Experimental studies of fetal asphyxia in the Guinea pig

In 1944 a Professor of Anatomy at the University of Pennsylvania, William F. Windle, came to Louisville to give a lecture on Asphyxia Neonatorum ¹. I missed the lecture since it was four years before my birth, and my father was in the middle of World War II, but I wish I had been there. The lecture recounted some experiments done on guinea pigs (the actual animal, not human volunteers) that gave some new and solid insight into the effects of intrauterine asphyxia on the fetus. Dr. Windle published two papers on this series of experiments^2,3. The paper by Windle and Becker in 1943 states that it was in direct response to Dr. Galloway’s critique of Dr. Schreiber’s paper (see previous section).

I am going to present these studies in some detail first to give a sense of the messiness of their methods but also because these studies unequivocally established the principle that now seems self evident, namely that interference with respiratory exchange of the fetus either by blocking the blood flow to the placenta or blood flow through the umbilical cord causes brain injury.

Their studies used near term guinea pigs because these animals have a hemochorial placenta, that is the placenta has direct maternal blood flow around the fetal placenta tissue like the human, although the placenta has a different microanatomy. Guinea pigs have multiple pups of a size that can be handled (approximately 90g). The experimental asphyxia was produced by surgically entering the maternal abdomen and either clamping the uterine arteries or the individual umbilical cords of some fetal pups. The clamping caused violent movements of the pups including ineffectual respiration within seconds to a couple minutes, followed by a period of quiescence, followed by deep rhythmic respirations that aspirated amniotic fluid, and finally followed by cessation of respiratory motion. The pup’s heart rate dropped to one half its previous rate in 2 minutes or less. A resuscitation of the pups was started either with the cessation of respiration or the loss a good heart beat after 4.5 to 23 minutes of asphyxia. A variety of resuscitation maneuvers were tried but the most effective was a needle and tube bagging respiration device using a mix of oxygen and 10% carbon dioxide. Sixty seven pups survived 30 minutes to 2 weeks, and they were compared to 50 control pups that were not asphyxiated. Despite these varied experimental procedures , the authors were able to conclude that asphyxia did cause motor, sensory and behavioral abnormalities. However, they found little correlation between the extent of asphyxia and the neurologic or neuropathological outcomes. They warned:“ One can not safely predict that a brief period of anoxia will fail to affect the infant nor that prolonged anoxia will surely lead to total destruction.” While it is obvious today, the authors noted the failure of the mechanical, chemical, and electrical methods to resuscitate fetal apnea. “We are certain that many if not all [apneic pups] would have responded to oxygen had proper measures been taken to inflate the lungs with it.” In other words, slapping them on the bottom is just not as effective as establishing ventilation.

Windle and colleagues description of the neuropathology made several key points: First, the same changes of neuronal injury and vascular proliferation occurred whether or not there was hemorrhage². Some older theories had attributed neonatal brain injury directly to the effects of hemorrhage. Second, all but one premature fetus demonstrated gross and microscopic evidence of brain edema (swelling), if autopsied between 8 hours and 4 days after the asphyxia. There was some evidence of edema even two hours after asphyxia. Control brains and those examined after 4 days of asphyxia did not demonstrate edema. Edema will prove to be a very important observation in explaining the massive destruction of brain tissue in some types of brain injury in the primate and human infant. Third as stated by the authors “Such wide variations in location and degree of destructive processes were encountered that it is difficult to give a generalized description.” In the discussion, they did suggest some of the cause of such variation “ “All we can say is that all animals whose placental circulation was occluded for 8 minutes or more and which required more than 5 minutes to resuscitate showed definite and often marked pathological changes. Those whose vessels were occluded only until intrauterine efforts, induced thereby, had become weak and which required little or no resuscitation, thereafter showed as a rule indefinite or only slight pathological alterations. Variations were so great, however, that it is impossible to state categorically that asphyxia of short duration will cause no nervous changes, or that more prolonged asphyxia will be certain to lead to significant permanent effects.”

From hindsight, there were many important uncontrolled variables in Windle’s experiments. The potential importance of these variables will become clear in the later work by Dr. Ron Myers. These studies from the 1940’ established a basic model for studying the effect of fetal asphyxia on subsequent brain injury.

References:

1. Windle WF. Asphyxia Neonatorum. Springfield, IL: Charles C Thompson; 1950.
2. Windle WF, Becker R, Weil A. Alterations in brain structure after asphyxiation at birth An experimental study in the Guinea pig. J Neuropathol Exp Neurol 1944;3:224-38.
3. Windle WF, Becker R. Asphyxia Neonatorum an experimental study in the Guinea pig. Am J Obstet Gynecol 1943;45:183-200.