An immortalized rat ventral mesencephalic cell line, RTC4, is protective in a rodent model of stroke.

One therapeutic approach to stroke is the transplantation of cells capable of trophic support, reinnervation, and/or regeneration. Previously, we have described the use of novel truncated isoforms of SV40 large T antigen to generate unique cell lines from several primary rodent tissue types. Here we describe the generation of two cell lines, RTC3 and RTC4, derived from primary mesencephalic tissue using a fragment of mutant T antigen, T155c (cDNA) expressed from the RSV promoter. Both lines expressed the glial markers vimentin and S100beta, but not the neuronal markers NeuN, MAP2, or beta-III-tubulin. A screen for secreted trophic factors revealed substantially elevated levels of platelet-derived growth factor (PDGF) in RTC4, but not RTC3 cells. When transplanted into rat cortex, RTC4 cells survived for at least 22 days and expressed PDGF. Because PDGF has been reported to reduce ischemic injury, we examined the protective functions of RTC4 cells in an animal model of stroke. RTC4 or RTC3 cells, or vehicle, were injected into rat cortex 15-20 min prior to a 60-min middle cerebral artery ligation. Forty-eight hours later, animals were sacrificed and the stroke volume was assessed by triphenyl-tetrazolium chloride (TTC) staining. Compared to vehicle or RTC3 cells, transplanted RTC4 cells significantly reduced stroke volume. Overall, we generated a cell line with glial properties that produces PDGF and reduces ischemic injury in a rat model of stroke.

Increased reactivity of platelets induced by fibrinogen independent of its binding to the IIb-IIIa surface glycoprotein: a potential contributor to cardiovascular risk.

OBJECTIVES: To determine whether augmented activation (degranulation) of platelets might contribute to the association between higher concentrations of fibrinogen and risk of myocardial infarction, we characterized adenosine diphosphate (ADP)-induced expression of P-selectin by platelets in whole blood as a function of this exposure to selected concentrations of fibrinogen. BACKGROUND: An increased risk of myocardial infarction has been associated with increased concentrations of fibrinogen. METHODS: Fibrinogen was added to blood anticoagulated with corn trypsin inhibitor (a specific inhibitor of Factor XIIa without effect on other coagulation factors). Degranulation of platelets was identified by flow cytometry. RESULTS: Addition of fibrinogen to blood did not activate platelets under basal conditions (without ADP). By contrast, a concentration-dependent increase in ADP and thrombin receptor agonist peptide (TRAP)-induced activation occurred with increasing concentrations of fibrinogen. Increased ADP-induced degranulation was apparent with the addition of 100 mg/dl of fibrinogen (p < or = 0.001 for 1.5 micromol/liter ADP, n=10 subjects). Inhibition by abciximab of binding of fibrinogen to the surface glycoprotein IIb-IIIa did not attenuate the observed augmentation of reactivity induced by fibrinogen. Augmented degranulation was associated with uptake of fibrinogen into alpha-granules without surface binding despite pretreatment with abciximab as shown by laser scanning confocal microscopy. CONCLUSIONS: Fibrinogen in blood augments degranulation of platelets in response to ADP and is accompanied by uptake of fibrinogen into alpha-granules. Thus, elevated concentrations of fibrinogen secondary to inflammation implicated in cardiovascular risk may operate, in part, by increasing reactivity of platelets.

Examination of data normalization procedures for expressing peak VO2 data.

Peak O2 uptake (VO2) has traditionally been compared among individuals differing in body composition by dividing measured values (1/min) by fat-free mass (FFM) (i.e., ratio method). However, the ability of the ratio method to mathematically remove the confounding influence of FFM from peak VO2 has recently been questioned. Therefore, we compared the effectiveness of the ratio method vs. regression modeling to normalize peak VO2 in a large cohort of males and females for differences in FFM. Regression modeling adjusts peak VO2 according to the relationship derived from the regression of peak VO2 on FFM. Results showed that peak VO2 was 60% higher in males (3.53 +/- 1.01/min) than in females (2.22 +/- 0.6 l/min; P < 0.01). With the ratio method (i.e., peak VO2/FFM), peak VO2 was 15% higher in males (54.6 +/- 12 ml.kg FFM-1.min-1) than in females (47.4 +/- 11 ml.kg FFM-1.min-1; P < 0.01). In contrast, when a regression-based approach was employed to normalize values, no significant difference in adjusted peak VO2 was observed between males and females (3.04 +/- 0.9 vs. 3.01 +/- 1.0 l/min). In conclusion, dividing peak VO2 by FFM can produce spurious results, because this approach does not take into account the nonzero intercept. Therefore, a regression-based approach should be used to normalize peak VO2.

Differences between activation thresholds for platelet P-selectin glycoprotein IIb-IIIa expression and their clinical implications.

Increased platelet reactivity is a descriptor of the risk of cardiovascular events in healthy men and in patients with overt coronary artery disease. We sought to determine if differential thresholds exist for activation of platelets with respect to alpha-granule degranulation and fibrinogen binding in healthy volunteers and in patients with acute coronary syndromes. We also sought to characterize the effect of aspirin on activation. Platelet activation was assessed with flow cytometry in whole blood anticoagulated with corn trypsin inhibitor and incubated with fluorescein isothiocyanate conjugated fibrinogen (to define activation of glycoprotein IIb-IIIa), a phycoerythrin conjugated antibody to P-selectin (a marker of alpha-granule degranulation), and selected concentrations of adenosine diphosphate (ADP) or thrombin receptor agonist peptide. ADP-induced fibrinogen binding was found to be a low threshold activation event (40% of platelets bound fibrinogen in response to 0.2 microM ADP). Alpha-granule degranulation was a higher threshold event (33% of platelets expressed P-selectin in response to 1.0 microM ADP). Intra- and interindividual variability were most apparent with low concentrations of agonist (0.2 microM ADP). Patients with acute coronary syndromes (on aspirin) had significantly increased P-selectin expression in response to ADP compared with healthy subjects (on aspirin), but no difference in ADP-induced fibrinogen binding was observed. Daily ingestion of 325 mg of aspirin had no effect on either P-selectin expression or fibrinogen binding in healthy subjects. Analysis of platelet reactivity with flow cytometry characterizes activation with respect to specific components of the process and should facilitate development and optimal titration of antiplatelet therapy.

Delayed tissue-plasminogen activator therapy in a rabbit model of thromboembolic stroke.

This study investigated the efficacy and safety of delayed therapy with tissue-plasminogen activator (t-PA) in a rabbit model of thromboembolic stroke. The t-PA therapy was started 3, 4, or 5 hours after autologous clot embolization. New Zealand rabbits were randomized to receive a 2-hour intravenous infusion of either t-PA (6.3 mg/kg) or a saline solution (0.9% saline) after an autologous clot had embolized the anterior cerebral circulation. Regional cerebral blood flow (rCBF), intracranial pressure (ICP), and infarct size were measured to determine the effects of the delayed administration of the t-PA after intracranial embolization. Additionally, the following physiological parameters were monitored throughout the protocol: mean arterial pressure, hematocrit, arterial blood gases, glucose, and core and brain temperatures. All animals were studied for 4 hours after the administration of the t-PA or control solution; thus, the duration of each experiment was 7, 8, or 9 hours after autologous clot embolization. In control animals, brain infarct size and final ICP values were directly related to the length of time studied after clot embolization; among control animals, the largest infarct size and greatest rise in ICP were seen 9 hours after embolization.(ABSTRACT TRUNCATED AT 250 WORDS)

16(R)-hydroxyeicosatetraenoic acid, a novel cytochrome P450 product of arachidonic acid, suppresses activation of human polymorphonuclear leukocyte and reduces intracranial pressure in a rabbit model of thromboembolic stroke.

OBJECTIVE: Activated polymorphonuclear leukocytes (PMNs) have been suggested to contribute to the development of increased intracranial pressure (ICP). We recently demonstrated that human PMNs produce a novel cytochrome P450-derived arachidonic acid metabolite, 1 6(R)-hydroxyeicosatetraenoic acid [16(R)-HETE], that modulates their function. It was thus of interest to examine this novel mediator in an acute stroke model. METHODS: 16-HETE was assessed initially in a variety of human PMN and platelet in vitro assays and subsequently in an established rabbit model of thromboembolic stroke. A total of 50 rabbits completed a randomized, blinded, four-arm study, receiving 16(R)-HETE, tissue plasminogen activator, both, or neither. Experiments were completed 7 hours after autologous clot embolization. The primary end point for efficacy was the suppression of increased ICP. RESULTS: In in vitro assays, 16(R)-HETE selectively inhibited human PMN adhesion and aggregation and leukotriene B4 synthesis. In the thromboembolic stroke model, animals that received 16(R)-HETE demonstrated significant suppression of increased ICP (7.7 +/- 1.2 to 13.1 +/- 2.7 mm Hg, baseline versus final 7-h time point, mean +/- standard error), compared with either the vehicle-treated group (7.7 +/- 0.9 to 15.8 +/- 2.6 mm Hg) or the tissue plasminogen activator-treated group (7.6 +/- 0.6 to 13.7 +/- 2.1 mm Hg). The group that received the combination of 16(R)-HETE plus tissue plasminogen activator demonstrated no significant change in ICP for the duration of the protocol (8.6 +/- 0.6 to 11.1 +/- 1.2 mm Hg). CONCLUSION: 16(R)-HETE suppresses the development of increased ICP in a rabbit model of thromboembolic stroke and may serve as a novel therapeutic strategy in ischemic and inflammatory pathophysiological states.

Neutrophil activation in acute human central nervous system injury.

The hypothesis that neutrophil activation exacerbates brain injury in acute stroke is currently receiving wide acceptance. However, the temporal relationship of neutrophil activation to the ischemic event in clinical states is not clear. Therefore, this study was undertaken to examine human neutrophil activation by the technique of luminol-dependent chemiluminescence in both acute bland and hemorrhagic stroke. Patients (bland, n = 18; hemorrhagic, n = 16) were entered into this study within six hours of the ictus. These results were compared to other clinical central nervous system insults: subarachnoid hemorrhage (n = 11), spinal trauma (n = 9) and isolated closed head injury (n = 19). All subjects were sampled upon presentation to the emergency room and 0.5, 1, 2, 3, 4 and 5 days following the event. Neutrophil activation, as determined by luminol-dependent chemiluminescence, was evident at day 1 following the ictus in the bland stroke group (p < 0.05), although this trend was not demonstrated for hemorrhagic stroke. Patients suffering a closed head injury demonstrated greater initial neutrophil activation with values being significantly lower than baseline at days 0.5 (p = 0.01) and 1 (p = 0.05). No significant change was demonstrated for the groups with spinal trauma or subarachnoid hemorrhage. These results support a role for neutrophil activation during various central nervous system insults and provide a temporal framework for considering drug therapy directed at transient suppression of neutrophil function.

The effect of vasodilators on aspirin-induced antagonism of t-PA thrombolysis.

Although i.v. t-PA has proven successful in reducing neurologic deficits in acute ischemic stroke, the disadvantages of a narrow therapeutic time window and the failure of thrombolysis in more than 50% of patients treated have necessitated an examination of adjuvant therapies to improve the rate of thrombolysis. Experimentally, the combination of aspirin therapy with t-PA has resulted in a paradoxical antagonism of thrombolysis. Reversal of this antagonism with nitric oxide (NO) donors suggested that aspirin may inhibit/ antagonize NO-related mechanisms. Using this rabbit model of thromboembolic stroke, this hypothesis is now expanded to compare two clinically relevant anti-hypertensive agents, atenolol (NO-dependent) and hydralazine (NO-independent), for their ability to improve t-PA-mediated clot lysis following aspirin pre-treatment. Thirty rabbits (10 per group) were pre-treated with aspirin (20mg kg(-1), i.v.) and then randomized to receive either vehicle, atenolol (20 microg kg(-1) h(-1), i.v.) or hydralazine (10 microg kg(-1) min(-1), i.v.) beginning 30 min following autologous clot embolization. All rabbits then received t-PA (6.3 mg kg(-1), i.v.) beginning 1 h after embolization, with completion of the protocol 4 h after embolization. Aspirin therapy reduced regional cerebral blood flow (rCBF) from 82.8m +/- 4.7 to 62.5 +/- 6.6 (n = 30; p = 0.0005). In the aspirin control group only 30% (3 of 10) rabbits demonstrated complete clot lysis, whereas the combined atenolol (60%) and hydralazine (70%) groups experienced a clot lysis rate of 65% (13 of 20 rabbits), similar to clot lysis rates previously observed with t-PA alone. In a separate series of experiments, all agents able to reverse aspirin antagonism of thrombolysis demonstrated an improvement in rCBF, suggesting a common mechanism for this diverse group of agents in reversing aspirin's antagonism of thrombolysis.

TGF-beta 1 post-treatment in a rabbit model of cerebral ischaemia.

Transforming growth factor-beta 1 (TGF-beta 1), suggested in some studies to suppress astrocyte and neutrophil function, has also reduced ischaemic brain injury when administered immediately prior to clot embolization in models of thromboembolic stroke. The effect of TGF-beta 1 as a post-treatment paradigm was investigated in a rabbit model of thromboembolic stroke. Following clot embolization, regional cerebral blood flow fell to < 10 cc 100 g-1 min-1 in all animals. TGF-beta 1 (10 micrograms) or vehicle (n = 5 each group) was infused via the contralateral carotid artery. TGF-beta 1 administration resulted in a rapid and selective reduction in the peripheral neutrophil count as compared to a significant (p < 0.05) increase in control values (2336 +/- 817 vs 4320 +/- 928 neutrophils mm3, mean +/- SEM). Neutrophil aggregation was increased within 30 min of TGF-beta 1 infusion when compared to control (2.07 +/- 0.70 vs 1.09 +/- 0.17 ohms, p < 0.05); neutrophil chemiluminescence, an index of the oxygen respiratory burst was not significantly affected by TGF-beta 1 administration. No difference in platelet counts or aggregation was noted. There was no significant difference between the two groups regarding brain infarct size (47.5 +/- 10.9 vs 56.5 +/- 10.4, n = 4, TGF-beta vs control, mean +/- SEM), intracranial pressure, or brain excitatory amino acid levels (aspartate and glutamate) within ischaemic regions.(ABSTRACT TRUNCATED AT 250 WORDS)

The influence of panic attacks on response to phenelzine and amitriptyline in depressed outpatients.

A total of 169 depressed outpatients completed a 6-week double-blind study designed to compare the relative efficacy of a tricyclic antidepressant (amitriptyline) with a monoamine oxidase inhibitor (phenelzine). Various "target" symptoms reported to predict preferential response to monoamine oxidase inhibitors were assessed. The major finding within the whole patient sample, based on results from serial self-report and interviewer-rated scales, was that phenelzine-treated patients showed greater improvements in anxiety symptoms than did patients treated with amitriptyline. Because of the heterogeneity of the sample, patients were classified into homogeneous subgroups of clinical interest. Data analyses of these subgroups detected important drug treatment differences not discernible by analysis of data from the overall sample. Panic attacks and corresponding anxiety symptoms were reported by about one third of the patients, more often by patients with major depression than with minor depression. Patients who reported "spells of terror or panic" responded preferentially to phenelzine on several measures, particularly on items measuring anxiety. Results suggest that phenelzine may be a preferred drug for treating depressed patients with panic attacks.

Transforming growth factor-beta 1 reduces infarct size after experimental cerebral ischemia in a rabbit model.

BACKGROUND AND PURPOSE: The aim of this study was to examine the effect of transforming growth factor-beta 1, a cytokine shown to amelioriate cardiac ischemia, in a rabbit model of thromboembolic stroke. METHODS: An autologous clot embolus was introduced intracranially through the right internal carotid artery in 21 New Zealand White rabbits, with seven in each group receiving either vehicle control (albumin) or 10 or 50 micrograms transforming growth factor-beta 1 administered as an intracarotid bolus immediately before autologous clot embolization. Multiple physiological parameters were monitored, including regional cerebral blood flow, arterial blood gases, hematocrit, glucose, core temperature, and mean arterial pressure. The brain was harvested 4 hours after embolization, and infarct size was determined planimetrically as a percentage of the entire hemisphere. RESULTS: Brain infarct size was reduced in both the 10-microgram (16.7 +/- 4.0% [mean +/- SEM], p < 0.05) and 50-microgram (21.7 +/- 4.5%) transforming growth factor-beta 1-treated groups when compared with the control group (31.9 +/- 6.6%). Regional cerebral blood flow did not show any significant intergroup or intragroup variation over time, although the 10-microgram transforming growth factor-beta 1 group experienced a greater return of cerebral blood flow in the first 2 hours after embolization. CONCLUSIONS: Transforming growth factor-beta 1 reduced brain infarct size in a rabbit model of thromboembolic stroke. This effect was not related to a direct effect on blood flow. Studies are ongoing to determine the mechanism by which transforming growth factor-beta 1 salvages ischemic brain.

Survey of Bulk Tank Milk using Blood-Esculin Agar Counts 1.

Milk from bulk tanks of 2,931 dairy herds were sampled and evaluated using trypticase blood-esculin agar, somatic cell, standard plate and preliminary incubation counts. Percent samples with trypticase blood-esculin agar counts >1 × 102 colony forming units/ml by organisms were Staphylococcus aureus , 33; Staphylococcus spp., 84; Streptococcus agalactiae , 47; esculin-positive streptococci, 72; coliforms, 73; and other microbes, 89. Trypticase blood-esculin agar counts were useful for identifying primary bacterial contaminants. Correlations were low between trypticase blood-esculin agar counts of specific bacterial groups and somatic cell, standard plate and preliminary incubation counts.

Increased usage of V beta 2 and V beta 6 in rheumatoid synovial fluid T cells.

OBJECTIVE: To determine if the T cell antigen receptor V beta usage of unstimulated rheumatoid arthritis (RA) synovial fluid (SF) T cells is biased compared with those in peripheral blood (PB). METHODS: Freshly isolated, matched synovial fluid and peripheral blood T cells were analyzed for V beta gene expression using quantitative polymerase chain reaction (PCR) methods. Ten synovial fluid samples from the knees of 7 patients with RA were studied. The PCR assay used 26 V beta primers with a constant region C beta primer, and 2 C alpha primers that co-amplified a product that served as an internal standard. Cycle number and complementary DNA content were controlled to ensure the linear accumulation of PCR products. Labeled products were separated on 10% polyacrylamide gels and counted with a Betascope blot analyzer. RESULTS: There were consistent differences between the V beta gene usage of SF and PB T cells directly isolated from patients with RA, regardless of HLA-DR haplotype. In all synovial specimens, V beta 2 was increased relative to the peripheral blood, while V beta 13.1 and V beta 13.2 were decreased. V beta 6 and V beta 21 were increased in 9 of the 10 synovial samples. Analyses of bilateral SF specimens from 2 subjects and serial specimens from the same knee of 1 subject revealed virtually identical patterns in each patient. The SF V beta bias was not solely due to differences in the proportion of CD4+ and CD8+ cells, because the CD4:CD8 ratios in SF and PB were similar. However, V beta gene usage of separated CD4+ and CD8+ synovial T cells showed that V beta 2 and V beta 6 were more highly expressed on CD4 cells. CONCLUSION: Freshly isolated synovial T cells from inflamed (not end-stage) knees of patients with RA have a remarkably consistent biased V beta gene usage compared with PB T cells. V beta 2 and V beta 6 are uniformly increased, and this increase is primarily in CD4+ T cells. The same V beta bias in the SF T cells of several RA patients suggests that shared antigens may be stimulating the T cell response.

The influence of intensively managed rotational grazing, traditional continuous grazing, and confinement housing on bulk tank milk quality and udder health.

Monthly bulk tank milk samples and veterinary records were analyzed for 1 yr on 15 Vermont dairy farms. Data were evaluated using ANOVA to compare effects of grazing management systems on milk quality and udder health. Systems evaluated were intensively managed rotational grazing, traditional continuous grazing, and confinement housing. Bulk tank samples were evaluated for standard plate count, bacterial type counts on tryptose-blood-esculin agar, and SCC. Veterinary records were evaluated for incidence of clinical mastitis, udder edema, and teat injuries. Within- and between-treatment group analyses were conducted by season, herd size, and udder sanitation systems. Mean standard plate counts were lower in rotationally grazed herds than counts of confined herds during the grazing season. Similarly, rotationally grazed herds with fewer than 60 cows had lower standard plate counts than confined herds of similar size. Mean bulk tank counts of streptococci other than Streptococcus agalactiae during the grazing season differed among treatments. The lowest counts occurred in rotationally grazed herds. Among herd using predip products recognized as efficacious, fewer streptococci other than S. agalactiae were isolated from bulk tank milk of rotationally grazed herds than confined herds. Rotationally grazed herds using postdips recognized as efficacious had lower SCC than those using unrecognized postdips. No udder health differences were observed among grazing treatments.

Nitric oxide reverses aspirin antagonism of t-PA thrombolysis in a rabbit model of thromboembolic stroke.

Randomized trials of thrombolytic therapy in stroke have reported an improvement in neurologic outcome; however, the addition of aspirin has resulted in a significant increase in mortality and antagonism of clot lysis in clinical and animal studies, respectively. This finding is in contradistinction to the known synergy in mortality reduction for aspirin and thrombolytics in myocardial infarction. It is hypothesized that aspirin antagonism of clot lysis is related to inhibition of nitric oxide (NO) and may be reversed by providing a source of NO. Twenty rabbits were treated with aspirin (20 mg/kg, i.v.) prior to internal carotid clot embolization. One-half hour following embolization, rabbits were randomized to receive vehicle (n = 5), the NO precursor L-arginine (300 mg/kg, i.v. bolus at 0.5 and 2.5 h postembolus; n = 5), or a nitric oxide donor (nitroprusside, 1 mg/kg/h, i.a., or nitroglycerin, 10 microg/kg/min, i.v., n = 5 each agent). Tissue plasminogen activator (t-PA) (6.3 mg/kg) was administered from 1 to 3 h after embolization. Lysis of the tin-tagged clot was followed with serial X rays and gross examination. No rabbit in the control group experienced complete clot lysis. However, 2 of 5 rabbits in the L-arginine group and 6 of 10 rabbits in the nitric oxide donor (nitroprusside and nitroglycerin) groups noted complete clot lysis (P < 0.05, Fisher exact test). Thus, administration of an NO donor (nitroglycerin or nitroprusside) and, to a lesser extent L-arginine, reversed aspirin's antagonism of t-PA thrombolysis. This study may help explain the discrepant results seen with aspirin and thrombolytics.

Major depression and external stressors: the Lebanon Wars.

This article examines the effect of war events and pre-war depression on the prevalence of major depression during war. A total of 658 subjects aged 18-65 years were randomly selected from four Lebanese communities differentially exposed to the Lebanon Wars and were interviewed using the Diagnostic Interview Schedule (Arabic version). The individual levels of exposure to war events were assessed through a War Events Questionnaire. The lifetime prevalence of the DSM-III-R-defined major depression varied across the four communities from 16.3 to 41.9%; the final parameters predicting major depression since the onset of the wars were: depression before the wars and exposure to the wars. Both, individual levels of exposure to war and a history of pre-war depression, predict the development of depression during war.

Stroke risk factor knowledge in Hispanic and non-Hispanic white women in New Mexico: implications for targeted prevention strategies.

Hispanic women in New Mexico have recently experienced an increase in age-adjusted mortality compared with non-Hispanic white women. Since patients' knowledge of stroke risk factors may affect risk factor control, the present study was undertaken to characterize stroke risk factor understanding in Hispanic and non-Hispanic white women in New Mexico. We administered a stroke risk factor knowledge survey to 215 women hospitalized in Albuquerque, New Mexico. Patients were classified by each of three dichotomous groupings: stroke or nonstroke diagnosis; Hispanic or non-Hispanic white ethnicity; history of cardiovascular risk factors. The frequency of specific item responses was determined for each patient grouping. Two-way analysis of variance was used to determine whether composite knowledge score differed among patient groups. Stress was the attribute most commonly thought to be a risk factor for stroke. Although no ethnic differences were found on composite knowledge score, Hispanic women were significantly less likely to report hypertension as a stroke risk factor than non-Hispanic white women. We suggest that stroke risk factor understanding in Hispanic and non-Hispanic white women in New Mexico is inadequate. Insufficient understanding of the consequences of hypertension, including stroke, may diminish the degree of hypertension control that patients achieve. Further study of the relationship between stroke risk factor understanding and health behavior could enhance prevention efforts.