William F. Windle

A Physiological Approach to Perfusion-Flxation of Tissues with Formalin

A method of perfusion-fixation with formalin is here presented which is based upon well established physiological principles. It represents an attempt to preserve more accurately the structure and relationships of nervous tissue found in the living state. The essential points include the following: (1) Perfusion at an hydrostatic pressure equivalent to the mean arterial pressure of the animal to be fixed to maintain the normal patency of the vascular bed. For the guinea pig, it is approximately 70 mm. Hg; for the cat and monkey, it is approximately 120 mm. Hg. (2) The addition of a colloid to the perfusion fluid which will exert an osmotic pressure equal to the hydrostatic pressure in the capillaries and thus prevent edema. Gum acacia is used in this method. Its concentration depends upon the hydrostatic pressure to be used: for 70 mm. Hg, in the guinea pig it is 2.4%; for 122 mm. Hg, in the cat and the monkey, it is 5.6% gum acacia. (3) The addition of an electrolyte to the perfusion fluid to make it iso...

A Nissl Method Using Buffered Solutions of Thionin

Experiments were performed in an attempt to obtain a rapid method for staining the chromophilic substance of formalin-fixed nerve cells differentially without resorting to over-staining and subsequent acid-decolorizing. A satisfactory procedure using thionin in dilute buffered solutions was developed: Prepare a stock solution of the dye using 1 g. in 100 ml. of distilled water. Prepare veronal-acetate buffers (Michaelis, 1931) in the range of pH 5 to pH 3.S. To each 10 ml. of the buffer add 0.5 ml. of the stock dye solution. After rinsing in CO2-free distilled water place mounted or unmounted sections in this solution. (Freshly fixed material, 10μ to 20μ thick, is completely stained in 10 to 20 minutes but over-staining does not occur when longer times are allowed.) Return sections to distilled water (2 changes) and wash until diffusion of excess dye is no longer visible. Wash in 70% ethyl alcohol (2 changes) until diffusion of excess dye is no longer visible. Dehydrate in 95% ethyl alcohol and normal but...

Genesis of Somatic Motor Function in Mammalian Embryos: A Synthesizing Article

IN the genesis of motor functions I intend to consider the subject critically and mainly in terms of recent investigations. It is not my purpose to write a comprehensive review. I shall try to reconstruct the train of thoughts which have led to my present views and discuss the relationship of these to opinions of others. I hope this synthesis will help clarify the subject and that, when the time again arrives to renew studies of this character, workers will be able to proceed in greater harmony. It is nearly twenty years since concerted efforts to study motility of mammalian embryos and genesis of nervous activity were begun. The investigations of Coghill, reported first in 1908 and in a long series of articles since that time, stimulated search for correlations be-

Some observations on genesis of somatic movements in sheep embryos

ALTHOUGH a number of studies in development of early foetal movements have been reported [Windle and Griffin, 1931; Angulo, 1932; Windle, Orr and Minear, 1934; Windle, Minear, Austin and Orr, 1935; Bridgman and Carmichael, 1935] complete agreement regarding -the way behaviour becomes established has not been reached [A n gulo, 1935]. The opportunity to investigate the sheep was welcomed because it offered hope that observations on large embryos might lead to better understanding of the phenomena seen in smaller laboratory specimens. A few representative experiments between the 32nd and the 38th day of the gestation period will be described.

Block Staining of Nervous Tissue with Silver IV. Embryos

Procedures having enhanced reliability over older methods for both Bielschowsky and Cajal types of stain are described.Fixation of embryos in a solution containing 4% formaldehyde and 0.5% trichloracetic acid greatly improved the staining of neural elements by Bielschowskys method.Among the variations of Cajals type of staining tried, a modification of Ransons pyridin-silver method gave the most complete staining of neurofibrillar elements. Washing for 0.5 to 1 hour after silver impregnation and shortening of the reduction time from 24 to 4 hours corrected the tendency of the method to overstain.

Effects of Anoxia at Birth on Central Nervous System of the Guinea Pig

Summary Anoxia induced at birth invariably produced symptoms of neural damage. Transient shock, tremors, ataxia, and incoördination were not usually associated with impaired behavior or brain pathology. More than 8 min of anoxia lead to such symptoms as decerebrate states, marked ataxia, convulsions, paralysis, hyper- and hypoesthesia and somnolence, correlated in some cases with behavioral changes. Highly variable nonspecific devastation of brain and cord tissues, occasional hemorrhages, gliosis and generalized atrophy were encountered. All experiments were controlled with healthy litter-mates and permit the conclusion that neonatal asphyxia (asphyxia pallida) may induce irreparable fortuitous destruction of large or small regions of the brain.

Effect of Anoxia on Trigeminal Reflexes in Cat Fetuses

M AMMALIAN fetuses can be made to respond to changes in an artificial external environment before the middle of the gestation period. Reflex movements of the forelimbs and head are elicitable in cat embryos at experimental Cesarean section on the twenty-fourth day after insemination (Windle, Orr, and Minear, 1934). The first clear evidence that fetuses respond to induced alteration of their internal environment is the respiratory movement occurring when anoxial conditions are produced experimentally at about 32 days of gestation (Windle, Monnier, and Steele, 1938). Previous to this, internal environmental changes can alter responsiveness to external stimuli (Windle and Becker, I94oa). Various investigators have proposed that oxygen want, or asphyxia with carbon dioxide accumulation, may stimulate groups of neurons, release them from higher inhibitory influences, or simply depress their activity altogether. Angulo y Gonzalez (1930) believed that asphyxia caused the fetus to recapitulate inversely its behavioral ontogeny by stimulating growing neurons. However, it may be doubted that the responses he observed were reflexes, because they have been reproduced in dying embryos placed in solutions of ionic imbalance (Windle, 1939). Barcroft and Barron (Barron, 1941) rejected the concept that it is metabolic carbon dioxide which acts as a stimulating agent. Experiments in sheep fetuses led them to suggest that anoxemia depressed newly developed inhibitory centers of the brain, rendered them nonfunctional, and left older motor neuron groups to function in a manner characteristic of younger fetuses (Barcroft and Barron, 1939). Windle and Becker (I94oa) found that anoxemia first enhanced, then suppressed, individual fetal reflexes. Appendicular movements-as early as any reflexes to appear under reasonably good physiologic conditions-were abolished before more massive trunk and neck reactions. There was little correlation between age of a pattern of movement and length of time the response persisted after clamping the umbilical cord. Anoxia appeared to convert unit reflexes into mass reactions. Porter, Blair, and Bohmfalk (1938) found that slight anoxia and even asphyxia increased the number of adult motor units responding to a given stimulus, and transient stimulation of the nervous system by progress anoxia has been demonstrated by Sugar and Gerard (1938). From time to time it has been observed that embryos or small fetuses respond in one way during the early part of an experiment and in another way later in the same experiment as the reaction changes as anoxia develops. This led to a further study of reflex reversals and their relation to anoxemia.

Effect of Depriving Newborn of Placental Blood upon Early Postnatal Blood Picture

Within the last few years, the collection of postpartum placental blood for “blood banks” has been strongly advocated not only in Russia 1 but also in Canada 2 and in this country. 3 4 5 It has been pointed out that this is an inexhaustible and “lucrative” source of blood, satisfactory for transfusion purposes, and that its collection has no deleterious effect upon the mother. Possible effects upon the child have been disregarded because it has already become a rather common obstetrical practice to clamp the umbilical cord promptly at birth in spite of the fact that most of the placental blood normally drains into the body of the infant within a few minutes when the umbilical cord is not clamped immediately after delivery. 6 When the cord is clamped immediately, the infant is deprived of an alarming proportion of its total blood volume and usable iron at the very beginning of extra-uterine life. We have obtained proof that this blood-letting at birth affects the blood picture of the newborn significantly. The practice should be strongly condemned. We have determined the amount of hemoglobin and number of red blood corpuscles in blood taken from the mother on the day of birth, in cord blood at birth, in blood from the newborn (heel) 15 to 75 minutes after birth, and from the infant at one, 3 to 4, and 6 to 7 days after birth in 2 series of patients. In one series of 25, the umbilical cord was clamped within 30 seconds after delivery; in another series of 29, clamping was delayed until pulsations had ceased and the placenta had separated. Hemoglobin was determined in grams percent with a calibrated Hellige-Sahli instrument. Red blood corpuscle counts were made with a Spencer hemo-cytometer (N.B.S.).

Effect of Picrotoxin on Electrical Excitability of the Respiratory Center

Summary A technique has been described whereby electrical stimulation of the respiratory center can be employed to directly determine the influence on the respiratory center of various drugs affecting respiration. Picrotoxin has been shown to produce a marked and prolonged increase in the sensitivity of the inspiratory center of the cat to direct electrical stimulation. It is believed that the technique which has been described can be used to advantage to contribute additional information to our knowledge of drugs affecting respiration.