Mild to Moderate Hypothermia Prevents Microvascular Basal Lamina Antigen Loss in Experimental Focal Cerebral Ischemia
Background and Purpose— Microvascular basal lamina damage occurs after cerebral ischemia and is important for the development of hemorrhage. The aim of this study was to determine whether hypothermia could maintain microvascular integrity in ischemic stroke.
Methods— Using the suture model, we subjected 12 rats to 3 hours of focal ischemia and 24 hours of reperfusion. Six rats received postischemic normothermia (37°C) and 6 received hypothermia (32°C to 34°C) for the reperfusion period; a group of 6 sham-operated animals without ischemia was used as control. Collagen type IV and hemoglobin were measured by Western blot analysis, matrix metalloproteinase (MMP)-2 and MMP-9 by gelatin zymography, and urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA) by plasminogen-casein zymography.
Results— Hypothermia reduced basal lamina collagen type IV loss: 87±16% (hypothermia) versus 43±4% (normothermia) in basal ganglia and 74±16% versus 64±4% in cortex; hypothermia reduced hemorrhage from 431±65% (normothermia) to 241±28% (basal ganglia) (P<0.05). Hypothermia also reduced MMP-2, MMP-9, uPA, and tPA (basal ganglia: MMP-2: 71±20% [hypothermia] versus 109±3% [normothermia]; MMP-9: 38±12% versus 115±4%; uPA activity: 310±86% versus 1019±22%; tPA activity: 61±17% versus 111±13%; cortex: MMP-2: 53±6% versus 116±1%; MMP-9: 16±4% versus 123±3%; uPA: 180±27% versus 176±10%; tPA: 91±15% versus 101±8%; each difference: P<0.001) (nonischemic control side=100%).
Conclusions— Hypothermia maintains microvascular integrity and reduces hemorrhage and the activities of MMP-2, MMP-9, uPA, and tPA.
- basement membrane
- cerebral ischemia
- plasminogen activators
- tissue plasminogen activator
Interendothelial tight junctions, the basal lamina, and perivascular astrocytes constitute the blood-brain barrier.1 After disruption of the endothelium in focal cerebral ischemia, the basal lamina prevents extravasation of cellular blood elements. Loss of basal lamina integrity results in hemorrhage.2,3 The components of the basal lamina (type IV collagen, laminins, and fibronectin4) were shown to be degraded in a baboon stroke model.3 The mechanisms involved are not entirely understood.1,5,6 Noncellular proteolytic systems, eg, matrix metalloproteinases (MMPs) and the plasminogen-plasmin system, hydrolyze the basal lamina. MMP-2 (gelatinase A, 72 kDa) and MMP-9 (gelatinase B, 92 kDa) are known to degrade type IV collagen and laminin,5 and both have been reported to be increased in experimental cerebral ischemia.7
Treatment with intravenous recombinant tissue-type plasminogen activator (tPA) within 3 hours after stroke onset improves clinical outcome but carries the risk of hemorrhage.8 Clinical trials with mild to moderate hypothermia in acute stroke patients are under way,9 but its benefit has not yet been proven.10
Hypothermia limits ischemic damage by decreasing metabolism, suppressing blood-brain barrier breakdown,11 and reducing free radical formation12 and inflammation.11 In animal stroke models, hypothermia was also shown to decrease infarct size.12–16
The aim of this study was to evaluate the effect of postischemic mild to moderate hypothermia on the microvascular basal lamina during focal cerebral ischemia.
Materials and Methods
All experimental procedures were approved by the government of Upper Bavaria (211-2531/48/98) and were in accordance with animal protection guidelines. For additional details, see the Appendix, which is available online at http://stroke.ahajournals.org.
All experiments used male Wistar rats (weight, 250 to 300 g) (Charles River Laboratories, Sulzfeld, Germany) (a total of 18 rats: 12 for ischemia/reperfusion, 6 for sham-operated controls). A period of 3 hours of ischemia was followed by 24 hours of reperfusion. Six of these animals were kept at a body temperature of 37°C as the normothermic group, and the remaining 6 were kept at a body temperature of 32°C to 34°C for the reperfusion period as the hypothermic group.
Mild to moderate hypothermia (32°C) was induced 30 minutes before reperfusion by applying active external cooling. Both the temperature control and the method of inducing hypothermia were adapted from Yanamoto et al.19 The body temperature was measured by a small thermistor in the right temporal muscle. The hypothermic rats were kept in a refrigerated cage at 4°C to 8°C. Cage and body temperatures were continuously monitored; body temperature was kept at 32°C by feedback. The normothermic animals were also continuously monitored, and a heating pad was adjusted to maintain normothermia.
Control animals were sham-operated by advancing the thread only 12 mm toward the intracranial part of the internal carotid artery so that it did not occlude the middle cerebral artery.
Preparation of Cryostat Sections
Cryostat sections of 10-μm thickness were taken from regions 0 to 1 mm behind the bregma20 and stored at −80°C.
Protein Isolation and Western Blot for Collagen Type IV and Hemoglobin
For details, see Hamann et al.17 The antibodies used were goat anti-collagen type IV at a dilution of 1:500 (Southern Biotechnology) and a polyclonal rabbit anti-hemoglobin antibody at a dilution of 1:200 (DPC Biermann).
Gelatin Zymography for MMPs
For details, see Burggraf et al.21 Molecular standards and recombinant human MMP-2 and MMP-9 standards (Sigma) were used to calibrate molecular weights.
Plasminogen-Dependent Casein Zymography
Gel zymography was adapted from the procedure described.22 Transparent zones of lysis at 64 and 46 kD correspond to tPA and urokinase-type plasminogen activator (uPA), respectively. Molecular standards were used to calibrate molecular weights.
Analysis of Blotting Results
The bands of the Western blot and zymography were scanned and analyzed with an optical analysis program (TINA, version 2.08, Raytest Isotopenmeβgeräte GmbH) by optical densitometry. To allow comparison over multiple samples run in different gels, the amount of the proteins in the ischemic side was normalized by dividing it by the nonischemic side. For details, see Hamann et al.17
Data were expressed as mean±SEM. All analyses were done by ratios of the ischemic to the nonischemic sides. Comparisons between the experimental groups were made with the Mann-Whitney U test (level of significance of 5%). MMP-2, MMP-9, uPA, and tPA analyses were performed with an ANOVA test (Kruskal-Wallis analysis).
Reduced Infarct Volume
Rats undergoing hypothermia had significantly lower infarct volumes: 153±42 mm3 after hypothermia versus 192±43 mm3 in normothermic animals (Table 1).
Protection of Basal Lamina
The Western blot analysis revealed a significant loss of collagen type IV. In the normothermic group, collagen type IV was reduced to 64±4% in the cortex and to 43±4% in the basal ganglia compared with the nonischemic control side. The breakdown of collagen was significantly less in the hypothermic animals. Collagen was reduced to 74±16% in the cortex and to 87±16% in the basal ganglia (Figure 1). In the sham-operated animals, no difference of the collagen type IV content of both hemispheres could be seen (Table 2). The reduced loss of collagen type IV was significant (cortex and basal ganglia) (P<0.05).
Reduction of Hemoglobin Extravasation
Table 2 shows the hemoglobin data of the normothermic and the hypothermic groups. Ischemia led to the extravasation of hemoglobin (431±65% basal ganglia, 197±25% cortex), which was dramatically reduced by hypothermia (to 241±28% and 163±10%, respectively; P<0.05).
Concentration of MMPs
Ischemia/reperfusion increased the MMP-2 concentration. Cortical areas in normothermic animals exhibited concentrations of 116±1% of the nonischemic control side, and the basal ganglia reached 109±3%. MMP-9 was also elevated after ischemia/reperfusion. Cortical areas in normothermic animals showed MMP-9 of 123±3% of the nonischemic control side, whereas basal ganglia regions exhibited 115±4%. Sham-operated animals showed no change in the MMP activities (Table 3). Both MMP-2 and MMP-9 were significantly reduced by hypothermia (MMP-2 to 53±6% of the nonischemic side in cortical areas and to 71±20% in the basal ganglia, P<0.001; MMP-9 to 16±4% in cortical and 38±12% in basal ganglia regions, P<0.001) (Figure 2).
Activity of Endogenous Plasminogen Activators
The activity of tPA in ischemic cortical areas compared with the nonischemic side was 101±8% in normothermic animals; the respective value for the basal ganglia was 111±13%. Hypothermia reduced these activities to 91±15% in cortex and to 61±17% in basal ganglia (P<0.001). Animals with no ischemia showed no change in the activity of tPA.
The uPA activity for the cortex was not significantly different between hypothermia (180±27%) and normothermia (176±10%), but there was a significant increase (P<0.0001) relative to sham-operated animals: 95±7% (cortex) and 101±8% (basal ganglia). In the basal ganglia there was a striking increase of uPA activity in normothermic controls (1019±22%); this was significantly reduced by hypothermia (310±86%; P<0.001) (Figure 3; Table 3).
The main finding of this study is that postischemic mild to moderate hypothermia prevents microvascular basal lamina antigen loss and subsequent hemoglobin extravasation during focal ischemia. The degradation of the basal lamina probably begins very early in cerebral ischemia.1–3,5,17 A similar degree of microvascular damage in normothermic controls was reported previously.3,5,17 However, we have now shown for the first time that postischemic mild to moderate hypothermia after 3 hours of ischemia and 24 hours of reperfusion significantly reduces the loss of collagen type IV from the basal lamina of cerebral microvessels in the rat. Accordingly, infarct size was also reduced, as expected. It could also be shown that subsequent hemorrhage was significantly reduced. We did not examine the effects on edema in this study. The protective effect of hypothermia on the basal lamina may be explained by the reduction of the proteases MMP-2, MMP-9, tPA, and uPA.
Interendothelial tight junctions, the basal lamina, and perivascular astrocytes are jointly referred to as the blood-brain barrier.1 The endothelial barrier regulates substrate transfer. The basal lamina provides a structural barrier to extravasation of cellular blood elements and anchors endothelial cells and astrocytes. Intact microvascular basal lamina and integrin-mediated matrix adhesion are essential for cellular function.4,5 During cerebral ischemia, the functional and structural integrity of the endothelial barrier rapidly disintegrates.24 The only barrier that protects the brain from protein-rich fluids and cellular blood elements in this situation is the basal lamina,1 which is embedded in the extracellular matrix and consists of a sheet of collagen type IV and a net of laminin interconnected by entactin.4,25 Proteolytic enzymes from both the blood and brain tissue instantly start digesting the basal lamina. The brain parenchyma is then exposed to the blood.5,26
Microvascular basal lamina changes under ischemic conditions involve the plasminogen-plasmin system, various MMPs, and leukocyte activation.1 Our data suggest that mild to moderate hypothermia is accompanied by reduced activity and concentration of proteinases, including MMP-2, MMP-9, uPA, and tPA. Several studies have demonstrated the critical role of protease degradation after cerebral ischemia.
Endothelial cells produce endogenous tPA to prevent wall thrombosis.27 Compelling uPA activation is seen in cerebral ischemia.28 A balanced fibrinolytic activity is essential for microvascular function.29 Besides its thrombolytic activity, which provides microvascular patency, plasmin hydrolyzes extracellular matrix proteins and activates MMP-9.27–30 The balance between proteases and their inhibitors determines whether there is breakdown or buildup of the extracellular matrix.31–33 At least 2 MMPs (MMP-2 and MMP-9) are involved in these processes. MMP-2 and MMP-9 activities were increased in neutrophils, endothelial cells, and macrophages in a permanent stroke model.34 MMP-2 is constitutively expressed and activated by a membrane-type metalloproteinase. Activation of MMP-2 in turn activates pro-MMP-9 to MMP-9. Activated MMP-9 appears as early as 3 hours after transient focal cerebral ischemia.35 Rosenberg et al36 have shown increases of pro-MMP-9 after transient ischemia. In the present study both MMP-2 and MMP-9 were significantly decreased by postischemic hypothermia. MMP gel activity reflects the overall MMP concentration rather than an in vivo activity since the SDS from the gel also activates inactive pro-MMPs. One can speculate that the reduction in proteolytic activities below 100% on the ischemic side of hypothermic animals reflects an earlier activity (by the regularly available proteolytic systems, as in nonischemic brain tissue), which is used up after longer reperfusion. The later, more severe, and pronounced proteolytic activation (as seen in normothermic controls) is inhibited by hypothermia. The reduction in MMP activity may also at least partly reflect decreased ischemic injury after hypothermia.
This study also revealed that endogenous plasminogen activation is significantly reduced by hypothermia. In particular, the strong activation of uPA, which is thought to play a key role in the brain after ischemia,28 was reduced to approximately 20% of the level of the normothermic controls. The main change of uPA is seen in basal ganglia in contrast to the cortex. This may reflect the occurrence of hemorrhagic complications, which are known to show a preference for the basal ganglia.
Like others, we also found the most robust effects in uPA and MMP-9 expression.28,37 One could argue that the cutback in ischemic uPA upregulation reduces secondary MMP activation and prevents collagen IV loss in the basal lamina.
Although most protective mechanisms of hypothermia remain elusive, there is evidence that hypothermia can reduce the consequences of cerebral ischemia in experimental15 and clinical settings.38,39 Our results demonstrate that mild to moderate hypothermia protects the basal lamina, in addition to reducing the infarct size, as was expected. Therefore, hypothermia may reduce hemorrhage after thrombolysis for acute ischemic stroke. Hypothermia could influence the delicate risk/benefit ratio of thrombolysis and allow more aggressive or delayed restoration of blood flow at a reduced hemorrhagic risk.
Supplemental Material and Methods
All animals were allowed to free access to food and water.
Rats subjected to 24-hour reperfusion were intubated and anesthetized with isoflurane inhalation. Rats were not allowed to wake up and were kept in narcosis until reperfusion started. Rectal temperature was kept at 37.0±0.5°C by means of a feedback-controlled heating pad. All animals were subjected to 3 hours of ischemia. Transient ischemia and reperfusion were induced with the use of a modified intravascular filament model, according to the surgical procedure of Longa and colleagues.17,18 Briefly, a midline incision was made at the neck. The left common carotid artery (CCA), its bifurcation into the external carotid artery (ECA), and the internal carotid artery (ICA) were exposed under an operating microscope. Special care was taken to avoid injury of the vagus nerve. After the CCA was ligated distally, blood flow within the CCA was interrupted up to the bifurcation. To occlude the MCA, a surgical nylon monofilament thread (Ethicon 4/0; Ethicon), its tip covered by a silicone end, was inserted in a guide sheath made from a polyethylene catheter (inner diameter 58 mm, outer diameter 96 mm; Portex Co). A small incision was made in the CCA to insert the catheter; the tip of the sheath was placed just before the bifurcation into the ECA and ICA. Then the filament was gently advanced approximately 16 to 18 mm into the intracranial part of the ICA.
The suture thread was no longer advanced as soon as a remarkable increase in vascular resistance was detected. After onset of reperfusion by withdrawing the thread by approximately 4 to 5 mm, rats were allowed to awake ad libitum. At the end of the reperfusion period, rats were killed by transcranial perfusion with cold physiological saline containing bovine serum albumin (10 g/L), heparin (10 iE/L), and 2 mL/L nitroprusside solution (1.8 g/L) diluted in 1 L of NaCl isotonic solution (9 g/L) under deep anesthesia. Brains were removed, and the skull base was inspected to exclude hemorrhage.
Consecutive coronary cryosections (10 μm thick) in the region 0.0 mm up to +1.0 mm distant from bregma, with stereotaxic coordinates following those of Paxinos and Watson,20 were prepared for further analysis.
Four animals (2 in each group) died before the end of the experiment; they were not included in the evaluation and replaced.
All steps for brain tissue homogenization and centrifugation were performed at 4°C. Brain areas in regions of the cortex and the basal ganglia having the same size in the ischemic and nonischemic hemispheres were excised from cryosections of 10-μm thickness. For the following Western blot analysis, brain material was homogenized in a lysis solution of 20 mmol/L Tris, pH 7.3, 1 mmol/L EDTA, 2% sodium dodecyl sulfate (SDS). Samples were then sonicated for 10 seconds and spun at 13 000 rpm for 10 minutes. For the following zymographic analysis, brain material was homogenized in a homogenization solution of 5 mmol/L Tris, pH 7.5, 75 mmol/L NaCl. Protease inhibitor phenylmethylsulfonyl fluoride (100 μg/mL) was added to prevent protein degradation. Samples were then sonicated for 2 seconds and spun at 9000 rpm at 4°C for 10 minutes.
Protein concentrations were routinely determined by a modified Bradford protein assay (TEBU GmbH) with bovine serum albumin (BSA) used as a standard.
For the following SDS–polyacrylamide gel electrophoresis (PAGE),21 equal amounts of protein were diluted in 2× SDS-PAGE loading buffer containing 25 mmol/L β-mercaptoethanol to a final loading amount of 20 μg. The samples were heated to 95°C for 5 minutes, loaded on a 7.5% polyacrylamide gel, and run for 2 hours at 100 V. The proteins were transferred at 4°C to a PVDF membrane (Sequi-Blot; Biorad) for 1 hour at 400 mmol/L in a transfer buffer containing 10 mmol/L CAPS at pH 11.0 with 10% methanol. After immobilization, membranes were stained with Ponceau S (Sigma) to confirm equal loading and transfer of proteins in all steps.
The blots were blocked in 3% BSA in Tris-buffered saline (TBS) with 0.2% Tween 20 (TBST) at room temperature for 1 hour. Blots were incubated in goat anti-collagen type IV at a dilution of 1:500 (Southern Biotechnology) or in a polyclonal rabbit anti-hemoglobin antibody at a dilution of 1:200 (DPC Biermann) made up in blocking solution (3% BSA in TBST) overnight at 4°C. The blots were then washed 3 times for 10 minutes with TBST and incubated at room temperature for 1 hour with biotinylated anti-goat/anti-rabbit antibody (Vector Laboratories) in TBST. Vectastain ABC reagent was added for 30 minutes at room temperature after the sections were rinsed with TBST. After additional washing (3×10 minutes with TBST), the Western blot procedure was completed with an ECL developing kit (Amersham).
For the nonreducing SDS-PAGE, equal amounts of protein (20 μg) were run without β-mercaptoethanol on a 10% polyacrylamide gel containing 0.5% gelatin (Biorad). After the enzymes were renatured in the gel with 2.5% Triton X-100 (1 hour, room temperature) the enzymatic digestion in the gel proceeded in an incubation buffer containing 5 mmol/L CaCl2, 50 mmol/L Tris-HCl [pH 7.4], 200 mmol/L NaCl, and 0.2% Brij 35 for 24 hours at 37°C.
To visualize the enzymatic digestion, the gels were stained with Brilliant Blue R. The lysis zones representing the enzymatic digest appeared as clear zones in the gel. Molecular standards (BioRad) and recombinant human MMP-2 and MMP-9 standards (Sigma) were used to calibrate molecular weights.
Plasminogen-Dependent Casein Zymography
Gel zymography was adapted from the procedure described by Heussen and Dowdle.22 Ten percent polyacrylamide-SDS gels were copolymerized with casein (5 mg/mL; Sigma) and plasminogen (0.01 U/mL; Sigma). For the following SDS-PAGE, equal amounts of protein were diluted in 2× SDS-PAGE loading buffer without β-mercaptoethanol to a final loading amount of 20 μg. After electrophoresis, the SDS was extracted from the gel with the use of 2.5% Triton X-100 (1 hour, room temperature), and the gel was incubated for 20 hours in 0.1 mol/L Tris [pH 8.1] at 37°C, followed by staining with 0.4% Coomassie blue in 35% MeOH/10% acetic acid. Destaining with the same solvent revealed transparent zones of lysis against the dark protein background at 64 and 46 kDa, which corresponded to tPA and uPA, respectively. Molecular standards were used to calibrate molecular weights.
Analysis of Blotting Results
The bands of the Western blot and zymography were scanned with the use of an Epson Perfection 124OU scanner and analyzed with an optical analysis program. Results were displayed on an arbitrary optical density scale. The data were expressed as relative lysis zone (pixels) per microgram protein and per millimeter lane width. The relation between the data on the ischemic and the nonischemic area was determined.
Sections were fixed with cold acetone and chloroform (1:1) for 5 minutes at room temperature, immersed in 10 mmol/L glycine in PBS for 5 minutes, and rinsed 3 times for 5 minutes in PBS before a 20-minute incubation at 4°C with Blotto (50 g nonfat dry milk, 1 mL horse serum, and 0.3 mmol/L sodium azide diluted in 1 L Tris-saline stock, 38.5 mmol/L Tris, and 150 mmol/L sodium chloride diluted in 1 L distilled water) to reduce nonspecific binding.
The sections were incubated with the primary antibody against MAP-2 (Chemicon). The antibody was diluted 1:800 in reagent (400 mg BSA and 0.06 mmol/L thimerosal diluted in 1 L Tris-saline stock), first for 2 hours at 37°C and then additionally for 12 hours at 4°C. Sections were washed several times in PBS between the different incubation steps. The biotinylated secondary antibodies (anti-goat, Jackson Immunoresearch Laboratories) were further in cubated for 30 minutes at 37°C. They were diluted 1:200 in a solution of PBS, horse serum, and 10% Tween (1000:15:1). For subsequent development with the peroxidase technique, it was necessary to block the activity of endogenous peroxidases by incubating them with hydrogen peroxide (1 mL PBS, 30% hydrogen peroxide) for 20 minutes at room temperature. The Vectastain-Elite Kit (Vector Laboratories) was used to incubate sections with the avidin-biotin complex (according to the manufacturers’ instructions) for 30 minutes at 37°C. Subsequently, the peroxidase activity was detected with 3,3′-diaminobenzidine (concentrations according to the manufacturers’ instructions; Vector Laboratories).
This study was supported by the Kompetenznetzwerk Schlaganfall of the Germany Ministry of Education and Research–BMBF (B3) and Radiant Medical Incorporation, Redwood City, Calif. Drs Krieger and DeGeorgia serve as consultants for Radiant Medical Incorporation. We thank Judy Benson for copyediting the manuscript.
- Received July 14, 2003.
- Revision received September 30, 2003.
- Accepted November 6, 2003.
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