Mobile Stroke Unit Reduces Time to Image Acquisition and Reporting.

Timely administration of thrombolytic therapy is critical to maximizing the likelihood of favorable outcomes in patients with acute ischemic stroke. Although emergency medical service activation overall improves the timeliness of acute stroke treatment, the time from emergency medical service dispatch to hospital arrival unavoidably decreases the timeliness of thrombolytic administration. Our mobile stroke unit, a new-generation ambulance with on-board CT scanning capability, reduces key imaging time metrics and facilitates in-the-field delivery of IV thrombolytic therapy.

Recombinant human platelet-activating factor acetylhydrolase reduces the frequency of diabetes in the diabetes-prone BB rat.

Platelet-activating factor (PAF) has been implicated in the development of type 1 diabetes. Our previous studies have suggested that PAF inhibitors reduce insulitis and the frequency of diabetes in BB rats. In this study, serum PAF levels were reduced to address the hypothesis that PAF is important for the development of insulitis. From the age of 35 days on, DP-BB rats were treated with human recombinant PAF acetylhydrolase (rPAF-AH), which efficiently inactivates PAF. Our data indicate that intraperitoneal injections of rPAF-AH reduce the incidence of diabetes in the DP-BB rat. Daily intraperitoneal injections of 6.0 mg/kg body wt rPAF-AH reduced the frequency of diabetes in saline-injected rats from 90% (27/30) to 57% (17/30) (P = 0.004). As found by morphometric analysis on pancreatic islets, DP-BB rats protected from diabetes had less severe degrees of insulitis in a dose-dependent manner. DP-BB rats protected by rPAF-AH also had a higher percentage of insulin-positive cells in pancreas sections compared with those from diabetic animals. We therefore speculated that the beta-cells were protected from insulitis by rPAF-AH.

Anti-inflammatory properties of a platelet-activating factor acetylhydrolase.

Platelet-activating factor (PAF) is a potent pro-inflammatory phospholipid that activates cells involved in inflammation. The biological activity of PAF depends on its structural features, namely an ether linkage at the sn-1 position and an acetate group at the sn-2 position. The actions of PAF are abolished by hydrolysis of the acetyl residue, a reaction catalysed by PAF acetylhydrolase. There are at least two forms of this enzyme--one intracellular and another that circulates in plasma and is likely to regulate inflammation. Here we report the molecular cloning and characterization of the human plasma PAF acetylhydrolase. The unique sequence contains a Gly-Xaa-Ser-Xaa-Gly motif commonly found in lipases. Recombinant PAF acetylhydrolase has the substrate specificity and lipoprotein association of the native enzyme, and blocks inflammation in vivo: it markedly decreases vascular leakage in pleurisy and paw oedema, suggesting that PAF acetylhydrolase might be a useful therapy for severe acute inflammation.

Post-ischemic acute renal failure protects proximal tubules from O2 deprivation injury, possibly by inducing uremia.

Rats within the early maintenance phase of post-ischemic acute renal failure (ARF) can resist additional ischemic insults. This study assessed whether this protection exists directly at the tubular cell level, and if so, whether it is a consequence of prior cell injury (for example, due to heat-shock protein synthesis; HSP), or if it arises in response to reductions in functional renal mass and/or the uremic environment. Rats were subjected to either 15 or 35 minutes of unilateral or bilateral renal ischemia, and after 15 minutes to 24 hours of reflow, proximal tubular segments (PTS) were isolated for study. Their viability following oxygenation and hypoxic/reoxygenation injury (H/R) was tested (LDH release). The influence of uremia/reduced renal mass was determined by studying PTS extracted 24 hours after 1 1/2 nephrectomy, and by determining whether PTS exposure to a "uremic milieu" (urine addition) blocks H/R damage. HSP effects were gauged by correlating renal cortical HSP-70 expression with degrees of in vitro protection, and by ascertaining whether in vivo hyperthermia (42 degrees C; 15 min) mitigates subsequent PTS H/R damage. Results were compared with those obtained from normal PTS. The in vivo experimental protocols did not substantially alter PTS isolation or their viability during oxygenation. Fifteen minutes of ischemia induced neither azotemia nor PTS cytoprotection. In contrast, 35 minutes of ischemia conferred marked protection against subsequent H/R, but only when azotemia was permitted to develop (protection seen after 24 hr, but not at 4 hr of reflow; protection abrogated by retention of 1 normal kidney). Renal failure in the absence of tubular necrosis (1 1/2 uninephrectomy) protected PTS from H/R damage.(ABSTRACT TRUNCATED AT 250 WORDS)

Phospholipase A2 activity can protect renal tubules from oxygen deprivation injury.

During hypoxic or ischemic renal tubular injury, phospholipase A2 (PLA2) induces membrane deacylation, causing fatty acid accumulation and phospholipid breakdown. Because these changes can compromise cellular integrity, PLA2 activity has been widely proposed as a critical mediator of hypoxic renal tubular injury and, hence, of ischemic acute renal failure. To explore this hypothesis, isolated rat proximal tubules were subjected to continuous oxygenation or to hypoxic injury with or without exogenous PLA2 addition (porcine or bovine pancreatic PLA2; bee or snake venom PLA2). Cell death was quantified by lactic dehydrogenase (LDH) release. Pancreatic PLA2 (0.4 unit/ml) caused no LDH release under oxygenated conditions, and it dramatically attenuated hypoxic cell death (e.g., no PLA2, 55 +/- 3% LDH release; porcine pancreatic PLA2, 22 +/- 1% LDH release; P < 0.001). Bee and snake venom PLA2 (0.4 unit/ml) were directly toxic to tubules under oxygenated conditions, and this injury was additive with that induced by hypoxia. However, when these venoms were serially diluted (removing their overt toxicity), they, too, mitigated hypoxic cell death (LDH release with PLA2, 33 +/- 2%; without PLA2, 60 +/- 1% LDH release; P < 0.001). PLA2-mediated cytoprotection was Ca2+ dependent (negated by Ca2+ chelation), and it was expressed despite worsening hypoxia-associated membrane deacylation/fatty acid accumulation (12 times) and ATP depletion. These results indicate that PLA2 activity can exert both beneficial and deleterious effects on O2-deprived renal tubules, the net result of which can be a salvaging of cells from hypoxic cell death.

Physiological pH. Effects on posthypoxic proximal tubular injury.

After O2 deprivation, tissue acidosis rapidly self-corrects. This study assessed the effect of this pH correction on the induction, and pathways, of posthypoxic proximal tubular injury. In addition, ways to prevent the resultant injury were explored. Isolated rat proximal tubular segments (PTSs) were subjected to hypoxia/reoxygenation (50/30 or 30/50 minutes) under the following incubation conditions: 1) continuous pH 7.4, 2) continuous pH 6.8, or 3) hypoxia at pH 6.8 and reoxygenation at pH 7.4 (NaHCO3 or Tris base addition). Continuously oxygenated PTSs maintained under these same pH conditions served as controls. Lethal cell injury was assessed by lactate dehydrogenase (LDH) release. pH effects on several purported pathways of hypoxia/reoxygenation injury were also assessed (ATP depletion, lipid peroxidation, and membrane deacylation). Acidosis blocked hypoxic LDH release (pH 7.4, 50 +/- 2%; pH 6.8, 6 +/- 1%) without mitigating membrane deacylation or ATP depletion. During reoxygenation, minimal LDH was released (3-5%) if pH was held constant. However, if posthypoxic pH was corrected, immediate (< or = 5 minutes) and marked cell death (e.g., 55 +/- 3% with Tris) occurred. This was dissociated from lipid peroxidation or new deacylation, and it was preceded by a depressed ATP/ADP ratio (suggesting an acidosis-associated defect in hypoxic/posthypoxic cell energetics). Realkalinization injury was not inevitable, since it could be substantially blocked by 1) posthypoxic glycine addition, 2) transient posthypoxic hypothermia, or 3) allowing a 10-minute reoxygenation (cell recovery) period before base addition. Neither mannitol nor graded buffer Ca2+ deletion conferred protection. Acute pH correction caused no injury to continuously oxygenated PTSs. Conclusions are as follows: 1) Posthypoxic "pH shock" causes virtually immediate cell death, not by causing de novo injury but, rather, by removing the cytoprotective effect of acidosis. 2) This injury can be prevented by a variety of methods, indicating a great potential for salvaging severely damaged posthypoxic PTSs.

Inorganic iron effects on in vitro hypoxic proximal renal tubular cell injury.

Iron-dependent free radical reactions and renal ischemia are believed to be critical mediators of myohemoglobinuric acute renal failure. Thus, this study assessed whether catalytic iron exacerbates O2 deprivation-induced proximal tubular injury, thereby providing an insight into this form of renal failure. Isolated rat proximal tubular segments (PTS) were subjected to either hypoxia/reoxygenation (H/R: 27:15 min), "chemical anoxia" (antimycin A; 7.5 microM x 45 min), or continuous oxygenated incubation +/- ferrous (Fe2+) or ferric (Fe3+) iron addition. Cell injury (% lactic dehydrogenase [LDH] release), lipid peroxidation (malondialdehyde, [MDA]), and ATP depletion were assessed. Under oxygenated conditions, Fe2+ and Fe3+ each raised MDA (approximately 7-10x) and decreased ATP (approximately 25%). Fe2+, but not Fe3+, caused LDH release (31 +/- 2%). During hypoxia, Fe2+ and Fe3+ worsened ATP depletion; however, each decreased LDH release (approximately 31 to approximately 22%; P < 0.01). Fe(2+)-mediated protection was negated during reoxygenation because Fe2+ exerted its intrinsic cytotoxic effect (LDH release: Fe2+ alone, 31 +/- 2%; H/R 36 +/- 2%; H/R + Fe2+, 41 +/- 2%). However, Fe(3+)-mediated protection persisted throughout reoxygenation because it induced no direct cytotoxicity (H/R, 39 +/- 2%; H/R + Fe3+, 25 +/- 2%; P < 0.002). Fe3+ also decreased antimycin toxicity (41 +/- 4 vs. 25 +/- 3%; P < 0.001) despite inducing marked lipid peroxidation and without affecting ATP. These results indicate that catalytic iron can mitigate, rather than exacerbate, O2 deprivation/reoxygenation PTS injury.

Direct amphotericin B-mediated tubular toxicity: assessments of selected cytoprotective agents.

Amphotericin B (AB) may induce acute renal failure by vasoconstrictive and tubulo-toxic effects. Although mannitol, Ca2+ channel blockers, and lipid-based AB preparations have been suggested to mitigate in vivo AB nephrotoxicity, whether they confer direct tubular cytoprotection has not been defined. Therefore, this study assessed the impact of mannitol, verapamil/extracellular Ca2+, and cholesteryl sulfate (CS) AB binding on AB cytotoxicity, employing an isolated rat proximal tubular segment (PTS) preparation. After 30 to 60 minutes of incubation, 0.2 mg/ml of AB (Fungizone) caused marked toxicity, as assessed by LDH release (29 to 44%) and ATP depletion (greater than 90%). Approximately 40% of the LDH release could be attributed to deoxycholate, the standard AB (Fungizone) solubilizing agent. Both 100 mM mannitol and 100 mM glucose decreased AB-mediated LDH release, despite having a quantitatively trivial impact on ATP concentrations (increments of less than or equal to 1% at normal values). Dimethylthiourea (25 mM; equipotent to 100 mM mannitol/glucose as a hydroxyl radical scavenger) did not decrease LDH release. Neither verapamil addition (100 microM) nor Ca2+ removal from the PTS buffer had a protective effect. CS binding completely eliminated AB's toxicity (no LDH or ATP losses). The effect of AB and CS-AB on concomitant O2 deprivation/reoxygenation (30 min/15 min) PTS injury was also assessed. AB and hypoxia/reoxygenation caused additive, not synergistic, LDH release whereas CS-AB had no adverse effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence against increased hydroxyl radical production during oxygen deprivation-reoxygenation proximal tubular injury.

The purpose of this study was to assess whether proximal renal tubules generate excess hydroxyl radical (.OH) during hypoxia/reoxygenation or ischemia/reperfusion injury, thereby supporting the hypothesis that reactive oxygen species contribute to the pathogenesis of postischemic acute renal failure. In the first phase of the study, rat isolated proximal tubular segments (PTS) were subjected to hypoxia (95% N2- 5% CO2) for 15, 30, or 45 min, followed by 15 to 30 min of reoxygenation in the presence of sodium salicylate, a stable .OH trap. Cellular injury after hypoxia and reoxygenation was assessed by lactate dehydrogenase release; .OH production was gauged by hydroxylated salicylate by-product generation (2,3-, 2,5-dihydroxybenzoic acids (DHBA); quantified by HPLC/electrochemical detection). Continuously oxygenated PTS served as controls. Despite substantial lactate dehydrogenase release during hypoxia (8 to 46%) and reoxygenation (8 to 11%), DHBA production did not exceed that of the coincubated, continuously oxygenated control PTS. In the second phase of the study, salicylate-treated rats were subjected to 25 or 40 min of renal arterial occlusion +/- 15 min of reperfusion. No increase in renal DHBA concentrations occurred during ischemia or reperfusion, compared with that in sham-operated controls. To validate the salicylate trap method, PTS were incubated with a known .OH-generating system (Fe2+/Fe3+); in addition, rats were treated with antioxidant interventions (oxypurinol plus dimethylthiourea). Fe caused marked DHBA production, and the antioxidants halved in vivo DHBA generation. In conclusion, these results suggest that exaggerated .OH production is not a consequence of O2 deprivation/reoxygenation tubular injury.

Target biting associated with a gustatory avoidance response.

Gustatory avoidance studies normally focus on the diminished occurrence of a consummatory behavior as the primary dependent variable. Several reports have described behaviors which accompany this gustatory avoidance response but no attempt has been made to automate collection of such data. In the following studies lithium chloride (LiCl) was administered to rats following saccharin consumption in a standard gustatory avoidance paradigm. The rats also had the opportunity to make noncontingent target biting responses on an inanimate target. It was observed that there was a significant decrease in saccharin intake following its pairing with LiCl. High target biting rates were associated with this avoidance response and, to a lesser degree, with initial target exposure. These observations are discussed in terms of paradigm contingencies.