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The Journal of Neuroscience, July 15, 1998, 18(14):5151-5159
Cyclosporin A, But Not FK 506, Protects Mitochondria and Neurons
against Hypoglycemic Damage and Implicates the Mitochondrial
Permeability Transition in Cell Death
Hans
Friberg1, 2,
Michel
Ferrand-Drake1,
Finn
Bengtsson3,
Andrew P.
Halestrap4, and
Tadeusz
Wieloch1
1 Laboratory for Experimental Brain Research,
Wallenberg Neuroscience Center, Departments of
2 Anesthesiology and 3 Clinical Pharmacology,
University Hospital, S-221 85 Lund, Sweden and 4 Department
of Biochemistry, University of Bristol, Bristol BS8 1TD, United
Kingdom
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ABSTRACT |
Induction of the mitochondrial permeability transition (MPT) has
been implicated in cellular apoptosis and in ischemia-reperfusion injury. During MPT, a channel in the inner mitochondrial membrane, the
mitochondrial megachannel, opens and causes isolated mitochondria to
swell. MPT and mitochondrial swelling is inhibited by cyclosporin A
(CsA), which may also inhibit apoptosis in some cells.
Treatment with CsA (50 mg/kg, i.v.) showed a robust reduction of brain
damage when administered 30 min before insulin-induced hypoglycemic
isoelectricity of 30 min duration. Ultrastructural examination of the
dentate gyrus revealed a marked swelling of dendrites and mitochondria
during the hypoglycemic insult. In CsA-treated animals, mitochondria
resumed a normal and contracted appearance during and after the
hypoglycemic insult. Treatment with FK 506 (2 mg/kg, i.v.), a compound
with immunosuppressive action similar to that of CsA, was not
protective. Studies on the swelling kinetics of isolated mitochondria
from the hippocampus showed that CsA, but not FK 506, inhibits calcium
ion-induced MPT.
We conclude that CsA treatment during hypoglycemic coma inhibits the
MPT and reduces damage and that mitochondria and the MPT are likely to
be involved in the development of hypoglycemic brain damage in the
rat.
Key words:
Cyclosporin A; hippocampal mitochondria; hypoglycemia; mitochondrial permeability transition; brain damage; cell death
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INTRODUCTION |
When isolated mitochondria are
exposed to high concentrations of calcium ions, a mitochondrial
megachannel (MMC) opens in the inner mitochondrial membrane (Haworth
and Hunter, 1979 ). This event, also named the mitochondrial
permeability transition (MPT), is considered to be an early event in
apoptosis in some cells (Marchetti et al., 1996a; Kroemer, 1997) and
possibly a trigger of cell death in ischemia-reperfusion damage
(Griffiths and Halestrap, 1993a; Uchino et al., 1995).
Cyclosporin A (CsA) and FK 506 are immunosuppressive compounds,
believed to exert their action through binding to small intracellular regulatory proteins, the cyclophilins (CyPs) or FK binding proteins (FKBPs), also known as immunophilins (Galat and Metcalfe, 1995). When
complexed with CsA and FK 506, the properties of the immunophilins change, leading for example to the inhibition of the phosphatase calcineurin (Liu et al., 1991) and subsequent inhibition of T-cell activation (Clipstone and Crabtree, 1992; O'Keefe et al., 1992).
Cyclosporin A, but not FK 506, prevents MPT by blocking translocation
of the mitochondrial matrix-specific cyclophilin-D (CyP-D) to the inner
membrane of liver mitochondria, thereby decreasing MMC sensitivity to
calcium ions (Connern and Halestrap, 1994). Several other modulators of
the MMC have been characterized in liver and heart mitochondria, among
them free radicals, adenine nucleotide depletion, a low mitochondrial
membrane potential, and increased matrix pH, which all stimulate the
calcium-induced MPT, whereas decreasing the matrix pH below 7 is
inhibitory (Halestrap, 1991; Bernardi et al., 1992; Zoratti and Szabo,
1995).
Although MPT is clearly involved in apoptosis in thymocytes (Marchetti
et al., 1996b), its role in neuronal death attributable to energy
shortage is still elusive. Early work by Shiga et al. (1992) showed
brain protection by CsA in a model of focal ischemia. In other studies,
FK 506, which readily penetrates the blood-brain barrier (BBB), and
high doses of CsA showed a significant reduction of infarct volume in a
model of endothelin-induced focal ischemia (Sharkey and Butcher, 1994;
Butcher et al., 1997). In a rat model of global brain ischemia, FK 506 is neuroprotective when administered before the insult (Drake et al.,
1996). Protection by FK 506 was also attained when administered after
global ischemia in the gerbil, which may have been caused partially by
hypothermia induced by the drug (Ide et al., 1996). Cyclosporin A, on
the other hand, which has a limited ability to penetrate an undamaged
BBB (Begley et al., 1990), has been shown to protect from damage after
global ischemia only if combined with a needle lesion into the brain parenchyma (Uchino et al., 1995).
Severe insulin-induced hypoglycemia causes selective neuronal damage to
certain brain regions, such as the outer layers of the cortex, the
striatum, the medial CA1 region, and the crest of the dentate gyrus
(Auer and Siesjo, 1993). Hypoglycemia is associated with severe energy
failure and loss of ion homeostasis, which includes a massive influx of
calcium ions into brain cells (Siesjo and Bengtsson, 1989). In contrast
to ischemia, hypoglycemia is not accompanied by acidosis but rather by
a mild alkalosis, because of reduced glycolytic flux and combustion of
glycolytic metabolites in the tricarboxylic cycle. Early
ultrastructural changes associated with the hypoglycemic insult include
axon-sparing dendritic lesions with swollen dendrites and mitochondria
(Auer et al., 1985). During hypoglycemic coma, conditions apparently favor the activation of MPT.
This study was performed to determine whether there are ultrastructural
signs of MPT during hypoglycemia. Also, we aimed to assess whether CsA
prevents calcium-induced swelling of isolated hippocampal mitochondria
and whether treatment with CsA protects against hypoglycemic brain
damage in vivo.
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MATERIALS AND METHODS |
Animal surgery. All animal experiments were approved
by the ethical committee at the University of Lund. Male Wistar rats from Moellegard avlslaboratorium (Copenhagen, Denmark), weighing 300-340 gm, were fasted overnight with access to water. One hour before surgery, the rat received an intraperitoneal injection of 2 IU/kg of insulin (Actrapid, Novo Nordisk A/S, Copenhagen, Denmark).
Induction of anesthesia was performed with 3% halothane in a 70:30
mixture of nitrous oxide and oxygen. The animal was intubated with
polyethylene tubing and connected to a small animal respirator.
Halothane concentration was reduced and kept at 1.5% during surgery.
For glucose infusion, a venous line from a tail vein was used, and for
blood sampling and recording of the mean arterial blood pressure
(MABP), an arterial line from a tail artery was used. The jugular vein
was exposed, and a soft SILASTIC catheter was placed into the inferior
vena cava to control blood pressure by exsanguination or reinfusion of
blood. After the surgical procedure, the halothane concentration was
lowered to 0.5%, and the animals were allowed a steady-state period of
~30 min before the induction of hypoglycemia. Heparin (50 IU) was
given as an anticoagulant, temperature and MABP were measured
continuously, and blood gases were controlled. An infusion of
vecuronium (Norcuron, Organon Teknika, Amsterdam, The Netherlands) in
Krebs' solution was given. The arterial PCO2
was adjusted to 35-40 mmHg, and the rectal temperature was kept at
37°C by external heating. Two subcutaneous biparietal needle
electrodes were used to record a bipolar EEG, which was monitored
continuously. At the onset of cerebral isoelectricity, blood pressure
rose, and blood was therefore drawn from the central venous catheter to
keep the blood pressure between 140 and 160 mmHg during the entire
isoelectric period. Isoelectricity was terminated as 0.2 ml of 50%
glucose was given at a rate of 1.5 ml/hr, followed by the infusion of
1:1 solution of 50% glucose and Krebs' solution given at 0.5-1.0
ml/hr to maintain plasma glucose levels between 5 and 10 mmol/l. The
animal was extubated by the time it recovered spontaneous respiration
and made active movements to remove the endotracheal tube. Before the
tail catheters were removed, blood glucose and blood gases were checked
in the awake, conscious animal. The animals were allowed access to
water and food pellets in the cage after surgery. Rectal temperature was monitored daily.
Treatment paradigms. Cyclosporin A, 50 mg/ml (Sandimmune),
was diluted six times in NaCl. FK 506 (10 mg) was dissolved in 0.5 ml
of a 9:1 mixture of ethanol/Tween 80 and further diluted once in water,
yielding a 10 mg/ml stock solution. The vehicle for CsA and FK 506 was
the same 9:1 mixture of ethanol/Tween 80, diluted once in
H2O, and further diluted six times in NaCl, yielding a
7.5% ethanol, 0.8% Tween 80 solution.
Animals prepared for histopathological investigation were divided into
six groups. Cyclosporin A [20 mg/kg (n = 6)],
cyclosporin A [50 mg/kg (n = 8)], and vehicle
(n = 8) were given intravenously as a single dose ~30
min before the onset of isoelectric EEG. FK 506 [2 mg/kg
(n = 4)] and vehicle (n = 4) were
given intravenously as a single dose in the same manner. Finally,
sham-operated control rats were given glucose after insulin
administration to maintain a plasma glucose level between 5 and 10 mmol/l (n = 4).
Histopathology. After 1 week, animals were again
anesthetized and perfused with a 4% formaldehyde solution. Brains were
allowed to post-fix and later were sectioned into 2.8-mm-thick slices and processed in graded ethanol. They were embedded in paraffin, sectioned at 6 µm, and finally stained with 1% acid fuchsin and 0.05% toluidine blue. Damage in the dorsal dentate gyrus was assessed by a graded damage score: 0 = no damage, 1 = damage to the
tip of the dentate gyrus, 2 = damage extended to half the ventral or dorsal blades of the dentate gyrus, and 3 = damage to the
entire dentate gyrus. Damage in the CA1 region was determined at the same level as the dentate gyrus in a visual field of 400 µm diameter, using the following damage score scale: 0 = no damage, 1 = <30% damage, 2 = 30-60% damage, and 3 = 60-100% damage.
Damage in the parietal cortex was assessed by neuronal counting in an
area 400 µm wide, on the same brain slice where damage to the
dentate gyrus was assessed. Striatal damage was assessed using the
following damage score: 0 = no damage, 1 = scattered damage
to the dorsal crest of the striatum, 2 = damage to the dorsal and
ventral crest of the striatum, and 3 = dense damage to the
ventral, dorsal, and medial striatum. Damage was assessed in two
consecutive sections and is the mean of the two hemispheres. All
histopathological assessments were performed by an observer blinded to
treatment conditions.
Electron microscopy. Animals prepared for the electron
microscopy (EM) study were divided into five groups. Cyclosporin A (50 mg/kg), its vehicle, or FK 506 (2 mg/kg) was given intravenously ~30
min before the onset of isoelectric EEG, in the same manner as for
animals prepared for the histopathological investigation. Animals were
killed at either 30 min of isoelectric hypoglycemic coma [CsA
(n = 4), vehicle (n = 4), FK 506 (n = 3)] or after 30 min of recovery after 30 min of
hypoglycemic coma [CsA (n = 4), vehicle
(n = 4)]. Animals were fixed with glutaraldehyde (2%) by intra-aortic perfusion, and brains were immediately removed and
placed in fixation solution. The dentate gyrus was dissected and
allowed to post-fix in glutaraldehyde solution for 1 hr, rinsed in
Soerensen buffer overnight, and then further processed for EM.
To assess mitochondrial swelling at 30 min of isoelectric hypoglycemic
coma, EM pictures of the distal stratum moleculare, superjacent to the
crest of the dentate gyrus, were analyzed at 3800× magnification. The
number of swollen mitochondria in an area measuring 26 µm × 26 µm was counted, and the mean from three areas calculated. We defined
a swollen mitochondrion as being rounded, with a diameter >0.8 µm.
This definition is based on the size of mitochondria in control animals
and on data from the literature (Auer et al., 1985).
Measurements of cyclosporin A concentrations in vivo. Rats
were given CsA [20 mg/kg (n = 4) or 50 mg/kg
(n = 3)] intravenously, ~30 min before the induction
of hypoglycemic coma. Control animals (n = 6) that were
not undergoing insulin-induced hypoglycemia were also given CsA (50 mg/kg, i.v.). The brains of three of the control animals were perfused
with isotonic saline for 1 min through an intra-aortic catheter before
decapitation. Blood samples were taken 45 min after the intravenous CsA
injection, and the animals were then decapitated. The hippocampus (180 mg) was dissected out and homogenized in 350 µl of water. Samples
were stored on ice and further analyzed for CsA content using a
commercial EMIT kit (Behring Diagnostics, Cupertino, CA) on a Cobas
Mira S equipment (Roche Products, Hertforshire, UK). Negative controls
were rat blood and brain homogenate from animals not given CsA that
showed CsA values of <50 ng/ml and 20 ng/ml, respectively. Addition of known amounts of CsA to brain homogenate yielded the expected concentrations. All samples were diluted with methanol to obtain values
within the 100-500 ng/ml range.
Preparation of isolated mitochondria. Male Wistar rats
(300-350 gm) were decapitated, and the brain were transferred to
ice-cold isolation buffer (0.32 M sucrose, 2 mM
EGTA, 10 mM Tris-base, pH 7.4). The hippocampus was
dissected out from each hemisphere in ice-cold buffer in a Petri dish.
Isolated mitochondria were prepared according to Sims, method B (Sims,
1990). Approximately 180 mg of hippocampal tissue was dissected out and
homogenized in 12% Percoll, using a 2 ml Kontes Teflon homogenizer.
The homogenate was added to a Percoll gradient, 40% and 26%, using a
Pasteur pipette, and centrifuged according to published procedures
(Sims, 1990).
Measurements of respiratory control ratio and MPT pore
opening. Respiratory activity of mitochondrial preparations was
measured by determining oxygen consumption in an airtight chamber in
the presence of 5 mM malate and 5 mM glutamate
and after the addition of ADP (Sims, 1990). Respiratory control ratio
(RCR) was calculated as the ratio of state 3/state 4 respiration.
In subsequent preparations for mitochondrial swelling experiments, the
final pellet was resuspended in isolation buffer and kept on ice.
Measurements of the calcium-induced mitochondrial swelling under
de-energized conditions were performed in a Perkin-Elmer (Emeryville,
CA) fluorometer by measuring the decrease in light-scattering at 520 nm. A fixed volume (10 µl) of mitochondrial suspension was added to
1.1 ml of isotonic buffer, pH 7.0, containing 150 mM KCl,
20 mM Mops, 10 mM Tris, 2 mM
nitrilotriacetic acid (NTA), 0.5 µM rotenone, 0.5 µM antimycin, and 2 µM A23187 calcium
ionophore, added to ensure complete equilibration of calcium ions
across the mitochondrial membrane under de-energized conditions
(Halestrap, 1991). Calcium ion concentrations were calculated from the
NTA buffering (Connern and Halestrap, 1994). Protein concentrations were determined using a Bio-Rad assay (Bio-Rad, Richmond, CA). The
protein concentration in the cuvette was ~25 µg/ml, and the experiments were run at a temperature of 26°C.
Calcium dose-response measurements were performed, and the initial
swelling rate, excluding the first 10 sec attributable to mixing
artifacts, was calculated as the decrease in light scattering per time
unit and per microgram of protein. When the effect of CsA or FK 506 on
mitochondrial swelling was studied, the compound was added at 200 nM final concentration, before the addition of calcium
chloride (see Fig. 3).
All chemicals were from Sigma (St. Louis, MO), and the Percoll solution
was from Pharmacia and Upjohn (Bedminster, NJ). Cyclosporin A
(Sandimmune) was from Novartis, and FK 506 (tacrolimus) was a gift from
Fujisawa (Tokyo, Japan).
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RESULTS |
The effect of cyclosporin A and FK 506 on hypoglycemic
brain damage
Plasma glucose levels before, during, and after the hypoglycemic
insult were not significantly different between the groups (Table
1). Neither was there any significant
difference in the average time span between injection of drugs or
vehicle and the initiation of isoelectric EEG (data not shown). Body
temperature in the recovery phase was not significantly different
between the experimental groups, although in the CsA-treated rats a
tendency to a slightly lower temperature was seen (Table
2).
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Table 1.
Plasma glucose levels at different time points after the
induction of insulin-induced hypoglycemic coma in animals given either
cyclosporin A or its vehicle
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There was a dose-dependent neuroprotective effect of CsA in the dentate
gyrus and in the CA1 area of the hippocampus (Fig. 1). In vehicle-treated rats, neuronal
damage was seen in the outer layers of the cortex, the striatum, the
medial CA1 region, and the dentate gyrus granule cells at 1 week after
30 min of hypoglycemic isoelectricity. In all eight rats treated with
vehicle, damage to the crest and blades of the dentate gyrus was seen.
Also, ~50% of neurons in the CA1 region had succumbed. In the
cortex, a mean of eight damaged neurons were counted, and in the
striatum, damage to the ventral and dorsal part was quite extensive
(damage score = 3.0). In all rats treated with 50 mg/kg CsA, no
damage to the dentate gyrus or the cortex was observed. In the CA1
pyramidal neurons, <30% damage was noted, whereas in the striatum
damage was less effectively diminished (damage score = 2.4). When
rats were treated with 20 mg/kg CsA, a variable degree of
neuroprotection was seen, whereas treatment with 2 mg/kg FK 506 did not
yield protection in any brain region (Fig. 1).
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Figure 1.
Damage score in the dentate gyrus and in the
hippocampus CA1 region in rats, assessed at 7 d of recovery after
30 min of hypoglycemic coma. Rats were treated with a single dose of
CsA (CsA20; 20 mg/kg, i.v.), CsA (CsA50;
50 mg/kg, i.v.), FK 506 (FK506; 2 mg/kg, i.v.), or
vehicle (Vh). There was a significant
(p < 0.05) decrease in damage in both
regions in animals treated intravenously with 50 mg/kg, when compared
with the control group. Kruskal-Wallis test.
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Figure 2 shows a photomicrograph of the
dentate gyrus of a sham-operated rat (Fig. 2A), a rat
treated with vehicle (Fig. 2B), and a rat treated
with 50 mg/kg CsA (Fig. 2C). No damage is seen in the
CsA-treated rat, whereas dense neuronal damage is seen in the tip of
the dentate gyrus of the vehicle-treated animal.
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Figure 2.
Photomicrographs of the dentate gyrus at 7 d
of recovery after 30 min of hypoglycemic coma. A control section
(toluidine blue stain) is shown (A) with normal
neurons with owl eye-appearing nuclei. Widespread neuronal death is
seen in the vehicle-treated rat (B) with dark
pyknotic neurons. In rats treated with a single dose of CsA (50 mg/kg,
i.v.), no neuronal damage is seen (C). Scale bar,
10 µm.
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Cyclosporin A concentrations in blood and brain
Concentrations of CsA in blood and hippocampal parenchyma, 45 min
after a 20 mg/kg or 50 mg/kg intravenous dose, are shown in Table
3. Hypoglycemic animals and control
animals, not subjected to hypoglycemia, showed similar CsA
concentrations and blood/brain parenchyma ratios at the given dose. At
a dose of 20 mg/kg, the CsA concentration in the brain was 2.5 µg/g
(2 µM) and at 50 mg/kg the concentration increased
multifold (10-12 µM). The ratios between blood and brain
CsA concentrations at 45 min after the injected dose of 20 mg/kg or 50 mg/kg were 0.36 and 0.26, respectively. There was no significant
difference in the levels of CsA in brains that were perfused with
saline and those that were not (data not shown), indicating that the
measured CsA concentrations in brain were not attributed to blood
remaining in the intracerebral vessels.
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Table 3.
Concentrations of cyclosporin A in blood and hippocampal
parenchyma, 45 min after an intravenous injection
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Respiratory rate and mitochondrial swelling
Mitochondria isolated from the hippocampus were well-coupled with
an RCR of 5.5 ± 0.4 (n = 3).
When the mitochondrial suspension was added to the isotonic KCl buffer,
a low basal mitochondrial swelling was seen (Fig. 3, left). On addition of
calcium ions the swelling rate increased in a dose-dependent manner,
hence the initial swelling rate at 100 µM calcium
chloride was ~10 times faster than at 20 µM. Addition of 200 nM CsA effectively inhibited swelling at 20 µM calcium chloride and decreased significantly the
swelling rate induced by 100 µM calcium chloride (Fig.
3). Addition of 200 nM FK 506 did not affect the
calcium-induced mitochondrial swelling (Fig. 3).
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Figure 3.
Left, Calcium ion-induced
de-energized mitochondrial swelling measured as a decrease in light
scattering. Mitochondria were isolated from the rat hippocampus and
added to isotonic KCl buffer. The addition of 100 µM Ca
(arrow) initiated rapid swelling, whereas swelling
decreased significantly when 200 nM CsA was present.
Right, The effect of 20 and 100 µM calcium
chloride on de-energized mitochondrial swelling, in the absence and
presence of 200 nM CsA or 200 nM FK 506. Swelling was measured as the decrease in light scattering at 520 nm
initiated by the addition of calcium ions. A numerical value was
calculated from the initial swelling (absorbency/time unit) divided by
protein concentration. ** denotes significant differences from
untreated samples with p < 0.01; Dunnett's
test.
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Ultrastructural changes during hypoglycemic coma
Ultrastructural morphology of the stratum moleculare superjacent
to the crest of the dentate gyrus in a sham-operated animal is shown in
Figure 4A. The
morphology displays a compact neuropil. At higher magnification,
dendrites containing neurofilaments and axonal endings with synaptic
vesicles are seen embutting on dendrites with clear postsynaptic
densities (Fig. 4B). The mitochondria are compact and
contracted. In vehicle-treated animals exposed to 30 min of isoelectric
EEG induced by hypoglycemia, dendrites are markedly swollen (Fig.
4C). Dendritic mitochondria are also swollen, and some have
disrupted cristae (Fig. 4C,D). In animals treated with 50 mg/kg CsA intravenously before 30 min of hypoglycemic isoelectricity,
dendrites appear similarly swollen as in the vehicle-treated rats (Fig.
4E). However, dendritic mitochondria are contracted and appear to be morphologically normal (Fig.
4F) and similar to those in the sham-operated
animals (Fig. 4B). In untreated and vehicle-treated
animals, the presynaptic structures seem unaltered, with mitochondria
appearing morphologically intact (Fig.
5). At 30 min of recovery after 30 min of
isoelectric EEG, the neuropil becomes loose and structurally disordered
(Fig. 6A). The
dendrites collapse, shrink, and become dark, whereas mitochondria are
swollen with disrupted or electron-dense matrix (Fig.
6A). In the CsA-treated animal, dendrites are still
swollen, but the mitochondria appear normal and compact (Fig.
6B). The neuropil is also remarkably compact. The
ultrastructural changes in FK 506-treated animals were not different
from vehicle-treated ones (data not shown).
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Figure 4.
Ultrastructural changes in the stratum moleculare
superjacent to the crest of the dentate gyrus in a sham-operated animal
(A, B), showing a compact structure with normal
dendrites (arrowhead or d), normal axons,
and compact and contracted mitochondria (arrow). At
higher magnification (B), the postsynaptic
densities are evident as dark electrondense structures on the dendritic
membrane (*). In the vehicle-treated animals (C, D),
dendrites (arrowhead or d) are markedly
swollen at 30 min of isoelectric EEG, whereas axonal endings (*)
impinging on dendrites are unremarkable. Mitochondria
(arrow) in the dendrites of the vehicle-treated animals
are swollen, and some have disrupted cristae. In animals treated
intravenously with 50 mg/kg CsA (E, F), dendritic
mitochondria (arrow) are contracted and appear to be
morphologically normal and similar to those in the sham-operated
animals (A, B). Still, dendrites
(arrowhead or d) appear similarly swollen
as in the vehicle-treated rats. Scale bars; A, C, E, 2.7 µm; B, D, F, 0.54 µm.
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Figure 5.
Ultrastructural changes in the stratum moleculare
superjacent to the crest of the dentate gyrus in a vehicle-treated
animal at 30 min of hypoglycemic coma. Presynaptic structures appear
normal with normal mitochondria in nerve endings (white
arrow). Dendrites (d), on the other hand,
are markedly swollen, containing swollen mitochondria with disrupted
cristae (arrow). Scale bar, 0.68 µm.
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Figure 6.
Ultrastructural changes in the stratum moleculare
superjacent to the crest of the dentate gyrus at 30 min recovery after
30 min of isoelectric EEG. A, The dendrites are
disrupted in the vehicle-treated animals, and the mitochondria
(arrow) appear swollen with disrupted or electrondense
matrix. Some cells (arrowhead) are condensed.
B, In the animal treated intravenously with 50 mg/kg
CsA, dendrites (d) are still swollen, whereas the
mitochondria (arrow) appear normal and contracted. Scale
bar, 0.54 µm.
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The number of swollen mitochondria was significantly larger in the
vehicle and in FK 506-treated animals compared with the CsA-treated
ones (Fig. 7). In vehicle and FK
506-treated animals, the number of swollen mitochondria was 38.5 ± 6.6 and 36 ± 4.4, respectively, whereas in the CsA-treated
group the number was 4.5 ± 3.8 (p < 0.01).
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Figure 7.
Mean number of swollen mitochondria (>0.8 µm in
diameter) in an area measuring 26 × 26 µm in the distal stratum
moleculare, superjacent to the crest of the dentate gyrus. The animals
were subjected to 30 min of hypoglycemic isoelectricity and treated
with 50 mg/kg CsA (n = 4), 2 mg/kg FK 506 (n = 3), or vehicle (n = 4). **
denotes significant difference in CsA-treated animals, compared with
vehicle and FK 506-treated animals. p < 0.01;
Scheffe's test.
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DISCUSSION |
The main finding of this investigation is a marked neuroprotective
effect of cyclosporin A in a model of severe insulin-induced hypoglycemia. Our data show that mitochondria swell noticeably during
and after hypoglycemic coma and that this swelling is prevented by
cyclosporin A. We also show that calcium-induced swelling in isolated
mitochondria from the hippocampus is depressed by cyclosporin A but not
by FK 506. Because calcium-induced pathophysiological swelling of
mitochondria is believed to be caused by the activation of the MPT and
because MPT may be involved in cell death, the following discussion
will be focused on the possible involvement of MPT in the development
of hypoglycemic brain damage.
Mitochondrial permeability transition
When mitochondria are exposed to high levels of calcium ions and
oxidative stress, MPT is induced, caused by the opening of a large
proteinaceous pore, the MMC. The opening of the MMC and subsequent
collapse of the mitochondrial potential allows solutes of up to 1500 Da
size to flow across the membrane, and when studied in vitro,
swelling of the isolated mitochondria ensues (Haworth and Hunter, 1979;
Crompton et al., 1987; Gunter and Pfeiffer, 1990). Reactive oxygen
species (ROS), adenine nucleotide depletion, and a low mitochondrial
membrane potential all facilitate MPT in vitro, seemingly by
increasing pore sensitivity to calcium ions (Bernardi et al., 1994;
Halestrap, 1994). In addition, calpain-like proteases (Aguilar et al.,
1996) and ICE-like caspases (Susin et al., 1997) have been found to
induce MPT. Low matrix pH, on the other hand, is a potent inhibitor of
MPT (Halestrap, 1991; Bernardi et al., 1992). The Bcl-2 family of
proteins also influences MPT through several possible mechanisms
(Kroemer, 1997).
Recently, the matrix-specific rat CyP-D showing striking homology to
the human hCyp-3, was cloned (Woodfield, 1997). Cyclophilin-D has been
shown to be of importance in the regulation of the mitochondrial permeability transition, and it has been suggested that CyP-D interacts
with an integral membrane protein, possibly the adenine nucleotide
translocase, thereby facilitating MPT on calcium binding (Halestrap and
Davidson, 1990; Connern and Halestrap, 1992, 1994). Nanomolar
concentrations of CsA inhibit the MPT by blocking translocation of
CyP-D to the mitochondrial inner membrane in isolated liver mitochondria, decreasing MMC sensitivity to calcium ions (Connern and
Halestrap, 1994).
The activation of MPT by calcium ions and the regulation of the pore by
different modulators has been studied extensively in isolated
mitochondria from the liver and the heart (Crompton et al., 1987;
Gunter and Pfeiffer, 1990; Halestrap and Davidson, 1990) and recently
in isolated mitochondria from whole brain (Kristal and Dubinsky, 1997).
Variations in sensitivity to calcium ion-induced MPT in different
species and tissue, which have been attributed to differences in the
content of MPT modulators (Griffiths and Halestrap, 1993b; Jurkowitz
and Brierley, 1982), have been demonstrated (Zoratti and Szabo, 1995).
The data presented here show that MPT is induced in isolated
hippocampal mitochondria by calcium ions in a dose-dependent manner. We
also demonstrate that cyclosporin A, but not FK 506, is a potent
inhibitor of hippocampal mitochondrial swelling. We conclude that
hippocampal mitochondria undergo MPT during exposure to
supraphysiological levels of calcium ions. This process is inhibited by
cyclosporin A, in a manner similar to that reported in other studies
(Crompton et al., 1988; Griffiths and Halestrap, 1991; Kristal and
Dubinsky, 1997).
MPT and hypoglycemic brain damage
The pathophysiological conditions prevailing during hypoglycemia
favor MPT, and the effect of CsA on the ultrastructural changes of
dentate gyrus granule cells during hypoglycemia supports this notion.
The ultrastructural data presented here demonstrate that dendrites and
mitochondria swell considerably during the hypoglycemic insult. This is
presumably caused by the energy failure, loss of ion homeostasis, and
excitotoxic stress with concomitant massive influx of sodium and
calcium ions and water. During hypoglycemic coma, the tissue ATP levels
drop to below 1 mM (Wieloch et al., 1984), which indicates
that mitochondrial ATP production is limited, most probably because of
a decrease in mitochondrial membrane potential secondary to the
shortage in glucose supply. The mitochondria therefore can be
considered to be at least partly de-energized. The extracellular
calcium ion concentration in the brain decreases from ~1
mM to below 100 µM (Harris et al., 1984).
Because the plasma membrane is depolarized, an equilibration of the
ionic gradients across the cell membrane occurs. Therefore, an
intracellular calcium ion concentration of ~100 µM can
be expected, which is in the range where we observe extensive swelling
of isolated de-energized mitochondria.
Cyclosporin A dramatically prevents the mitochondrial swelling in
granule cells but not the swelling of dendrites, which are still
swollen 30 min after the hypoglycemic insult. Apparently, membrane
depolarization seems to occur also in cyclosporin A-treated rats during
the insult, leading to dendritic swelling. This ensues during early
recovery after hypoglycemia because dendrites still may experience
glutamate receptor overactivation (Sandberg et al., 1986).
Mitochondria, on the other hand, are still compact during recovery in
CsA-treated animals. Hence there is a clear correlation between
preservation of mitochondrial ultrastructure and neuronal survival
after CsA treatment, which suggests strongly that CsA-sensitive
mitochondrial swelling is associated with the activation of a
deleterious process.
The activation of the MMC has several consequences. First, loss of ion
gradients and depolarization of the mitochondrial membrane potential
uncouples oxidative phosphorylation. Second, a burst of ROS production
is induced (Zamzami et al., 1995), leading to depletion of reduced
glutathione and NADPH. Third, mitochondrial proteins activating the
initial steps of apoptosis may be released (Susin et al., 1996).
However, it is not clear how the release of these factors, which
requires an increase in the permeability of the outer membrane to large
molecules, is related to the MPT, which is associated with an increase
in the permeability of the inner membrane to smaller molecules. Indeed,
in some systems, the release of apoptogenic proteins occurs before the
initiation of MPT (Kluck et al., 1997; Yang et al., 1997; Zou et al.,
1997). In a yet unidentified manner, these proteins encompassing
apoptosis-inducing factor, cytochrome c, and apoptosis
protease-activating factor, in turn activate caspases, the executioners
of apoptotic cell death (Nicholson and Thornberry, 1997). During
hypoglycemia, the induction of MPT is followed by a series of events
leading to DNA fragmentation (M. Ferrand-Drake, H. Friberg, T. Wieloch,
unpublished observations).
Cyclosporin A, FK 506, and neuroprotection
A decrease in brain temperature to 33°C during or after ischemia
has repeatedly been shown to markedly diminish brain damage (Ginsberg,
1997). Neuroprotection observed by some pharmacological interventions
has been attributed to hypothermia that accompanies drug treatment,
rather than to the drug effect (Buchan and Pulsinelli, 1990). In
contrast, hypothermia during or after a hypoglycemic insult does not
affect the neuronal damage incurred (Agardh et al., 1992). Therefore,
the tendency to a slightly lower body temperature after hypoglycemia
observed in CsA-treated rats compared with vehicle-treated ones in the
present investigation could not have contributed to the observed
dramatic neuroprotective effect.
Cyclosporin A has a restricted passage into the brain parenchyma
(Begley et al., 1990; Sakata et al., 1994), but it accumulates to
pharmacological concentrations in the brain at high blood levels (Bernareggi and Rowland, 1991). We also found a dose-dependent increase
of CsA levels in brain parenchyma after single intravenous injections,
clearly showing permeability of CsA across the BBB at high doses.
Although it is highly probable that the accumulated CsA is found mainly
in the parenchyma and therefore also in neurons, accumulation of CsA in
the endothelium cannot be excluded. Cyclosporin A concentrations in the
brain obtained in the present investigation were higher than those
needed to inhibit MPT in isolated hippocampal mitochondria, but were
comparable with those shown to delay and block the MPT in mixed
neuronal cultures after NMDA exposure (Nieminen et al., 1996; Schinder
et al., 1996; White and Reynolds, 1996). It is therefore reasonable to
believe that the concentrations of CsA attained in the brain in this
study are sufficient to inhibit MPT during hypoglycemia. In previous
investigations in models of brain ischemia, CsA was neuroprotective at
doses generally lower than those used in the present study (Shiga et
al., 1992; Butcher et al., 1997). For example, in models of focal
ischemia the CsA dose showing neuroprotection was in the range of
15-20 mg/kg, which is similar to the dose where neuroprotection is
seen in the present study, whereas in global ischemia, a 10 mg/kg dose was not sufficient to provide protection (Uchino et al., 1995). Tissue
CsA concentrations, however, were not measured in these studies, and
the effective pharmacological doses can therefore not be compared with
those reported here.
FK 506, on the other hand, has been shown to readily penetrate the BBB
(Dawson et al., 1993; Butcher et al., 1997). The 2 mg/kg dose used in
this study is in the same order of magnitude as published earlier,
showing neuroprotection in a model of transient global ischemia (Drake
et al., 1996) and in a model of focal ischemia (Sharkey and Butcher,
1994).
Several different mechanisms have been proposed to be responsible for
the neuroprotective properties of immunophilin ligands, such as CsA, FK
506, or their analogues. In some models, the immunosuppressive action
of CsA and FK 506 via calcineurin inhibition may be of importance
(Butcher et al., 1997), especially if an inflammatory component is
obvious in neuronal death. Other neuroprotective effects may involve
the regulatory role of immunophilins at the ryanodine receptor or the
IP3 receptor (Brillantes et al., 1994; Cameron et al.,
1995). A direct neurotrophic effect by immunophilin ligands has been
proposed as well, although the cellular mechanisms behind this
remain unclear (Steiner et al., 1997). Patch-clamp studies in
hippocampal neurons have shown that CsA action is not mediated through
NMDA-receptor blockade (V. S. Vorobjev, personal communication).
On the basis of our experiments and earlier published data, we propose
that the decreased flux in the electron transport chain caused by
glucose deprivation and the ensuing loss of membrane potentials, as
well as the calcium ion flow into neurons concomitant with mild
alkalosis, enhance MPT during hypoglycemia. This is evident as
mitochondrial swelling, which is blocked by CsA. In animals treated
with CsA, MPT is therefore not initiated, mitochondrial integrity is
preserved, and the cascade leading to cell death is prevented. Although
the lack of a protective effect in FK 506-treated animals in this study
implies that calcineurin is not involved in the process leading to
hypoglycemic cell death, we cannot exclude a combinatorial protective
effect by CsA through the blockade of MPT and calcineurin inhibition
(Shibasaki et al., 1997).
| |
FOOTNOTES |
Received Oct. 2, 1997; revised April 23, 1998; accepted April 28, 1998.
This work was supported by the Swedish Medical Research Council (Grant
8644), the European Union BIOMED II (Grant BMH4-CT96-0851), and The
Bergendahl Foundation. We thank Kerstin Beirup for excellent technical
assistance and Chief Chemist Henrik Björk at the Department of
Clinical Pharmacology for skillfully analyzing CsA.
Correspondence should be addressed to Hans Friberg, Laboratory for
Experimental Brain Research, Wallenberg Neuroscience Center, University
Hospital, S-221 85 Lund, Sweden.
| |
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Top
Abstract
Introduction
