Threshold duration of ischemia for myogenic tone in middle cerebral arteries: effect on vascular smooth muscle actin.

BACKGROUND AND PURPOSE: We investigated the effect of different periods of ischemia on the myogenic tone of middle cerebral arteries (MCAs) and tested the hypothesis that ischemia disrupts the actin cytoskeleton in vascular smooth muscle. METHODS: The MCA occlusion model was used in male Wistar rats (n=27) to induce different periods of ischemia (15, 30, and 120 minutes) with 24 hours of reperfusion. Successful occlusion was determined by laser-Doppler flowmetry. MCAs were then studied in vitro with a specialized arteriograph system that allowed control of transmural pressure and measurement of lumen diameter. After equilibration for 1 hour at transmural pressure of 75 mm Hg, lumen diameter was measured, and the amount of spontaneous myogenic tone was determined. Arteries were then fixed with 10% formalin while still pressurized in the arteriograph bath and stained for filamentous (F-) actin with fluorescently labeled phalloidin, a specific probe for F-actin. The amount of F-actin was quantified by confocal microscopy. RESULTS: The amount of tone was similar between control and 15 minutes of ischemia (27.0+/-2.0% and 25.3+/-1.7%, respectively; P>0.05) but was significantly diminished after 30 and 120 minutes (11.7+/-2.0% and 8.5+/-2.0%, respectively; P<0.01 versus control). F-actin content also decreased at the longer ischemic periods and correlated significantly with vascular tone (P=0.04) such that the lesser the tone, the lesser was the F-actin content. Fluorescence intensity for control and 15, 30, and 120 minutes of ischemia was (x10(7)) 3.21+/-0.25, 2.54+/-0.32 (P>0.05), 2.32+/-0.15 (P<0.01), and 2.22+/-0.16 (P<0.01), respectively. CONCLUSIONS: These results demonstrate that ischemia disrupts the actin cytoskeleton in smooth muscle and diminishes vascular tone of MCAs in a threshold-dependent manner. This effect likely exacerbates brain tissue damage during stroke, including infarction and edema formation.

Efficacy of antioxidant therapies in transient focal ischemia in mice.

BACKGROUND AND PURPOSE: Ginkgo biloba extract (EGb) and alpha-lipoic acid (LA) are commercially available "antioxidant supplements" with a variety of actions that may be beneficial during acute stroke. These actions include inhibiting platelet and leukocyte activation and adhesion, reducing free radical generation, and increasing cerebral blood flow. Both EGb and LA have been shown to be neuroprotective in cell culture and global central nervous system ischemia models. In this study we investigated the neuroprotective efficacy of EGb and LA in a clinically relevant, transient focal central nervous system ischemic model. METHODS: In the EGb study, 60 adult C57blk mice were randomized to treatment with EGb given orally (via gavage) for 7 days: low dose, 50 EGb mg/kg daily; high dose, 100 mg/kg daily; matched placebo. On day 7, reversible middle cerebral artery occlusion was produced by advancing a silicone-coated 8-0 filament into the internal carotid artery for 45 minutes followed by reperfusion. At 24 hours, the animals were evaluated on a 28-point clinical scale, and infarct volume was determined with the use of triphenyltetrazolium chloride. In the LA study, 24 C57blk mice were treated with 100 mg/kg SC of LA or placebo 1.5 hours before transient MCAO, as in the EGb study. RESULTS: In the EGb study, values for infarct volume at 24 hours were as follows (mean+/-SD): low dose (n=18), 13+/-5 mm(3); high dose (n=22), 22+/-12 mm(3); placebo (n=20), 20+/-10 mm(3) (P:=0.03 overall; P=0.02, low dose versus placebo). Infarct percentage of hemisphere values were as follows: low dose, 14+/-5%; high dose, 21+/-11%; placebo, 20+/-9% (P=0.03 overall; P=0.02, low dose versus placebo). Ten percent of the high-dose group showed significant intracerebral hemorrhage (ICH) within the infarct, while no ICH was seen in the other groups. Neurological function scores were as follows: low dose, 11.8+/-1.5; high dose, 11.4+/-1.7; placebo, 11.3+/-1.8 (P=NS). In the LA study, infarct volume was as follows: 100 mg/kg LA (n=12), 16.8+/-8.3 mm(3); placebo (n=12), 27.2+/-14.6 mm(3) (P<0.05). LA also produced a significant improvement in neurological function at 24 hours: LA, 9.5+/-1.2; placebo, 11.2+/-1.8 (P=0.02). There was no evidence of ICH in any of the animals. CONCLUSIONS: Both oral EGb and LA therapies produced significant reductions in stroke infarct volume. However, for EGb this beneficial effect appears to be dose related, with higher doses potentially increasing the risk of ICH.

Lack of interleukin-6 expression is not protective against focal central nervous system ischemia.

BACKGROUND AND PURPOSE: Interleukin-6 (IL-6) appears to be involved in the inflammatory response associated with central nervous system (CNS) ischemia. Although IL-6 levels increase after stroke, it is not known whether IL-6 directly influences CNS ischemic injury. In this study, we used a focal reversible stroke model to investigate whether mice lacking IL-6 were protected against acute ischemic injury. METHODS: We bred IL-6-deficient C57 black mice (I-129 IL-6 KO back-crossed with C57), including homozygous knockouts (IL-6 -/-), heterozygous littermates (IL-6 +/-), and normal littermates (IL-6 +/+). The status of all animals was confirmed by DNA sampling and polymerase chain reaction analysis. Reversible middle cerebral artery occlusion was produced by advancing a silicone-coated 8-0 filament into the internal carotid artery for 2 hours (experiment 1) or 45 minutes (experiment 2). At 24 hours, animals were evaluated on a 28-point clinical scale, blood and cerebrospinal fluid were obtained, and the brains were evaluated for infarct volume and IL-6 mRNA levels. RESULTS: In experiment 1 (severe ischemia), no differences were seen in lesion size or neurological function between the groups: lesion volume was IL-6 -/- (n=15), 57+/-13 mm(3); IL-6 +/- (n=15), 58+/-23 mm(3); and IL-6 +/+ (n=15), 58+/-18 mm(3) (P=NS). ELISA testing confirmed very low to absent levels of IL-6 in the serum and cerebrospinal fluid of knockout animals. Brain mRNA levels of the other proinflammatory cytokines, including tumor necrosis factor-alpha, IL-1beta, and IL-1 receptor antagonist, were 50% lower in IL-6-deficient ischemic animals than in normal animals. In experiment 2 (mild ischemia), no differences were seen in lesion size or neurological function between the groups: lesion volume was IL-6 -/- (n=10), 16+/-8 mm(3); IL-6 +/- (n=10), 14+/-4 mm(3); and IL-6 +/+ (n=10), 19+/-12 mm(3) (P=NS). CONCLUSIONS: In this study, infarct size and neurological function at 24 hours were not different in animals deficient in IL-6 after transient CNS ischemia. This suggests that IL-6 does not have a direct influence on acute ischemic injury. Further study investigating the role of IL-6 on long-term recovery after stroke is in progress.

Postischemic attenuation of cerebral artery reactivity is increased in the presence of tissue plasminogen activator.

BACKGROUND AND PURPOSE: We investigated the combined effect of tissue plasminogen activator and ischemia on middle cerebral artery (MCA) reactivity to determine whether abnormal MCA function after 2 hours of ischemia was worse in arteries perfused with recombinant tissue plasminogen activator (rtPA). METHODS: The intraluminal suture model of focal cerebral ischemia was used to induce 2 hours of ischemia in rats, after which occluded MCAs were removed and studied in vitro with an arteriograph system that allowed control of transmural pressure (TMP) and measurement of lumen diameter. Arteries were either nonischemic (control; n=8), nonischemic and perfused with 400 microg/mL rtPA (rtPA; n=5), ischemic (ISC; n=6), or ischemic and perfused with 400 microg/mL rtPA (ISC-rtPA; n=6). After a 1-hour equilibration at 75 mm Hg, TMP was increased to 125 mm Hg and lumen diameter was recorded at each pressure. Reactivity to acetylcholine (ACh, 0.1 to 10.0 micromol/L) and serotonin (0.01 to 10 micromol/L) was then determined. RESULTS: Control arteries responded myogenically to pressure and increased the amount of tone from 18.5+/-3.8% at 75 mm Hg to 24.8+/-3.0% at 125 mm Hg (P<0.05), which decreased diameter from 241+/-7 to 232+/-6 microm. In contrast, all other groups decreased tone at 125 mm Hg, which demonstrated a loss of myogenicity. The percent tone in each group at 75 versus 125 mm Hg was rtPA, 16.0+/-4.5% versus 11.8+/-3.8%; ISC, 23.5+/-4.5% versus 13. 5+/-3.1%; and ISC-rtPA, 23.5+/-4.2% versus 12.3+/-3.2% (P<0.05 for all). The percent increase in lumen diameter at each concentration of ACh was diminished in all groups compared with control; ISC-rtPA arteries responded the least, which suggests an additive effect of rtPA in ischemic arteries. The percent increase in lumen diameter at 10(-5)mol/L ACh was 23+/-4% for control versus 15+/-2% for rtPA; 17+/-3% for ISC arteries (P<0.05), and 8+/-2% for ISC-rtPA arteries (P<0.01). Sensitivity to serotonin was equally diminished in all groups compared with control: EC(50) (micromol/L) was 0.06+/-0.01 for control, 0.17+/-0.02 for rtPA, 0.22+/-0.07 for ISC, and 0.16+/-0. 04 for ISC-rtPA (P<0.05). CONCLUSIONS: These results demonstrate that both ischemia and rtPA perfusion diminish cerebral artery reactivity and that the combination may produce an additive effect. This impaired reactivity may contribute to reperfusion-induced injury during or after thrombolysis by altering upstream cerebrovascular resistance.

Time course of IL-6 expression in experimental CNS ischemia.

Interleukin-6 (IL-6) appears to be an important modulator of the inflammatory response associated with CNS ischemia. Clinically, IL-6 values obtained in the first week post-stroke have been shown to correlate with infarct size and outcome. In this study we used a focal reversible stroke model to investigate the time course and relationship to outcome of IL-6 production in plasma, brain and CSF. Reversible middle cerebral artery occlusion or sham surgery was produced in 50 adult Swiss Webster mice by advancing an 8-0 filament into the internal carotid artery for 2 h (sham 1 min). At 3, 6, 12, 24, and 72 h (8 each ischemia; 2 each sham) groups of animals were evaluated on a 28 point clinical scale, blood and CSF obtained, and the brains were evaluated for infarct volume and IL-6 mRNA levels. Serum levels of IL-6 (ELISA mean +/- SD; undetectable in controls) overall sham group, 102 +/- 87; 3 h, 908 +/- 494* pg ml-1; 6 h, 1079 +/- 468* pg ml-1; 12 h, 980 +/- 221* pg ml-1; pg ml-1; 24 h, 320 +/- 314* pg ml-1; 72 h, 20 +/- 30* pg ml-1 (*p < or = 0.05 to sham). CSF levels (ELISA) overall sham group, 10 +/- 18; 3 h, 379 +/- 210* pg ml-1; 6 h, 157 +/- 61* pg ml-1; 12 h, 136 +/- 88* pg ml-1; 24 h, 127 +/- 99 pg ml-1; 72 h, 72 +/- 9* pg ml-1 (*p < or = 0.05 to sham). Brain IL-6 mRNA levels overall sham group, 20; 3 h, 480; 6 h, 599; 12 h, 7960; 24 h, 20267; 72 h, 0. There was an overall R2 of 0.20 between plasma and CSF IL-6. There was an overall R2 of 0.13 and 0.20 between infarct size and serum and CSF IL-6 level respectively, and an overall R2 of 0.10 and 0.17 between neurologic function and serum and CSF IL-6 level respectively. These findings confirm that IL-6 values increase following CNS ischemia with peak serum and CSF levels occurring before brain values. CSF IL-6 levels had a stronger correlation with neurologic function and infarct size than serum.

Temporal modulation of cytokine expression following focal cerebral ischemia in mice.

There is increasing evidence that the inflammatory response plays an important role in CNS ischemia. The murine model of focal ischemia, however, remains incompletely characterized. In this study we examined expression of several cytokines and the vascular adhesion molecule E-selectin, in order to characterize the molecular events following stroke in the C57BL/6J mouse. Using a multi-probe RNAse protection assay (RPA), mRNA for 19 cytokines was analyzed following permanent and transient occlusion of the middle cerebral artery in mice. In addition, samples from the same mice were analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) to evaluate E-selectin mRNA expression levels. Several cytokine mRNAs showed a similar expression pattern in both permanent and transient CNS ischemia while others showed a temporal expression pattern that was dependent on the type of stroke. For both models, mRNA levels of TNFalpha rose early (4 h) followed by IL-6 (10-18 h) and a comparatively late increase (96 h) in TGFbeta1. IL-1alpha, IL-1beta and IL-1ra levels showed a model dependent shift in temporal expression. Reperfusion appeared to delay the induction of these cytokines. Temporal changes in cytokine mRNA expression in the mouse CNS occur following ischemic damage. Our findings demonstrate the utility and power of multi-probe RPA for evaluation of changes in cytokine mRNA levels. Moreover, this study is, to our knowledge the first to show temporal changes in cytokine mRNA in mouse cerebral ischemia, forming a basis for further exploration of the roles of these cytokines in modulating ischemic neuronal damage in this model.

Retention of nociceptor responses during deep barbiturate anesthesia in frogs.

Bullfrogs (Rana catesbeiana) anesthetized with a large dose of thiopental (42.8 mg/kg) retained movement responses to nociceptor stimuli despite an average plasma drug level of 51 mg/l, of which 63% was bound to plasma proteins. This concentration, when corrected to include only unbound and uncharged drug, was 2-fold greater than those reported to abolish nociceptor response (NR) during surgical anesthesia in man. The median anesthetic dose (AD50) for loss of the righting reflex was 11.2 mg/kg by s.c. injection into the abdominal lymph sac; however, at 54.0 mg/kg, all frogs retained NRs, although otherwise deeply anesthetized. The ratio of NR-blocking dose to light AD was thus > 4.8, as compared to < 2 in mammalian studies. Whole body levels of thiopental determined at 3 h after intralymphatic injection showed that about half the injected drug had been eliminated by this time and that termination of anesthesia was chiefly due to drug elimination. Even though the pharmacokinetics of thiopental appears to differ markedly in frogs and men, the poor analgesia seen in the present study frequently has been reported during clinical barbiturate anesthesia. Since this deficiency is much more pronounced in the bullfrog than in man, its neurophysiological basis might profitably be studied using the bullfrog as a model; however, the high mortality associated with deep thiopental anesthesia in the frog should preclude its use as a practical anesthetic in amphibia.

Citicoline treatment for experimental intracerebral hemorrhage in mice.

BACKGROUND AND PURPOSE: Citicoline sodium (cytidine-5'-diphosphocholine) has been shown previously to reduce ischemic injury in focal central nervous system models. Intracerebral hemorrhage (ICH) appears to be associated with an area of edema and ischemic injury surrounding the hematoma that may be reduced by neuroprotective therapy. The present study was designed to test whether treatment with citicoline reduces ischemic injury and improves functional neurological outcome in an experimental model of ICH. METHODS: In 68 Swiss albino mice (26 to 36 g), ICH was induced by collagenase injection into the caudate nucleus. Animals were randomized to receive either: citicoline 500 mg/kg or saline IP prior to collagenase and at 24 and 48 hours. Animals were rated on a 28-point neurological scale and sacrificed at 54 hours. The brains were sectioned, and the volume of hematoma, total lesion, and surrounding ischemic injury was determined. RESULTS: In terms of functional outcome, animals treated with citicoline had improved neurological outcome scores compared with placebo-treated animals: 10.4+/-2.0 versus 12.1+/-2.4 (P<0.01). Regarding ischemic injury, although there was no difference in the underlying hematoma volumes, animals treated with citicoline had a smaller surrounding volume of ischemic injury than placebo-treated animals: citicoline, 13.8+/-5.8 mm3 (10.8+/-4.3% of hemisphere); placebo, 17.0+/-7.1 mm3 (13.3+/-5. 1%) (P<0.05). CONCLUSIONS: In this animal model of ICH, treatment with citicoline significantly improved functional outcome and reduced the volume of ischemic injury surrounding the hematoma. This study supports a potential role for citicoline in clinical ICH treatment.

Reperfusion decreases myogenic reactivity and alters middle cerebral artery function after focal cerebral ischemia in rats.

BACKGROUND AND PURPOSE: After focal cerebral ischemia, the function of cerebral arteries is critical to maintain cerebrovascular resistance and minimize damage to ischemic brain regions during reperfusion. In this study we examined the contractile function of isolated and pressurized middle cerebral arteries (MCAs) after 2 hours of occlusion with either 1 to 2 minutes or 24 hours of reperfusion using the intraluminal suture model of transient focal ischemia in rats. METHODS: MCAs were dissected after 2 hours of occlusion with either 1 to 2 minutes (OCC, n = 8) or 24 hours (RPF, n = 5) of reperfusion and compared with those of controls that did not have surgery (n = 5). Isolated MCAs were mounted on two glass cannulas in an arteriograph chamber that allowed control over transmural pressure (TMP) and measurement of lumen diameter. Responses to changes in TMP (including myogenic reactivity, basal tone, and passive distensibility) and sensitivity to serotonin and acetylcholine were compared. RESULTS: Increasing TMP from 25 to 75 mm Hg caused vasoconstriction and development of tone that was similar in control and OCC arteries: percent tone was 33 +/- 5% versus 25 +/- 7% (P > .05). In contrast, tone was severely diminished in RPF MCAs after 24 hours of reperfusion: percent tone = 8 +/- 4% (P < .01). Sensitivity to serotonin was reduced in OCC arteries, increasing the EC50 value from 0.04 +/- 0.1 to 0.11 +/- 0.02 mumol/L (P < .05); after 24 hours of reperfusion, sensitivity of RPF MCAs was similar to control. Vasodilation to 10.0 mumol/L acetylcholine was significantly impaired only in RPF arteries: percent increased lumen diameter was 19 +/- 3% (control) and 13 +/- 4% (OCC, P > .05) versus 9 +/- 2% (RPF, P < .01). Passively, OCC MCAs were more distensible, which was reversed after 24 hours of reperfusion; RPF vessels had distensibility similar to that of control arteries but thicker arterial walls. CONCLUSIONS: Abnormal structure and function of MCAs occur after 2 hours of ischemia, with diminished myogenic reactivity and tone associated with longer reperfusion.

Doxycycline treatment reduces ischemic brain damage in transient middle cerebral artery occlusion in the rat.

Agents that inhibit leukocyte adhesion including intercellular adhesion molecule-1 antibodies (anti-ICAM-1) have shown beneficial effects in experimental central nervous system (CNS) ischemia. Doxycycline inhibits leukocyte function in vitro by binding divalent cations and reduces spinal cord reperfusion injury. The authors used a clinically relevant model of focal CNS reperfusion injury to test whether treatment with doxycycline would reduce cerebral ischemic damage and improve functional outcome. Reversible middle cerebral artery occlusion was produced in adult Sprague-Dawley rats by advancing a filament into the internal carotid artery for 2 h. Animals received either i.p. doxycycline (10 mg/kg) (N = 13) or saline (N = 11) 30 min before ischemia, followed by 10 mg/kg every 8 h x 6. Both functional assessment (5 point neurologic scale) and infarct volume was evaluated at 48 h. Functional efficacy: doxycycline 0.5 +/- 0.2 (mean +/- SE) vs control 1.3 +/- 0.3 (p = 0.03). Infarct volume: doxycycline 56 +/- 18 mm3 vs control 158 +/- 44 mm3 (p = 0.03); This protective effect supports the role of doxycycline in reducing CNS reperfusion injury.

Monofilament intraluminal middle cerebral artery occlusion in the mouse.

The rat middle cerebral artery (MCA) occlusion model with an intraluminal filament is well characterized with a two hour period of occlusion in widespread use. The recent availability of transgenic animals has led to an interest in adapting the MCA model in the mouse. To date the model has not been well characterized in the mouse. We performed the present study to compare different durations of MCA occlusion and to validate new functional assessments in this model. The MCA occlusion model (5-0 filament) was used. Swiss-Webster mice, 24-44 g, were randomly assigned to four groups: one hour of occlusion; two hours of occlusion; three hours of occlusion; or permanent occlusion. At 48 hours post-ischemia, the animals were rated on three neurologic function scales, and then the brains were removed for lesion size determination. Overall, there was a significant difference in lesion volume (p < 0.001) between the groups. In the permanent group of mice, the average lesion volume was 78.41 +/- 17.47 mm (n = 12); two and three hours of ischemia produced 51.29 +/- 29.82 mm3 (n = 11) and 54.85 mm3 (n = 13), respectively, significantly different than the one hour group 14.84 +/- 31.34 mm3 (n = 11). All three functional scoring systems found significant overall differences between the four groups with our detailed General and Focal scores producing more robust between group treatment differences and showing correlation coefficients of r = 0.766 and r = 0.788, respectively to infarct volume. The MCA filament occlusion model can be successfully adapted in the mouse with either two or three hour occlusions producing reliable infarcts. New functional scoring systems unique to the mouse appear to add additional information.

Forebrain ischemia increases GLUT1 protein in brain microvessels and parenchyma.

Glucose transport into nonneuronal brain cells uses differently glycosylated forms of the glucose transport protein, GLUT1. Microvascular GLUT1 is readily seen on immunocytochemistry, although its parenchymal localization has been difficult. Following ischemia, GLUT1 mRNA increases, but whether GLUT1 protein also changes is uncertain. Therefore, we examined the immunocytochemical distribution of GLUT1 in normal rat brain and after transient global forebrain ischemia. A novel immunocytochemical finding was peptide-inhibitable GLUT1 immunoreactive staining in parenchyma as well as in cerebral microvessels. In nonischemic rats, parenchymal GLUT1 staining co-localizes with glial fibrillary acidic protein (GFAP) in perivascular foot processes of astrocytes. By 24 h after ischemia, both microvascular and nonmicrovascular GLUT1 immunoreactivity increased widely, persisting at 4 days postischemia. Vascularity within sections of brain similarly increased after ischemia. Increased parenchymal GLUT1 expression was paralleled by staining for GFAP, suggesting that nonvascular GLUT1 overexpression may occur in reactive astrocytes. A final observation was a rapid expression of inducible heat shock protein (HSP)70 in hippocampus and cortex by 24 h after ischemia. We conclude that GLUT1 is normally immunocytochemically detectable in cerebral microvessels and parenchyma and that parenchymal expression occurs in some astroglia. After global cerebral ischemia, GLUT1 overexpression occurs rapidly and widely in microvessels and parenchyma; its overexpression may be related to an immediate early-gene form of response to cellular stress.

Time course of ICAM-1 expression and leukocyte subset infiltration in rat forebrain ischemia.

The time course of ICAM-1 expression and leukocyte subset infiltration was studied in a model of CNS reperfusion injury in adult rats. Leukocyte adhesion and infiltration, mediated in part by intercellular adhesion molecule-1 (ICAM-1), appears to potentiate CNS reperfusion injury. The timing and relationship between ICAM-1 staining and leukocyte infiltration postglobal CNS ischemia is unknown. Reversible forebrain ischemia was produced in 32 adult Sprague-Dawley rats using the two-vessel occlusion model with histologic analysis performed at specific intervals postischemia: 1, 3, 6, 12, and 24 h, 4 and 7 d, or sham-operated controls (n = 4 each group). Monoclonal antibodies against ICAM-1 (1A29 and TM8), a specific granulocyte (PMN) (HIS48), and a specific monocyte/macrophage (M phi) (ED1) were used. No specific leukocyte and only rare ICAM-1 vessel immunoreactivity was observed in sham controls. ICAM-1: Significant expression in microvessels beginning at 1 h with additional diffuse CA1 pyramidal layer staining beginning at 4 d. Leukocytes: No PMN cells and rare M phi identified at 6 and 12 h. By 24 h: moderate infiltrate in areas of ICAM-1 expression of PMN and M phi. At 4 and 7 d: only M phi accumulation, cellular morphology now similar to microglia. The results of this study indicate that early and persistent ICAM-1 expression occurs following CNS ischemia with associated leukocyte infiltration.

The influence of antiadhesion therapies on leukocyte subset accumulation in central nervous system ischemia in rats.

Although treatment with agents that block leukocyte function, including anti-ICAM-1 and doxycycline, reduces experimental central nervous system (CNS) ischemic injury, it is not known how leukocyte subset accumulation is affected by these agents. Using the rat two-vessel occlusion model and immunohistochemistry, we investigated granulocyte (PMN) and monocyte/macrophage (M phi) accumulation at 1 and 4 d postischemia. A total of 24 animals were randomized to sham surgery, or to ischemia with saline, anti-ICAM-1, or doxycycline treatments. No leukocytes were observed in sham animals. At 24 h postischemia, there was a moderate infiltration of PMN and M phi in untreated animals that was significantly decreased with either treatment. At 4 d after ischemia no PMN were identified, with extensive M phi accumulation occurring in untreated animals that was only partially reduced with doxycycline treatment. These results confirm that both anti-ICAM-1 and doxycycline treatments reduce PMN and M phi infiltration at 24 h. Delayed M phi accumulation occurs despite treatment, suggesting that some of these cells represent transformed resident microglia.

Progressive hippocampal loss of immunoreactive GLUT3, the neuron-specific glucose transporter, after global forebrain ischemia in the rat.

Brain damage after global forebrain ischemia is worsened by prior hyperglycemia and ameliorated by antecedent hypoglycemia. To assess whether GLUT3, the neuron specific glucose transporter and its mRNA, are affected by cerebral ischemia, we investigated the hippocampal pattern of GLUT3 immunoreactivity and GLUT3 gene expression 1, 4 and 7 days after global forebrain ischemia in a rat 2-vessel occlusion model. We used a newly generated, specific, C-terminally directed polyclonal antiserum against GLUT3 to stain coronal frozen sections. Thionin staining and the microglial marker, OX42, indicated the extent of ischemic damage in hippocampus and correlated with GLUT3 loss. One day after ischemia, no significant change in hippocampal GLUT3 immunoreactivity was observed; by 4 days however, there was consistent and pronounced loss; and at 7 days the loss of GLUT3 staining was maximal. The greatest loss of GLUT3 staining was in the CA1 region, especially the strata oriens and radiatum of Ammon's horn. By contrast, GLUT3 staining was undiminished in the stratum lacunosum moleculare, in the mossy fibers of the lateral aspect of CA3 and in all but the inner-most portion of the molecular layer of the dentate gyrus, immediately adjacent to the granule cells. GLUT3 mRNA levels were not significantly altered at 24 hours and significantly declined at 4 and 7 days after ischemia in the CA1 pyramidal layer. These data are consistent with the pattern of neuronal loss and microglial activation in hippocampus. Loss of GLUT3 may affect the availability of glucose, and possibly the viability of ischemically damaged neurons.