(Stroke. 1996;27:712-719.)
© 1996 American Heart Association, Inc.
Articles |
Brain Capillary Tissue Plasminogen Activator in a Diabetes Stroke Model
From the Departments of Neurosurgery (M.K., L.W., B.V.Z.) and Neurology (N.S., S.S.S., M.F.) and Division of Neurosurgery, Children's Hospital (B.V.Z.), University of Southern California School of Medicine, Los Angeles, and the Neuroscience Center, University of Colorado, Health Sciences Center (N.W.S.), Denver, Colo.
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Abstract |
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Background and Purpose Tissue plasminogen activator (TPA) is normally expressed in rat brain capillaries. This study examines the expression of TPA in brain capillaries of diabetic rats in relation to focal ischemic brain injury.
Methods Diabetes type 1 was induced by streptozotocin for 7 days. Acute hyperglycemia was induced by 50% dextrose. Expression of TPA in brain capillaries was determined by Western blot and reverse transcription-polymerase chain reaction analyses. Focal stroke was produced by 1 hour of reversible middle cerebral artery occlusion. Physiological variables and cerebral blood flow were monitored during occlusion and within 1 hour of reperfusion. Neurological and neuropathologic examinations were performed after 24 hours of reperfusion.
Results All rats developed comparable hyperglycemia (
15
mmol/L). A complete depletion of TPA protein and 6.5-fold decrease in
TPA mRNA were found in brain capillaries of diabetic rats, in contrast
to normal TPA capillary levels in hyperglycemic rats. The blood flow in
the periphery of the ischemic core was significantly reduced
during reperfusion by 52% to 62% (P<.001) in diabetic
rats and by 23% to 25% (P<.05) in hyperglycemic rats. The
neurological score was worsened by 3.2-fold (P<.0003) by
diabetes and by 24% by hyperglycemia only. Significant 41%
(P<.007) and 29% (P<.05) increases in infarct
volume and 163% (P<.007) and 60% increases in edema
volume were found in diabetic rats relative to control and
hyperglycemic rats, respectively.
Conclusions Diabetes type 1, but not acute hyperglycemia, produces downregulation of TPA in rat brain capillaries. This TPA reduction is associated with impaired restoration of blood flow after an ischemic insult, poor neurological outcome, and enhanced ischemic brain injury.
Key Words: cerebral blood flow • cerebral ischemia • diabetes mellitus • hyperglycemia • tissue plasminogen • rats
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Introduction |
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Tissue plasminogen activator is a key enzyme in the control of fibrinolysis within the vascular system.1 2 In vitro studies have demonstrated that TPA is synthesized in a transcriptionally controlled manner by endothelial cells from a variety of systemic vessels (umbilical vein and artery, pulmonary artery, aorta, vena cava),3 4 as well as by brain microvascular endothelial cells.5 Recently, we provided evidence that TPA is normally expressed in vivo in cerebral capillaries in rodents,6 suggesting that microvascular endothelium, ie, the blood-brain barrier, may have a role in promoting plasmin-dependent fibrinolysis in brain microcirculation.
Thrombolytic therapy with agents such as TPA is currently the focus of much attention for the treatment of cerebral ischemia in both clinical7 and experimental8 studies. In this regard, altered coagulation with abnormal fibrinolysis has been demonstrated in ischemic stroke patients.9 However, the role of endogenous cerebromicrovascular TPA in an ischemic brain insult is less well understood. It has been suggested that the blood-brain barrier, when exposed to stroke risk factors including diabetes, may be transformed from a normally anticoagulant into a procoagulant surface.10 In the present study this hypothesis was tested by examining the effect of streptozotocin-induced diabetes type 1 on expression of TPA in brain capillaries in relation to focal ischemic brain injury. To eliminate the effect of hyperglycemia in this diabetes model, normal rats were rendered hyperglycemic to a comparable level by dextrose administration. Preliminary results have been reported.11
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Materials and Methods |
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Studies were performed with the use of 81 male Sprague-Dawley rats weighing 260 to 300 g obtained from Charles River Breeders. Animals were housed under diurnal light conditions with unlimited access to food and water and were allowed a minimum of 3 days for acclimation. All procedures were done in accordance with the Animal Care Guidelines at the University of Southern California and approved by the National Institutes of Health.
Experimental Diabetes and Hyperglycemia
The effects of streptozotocin-induced diabetes type 1 were
examined in 32 rats. Streptozotocin (65 mg/kg IP; Sigma) was injected 7
days before determination of TPA expression in brain capillaries and
MCA occlusion. A hyperglycemic group of 17 rats was given 2.5 mL of
50% dextrose IP 15 minutes before TPA determination and MCA occlusion.
A control group of 32 rats received only vehicle. Blood glucose was
determined before streptozotocin, dextrose, or vehicle; before
occlusion; and during both occlusion and reperfusion.
Expression of Brain Capillary TPA
Isolation of Brain Capillaries
Brain capillaries were isolated from 20 diabetic, 5
hyperglycemic, and 20 control rats by a modified mechanical
homogenization technique.12 Briefly,
brains were immediately removed from the skull and immersed in
ice-cold buffer B (mmol/L: NaCl 103, KCl 4.7, CaCl2
2.5, KH2PO4 1.2, MgSO4 1.2, HEPES
15, pH 7.4). The cerebral cortical mantles were rapidly freed of
meninges (ie, pial vessels); the arteries of the circle of Willis,
veins, and choroid plexi were discarded; and cerebral cortices were
used to isolate the capillaries. Brain
homogenization in buffer A (buffer B+25 mmol/L
HCO3, 10 mmol/L glucose, 1 mmol/L sodium pyruvate,
and 1 g/100 mL dextran, Mr=64 000) with a
handheld polytetrafluoroethylene
homogenizer was followed by dextran density
centrifugation at 5800g at 4°C. The pellet
was resuspended in buffer A and passed over an 85-µm nylon mesh.
Arterioles and venules remained on top of the mesh, while the
capillaries, red cells, nuclei, and other debris were collected in the
filtrate passing through the mesh. This filtrate was then passed over a
3x4-cm glass bead column (0.45-mm glass beads) with 44-µm nylon mesh
at the bottom, and the column was washed with buffer B. The brain
capillaries adhere to the glass beads while the other contaminants pass
unimpeded. Capillaries were recovered by repeated gentle agitation of
the glass beads in buffer A; the supernatant with capillaries was
decanted and spun at 500g for 5 minutes to obtain the final
pellet. The purity of the cerebral capillary preparation was checked by
light and phase microscopy. The cerebral capillaries were free of
adjoining brain tissue; preparations consisted primarily of capillaries
but also contained minor amounts (5% to 10%) of small arterioles, as
described.6 12 Capillary-depleted brain
homogenates were centrifuged again at
5800g at 4°C (second centrifugation) to
obtain a final capillary-free brain supernatant. The presence of no
more than minimal detectable activity of a specific cerebrovascular
marker, 
-glutamyl transpeptidase, was used to confirm
absence of contamination of capillary-depleted brains with
microvessels.
Western Blot Analysis
Brain capillaries and capillary-depleted brains from 8
diabetic, 5 hyperglycemic, and 8 control rats were
homogenized by a motor-driven Potter-Elvehjem
polytetrafluoroethylene
homogenizer in a buffer containing 5% sorbitol, 5
mmol/L histidine, 25 mmol/L imidazole, 1 mmol/L EDTA at pH 7.5 with
inclusion of proteolytic enzyme inhibitors, 0.5 mmol/L
phenylmethylsulfonyl fluoride, 1 µg/mL leupeptin, and 1
mmol/L aminobenzamidine dihydrochloride, as reported.6 12
A rabbit polyclonal anti-mouse TPA IgG fraction that detects TPA
only and not TPA-plasminogen activator
inhibitor-1 complexes6 13 was used. The
specificity of this TPA antibody was confirmed previously on Western
blots containing conditioned medium from developing mouse cerebellar
cells that actively secrete TPA,13 1 ng human melanoma TPA
(World Health Organization international standard),6 and
rat kidney extracts rich in TPA (not shown). Preabsorption of this TPA
antibody with mouse TPA resulted in loss of immunoreactivity in
TPA-rich kidney tissue and abolished detection of the material on
Western blots6 13 (data not shown). Gels were blotted onto
diazotized paper and incubated overnight with polyclonal anti-TPA
antibodies (1:600 dilution). The enhanced chemiluminescence Western
blotting detection system (Amersham) was used to detect TPA and expose
it to hyperfilm. The migration of TPA from tissue samples was compared
with the relative mobilities of molecular weight standards run on the
same gel. Chemilumigrams were scanned with a Hoefer GS 300 scanning
densitometer, each sample was analyzed at least two times, and
each lane was scanned three times. The linearity of the system
(standard curve) was demonstrated to be between 1 and 10 µg of total
capillary proteins.6
RT-PCR
Total RNA (4 µg) was extracted with guanidium
isothiocyanate from pooled capillary samples obtained from either 4
diabetic or 4 control rats and from capillary-depleted
brains.14 This experiment was repeated three times with 12
diabetic and 12 control rats. The RNA was reverse transcribed with the
use of 1 µg oligo(dT)15 in 20 µL reaction buffer
containing the following (mmol/L): each dNTP 1, Tris-HCl 25 (pH 8.3),
KCl 25, MgCl2 5, dithiothreitol 5, spermidine 0.25, plus 10
U RNasin (Boehringer Mannheim) and 10 U AMV reverse
transcriptase (Promega). The mixture was incubated at 42°C for 1 hour
and then at 52°C for 40 minutes, followed by the addition of 160 µL
of 10 mmol/L Tris-HCl (pH 7.4) and 1 mmol/L EDTA.
PCR was performed with 10 µL of the RT reaction mixture, 50 mmol/L KCl, 10 mmol/L Tris-HCl (pH 9.0), 0.1% Triton X-100, 1 mmol/L MgCl2, 400 nmol/L of each TPA primer, 200 µmol/L each dNTP, and 5 U Taq DNA polymerase (Boehringer Mannheim) in a final volume of 50 µL. The sample was subjected to 30 cycles (94°C, 1 minute; 58°C, 1 minute; 72°C, 3 minutes) in a DNA Thermal Cycler (Perkin Elmer), and 5 µL of the PCR product was analyzed by electrophoresis on a 1% agarose gel. Primers for TPA were as follows: sense (5'-TGGCACCCACAGCTTTACCACATCCAAGG-3') and antisense (5'-CTCCTGAGTCACCTGGCACGCGTCATGG-3'), corresponding to nucleotides 699 to 727 and 1540 to 1568, respectively, of the published sequence for rat TPA.15 Amplification of ß-actin by PCR served as an internal control for PCR reaction. Mouse ß-actin primers were obtained from Stratagene.
Focal Brain Ischemia
General Preparation
Rats were deprived of food 12 hours before surgery. Reversible
MCA occlusion was performed in 12 diabetic, 12 hyperglycemic, and 12
control rats with a modified intraluminal thread
technique.16 The procedure involved initial
anesthesia with metofane and maintenance with 50
mg/kg pentobarbital IP. Atropine methonitrate (0.18 mg/kg IP) was given
as premedication to prevent airway obstruction by mucus formation. The
animals were allowed to breath spontaneously. Rectal temperature was
maintained at 37°C by a thermostatically regulated heating pad. A
polyethylene catheter (PE-50) was introduced into the right femoral
artery for continuous monitoring of mean arterial blood
pressure and repeated sampling of blood for serial measurements of
PaO2,
PaCO2, and pH (ABL 30 Acid-Base
Analyzer, Radiometer) as well as hematocrit and blood
glucose.
Operative Technique
Under the operating microscope, the right CCA was exposed
through a ventral midline incision, and the ECA and ICA were isolated.
The CCA was tied at approximately 8 mm from the bifurcation. The ECA
was tied at approximately 5 mm from the bifurcation (permanent double
knot), and a second loose knot was placed around the ECA origin. After
a microvascular clip was placed across the ICA adjacent to the ECA
origin, a partial incision was made in the ECA midway between the
permanent double knot and second loose knot. A 3-0 monofilament nylon
suture with rounded tip was introduced into the ECA lumen, the ECA
stump was tightened around the intraluminal nylon suture to prevent
bleeding, and the microvascular clip was removed. The nylon suture was
then gently advanced from the ECA into the ICA lumen, and the tip was
advanced 17.5 mm from the bifurcation to occlude the MCA at its origin
from the circle of Willis. After 1 hour of occlusion, the thread was
pulled out, the ECA was permanently tied at the level of bifurcation,
and the CCA was opened to allow reperfusion.
Blood Flow and Head Temperature
Cortical CBF was monitored before occlusion, during occlusion,
and within 1 hour of reperfusion by laser-Doppler
flowmetry. Laser-Doppler flow probes (0.8 mm in diameter)
positioned at 0.1 mm above the dura over the cortical surface were
connected to a tissue perfusion monitor (Transonic BLF21). In the
hemisphere ipsilateral to the MCA occlusion, coordinates were as
follows: point A, 1 mm posterior to the bregma and 5.4 mm lateral to
the midline; point B, 1 mm posterior to the bregma and 2.1 mm lateral
to the midline; and point D, 1 mm anterior to the bregma and 3.4 mm
lateral to the midline. Point C in the contralateral hemisphere was 1
mm posterior to the bregma and 5.4 mm from the midline. At each point a
small burr hole was drilled in the skull and the bone carefully removed
to prevent damage to the cortex. Steady-state baseline values were
recorded before occlusion, and CBF during occlusion and reperfusion
was expressed as a percentage of the baseline values. Head temperature
was monitored with a 36-gauge thermocouple temperature probe in the
temporalis muscle connected to a digital Thermometer/Thermoregulator
(model 9000, Omega).
Neurological Examination
Neurological examination was performed 24 hours after
reperfusion before the animals were killed. Neurological outcome was
scored on a six-point scale as described16 : a score of
0 indicated no neurological deficit (normal); 1 (failure to extend left
forepaw fully), mild focal neurological deficit; 2 (circling to the
left), moderate focal neurological deficit; 3 (falling to the left),
severe focal deficit; 4, rat did not walk spontaneously and had a
depressed level of consciousness; and 5, stroke-related death (not
observed in the present study).
Quantification of Ischemic Brain Damage
The brain was placed into ice-cold phosphate-buffered
saline; after 20 minutes of chilling, it was sliced into 2-mm coronal
sections. The infarct area was delineated by incubation of unfixed 2-mm
coronal brain slices in 2%
triphenyltetrazolium chloride in 0.173
mol/L sucrose and 50 mmol/L K+ phosphate buffer (pH 7.4)
for 20 minutes at 37°C and then stored in 10% formalin, as reported.
Serial coronal sections were displayed on a digitizing video screen
with the use of the imaging system Jandel Scientific, and the areas of
nonstaining tissue were determined in each section. The infarct volume
was calculated by summing affected areas from each coronal section and
multiplying by the thickness of each section. The volumes of the
control and lesioned hemispheres were calculated, and the amount of
infarct was expressed as a percentage of total cerebral volume (%
infarct volume) and in absolute terms in cubic millimeters. The infarct
volume of gray matter structures was corrected for brain edema in the
lesioned hemisphere by subtracting the volume of the normal tissue in
the lesioned hemisphere from the volume of the control (normal)
hemisphere, based on described methods.17 The edema volume
was calculated by subtracting the volume of the normal gray matter in
the control hemisphere from the volume of gray matter in the lesioned
hemisphere.17 Measurements of infarct volume were done
separately for the pallium and striatum.
A coronal section from each brain was obtained at the level of the optic chiasm. Representations of the infarct areas of individual sections from each animal in control and diabetic groups were superimposed to gain an impression of the topography and incidence of infarction. The boundary of infarction was redrawn for each rat on the corresponding coronal section, and the following areas were delineated17 : the infarct area where 100% of rats were affected, the infarct area where at least 50% of rats were affected, and the infarct area where less than 50% of rats were affected.
Statistical Analysis
Physiological variables and infarct and
edema volumes were compared between groups by Student's t
test. Nonparametric data (neurological outcome scores) were
subjected to the Kruskal-Wallis test. A value of P<.05 was
considered statistically significant.
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Results |
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Expression of TPA
All streptozotocin-treated rats developed a stable hyperglycemia (
15 mmol/L) throughout the study. After 7 days, the
presence of a single TPA band of approximately 67 kD was detected only
in brain capillaries of control rats, and TPA signal was absent from
capillaries of diabetic rats (Fig 1
). Complete depletion
of TPA was found in all studied diabetic rats. In both groups, the TPA
signal was absent from capillary-depleted brains (data not shown),
as demonstrated.6 The relative mobility of rat TPA from
control capillary samples was comparable to that of TPA actively
secreted by developing mouse cerebellar cells or human melanoma cells.
Omission of primary anti-TPA antibodies resulted in loss of TPA signal
(data not shown). Preincubation of this anti-TPA antibody with
immobilized TPA on Affigel beads resulted in loss of
immunoreactivity in TPA-rich tissues such as kidney glomeruli and
abolished detection of the material on Western blots (data not
shown).6 13 Fig 2 illustrates that the TPA
signal remains on Western blots in capillaries of hyperglycemic (15
mmol/L) dextrose-treated rats, and scanning densitometry confirmed
the same intensity of bands in the control and these hyperglycemic
rats.
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The results in diabetic rats were corroborated at the mRNA level.
RT-PCR analysis revealed the presence of TPA cDNA in samples
derived from brain capillaries of control rats (Fig 3),
confirming recent findings.6 Only very low levels of TPA
expression were found in two groups of diabetic rats (Fig 3), while in
one group of diabetic rats the signal was almost undetectable (data not
shown). Scanning densitometry indicated that relative abundance of TPA
mRNA in two diabetic groups was 6.5 times lower than that in
corresponding controls, with ß-actin as an internal control.
After RT-PCR, ß-actin band density was similar for capillary
samples of control and diabetic rats. All capillary-depleted brains
exhibited minimal or no expression of TPA mRNA, as
reported.6
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Physiological Variables
Blood glucose levels were significantly elevated (15 mmol/L) in
both streptozotocin- and dextrose-treated rats (Table 1). No differences in physiological
variables (including the temporalis muscle temperature) were noted
between the diabetic, hyperglycemic, and control groups before MCA
occlusion, during occlusion, and during reperfusion.
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Fig 4 illustrates changes in CBF during MCA occlusion
and reperfusion. No significant differences in baseline tissue
perfusion units were observed between diabetic, hyperglycemic, and
control rats, indicating similar preocclusion CBF values. Reductions in
CBF during occlusion were comparable between the three groups, ie, 17%
to 18% (P<.001), 13% to 14% (P<.001), and
15% to 16% (P<.001) of baseline in the ischemic
core of control, diabetic, and hyperglycemic rats, respectively, and
34% to 39% (P<.001), 26% to 32% (P<.001),
and 36% to 38% (P<.001) of baseline in the periphery of
the ischemic core of control, diabetic, and hyperglycemic rats,
respectively. The trend toward CBF restoration during reperfusion was
greatly impaired in the periphery of the ischemic core of
diabetic rats, and only a moderate increase, ie, from 43% to 48%
(P<.001) of baseline, was recorded from 10 to 60
minutes of reperfusion. In contrast, during the same period of
reperfusion the CBF in hyperglycemic rats rose to 75% to 77%
(P<.05) of normal values and 80% to 88% in control rats.
In control rats, the CBF was restored to above 50%
(P<.001) in the ischemic core, while in diabetic
and hyperglycemic rats the restoration was up to 45%
(P<.001) and from 49% (P<.001) of preocclusion
values, respectively.
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Neurological Outcome
There was a marked difference in the outcome between control and
diabetic rats (Table 2). For example, in the control
group 5 of 12 rats did not have neurological symptoms, in contrast to
the diabetic group, in which all rats had some neurological deficit. A
typical score of 2 was found in 50% of diabetic rats and in only 1
control rat. One third of diabetic rats exhibited a severe focal
neurological deficit (score of 3). In hyperglycemic rats, a score of 2
was found in 3 rats, while 5 of 12 rats did not have neurological
symptoms, similar to the control group.
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Neuropathology
The total infarct volume corrected for the edema volume was
significantly increased in the diabetic group by 41% and 29% relative
to control and hyperglycemic rats, respectively (Table 3). The edema volume was also increased in diabetic rats
by 163% and 60% compared with control and hyperglycemic rats,
respectively. These increases were caused by significant increases in
the infarct and edema volumes in the pallium, indicating expansion of
injury into cortical regions. Changes in the striatum were not
statistically significant, although there was a trend toward an
increase in diabetic animals. The variability of the infarct volume was
larger in diabetic and hyperglycemic groups than in the control group.
Total area of brain injury determined at the level of the optic chiasm
(anterior coronal block) and expressed as a percentage of the coronal
sectional area was increased in diabetic rats by 65% and 43% relative
to control and hyperglycemic groups, respectively. Table 3 also shows
that hyperglycemic rats in comparison to control rats had increases in
brain edema by 64% and in infarct volumes (ie, total and pallium) by
approximately 21%, but these differences were not statistically
significant because of the relatively large variation between these two
groups. Fig 5 illustrates that 100% of control rats had
infarction only in the lateral striatum; 50% or more exhibited changes
in the dorsolateral, lateral, and ventrolateral cortex, and less than
50% showed changes in the medial cortex and striatum. All diabetic
rats exhibited changes in the dorsolateral, lateral, and ventrolateral
cortex in addition to the lateral striatum. All hyperglycemic rats had
somewhat larger infarction in the striatum than control rats, and 50%
or more of hyperglycemic rats exhibited expansion of the injury into
the dorsomedial cortex, similar to diabetic rats; less than 50% of
control rats had extension of the injury into the dorsomedial
cortex.
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Discussion |
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The present study demonstrates that streptozotocin-induced diabetes type 1 produces downregulation of TPA expression in rat brain capillaries that is not seen in rats acutely rendered hyperglycemic to a comparable level by dextrose treatment. Depletion of brain capillary TPA in diabetic rats is associated with impaired restoration of blood flow after reversible MCA occlusion, poor neurological outcome, and enhanced focal ischemic brain injury compared with both control and hyperglycemic rats.
This study confirmed TPA expression at the protein and mRNA levels in brain capillaries isolated from normal rats.6 The mobility of cerebromicrovascular TPA on SDS-PAGE was the same as recently reported6 and comparable to previously described mobility of rat TPA obtained from adrenal medulla and prostate,18 TPA secreted by granule neurons in cultures of developing mouse cerebellum,13 and human TPA from melanoma cells in culture.19 The presence of TPA mRNA in normal rat brain capillaries and absence or very low signal from capillary-depleted brains6 are in agreement with recent reports of TPA mRNA expression in bovine brain microvascular endothelial cells in culture5 and its limited expression in "mature" brain after central nervous system differentiation.13
It has been shown that TPA production and release from systemic endothelial cells are regulated by various physiological and pathological stimuli, including exercise, stress, infusion of drugs, shear stress,20 pulsatile stretch,21 and osmotic stress.22 In contrast, regulation of local TPA production and secretion at the blood-brain barrier is poorly understood. This study shows complete TPA depletion in brain capillaries of diabetic rats that is likely caused by downregulation of TPA synthesis at the transcriptional level. The exact mechanism for this downregulation of TPA mRNA in diabetic brain capillaries is presently unknown. An in vitro study indicated that elevated glucose does not affect TPA mRNA levels in bovine brain endothelial cells,5 and the present study confirms that hyperglycemia alone does not affect TPA levels in brain capillaries. Therefore, it may be that the direct effect of hyperglycemia in the present diabetes model is not responsible for the observed downregulation of TPA. Other factors besides hyperglycemia have been implicated in brain injury in in vivo models of experimental diabetes, including AGE and cytokines.23 Although AGE peptides are able to produce procoagulant transformation of endothelial cells in vitro,3 4 their significant accumulation in tissues has been typically associated with longer-term diabetic complications observed after 8 weeks in the streptozotocin model,24 compared with only 1 week in the present study.
In experimental models of thromboembolic stroke8 and MCA occlusion,25 thrombolytic therapy with exogenous TPA administered within the therapeutic window reduced size of infarct and edema and partially restored CBF.8 Using a model of reversible MCA occlusion, the present study demonstrates that depletion of endogenous brain capillary TPA in diabetes correlates with diminished restoration of CBF during reperfusion, poor neurological outcome, and larger volumes of brain infarct and edema compared with both control and hyperglycemic rats. During reperfusion, diabetic animals had an impaired trend toward CBF restoration in the periphery of the ischemic region, perhaps due to abnormal endogenous thrombolysis. Hyperglycemia has been shown in some ischemia models to accentuate postischemic brain edema,26 27 and this has been confirmed in the present study. The detrimental effects of preischemic hyperglycemia and subsequent lactic acidosis on cerebral blood vessels and postischemic blood flow have been documented in models of transient global brain ischemia.28 29 It has been reported that glucose pretreatment caused vascular endothelial swelling and luminal narrowing after 30 minutes of global ischemia in rats,29 while large glucose loads before ischemia dramatically impaired postischemic cerebral perfusion in cats.28 Significant but still moderate reductions in blood flow during reperfusion after glucose pretreatment in the present study could be related to less severe hyperglycemia than that in previous studies.28
Although hyperglycemia has been shown to invariably exacerbate
ischemic brain injury after global cerebral
ischemia,26 28 studies on hyperglycemia and focal
cerebral ischemia have been less consistent. It has
been reported that hyperglycemia reduces the extent of cerebral
infarction after irreversible photochemically induced
end-arteriolar thrombosis in rats,30 decreases acute
ischemic neuronal changes after permanent MCA occlusion in
cats,31 does not affect infarct volume after permanent MCA
occlusion in rats,32 increases infarct size after
permanent MCA occlusion in cats,33 and increases infarct
size in collaterally perfused territories but does not affect
end-arterial vascular territories after permanent MCA
occlusion in rats.34 In a reversible MCA model in cats,
severe hyperglycemia (4-fold increase in plasma glucose)
significantly enhanced infarct size after 4 hours of
occlusion,35 while moderate hyperglycemia (2-fold
increase in plasma glucose) did not cause significant increase in
infarct size after 1.5 and 2 hours of reversible MCA occlusion in fed
versus fasted rats.36 However, a remarkable increase in
infarct size was obtained in fed versus fasted rats36
after 45 minutes of reversible MCA occlusion.
The present study demonstrated a 21% increase in infarct volume in
hyperglycemic (3.5-fold increase in plasma glucose) versus
normoglycemic rats after 1 hour of reversible MCA occlusion, which was
similar to the approximately 15% increases in infarct size obtained
after 1.5 and 2 hours of reversible MCA in the presence of somewhat
lower hyperglycemia.36 Although the difference in infarct
volume between hyperglycemic and control rats in this study was not
statistically significant (possibly due to large variability within
groups), the infarct volume in diabetic rats was significantly higher
by 41% and 29% relative to both control and hyperglycemic rats,
respectively. Significant increases in cerebral infarction have been
previously shown in streptozotocin-induced short-term
diabetes after either transient (5 to 15 minutes of
occlusion)37 or permanent MCA occlusion.38
Thus, present results indicate that brain injury in diabetes may be
mediated by factors other than hyperglycemia, eg, AGE proteins,
cytokines,23 24 and TPA depletion.
It is unclear to what extent the present findings can be extrapolated to humans. It is possible that downregulation of local TPA production within the brain microcirculation may compromise brain fibrinolytic capacity, predisposing to larger and more disabling strokes. This would be similar to the recently reported association between depletion of TPA from the coronary vessels and the subsequent development of coronary artery disease.39 Little is known with regard to whether this mechanism can be considered universal across species. One study in primates would argue against the TPA hypothesis, since the expression of TPA in cerebral microvessels in the basal ganglia of baboons was found in a limited number of precapillary arterioles and postcapillary venules, while most capillaries were TPA negative.40 Recent findings in human cerebral microvascular endothelial cells suggest the possibility that urokinase-type plasminogen activator is a key catalyst of fibrinolysis.41 On the other hand, work from our laboratories (F. Hofman, unpublished data, 1995) and others41 indicates that TPA is secreted by human brain microvascular endothelial cells in culture. Along with the presence of TPA mRNA in bovine brain microvascular cells,5 this supports the concept that primates and other mammalian species may express TPA at the blood-brain barrier in a manner similar to rodents.6
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported by National Institutes of Health grant NS31945 and the Hoover Foundation.
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Footnotes |
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Reprint requests to Berislav V. Zlokovic, MD, PhD, 2025 Zonal Ave, RMR 506, Los Angeles, CA 90033.
Received September 1, 1995; revision received January 3, 1996; accepted January 11, 1996.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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- Collen D. On the regulation and control of fibrinolysis. Thromb Haemost. 1980;43:77-89. [Medline] [Order article via Infotrieve]
- Francis RB Jr. Clinical disorders of fibrinolysis: a critical review. Blut. 1989;59:1-14. [Medline] [Order article via Infotrieve]
- Gilboa N, Kaplan JE. Plasma fibronectin enhances fibrinolytic system in vitro. Thromb Haemost. 1985;54:639-644. [Medline] [Order article via Infotrieve]
- van Hinsbergh VW, Kooistra T, Emeis JJ, Koolwijk P. Regulation of plasminogen activator production by endothelial cells: role in fibrinolysis and local proteolysis. Int J Radiat Biol. 1991;60:261-272. [Medline] [Order article via Infotrieve]
- Kollros PR, Konkle BA, Ambarian AP, Henrikson P. Plasminogen activator inhibitor-1 expression by brain microvessel endothelial cells is inhibited by elevated glucose. J Neurochem. 1994;63:903-909. [Medline] [Order article via Infotrieve]
- Zlokovic BV, Wang L, Sun N, Haffke S, Verrall S, Seeds NW, Fisher M, Schreiber SS. Expression of tissue plasminogen activator in cerebral capillaries: possible fibrinolytic function of the blood-brain barrier. Neurosurgery. 1995;37:955-961. [Medline] [Order article via Infotrieve]
- Levine SR, Brott TG. Thrombolytic therapy in cerebrovascular disorders. Prog Cardiovasc Dis. 1992;34:235-262. [Medline] [Order article via Infotrieve]
- Gross CE, Raymond AS, Howard DB, Bednar MM. Delayed tissue-plasminogen activator therapy in a rabbit model of thromboembolic stroke. Neurosurgery. 1995;36:1172-1177. [Medline] [Order article via Infotrieve]
- Fisher M, Francis R. Altered coagulation in cerebral ischemia: platelet, thrombin, and plasmin activity. Arch Neurol. 1990;47:1075-1079. [Abstract]
- Zlokovic BV, Wang L, Schreiber SS, Fisher M. Hemostatic functions of the blood-brain barrier: possible implications in the pathogenesis of stroke. In: Greenwood J, Begley DJ, Segal MB, eds. New Concepts of the Blood-Brain Barrier. New York, NY: Plenum Press; 1995:141-145.
- Zlokovic BV, Wang L, Schreiber SS, Fisher M. Downregulation of tissue plasminogen activator at the blood-brain barrier in diabetic rats. Stroke. 1995;26:166. Abstract.
-
Zlokovic BV, Mackic BV, Wang L, McComb JG, McDonough
A. Differential expression of Na,K-ATP-ase
and ß subunit
isoforms at the blood-brain barrier and the choroid plexus.
J Biol Chem. 1993;268:8019-8025. [Abstract/Free Full Text] - Friedman GC, Seeds NW. Tissue plasminogen activator expression in the embryonic nervous system. Dev Brain Res. 1994;81:41-49. [Medline] [Order article via Infotrieve]
- Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987;162:156-159. [Medline] [Order article via Infotrieve]
- Ny T, Leonardsson G, Hsueh AJW. Cloning and characterization of a cDNA for rat tissue-type plasminogen activator. DNA. 1988;7:671-677. [Medline] [Order article via Infotrieve]
-
Zea Longa E, Weinstein PR, Carlson S, Cummins R.
Reversible middle cerebral artery occlusion without craniectomy in
rats. Stroke. 1989;20:84-91.
[Abstract/Free Full Text] - Swanson RA, Morton MT, Tsao-Wu G, Savalos RA, Davidson C, Sharp FR. A semiautomated method for measuring brain infarct volume. J Cereb Blood Flow Metab. 1990;10:290-295. [Medline] [Order article via Infotrieve]
- Freeman SN, Rennie PS, Chao J, Lund LR, Andreasen PA. Urokinase- and tissue-type plasminogen activators are suppressed by cortisol in the involuting prostate of castrated rats. Biochem J. 1990;269:189-193. [Medline] [Order article via Infotrieve]
- Pennica D, Holmes WE, Kohr WJ, Harkins RN, Vehar GA, Ward CA, Bennett WF, Yelverton E, Seeburg PH, Heyneker HL, Goeddel DV, Collen D. Cloning and expression of human tissue-type plasminogen activator cDNA in E. coli. Nature. 1983;301:214-220. [Medline] [Order article via Infotrieve]
- Diamond SL, Sharefkin JB, Dieffenbach C, Frasier-Scott K, McIntrie LV, Eskin SG. Tissue plasminogen activator messenger RNA levels increase in cultured human endothelial cells exposed to laminar shear stress. J Cell Physiol. 1990;143:364-371. [Medline] [Order article via Infotrieve]
- Iba T, Shin T, Sonoda T, Rosales O, Sumpio BE. Stimulation of endothelial secretion of tissue-type plasminogen activator by repetitive stretch. J Surg Res. 1991;50:457-460. [Medline] [Order article via Infotrieve]
-
Levin EG, Santell L, Saljooque F. Hyperosmotic
stress stimulates tissue plasminogen activator
expression by a PKC-independent pathway. Am J
Physiol. 1993;265:C387-C396.
[Abstract/Free Full Text] -
Zimmerman GA, Meistrell M III, Bloom O, Cockroft KM,
Bianchi M, Risucci D, Broome J, Farmer P, Cerami A, Vlassar H, Tracey
K. Neurotoxicity of advanced glycation end products during
focal stroke and neuroprotective effects of aminoguanidine.
Proc Natl Acad Sci U S A. 1995;92:3744-3748.
[Abstract/Free Full Text] - Huijberts MS, Wolffenbuttel BH, Crijns FR, Nieuwenhuijzen-Kruseman AC, Bemelmans MH, Struijker-Boudier HA. Aminoguanidine reduces regional albumin clearance but not urinary albumin excretion in streptozotocin-diabetic rats. Diabetologia. 1994;37:10-14. [Medline] [Order article via Infotrieve]
- Nakajima S, Yoshimine T, Kato A, Kumura E, Takemoto O, Hayakawa T. The effect of tPA on the microcirculatory disturbance in experimental cerebral ischemia. J Cereb Blood Flow Metab. 1993;13:S183-S184.
-
Pulsinelli WA, Waldman S, Rawlinson D, Plum F.
Moderate hyperglycemia augments ischemic brain damage: a
neuropathologic study in the rat. Neurology. 1982;32:1239-1246.
[Abstract/Free Full Text] -
Warner DS, Smith M-L, Siesjo BK.
Ischemia in normo- and hyperglycemic rats: effects on brain
water and electrolytes. Stroke. 1990;21:758-764.
[Abstract/Free Full Text] -
Ginsberg MD, Welsh FA, Budd WW. Deleterious
effect of glucose pretreatment on recovery from diffuse cerebral
ischemia in the cat. Stroke. 1980;11:347-355.
[Abstract/Free Full Text] - Paljarvi L, Rechncrona S, Soderfeldt B, Olsson Y, Kalimo H. Brain lactic acidosis and ischemic cell damage: quantitative ultrastructural changes in capillaries of rat cerebral cortex. Acta Neuropathol (Berl).. 1983;60:232-240. [Medline] [Order article via Infotrieve]
-
Ginsberg MD, Prado R, Dietrich WD, Busto R, Watson
BD. Hyperglycemia reduces the extent of cerebral infarction in
rats. Stroke. 1987;18:570-574.
[Abstract/Free Full Text] -
Zasslow MA, Pearl RG, Shuer LM, Steinberg GK, Lieberson
RE, Larson CP. Hyperglycemia decreases acute neuronal
ischemic changes after middle cerebral artery occlusion in
cats. Stroke. 1989;20:519-523.
[Abstract/Free Full Text] - Nedergaard M, Diemer NH. Focal ischemia of the rat brain with special reference to the influence of plasma glucose concentration. Acta Neuropathol (Berl).. 1987;73:131-137. [Medline] [Order article via Infotrieve]
-
de Courten-Myers GM, Myers RE, Schoolfield L.
Hyperglycemia enlarges infarct size in cerebrovascular occlusion in
cats. Stroke. 1988;19:623-630.
[Abstract/Free Full Text] - Prado R, Ginsberg MD, Dietrich WD, Watson BD, Busto R. Hyperglycemia increases infarct size in collaterally perfused but not end-arterial vascular territories. J Cereb Blood Flow Metab. 1988;8:186-192. [Medline] [Order article via Infotrieve]
-
de Courten-Myers GM, Kleinholz M, Wagner KR, Myers
RE. Fatal strokes in hyperglycemic cats.
Stroke. 1989;20:1707-1715.
[Abstract/Free Full Text] -
Yip PK, He YY, Hsu CY, Garg N, Marangos P, Hogan
EL. Effect of plasma glucose on infarct size in focal cerebral
ischemia-reperfusion. Neurology. 1991;41:899-905.
[Abstract/Free Full Text] - Nedergaard M. Transient focal ischemia in hyperglycemic rats is associated with increased cerebral infarction. Brain Res. 1987;408:79-85. [Medline] [Order article via Infotrieve]
- Duverger D, Mackenzie E. The quantification of cerebral infarction following focal ischemia in the rat: influence of strain, arterial pressure, blood glucose concentration, and age. J Cereb Blood Flow Metab. 1988;8:449-461. [Medline] [Order article via Infotrieve]
-
Lasarsere CA, Pitts D, Nelson DR, Faulk PW.
Vascular tissue plasminogen activator and the
development of coronary artery disease in heart-transplant
recipients. N Engl J Med. 1995;333:1111-1116.
[Abstract/Free Full Text] - Levin EG, del Zoppo GJ. Localization of tissue plasminogen activator in the endothelium of a limited number of vessels. Am J Pathol. 1994;144:855-861. [Abstract]
-
Shatos MA, Orfeo T, Doherty JM, Penar PL, Collen D,
Mann KG. -Thrombin stimulates urokinase production
and DNA synthesis in cultured human cerebral microvascular
endothelial cells. Arterioscler Thromb
Vasc Biol. 1995;15:901-911.
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