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Direct evidence for calcium-induced ischemic and reperfusion injury.

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Direct Evidence for
Calcium-induced Ischemic
and Reperfusion Injury
Daisuke Uematsu, MD,*$ Joel H. Greenberg, PhD,*
Martin Reivich, MD,' and William F. Hickey, MDfl
Changes in cytosolic free calcium (ICaZf]i) in the cat
cortex were measured in vivo by indo-1 fluorometry
during cerebral ischemia and reperfusion and were correlated to the histopathological ischemic changes. These
changes were most pronounced in stroke cases with an
increase in [Ca2'Ii throughout the ischemic and reperfusion periods. Cases without a {CaZ+};increase showed no
histopathological change in the cortical gyms in which
[Ca2+jiwas measured. The data support the hypothesis
that an increase in [Ca2+Iiduring cerebral ischemia and
reperfusion leads to neuronal damage.
Uematsu D, Greenberg JH, Reivich M, Hickey
WF. Direct evidence for calcium-induced
ischemic and reperfusion injury.
Ann Neurol 1989;26:280-283
In the past decade, an increase in cytosolic free calcium
({Ca' 'li) has been hypothesized to have a causative
role in mediating neuronal damage during cerebral
ischemia and reperfusion 11, 2). Employing an in vivo
fluorometric technique with indo-1, a fluorescent Ca2+
indicator, we recently demonstrated in a focal ischemic
model that {Ca'+]i in the cortex increases only in
stroke cases with severe electroencephalographic
(EEG) changes {31. We also found that the recovery of
{Ca2+]i is closely related to that of EEG during the
reperfusion period {4]. Further studies have been undertaken to investigate a causal relationship between
the increase in {Ca' +}i and histopathological brain
The animal model and fluorometric technique used in this
study are described in detail in a separate communication
{4]. In brief, adult male cats were anesthetized by inhalation
of halothane and placed on a respirator. A burr hole was
From the 'Cerebrovascular Research Center, Department of Neurology, and ?Division of Neuropathology, Department of Pathology, University of Pennsylvania, Philadelphia, PA.
Received for publication Sep 27, 1988, and in revised form Jan 16,
1989. Accepted for publication Jan 18, 1989.
Address correspondence to Dr Greenberg, Cerebrovascular Research Center, 429 Johnson Pavilion, 36th Street & Hamilton Walk,
University of Pennsylvania, Philadelphia, PA 19104.
Present addresses: $Department of Neurology, Keio University
School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo, Japan;
and §Department of Pathology, Division of Neuropathology, Washington University, St. Louis, MO.
280 Copyright 0 1989 by the American Neurological Association
Histopathologicd Data
Histological Gradesa
No. of
Focal Damageb
0.54 t 0.23
4.22 0.6@
Total Damage'
Severe, type 1
Severe, type 2
2.23 ? 2.23
4.66 2 2.02
16.06 ? 5.87
"Histological ischemic changes were graded into 4 categories [b, 73: 0 = histologically normal, 1 = slight changes with a few scattered affected
neurons (shrunken cell bodies with triangular, darkly stained cytoplasm), 2 = moderate changes with a typical microscopic field containing
several affected neurons and an edematous neuropil, and 3 = severe changes with death of all parenchymal cell types, cytoplasmic retraction,
and an edematous neuropil.
bExpressed as percentage by dividing the area of damage in rhe cortical gyrus of the cytosolic free calcium measurement by the total area of the
left hemisphere.
'Calculated as percentage by dividing the total damaged area by the total area of the left hemisphere.
dSignificantdifferences in focal damage between types 1 and 2, p < 0.05.
EEG = electroencephalographic.
drilled in the left skull in the territory of the middle cerebral
artery (MCA), and a quartz cranial window equipped with a
pair of EEG electrodes was placed over the exposed cat
cortex. Indo-1 was loaded into the cortex by a 2-hour superfusion of 7 IJ.M membrane-permeant indo-1 acetoxymethyl
ester (indo-1-AM, Molecular Probes Inc, Eugene, OR) in
artificial cerebrospinal fluid. A small cortical area of interest
was illuminated intermittently with ultraviolet rays (340/12
nm), and changes in intracellular indo-l-Ca2 fluorescence at
400 and 506 nm, NADH fluorescence at 464 nm (a crossover point of indo-1 emission spectra), and reflectance at 340
nm were recorded by 4 photomultiplier tubes with appropriate barrier filters. The changes in reflectance are inversely
proportional to changes in local cortical blood volume
(ICBV). Percent changes in local cortical blood flow (ICBF)
were assessed from cerebrovascular hemodilution curves by
dividing ICBV by mean transit time as described elsewhere
I S ] . A {Ca2+fisignal ratio at 400 and 5 0 6 nm was utilized as
a measure of change in {Ca2']i 141. A trunk of the left MCA
was occluded microsurgically for 1 hour with a miniature
Mayfield clip via transorbital approach. Three hours after the
reperfusion, the brain was perfused with a solution containing 3% glutaraldehyde and 90 mM potassium oxalate via an
aortic cannula.
The animals were then killed and the brains postfixed with
10% formaldehyde for 5 days. Each brain was then sectioned
coronally through the plane of the optical measurement and
processed for microscopic examination. The degree of the
histopathological ischemic changes was evaluated according
to a semiquantitative grading system, described elsewhere 16,
7f (Table). The histological evaluation was performed by a
neuropathologist without knowledge of other experimental
data. The damaged area in the same cortical g y m of the
[Ca2+], measurement was also examined using an image analyzer with a video camera, and a ratio of the damaged area to
the total area of the left hemisphere was calculated and used
as an index of extent of ischemic damage.
During ischemia the threshold of lCBF was 20% of
the resting flow below which [Ca2+}i began to increase
(Figure, top). The a n i m a l s whose ICBF fell to less than
the threshold level also showed a reduction of EEG
amplitude by 93 & 1% (mean -+ SEM) at 30 minutes
after the MCA occlusion. Those animals with a large
depression in EEG amplitude (to less than 20% of
control animals) were considered to be severely affected. This group was further divided into 2 subtypes
according to the pattern of the EEG amplitude recovery during the first 30 minutes of reperfusion: type 1
with 10% or more recovery and type 2 with less than
10% recovery 141. Type 1 animals showed a significant
recovery in [Ca2+]; signal ratio during reperfusion
from 2.68 & 1.04 (mean f SEM) at the end of MCA
occlusion to 1.50 ? 0.19 30 minutes into the reperfusion ( p < 0.05, paired Student's t test), whereas type 2
animals showed no recovery of [Ca*+};. No significant
difference was noted in ICBF between the two subtypes 30 minutes into reperfusion. In all stroke groups
lCBF and {Ca2+}ilevels 30 minutes after reperfusion
were not correlated (Figure, bottom), indicating that
the level of lCBF is not a prime determinant of [Ca2+li
during the reperfusion period.
Histopathological data on the grading of ischemic
changes as well as the area of damage are summarized
in the Table. All animals with a marked reduction in
EEG amplitude, a low lCBF (< 20%), and a high
[Ca2+}i at 30 minutes after the MCA occlusion
showed histopathological changes in the gyrus in which
[Ca2+}iwas measured. The area of damage in the cortical gyrus of the {Ca2+};measurement was significantly
larger in the type 2 than in the type 1 subgroup ( p <
0.05, Student's t test). In both severe groups a significant correlation was seen between the [Ca2'Ii signal
ratio at 30 minutes after reperfusion and the histological grading (Spearman rank correlation coefficient, p;
= 0.88, p < 0.001). The damage seen in the type 2
subgroup was both greater in extent and of a higher
grade than in type 1. One of 6 animals with a moderate
Brief Communication: Uematsu et al: Calcium and Ischemic Injury
{lo], since the excitatory amino acids such as glutamate and aspartate increase extracellularly during cerebral ischemia { 111.
After reperfusion, in the type 2 severe subgroup,
[Ca2 '1, remained elevated despite recovery of 1CBF.
The histopathological changes were significantly
greater in the type 2 subgroup than in the type 1
subgroup, which showed much greater recovery in
both EEG and [Ca2+li during the reperfusion period.
It was the level of {Ca2 li during the reperfusion period, not the level of ICBF, that appeared responsible
for both recovery of EEG and severity of histopathological changes. We assume that Ca2 ' extrusion mechanisms in the plasma membrane, such as calciummagnesium ATPase and the sodium-calcium exchange
system 1121, were rapidly restored after reperfusion in
the type 1 subgroup but not in the type 2 subgroup.
Consequently a further accumulation of Ca2 would
occur in the latter cases, triggering numerous Caz+dependent enzymatic processes [ 131 as well as superoxide radical reactions {14],which would lead to irreversible cellular damage.
We conclude that alterations of cytosolic free calcium during cerebral ischemia and reperfusion are
closely related to the histopathological neuronal damage.
5Severe, type I
Severe, type 2
A Moderate
ICBF (% of Control1
Cowelation between local cortical blood flow(ICBF ) and cytosolic
f e e calcium ({CaZ+};)at 30 minutes into i.irbernia (top) and
30 minutes into repefusion (bottom). During ischemia
{Ca'-}, signal ratio started to increase below a certain lez!eI
(20%) of ICBF. N o correlation waj seen between ICBF and
{Ca' 7 } t /evel30 minutes into reperfusion. Halftone area itidirates a norma( range of {Ca2-), signai ratio (mean 2.5 standard dmiation).
or mild EEG deterioration, a higher ICBF (> 20%),
and normal [Ca2 +lirevealed a small focus of grade 1
histological change; this damage, however, was outside
the focus of the {CaLflimeasurement.
We have shown that during cerebral ischemia the
blood flow threshold is at 20% of the resting level
below which [Ca2 '1i starts to increase. Calcium ion can
enter the neurons through voltage-dependent Ca2
channels during depolarization that occurs during severe ischemia caused by ATP depletion [81. Our data
are in good agreement with those of Harris and colleagues [ 9 ] ,who showed that there was a flow threshold below which extracellular Ca2- began to decrease.
Calcium entry into the neurons can also occur via Nmethyl-D-aspartate (NMDA)-operated Ca2+ channels
This work was supported by National Institutes of Health grant NS
ICBF (% of Control)
Annals of Neurology Vol 26 No 2 August 1989
1. Siesjo BK. Cell damage in thc brain: a speculative synthesis. J
Cereb Blood Flow Metab 1981;1:155-185
2. Siesjo BK. Calcium and ischemic brain damage. Eur Neurol
3. Grynkiewicz G, Poenie M, Tsien RY. A new generation of
Ca" indicators with greatly improved fluorescence properties. J
Biol Chem 1985;260:3440-3450
4. Uematsu D, Greenberg JH, Reivich M, Karp A. In vivo measurement of cytosolic-free calcium during cerebral ischemia and
reperfusion. Ann Neurol 1988;24:420-428
5. Dora E, Tanaka K, Greenberg JH, et al. Kinetics of microcircdatory, NADINADH, and electrocorticographic changes in
cat brain cortex during ischemia and recirculation. Ann Neurol
6. Ginsberg MD, Graham DI, Welsh FA, Budd WW. Diffuse
cerebral ischemia in the cat: 111. Neuropathological sequelae of
severe ischemia. Ann Neurol 1978;5:350-358
?. Tanaka K, Greenberg JH, Gonatas NK, Reivich M. Regional
flowmetabolism couple following middle cerebral artery occlusion in cars. J Cereb Blood Flow Metab 1985;5:241-252
8. Harris RJ, Symon L, Branston NM, Bayhan M. Changes in
extracellular calcium activity in cerebral ischemia.J Cereb Blood
Flow Metab 1981;1:203-209
9. Harris RJ, Symon L Extracellular pH, potassium, and calcium
activities in progressive ischema of rat cortex. J Cereb Blood
Flow Metab 1984;4:178-186
10. MacDermott AB, Mayer JL, Westbrook GL, et al. NMDAreceptor activation increases cytoplasmic calcium concentration
in cultured spinal cord neurones. Nature 1986;321:519-522
11. Benveniste H, Drejer J, Schousboe A, Diemer N H . Elevation
of the extracellular concentrations of glutamate and aspartate in
rat hippocampus during transient cerebral ischemia monitored
by intracerebral microdialysis. J Neurochem 1984;43:13691374
12. Peters T. Calcium in physiological and pathological cell function. Eur Neurol 1986;25(suppl 1):27-44
13. Raichle ME. The pathophysiology of brain ischemia. Ann Neurol 1983;13:2-10
14. Kogure K, Arai H, Abe K, Nakano M. Free radical damage of
brain following ischemia. In: Kogure K, Hossmann K-A, Siesjo
BK, Welsh FA, eds. Molecular mechanisms of ischemic brain
damage. Progress in brain research, vol 63. Amsterdam: Elsevier, 1985:237-259
Isolation of Herpes
Simplex Virus Type 1
During First Attack of
Multiple Sclerosis
Tomas Bergstrom, MD,” Oluf Andersen, MD, PhD,?
and Anders Vahlne, MD, PhDX
Herpes simplex virus type 1 was isolated from the cerebrospinal fluid of a patient during the first attack of
multiple sclerosis. This is the first virus to be isolated
from the central nervous system of a living patient with
MS. The virus was identified as herpes simplex virus
type 1 by restriction endonuclease analysis and by an
enzyme immunoassay using monoclonal antibodies.
Antibodies against type 1 but not type 2 were detected
in consecutive samples of serum and cerebrospinal fluid.
The patient has since entered a progressive phase of
multiple sclerosis. The isolated type 1strain might be of
pathogenetic relevance to the development of multiple
sclerosis in this patient.
Bergstrom T, Andersen 0, Vahlne A. Isolation of
herpes simplex virus type 1 during first attack of
multiple sclerosis. Ann Neurol 1989;26:283-285
Although epidemiological studies on multiple sclerosis
(MS) have indicated an etiological role of viral infection, attempts to isolate virus from the central nervous
From the “Department of Chnical Virology, and the ?Department of
Neurology, University of Goteborg, Sahlgrens Hospital, Goteborg,
Received Dec 9, 1988, and in revised form Jan 27, 1989 Accepted
for pubhation Jan 28, 1787.
Address correspondence to Dr Bergstrom, Department of Clinical
Virology, Guldhedsgatan 10B,S-413 46 Goteborg, Sweden
system (CNS) or cerebrospinal fluid (CSF) in patients
with MS have been unsuccessful [lJ. Indirect evidence, such as the presence of intrathecally produced
antibodies and positive findings of RNA by in situ
hybridization of CSF and brain cells, has been presented in support of both paramyxovirus and retrovirus as causative agents 12-47, but these findings
await confirmation {5}. In recurrent demyelinating diseases of the CNS, such as MS, viruses that can establish persistent or latent infections have attracted particular interest as possible etiological agents. Herpes
simplex viruses type 1 (HSV-1) and type 2 (HSV-2)
display these properties and can induce CNS demyelination as demonstrated experimentally {GI. Attacks
in both reactivated HSV infection and MS may be
precipitated by emotional stress, other infections,
trauma, and irradiation {?}. The anatomical locations of
sequential MS foci suggest axonal transport of an etiological agent [a], a route that is established for HSV
[9].HSV-2 has been isolated at autopsy from the brain
of a patient with MS {lo].
We have isolated an HSV-1 strain from the CSF of a
previously healthy woman during the first episode of
neurological disease. She fulfilled the criteria for MS
according to accepted guidelines [ll], and after 4 years
she was in a progressive phase of the disease. CSF
laboratory measures consistent with the diagnosis of
MS, such as elevated IgG index, normal albumin ratio,
and presence of oligoclonal bands in isoelectric focusing, were recorded repeatedly since the onset of disease in the patient. We analyzed the patient’s antibody
response in serum and CSF to HSV-1 and to other
viral and bacterial agents of potential interest.
Patient and Samples
The patient was a previously healthy 52-year-old unmarried
nurse assistant. In January 1984, after 3 weeks of sensory
symptoms including coldness of the left leg to the left side of
the waist, she had acute attacks of dizziness elicited by movement. On left gaze, eye movements were uncoordinated and
unstable. When she was admitted to the hospital, ophthalmological and neurological examination revealed bilateral internuclear ophthalmoplegia and weaker pharyngeal contraction on the left. Findings on audiology testing were normal.
She recovered within 2 months and was well until June 17,
when she again noted some dizziness, and diplopia on left
gaze. One week later she developed left peripheral facial
palsy with associated disturbance of taste and hyperacusis.
Again, recovery was complete. At follow-up on March 26,
1985, slight imbalance and subtle increase in tone with a
right-sided hyperreflexia were found. On follow-up visits
through 1988 transient nystagmus on downgaze and the
g r a d d development of a moderate spastic paraparesis were
noted. The diagnosis was clinically definite MS. Paired samples of serum and CSF were drawn repeatedly and analyzed
immediately, except for samples undergoing enzyme-linked
immunosorbent assay (ELISA), which were stored at - 20°C
for later simultaneous quantification of antibodies.
Copyright t
3 1989 by the American Neurological Association 283
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reperfusion, ischemia, induced, evidence, direct, injury, calcium
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