| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Departments of 1Environmental Health Sciences, and 2Pulmonary and Critical Care Medicine, Bloomberg School of Public Health and School of Medicine, The Johns Hopkins University, Baltimore, Maryland 21205
Submitted 24 February 2003 ; accepted in final form 25 May 2003
 |
ABSTRACT |
|---|
 TOP ABSTRACT METHODSRESULTSDiscussionDISCLOSURESREFERENCES |
|---|
physiological aging; particulate matter; air pollution susceptibility; diethylenetriamine pentaacetic acid clearance in mice; epithelial-endothelial barrier dysfunction
Recent animal models of aging have been developed in which physiological deficiencies in vital organ systems forecast imminent death better than chronological age (10, 20, 21, 30). Homeostatic instability has been suggested in rodents showing loss of body weight and disintegration of heart rate (HR) and temperature regulation. In one study (30), an abrupt loss of body weight occurs in AKR/J mice 5-6 wk before death. Failure to maintain body weight coincides with a decline in average deep-body temperature and the disintegration of circadian rhythm in activity and HR (30). Because the lung is the target organ of air-pollutant exposure, we surmised that deficiencies in lung function and structure may also be involved and contribute to increased PM susceptibility with aging. However, there is little evidence concerning senescent-dependent changes in epithelial-endothelial barrier function that may contribute to overall susceptibility to ambient PM.
The purpose of the present study was to investigate senescent-dependent changes in lung permeability. Although a decrease in lung epithelial-endothelial barrier function with terminal senescence serves as a contributing factor for soluble components of PM or lung mediators of PM exposure to reach the systemic circulation and other organs, present data in this regard are unavailable. Therefore, this study was designed to examine short-term clearance rates of the inert tracer particle technetium-99m (99mTc)-diethylenetriamine pentaacetic acid (DTPA) from the lung in anesthetized mice by scintigraphy. This technique was used to test for differences in lung clearance rates between older, healthy AKR/J mice and similar mice manifesting signs of homeostatic instability. Because clearance of soluble particles is proportional to lung surface area (22), we indirectly assessed senescent-dependent changes in surface area by measuring changes in the lung pressure-volume (P-V) curves. Obtaining these curves also allowed us to assess the potential role of changes in pulmonary surfactant on DTPA clearance (5, 8) in this mouse model. Our results indicate that lung structural changes do occur with terminal senescence, consistent with a decrease in total lung volume and a decrease in epithelial-endothelial permeability barrier function.
|
METHODS |
|---|
|
TOPABSTRACTMETHODSRESULTSDiscussionDISCLOSURESREFERENCES |
|---|
180 days of age. The mice were individually housed in an animal facility at Johns Hopkins Bloomberg School of Public Health. All animals were provided water and chow (Agway Pro-Lab RMH 1000) ad libitum. The light-dark phases of the animal facility were controlled by a 12-h cycle. All animal protocols were reviewed and approved by the Animal Care and Use Committees of the Johns Hopkins Schools of Medicine or Public Health.
The AKR/J strain is relatively short-lived compared with other inbred strains (32). The mean life span is 319 ± 14 days with a range between 197 and 548 days. Body weight gradually increases or remains stable in older healthy AKR/J mice. In contrast, an abrupt decline in body weight of >1.5 g occurs between weekly measurements at 5 wk before the end of life. This abrupt decline in body weight coincides with deficiencies in other vital physiological systems, including HR and temperature regulation (30).
Lung permeability. Animals were divided into two groups on the basis of weekly body weight measurements. Mice were classified as stable (control group) if they showed an increase or no change in weekly body weight. A second group of mice was defined as terminally senescent on the basis of a weekly body weight loss of >1.5 g, although these mice were not always studied immediately after this first weight loss determination. Several animals were housed for additional weeks to monitor greater decrements in body weight. The average weight loss for the terminally senescent animals was 6.7 ± 1.5 g, which was significantly (P < 0.01) different from the control group.
Soluble particle clearance from the lung was measured by using the small (molecular weight 492; 0.57-nm diameter), soluble, hydrophilic tracer 99mTc-DTPA (CIS-US, Bedford, MA). Two groups of AKR/J mice, one group of >260 days of age showing stable body weight and the other group showing signs of terminal senescence (i.e., body weight loss of >1.5 g between weekly measurements), were anesthetized (30 µl of 10:1 ketamine/acepromazine) and intubated with an endotracheal tube. The endotracheal tube was used to slowly introduce a 25-µl 99mTc-DTPA (activity: 5 µCi) aspirate solution into the lungs of each mouse. The mouse was then placed onto an imaging table from which images were acquired continuously for a 30-min period by using a gamma camera with a pinhole collimator (GE Medical Systems, Hanover, MD). To verify the labeling procedure, the tracer was periodically sampled predelivery and assayed for unbound 99mTc-DTPA by using a silica gel media and thin-layer chromatography (3).
Radioisotope delivery and clearance data were quantified with modified techniques of Foster and Stetkiewicz (9). An initial lung image was acquired immediately after 99mTc-DTPA delivery and was imaged on the computer screen to enable the region of interest (ROI) to be selected by cursor manipulation. Pixel area evaluated was the same for all studies. That is, the largest ROI was initially determined and then used as the standard pixel area for all the studies. Adjustments in the ROI were not made to compensate for the smaller lung in the terminally senescent mice. To determine the clearance of 99mTc-DTPA, activity time plots were measured with the ROI every 2 min for the subsequent 30 min. The curves were corrected for radioactive decay and expressed as a percentage of the 99mTc-DTPA delivered at time 0. To quantify the clearance, the retention half-time was calculated as the time for retention curve in each mouse to reach 50% of its initial value. In three animals, where the retention had not quite fallen to 50% at 30 min, a linear extrapolation was done from the last three recorded points. Figure 1 shows a representative series of scintigraphic images in a clearance sequence depicting the time-dependent changes in 99mTc-DTPA radioactivity. At 2 min after the instillation of the radiolabel, radioactivity resides predominantly in lung and is absent in other regions of the image. With time, the 99mTc-DTPA activity moves from the lung to other organs with a concentration in the kidneys. At the end of 30 min, radioactivity is most evident in the bladder, and lung radioactivity is substantially reduced.
|
Quasistatic P-V curves. In a separate group of mice, animals were anesthetized with intraperitoneal injections of pentobarbital sodium at a dose of 70-80 mg/kg body wt. While animals were in a supine position, the trachea was cannulated, and each animal was ventilated with 100% oxygen for 10 min. The cannula was sealed with a stopcock to degas the lung (11). Two complete loops of the quasistatic P-V curve were then immediately performed in situ. Previous work (31) has shown that the mouse chest wall has a negligible influence on the intact P-V curve. A dual infusion-withdrawal pump (model 900-610, Harvard Appartus, Dover, MA) was used to standardize the rate of inflation and deflation. The airway pressure was measured by using a differential pressure transducer (model 8510B-2, Endevco, San Juan Capistrano, CA). The initial inflation rate was very slow (0.5 ml/min) to ensure opening of all lung regions. Once a pressure of 30 cmH2O was reached on the first inflation, the flow rate was increased to 2.1 ml/min for the remaining inflation/deflation maneuvers. The limits of the inflation and deflation airway pressures were 30 and -5 cmH2O, respectively.
Residual volume (RV) and total lung capacity (TLC) were assessed from the deflation curves at -5 and 30 cmH2O, respectively. Lung compliance was computed from a linearized slope of the P-V relationships between 0 and 10 cmH2O on deflation. To obtain indirect estimates of surface properties of the lung, we measured the fractional volume remaining on deflation at 10 cmH2O (16, 19) and P-V hysteresis. The hysteresis was quantified by integrating the area bordered by the inflation and deflation curves and dividing by the rectangular area bounded by the TLC and RV endpoints (1).
Wet and dry lung weights. In a separate series of studies, a third group of stable (n = 5) and declining (n = 4) AKR/J mice were anesthetized (2% isoflurane in O2) and then killed by cervical dislocation. The lungs were dissected and removed, quickly rinsed and then blotted dry, and the wet weight recorded. Subsequently, the lungs were dried on a heating block (70°C) for 3 consecutive days, and dry weights were obtained.
Data analysis. Data reported in the figures and tables are expressed as means ± SE. Values of clearance half-time, lung volume, compliance, hysteresis, and fractional volume were evaluated by using a one-way analysis of variance to determine senescent-dependent differences. An 
-level of 0.05 was used to establish statistical significance.
|
RESULTS |
|---|
|
TOPABSTRACTMETHODSRESULTSDiscussionDISCLOSURESREFERENCES |
|---|
|
|
|
Lung mechanics. Figure 5 summarizes the effects of terminal senescence on mouse lung mechanical properties. Changes in TLC (i.e., volume at 30 cmH2O), RV, compliance, and P-V hysteresis are plotted. Lung compliance and TLC in the group of terminally senescent mice showed a significant (P < 0.05) decline compared with the control group of mice. Between the two groups, there were no significant differences in RV or in measures linked to lung surface tension, the lung volume at 10 cmH2O, and the lung P-V hysteresis. Figure 6 shows the correlations between individual weight loss, TLC, and compliance. There were significant negative correlations between these variables (P < 0.05).
|
|
Lung wet weight-to-dry weight ratio was quantified in separate groups of AKR/J mice (n = 6; weight loss: 1.8 ± 0.3 g) and AKR/J mice of similar age range (240-310 days; n = 5; average weight loss: 13.1 ± 2 g). Average lung wet/dry weight for the stable AKR/J mice was 5.17 ± 0.06 g and was not different in the senescent AKR/J mice (5.25 ± 0.17 g; P > 0.05).
|
Discussion |
|---|
|
TOPABSTRACTMETHODSRESULTSDiscussionDISCLOSURESREFERENCES |
|---|
Experimental studies of particle transits in the lung have relied extensively on measurement of radiolabeled tracer kinetics, specifically the small, soluble, hydrophilic 99mTc-DTPA. 99mTc-DTPA, delivered through the airways, has been used primarily for physiological studies of epithelial integrity and is favored because of its small molecular size (molecular weight = 492) and radius (0.57 nm). On the basis of these properties, it is generally assumed that this small soluble particle is freely diffusible into the vasculature (23). In vivo studies of inhaled 99mTc-DTPA evaluate the penetration of tracer through the alveolar epithelium via paracellular pathways, the interstitial space of extra-cellular matrix, and the endothelial barrier. Although this technique has been widely used in human studies as well as many animal models, it has not been previously utilized in mice.
Several studies have investigated the effects of aging on 99mTc-DTPA clearance in human subjects and other animal models. In healthy human volunteers, lung clearance of 99mTc-DTPA has been shown to decrease or remain unchanged with age through six to seven decades (14, 25). In contrast, human subjects with lung disease such as asthma (15) and adult respiratory distress syndrome (6) demonstrate increased 99mTc-DTPA clearance. Similar findings are demonstrated in subgroups of smokers (22, 29). Thus it appears that the lung permeability barrier remains intact in healthy elderly populations; however, increased permeability is strongly correlated with lung disease. Our present findings in mice are consistent with these observations in larger species relative to the development of lung disease and may reflect a more general homeostatic decay coincident with a multiorgan system failure. It is noteworthy to mention, however, that the average rate of clearance in control mice reported in the present study is more rapid compared with other species, such as rats given a similar application of 99mTc-DTPA (25).
In several different models of endothelial and epithelial injury that result in pulmonary edema, 99mTc-DTPA has been shown to move more rapidly from airspaces into the blood after the generation of edema than during control conditions (2, 24, 27). We therefore considered whether the enhanced clearance that is observed in the declining AKR/J mice might be related to changes in lung water. However, based on conventional assessment of gross lung fluid balance (wet/dry weight), we detected no differences between stable and declining AKR/J mice. Thus the mechanism responsible for enhanced permeability of the soluble tracer particle is not likely related to lung edema.
Previous work using the AKR/J inbred mouse strain as a model of physiological aging demonstrated discrete stages of disintegration in multiple systems, which resulted in a predictable sequence of pathophysiological events with imminent death (30). These results showed instability in cardiac and thermal regulatory mechanisms in AKR/J mice that occurred coincident with abrupt decrements in body weight, attributes that accurately forecasted a remaining life span of only 5-6 wk. A second stage of disintegration was distinguished by an abrupt decline in daily mean HR, which occurred 3-4 wk before death. The present study suggests that lung homeostasis is also involved in this decline, showing pathophysiological changes coincident with terminal senescence. The changes in lung structure and function are characterized by significantly reduced lung volumes and compliance relative to weight-stable aging mice. The decrease in lung compliance with loss of lung homeostasis indicates a greater work of breathing required to maintain stable alveolar ventilation. Kurozumi et al. (18) studied age-dependent changes in lung structure in a mouse model of variable senescence by using substrains derived from the AKR/J strain. These investigators showed that lung volume at 30 cmH2O and alveolar size increased similarly in strains resistant (SAM-R/1) and prone (SAM-P/1) to accelerated senescence. We also observed an increase in lung volume in the AKR mice at younger ages than were studied here (data not shown). However, the focus of our present study is on the changes that occur during the period of terminal senescence, a time period that Kurozumi et al. did not investigate. Our most striking finding, which extends the studies of Kurozumi et al., is the substantially more rapid lung clearance in terminally senescent mice, indicating a disintegration of the permeability barrier across the alveolar epithelium and endothelium. Likewise, the present study demonstrates that vital capacity and lung compliance significantly decline with terminal senescence without influencing properties of lung surface tension (16, 19).
The demonstrated change in lung permeability in senescent mice may possibly be related to the epidemiology linking increased daily mortality rates with abrupt elevations in PM as an air pollutant. One hypothesis suggests that individuals susceptible to PM-induced mortality are the elderly and patients with cardiopulmonary disease generally characterized by extreme fragility (10, 12, 13). By defining terminal senescence using an inventory of physiological indicators, we can precisely time a PM intervention to test whether acute exposure leads to premature mortality. Although we did not study the effect of an environmental sample of PM, we did study the dynamics of an inert tracer with properties consistent with that of the water-soluble component of PM. A rational hypothesis to explain PM epidemiology predicts that PM components in real-world mixtures would be similarly able to follow the pathway of the DTPA used in the present study. In this regard, the present results support a contributing factor for PM-induced mortality risk by demonstrating tracer access to the systemic circulation at greater rates in terminally senescent animals.
In conclusion, the present results demonstrate a new use of 99mTc-DTPA lung clearance as a marker of changes in lung permeability in the mouse. Our motivation for these studies is to develop a model of PM-induced susceptibility and to show decay in lung homeostasis resulting in greater movement of soluble components of PM or lung mediators of PM exposure to the systemic circulation. The results support the hypothesis that the disintegration of the epithelial-endothelial permeability barrier is a feature of lung homeostatic loss during a period of terminal senescence in AKR/J mice. The structural mechanisms underlying this increased permeability, as well as the relation to the increased risk of PM-induced morbidity or mortality, however, remain unknown.
|
DISCLOSURES |
|---|
|
TOPABSTRACTMETHODSRESULTSDiscussionDISCLOSURESREFERENCES |
|---|
|
FOOTNOTES |
|---|
Original submission in response to a call for papers on "Physiology of Aging."
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
|
REFERENCES |
|---|
|
TOPABSTRACTMETHODSRESULTSDiscussionDISCLOSURESREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  S. Skrovanek, M. C. Valenzano, and J. M. Mullin Restriction of sulfur-containing amino acids alters claudin composition and improves tight junction barrier function Am J Physiol Regulatory Integrative Comp Physiol, September 1, 2007; 293(3): R1046 - R1055. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Visit Other APS Journals Online |
) and terminally senescent (
) groups of mice, indicating that the increase in lung permeability is associated with the degree of lung homeostatic decay in terminal senescence.
P < 0.01 and *P < 0.05 between control and senescent groups.