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© 1997 American Sleep Disorders Association and Sleep Research Society
The History of Sleep Advances
'l'he Pioneering Experimental Studies on
Sleep Deprivation
*Marina Bentivoglio and tGigliola Grassi-Zucconi
*Institute of Anatomy and Histology, University of Verona, Verona, Italy; and tDepartment of Cell Biology,
University of Perugia, Perugia, Italy
Summary: The experimental studies on sleep deprivation were initiated by the Russian physician and scientist,
Marie de Manaceine, who studied sleep-deprived puppies kept in constant activity. She reported in 1894 that the
complete absence of sleep was fatal in a few days, pointing out that the most severe lesions occurred in the brain.
In 1898, the Italian physiologists Lamberto Daddi and Giulio Tarozzi also kept dogs awake by walking them; the
animals died after 9-17 days, and their survival was unrelated to food consumption. In the histological study
performed by Daddi, degenerative alterations, mainly represented by chromatolytic changes, were observed in
neurons of the spinal ganglia, Purkinje cells of the cerebellum, and neurons of the frontal cortex. Daddi ascribed
these changes to a state of autointoxication of the brain during insomnia. In 1898, the psychiatrist Cesare Agostini,
interested in the psychic phenomena caused by prolonged insomnia in humans, sleep deprived dogs by keeping
them in a metallic cage in order to avoid fatigue. The dogs survived about 2 weeks, and degenerative changes were
observed in their brains. In these experimental paradigms, the effect of sleep loss was confounded by motor
exhaustion and/or intense sensory stimulation. In spite of the absence of adequate controls, the pioneering studies
performed at the end of the 19th century represented the first experimental attempts to relate sleep with neural
centers and suggested that sleep is a vital function and that the brain may be affected by insomnia. Key Words:
Insomnia-Sleep disorder-Neuronal degeneration-History of neuroscience.
"While the observations on normal and artificial
sleep are becoming increasingly numerous, absolute
insomnia or complete sleep deprivation has not been
up to now subjected to experimental research. However, in order to understand the role of sleep in the
organic life, the influence of complete sleep deprivation should also be ascertained. It is known that in
China and in the antiquity there was, among different
kinds of tortures, death caused by sleep deprivation,
i.e. the condemned man was killed by being forbidden
to sleep and waking him up as soon as he started falling asleep. Facts of this kind clearly demonstrate that
sleep deprivation produces a most noxious influence".
This is the initial paragraph of a paper published in
1894 by Marie De Manaceine (1), in which she provided the first report of experimental sleep deprivation.
Her study was followed by the reports of Italian investigators. We present an overview of these first studAccepted for publication May 1997.
Address correspondence and reprint requests to Dr. M. Bentivoglio, Institute of Anatomy and Histology, Medical Faculty, Strada
Le Grazie, 37134 Verona, Italy.
ies on experimental insomnia, which were performed
in the last years of the 19th century.
Marie De Manaceine and the first experimental
study of sleep deprivation
Maria Michailovna Mana(s)seina (1843-1903; Marie De Manaceine in the authorship of her publication,
following the practice of the time of translating names
into French for an international audience) (Fig. 1) was
a remarkable woman physician and scientist in St. Petersburg (2,3). Growing up in an academic milieu and
daughter of the renowned archeologist M. A. Korkunov, she was very interested in the natural and medical
sciences. She entered medical school and graduated as
a medical doctor (one of the first female physicians in
Russia and certainly in Europe); immediately after her
degree, she married V. A. Manassein, professor at the
Medical Military Academy in St. Petersburg. Madame
De Manaceine started her career as a biochemist in the
Polytechnic Institute in Vienna and provided an outstanding contribution to the study of alcoholic fermen-
570
PIONEERS IN EXPERIMENTAL SLEEP DEPRIVATION
FIG. 1.
Marie De Manaceine. (Reproduced from ref. 2.)
tation. She published her first book (Child-Rearing
During the First Years of Their Life) in 1870 and was
a very prolific writer and active scientist, devoted to
child education, psychology, physiology, and science
popularization. Her interest in sleep was stimulated by
her collaboration with the physiologist I. Tarkhanoff.
Among the many volumes Madame De Manaceine
published in Russian, (he following titles testify to her
interests: Abnonnality of Brain Activity of Recent Cultured Man, On Writing, Mirror Writing and Above All
on the Functions of Brain Hemispheres, Fatigue, Sleep
as One-Third of Human Life, or Physiology, Pathology, Hygiene and Psychology of Sleep, and Principles
of Education From the First Years of Life to the End
of University Studies. In the last years of her life, Madame De Manaceine left St. Petersburg and lived in a
small town (Solyanyj gorodog) where she lectured on
psychology, pedagogy, and brain physiology. She also
translated into Russian many scientific works and popular science papers.
The first experimental study on sleep deprivation,
571
presented by Marie De Manaceine at the International
Congress of Medicine in Rome in 1894 0), was
prompted not only by the knowledge that forced insomnia was torture but also by previous clinical reports of mental disturbances after partial insomnia
(4,5) and reports of a subject who had survived only
9 days of total insomnia (5). Madame De Manaceine
performed her experimental investigation on 10 puppies (2, 3, or 4 months old), fed by their mothers, by
keeping the animals in constant activity. The experiment came to the straightforward conclusion that "the
total absence of sleep is more fatal for the animals than
the total absence of food", since the dogs could be
rescued after 20-25 days of starvation, but they were
"irreparably lost" after a sleep deprivation of 96-120
hours. De Manaceine also noted that older dogs were
more resistant to insomnia than younger ones and that
body temperature decreased from the second day of
sleep deprivation on and was 4,5, or even 5.8°C lower
than normal before the animal's death. After the initial
decrease of body temperature (0.5-0.9°C), locomotor
activity had started becoming "slower and weaker",
and red blood cell counts had decreased. The weight
loss of the animals before death was relatively mild
(5-13%). The "histological study" of body organs
(apparently limited, however, to a macroscopic examination) clearly demonstrated that "the brain was the
site of predilection of the most severe and most irreparable changes" (the italics are in the original text):
"fat degeneration" in many brain "ganglia", abnormalities of blood vessels (probably including perivascular infiltrates), and small hemorrhages. These
changes were very different from those De Manaceine
had observed in animals that died of starvation, in
which the brain was "remarkably spared". She concluded that her findings provided "a proof of the great
importance of sleep for the organic life of animals
equipped with a cerebral system, and also entitle to
consider a bad paradox the strange opinion regarding
sleep as a useless, stupid and even noxious habit".
Experimental sleep deprivation studies in the
Laboratory of Physiology of Pisa: Lamberto Daddi and Giulio Tarozzi
Stimulated by De Manaceine's report and by the interest of V. Aducco, director of the Laboratory of
Physiology of the University of Pisa, two young Italian
investigators provided very detailed reports of experimental insomnia, achieved by walking animals, especially during the night hours.
In order to perform a more detailed anatomical
study than that reported by De Manaceine, in January
1898 Lamberto Daddi published the histopathological
description of the brains of three of the dogs (6) that
Sleep, Vol. 20, No.7, 1997
M. BENTlVOGLIO AND G. GRASSI-ZUCCONI
572
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FIG. 2. Degenerative changes in the brain of sleep-deprived dogs. Chromatolytic changes and "protoplasmic vacuolization" in pyramidal
neurons from the "anterior lobe" (top) and in a Purkinje cell of the cerebellum (bottom right); chromatolysis, nuclear eccentricity, and
degeneration of the nuclear membrane in a cortical neuron of the "anterior lobe" (bottom left). (Reproduced from ref. 6.)
were also used for Tarozzi's study (7). One of these
dogs had been starved and survived insomnia for 17
days, whereas the other two had been normally fed
and survived total sleep deprivation for 9 and 13 days,
respectively. The brains were paraffin-embedded and
stained with thionin, hematoxylin, carmin, safranin,
and Golgi impregnation. The most severe changes
were found in the spinal ganglia and cerebellar and
cerebral cortices, whereas the spinal cord and the brain
stem appeared normal. The alterations were representSleep, Vol. 20, No.7, 1997
ed by varying degrees of chromatolysis ("the so-called
chromatic part is fragmented in very minute granules
similar to fine dust and tends to disappear"), "rarefaction" and vacuolization of the cytoplasm, and nuclear
eccentricity and fragmentation of the nuclear membrane, with relative sparing of the nucleolus (Fig. 2).
Such degenerative changes were detected in neurons
of the spinal ganglia, in which altered cells were intermingled with normal ones, in the Purkinje cells of
the cerebellum, in neurons of the cerebral cortex,
573
PIONEERS IN EXPERIMENTAL SLEEP DEPRNATION
FIG. 3. Neuronal degenerative changes after intoxication with lead
or arsenic, which Daddi (6) and Agostini (II) considered similar to
those they observed in the brains of sleep-deprived dogs. Cortical
pyramidal neuron (top right) and the "peripheral chromatolysis" of
spinal ganglia neurons that proceeds toward severe degeneration
(bottom left). (Reproduced from ref. 8.)
were very similar to those observed in the nervous
system of dogs after experimental intoxication with arsenic and lead (8) (Fig. 3). In his interpretation of the
experimental data, Daddi (6) insisted on the "excessive fatigue of the nervous system" during insomnia
and argued that the alterations observed in the "psychomotor zone" of the cerebral cortex could be attributed to its "frequent stimulation" during wake, thus
supporting the theory of a restorative effect of sleep:
"the daily alternation between sleep and wake should
be searched in the consumption of nervous elements,
due to all the excitation they receive and to their activity during wake".
In a subsequent study on the effects of experimental
starvation lasting from 9 to 59 days, Daddi (9) described in the dog nervous system changes similar to
those he had observed after experimental insomnia but
that were much less numerous and severe.
Giulio Tarozzi investigated the metabolic changes
(analyzing nitrous compounds in the urine, white and
red blood cell counts, body temperature, and heart and
breathing rates) in four sleep-deprived dogs, one of
which was kept in starvation (7). The conclusions of
this study, which provided very detailed protocols of
the analyzed parameters, were that the elimination of
nitrous compounds increased only in a very advanced
stage of insomnia and kept increasing until the animal's death; the initial increase was sudden and accompanied by a sudden hyperthermia. The body temperature then progressively decreased, and "general
depressive phenomena", lasting 1-4 days, preceded
death.
Cesare Agostini and forced insomnia
where degenerating cells were grouped in clusters, and
were most frequently observed in the "anterior lobe".
The largest pyramidal neurons and the posterior cortical fields were relatively spared from degenerative
changes. No perivascular lymphocytic infiltrates were
evident throughout the nervous system, ruling out an
inflammatory reaction. Golgi impregnation revealed a
normal morphology of glial cells, whereas the nerve
cells displayed "irregular contours", thinning of the
protoplasmic processes, and the features of the "socalled varicose atrophy". All these alterations were
consistent in the three examined brains and were more
severe in the starved animal than in the other two dogs.
Daddi clearly stated that the intermingling of normal
and altered cellular elements argued against fixation or
staining artefacts. Discussing his findings in relation to
literature data on the effects of several toxic and infectious agents, Daddi concluded that the changes he
had observed after total insomnia did not display any
specific feature. In particular, he stated that the findings
Clinical practice, personal experience, and the lack
of details in De Manaceine's report also stimulated the
interest of the Italian psychiatrist Cesare Agostini, who
performed an experimental study on sleep deprivation
in dogs and communicated his findings to the Medical
Academy of Perugia in February 1898, shortly after
Daddi's report.
Cesare Agostini (1864-1942) (Fig. 4) worked as an
intern student (and published a study on the origin of
the brachial plexus) at the Institute of Anatomy of the
University of Florence, where he graduated in Medicine in 1889. Very interested in psychiatry, he pursued
his training at the Institute of Psychiatry of Reggio
Emilia and in 1891 moved to Heidelberg, where he
worked under E. Kraepelin's guidance. In 1893, Agostini was appointed to the asylum of Rome and then to
that of Perugia, where he became director. He also
gave courses on criminal anthropology and forensic
psychiatry at the University of Perugia. During the
First World War, Agostini served in the army and
Sleep, Vol. 20, No.7, 1997
M. BENTIVOGLIO AND G. GRASSI-ZUCCONI
574
FIG. 4.
Cesare Agostini.
worked actively at establishing an efficient neuropsychiatric assistance. In 1928, he left the direction of the
asylum of Perugia and was appointed (for "exceptional merit") director of the Clinic of Nervous and Mental Diseases of the University of Perugia; he retired in
1935.
Following previous clinical observations on the
physical and psychic disturbances provoked by insomnia (4,5,10), Agostini reported two cases in which a
long and persistent sleep deprivation represented the
only etiology of sudden episodes of delirium (11). The
first involved a 45-year-old healthy railway engineer,
who, due to the illness of his colleague on duty, had
been obliged to work for 6 days and 6 nights, after
which, having reached a station, he suddenly started
to behave strangely. Admitted to the asylum in an obvious confusional state, presenting extreme excitation,
hallucinations, severe attention deficit, and inadequate
emotional attitudes, the patient was put to bed, slept
for 15 hours, and woke up in an absolutely normal
state with no memory of the previous confusion. The
second case involved a healthy young woman, working as a chambermaid, who had to assist her ill, ailing
mistress for several nights, also having to attend to the
housework during the day. On the 10th day, she began
to show a confused and maniac behavior; once sedated, she slept for several hours and woke up in a normal
state with only vague memories of the preceding episode. After a day of rest, she returned to assist the sick
lady and suffered during the night from another episode of confusion and delirium. Forced to rest for several days, she then completely recovered. Agostini also
Sleep, Vol. 20, No.7, 1997
reported a personal experience during an excursion in
the mountains: forced to continue walking for 3 days,
with only a few hours of rest, not having found any
convenient lodging, he and his friends started having
hallucinations during the night.
On the basis of these episodes, Agostini stated that
"the continuous sensory excitation due to prolonged
lack of rest, together with the effort in maintaining
alertness, induces necessarily, due to exhaustion of the
central nervous system, some kind of open-eye sleep,
an intermediate state between the consciousness of
wake and that of sleep, in which a voluntary order of
thoughts and reflections cannot be followed, but the
ego instead assists passively to the kaleidoscopic recollection of increasingly strange and fantastic series of
representations, that appear and disappear as the waves
of an agitated sea".
In those years, the neuronal theory maintained that
the nervous system is composed of functionally and
anatomically distinct cells (which W. Waldeyer had
named neurons in 1891), as opposed to the reticular
theory, which maintained that the nervous system was
represented by a continuous network (12,13). Convinced that the "modern" neuron doctrine was correct,
Agostini (11), in line with Duval (14), hypothesized
that "sleep mechanism could be due to a decreased
contiguity among neuronal dendrites, caused by a decrease in the cell body size and weakening of the nervous wave transmission, due to the altered cell chemical set-up produced by wake ... , to fatigue of the
cell body causing an increase of interneuronal intervals" .
Assisted by "conscientious coworkers", Agostini
(11) devised a "fairly simple but immensely tiring"
experimental paradigm of sleep deprivation, criticizing
De Manaceine's and Daddi's studies, in which fatigue
could have represented a confounding factor. In Agostini's laboratory, two adult dogs were kept in a large
metallic cage with a floor made of tin and with bells
at each corner, so that each movement of the animal
was accompanied by an "ear-splitting jangling noise".
The animal was under the continuous surveillance of
personnel assigned to 6-hour shifts, who moved the
cage as soon as the animal seemed to fall asleep. One
of the dogs survived 17 days and the other 12 days.
Histopathological examination of the brains revealed
changes very similar to those described by Daddi (6),
and with the same distribution. On the basis of the
animals' behavior during the forced insomnia, Agostini (11) argued that total sleep deprivation induced a
"progressive exhaustion of psychic activity", and the
experimental findings suggested that total insomnia per
se may cause death.
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PIONEERS IN EXPERIMENTAL SLEEP DEPRIVATION
Concluding remarks
The pioneering studies on experimental insomnia
represent the first attempts, in the neuronal era, to relate sleep and neural centers. In these experiments, the
stimuli used to prevent sleep implied motor exhaustion
and/or excessive sensory stimulation in the absence of
careful controls both in the design of the experimental
procedures and in the evaluation of the histological
findings. However, these studies suggested a vital role
for sleep and drew attention to the brain as the site of
damage induced by sleep deprivation.
In the first years of the 20th century, Kuniomi Ishimori (15), searching for a "hypnogenic substance" in
the brain tissue, and Henri Pieron and Rene Legendre,
in a series of studies aimed at verifying that a "hypnotoxin" circulating in the blood of an insomniac animal would have induced the need of sleep in a normal
one, forwarded the hypothesis that the brain was intoxicated during insomnia. Legendre and Pieron again
performed sleep deprivation experiments in dogs by
keeping them in constant activity. In their histological
study of the nervous system of the sleep-deprived animals (16), chromatolytic alterations were reported in
pyramidal neurons of the prefrontal cortex and sigmoid gyrus (the "somatomotor cortex"), whereas no
degenerative changes were found in the occipital cortex; minor changes were observed in the Purkinje cells
of the cerebellum, and no abnormalities were detected
in the spinal ganglia.
In the study of the central nervous system performed by Kleitman (17) in puppies sleep deprived by
gentle stimulation and by walking them, the only
marked change after a period of experimental insomnia
of 2-7 days was a decrease in red blood cell count; at
the histological examination, abnormalities of nerve
cells were detected, but these were also observed in
the control litter mates.
Sleep-deprivation experiments were also performed
on rabbits by Crile (18) by using gentle stimulation;
at the histological examination, lesions were detected
in the liver, adrenal gland, and central nervous system.
In their study of the spinal cord and medulla of sleepdeprived rabbits, Bast and colleagues (19,20) observed
that "the majority of cells appeared normal" and stated that "it is not easy to detect nerve cell changes
following prolonged periods of sleeplessness" (19). In
these latter investigations, however, chromatolytic
changes were found in neurons, and they were especially pronounced in the "completely exhausted"
animals (19,20).
A review of the subsequent experimental work on
the effects of sleep deprivation goes far beyond the
scope of the present article. It should, however, be
pointed out that a wealth of studies have been devoted
to the effects of sleep deprivation in humans and in
animals (see 21-23 inter alia). In particular, the biochemical and neurochemical effects of total sleep loss,
the effects on the immune system, body metabolism,
and physiological measures including electroencephalogram (EEG), energy metabolism, and thermoregulation have been thoroughly analyzed. Sleep-loss effects in rats were not found to be part of a nonspecific
stress response, implying a generalized impairment of
physiological parameters; instead, sleep deprivation
was found to result in reliable physiological effects
represented by an increase in food intake, weight loss,
disturbed thermoregulation, severe ulcerative and keratotic skin lesions, alterations in endocrine parameters,
and eventual death (22,23). Experimental insomnia,
however, did not produce definite evidence of impairment of brain function and did not provide clues of
the proximal cause of death of the insomniac animals
(23). After sleep deprivation in rats, based on controlled experimental parameters, the evaluation of the
histological features performed by investigators unaware of the experimental group assignment pointed
out that alterations of glands and viscera and nerve
cell abnormalities, such as vacuolization and shrinkage
of neuronal perikarya, could be seen with similar frequency in sleep-deprived animals and yoked controls
(24). However, these studies pointed out that the normal-functioning brain needs sleep (21) and supported
"the view that sleep does serve a vital physiological
function" (22).
Marie De Manaceine (25) had wondered: "We all
love life and we all wish to live as long as possible
but, in spite of this, we sacrifice one-third, sometimes
even half, of our life in sleeping .... What is sleep?
In what respect does a sleeping person differ from a
waking individual? It is difficult to find any part of
life which has been subjected to so much injustice and
ingratitude as sleep". During the 20th century, the "injustice and ingratitude" toward sleep may have diminished, but these crucial questions have remained unanswered.
Acknowledgements: This work was supported by
grants of the Italian MURST. The authors are very grateful
to Professor Krister Kristensson, Ms. Teodora Oker-Blom,
Ms. Rita Capecchi, Dr. Jama Mhlanga, and Dr. Giuseppe
Bertini for their help in collecting some of the bibliographic
material, to anonymous reviewers of their suggestions, and
to Professor Rastislav Druga for his help in the translation
of a Russian text.
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