LLU&MC Scope, Autumn 1997, editors

by Lawrence D. Longo, MD
Director, Center for Perinatal Biology,
departments of obstetrics and gynecology and physiology, School of Medicine

Lawrence D. Longo, MD, spearheads studies by Center for Perinatal Biology researchers who use the high altitudes of California's White Mountain to explore acclimatization responses in the fetus and adult.

As mountain climbers and skiers know all too well, dire results may strike even the fittest when they leave their lowland turfs for a mountaintop experience. The diminished oxygen pressure at high altitudes may cause them to lose their appetite and incur breathlessness, dizziness, headaches, insomnia, and nausea. The brain may swell and lungs fill with fluid. Extreme cases may cause disorientation, vomiting, convulsions, and death.

For years, the mystery of how unborn babies escape these consequences when they exist in an environment known to have low levels of oxygen has intrigued scientific researchers.

Nearly 140 million people live at high altitudes worldwide, and more than 35 million travel to high altitudes each year. High altitudes are defined as elevations above 8,000 feet (or about 2,500 meters) since, at these levels, oxygen saturation of hemoglobin in the blood usually begins to fall below sea-level values. Air travel exposes even more people to moderate altitudes, as airplane cabins are routinely maintained at about 8,000 feet to conserve fuel. Populations reside permanently at high altitudes in the Rocky Mountains of North and Central America, the Andes of South America, the East African highlands, and the Asian Himalayan plateau.

The predominant characteristic of the high-altitude environment, hypoxia, results from the decreased partial pressure of oxygen in the atmosphere. The health effects of high altitude are of interest from public health, clinical, and basic science perspectives. Historically, decreased fertility and fecundity have been reported from high altitude. Current data indicate that intrauterine growth retardation, neonatal stroke, and intraventricular hemorrhage, preeclampsia, and other complications of fetal and maternal life are more common at high altitude.

Thus, studies of the effects of high altitude on reproduction have relevance for understanding fetal and maternal complications of pregnancy as well as other conditions involving the heart, lungs, blood, and metabolic pathways involved in oxygen transport or utilization. A broad range of disciplines--including physiology, biochemistry, genetics, and anthropology--are actively involved in investigating the mechanisms by which human populations respond and adapt to the conditions of reduced oxygen at high altitude.

Under normal conditions, the developing fetus in utero exists in an environment which traditionally has been considered hypoxic, or lacking in oxygen. For instance, fetal arterial oxygen tension (or partial pressure) is about 25 Torr (the value for a healthy adult is about 100 Torr), equivalent to that of a person on the summit of Mt. Everest. This fact has given rise to the concept of the fetus being at "Mt. Everest in utero."

Thus, the question arises, if in a pregnant woman at sea level the fetal oxygen tension is similar to that of an adult at high altitude, how can the fetus grow and develop normally when the mother is at high altitude, where the oxygen supply is less than normal? This question, and the related issues of how placental exchange of respiratory gases, the fetal cardiovascular system, and some of its endocrine organs adapt to supply the fetal brain with oxygen and other nutrients, has occupied the attention of investigators in the Center for Perinatal Biology.

During the past decade, Loma Linda University has emerged as one of the world centers for studying both maternal and fetal responses to the hypoxia of sustained exposure to high altitude (so-called "acclimatization"). While applicable to the study of high altitude per se, this work is particularly germane to pregnancy in which hypoxia is a feature, such as the fetuses of women who smoke, those who are anemic or malnourished, those with certain heart or lung diseases, and those in which the mothers engage in strenuous physical activity or heavy work, or have preeclampsia or other diseases. In many such cases the fetuses and newborns are small for gestational age, and may suffer severe neurological or cardiovascular problems.

Thus, the problems of chronic hypoxia, and the adaptive mechanisms whereby the mother and fetus attempt to maintain oxygen delivery to the tissues and cells, is of great clinical relevance as well as being of fundamental biologic importance. Although a number of investigators have explored the various acclimatization responses in the adult, for pregnancy, and particularly in regards to fetal development, there have been few such studies.

Over the short-term, the healthy adult deals with high altitude or other forms of hypoxia by hyperventilating, and to a lesser extent by increasing cardiac output (the amount of blood the heart pumps to the tissues). More long-term acclimatization responses include increasing the erythrocyte mass (e.g., the concentration of hemoglobin in the circulating red blood cells), decreasing hemoglobin's affinity for oxygen, and alteration of excretion of certain ions and metabolites by the kidney. For the fetus in utero which does not respire air, and which cannot increase its cardiac output which is normally above a level several times that of the adult, these options are not available.

For the past decade, in studies supported by the National Institutes of Health and the American Heart Association, our group has explored some of these acclimatization responses in the fetus, and the mechanisms by which they operate. Although ideally we would have liked to perform studies at very high altitudes, such as in Tibet or the Himalayas, that was not feasible. Also, it was not practical for us to work at altitudes of 15,000 to 16,000 feet elevation, such as on the altiplano of Peru or Chile. Nonetheless, California is blessed by having a high-altitude research station on White Mountain, just east of Bishop. White Mountain itself is 14,246 feet (4,342 meters) high. The research station is at 12,532 feet (3,820 meters), just north of the Patriarch Grove area where ancient bristlecone pine trees grow. The station, operated by the University of California under the directorship of Frank L. Powell, Jr., PhD, is used by astrophysicists, geologists, botanists, zoologists, and other biologists for research studies.

In 1988, we first transported some pregnant sheep up to the research station to study the maternal and fetal responses to this altitude. Sheep were chosen, in part, because they thrive at high altitude, but also because the near-term fetus is of a size (7 to 8 pounds, ~ 3 to 4 kilograms) that allows physiologic measurements. In addition, they are a species long used for studies of developmental physiology, and on which considerable baseline information is available.

Beginning in early June, when the roads are first passable following the winter snows, we transport early pregnant (~30 days) and nonpregnant ewes to the White Mountain Research Station. Near the end of gestation (~ 140 days), the ewes are transported to Loma Linda University where they are maintained in an oxygen environment equivalent to that at high altitude.

Under general anesthesia, we place small polyvinyl catheters in certain blood vessels of the ewe and fetus, as well as in the amniotic fluid compartment which surrounds the fetus. Then, after five to seven days when physiologic functions have returned to normal, we commence the studies.

One of the questions we initially asked was: to what extent is placental function altered in these hypoxic animals? We reasoned that, perhaps in an effort to supply the fetus with adequate oxygen, the placental diffusing capacity (its ability to exchange respiratory gases) was increased above normal. Working with Dr. Gilbert, we examined this issue.

To our surprise, the diffusing capacity of the high-altitude placenta was similar to the value we had found in earlier studies in sea-level control animals. In subsequent studies, we have shown that the placenta does this by markedly altering the anatomical arrangement of both maternal and fetal blood vessels in the placental exchange area.

In collaboration with Drs. Krebs and Leiser, placental anatomists of the University of Giessen, Germany, and by using the techniques of both two-dimensional and three-dimensional morphometry, we have demonstrated a marked increase in density and tortuosity of small blood vessels in the placental exchange area. This provides a shorter distance for oxygen molecules to diffuse between the maternal and fetal blood, thereby helping to maintain a normal supply of oxygen and other nutrients.

In a more recent study with Luit Penninga, a fourth-year medical student from the University of Groningen, the Netherlands, we have shown that these vascular changes are accompanied by significant changes in the morphology of the placental cotyledons. These are remarkable adaptations. What is unclear at this time are the cellular and molecular bases of this morphological remodeling, which we are now exploring.

Another series of questions has involved the function of the fetal heart under conditions at high altitude. Dr. Gilbert has led out in these studies. Initially, we examined cardiac function in the fetuses of ewes which we made hypoxemic for two to four weeks at Loma Linda. We did this by infusing nitrogen gas into the mother's trachea so that her arterial oxygen tension decreased from ~100 Torr to ~60 Torr, that value measured in ewes at the White Mountain Research Station. In these chronically catheterized animals, fetal cardiac function decreased significantly during the first three days of hypoxia. After that it slowly increased, so that by seven days they were near normal again, albeit on a different portion of their cardiac function curve.

Then we studied fetuses of ewes which had been acclimatized to high altitude for three to four months. In these animals, a striking finding was that while total fetal cardiac output was decreased ~27 percent, blood flows to the brain and heart were similar to that of sea-level control animals. In contrast, blood flow to the rest of the body was decreased about 40 percent. At the end of pregnancy, this results in a fetus with a small body but relatively normal sized brain and heart. Later, we would show that the decrease in cardiac output was due to a significant decrease in the output of the right ventricle only, with both "preload" and "afterload" being decreased.

Subsequently, Dr. Gilbert and his colleagues have been investigating the cellular mechanisms in the heart which might be responsible for the decrease in its ability to contract and have a normal output under these circumstances.

The heart beats when calcium--which quickly moves from outside a muscle cell into the cell and is also released from storage sites within the cell--interacts with the contractile fibers in the cell. Surprisingly, we found that the movement of calcium into the cell and its release from storage sites within the cell is not impaired in fetuses at high altitude. We have preliminary evidence that the problem may be with the number of contractile fibers within each cell or in the way calcium interacts with them.

We also are investigating the regulation of blood flow to the fetal heart through the coronary circulation, to try to understand how blood flow to the high-altitude fetal heart can be maintained at a normal level, while blood flow to the rest of the body is reduced to one-half normal.

The research station, situated at an elevation of 12,532 feet, is operated by the University of California for the use of astrophysicists, geologists, botanists, zoologists, and biologists in their studies.

From my own standpoint, my chief area of investigation of the high-altitude animals has been cerebral blood flow, and the mechanisms whereby the fetus and the adult optimize blood flow to the brain in the face of decreased cardiac output. In the developing fetus and newborn infant, the brain is rapidly growing and maturing. During this period, the changing energy demands of the immature brain must be carefully matched to its supply of blood and nutrients which are delivered by the arteries. Any disturbance or injury which interferes with the balance between the energy needs of the brain and its supply of blood and nutrients can result in cerebrovascular complications leading to brain damage, permanent disability, or even death.

Unfortunately, such problems are all too common--as indicated by the large number of babies each year in neonatal intensive care units because of asphyxia and many other insults. For pediatricians,
it is often quite difficult to manage these young patients due to the fact that the cerebral arteries of infants are quite immature, as compared with those in adults.

Although a great deal of scientific research has explored the properties and regulation of adult cerebral arteries, relatively little is known about the characteristics and function of immature cerebral arteries. Thus, the main focus of our research is to study immature cerebral arteries, with particular emphasis on how these arteries respond to certain stimuli, and how these responses are modulated by environmental factors such as long-term oxygen deficiency.

The cerebral vasculature is richly innervated by sympathetic nerves, and the neurotransmitter norepinephrine acts post-synaptically on smooth muscle adrenergic receptors and is a principal determinant of vascular contractility. In response to high-altitude, long-term hypoxia, the cerebral arteries of both adult and fetal sheep show decreased responses to norepinephrine and other agonists.

Working with Dr. Pearce, we have explored how maturation and oxygen lack alter signaling pathways that regulate how vigorously cerebral arteries contract in response to well-known biological signals. In particular, these experiments explore the hypothesis that the mechanisms which regulate the role of calcium in cerebral artery contractions differ between neonates and adults.

We have attempted to dissect out the hypoxic-mediated responses in terms of pre-synaptic vs. post-synaptic mechanisms. In these vessels, high-altitude hypoxia is associated with altered pre-synaptic function, e.g., decreased norepinephrine content, suggesting decreased numbers of adrenergic nerve terminals or norepinephrine per nerve. In contrast, the vessels show increased norepinephrine uptake suggesting higher nerve density.

From a standpoint of post-synaptic function, we have shown significant decreases in several aspects of signal transduction, by which a given agonist effects contractile response. For instance, there is a marked decrease in the density of

1-adrenergic receptors (to which norepinephrine binds), as well as decreased response of the second messenger inositol 1,4,5-trisphosphate.

Perhaps most surprisingly, we found that the density of the sarcoplasmic reticulum inositol 1,4,5-trisphosphate receptor, to which the second messenger binds (and which acts as a channel for the release of calcium to effect vascular contraction), is significantly decreased in the vessels from the high-altitude animals. In other studies, we have shown that the high-altitude vessels have markedly depressed relaxation responses to various agents. Our most recent studies indicate that the function of calcium channels on the vascular smooth muscle cell membrane is also significantly altered in the high-altitude fetus and adult.

In addition, Dr. Buchholz and Dr. Duckles have found that the cerebral arteries of high-altitude animals (both adult and fetus) have depressed mechanisms of norepinephrine release and re-uptake.

Taken together, these studies demonstrate significant alterations in various aspects of the signal transduction cascade in response to high-altitude hypoxemia. It may be that with chronic stress, and elaboration of excessive norepinephrine (a potent vasoconstrictor), the noradrenergic system of the cerebral blood vessels is depressed or down regulated to help maintain normal cerebral blood flow, oxygen consumption, and metabolism.

In addition, studies show that these factors are regulated independently. The changes are not the same for the extracranial vessels, and they may differ significantly between fetus and adult. Perhaps most importantly, the studies provide useful leads to examine the critical issue of how high-altitude hypoxia regulates gene transcription, and thus elaboration
of new proteins to affect vessel function.

It is hoped that expanded knowledge of these differences will help neonatologists better manage infants with cerebrovascular complications and possibly also help identify practices which can help minimize chances for such complications in all infants.

In a related series of studies, Dr. Zhang is exploring how the regulation of uterine blood flow is affected by high altitude. His studies have demonstrated that chronic hypoxia changes receptor-mediated excitation-contraction coupling and/or signal transduction in the vascular smooth muscle of the uterine blood vessels.

This attenuation of uterine artery contractility probably represents one of the mechanisms for maternal and fetal adaptation to mild chronic hypoxia, and may lead to an adjustment of blood flow to the placenta under this stress. Dr. Zhang's present studies focus on cellular and molecular mechanisms underlying chronic hypoxic-induced changes.

Understanding of the mechanisms in regulation of uterine blood flow is of fundamental physiologic importance in its own right, as well as having considerable clinical relevance. For instance, these studies will provide a basis for and facilitate future studies of pregnancy-associated diseases such as pregnancy-induced hypertension and essential hypertension in which uterine blood flow can be significantly altered. They will also improve our understanding of the mechanisms underlying fetal intrauterine growth restriction, birth defects, and other fetal abnormalities associated with maternal hypoxia.

Another series of studies is being conducted by Dr. Ducsay on uterine muscle and fetal hypothalamic-pituitary adaptations to chronic hypoxia. One of the adaptations of mother and fetus to long-term hypoxia appears to be in the mechanisms that prevent premature delivery despite chronic stress.

Dr. Ducsay's studies have revealed that the fetal adrenal gland is less responsive to stimulation by adrenocorticotrophic hormone (ACTH). This results in a decreased production of the stress hormone cortisol. His most recent studies suggest that this results from altered function of the enzyme which produces cortisol. Because this hormone is responsible for stimulating birth in the sheep, its suppression may prevent a hypoxic-induced premature rise in cortisol that would normally trigger labor and delivery.

There also appears to be a decrease in sensitivity of the uterine muscle, the myometrium, to stimulation. There is a measurable reduction in oxytocin receptors which diminishes the ability of the uterus to contract. Taken together, these data indicate an adaptive response by both mother and fetus to prevent preterm delivery in the face of a chronic stress which would be anticipated to stimulate uterine activity.

During the past several years, we have presented the results of this research at a number of scientific meetings, including the Second World Congress of High Altitude Physiology and Medicine in Cuzco, Peru; the Society for the Study of Fetal and Neonatal Physiology in Arica, Chile; and many others. Results of these studies have been published in over 50 reports in scientific journals.

In addition to these studies by our own research group, at least five other groups of investigators are planning to collaborate with us during the next several years to explore fundamental questions of maternal and fetal adaptation to hypoxia. These include scientists from Cambridge University, the University of London, the University of Giessen (Germany), and the University of Adelaide and Monash University (Australia). Truly, Loma Linda University is emerging as a major center in this important area of high altitude research.

It is our goal to continue to pursue this research at the cellular and molecular level, in an attempt to understand better the miraculous manner in which the mother and fetus acclimatize to long-term hypoxia. We would like to think that this is yet another way in which Loma Linda University works "to make man whole."