Rapid loss of microvascular integrin expression during focal brain ischemia reflects neuron injury.

The integrity of cerebral microvessels requires the close apposition of the endothelium to the astrocyte endfeet. Integrins alpha1beta1 and alpha6beta4 are cellular matrix receptors that may contribute to cerebral microvascular integrity. It has been hypothesized that focal ischemia alters integrin expression in a characteristic time-dependent manner consistent with neuron injury. The effects of middle cerebral artery occlusion (MCAO) and various periods of reperfusion on microvasclar integrin alpha1beta1 and alpha6beta4 expression were examined in the basal ganglia of 17 primates. Integrin subunits alpha1 and beta1 colocalized with the endothelial cell antigen CD31 in nonischemic microvessels and with glial fibrillary acidic protein on astrocyte fibers. Rapid, simultaneous, and significant disappearance of both integrin alpha1 and beta1 subunits and integrin alpha6beta4 occurred by 2 hours MCAO, which was greatest in the region of neuron injury (ischemic core, Ic), and progressively less in the peripheral (Ip) and nonischemic regions (N). Transcription of subunit beta1 mRNA on microvessels increased significantly in the Ic/Ip border and in multiple circular subregions within Ic. Microvascular integrin alpha1beta1 and integrin alpha6beta4 expression are rapidly and coordinately lost in Ic after MCAO. With loss of integrin alpha1beta1, multiple regions of microvascular beta1 mRNA up-regulation within Ic suggest that microvessel responses to focal ischemia are dynamic, and that multiple cores, not a single core, are generated. These changes imply that microvascular integrity is modified in a heterogeneous, but ordered pattern.

Rapid differential endogenous plasminogen activator expression after acute middle cerebral artery occlusion.

BACKGROUND AND PURPOSE: During focal cerebral ischemia, the microvascular matrix (ECM), which participates in microvascular integrity, is degraded and lost when neurons are injured. Loss of microvascular basal lamina antigens coincides with rapid expression of select matrix metalloproteinases (MMPs). Plasminogen activators (PAs) may also play a role in ECM degradation by the generation of plasmin or by MMP activation. METHODS: The endogenous expressions of tissue-type plasminogen activator (tPA), urokinase (uPA), and PA inhibitor-1 (PAI-1) were quantified in 10-microm frozen sections from ischemic and matched nonischemic basal ganglia and in the plasma of 34 male healthy nonhuman primates before and after middle cerebral artery occlusion (MCA:O). RESULTS: Within the ischemic basal ganglia, tissue uPA activity and antigen increased significantly within 1 hour after MCA:O (2P<0.005). tPA activity transiently decreased 2 hours after MCA:O (2P=0.01) in concert with an increase in PAI-1 antigen (2P=0.001) but otherwise did not change. The transient decrease in free tPA antigen was marked by an increase in the tPA-PAI-1 complex (2P<0.001). No significant relations to neuronal injury or intracerebral hemorrhage were discerned. CONCLUSIONS: The rapid increase in endogenous PA activity is mainly due to significant increases in uPA, but not tPA, within the ischemic basal ganglia after MCA:O. This increase and an increase in PAI-1 coincided with latent MMP-2 generation and microvascular ECM degeneration but not neuronal injury.

Integrin alpha(IIb)beta(3) inhibitor preserves microvascular patency in experimental acute focal cerebral ischemia.

BACKGROUND AND PURPOSE: Platelets become activated and accumulate in brain microvessels of the ischemic microvascular bed after experimental focal cerebral ischemia. The binding of glycoprotein IIb/IIIa (integrin alpha(IIb)beta(3)) on platelets to fibrinogen is the terminal step in platelet adhesion and aggregation. This study tests the hypothesis that inhibition of platelet-fibrin(ogen) interactions may prevent microvascular occlusion after experimental middle cerebral artery occlusion (MCA:O). METHODS: TP9201 is a novel Arg-Gly-Asp (RGD)-containing integrin alpha(IIb)beta(3) inhibitor. Microvascular patency after 3-hour MCA:O and 1-hour reperfusion within the ischemic and nonischemic basal ganglia was compared in adolescent male baboons who received high-dose TP9201 (group A: IC(80) in heparin, n=4), low-dose TP9201 (group B: IC(30) in heparin, n=4), or no treatment (group C: n=4) before MCA:O. RESULTS: After MCA:O, microvascular patency decreased significantly in group C. However, in the ischemic zones of groups A and B compared with group C, patencies were significantly greater in the 4.0- to 7. 5-microm-diameter (capillary) and 7.5- to 30.0-microm-diameter vessels (2P<0.05). A dose-dependent increase in hemorrhagic transformation was seen in group A (3 of 4 animals) compared with group B (1 of 4 animals), and no hemorrhage was visible in group C (chi(2) analysis for trend, P<0.05). CONCLUSIONS: Platelet activation contributes significantly to ischemic microvascular occlusion. Occlusion formation may be prevented by this RGD-integrin alpha(IIb)beta(3) inhibitor at a dose that does not produce clinically significant parenchymal hemorrhage. The effect of microvascular patency on neuron recovery can now be tested.

Activated microvessels express vascular endothelial growth factor and integrin alpha(v)beta3 during focal cerebral ischemia.

Both vascular endothelial growth factor (VEGF) and integrin alpha(v)beta3 play roles in angiogenesis. In noncerebral vascular systems, VEGF can induce endothelial integrin alpha(v)beta3 expression. However, it is unknown whether VEGF, like integrin alpha(v)beta3, appears in the initial response of microvessels to focal brain ischemia. Their coordinate expression in microvessels of the basal ganglia after middle cerebral artery occlusion (MCAO) in the nonhuman primate model was examined quantitatively. Cells incorporating deoxyuridine triphosphate (dUTP+) by the polymerase I reaction at 1 hour (n = 3), 2 hours (n = 3), and 7 days (n = 4) after MCAO defined the ischemic core (Ic) and peripheral regions. Both VEGF and integrin alpha(v)beta3 were expressed by activated noncapillary (7.5- to 30.0-microm diameter) microvessels in the Ic region at 1 and 2 hours after MCAO. At 7 days after MCAO, the number of VEGF+, integrin alpha(v)beta3+, or proliferating cell nuclear antigen-positive microvessels had decreased within the Ic region. The expressions of VEGF, integrin alpha(v)beta3, and proliferating cell nuclear antigen were highly correlated on the same microvessels using hierarchical log-linear statistical models. Also, VEGF and subunit alpha(v) messenger ribonucleic acids were coexpressed on selected microvessels. Here, noncapillary microvessels are activated specifically early during a focal cerebral ischemic insult and rapidly express VEGF and integrin alpha(v)beta3 together.

Matrix metalloproteinases increase very early during experimental focal cerebral ischemia.

Microvascular integrity is lost during focal cerebral ischemia. The degradation of the basal lamina and extracellular matrix are, in part, responsible for the loss of vascular integrity. Matrix metalloproteinases (MMPs) may play a primary role in basal lamina degradation. By using a sensitive modification of gelatin zymography, the authors investigated the activity of MMP-2 and MMP-9 in frozen 10-microm sections of ischemic and nonischemic basal ganglia and plasma samples of 27 non-human primates after middle cerebral artery occlusion/reperfusion (MCAO/R) for various periods. The gelatinolytic activities were compared with parallel cell dUTP incorporation in the ischemic zones of adjacent sections. In the brain, the integrated density of MMP-2 increased significantly by 1 hour after MCAO and was persistently elevated thereafter. Matrix metalloproteinase-2 expression was highly correlated with the extent of neuron injury and the number of injured neurons (r = 0.9763, SE = 0.004, 2P < 0.0008). Matrix metalloproteinase-9 expression only was significantly increased in subjects with hemorrhagic transformation. In plasma, only MMP-9 increased transiently at 2 hours of MCAO. These findings highlight the early potential role of MMP-2 in the degradation of basal lamina leading to neuronal injury, and an association of MMP-9 with hemorrhagic transformation after focal cerebral ischemia.

Integrin alphavbeta3 is expressed in selected microvessels after focal cerebral ischemia.

The endothelial and smooth muscle integrin alphaVbeta3, a receptor for vitronectin and fibrinogen, participates in angiogenesis associated with wound healing and tumorigenicity. The microvascular expression of alphavbeta3 and fibrin during experimental middle cerebral artery occlusion and reperfusion in a nonhuman primate model was examined by computer-assisted video imaging microscopy. No microvascular expression of alphavbeta3 was seen in the control subjects (n = 3) or the non-ischemic basal ganglia of subjects undergoing 2-hour MCA:O (middle cerebral artery occlusion) or 3-hour occlusion with 1-hour (n = 3), 4-hour (n = 3), and 24-hour (n = 3) reperfusion. In the ischemic territory, alphavbeta3 appeared initially at 2 hours of middle cerebral artery occlusion. Up-regulation of alphavbeta3 was confined to the media of 30.0- to 50.0-micron-diameter arterioles in the ischemic core and correlated significantly with fibrin deposition in those vessels (P < 0.0005). Integrin alphavbeta3 and its ligand fibrinogen appear in a subpopulation of microvessels after focal cerebral ischemia.

The structure-function relationship and reduction potentials of high oxidation states of myoglobin and peroxidase.

In these studies, we substitute electron-withdrawing (diacetyl) or -donating (diethyl) groups at the 2- and 4-positions of the heme in sperm whale Mb and HRP, and examine the structural and biochemical consequences. X-ray absorption spectroscopy shows that increased electron density at the heme results in an increased iron-pyrrole nitrogen average distance in both HRP and Mb, while decreased electron density results in shorter average distances. In HRP, the proximal ligand is constrained by a H-bonding network, and axial effects are manifested entirely at the distal site. Conversely, in Mb, where the proximal ligand is less constrained, axial effects are seen at the proximal side. In HRP, electron density at the heme iron depends linearly on pK3, a measure of the basicity of the porphyrin pyrrole nitrogens [Yamada, H., Makino, R., & Yamazaki, I. (1975) Arch. Biochem. Biophys. 169, 344-353]. Using diethyl substitution (pK3 = 5.8) and diacetyl substitution (pK3 = 3.3) in HRP and Mb, we measured the one-electron reduction potentials (E(O)') of HRP compounds I and II and ferryl Mb. Compound I showed a decreased E(O)' with increasing electron density at the heme (pK3), similar to E(O)' of ferric HRP. E(O)' of HRP compound II and ferryl Mb showed an opposite dependence. This behavior of E(O)', while initially surprising, can be explained by the apparent net positive charge on the iron porphyrin in each oxidation state of the hemoproteins.

Polymorphonuclear leukocyte behavior in a nonhuman primate focal ischemia model.

There is increasing interest in the role of polymorphonuclear (PMN) leukocytes in the evolution of focal cerebral infarction. Surgical preparation of focal cerebral ischemia models may alter leukocyte reactivity and thereby make interpretation of leukocyte function following ischemia/reperfusion difficult. The effects of surgical preparation and of experimental ischemia/reperfusion on granulocyte function have been examined prospectively in a baboon model. Twenty-six adolescent male baboons underwent surgical preparation, of which 21 underwent middle cerebral artery occlusion/reperfusion. Four additional animals served as nonsurgical controls. Peripheral venous blood specimens were taken for performing assays of leukocyte function at defined intervals before and after both the surgical preparation (i.e., the overall procedure for implantation of the middle cerebral artery occlusion device) and occlusion/reperfusion. A stress-related elevation in total leukocyte number was attributed mainly to an increase in the number of circulating PMN leukocytes. Values rose from 13.9 +/- 4.9 x 10(3) to 27.8 +/- 5.8 x 10(3)/microliters, (+/- SD; n = 21) for total leukocyte number, with p < 0.001, and from 4.3 +/- 2.1 x 10(3) to 15.9 +/- 4.7 x 10(3)/microliters (n = 21) for PMN leukocytes, with p < 0.001. Surgical preparation had no effect (p > or = 0.4) on the ability of PMN leukocytes, isolated 24 h after the implantation procedure, to display polarization, O2.- production, or beta-glucuronidase release when stimulated with human C5a. A moderate decrease in the chemotactic response to C5a resolved within the 7-day postsurgery (preocclusion) period. Three-hour middle cerebral artery occlusion and 1-h reperfusion resulted in a significant reduction in C5a-induced polarization.(ABSTRACT TRUNCATED AT 250 WORDS)

Fibrin contributes to microvascular obstructions and parenchymal changes during early focal cerebral ischemia and reperfusion.

BACKGROUND AND PURPOSE: Ischemic cerebral injury is associated with activation of the blood coagulation cascade. To elucidate the contribution of fibrin formation to microvascular injury during focal cerebral ischemia and reperfusion, we have studied the time course and the localization of fibrin deposition in cerebral microvessels and the surrounding tissues during ischemia/reperfusion in a well-described nonhuman primate model. METHODS: Cerebral tissues from adolescent male baboons were examined after 2-hour middle cerebral artery occlusion (n = 3) and after 3 hours of middle cerebral artery occlusion and 1-hour (n = 6), 4-hour (n = 3), and 24-hour (n = 4) reperfusion; tissues from control primates (n = 3) also were examined. Fibrin deposition was detected by immunohistochemical techniques using the fibrin-specific monoclonal antibody MH-1. The number and size distribution of microvessels associated with fibrin were quantified by video-imaging microscopy. RESULTS: Fibrin was associated with microvessels only in the ischemic zone where severe neuronal injury was documented, its frequency increasing with the reperfusion period (F4,26 = 3.80, P < .05). Extravascular fibrin deposition was significantly increased by 24-hour reperfusion compared with the other subjects (P < .05). Preischemia infusion of the anti-tissue factor monoclonal antibody TF9-6B4 resulted in significant reduction of intramicrovascular fibrin (P < .038 versus no intervention) at 1-hour reperfusion but had no effect on extravascular fibrin deposition. CONCLUSIONS: These results suggest that microvascular fibrin deposition accumulates in a time-dependent manner during focal cerebral ischemia/reperfusion and that exposure of focal cerebral ischemia/reperfusion and that exposure of plasma to perivascular tissue factor is partially responsible for occlusion formation. During ischemia the large plasma protein fibrinogen extravasates and interacts with parenchymal tissue factor, forming significant extravascular fibrin by 24 hours of reperfusion.

Oxidation-reduction properties of compounds I and II of Arthromyces ramosus peroxidase.

At neutral pH, compound I of Arthromyces ramosus peroxidase (ARP) was stable and was reduced to ferric ARP without apparent formation of compound II upon titration with ascorbate or hydroquinone. In the titration experiments, compound II was seen as an intermediate only at alkaline pH. However, measuring a difference spectrum in the Soret region by a stopped-flow method, we found that compound II was formed during the catalytic oxidation of ascorbate even at neutral pH. Using an EPR spectrometer with a microflow system, we measured the steady-state concentration of benzosemiquinone formed in the ARP-catalyzed oxidation of hydroquinone. The results clearly showed that ARP catalyzes the oxidation of hydroquinone by a one-electron-transfer mechanism, as does horseradish peroxidase. These observations led to the conclusion that compound I is reduced to compound II through a one-electron reduction by ascorbate or hydroquinone. Therefore, we concluded that ARP compound II is unusually unstable and is rapidly reduced to ferric enzyme without accumulation in the titration experiment. The unusual instability of ARP compound II is explained in terms of the high reduction potential of compound II. The reduction potentials (E0') of compounds I and II were measured at several pH values from redox equilibria with potassium hexachloroiridate on the basis of E0' = 0.90 V for the IrCL6(2)-IrCl6(3)- couple. These values were determined to be 0.915 and 0.982 V at pH 7, respectively, and decreased with increasing pH. This pH dependence was markedly changed by the buffer concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

P-selectin and intercellular adhesion molecule-1 expression after focal brain ischemia and reperfusion.

BACKGROUND AND PURPOSE: Polymorphonuclear leukocytes have been implicated in the development of the "no-reflow" phenomenon after focal cerebral ischemia and reperfusion. To further understand the role of granulocytes in microvascular occlusions, the responses of the granulocyte-endothelial cell adhesion molecules P-selectin and intercellular adhesion molecule-1 during middle cerebral artery ischemia and reperfusion were examined in a primate model. METHODS: Twelve adolescent male baboons were used for 2-hour middle cerebral artery occlusion (n = 3) or for 3-hour occlusion with 1-hour (n = 3), 4-hour (n = 3), and 24-hour (n = 3) reperfusion, and three separate unoperated primates served as controls. A quantitative immunohistochemical study of the microvascular distribution of P-selectin and intercellular adhesion molecule-1 was performed using 10-microns frozen sections from basal ganglia analyzed with computerized light microscopy video imaging. RESULTS: Significant (P < .05) persistent upregulation of P-selectin (beginning during ischemia) and transient upregulation of intercellular adhesion molecule-1 (at 1 and 4 hours of reperfusion) were observed on endothelium of selected post-capillary microvessels of the ischemic lenticulostriate artery territory. Platelet accumulation also occurred in this territory and was responsible for a significant proportion of the nonendothelial P-selectin signal at 24 hours after reperfusion. CONCLUSIONS: Focal cerebral ischemia/reperfusion stimulates endothelial P-selectin and intercellular adhesion molecule-1 expression in brain microvessels in the ischemic zone, which may contribute to enhanced leukocyte adherence and persistent activation.

Tissue factor contributes to microvascular defects after focal cerebral ischemia.

BACKGROUND AND PURPOSE: Microvascular perfusion defects occur after occlusion and reperfusion of the middle cerebral artery in examples of focal cerebral ischemia. In addition to cellular (eg, polymorphonuclear leukocyte) contributors to the focal "no-reflow" phenomenon, activation of coagulation may also play a role. We have tested a potential role of tissue factor-mediated coagulation in the microvascular perfusion defects seen after focal cerebral ischemia-reperfusion in a baboon model of reversible middle cerebral artery occlusion with the murine anti-tissue factor monoclonal antibody TF9-6B4. Tissue factor is the principal resident procoagulant substance in cerebral tissues and has a distinct perivascular distribution. METHODS: Microvascular patency in the basal ganglia after 3-hour middle cerebral artery occlusion and 1-hour reperfusion was quantified by computerized video imaging of carbon-tracer perfused tissues. Animals were randomized to receive intravenous TF9-6B4 (10 mg/kg) 10 minutes before middle cerebral artery occlusion (n = 6) or no treatment (n = 6) in an open study. RESULTS: In the control animals, a significant decrease in patency was confirmed in microvessels less than 30 microns in diameter. Infusion of TF9-6B4 before middle cerebral artery occlusion produced a stable maximal level of circulating antibody within 10 minutes, which lasted the duration of ischemia and reperfusion. An increase in reflow in microvessels of all size classes occurred after TF9-6B4 infusion, which was significant in those 7.5 to 30 microns (P = .038) and 30 to 50 microns (P = .013) in diameter. CONCLUSIONS: These results indicate that tissue factor-mediated events may also contribute to no-reflow in noncapillary microvessels after focal cerebral ischemia.

Tissue factor localization in non-human primate cerebral tissue.

Tissue factor (TF), the principal procoagulant of human brain, resides in specific regions of the non-human primate central nervous system. Immunohistochemical studies employing murine anti-human TF monoclonal antibodies (MoAbs) detected TF antigen in the cortex, basal ganglia, cerebellum, and cervical spinal cord in three normal baboon subjects. Although significantly less prominent than human cortical gray matter, a distinct partition of TF in gray matter > white matter was noted. The gray matter predilection of TF was confirmed in primate temporal and parietal lobe cortex by both sandwich ELISA and one-stage coagulation assay. Variation in the relative quantity of TF antigen was observed by ELISA among the three subjects studied. Procoagulant activity followed the pattern of TF antigen (cortical gray matter > basal ganglia > or = cerebellum > cortical white matter), and was 96.5-98.5% inhibitable by a function inhibiting anti-human TF MoAb combination. TF antigen was associated with the microvasculature of all cerebral tissues studied, and spared capillaries most selectively in the cerebral cortex, basal ganglia, and cerebellum. These findings suggest a highly specific ordering of TF antigen and related procoagulant activity in the central nervous system of the baboon, confined primarily to gray matter parenchyma, and to the non-capillary microvasculature.

Inhibition of polymorphonuclear leukocyte adherence suppresses no-reflow after focal cerebral ischemia in baboons.

BACKGROUND AND PURPOSE: While polymorphonuclear leukocytes may contribute to the "no-reflow" phenomenon after focal cardiac and skeletal muscle ischemia/reperfusion, their contribution to acute focal cerebral ischemia is unresolved. We have examined the role of polymorphonuclear leukocytes in microvascular perfusion defects after focal cerebral ischemia/reperfusion in a baboon model of reversible middle cerebral artery occlusion with the anti-CD18 monoclonal antibody IB4, which inhibits neutrophil adherence to endothelium. METHODS: Microvascular patency in the basal ganglia after 3-hour middle cerebral artery occlusion and 1-hour reperfusion (by india ink tracer perfusion) was quantified by computerized video imaging. Animals were randomized to receive intravenous IB4 infusion 15 minutes before reperfusion (n = 7) or to receive no treatment (n = 6). Binding of IB4 to baboon leukocytes was maximal within 5 minutes of infusion. RESULTS: In the untreated group, a significant reduction in patency was observed in microvessels less than 30 microns diameter: mean percent reflow was 51% in the capillary diameter class (4.0-7.5 microns) and 39% in the precapillary arteriole and postcapillary venule diameter class (7.5-30 microns). Infusion of IB4 before middle cerebral artery reperfusion increased reflow in microvessels of all size classes, most significantly in those 7.5-30 microns (p = 0.049) and 30-50 microns (p = 0.034) in diameter. CONCLUSIONS: These results suggest that CD18-mediated polymorphonuclear leukocyte-endothelium adherence contributes to no-reflow predominantly in noncapillary microvessels and at least partially to that in capillaries.

Polymorphonuclear leukocytes occlude capillaries following middle cerebral artery occlusion and reperfusion in baboons.

BACKGROUND AND PURPOSE: Microvascular perfusion defects may accompany sustained occlusion and subsequent reperfusion of the middle cerebral artery; however, the nature of such "no-reflow" defects remains unclear. METHODS: In the absence of antithrombotic pretreatment, we documented lenticulostriatal microvascular flow integrity following 3-hour middle cerebral artery occlusion and 1-hour reperfusion in a baboon occlusion/reperfusion model by two methods identifying 1) microvascular occlusion and 2) microvascular patency. RESULTS: Microvascular "no-reflow" involved capillaries (vessels of 4.0-7.5 microns diameter) of the lenticulostriatal territory. Capillary reflow included 27-39% of all capaillaries in two subjects, indicating a significant reduction of perfusion from normal (2p = 0.045). In identical experimental preparations, single polymorphonuclear leukocytes completely occluded 4.7% of microvessels of capillary diameter in randomly selected fields, partially occluded 3.5% of postcapillary venules, and occluded 40% (four of 10) of capillaries in linear reconstruction along a 110 microns length. Circumferential contact between polymorphonuclear leukocytes and the luminal endothelial cell membranes was documented, with an intrecellular gap of, at most, 160 nm. Fibrin was found with degranulated platelets when the latter were associated with granulocytes, but not with polymorphonuclear leukocytes alone. CONCLUSIONS: The finding of capillary-obstructing polymorphonuclear leukocytes in the microvascular bed following middle cerebral artery reperfusion in focal ischemia in this model satisfies an essential requirement for postulating their role in early microvascular injury and the "no-reflow" phenomenon.

Hemorrhagic transformation following tissue plasminogen activator in experimental cerebral infarction.

The effect of an intravenous infusion of recombinant tissue plasminogen activator on hemorrhagic transformation early after middle cerebral artery territory ischemia was studied in an established awake nonhuman primate (baboon) model. Following 3 hours' occlusion of the middle cerebral artery and 30 minutes' reperfusion in each of 30 baboons, a 60-minute infusion of recombinant tissue plasminogen activator (at three doses: Group A, 0.3 mg/kg, n = 6; Group B, 1.5 mg/kg, n = 6; Group C, 10 mg/kg, n = 6) or normal saline (n = 12) was undertaken. The frequency and volume of intracerebral hemorrhage, the volume of infarction, and clinical alterations were determined by computed tomography at 24 hours and 10 days, neuropathology at 14 days, and serial daily neurologic evaluations, respectively. Peripheral (nonintracranial) hemorrhage (Group A, p = 0.46; Group B, p = 0.015; Group C, p = 0.002) and peak plasma tissue plasminogen activator levels varied directly with the dose of recombinant tissue plasminogen activator. Petechial hemorrhagic infarction was a common finding among the 30 baboons. No significant differences in the incidences or volumes of infarction-related hemorrhage were apparent in any group compared with the respective saline-treated baboons. In pooled data, no significant relation between the volume of hemorrhage and the volume of infarction could be established. We conclude that the incidence and severity of hemorrhagic transformation are not related to infarction size and that recombinant tissue plasminogen activator does not increase the incidence or severity (volume) of hemorrhage when given early (less than or equal to 3.5 hours) after the onset of focal cerebral ischemia in this model.

Experimental acute thrombotic stroke in baboons.

To study the effects of antithrombotic therapy in experimental stroke, we have characterized a baboon model of acute cerebrovascular thrombosis. In this model an inflatable silastic balloon cuff has been implanted by transorbital approach around the right middle cerebral artery (MCA), proximal to the take-off of the lenticulostriate arteries (LSA). Inflation of the balloon for 3 hours in six animals produced a stereotypic sustained stroke syndrome characterized by contralateral hemiparesis. An infarction volume of 3.2 +/- 1.5 cm3 in the ipsilateral corpus striatum was documented by computerized tomographic (CT) scanning at 10 days following stroke induction and 3.9 +/- 1.9 cm3 (n = 4) at 14 days by morphometric neuropathologic determinations of brain specimens fixed in situ by pressure-perfusion with 10% buffered formalin. Immediate pressure-perfusion fixation following deflation of the balloon was performed in 16 additional animals given Evans blue dye intravenously prior to the 3 hour MCA balloon occlusion. Light microscopy and transmission electron microscopy consistently confirmed the presence of thrombotic material occluding microcirculatory branches of the right LSA in the region of Evans blue stain, but not those of the contralateral corpus striatum. When autologous 111In-platelets were infused intravenously in four animals from the above group prior to the transient 3 hour occlusion of the right MCA, gamma scintillation camera imaging of each perfused-fixed whole brain demonstrated the presence of a single residual focus of 111In-platelet activity involving only the Evans blue-stained right corpus striatum. Focal right hemispheric activity was equivalent to 0.55 +/- 0.49 ml of whole blood, and the occlusion score derived from histologic examination of the microcirculation of the Evans blue-stained corpus striatum averaged 34.8 +/- 2.8. Similar 111In-platelet imaging and histologic scoring experiments carried out in four animals pretreated with the antithrombotic combination heparin and ticlopidine showed marked reduction of both 111In-platelet activity (0.01 +/- 0.03 ml vs. 0.55 +/- 0.49 ml; p less than 0.01) and thrombotic occlusion of the microcirculation (10.8 +/- 7.4 units vs. 34.8 +/- 2.8 units; p less than 0.01) in the right corpus striatum following 3 hours of MCA occlusion. In separate control experiments 111In-labeled autologous platelets were infused after the 3 hour period of right MCA occlusion and subsequent balloon deflation in two animals; no focus of 111In-platelet activity was demonstrated in fixed whole brain.(ABSTRACT TRUNCATED AT 400 WORDS)

The beneficial effect of intracarotid urokinase on acute stroke in a baboon model.

The capacity of intracarotid infusion of urokinase to salvage neurologic function in a baboon model of acute thrombotic stroke has been studied. The model consists of reversible eccentric balloon compression (3 hours) of the right middle cerebral artery (MCA) proximal to the take-off of the lenticulostriate arteries (LSA), resulting in in situ thrombosis of perforating branches supplying the right corpus striatum. Neurologic endpoints included quantitative assessment of neurologic function (NE), estimation of cerebral infarction volume by computerized tomographic (CT) scan, and carotid angiography. In untreated acute stroke control animals (n = 6), a persistent decrease in functional score (from 100 to 36 +/- 11) at 14 days and a defined region of cerebral infarction (volume = 3.2 +/- 1.5) were detected at 10 days. Intracarotid urokinase administered to five animals (1.2 X 10(6) IU over 60 min) following the 3 hour period of MCA occlusion improved neurologic function (NE = 50, 55, 85, 100, 100) and reduced infarction size (0.3, 0.5, 0.8, 0.7, 1.1 cm3, respectively) without evidence of intracranial hemorrhage. Systemic fibrinogenolysis was produced in all five treated animals. We conclude that thrombolytic therapy given within 3 hours of experimental thrombotic occlusion may salvage neurologic function and reduce cerebral infarction volume without CT scan detectable intracranial bleeding.

Cystic supratentorial gliomas: natural history and evaluation of modes of surgical therapy.

The management of cystic supratentorial gliomas is hampered by lack of documentation of the natural history of these lesions and by a lack of evaluation of modes of surgical therapy. We analyzed these factors in 25 patients with solitary cysts operated upon over a 20-year period. Two distinctive patterns of symptoms were seen: short duration (increased pressure and hemiparesis), most often heralding a malignant lesion, and long duration (commonly seizure disorder), associated more often with a benign pathological condition. Large solitary cysts were found in tumors of all histological grades. Surgical procedures included extirpation, biopsy/partial resection, cyst communication to ventricle or marsupialization, burr hole aspiration, aspiration via an indwelling reservoir, and cyst-peritoneal shunting. Radiotherapy, given in all cases, did not prevent cyst recurrence. Of the 25 patients, 76% are alive and remain cyst free at follow-up intervals of 1 to 16 years (mean, 3.2). Five patients died from their tumors, with a mean survival of 33 months after decompression. In 7 of 8 patients with cysts largely or entirely within the basal ganglia or thalamus, successful operative cyst control was achieved. Patients with solitary cystic gliomas seem to have a favorable prognosis, and vigorous efforts to control cyst recurrence and limit disability are warranted. Analysis of our data suggests that craniotomy for tumor resection, cyst decompression, and tissue diagnosis is the initial procedure of choice. Cyst recurrence without major solid tumor should be controlled by computed tomography-guided tap or shunt drainage. Reexploration is indicated when cyst reaccumulation is accompanied by clear regrowth of a solid component.

Secretion and degradation of mutant leucine-specific binding protein molecules containing C-terminal deletions.

The leucine-specific binding protein (LS-BP), a periplasmic component of the Escherichia coli high-affinity leucine transport system, is initially synthesized in a precursor form with a 23 amino acid N-terminal leader sequence that is removed during secretion of the protein into the periplasm. Using in vitro mutagenesis, deletion mutants of the LS-BP gene have been constructed with altered or missing amino acid sequences in the C-terminal portion of the protein. These altered binding proteins exhibited normal processing and secretion but were rapidly degraded in the periplasmic space. In the presence of an uncoupler of the transmembrane potential (CCCP) the precursor forms accumulated in the membrane and were protected from degradation. The altered binding proteins also were secreted by spheroplasts of E coli, after which they were easily detected.