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Sepsis is a major health problem in the United States with high incidence and elevated patient care cost. Using an animal model of sepsis, cecum ligation, and puncture, we observed that mice became rapidly hypothermic reaching a threshold temperature of 28 °C within 5-10 h after initiation of the insult, resulting in a reliable predictor of mortality, which occurred within 30-72 h of the initial procedure. We also observed that the inflammatory gene expression in lung and liver developed early within 1-2 h of the insult, reaching maximum levels at 6 h, followed by a decline, approaching basal conditions within 20 h. This decrease in inflammatory gene expression at 20 h after cecal ligation and puncture was not due to resolution of the insult but rather was an immune dysfunction stage that was demonstrated by the inability of the animal to respond to a secondary external inflammatory stimulus. Removal of the injury source, ligated cecum, within 6 h of the initial insult resulted in increased survival, but not after 20 h of cecal ligation and puncture. We concluded that the therapeutic window for resolving sepsis is early after the initial insult and coincides with a stage of hyperinflammation that is followed by a condition of innate immune dysfunction in which reversion of the outcome is no longer possible.

Background and Purpose — It has been proposed that the novel spin trap agent disodium-[(tert-butylimino)methyl]benzene-1,3-disulfonate N -oxide (NXY-059) may be useful in the treatment of ischemic stroke. However, there is little information concerning the neuroprotective properties of NXY-059 when administered after an embolic stroke. Moreover, there is no information available concerning the combination of NXY-059 with the only Food and Drug Administration–approved pharmacological agent for the treatment of acute stroke, the thrombolytic tissue plasminogen activator (tPA). Thus, we determined the effects of NXY-059G, a generic form of NXY-059, on behavioral outcome after an embolic stroke when administered alone or in combination with tPA. Methods — Male New Zealand White rabbits were embolized by injecting a suspension of small blood clots into cerebral circulation via a carotid catheter. NXY-059G (100 mg/kg) was infused intravenously 5 minutes or 3 hours after embolization, whereas control rabbits received infusions of the saline vehicle. In tPA studies, the thrombolytic was administered intravenously starting 60 minutes or 3 hours after embolization (3.3 mg/kg). In combination studies, NXY-059G was given 5 minutes after embolization, followed by the administration of tPA beginning either 60 minutes or 3 hours after embolization. Behavioral analysis was conducted 24 hours after embolization. Results — In the vehicle control group, the ES 50 value (calculated as the amount of microclots [milligrams] that produce neurological dysfunction [impairment] in 50% of the rabbits within a specific treatment group) measured 24 hours after embolism was 1.04±0.31 mg, and this was increased by 153% to 2.54±0.72 mg if NXY-059G was administered beginning 5 minutes after embolization. However, if NXY-059G was administered beginning 3 hours after embolization, the ES 50 was 2.01±0.40 mg. The rabbits treated with tPA alone had an ES 50 of 2.64±0.66 or 0.63±0.35 mg if tPA administration started 60 minutes or 3 hours after embolization, respectively. If tPA was administered after NXY-059G (started at 5 minutes), the ES 50 values were 3.15±0.50 or 2.66±0.82 if tPA administration started 60 minutes or 3 hours after embolization, respectively. Conclusions — This study suggests that NXY-059G is neuroprotective and can increase behavioral ratings if administered early after an embolic stroke. In addition, the study shows that NXY-059G can be used in combination with tPA without negative side effects. The drug combination can improve behavioral function and increase ES 50 values. However, during the short time course of the behavioral analysis, the combination was not statistically better than either drug alone.

Background Sepsis is a major health problem in the United States that affects more than three-quarters of a million people every year. Previous studies have shown that scavenger receptor A (Sra), also known as macrophage scavenger receptor 1 (Msr1), is a modifier of interleukin 10 (IL-10) expression after injection of bacterial lipopolysaccharide (LPS). Therefore, we investigated the response to sepsis in Sra knock out mice. Materials and Methods C57BL/6J (B6) ( n = 88) and Sra (–/–) mice ( n = 88) were subjected to cecal ligation and puncture (CLP) using 18G or 16G needles, sham operation, or non-operated controls. At the end, mice were autopsied for the determination of abnormalities after the procedure. Cytokine gene expression was examined in lung and liver samples by quantitative RT-PCR (qRT-PCR), and circulating cholesterol levels were also measured. Results Sra (–/–) mice displayed an enlargement of the gallbladder after CLP that was not detected in sham or non-operated mice or in B6 mice (wild-type) after CLP. The enlarged gallbladder resembles a condition of acute acalculous cholecystitis observed in humans. Sra (–/–) mice presented high cholesterol levels in circulation as opposed to wild type B6 mice. Moreover, Sra (–/–) mice exhibited a reduction in IL-10 mRNA levels in lungs compared to wild-type B6 mice after CLP. Conclusions The development of acute acalculous cholecystitis may be the combination of pre-existing conditions, such as hypercholesterolemia associated with a defect in Sra (Msr1) and a robust inflammation induced by sepsis.

Caffeinol is currently being tested in acute ischemic stroke patients. However, little is known about the pharmacology or safety of caffeinol in preclinical embolic stroke models. We determined the pharmacological effects of caffeinol administration on clinical rating scores in rabbits following small clot embolic strokes (RSCEM). Male New Zealand white rabbits were embolized by injecting blood clots into the cerebral circulation via a carotid catheter. Behavioral analysis was conducted 24 h following embolization, allowing for the determination of the effective stroke dose (P50) or clot amount (mg) that produces neurological deficits in 50% of the rabbits. In the current study, the P50 values for the control groups were 1.32 +/- 0.23 and 1.66 +/- 0.29 mg for the bolus-injected and infused groups, respectively. Rabbits treated with caffeinol (bolus) starting 15 min following embolization had a P50 value of 1.70 +/- 1.18 mg. Caffeinol-infused rabbits had a P50 value of 2.05 +/- 0.47 and 1.67 +/- 0.48 mg for low- and high-dose ethanol, respectively. In tPA-treated rabbits (0.9 mg/kg), the group P50 was 1.58 +/- 0.43 mg. In caffeinol (bolus) and tPA-treated rabbits, we measured a decrease in the P50 value to 0.70 +/- 0.30 mg and an increase in the rate of intracerebral hemorrhage compared to control. This primary finding of this study indicates that neither bolus-injected nor infused caffeinol affects behavioral deficits following embolic strokes in rabbits. Moreover, the combination of caffeinol plus low-dose tPA does not improve behavioral deficits. However, our study suggests that there is the potential for exacerbation of stroke-induced behavioral deficits following caffeinol administration in combination with a thrombolytic that may be related to increased intracerebral hemorrhage.

Background and Purpose— It has been proposed that the novel thrombolytic microplasmin may be useful in the treatment of ischemic stroke. In the present study the effects and safety profile of microplasmin were evaluated in 2 rabbit embolic stroke models that have been used successfully to develop tissue plasminogen activator (tPA) as the only Food and Drug Administration-approved treatment for stroke. The rabbit small clot embolic stroke model (RSCEM) and rabbit large clot embolic stroke model (RLCEM) were used to determine the potential neuroprotective properties and safety profile of microplasmin, respectively, after an embolic stroke. Methods— Rabbits were embolized by injecting small blood clots (RSCEM) or large blood clots (RLCEM) into the cerebral circulation. For the RSCEM, 126 rabbits were included, with behavioral analysis conducted 24 hours later, allowing for determination of the effective stroke dose (ES 50 ) or clot amount (milligrams) that produces severe neurological deficits in 50% of rabbits. For RLCEM safety study analysis, 47 rabbits were included, with postmortem analyses consisting of assessment of hemorrhage and infarct rate and size. In test animals microplasmin was infused intravenously 60 minutes after embolization, whereas control rabbits were given infusions of the saline/Plasma-Lyte vehicle with all assessments performed in a blinded fashion. Results— In the RSCEM, a drug is considered neuroprotective if it significantly increases the ES 50 compared with the vehicle-treated control group. The ES 50 of the vehicle-treated control group 24 hours after embolization was 1.36±0.42 mg (n=38). Microplasmin, infused starting 60 minutes after embolization, increased the ES 50 to 2.32±0.57 (n=21), 1.89±0.48 (n=21), 2.81±0.55 (n=22), and 1.89±0.28 mg (n=24) for the 1-, 2-, 4-, and 8-mg/kg doses, respectively. There was a statistically significant behavioral improvement in the 4-mg/kg dose arm ( P =0.040). The microplasmin dose of microplasmin that was statistically significant (4 mg/kg) was subsequently determined to be safe in the RLCEM because it did not increase the incidence of hemorrhages (56%) compared with vehicle-treated rabbits (63%), nor did it significantly alter hemorrhage volume, infarct rate, or infarct volume. Conclusions— The present study shows that microplasmin improves behavioral rating scores in the RSCEM when administered 60 minutes after embolization, at a dose that does not increase hemorrhages in the RLCEM. This is in contrast to tPA, which significantly enhances the hemorrhage rate in the RLCEM.

Background and Purpose — It has been proposed that the novel spin trap agent disodium-[(tert-butylimino)methyl]benzene-1,3-disulfonate N -oxide (NXY-059) may be useful in the treatment of ischemia and stroke. To date, there is little information concerning the safety of NXY-059 when administered in combination with the only Food and Drug Administration–approved pharmacological agent for the treatment of stroke, the thrombolytic tissue plasminogen activator (tPA). Thus, we determined the effects of NXY-059G, a generic form of NXY-059, on hemorrhage and infarct rate and volume when administered alone or in combination with tPA. In addition, we determined whether NXY-059G affected 2 physiological variables, blood glucose levels and body temperature. Methods — Male New Zealand White rabbits were embolized by injecting a large blood clot into the middle cerebral artery via a catheter. Five minutes after embolization, NXY-059G (100 mg/kg) was infused intravenously; control rabbits received infusions of saline, the vehicle required to solubilize NXY-059G. In tPA studies, the thrombolytic was administered intravenously starting 60 minutes after embolization (20% bolus injection/80% infusion over 30 minutes). Body temperature and blood glucose levels were measured throughout the study. Postmortem analysis included assessment of hemorrhage and infarct rate, size, and location. Results — In the vehicle control group, the hemorrhage rate after a thromboembolic stroke was 52% (n=23), and this was increased by 67% if tPA was administered (n=15). The rabbits treated with NXY-059G in the absence of tPA had a 79% incidence of hemorrhage (n=19), an increase of 52% over the control group. In the combination drug–treated groups, the NXY-059G/tPA group had a 47% incidence of hemorrhage (n=15). There was a decrease of hemorrhage volume in the NXY-059G+tPA group compared with the other 3 groups included in the study. There was no significant effect of NXY-059G either alone or in combination with tPA on infarct rate or volume. NXY-059G did not significantly alter the physiological variables that were measured. Conclusions — This study suggests that NXY-059G may affect the integrity of the cerebral vasculature when administered immediately after an embolic stroke, as evidenced by an increase in hemorrhage rate. However, when NXY-059G is administered in combination with tPA, it may improve the safety of tPA by reducing the incidence of tPA-induced hemorrhage. The mechanism(s) involved in the NXY-059G–induced increase in hemorrhage rate and reduction of tPA-induced hemorrhage rate remains to be elucidated.

Background and Purpose — Platelet activation and deposition in brain microvessels appear to be key events in the pathogenesis of ischemia-induced neuronal degeneration and behavioral deficits. It has been hypothesized that activated platelets in combination with polymorphonuclear leukocytes and fibrin may play a role in vessel reocclusion leading to the “no-reflow” phenomenon after administration of the thrombolytic tissue plasminogen activator (tPA). We studied the effects of the novel glycoprotein IIb/IIIa platelet receptor antagonist SM-20302 when administered in combination with tPA on infarct and hemorrhage rate and volume to determine whether activated platelets are involved in either infarct or hemorrhage generation after a thromboembolic stroke. Methods — One hundred thirty-two male New Zealand White rabbits were included in the present study. Rabbits were embolized by injecting a blood clot into the middle cerebral artery via a catheter. Five or 65 minutes after embolization, SM-20302 (5 mg/kg) was infused intravenously. In drug combination studies, tPA was infused intravenously for 30 minutes starting 60 minutes after embolization, and SM-20302 was administered 5 or 65 minutes after embolization. Postmortem analysis included assessment of hemorrhage, infarct size and location, and clot lysis. Results — In the vehicle control group, the hemorrhage rate after a thromboembolic stroke was 33%. There was a significant increase (109%) in the hemorrhage rate in the group of rabbits treated with the thrombolytic tPA. SM-20302 by itself did not significantly alter the embolism-induced hemorrhage rate when administered either 5 or 65 minutes after embolism. The SM-20302 groups had a 42% and 33% incidence of hemorrhage in the 5- and 65-minute groups, respectively. In groups treated with a combination of drugs, the SM-20302/tPA group had a 31% and 71% incidence of hemorrhage when SM-20302 was administered 5 and 65 minutes after embolization, respectively. SM-20302 in combination with tPA also significantly increased infarct rate, but not hemorrhage or infarct volume. Conclusions — This study suggests that treatment of thromboembolic stroke with the combination of a platelet inhibitor and tPA may have a beneficial outcome on the basis of the following: First, acute administration of SM-20302 did not significantly increase hemorrhage rate. Second, SM-20302 in combination with tPA significantly reduced tPA-induced intracerebral hemorrhage. Third, there appears to be a specific window of opportunity when a platelet inhibitor must be administered to produce a beneficial effect. Overall, on the basis of our results, we hypothesize that the increased rate of intracerebral hemorrhage observed after tPA administration may be partly due to increased reocclusion of cerebral vessels following lysis of the emboli and that reocclusion can be controlled by administration of a platelet inhibitor.