 35. Immune cell clusters were detected as early as day 14 and persisted through day 35, and stained positive for B, T, and follicular dendritic cells, indicating these clusters were inducible bronchus associated lymphoid tissues (iBALTs). Classically activated inflammatory M1 macrophages were the predominant subtype early on while alternatively activated M2 macrophages increased later during infection. Adoptive transfer of M1 but not M2 macrophages intratracheally 1 week after infection resulted in greater lung inflammation, severe fibrosis, and increased numbers of iBALTS 35 days after infection. In summary, we show that CP lung infection in mice induces chronic inflammatory changes including iBALT formations as well as fibrosis. These observations suggest that the M1 macrophages, which are part of the normal response to clear acute C. pneumoniae lung infection, result in an enhanced acute response when present in excess numbers, with greater inflammation, tissue injury, and severe fibrosis.     

Milano Summer Particulate Matter (PM10) Triggers Lung Inflammation and Extra Pulmonary Adverse Events in Mice

Recent studies have suggested a link between particulate matter (PM) exposure and increased mortality and morbidity associated with pulmonary and cardiovascular diseases; accumulating evidences point to a new role for air pollution in CNS diseases. The purpose of our study is to investigate PM10sum effects on lungs and extra pulmonary tissues. Milano PM10sum has been intratracheally instilled into BALB/c mice. Broncho Alveolar Lavage fluid, lung parenchyma, heart and brain were screened for markers of inflammation (cell counts, cytokines, ET-1, HO-1, MPO, iNOS), cytotoxicity (LDH, ALP, Hsp70, Caspase8-p18, Caspase3-p17) for a putative pro-carcinogenic marker (Cyp1B1) and for TLR4 pathway activation. Brain was also investigated for CD68, TNF-α, GFAP. In blood, cell counts were performed while plasma was screened for endothelial activation (sP-selectin, ET-1) and for inflammation markers (TNF-α, MIP-2, IL-1β, MPO). Genes up-regulation (HMOX1, Cyp1B1, IL-1β, MIP-2, MPO) and miR-21 have been investigated in lungs and blood. Inflammation in the respiratory tract of PM10sum-treated mice has been confirmed in BALf and lung parenchyma by increased PMNs percentage, increased ET-1, MPO and cytokines levels. A systemic spreading of lung inflammation in PM10sum-treated mice has been related to the increased blood total cell count and neutrophils percentage, as well as to increased blood MPO. The blood-endothelium interface activation has been confirmed by significant increases of plasma ET-1 and sP-selectin. Furthermore PM10sum induced heart endothelial activation and PAHs metabolism, proved by increased ET-1 and Cyp1B1 levels. Moreover, PM10sum causes an increase in brain HO-1 and ET-1. These results state the translocation of inflammation mediators, ultrafine particles, LPS, metals associated to PM10sum, from lungs to bloodstream, thus triggering a systemic reaction, mainly involving heart and brain. Our results provided additional insight into the toxicity of PM10sum and could facilitate shedding light on mechanisms underlying the development of urban air pollution related diseases.     

慢性阻塞性肺病大鼠模型的肺功能及肺组织病理学变化评估

目的:探讨被动吸烟的时间长短与慢性阻塞性肺病(COPD)大鼠模型的肺功能及肺组织病理学的变化之间的关系.方法:采用被动吸烟法建立COPD大鼠模型,随机分为被动吸烟4周组、6周组、8周组和对照组,分别测定和评估不同时间段的各组大鼠模型的肺功能和气道肺组织的病理变化.结果:6周组及8周组大鼠模型的呼气峰流速(PEF)显著降低、气道内压上升坡度(IP-slope)显著增高,与对照组比较P＜0.01.以PEF值小于对照组的大鼠80%PEF值为存在气流受限的界线,则4周组、6周组、8周组出现气流受限的鼠分别为0只(0)、6只(75%)及8只(100%),6周组及8周组的大鼠气流受限发生率显著高于4周组(P＜0.01).8周组大鼠出现明显气道壁增厚、气道狭窄及显著肺气肿改变,其气道壁炎细胞浸润、杯状细胞化生、气道壁坏死糜烂、纤维结缔组织及平滑肌增生等评分均显著高于对照组(P＜0.01),气道坏死糜烂、小气道阻塞发生率及气道平滑肌增生等指标显著高于6周组(P＜0.01).6周组中有6只大鼠出现气道狭窄及肺气肿改变,气道壁炎细胞浸润、杯状细胞化生、气道壁坏死糜烂、纤维结缔组织及平滑肌增生等评分均显著高于对照组(P＜0.01).6周组及8周组大鼠PEF值与气道壁纤维组织和平滑肌增生呈显著负相关(P＜0.05);而4周组大鼠未出现典型肺气肿及气道狭窄.结论:COPD的形成与吸烟时间有关,在吸烟量相同条件下,吸烟时间越长,气道和肺组织病变越重,气流受限的发生率越高.     

Increase of Lipid Hydroperoxides in Tissues of Vitamin E-Deficient Rats

The level of lipid hydroperoxides was determined by a newly developed method in rat tissues of vitamin E deficiency, which was a good in viuo model of enhanced radical reactions. In the heart, lung and kidney, the level of lipid hydroperoxides increased significantly as early as 4 weeks after feeding on a tocopherol-deficient diet compared with that of the control group. After 8 weeks of the deficiency, similar results were obtained. These results indicate that the lipid hydroperoxide is available as an extremely sensitive indicator of lipid peroxidation in these organs, because it takes several months to detect manifestations of the vitamin deficiency based on conventional indices.     

Chilling Injury Induces Lipid Phase Changes in Membranes of Tomato Fruit

Wide-angle x-ray diffraction has provided evidence for lipid phase separations in microsomal membranes from chill-injured tomato (Lycopersicon esculentum Mill. cv Caruso) fruit. Mature-green fruit stored for 20 d at 5[deg]C had not begun to ripen and were essentially free of chilling injury symptoms. Within 4 d of being returned to 25[deg]C, however, the fruit displayed characteristic symptoms of chilling injury, including translucent water-soaked patches, surface pitting, and irregular pigmentation. Membrane damage measured as electrolyte leakage from pericarp discs intensified after the fruit were returned to ambient temperature. Wide-angle x-ray diffraction patterns recorded at 25[deg]C for microsomal membranes isolated from untreated, mature-green fruit indicated that the membrane bilayers were exclusively liquid-crystalline. Diffraction patterns for microsomal membranes from fruit stored for 20 d at 5[deg]C showed only trace amounts of gel phase lipid, but within 4 d of subsequent exposure of the fruit to ambient temperature, there was evidence for a pronounced lateral phase separation of lipids within the membranes that would render them leaky. Inas-much as the phase separations were detectable at 25[deg]C and became pronounced only subsequent to the chilling episode, they appear to be an indirect rather than direct effect of exposure to low temperature. The diffraction data thus support the notion that the lipid phase changes observed here are not directly induced by low temperature but rather reflect subsequent biochemical changes in the bilayers that may contribute to the development of chilling symptoms.     

Cocaine Induces Sex-Associated Changes in Lipid Profiles of Brain Extracellular Vesicles

Neurochemical Research - Cocaine is a highly addictive stimulant with diverse effects on physiology. Recent studies indicate the involvement of extracellular vesicles (EVs) secreted by neural cells...     

Repeated Cocaine Exposure Facilitates the Expression of Incentive Motivation and Induces Habitual Control in Rats

There is growing evidence that mere exposure to drugs can induce long-term alterations in the neural systems that mediate reward processing, motivation, and behavioral control, potentially causing the pathological pursuit of drugs that characterizes the addicted state. The incentive sensitization theory proposes that drug exposure potentiates the influence of reward-paired cues on behavior. It has also been suggested that drug exposure biases action selection towards the automatic execution of habits and away from more deliberate goal-directed control. The current study investigated whether rats given repeated exposure to peripherally administered cocaine would show alterations in incentive motivation (assayed using the Pavlovian-to-instrumental transfer (PIT) paradigm) or habit formation (assayed using sensitivity to reward devaluation). After instrumental and Pavlovian training for food pellet rewards, rats were given 6 daily injections of cocaine (15 mg/kg, IP) or saline, followed by a 10-d period of rest. Consistent with the incentive sensitization theory, cocaine-treated rats showed stronger cue-evoked lever pressing than saline-treated rats during the PIT test. The same rats were then trained on a new instrumental action with a new food pellet reward before undergoing a reward devaluation testing. Although saline-treated rats exhibited sensitivity to reward devaluation, indicative of goal-directed performance, cocaine-treated rats were insensitive to this treatment, suggesting a reliance on habitual processes. These findings, when taken together, indicate that repeated exposure to cocaine can cause broad alterations in behavioral control, spanning both motivational and action selection processes, and could therefore help explain aberrations of decision-making that underlie drug addiction.     

Impact of Statins on Gene Expression in Human Lung Tissues

Statins are 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors that alter the synthesis of cholesterol. Some studies have shown a significant association of statins with improved respiratory health outcomes of patients with asthma, chronic obstructive pulmonary disease and lung cancer. Here we hypothesize that statins impact gene expression in human lungs and may reveal the pleiotropic effects of statins that are taking place directly in lung tissues. Human lung tissues were obtained from patients who underwent lung resection or transplantation. Gene expression was measured on a custom Affymetrix array in a discovery cohort (n = 408) and two replication sets (n = 341 and 282). Gene expression was evaluated by linear regression between statin users and non-users, adjusting for age, gender, smoking status, and other covariables. The results of each cohort were combined in a meta-analysis and biological pathways were studied using Gene Set Enrichment Analysis. The discovery set included 141 statin users. The lung mRNA expression levels of eighteen and three genes were up-regulated and down-regulated in statin users (FDR < 0.05), respectively. Twelve of the up-regulated genes were replicated in the first replication set, but none in the second (p-value < 0.05). Combining the discovery and replication sets into a meta-analysis improved the significance of the 12 up-regulated genes, which includes genes encoding enzymes and membrane proteins involved in cholesterol biosynthesis. Canonical biological pathways altered by statins in the lung include cholesterol, steroid, and terpenoid backbone biosynthesis. No genes encoding inflammatory, proteases, pro-fibrotic or growth factors were altered by statins, suggesting that the direct effect of statin in the lung do not go beyond its antilipidemic action. Although more studies are needed with specific lung cell types and different classes and doses of statins, the improved health outcomes and survival observed in statin users with chronic lung diseases do not seem to be mediated through direct regulation of gene expression in the lung.     

Spermidine Feeding Decreases Age-Related Locomotor Activity Loss and Induces Changes in Lipid Composition

Spermidine is a natural polyamine involved in many important cellular functions, whose supplementation in food or water increases life span and stress resistance in several model organisms. In this work, we expand spermidine’s range of age-related beneficial effects by demonstrating that it is also able to improve locomotor performance in aged flies. Spermidine’s mechanism of action on aging has been primarily related to general protein hypoacetylation that subsequently induces autophagy. Here, we suggest that the molecular targets of spermidine also include lipid metabolism: Spermidine-fed flies contain more triglycerides and show altered fatty acid and phospholipid profiles. We further determine that most of these metabolic changes are regulated through autophagy. Collectively, our data suggests an additional and novel lipid-mediated mechanism of action for spermidine-induced autophagy.