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Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, Kansas 66160
Submitted 15 July 2003 ; accepted in final form 23 August 2003
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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microvascular inflammation; microvascular PO2; muscle microcirculation; compound 48/80; cromolyn
Mast cells play an early, key role in the microvascular effects of hypoxia. Activation of mast cells with the use of compound 48/80 in normoxic animals produces an inflammatory response similar to that elicited by hypoxia; in addition, mast cells degranulate during hypoxia, and blockade of hypoxia-induced degranulation with the mast cell stabilizer cromolyn prevents or attenuates the hypoxic microvascular inflammatory response (19). An alteration of the ROS-NO balance appears to be involved in the activation of mast cells during hypoxia, since both antioxidants and NO donors prevent the activation of mast cells (19) as well as the microvascular inflammation that accompanies hypoxia (20).
The mechanism by which a reduction in the inspired PO2 leads to a rapid and widespread microvascular inflammation is not clear. Hypoxia results in increased ROS generation in isolated cardiomyocytes (6) and endothelial cells (1, 13), elevated expression of adhesion molecules in isolated leukocytes (12, 15, 17), and increased adherence of leukocytes to cultured human umbilical vein endothelial cells (2, 3), suggesting that the local reduction in PO2 sets in motion the processes involved in the microvascular inflammation seen in the intact animal. On the other hand, a dissociation between cremaster microvascular PO2 (PmO2) and hypoxia-induced leukocyte adherence to cremaster venules has been shown in intact rats (16). Selective reduction of cremaster muscle PmO2 under conditions of normal systemic arterial PO2 (PaO2) was not accompanied by increased adherence of leukocytes to cremaster venules in intact rats; in contrast, leukocyte adherence occurred when cremaster PmO2 was maintained elevated during systemic hypoxia (16). Absence of leukocyte-endothelial adherence during selective cremaster hypoxia could be due to insufficient time for leukocytes to become fully activated during their transit through the hypoxic cremaster. However, if this is the case, the observation that leukocytes adhere to normoxic cremaster venules when rats breathe a hypoxic gas mixture (16) would suggest that only leukocytes, and not endothelial cells, need to become hypoxic for leukocyte-endothelial cell adherence to occur. Alternatively, the dissociation between PmO2 and leukocyte-endothelial adherence could be explained by the release of a mediator triggered by the reduction of PO2 at some central site. The microvascular response to this hypothetical mediator would be independent of the PmO2 prevalent at the site where inflammation develops.
The present study continues our investigation of the relationship between local PmO2 and hypoxia-induced microvascular inflammation in the cremaster muscle. Experiments were designed to further explore the possibility that systemic hypoxia results in the release of an intermediary substance, which sets in motion the microvascular inflammatory response. Based on the observation that mast cell activation is a necessary event in the development of hypoxia-induced inflammation, we reasoned that if a mediator released from a distant site is responsible for the initiation of the microvascular inflammation in the cremaster, cremaster mast cells would be activated during reductions in PaO2, even if local PmO2 is elevated. Conversely, selective cremaster hypoxia in the presence of systemic normoxia would not be accompanied by mast cell activation.
Evidence of mast cell activation was obtained indirectly from the effects of known mast cell stimulants and stabilizers on leukocyte-endothelial adherence and confirmed by direct observation of mast cell degranulation.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Surgical preparation. Male Sprague-Dawley rats, 175–225 g, were anesthetized with urethane (1.5 g/kg im) after an overnight fast with free access to water. Body temperature was maintained at 36–38°C by using a homeothermic blanket system connected to an intrarectal temperature probe. PE-50 catheters were inserted in the jugular vein and the carotid artery. Lactated Ringer solution was infused continuously via the jugular vein at a rate of 2 ml/h. Arterial blood pressure was continuously monitored with a digital blood pressure monitor connected to the carotid artery catheter. A tracheotomy was performed, and PE-240 tubing was connected to a rodent nonrebreathing two-way valve. The animals breathed spontaneously throughout the experiment.
Intravital microscopy. The right cremaster muscle was prepared for intravital microscopy as described previously (4). The rat was placed on the platform of a Nikon E600 FN microscope, and the cremaster was spread over a hollow Lucite cylinder, the top of which was sealed with a glass slide. Water was circulated through the cylinder to maintain muscle temperature at 37°C. Muscle temperature was monitored continuously via a thermistor placed underneath the muscle. The cremaster was covered with Saran wrap throughout the experiment.
In experiments in which cremaster PmO2 was altered independently of systemic PO2, the cremaster was spread over a hollow plastic cylinder, through which warm, humidified gas of the desired PO2 was circulated, and covered by a plastic dome, through which the same gas was flushed. The muscle was not covered with Saran wrap in these experiments. Muscle temperature was maintained at 37°C by means of a heating lamp.
Images of the cremaster microcirculation (x40 objective) were recorded on a videocassette recorder with a time-date generator. Straight, unbranched venules of 100 µm in length and 20–40 µm in diameter, with fewer than three adherent leukocytes in a 100-µm segment and no adjacent lymphatics, were selected for microscopic observation. Venular diameter was measured by using a video caliper. An optical Doppler velocimeter was used to measure venular centerline red blood cell velocity. Average red blood cell velocity was calculated as centerline velocity/1.6 (5). Wall shear rate, which represents the force generated at the vessel wall by the movement of blood, was calculated as 8 x (average red blood cell velocity/venular diameter) (8). Adherent leukocytes were defined as leukocytes that remained stationary for >30 s. Leukocyte adherence was expressed as the number of adherent leukocytes per 100 µm of vessel length.
Measurement of PmO2. A method based on the PO2 dependence of phosphorescence lifetime was used to determine PmO2 (14). The measurement of PmO2 was carried out in separate experiments in which the cremaster was prepared as described above, but the microcirculation was not visualized with intravital microscopy. The oxyphor Pd-porphyrin dendrimer (R2) was injected intravenously (16 mg/kg). At this concentration, R2 binds completely to albumin (9); furthermore, R2 has a negative net charge, which facilitates restriction to the vascular space. Phosphorescence was measured by using a phosphorometer (Oxyspot, Medical Systems, Greenvale, NY) with a bifurcated light guide positioned 2–4 mm above the cremaster. The excitation light emitted from the light guide reached a circular area of the cremaster of 
1 mm in diameter and 500 µm deep. The phosphorescence signal was averaged over 200 ms for each measurement. PmO2 was measured every minute. The value of PmO2 obtained is a weighted average determined by the relative proportion of blood contained in the arterioles, capillaries, and venules of the muscle. An excellent correlation was observed between PO2 values obtained by using a PO2 electrode and the phosphorescence quenching method in the same blood sample (16).
Assessment of mast cell activation. In some experiments, the cremaster was prepared for histological visualization of mast cells. The muscle was excised and fixed in Zamboni's solution for 24–48 h, after which it was transferred to a 30% sucrose solution, frozen in dry ice, sliced in 20-µm-thick slices, and stained in 1% toluidine blue solution.
Experimental protocols. In all protocols, 45 min were allowed for the animals to recover from surgery. Approximately half of the experiments were directed to study leukocyte-endothelial interactions via intravital microscopy; in the remaining experiments, PmO2 was measured as described above. Arterial blood samples for measurement of pH, PO2, and PCO2 were obtained at the end of each experimental period.
Systemic hypoxia. These experiments consisted of a 20-min normoxic control period, a 10-min hypoxia period in which the animals spontaneously breathed 10% O2-90% N2, and a 10-min normoxic recovery period. PmO2 was measured every minute.
In a subset of experiments using this protocol, the mast cell stabilizer cromolyn (2 ml, 0.11 mg/ml) was applied topically to the cremaster muscle at 10 min of the normoxic control period, and the application was repeated immediately before the hypoxic and normoxic recovery periods.
Cremaster hypoxia, systemic normoxia. In these experiments, the animals breathed room air throughout the experiment. The cremaster was equilibrated as described above with a gas mixture of 10% O2-5% CO2-85% N2. After a 20-min control period, local hypoxia of the cremaster was produced by changing the gas mixture equilibrating the muscle to 95% N2-5% CO2 while the animal continued breathing room air. After 10 min of cremaster hypoxia, the gas mixture equilibrating the muscle was returned to 10% O2-5% CO2-85% N2.
In a second series of experiments using this protocol, the mast cell activator compound 48/80 (2 ml, 15 µg /ml) was applied topically immediately before the onset of cremaster hypoxia.
In a third series of experiments, cromolyn (2 ml, 0.11 mg/ml) was applied topically at 10 min of the normoxic control and immediately before hypoxia and recovery. Compound 48/80 (2 ml, 15 µg /ml) was applied topically immediately before the onset of local hypoxia.
Cremaster normoxia, systemic hypoxia. In these experiments, the cremaster was equilibrated with a gas mixture of 10% O2-5% CO2-85% N2 throughout the experiment. After a 20-min normoxic control period, the animal breathed 10% O2-90% N2 while the muscle continued to be equilibrated with 10% O2-5% CO2-85% N2. The period of systemic hypoxia lasted 10 min and was followed by a 10-min normoxic recovery period.
In a second series of experiments with this protocol, the mast cell stabilizer cromolyn (2 ml, 0.11 mg/ml) was applied topically at 10 min of the normoxic control and immediately before the hypoxia and recovery periods.
In a few experiments of each protocol, the muscles were prepared for histological examination of mast cells. In these cases, the cremaster was removed at the end of the experimental period (i.e., systemic hypoxia, cremaster hypoxia, etc.) before the onset of the recovery period.
Statistics. Data are presented as means ± SE. Data after a given treatment were compared with the corresponding pretreatment data by using a t-test for paired samples. Intergroup comparisons were made with a one-way ANOVA followed by the Bonferroni test for multiple comparisons. A P value of 
0.05 was considered to indicate a significant difference.
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RESULTS |
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Selective cremaster hypoxia in the presence of systemic normoxia was not associated with increased leukocyte-endothelial adherence (Fig. 3, top, n = 6). Equilibration of the cremaster with 95% N2-5% CO2 while the animals breathed room air produced a rapid and sustained decrease in cremaster PmO2, which reached 9 ± 2 Torr at 1 min and remained at 7 ± 1 Torr at 10 min of hypoxia (Fig. 3, bottom, n = 6). PaO2 values (Fig. 3, bottom) remained essentially unchanged during cremaster hypoxia. Topical application of the mast cell activator compound 48/80 during cremaster hypoxia resulted in a rapid and sustained increase in leukocyteendothelial adherence (Fig. 3, top, n = 6), which was effectively blocked by pretreatment with the mast cell stabilizer cromolyn applied topically (Fig. 3, top, n = 6). Neither compound 48/80 nor cromolyn modified the Pm O2 response to cremaster equilibration with 95% N2-5% CO2 (Fig. 3). Cremaster mast cells did not show evidence of degranulation during cremaster hypoxia at normoxic systemic PO2 (Fig. 4, left panel); however, topical application of compound 48/80 under the same conditions resulted in mast cell degranulation (Fig. 4, middle), which was blocked by cromolyn (Fig. 4, right).
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Systemic hypoxia produced a significant increase in leukocyte-endothelial adherence in cremaster venules (Fig. 5, top) even though cremaster PmO2 was maintained between 62 and 68 Torr throughout the experiment by equilibration of the cremaster with 10% O2-5% CO2-85% N2 (Fig. 5, bottom). Topical application of cromolyn completely prevented the increase in leukocyte-endothelial adherence (Fig. 5, top). Cremaster PmO2 values during cromolyn treatment (Fig. 5, bottom) were not significantly different from those seen in the untreated rats. Cremaster mast cells showed degranulation in rats of the systemic hypoxia/cremaster normoxia group (Fig. 6, left), which was prevented by topical pretreatment with cromolyn (Fig. 6, right).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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In this study, activation or inhibition of mast cells was inferred from the effects of cromolyn, a mast cell stabilizer, and compound 48/80, a mast cell activator, on the changes in leukocyte-endothelial adherence produced by the various experimental interventions. Both of these agents have been used widely for these purposes. Both cromolyn and compound 48/80 were applied topically so that only cremaster mast cells were influenced. The effects of cromolyn and compound 48/80 on leukocyte-endothelial adhesive interactions were correlated with histological images of mast cells obtained when the cremaster muscles were exposed to the different experimental conditions and the tissue samples obtained before the recovery periods were initiated.
We have previously observed that mast cells play a key role in the hypoxia-induced microvascular inflammation in the mesentery (19): mesenteric mast cells degranulate during hypoxia, and blockade of mast cell degranulation prevents or attenuates the inflammatory response. The data presented here show that mast cells play a similar role in the microvascular response to hypoxia in skeletal muscle. Recent preliminary data (10) show that brain mast cells also degranulate during reduction of inspired PO2. Taken together, these results suggest that mast cell activation is a generalized response to systemic hypoxia. In the present experiments, cromolyn prevented the increase in leukocyteendothelial adherence produced during systemic hypoxia (Fig. 1), suggesting that cremaster mast cells are activated in this condition and that mast cell activation mediates the increase in leukocyte-endothelial adhesive interactions of systemic hypoxia. This is supported by the observation of cremaster mast cell degranulation in the hypoxic untreated rats and its absence in the hypoxic rats pretreated with cromolyn (Fig. 2).
The absence of leukocyte-endothelial adherence during local cremaster hypoxia, its increase with topical application of compound 48/80 (Fig. 3), and the blockade of the effects of compound 48/80 with cromolyn indicate that, under the experimental conditions required to produce local cremaster hypoxia and systemic normoxia, mast cells are inactive but can be activated if adequately stimulated. The histological images shown in Fig. 4 are consistent with this conclusion: mast cells show no evidence of degranulation during local cremaster hypoxia and degranulate in response to topical application of compound 48/80; furthermore, topical cromolyn blocks this effect of compound 48/80 on mast cell degranulation (Fig. 4). The correlation between changes in mast cell activation and leukocyte-endothelial adherence indicate that the effects of compound 48/80 and of cromolyn on leukocyte-endothelial adhesive interactions are mediated through changes in mast cell activity. When cremaster PmO2 was maintained elevated during systemic hypoxia, leukocytes adhered to the venular endothelium to the same extent as when cremaster PmO2 was reduced in systemic hypoxia (compare Figs. 1 and 5). This response was also blocked by cromolyn, suggesting that cremaster mast cell activation during systemic hypoxia occurs independently of whether cremaster PmO2 is elevated or reduced.
The overall weight of the data presented in this study supports the idea that local PO2 in the cremaster is not the specific stimulus that sets in motion the microvascular inflammatory cascade that follows a reduction in inspired PO2, since the early steps of this process, i.e., stimulation of mast cells and subsequent increase in leukocyte-endothelial adhesive interactions, can be observed when cremaster Pmo2 is elevated; conversely, reduction of local PmO2 fails to set the inflammatory cascade in motion if PaO2 remains within the normal range. These results are consistent with our previous observations that a reduction in cremaster PmO2, produced by mechanical restriction of blood flow to the cremaster in the presence of normal PaO2, also failed to elicit increased leukocyte-endothelial adherence (15).
Our data are consistent with the idea that the microvascular inflammatory response to systemic hypoxia is mediated through the release of an intermediary substance, which starts the inflammatory cascade by inducing activation of mast cells. This study, however, does not provide evidence regarding the nature of such substance. The time course of the response suggests an agent that is stored or is rapidly synthesized; the widespread nature of the microvascular inflammation suggests a central site of release such as the lungs. It is clear that further study is necessary to determine the existence and nature of this mediator.
The data presented in this study are in apparent conflict with in vitro observations of increased leukocyteendothelial interactions in response to hypoxia. The microvascular response observed when intact animals are exposed to systemic hypoxia is quite rapid, with significant increases in ROS levels (20), mast cell degranulation (19), and leukocyte rolling and adherence to venular endothelium (22) occurring within 5 min of hypoxic exposure. In general, in vitro studies of leukocyte-endothelial adhesive interactions have been carried out by using longer hypoxic exposure periods (2, 3, 11, 12, 15, 17). It is possible that different mechanisms operate at various times after the onset of hypoxia and that low PO2 could influence slowly developing mechanisms, which do not operate within the time frame of this study.
In summary, the present studies indicate that mast cells participate in the cremaster microvascular inflammation that follows a reduction of inspired PO2 in the rat. The results indicate that mast cell stimulation occurs only when systemic PaO2 is reduced and is independent of the local PmO2. These data are consistent with the presence of an intermediary released from a distant site under conditions of systemic hypoxia, and which sets in motion the initial microvascular inflammatory response to hypoxia.
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DISCLOSURES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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FOOTNOTES |
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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.
Present address of R. Dix: Department of Biology, Olathe North High School, Olathe, KS 66061.
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REFERENCES |
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, Untreated animals, n = 6; 
, cromolyn-treated animals, n = 6. Cromolyn (2 ml, 0.11 mg/ml) was topically applied on the cremaster at 10 min of the normoxic control period and immediately before the hypoxia and the normoxic recovery periods. Bottom: time course of microvascular PO2 (PmO2). Untreated animals (
, compound 48/80 (C48/80; 2 ml, 15 µg/ml) applied topically immediately before the onset of hypoxia (n = 6); 
