The Journal of Neuroscience, May 1989, g(5): 1705-1711
Damage Associated with Prolonged and Fatal Stress in
Hideo Uno,’ Ross
‘Regional Primate Research
Research, National Museums
Regional Primate Research
Tarara,2,3 James G. Else ,2,4 Mbaruk A. Suleman,* and Robert M. Sapolsky2-5
537151299, Center, The University
of Kenya, Nairobi, Kenya, 3California Primate Center, Davis, California 95616, 4Yerkes
Center, Emory University, Atlanta, Georgia 30322, and 5Department
Stanford, California 94305
of Wisconsin, Madison, Wisconsin *Institute of Primate
of Biological Sciences,
ation in the rat. This is particularly
a principal neural
acerbate the rate of neuron
well as the severity
gests a similar association
keys, housed in a primate
spontaneously from 1964 to 1966, were
to have multiple gastric
ological study was then done
tion. Compared with controls
generation that was apparent
itatively, and both ultrastructurally
scopic level. Minimal
agonal or post-mortem
appear to have been subject
haps in the form of social
groups: most came from social groups,
incidences of bite wounds
adrenal cortices, indicative
over, the specific hippocampal
cerated animals matched
suggesting that sustained
might be neurodegenerative
so in the hippocampus,
can cause degener-
target site for GCs, in which
of neuronal damage
Thus, stress can be a potent
in the rat. The present
in the primate.
GCs can ex-
Eight vervet mon-
in Kenya, that had died
of this opportunistic
euthanized for other research
had marked hippocampal
and on the light-micro-
was unlikely to have been due to an
artifact. Instead, ulcerated
subordinance in captive
at necropsy, and had hyperplastic
of sustained GC release.
Thus, this represents
stress, via GC hypersecretion,
in the primate.
social stress, per-
in ul- damaged
by GCs in the ro-
the first evidence
Glucocorticoids (GCs) are adrenal steroids secreted during stress,
whose numerous actions are central to the stress response (Munck
et al., 1984). As many of these actions are catabolic, prolonged
stress and/or GC exposure can be pathogenic (Krieger, 1982).
In the rat, recent work has shown these adverse effects to include
neural degeneration. This is particularly so in the hippocampus,
Disease and Related
July 11, 1988; revised
provided by NIH Grant
Sept. 21, 1988: accepted Sept. 26, 1988.
RR-00 167 (H.U.), the NIH-sponsored
and NIH Grant RR-000165
Foundation, NIH Grant AG-06699
Disorders Association (R.M.S.).
and Pam Brigham for technical assistance, and for neuropathologi-
from Drs. A. Hirano and G. Zurhein.
should be addressed to Robert
0 1989 Society for Neuroscience
M.A.S.), (R.T., J.G.E., the
and the Alzheimer’s
Thanks to Carol Thieme,
Sapolsky at the above address.
a principal neural target tissue for GCs, with high concentrations
of both types of corticosteroid receptors (McEwen et al., 1986).
Pharmacologic concentrations damage the structure (Aus der
Muhlen and Ockenfels, 1969). Moreover, normal loss of hip-
pocampal neurons with aging is paced by GC exposure, as mid-
age adrenalectomy retards the process, while prolonged expo-
sure to high (though physiological) concentrations of GCs
accelerates the neuron loss (Landfield et al., 198 1; Sapolsky et
al., 1985). GCs disrupt hippocampal glucose utilization (Ka-
dekaro et al., 1988); it has been suggested that this leaves neu-
rons vulnerable to metabolic insults (Sapolsky et al., 1986).
Thus, nontoxic concentrations of GCs exacerbate hippocampal
damage induced by insults as varied as hypoxia-ischemia, an-
timetabolites, epileptogenic excitotoxins, and superoxide radical
generators (Sapolsky, 1985a, b, 1986a, b; Sapolsky and Pulsi-
nelli, 1985; Theoret et -al., 1985; Koide et al., 1986; Sapolsky
et al., 1988); furthermore, the exacerbation can be prevented
by supplementing the neurons with alternatives to glucose (Sa-
These observations suggest that in the rat, stress, via GC
secretion, might be an important pacemaker of neuron loss dur-
ing aging as well as influence the neuropathologic outcome after
insults such as hypoxia-ischemia or seizure (Sapolsky et al.,
1986). Naturally, it is important to determine whether GCs
might also be neurotoxic in the primate brain. Were such the
case, prolonged stress should be associated with neural degen-
eration in the primate, with preferential vulnerability of the
hippocampus. In the present study, we have examined a pop-
ulation of monkeys in which these conditions occurred oppor-
tunistically, and we find evidence for the predicted pattern of
Materials and Methods
Subjects were adult vervet monkeys (C. aethiops).
exception of 1 colony-born individual
in Kenya after becoming agricultural
stitute of Primate Research in Nairobi,
month (in isolation, if trapped singly, or in group cages, if trapped with
other animals) and then assigned to either social breeding groups or
housed in isolation for research purposes. Isolates were housed in 0.6
x 0.6 x 0.68 m outdoor cages; social groups consisted of 1 male and
3-5 females in a 6 x 3 x 2.5 m outdoor cage.
Eight monkeys died spontaneously (“ulcerated group”). All had been
cachectic, with various illnesses (Table 1). At necropsy, all had multiple
gastric ulcers resembling early acute gastric ulcers in humans (Suleman
et al., 1984). Ulcerated monkeys had no sign of hemorrhagic
viruses or gastrointestinal parasites known to cause hemorrhagic lesions
in the primate stomach (Suleman et al., 1984). A control group consisted
(17F), all had been wild-trapped
pests. Upon transport to the In-
they were quarantined for 1
1706 Uno et al. - Stress and Neuropathology in Primate Hippocampus
Figure 1. a and b Representative photomicrographs
(b). Latter shows shrunken and sparse pyramidal neurons and reduced extent of CA3 region (between arrowheads).
from a control animal (c) compared with ulcerated monkey (4. Latter shows dispersed Nissl bodies and irregularly shaped perikarya with atrophic
dendritic branches in the stratum lucidum. x 500. e and f; Ultrastructure
from control (e) and ulcerated cf) monkeys. Latter shows marked atrophy and regressive changes in neuroplasma, nucleus, and dendrites of CA3
neurons and depletion of mossy fiber endings in the stratum lucidum. x 3380 (x 11,200, insert).
of Ammon’s horn neurons (CAl-4) of control animal (a) compared with ulcerated animal
x 85. c and d, CA3 neurons
of CA3 pyramidal neurons and mossy fiber terminals (arrows, insert)
of 5 monkeys that were euthanized
one animal that died shortly after experimental
Neuropathological analysis. All analyses were done without knowl-
edge of the groups from which subjects came. For light microscopy,
whole hemispheres of brains were fixed in 10% neutral buffered formalin
and hippocampal sections, cut coronally 1 mm caudal from the uncus,
for other research purposes, plus
were cut and embedded in glycol methacrylate resin (JB4 embedding
kit, Polysciences, Inc.). Other brain regions (striatal ganglia, frontal, pre-
and postcentral and temporal cortex) were cut and prepared similarly.
The plastic sections (3 pm thick) were stained with cresyl violet. For
cell counting, only cells with nuclei were included.
For ultrastructural studies, 1 x 1 x 2 mm slices (containing stratum
The Journal of Neuroscience, May 1989, 9(5) 1707
Figure I. Continued.
pyramidale and lucidum of CA3 region and cortical gray matter of the
postcentral gyrus) were cut and refixed with glutaraldehyde-parafor-
maldehyde solution (2.5 and l%, respectively) for 24 hr. Tissues were
processed routinely and observed by a Hitachi 200 electron microscope.
Adrenal tissue was prepared for light microscopy in the same way as
was the neural tissue; after embedding, 2 pm sections were cut and tissue
stained in methylene blue and basic fuscin.
Statistical comparisons. Comparison of adrenal cortical measures be-
1708 Uno et al. * Stress and Neuropathology in Primate Hippocampus
Table 1. Experimental history and pathologic findings of study animals
SD, d, e
SD, c, nd
SD, d, dhy
MGU, gu, e-c
MGU, sch, sld
MGU, g, n
MGU, sld, acd
lesions were in the gastric body and fundus,
congestion); “gu” = gingival
P-M time indicates post-mortem
NP indicates grade of neuropathologic
showing shrinkage of perikarya,
indicates severe changes, with more than 80% of neurons
physiology = reproductive experiments; “schisto” = experimental schistosomiasis; “survey” = parasite survey: “quar” = quarantine
= nasal discharge.
= death following experimental surgery; “SD” = spontaneous death; “d” = diarrhea; “e” = emaciated; “dhy” = dehydrated, “c” =
“sch” = liver schistosomiasis; “e-c” = entero-colitis;
of a focal absence of glandular
“gut necr” = acute necrosis of gut following
= adrenocortical degeneration;
= colitis; “g”
gastric ulcers (most
= splenic lymphoid
changes in the CA regions
dispersed Nissl bodies, and zonal disarray;
of hippocampus. - indicates
changes, with 20-80%
* indicates mild changes, with less than 20% of neurons
of neurons showing ** indicates such degeneration; ***
with such degeneration.
tween groups was made by unpaired t tests. Comparison
counts between groups in different hippocampal
2-way ANOVA followed by Newman-Keuls
regions was made with
post hoc tests.
Compared with control tissue, pronounced neuronal degener-
ation was observed in the hippocampi of ulcerated animals. The
circumferential extent of the CA3 region was generally reduced,
and the pyramidal neurons throughout the CA regions were
shrunken and sparse (Fig. 1, a, b). At higher magnification (Fig.
1, c, d), CA3 pyramidal neurons had reduced and irregularly
shaped perikarya associated with dispersed Nissl bodies and
atrophic dendritic branches. The stratum lucidum lost its clear
interlaced texture made of the dendritic branches of CA3 py-
ramidal neurons and of the mossy fiber endings of the dentate
granular neurons. Numerous swollen oligodendrocytes were ob-
served (as round clear cells with pyknotic nuclei).
Hippocampal CA3 pyramidal neurons were then examined
ultrastructurally (Fig. 1, e, J>. Among ulcerated animals, Nissl
bodies were dispersed and vesicle number increased. In more
severe cases, in addition, perikaryon size was decreased and
dendritic processes were narrowed. The mossy fiber terminals
were depleted in the stratum lucidum, and preterminal fibers
were dispersed. Moreover, synaptic vesicle number was re-
duced, and dendritic branches were edematous, with few neu-
rotubules. Swollen oligodendrocytes had edematous cytoplasma
containing dispersed ribosomes, many irregularly shaped vesi-
cles, myelin sheaths, and lysosome-like dense bodies.
Similar changes occurred in CA1 and CA4 pyramidal neu-
rons, but not in granular neurons of the dentate gyrus. Swollen
oligodendrocytes were noted in the cerebral cortex, thalamus,
hypothalamus, caudate, and pontine nuclei. No neuronal ab-
normalities were noted in these regions, except in the cerebral
cortex, where varying degrees of shrinkage of pyramidal neurons
occurred in layers 3-5 of the frontal, pre-, postcentral, and cin-
gulate gyri (Fig. 2~).
Ultrastructural examination of the cerebral cortices of ulcer-
ated monkeys showed pyramidal neurons with condensed peri-
karyonic cytoplasma, densely distributed ribosomes, many mi-
tochondria, and irregularly
oligodendrocytes were usually attached to shrunken neurons
(satellitosis) (Fig. 2b).
The hippocampal degeneration among ulcerated male mon-
keys was also apparent quantitatively, with fewer pyramidal
neurons in Ammon’s horn cell regions than in control monkeys
The adrenal cortices of ulcerated animals showed substantial
hyperplasia in the zona fasciculata and lesser amounts of hy-
perplasia in the other 2 cortical zones; hypertrophy of individual
cortical cells was also noted (Fig. 4, a, b). The average thickness
of cortex was increased in ulcerated animals: 0.7 f 0.03 versus
1.2 + 0.05 for control and ulcerated, respectively (p < 0.01,
unpaired t test).
shaped vesicles. Swollen
The Journal of Neuroscience, May 1989, 9(5) 1709
Figure 2. a, Shrinkage of pyramidal neurons with hydropic swelling and accumulation
x 500. b, Ultrastructure of a cortical pyramidal neuron and satellite cell showing condensation
satellite cell. x 4000.
of satellite cells in postcentral gyrus in ulcerated animals.
of neuron perikaryon and hydropic swelling of
We have observed marked differences in the neuropathological
profiles of ulcerated monkeys compared with healthy controls.
The neuropathology in ulcerated monkeys was unlikely to have
been an artifact of post-mortem decay since control animal 3531;
had a post-mortem delay at least equal to half the ulcerated
animals, yet had no degeneration, while all ulcerated animals
necropsied immediately had damage. Moreover, the neuro-
pathology was unlikely to have resulted from the agonal state
of spontaneous death (versus euthanization) since control ani-
mal 353F was not euthanized, yet had no degeneration. An
exception to this is likely to have been the swollen oligoden-
drocytes observed throughout the brains of the ulcerated ani-
mals. This is generally considered to be a nonspecific artifact of
either agonal state or post-mortem autolysis (Penfield and Cone,
1926; Brierley and Graham, 1984).
A number of observations suggest that the ulcerated animals
were socially subordinate and under prolonged stress. Among
captive groups of vet-vet monkeys, social behavior often in-
volves persistent and escalating aggression culminating in se-
rious injury, as well as an absence of ritualized gestures of social
submission that serve to terminate aggression in other primate
species; it is a typical aspect of social behavior that, under those
conditions, it is subordinate animals that are subject to the
highest rate of these social stressors (Rowell, 197 1; Wolfheim
and Rowell, 1972; Else, 1985; Kaplan, 1988). Six of 8 ulcerated
monkeys came from social groups, whereas only 1 of 6 control
animals did. Ulcerated animals from social groups appeared to
have suffered higher rates of social harassment and attack than
did control animals: 4 of 6 had bite scars at necropsy. In contrast,
5 other animals from social groups were examined as controls
during that period, and none had bite wounds. Moreover, while
GC concentrations could not be determined post-mortem, ul-
cerated animals had hyperplastic zona fasciculata of their ad-
renal cortices. As noted, this is the zone in which GCs are
synthesized, and its hyperplasia is indicative of sustained GC
hypersecretion (Bondy, 1985). In support of this view of ulcer-
ated animals as chronically stressed, psychologic or social stress
can be highly ulcerogenic in primates (Brady et al., 1958).
The anatomical and cellular patterns of damage in the ulcer-
ated animals are in accord with what is known about GC-in-
duced neurotoxicity. In the rat, it is the hippocampus that is
preferentially damaged by elevated GC concentrations (see Sa-
polsky et al., 1986); moreover, it is the hippocampus in which
normative, age-related neuron loss is halted by adrenalectomy
or behavioral diminution ofGC secretion (Landfield et al., 198 1;
Meaney et al., 1988). This hippocampal vulnerability is thought
to arise from the extremely high concentrations of corticosteroid
receptors in the structure (Sapolsky et al., 1986). The primate
hippocampus is also a major GC-target tissue (McEwen et al.,
1986), and ulcerated monkeys had preferential (although not
1710 Uno et al.
l Stress and Neuropathology in Primate Hippocampus
Figure 3. Numbers of pyramidal neu-
rons/coronal section through Ammon’s
horn. Data for each animal were de-
rived by averaging cell counts from 5
successive sections taken beginning 2
mm caudal to the uncus. *, ** indicate
p < 0.05, 0.002 compared with same-
sex controls (Newman-Keuls test fol-
lowing 2-way ANOVA).
dentate CA4 CA3 CA1
exclusive) hippocampal damage. Anatomical patterns of vul-
nerability within the hippocampus also suggest a causative role
for GCs. In the rat, neuron loss during aging is minimal in the
dentate gyrus and most pronounced in Ammon’s horn pyrami-
da1 neurons (reviewed by Coleman and Flood, 1987). Further-
more, the dentate seems resistant to damage induced by pro-
longed GC exposure, while Ammon’s horn neurons are
particularly sensitive (Sapolsky et al., 1985). In the present case,
a similar pattern of vulnerability occurred (Fig. 2). [It should
be noted that the pattern of hippocampal damage induced, not
by prolonged exposure to GCs alone, but by shorter exposures
to GCs in synergy with various insults varies according to which
insult is used (Sapolsky, 1986b).] Finally, prenatal exposure to
GCs in the primate can damage the hippocampus. In that study,
Figure 4. Cross sections of adrenal
gland from control (a) and ulcerated(b)
monkeys. Arrows indicate corticomed-
ullary bouridary. x 10.
The Journal of Neuroscience, May 1989, 9(5) 1711 Download full-text
133 d fetal rhesus monkeys were exposed to very high (5 mgl
kg mother’s body weight) concentrations
dexamethasone; near term, pyramidal neuron number was de-
creased; dendritic branches and axon terminals were swollen;
vesicles, mitochondria, and neurotubules
edematous axoplasm; and perikarya were atrophic (Uno et al.,
Considerable variability in the severity of hippocampal dam-
age occurred among ulcerated animals. This seemed not to be
a function of the severity of peripheral pathologies at necropsy.
Instead, more severe damage was observed among males (Fig.
2; Table l), and among animals that had been captive a shorter
time (Table 1). Unfortunately, these 2 trends covaried, and with
the small sample size, it was not possible to determine whether
one factor was more important.
the meaningful variable, it could reflect that ulcerated males
were exposed to more severe stressors than were ulcerated fe-
males and/or were more vulnerable to the neurodegenerative
effects of such stressors. In support of the former possibility,
male vervets are often socially subordinate to females and are
subject to high rates of attacks by them (Bernstein, 1970; Lan-
caster, 197 1; Bramblett et al., 1982); to our knowledge, patterns
of ulceration in wild vervets have never been studied.
In conclusion, a body of studies suggest that prolonged stress,
via GC hypersecretion, can damage the rodent hippocampus,
and the present data suggest, tentatively, a similar pattern in
the primate. It may be beyond ethical bounds to attempt to
demonstrate this more directly in a primate with an intentional
experimental stressor. Nevertheless,
tunistic study are suggestive: Ulcerated animals showed patho-
logical and morphological indices of prolonged stress; anatom-
ical and cellular features of damage among such animals were
quite similar to those of rodent models of GC neurotoxicity;
the precedent exists for (pharmacologic concentrations
damaging the fetal primate hippocampus. The potential impli-
cations of these observations are considerable, as the conse-
quences of hippocampal damage for learning and memory are
profound (Squire, 1986). The present data suggest that in the
primate, hippocampal degeneration might constitute a “stress-
related” pathology, as well as a potential consequence of pro-
longed exogenous GC administration.
Note added in proof
We have since expanded this study to
include more than a dozen such ulcerated animals and continue
to see the same pattern of neuropathology. Previous investi-
gators have noted a high incidence of multiple gastric ulcers
among captive vervet monkeys and in one such case where
behavioral data had been collected on individuals, these were
found to be socially subordinate animals (F. Ervin, personal
of the synthetic GC,
were dispersed in
Were the sex difference to be
the findings in this oppor-
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