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Background and Purpose— Systemic administration of Toll-like receptor (TLR) 4 and TLR9 agonists before cerebral ischemia have been shown to reduce ischemic injury by reprogramming the response of the brain to stroke. Our goal was to explore the mechanism of TLR-induced neuroprotection by determining whether a TLR7 agonist also protects against stroke injury. Methods— C57Bl/6, TNF −/− , interferon (IFN) regulatory factor 7 −/− , or type I IFN receptor (IFNAR) −/− mice were subcutaneously administered the TLR7 agonist Gardiquimod (GDQ) 72 hours before middle cerebral artery occlusion. Infarct volume and functional outcome were determined after reperfusion. Plasma cytokine responses and induction of mRNA for IFN-related genes in the brain were measured. IFNAR −/− mice also were treated with the TLR4 agonist (lipopolysaccharide) or the TLR9 agonist before middle cerebral artery occlusion and infarct volumes measured. Results— The results show that GDQ reduces infarct volume as well as functional deficits in mice. GDQ pretreatment provided robust neuroprotection in TNF −/− mice, indicating that TNF was not essential. GDQ induced a significant increase in plasma IFNα levels and both IRF7 −/− and IFNAR −/− mice failed to be protected, implicating a role for IFN signaling in TLR7-mediated protection. Conclusions— Our studies provide the first evidence that TLR7 preconditioning can mediate neuroprotection against ischemic injury. Moreover, we show that the mechanism of protection is unique from other TLR preconditioning ligands in that it is independent of TNF and dependent on IFNAR.

Primate models are essential tools for translational research in stroke but are reportedly inconsistent in their ability to produce cortical infarcts of reproducible size. Here, we report a new stroke model using a transorbital, reversible, two-vessel occlusion approach in male rhesus macaques that produces consistent and reproducible cortical infarcts. The right middle cerebral artery (distal to the orbitofrontal branch) and both anterior cerebral arteries were occluded with vascular clips. Bilateral occlusion of the anterior cerebral artery was critical for reducing collateral flow to the ipsilateral cortex. Reversible ischemia was induced for 45, 60, or 90 mins ( n = 2/timepoint) and infarct volume and neurologic outcome were evaluated. The infarcts were located predominantly in the cortex and increased in size with extended duration of ischemia determined by T2-weighted magnetic resonance imaging. Infarct volume measured by 2,3,5-triphenyl tetrazolium chloride and cresyl violet staining corroborated magnetic resonance imaging results. Neurologic deficit scores worsened gradually with longer occlusion times. A subset of animals ( n = 5) underwent 60 mins of ischemia resulting in consistent infarct volumes primarily located to the cortex that correlated well with neurologic deficit scores. This approach offers promise for evaluating therapeutic interventions in stroke.

Preconditioning with lipopolysaccharide (LPS), a toll-like receptor 4 (TLR4) ligand, provides neuroprotection against subsequent cerebral ischemic brain injury, through a tumor necrosis factor (TNF)α-dependent process. Here, we report the first evidence that another TLR, TLR9, can induce neuroprotection. We show that the TLR9 ligand CpG oligodeoxynucleotide (ODN) can serve as a potent preconditioning stimulus and provide protection against ischemic brain injury. Our studies show that systemic administration of CpG ODN 1826 in advance of brain ischemia (middle cerebral artery occlusion (MCAO)) reduces ischemic damage up to 60% in a dose- and time-dependent manner. We also offer evidence that CpG ODN preconditioning can provide direct protection to cells of the central nervous system, as we have found marked neuroprotection in modeled ischemia in vitro. Finally, we show that CpG preconditioning significantly increases serum TNFα levels before MCAO and that TNFα is required for subsequent reduction in damage, as mice lacking TNFα are not protected against ischemic injury by CpG preconditioning. Our studies show that preconditioning with a TLR9 ligand induces neuroprotection against ischemic injury through a mechanism that shares common elements with LPS preconditioning via TLR4.

Background and Purpose— Mild hypothermia confers profound neuroprotection in ischemia. We recently discovered 2 natural derivatives of thyroxine, 3-iodothyronamine (T 1 AM) and thyronamine (T 0 AM), that when administered to rodents lower body temperature for several hours without induction of a compensatory homeostatic response. We tested whether T 1 AM- and T 0 AM-induced hypothermia protects against brain injury from experimental stroke. Methods— We tested T 1 AM and T 0 AM 1 hour after and 2 days before stroke in a mouse model of focal ischemia. To determine whether T 1 AM and T 0 AM require hypothermia to protect against stroke injury, the induction of hypothermia was prevented. Results— T 1 AM and T 0 AM administration reduced body temperature from 37°C to 31°C. Mice given T 1 AM or T 0 AM after the ischemic period had significantly smaller infarcts compared with controls. Mice preconditioned with T 1 AM before ischemia displayed significantly smaller infarcts compared with controls. Pre- and postischemia treatments required the induction of hypothermia. T 1 AM and T 0 AM treatment in vitro failed to confer neuroprotection against ischemia. Conclusions— T 1 AM and T 0 AM, are potent neuroprotectants in acute stroke and T 1 AM can be used as antecedent treatment to induce neuroprotection against subsequent ischemia. Hypothermia induced by T 1 AM and T 0 AM may underlie neuroprotection. T 1 AM and T 0 AM offer promise as treatments for brain injury.

Summary Background Molecular mechanisms of neuroprotection that lead to ischaemic tolerance are incompletely understood. Identification of genes involved in the process would provide insight into cell survival and therapeutic approaches for stroke. We developed a mouse model of neuroprotection in stroke and did gene expression profiling to identify potential neuroprotective genes and their associated pathways. Methods Eight mice per condition were subjected to occlusion of the middle cerebral artery for 15 min (preconditioning), 60 min (injurious ischaemia), or preconditioning followed 72 h later by injurious ischaemia. RNA was extracted from the cortical regions of the ischaemic and non-ischaemic hemispheres. Three pools per condition were generated, and RNA was hybridised to oligonucleotide microarrays for comparison of ischaemic and non-ischaemic hemispheres. Real-time PCR and western blots were used to validate results. Follow-up experiments were done to address the biological relevance of findings. Findings Microarray analysis revealed changes in gene expression with little overlap among the conditions of injurious ischaemia, ischaemic preconditioning, or both. Injurious ischaemia induced upregulation of gene expression; 49 (86%) of 57 genes regulated showed increased expression in the ischaemic hemisphere. By contrast, preconditioning followed by injurious ischaemia resulted in pronounced downregulation; 47 (77%) of 61 regulated genes showed lower expression. Preconditioning resulted in transcriptional changes involved in suppression of metabolic pathways and immune responses, reduction of ion-channel activity, and decreased blood coagulation. Interpretation Preconditioning reprogrammes the response to ischaemic injury. Similar changes reported by others support an evolutionarily conserved endogenous response to decreased blood flow and oxygen limitation such as seen during hibernation.

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 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 (×10 7 ) 3.21±0.25, 2.54±0.32 ( P >0.05), 2.32±0.15 ( P P 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.

Background and Purpose —Ginkgo biloba extract (EGb) and α-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 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.

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 3 (10.8±4.3% of hemisphere); placebo, 17.0±7.1 mm 3 (13.3±5.1%) ( P 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.

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.

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.

Background Toll-like receptor 4 (TLR4) is activated in response to cerebral ischemia leading to substantial brain damage. In contrast, mild activation of TLR4 by preconditioning with low dose exposure to lipopolysaccharide (LPS) prior to cerebral ischemia dramatically improves outcome by reprogramming the signaling response to injury. This suggests that TLR4 signaling can be altered to induce an endogenously neuroprotective phenotype. However, the TLR4 signaling events involved in this neuroprotective response are poorly understood. Here we define several molecular mediators of the primary signaling cascades induced by LPS preconditioning that give rise to the reprogrammed response to cerebral ischemia and confer the neuroprotective phenotype. Methods C57BL6 mice were preconditioned with low dose LPS prior to transient middle cerebral artery occlusion (MCAO). Cortical tissue and blood were collected following MCAO. Microarray and qtPCR were performed to analyze gene expression associated with TLR4 signaling. EMSA and DNA binding ELISA were used to evaluate NFκB and IRF3 activity. Protein expression was determined using Western blot or ELISA. MyD88-/- and TRIF-/- mice were utilized to evaluate signaling in LPS preconditioning-induced neuroprotection. Results Gene expression analyses revealed that LPS preconditioning resulted in a marked upregulation of anti-inflammatory/type I IFN-associated genes following ischemia while pro-inflammatory genes induced following ischemia were present but not differentially modulated by LPS. Interestingly, although expression of pro-inflammatory genes was observed, there was decreased activity of NFκB p65 and increased presence of NFκB inhibitors, including Ship1, Tollip, and p105, in LPS-preconditioned mice following stroke. In contrast, IRF3 activity was enhanced in LPS-preconditioned mice following stroke. TRIF and MyD88 deficient mice revealed that neuroprotection induced by LPS depends on TLR4 signaling via TRIF, which activates IRF3, but does not depend on MyD88 signaling. Conclusion Our results characterize several critical mediators of the TLR4 signaling events associated with neuroprotection. LPS preconditioning redirects TLR4 signaling in response to stroke through suppression of NFκB activity, enhanced IRF3 activity, and increased anti-inflammatory/type I IFN gene expression. Interestingly, this protective phenotype does not require the suppression of pro-inflammatory mediators. Furthermore, our results highlight a critical role for TRIF-IRF3 signaling as the governing mechanism in the neuroprotective response to stroke.

Osteopontin (OPN), a large secreted glycoprotein with an arginine, glycine, aspartate (RGD) motif, can bind and signal through cellular integrin receptors. We have shown previously that OPN enhances neuronal survival in the setting of ischemia. Here, we sought to increase the neuroprotective potency of OPN and improve the method of delivery with the goal of identifying a treatment for stroke in humans. We show that thrombin cleavage of OPN improves its ability to ligate integrin receptors and its neuroprotective capacity in models of ischemia. Thrombin-cleaved OPN is a twofold more effective neuroprotectant than the untreated molecule. We also tested whether OPN could be administered intranasally and found that it is efficiently targeted to the brain via intranasal delivery. Furthermore, intranasal administration of thrombin-treated OPN confers protection against ischemic brain injury. Osteopontin mimetics based on the peptide sequences located either N or C terminal to the thrombin cleavage site were generated and tested in models of ischemia. Treatment with successively shorter N-terminal peptides and a phosphorylated C-terminal peptide provided significant neuroprotection against ischemic injury. These findings show that OPN mimetics offer promise for development into new drugs for the treatment of stroke.

Lipopolysaccharide (LPS) preconditioning provides neuroprotection against subsequent cerebral ischemic injury. Tumor necrosis factor-alpha (TNFalpha) is protective in LPS-induced preconditioning yet exacerbates neuronal injury in ischemia. Here, we define dual roles of TNFalpha in LPS-induced ischemic tolerance in a murine model of stroke and in primary neuronal cultures in vitro, and show that the cytotoxic effects of TNFalpha are attenuated by LPS preconditioning. We show that LPS preconditioning significantly increases circulating levels of TNFalpha before middle cerebral artery occlusion in mice and show that TNFalpha is required to establish subsequent neuroprotection against ischemia, as mice lacking TNFalpha are not protected from ischemic injury by LPS preconditioning. After stroke, LPS preconditioned mice have a significant reduction in the levels of TNFalpha (approximately threefold) and the proximal TNFalpha signaling molecules, neuronal TNF-receptor 1 (TNFR1), and TNFR-associated death domain (TRADD). Soluble TNFR1 (s-TNFR1) levels were significantly increased after stroke in LPS-preconditioned mice (approximately 2.5-fold), which may neutralize the effect of TNFalpha and reduce TNFalpha-mediated injury in ischemia. Importantly, LPS-preconditioned mice show marked resistance to brain injury caused by intracerebral administration of exogenous TNFalpha after stroke. We establish an in vitro model of LPS preconditioning in primary cortical neuronal cultures and show that LPS preconditioning causes significant protection against injurious TNFalpha in the setting of ischemia. Our studies suggest that TNFalpha is a twin-edged sword in the setting of stroke: TNFalpha upregulation is needed to establish LPS-induced tolerance before ischemia, whereas suppression of TNFalpha signaling during ischemia confers neuroprotection after LPS preconditioning.

Osteopontin (OPN) is a secreted extracellular phosphoprotein involved in diverse biologic functions, including inflammation, cell migration, and antiapoptotic processes. Here we investigate the neuroprotective potential of OPN to reduce cell death using both in vitro and in vivo models of ischemia. We show that incubation of cortical neuron cultures with OPN protects against cell death from oxygen and glucose deprivation. The effect of OPN depends on the Arg–Gly–Asp (RGD)-containing motif as the protective effect of OPN in vitro was blocked by an RGD-containing hexapeptide, which prevents integrin receptors binding to their ligands. Osteopontin treatment of cortical neuron cultures caused an increase in Akt and p42/p44 MAPK phosphorylation, which is consistent with OPN-inducing neuroprotection via the activation of these protein kinases. Indeed, the protective effect of OPN was reduced by inhibiting the activation of Akt and p42/p44 MAPK using LY294002 and U0126, respectively. The protective effect of OPN was also blocked by the protein synthesis inhibitor cycloheximide, suggesting that the neuroprotective effect of OPN required new protein synthesis. Finally, intracerebral ventricular administration of OPN caused a marked reduction in infarct size after transient middle cerebral artery occlusion in a murine stroke model. These data suggest that OPN is a potent neuroprotectant against ischemic injury.

Background and Purpose—Tolerance to ischemic brain injury is induced by several preconditioning stimuli, including lipopolysaccharide (LPS). A small dose of LPS given systemically confers ischemic protection in the brain, a process that appears to involve activation of an inflammatory response before ischemia. We postulated that LPS preconditioning modulates the cellular inflammatory response after cerebral ischemia, resulting in neuroprotection.Methods—Mice were treated with LPS (0.2 mg/kg) 48 hours before ischemia induced by transient middle cerebral artery occlusion (MCAO). The infarct was measured by 2,3,5-triphenyltetrazolium chloride staining. Microglia/macrophage responses after MCAO were assessed by immunofluorescence and flow cytometry. The effect of MCAO on white blood cells in the brain and peripheral circulation was measured by flow cytometry 48 hours after MCAO.Results—LPS preconditioning induced significant neuroprotection against MCAO. Administration of low-dose LPS before MCAO prevented the cellular inflammatory response in the brain and blood. Specifically, LPS preconditioning suppressed neutrophil infiltration into the brain and microglia/macrophage activation in the ischemic hemisphere, which was paralleled by suppressed monocyte activation in the peripheral blood.Conclusions—LPS preconditioning induces neuroprotection against ischemic brain injury in a mouse model of stroke. LPS preconditioning suppresses the cellular inflammatory response to ischemia in the brain and circulation. Diminished activation of cellular inflammatory responses that ordinarily exacerbate ischemic injury may contribute to neuroprotection induced by LPS preconditioning.

Corticotropin releasing hormone (CRH) and its family of related peptides are involved in regulating physiologic responses to multiple stressors, including stroke. Although CRH has been implicated in the exacerbation of injury after stroke, the mechanism remains unclear. After ischemia, both excitotoxic damage and inflammation contribute to the pathology of stroke. CRH is known to potentiate excitotoxic damage in the brain and has been shown to modulate inflammatory responses in the periphery. Here the present authors examine the relative contribution of the two known CRH receptors, CRH-R1 and CRH-R2, to ischemic injury using CRH receptor knockout mice. These results implicate CRH-R1 as the primary mediator of ischemic injury in this mouse model of stroke. In addition, the authors examine a potential role for CRH in inflammatory injury after stroke by identifying functional CRH receptors on astrocytes and microglia, which are cells that are known to be involved in brain inflammation. By single cell PCR, the authors show that microglia and astrocytes express mRNA for both CRH-R1 and CRH-R2. However, CRH-R1 is the primary mediator of cAMP accumulation in response to CRH peptides in these cells. The authors suggest that astrocytes and microglia are cellular targets of CRH, which could serve as a link between CRH and inflammatory responses in ischemic injury via CRH-R1.

Recent studies indicate that inflammation following cerebral ischemia contributes to neuronal damage. The local activation of resident cells and efficient recruitment of leukocytes into the central nervous system are critical steps in this inflammatory process. Here we describe studies using flow cytometry to examine the temporal pattern of inflammatory cell activation and infiltration following transient middle cerebral artery occlusion (MCAO) in mice. We found an increase in activated microglia/macrophages as early as 18 h post occlusion, which peaked at 48 h and remained abundant at 96 h post occlusion. Neutrophils were significantly increased by 48 h and remained elevated at 96 h post occlusion. T lymphocytes were increased relatively late (72 and 96 h) post occlusion. The flow cytometry data correlate well both quantitatively and qualitatively with immunohistochemistry analysis performed on the same mice. The present study demonstrates the power of flow cytometry in analyzing the inflammatory process following cerebral ischemia and offers temporal information on the cellular changes in mice following transient MCAO.

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-α, IL-1β, 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.

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.

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.

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.

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.

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 50 value from 0.04±0.1 to 0.11±0.02 μmol/L ( P P >.05) versus 9±2% (RPF, P Conclusions Abnormal structure and function of MCAs occur after 2 hours of ischemia, with diminished myogenic reactivity and tone associated with longer reperfusion.

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.