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J Appl Physiol 83: 25-31, 1997;
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Journal of Applied Physiology
Vol. 83, No. 1, pp. 25-31, July 1997
PULMONARY CIRCULATION AND LUNG FLUID BALANCE

Exaggerated pulmonary hypertension with monocrotaline in rats susceptible to chronic mountain sickness

Gene L. Colice, Nicholas Hill, Yan-Jie Lee, Hongkai Du, James Klinger, James C. Leiter, and Lo-Chang Ou

Departments of Medicine and Physiology, Dartmouth Medical School, Lebanon, New Hampshire 03756; and Rhode Island Hospital and Brown University, Providence, Rhode Island 02903

ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Colice, Gene L., Nicholas Hill, Yan-Jie Lee, Hongkai Du, James Klinger, James C. Leiter, and Lo-Chang Ou. Exaggerated pulmonary hypertension with monocrotaline in rats susceptible to chronic mountain sickness. J. Appl. Physiol. 83(1): 25-31, 1997.---Hilltop (H) strain Sprague-Dawley rats are more susceptible to chronic mountain sickness than are the Madison (M) strain rats. It is unclear what role pulmonary vascular remodeling, polycythemia, and hypoxia-induced vasoconstriction play in mediating the more severe pulmonary hypertension that develops in the H rats during chronic hypoxia. It is also unclear whether the increased sensitivity of the H rats to chronic mountain sickness is specific for a hypoxia effect or, instead, reflects a general propensity toward the development of pulmonary hypertension. Monocrotaline (MCT) causes pulmonary vascular remodeling and pulmonary hypertension. We hypothesized that the difference in the pulmonary vascular response to chronic hypoxia between H and M rats reflects an increased sensitivity of the H rats to any pulmonary hypertensive stimuli. Consequently, we expected the two strains to also differ in their susceptibility to MCT-induced pulmonary hypertension. Pulmonary arterial pressures in conscious H and M rats were measured 3 wk after a single dose of MCT, exposure to a simulated high altitude of 18,000 ft (barometric pressure = 380 mmHg), and administration of a single dose of saline as a placebo. The H rats had significantly higher pulmonary arterial pressures and right ventricular weights after MCT and chronic hypoxia than did the M rats. The H rats also had more pulmonary vascular remodeling, i.e., greater wall thickness as a percentage of vessel diameter, after MCT and chronic hypoxia than did the M rats. The H rats had significantly lower arterial PO2 than did the M rats after MCT, but the degree of hypoxemia was mild [arterial PO2 of 72.5 ± 0.8 (SE) Torr for H rats vs. 77.4 ± 0.8 Torr for M rats after MCT]. The H rats had lower arterial PCO2 and larger minute ventilation values than did the M rats after MCT. These ventilatory differences suggest that MCT caused more severe pulmonary vascular damage in the H rats than in the M rats. These data support the hypothesis that the H rats have a general propensity to develop pulmonary hypertension and suggest that differences in pulmonary vascular remodeling account for the increased susceptibility of H rats, compared with M rats, to both MCT and chronic hypoxia-induced pulmonary hypertension.

chronic hypoxia; ventilation; pulmonary vascular histology

INTRODUCTION

HILLTOP (H) strain Sprague-Dawley rats are more susceptible to chronic mountain sickness than are the Madison (M) strain rats (28). It is unclear what role pulmonary vascular remodeling, polycythemia, and hypoxia-induced vasoconstriction play in mediating the more severe pulmonary hypertension that develops in the H rats during chronic hypoxia. After 14 days of hypoxia, H rats have evidence of more pulmonary vascular remodeling than do M rats, i.e., greater medial thickness of intra-acinar and preacinar arteries and muscularity of intra-acinar arteries (15). The H rats also have more marked polycythemia (25) and a larger component of hypoxia-related pulmonary vasoconstriction (27) than do M rats with chronic hypoxia. It is also unclear whether the increased sensitivity of the H rats to chronic mountain sickness is specific for a hypoxia effect or, instead, reflects a general propensity toward the development of pulmonary hypertension.

We hypothesized that the difference in the pulmonary vascular response to chronic hypoxia between H and M rats reflects an increased sensitivity of the H rats to any pulmonary hypertensive stimuli. Consequently, we expected the two strains to also differ in their susceptibility to monocrotaline (MCT)-induced pulmonary hypertension. MCT is a plant-derived pyrrole alkaloid that causes pulmonary vascular remodeling and pulmonary hypertension. If this hypothesis were confirmed, comparisons between the H and M rats would be useful in the examination of the relationship between pulmonary vascular remodeling and pulmonary hypertension.

METHODS

General. All experiments were performed on paired groups of male Sprague-Dawley rats obtained from Harlan Laboratories (Madison, WI; M rats) and Hilltop Laboratories (Scottsdale, PA; H rats). The rats weighed between 300 and 380 g, and the paired groups were of similar age. The animals were housed in standard rat cages with three animals in each and were allowed several days rest after travel. They were randomly assigned to chronic hypoxia, MCT, or control exposures. The H and M rats chosen for the chronic hypoxia portion of the study were exposed to a simulated altitude of 18,000 feet (barometric pressure = 380 mmHg) in well-ventilated, temperature-controlled decompression chambers for 21 days. Free access to a standard rat diet and water was allowed throughout the exposure period. The chambers were opened every 2 days to replenish food and water supplies, weigh the animals, and provide clean cages. Venous blood was obtained at the end of the study for measurements of packed cell volume (hematocrit).

H and M rats randomized to MCT and control exposures received either a single subcutaneous injection of MCT (40 mg/kg body wt) or a single subcutaneous injection of 0.9% saline, equivalent in volume to the MCT injections. The MCT (Sigma Chemical) was dissolved in distilled water to a final concentration of 100 mg/ml, and the pH was adjusted with 0.5 N HCl to 7.40. The animals in the MCT and control groups were handled similarly to the chronic hypoxia group but under sea-level conditions. They were housed in the same laboratory, given free access to a standard rat diet and water, weighed, and provided clean cages every 2 days for 21 days before being studied.

Surgical preparation. Catheters were placed in the pulmonary and femoral arteries on the 19th or 20th day of hypoxic exposure or after MCT or 0.9% saline injection. The techniques for inserting catheters into the pulmonary artery have been well established in our laboratory (27). Briefly, after anesthesia with ketamine (60 mg/kg body wt im) and pentobarbital sodium (20 mg/kg body wt ip), the right external jugular vein was isolated under sterile conditions and cannulated with an introducer (PE-90). The introducer was advanced into the right ventricle. Position of the introducer in the right ventricle was confirmed by the characteristic pressure curves during continuous monitoring of vascular pressure (Grass model 7 polygraph and Statham transducer). A Silastic catheter (0.012 in. ID) was advanced through the introducer and carried by blood flow into the pulmonary artery. After position of the catheter in the pulmonary artery had been confirmed by identification of characteristic pressure tracings, the introducer was removed and the catheter was exteriorized to the neck and secured in place. On completion of all studies, position of the catheter in the pulmonary artery was confirmed at autopsy. The femoral artery was isolated under sterile conditions and cannulated with a catheter (Tygon tubing S-54-HL, 0.15 in. ID). Characteristic vascular pressure tracings confirmed appropriate position. The catheter was also exteriorized to the neck and secured in place. Animals were allowed to recover for at least 24 h before vascular pressure measurements were performed.

Pulmonary arterial pressure measurements. The rats randomized to chronic hypoxia were studied after 21 days at simulated high altitude. The rats assigned to the MCT and control groups were studied 21 days after the appropriate injections. Conscious rats were placed in plastic hooded restraining cages. Inspired gas supplied to these cages was either 10.5% inspired O2 fraction (FIO2) for the chronic hypoxia group or room air for the MCT and control groups. Inspired gas within the plastic hood was constantly measured (O2 analyzer, Beckman, Fullerton, CA) during hemodynamic measurements. Animals were allowed to rest quietly within the restraining cages for at least 20 min before pulmonary arterial pressure measurements were obtained (Grass model 7 polygraph and Statham transducer).

Right ventricle weight. On completion of the appropriate measurements, the rats were killed. The heart was removed and was placed in a 4% formaldehyde solution for 3 days. The atria were then trimmed away from the ventricles and discarded. The right ventricle was dissected from the left ventricle and septum, and these two portions were weighed separately. Right ventricular weight was expressed in proportion to the left ventricle plus septum weight and body weight.

Vessel histology. After completion of appropriate exposures, each rat was anesthetized (see Surgical preparation), and the chest was opened by median sternotomy. The rat was exsanguinated, and catheters were inserted into the pulmonary artery and trachea. The heart and lungs were removed en bloc. A double-fixation technique was used in which 10% buffered Formalin was infused intratracheally at 20 cmH2O and intraarterially at 75 cmH2O. After 1 h of infusion the left lung was removed and embedded with paraffin. Sagittal sections of the left middle and lower lung fields were taken and stained with hematoxylin and eosin or with elastic tissue stain.

Vessel histology was analyzed by using image-analysis techniques. Images of microscopic sections were digitized by using Biovision Software (Perceptics, Knoxville, TN). Vessel wall thickness and diameter were determined for vessels between 50 and 250 µm in diameter. Vessels were landmarked according to adjacent airways, terminal bronchiole, respiratory bronchiole, or alveolar duct (8). Percent wall thickness was determined as twice the average vessel wall thickness, divided by the vessel diameter, and multiplied by 100.

Ventilatory and arterial blood-gas measurements. After exposure periods, animals were placed individually in a Plexiglas plethysmographic box, and ventilatory measurements were performed by using the plethysmographic technique described by Bartlett and Tenney (2). A humidified gas mixture, either 10.5% FIO2 for the rats randomized to chronic hypoxia or room air for the control animals, was flushed through the box for 15-20 min. The MCT-treated animals were studied twice, first while breathing room air and again while breathing 10.5% FIO2. At the end of this equilibration period, while the animals were resting quietly, the inlet and outlet tubes of the box were clamped and pressure changes during respiration were recorded (Grass model 7 polygraph and Statham transducer). From these pressure recordings, respiratory rate was directly counted and tidal volume and minute ventilation were calculated. During these studies, the O2 concentration within the box was monitored (O2 analyzer, Beckman). Adequate gas flow and mixing throughout the box were provided to maintain the inspired CO2 fraction below 0.03%.

After ventilatory measurements, arterial blood samples of at least 300 µl were collected from the femoral arterial catheter in a heparinized syringe. Blood arterial PO2 (PaO2), arterial PCO2 (PaCO2), and pH were measured by using microelectrodes (Radiometer, Cleveland, OH) at 37°C. Arterial blood samples were obtained from chronically hypoxic rats while they were breathing 10.5% FIO2, control animals during room air inhalation, and MCT-treated animals while they were inspiring first room air and then 10.5% FIO2

Statistical analyses. All data are expressed as means ± SE. Student's t-test was used to compare differences between M and H rats and among the MCT, chronic hypoxia, and control groups. Bonferroni's correction was used when appropriate to adjust for multiple comparisons. Differences were considered statistically significant when P < 0.05.

RESULTS

General. All animals survived the exposure periods. Initial body weights were larger for M rats than H rats but were similar within strains for the three treatment groups (Table 1). At the end of the experiment, there were significant differences in weight within strains both between the MCT and chronic hypoxia groups and controls and between the MCT and chronic hypoxia groups. Baseline hematocrit was similar for M and H rats (Table 1). Hematocrit was significantly greater in MCT-treated rats than in controls and in chronic hypoxia-exposed animals than in both MCT and control animals. H rats had significantly higher hematocrits than did M rats after chronic hypoxia.

Table  1.   Effects of monocrotaline and chronic hypoxia on body weight and hematocrit in Madison and Hilltop rats
n Initial Body Wt, g Final Body Wt, g Final Hct, % 
Madison rats
  Saline 6 334.7 ± 2.8  424.5 ± 14.6  42.9 ± 0.3 
  MCT 6 341.2 ± 4.1  389.8 ± 8.3* 48.3 ± 0.8*
  Hypoxia 5 333.5 ± 2.2  336.5 ± 2.2*, dagger 64.4 ± 0.4*, dagger
Hilltop rats
  Saline 6 310.3 ± 3.6  416.2 ± 9.3  41.7 ± 0.8 
  MCT 6 316.3 ± 4.4  394.0 ± 9.2* 49.3 ± 0.5*
  Hypoxia 5 314.5 ± 4.1  357.8 ± 5.9*, dagger 77.0 ± 0.9*, dagger , Dagger
Values are means ± SE; n, no. of animals studied. MCT, monocrotaline; Hct, hematocrit. * P < 0.01 vs. saline within each strain. dagger P < 0.01 vs. MCT within each strain. Dagger P < 0.01 vs. Madison within a particular treatment.

Pulmonary arterial pressure. Chronic hypoxia and treatment with MCT caused significant increases in pulmonary arterial pressure compared with saline-treated control animals in both the M and H rats. Significant differences were not found between pulmonary arterial pressures in M rats randomized to chronic hypoxia and MCT. Significant differences were not found between pulmonary arterial pressures in H rats exposed to chronic hypoxia and MCT, but these pressure readings were significantly higher than those found in M rats given the same exposure. Pulmonary arterial systolic pressures for the different exposures are shown in Fig. 1. Pulmonary arterial diastolic pressures were 16.4 ± 0.7 (SE), 22.5 ± 0.9, and 27.7 ± 0.3 mmHg for the M rats after control, MCT, and chronic hypoxia exposures, respectively; they were 15.7 ± 0.8 36.6 ± 2.9, and 35.8 ± 1.4 mmHg for H rats after control, MCT and chronic hypoxia conditions, respectively.
Fig. 1. Pulmonary arterial systolic pressures for Madison (hatched bars) and Hilltop (open bars) rats obtained after 21 days exposure to simulated high altitude of 18,000 ft (hypoxia) and 21 days after subcutaneous injection of either saline (control) or monocrotaline (MCT) are shown. Similar changes (not shown) were seen for pulmonary arterial diastolic and mean pressures. Pulmonary arterial pressures were obtained while animals were conscious and resting quietly in hooded restraining cage and breathing either room air (control and MCT groups) or 10.5% inspired O2 fraction (hypoxia group). Values are means ± SE for 5 rats in each strain for saline, 6 rats in each strain for hypoxia, and 10 Hilltop rats and 6 Madison rats for MCT. * P < 0.01 vs. control within each strain. Dagger  P < 0.01 vs. Madison rats within a particular treatment.
[View Larger Version of this Image (29K GIF file)]

Right ventricular weight. Right ventricular weight measurements closely paralleled the pulmonary arterial pressure readings. Chronic hypoxia and MCT caused right ventricular hypertrophy, expressed as either the right ventricle-to-left ventricle plus septum (Fig. 2) or the right ventricle-to-body weight ratio (data not shown), in both M and H rats. Chronic hypoxia and MCT affected each strain to a similar degree, but the H rats had significantly greater right ventricular hypertrophy than did the M rats.
Fig. 2. Right ventricular (RV) weight-to-left ventricular (LV) plus septum (S) weight ratios (RV/LV+S) are shown for Madison (hatched bars) and Hilltop (open bars) rats after 21-day exposure to simulated high altitude of 18,000 ft (hypoxia) and 21 days after subcutaneous injection of either saline (control) or MCT. Values are means ± SE for 5 rats in each strain for saline, 6 rats in each strain for hypoxia, and 10 Hilltop rats and 6 Madison rats for MCT. * P < 0.01 vs. control within each strain. Dagger  P < 0.01 vs. Madison rats within a particular treatment.
[View Larger Version of this Image (26K GIF file)]

Vessel histology. Analysis of vessels landmarked to alveolar ducts (between 43 and 87 vessels counted from 3 to 5 lungs for each rat strain and exposure) reflected the patterns seen with pulmonary arterial pressure measurements and right ventricular weights (Fig. 3). Wall thickness as a percentage of vessel diameter was significantly greater after both MCT and chronic hypoxia than after saline in both rat strains. The results within strains were similar for MCT and chronic hypoxia, but the H rats had greater wall thicknesses than did the M rats for each exposure. Similar trends were seen for analyses of vessels landmarked to respiratory and terminal bronchioles (between 17 and 35 vessels counted from 3 to 5 lungs for each rat strain and exposure).
Fig. 3. Wall thickness, expressed as percentage of vessel diameter, is shown for vessels landmarked to terminal bronchioles, respiratory bronchioles, and alveolar ducts after 21 days at simulated altitude of 18,000 ft (hypoxia) and 21 days after a subcutaneous injection of either saline (control) or MCT. Values are means ± SE. Hatched bars, Madison rats; open bars, Hilltop rats. * P < 0.01 vs. control within each strain. Dagger  P < 0.01 vs. Madison rats within a particular treatment.
[View Larger Version of this Image (36K GIF file)]

Ventilation and arterial blood gases. The H rats randomized to saline control had higher respiratory rates than did the saline-treated M rats (Table 2). Respiratory rates and minute ventilations were significantly higher for H and M MCT-treated animals than for their saline controls during room air inhalation. The H rats receiving MCT had significantly greater respiratory rates and minute ventilations than did the MCT-treated M rats while they were breathing room air. The H rats randomized to MCT also had significantly larger tidal volumes and minute ventilations than did M rats treated with MCT under hypoxic gas inhalation conditions. As expected, both M and H rats randomized to chronic hypoxia and studied while they breathing hypoxic gas had higher respiratory rates, tidal volumes, and minute ventilations than did the saline-treated controls breathing room air. All ventilatory measurements, except tidal volume in the M rats, were significantly lower in chronically hypoxic H and M rats breathing hypoxic gas than in MCT-treated H and M rats exposed to hypoxic gas. The chronic hypoxia H rats had higher respiratory rates, lower tidal volumes, and similar minute ventilations than did the M rats in this exposure.

Table  2.   Effects of monocrotaline and chronic hypoxia on ventilatory parameters of Madison and Hilltop rats
Room Air
Hypoxic Gas
f, breaths/min VT, ml/100 g body wt  VE, ml · min-1 · 100 g body wt-1 f, breaths/min VT, ml/100 g body wt  VE, ml · min-1 · 100 g body wt-1
Madison rats
  Saline 95.0 ± 2.3 (7) 2.2 ± 0.1 (7) 69.8 ± 2.4 (7)
  MCT 137.0 ± 6.1* (6) 2.1 ± 0.1 (6) 84.0 ± 3.9* (6) 213.0 ± 5.7 (6) 2.7 ± 0.1 (6) 163.2 ± 4.6 (6)
  Hypoxia 137.0 ± 3.8*, dagger  (5) 3.1 ± 0.1*, dagger  (5) 136.8 ± 4.9*, dagger  (5)
Hilltop rats
  Saline 126.5 ± 9.5 (6) 1.9 ± 0.2 (6) 72.0 ± 4.7 (6)
  MCT 187.0 ± 10.1*, Dagger  (6) 2.3 ± 0.1 (6) 122.0 ± 13.4*, Dagger  (6) 219.0 ± 7.0 (6) 3.5 ± 0.1Dagger  (6) 212.9 ± 5.0Dagger  (6)
  Hypoxia 177.8 ± 3.2*, dagger , Dagger  (5) 2.4 ± 0.1dagger , Dagger  (5) 122.8 ± 6.4*, dagger  (5)
Values are means ± SE; nos. in parentheses are no. of animals studied. f, Breathing frequency; VT, tidal volume; VE, minute ventilation. * P < 0.01 vs. saline within each strain under room air conditions. dagger P < 0.01 vs. MCT within each strain under hypoxic gas conditions. Dagger P < 0.01 vs. Madison rats within a particular treatment and gas exposure.

The M and H rats randomized to MCT had significantly lower PaO2 values while breathing room air than did the saline controls (Table 3). Under room air conditions, the H rats receiving MCT had significantly lower PaO2 and PaCO2 values than did the M rats given MCT and lower PaCO2 values than did saline controls. The H and M rats receiving MCT and studied while hypoxic had similarly low PaO2 and PaCO2 values and high pH measurements as did the chronically hypoxic animals studied under the same conditions.

Table  3.   Effects of monocrotaline and chronic hypoxia on arterial blood-gas measurements of Madison and Hilltop rats
Room Air
Hypoxic Gas
PaO2, Torr PaCO2, Torr pH PaO2, Torr PaCO2 , Torr pH
Madison rats
  Saline 86.6 ± 1.7 (6) 38.8 ± 0.4 (7) 7.39 ± 0.01 (7)
  MCT 77.4 ± 0.8* (5) 36.9 ± 2.3 (5) 7.39 ± 0.03 (5) 38.9 ± 1.3 (6) 19.8 ± 1.3 (6) 7.50 ± 0.02 (5)
  Hypoxia 45.8 ± 2.0 (5) 23.5 ± 0.9 (5) 7.46 ± 0.01 (5)
Hilltop rats
  Saline 88.3 ± 1.2 (7) 38.7 ± 0.5 (7) 7.39 ± 0.01 (7)
  MCT 72.5 ± 0.8*, dagger  (6) 29.8 ± 1.4*, dagger  (6) 7.39 ± 0.01 (6) 40.7 ± 2.0 (6) 19.3 ± 1.8 (6) 7.48 ± 0.04 (6)
  Hypoxia 37.8 ± 1.1 (5) 25.0 ± 0.6 (5) 7.43 ± 0.01 (5)
Values are means ± SE; nos. in parentheses are no. of animals studied. PaO2, arterial PO2; PaCO2, arterial PCO2. * P < 0.01 vs. saline within each strain. dagger P < 0.01 vs. Madison rats within a particular treatment.

The saline control M and H rats had similar minute ventilation and PaO2 values while breathing room air (Fig. 4). The MCT-treated H rats had a significantly higher minute ventilation and lower PaO2 values under room air conditions than did the MCT-treated M rats. During hypoxic gas inhalation, both the M and H MCT- treated rats had similarly low PaO2 values, but the H rats had significantly higher minute ventilations.
Fig. 4. Arterial PO2 (PaO2; A) and minute ventilation (B) for Madison (black-square) and Hilltop (bullet ) rats from saline (control) and MCT groups are shown. Control animals were studied only while they were breathing room air. MCT-treated animals were studied while they were breathing room air (RA) and again while they were breathing 10.5% inspired O2 fraction (HY). Values are means ± SE. bw, Body wt. Dagger  P < 0.01 vs. Madison rats within a particular treatment.
[View Larger Version of this Image (15K GIF file)]

DISCUSSION

This and previous studies have shown that H rats develop more severe pulmonary hypertension with chronic hypoxia than do M rats (27, 28). The results of this study further indicate that H rats are more sensitive to the pulmonary hypertensive effects of MCT than are M rats. Both pulmonary arterial pressures and right ventricular weights were greater in the H rats after chronic hypoxia and MCT than in the M rats. Analysis of pulmonary vessel histology indicates more extensive pulmonary vascular remodeling in the H rats after both chronic hypoxia and MCT than in the M rats. These data support the hypothesis that the H rats have a general propensity to develop pulmonary hypertension and also suggest that greater pulmonary vascular remodeling accounts for the increased susceptibility of H rats, compared with M rats, to both MCT and chronic hypoxia-induced pulmonary hypertension.

There are differences and similarities between MCT- and hypoxia-induced pulmonary hypertension. Hypoxia causes acute pulmonary vasoconstriction, but pulmonary arterial pressure has not been reported to increase substantially within the first week or two after MCT administration (4, 18, 33). There are intriguing similarities, however, in the patterns of histological abnormalities found with each of these causes of pulmonary hypertension. These similarities are important because the enhanced sensitivity of the H rats to pulmonary hypertensive stimuli may be in a pathway common to the development of both hypoxia- and MCT-induced pulmonary hypertension.

Electron-microscopic studies have described similar acute injury patterns in pulmonary vascular endothelial cells after MCT administration and exposure to hypoxia. After MCT, endothelial cells of pulmonary arterioles and capillaries are described as swollen and containing numerous large vesicles. These swollen endothelial cells protrude into capillary lumens, possibly obstructing flow (12, 17, 35). After exposure to hypoxia, pulmonary capillary endothelial cells also swell and accumulate large vesicles (10, 19). Swollen endothelial cells, along with protrusion of large fluid-filled blebs from the basal lamina, appear to obstruct the lumen of small pulmonary vessels in hypoxic animals (5, 30, 32).

MCT administration appears to cause thrombus formation in pulmonary arterioles and capillaries. Endothelial cell swelling presumably reflects direct toxicity due to MCT (9) and may be the initiating factor for thrombus formation (14, 17). Consistent with these observations has been the correlation noted between the fall in circulating platelet count after MCT and the extent of endothelial cell damage (7). Electron microscopy also reveals accumulation of platelets along pulmonary arteriole endothelial cells within hours after the onset of hypoxia. Accumulation of platelets in hypoxic pulmonary vessels corresponds temporally with endothelial cell swelling (31).

Within days of MCT administration (4, 7, 18) and onset of chronic hypoxia (29), increased medial thickening of muscular pulmonary arteries and muscularization of normally nonmuscular pulmonary arteries may be recognized by using light microscopy. These histological changes are similar for the two different pulmonary vascular insults (8, 18, 20, 29). The degree of histological change has been found to directly correspond to the increase in pulmonary arterial pressure (30). This and previous work also shows a direct relationship between the extent of pulmonary vacular remodeling and pulmonary hypertension. Our laboratory has shown that the H rats have greater increases in pulmonary arterial pressure and more extensive medial thickening and muscularization after chronic hypoxia than do the M rats (15). Analysis of pulmonary vessel histology in this study confirms that finding and further shows that H rats have greater increases in pulmonary arterial pressure and vessel wall thickness than does the M strain after MCT. These histological findings were most apparant in the alveolar ducts.

The mediators of pulmonary vascular remodeling are not well understood, but presently available evidence suggests similar mechanisms may be involved in both MCT- and chronic hypoxia-induced histological changes. Polyamines are cell growth and differentiation factors. Pulmonary polyamine content increases after both MCT and chronic hypoxia. Administration of alpha -difluoromethylornithine, an inhibitor of ornithine decarboxylase, a critical enzyme in the polyamine biosynthetic pathway, attenuates the increase in pulmonary arterial pressures usually seen after MCT and chronic hypoxia (1, 22). Elastolytic activity in pulmonary arteries increases after MCT administration (34) and chronic hypoxia (16). Elastase inhibitors reduced the pulmonary arterial pressure and histological changes in both MCT- (37) and chronic hypoxia-induced pulmonary hypertension (16). Platelet-activating factor antagonists have also been found to reduce the pulmonary arterial pressure changes caused by MCT (23) and chronic hypoxia (24). Endothelin has been reported to play an important role in the pulmonary vascular remodeling after chronic hypoxia (3) and MCT (21) in the rat.

The hematopoietic responses of H and M rats in the present study of simulated high altitude are similar to those reported by our laboratory previously (25). The H rats had a more marked increase in hematocrit than did the M rats. This excessive polycythemia has been associated in the H rats with lower PaO2 values and with higher respiratory rates and lower tidal volumes than in M rats with chronic hypoxia (25). These differences in ventilatory responses, between the H and M strains, to chronic hypoxia were found in this study as well. Both rat strains had a small, but significant, increase in hematocrit after MCT. Previous studies have noted an increase (6) or no change (11, 18) in hematocrit after MCT administration. An explanation for the increase in hematocrit in this study is not readily apparent. The mild hypoxemia found in MCT-treated M and H animals should not have caused the increase in hematocrit. Because the increase in hematocrit was small and equivalent in the two rat strains, it is unlikely that changes in hematocrit contributed to the substantial differences in pulmonary arterial pressure found between the M and H rats.

The H rats had significantly lower PaO2 after MCT than did the M rats. Both the M and H MCT-treated rats had lower PaO2 values than did the saline-treated control groups. Previous studies have shown different effects of MCT on PaO2. Some investigators (11) found no significant change in PaO2, but these measurements were obtained within 2 wk of MCT administration. This may have been too early to evaluate the complete effect of MCT on pulmonary vessels and gas exchange. Although the MCT effect on pulmonary arterial pressure may begin within 2 wk, pulmonary hypertension becomes most evident 3 or more wk after MCT administration (4, 33). Three weeks after MCT, PaO2 declines significantly (6, 18). Hill et al. (6) suggested that the fall in PaO2 may have contributed to the MCT-induced pulmonary hypertension because supplemental O2 minimized changes in PaO2 and pulmonary arterial pressure in MCT-treated rats. Unfortunately, measurements of arterial blood gases in that study (6) were obtained in anesthetized rats and may have underestimated ambulatory values. Arterial blood gases in this study were obtained from conscious animals. Mean PaO2 values for both H and M rats were above 70 Torr. This degree of hypoxemia should not have contributed to pulmonary hypertension (36). This point further supports the conclusion that the enhanced sensitivity of the H rat to hypoxia and MCT-induced pulmonary hypertension is general and not specific for hypoxia alone.

Besides a greater fall in PaO2, the H rats also had lower PaCO2 values than did the M rats after MCT. This indicates a wider alveolar-arterial O2 difference in the H rats. Consistent with these findings, the H rats also had higher minute ventilation after MCT under both room air and hypoxic conditions than did the M rats. As shown in Fig. 4, H rats given MCT required a significantly larger minute ventilation to maintain their PaO2 during hypoxic gas inhalation than did M rats. Similarly, PaO2 was lower in the H rats than in the M rats during normoxic gas inhalation despite a greater minute ventilation. These observations are all consistent with more extensive damage after MCT at the gas-exchange interface, e.g., the pulmonary microcirculation, in the H rats than in the M rats. However, because we did not measure airway resistance and compliance, we cannot exclude an effect by MCT on pulmonary mechanical properties (13).

In summary, the results of this study show that H rats develop more severe pulmonary hypertension and pulmonary vascular remodeling after both MCT and chronic hypoxia than do M rats. The exaggerated pulmonary hypertensive effect of MCT in the H rats is not confounded by excessive polycythemia or hypoxemia, suggesting an enhanced susceptibility in the H rats to pulmonary vascular remodeling stimuli in general. Understanding mechanisms responsible for the greater development of pulmonary vascular remodeling in the H strain may provide insights into the pathogenesis of pulmonary hypertension. Similarities in the time course and magnitude of pulmonary arterial pressure change in the two rat strains after MCT and chronic hypoxia suggest that this rat model may be useful in future studies on the pathogenesis of pulmonary hypertension. The finding of a heightened pulmonary vascular response to various pulmonary hypertensive stimuli in a rat model with a propensity to chronic mountain sickness may prove useful in understanding the human form of this disease.

FOOTNOTES

Address for reprint requests: G. L. Colice, 3M Pharmaceuticals, 3M Center, 270-3A-01, St. Paul, MN 55144.

Received 28 May 1996; accepted in final form 24 February 1996.

REFERENCES

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