Hr Of Ischemia Research Articles

Initiation of Microvascular Protection by Nitric Oxide in Late Preconditioning

The authors hypothesized that nitric oxide is induced by a brief period of ischemia/reperfusion (ischemic preconditioning, IPC) on postoperative day (POD) 1, and that this released nitric oxide is responsible for initiating a delayed microvascular protection against a prolonged period of ischemia in skeletal muscle on POD day 2. The cremaster muscle of male Sprague-Dawley rats underwent 4 hr of ischemia, and then 60 min of reperfusion. IPC consisted of 45 min of ischemia but was done 24 hr before the prolonged ischemia. Local intraarterial infusion of sodium nitroprusside (SNP, a donor of nitric oxide) or Nw-nitro-L-arginine (L-NA, a nonselective nitric oxide synthase antagonist) were also given 24 hr before prolonged ischemia. Arteriole diameters and capillary perfusion were measured using intravital microscopy. Four groups were compared: 1) control; 2) IPC; 3) SNP + sham IPC; and 4) L-NA + IPC. Four hours of ischemia followed by reperfusion created a significant vasoconstriction and capillary no-reflow in the microcirculation of cremaster muscles. These alterations were largely prevented by IPC. Local intraarterial infusion of SNP without IPC created a similar microvascular protection to that induced by IPC alone. In contrast, intraarterial infusion of L-NA prior to IPC eliminated the IPC-induced microvascular protection. In conclusion, in late preconditioning, nitric oxide contributes to the initiation of a delayed microvascular protection against prolonged ischemia in skeletal muscle.

Effect of AMPA on cerebral cortical oxygen balance of ischemic rat brain.

We tested the hypothesis that the excitatory neurotransmitter receptor agonist, alpha amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA), would worsen cerebral cortical oxygen supply/consumption balance during focal ischemia. In this study, we compared regional cerebral blood flow, arterial and venous O2 saturation, O2 extraction and oxygen consumption of ischemic and AMPA treated ischemic and control regions of rat brain. Ischemia was induced by middle cerebral artery (MCA) occlusion in isoflurane (1.4%) anesthetized Wistar rats. Twenty minutes after MCA occlusion, 10(-5) M AMPA was applied to the ischemic cortex (IC) for a period of 40 min; the fluid was changed every 10 min. After 1 hr of ischemia, animals were sacrificed and regional cerebral blood flow (rCBF) was determined using the C14-iodoantipyrine autoradiographic technique. Regional arterial and venous oxygen saturation were determined microspectrophotometrically. In control, the cerebral blood flow and oxygen consumption of the IC were significantly lower than the contralateral cortex (rCBF: 46 +/- 20 vs. 81 +/- 39 ml/min/100g, O2 consumption: 2.8 +/- 1.4 vs. 3.6 +/- 1.4 ml O2/min/100g). 10(-5) M AMPA did not significantly alter regional cerebral blood flow and oxygen consumption of the IC, but did decrease the average venous O2 saturation of the IC from 50.2 +/- 3.9% to 46.7 +/- 1.6%. AMPA also significantly increased the frequency of small veins with less than 45% O2 saturation in the IC (8 out of 56 veins in IC vs. 18 out of 56 veins in AMPA treated IC). Thus, topical application of 10(-5) M AMPA to the ischemic area worsens cerebral O2 balance and suggests that excitatory amino acids contribute to the degree of cerebral ischemia.

Cerebral Microvascular Obstruction by Fibrin is Associated with Upregulation of PAI-1 Acutely after Onset of Focal Embolic Ischemia in Rats

The mechanisms underlying cerebral microvascular perfusion deficit resulting from occlusion of the middle cerebral artery (MCA) require elucidation. We, therefore, tested the hypothesis that intravascular fibrin deposition in situ directly obstructs cerebral microcirculation and that local changes in type 1 plasminogen activator inhibitor (PAI-1) gene expression contribute to intravascular fibrin deposition after embolic MCA occlusion. Using laser-scanning confocal microscopy (LSCM) in combination with immunofluorescent staining, we simultaneously measured in three dimensions the distribution of microvascular plasma perfusion deficit and fibrin(ogen) immunoreactivity in a rat model of focal cerebral embolic ischemia (n = 12). In addition, using in situ hybridization and immunostaining, we analyzed expression of PAI-1 in ischemic brain (n = 13). A significant (p < 0.05) reduction of cerebral microvascular plasma perfusion accompanied a significant (p < 0.05) increase of intravascular and extravascular fibrin deposition in the ischemic lesion. Microvascular plasma perfusion deficit and fibrin deposition expanded concomitantly from the subcortex to the cortex during 1 and 4 hr of embolic MCA occlusion. Three-dimensional analysis revealed that intravascular fibrin deposition directly blocks microvascular plasma perfusion. Vascular plugs contained erythrocytes, polymorphonuclear leukocytes, and platelets enmeshed in fibrin. In situ hybridization demonstrated induction of PAI-1 mRNA in vascular endothelial cells in the ischemic region at 1 hr of ischemia. PAI-1 mRNA significantly increased at 4 hr of ischemia. Immunohistochemical staining showed the same pattern of increased PAI-1 antigen in the endothelial cells. These data demonstrate, for the first time, that progressive intravascular fibrin deposition directly blocks cerebral microvascular plasma perfusion in the ischemic region during acute focal cerebral embolic ischemia, and upregulation of the PAI-1 gene in the ischemic lesion may foster fibrin deposition through suppression of fibrinolysis.

Redox regulation of NF-kappaB and AP-1 in ischemic reperfused heart.

Two redox-sensitive transcription factors, AP-1 and NF-kappaB, have been implicated in the regulation of apoptosis induced by myocardial ischemia and reperfusion. Hearts adapted to ischemic stress by cyclic episodes of short durations of ischemia and reperfusion attenuate apoptotic cell death. This study was designed to examine the pattern of expression of these transcription factors and the redox sensitive transacting molecule, AP-1, NF-kappaB, and- Bcl-2, during ischemia/ reperfusion and myocardial adaptation to ischemia. NF-kappaB binding activity was low in nonischemic control heart. Fifteen minutes of ischemia resulted in translocation of NF-kappaB from cytosol to nucleus followed by activation. The binding activity of NF-kappaB was further enhanced after 60 min of ischemia. An even higher degree of NF-kappaB binding was noticed in the ischemically adapted myocardium. In contrast, AP-1 binding activity was highest for the hearts subjected to 15 min of ischemia followed by 2 hr of reperfusion. AP-1 binding was higher in the ischemically adapted heart as compared to the control. The Bcl-2 gene, which was found to be present in the control hearts, had lowered expression after 15 min of ischemia and 2 hr of reperfusion. Significant upregulation of Bcl-2 mRNA was noticed in the ischemically adapted hearts. Apoptotic cardiomyocytes were found only in the hearts that were reperfused for at least 90 min. No apoptosis occurred in hearts subjected up to 1 hr of ischemia or ischemic adaptation. Prolonged reperfusion, and not ischemia up to 1 hr, can induce cardiomyocyte apoptosis. In concert, ischemic/reperfusion increases the nuclear binding of both AP-1 and NF-kappaB, but downregulates Bcl-2 gene. Ischemic adaptation attenuates apoptotic cell death, further increases NF-kappaB binding activity and Bcl-2 gene induction, but reduces AP-1 binding activity. These results suggest that AP-1, NF-kappaB, and Bcl-2 are differentially regulated by ischemia/reperfusion and ischemic adaptation.

Acute and chronic effects of nicotine on anastomotic patency following ischemia/reperfusion.

The effect of nicotine on anastomotic patency was studied using an isogenic rat-knee transplantation model. Animals were divided into experimental and control groups, with experimental animals exposed to either 40 days of twice-daily nicotine injections (Group 1-chronic exposure) or acute nicotine exposure by graft perfusion (Group 2-acute exposure). Four and 6 hr warm ischemia times were studied. Syngeneic rat knees were transplanted from donor to recipient by microvascular anastomosis of the femoral vessels. There was a statistically significant difference (p < 0.05) in anastomotic patency after 4 hr of ischemia in Group 1 animals exposed to nicotine (33 percent patency) vs. controls (89 percent patency). In addition, there was a statistically significant difference in anastomotic patency after 4 hr of ischemia in Group 2 animals exposed to nicotine (11 percent patency) vs. controls (67 percent patency). No significant differences between controls and Group 1 animals were noted in anastomotic patency after 6 hr of ischemia. Both acute and chronic nicotine exposure reduces anastomotic patency after short intervals of warm ischemia in vascularized composite tissue grafts. This detrimental effect is lost when the ischemic interval is prolonged to periods with poor anastomotic patency rates related to the ischemia/reperfusion itself.

Effectiveness of ischemic preconditioning on long-term myocardial preservation.

This study was designed to assess whether the protective effect of ischemic preconditioning can be adapted for myocardium undergoing 6 hr of ischemia. Eighteen isolated rat hearts were perfused with oxygen-bicarbonated Krebs-Henseleit buffer in the Langendorff mode for 35 min (group A, controls) or perfused in the Langendorff apparatus for 20 min, followed by 5 min of global normothermic ischemia and 10 min of buffer perfusion (group B, preconditioning) or followed by two cycles of 2.5 min of global normothermic ischemia plus 5 min of buffer perfusion (group C, preconditioning). The hearts were then arrested and preserved for 6 hr with Bretschneider's histidine-tryptophan-potassium cardioplegic solution at 4 degrees C, followed by 30 min of reperfusion. Recovery of cardiac function, postischemic enzyme leakage, and intracellular calcium concentration were compared. After 6 hr of ischemia, the hearts that underwent preconditioning in groups B and C showed better recovery of left ventricular developed pressure (P<0.05), a lower end-diastolic pressure level (P<0.05), less leakage of creatine kinase, and a lower intracellular calcium concentration than those in group A. There were no statistical differences in the rate of recovery of coronary flow. Our study demonstrated that ischemic preconditioning improves myocardial functional recovery after 6 hr of hypothermic preservation in the isolated rat heart. Preconditioning might be useful for preserving the heart against long-term ischemia/reperfusion injury.

Effect of ketorolac tromethamine (Toradol) on ischemia-reperfusion injury in skeletal muscle.

The eicosanoids, leukotriene B4 (LTB4) and thromboxane A2 (TXA2), contribute to neutrophil adhesion and arteriole vasoconstriction, important microcirculatory events in ischemia-reperfusion (I-R) injury. The purpose of this study was to evaluate the effect of ketorolac on I-R injury of skeletal muscle. A videomicroscopic preparation of gracilis muscle in male Wistar rats (n=7) in two experimental groups was evaluated: Group 1-4 hr global ischemia only (19 arterioles, 19 venules), and Group 2-4 hr ischemia plus ketorolac (13 arterioles, 14 venules). Ketorolac (0.86 mg/kg, i.m.) was given 30 min prior to reperfusion. The number of neutrophils, rolling and adherent, was counted in 100-micron venular segments, and arteriole diameters were measured at 5, 15, 30, 60 and 120 min of reperfusion. The I-R-induced increase in neutrophil adhesion was significantly reduced by ketorolac, which significantly increased arteriolar vasodilation in the first 30 min of reperfusion and significantly reduced the I-R-induced vasoconstriction in arterioles at 30 min; this effect was lost at 1 hr of reperfusion. Although ketorolac augments immediate arteriole vasodilation and blocks subsequent vasoconstriction, this effect appears to be transient. These findings suggest that ketorolac may have potential as a treatment for I-R injury.

Selective neutrophil depletion with monoclonal antibodies attenuates ischemia/reperfusion injury in skeletal muscle.

The purpose of this study was to determine whether depletion of circulating neutrophils, using an antineutrophil monoclonal antibody (RP3), would attenuate ischemia/reperfusion injury in rat skeletal muscle. A 3- and 5-hr period of ischemia was induced unilaterally into the hindlimbs of rats; the isolated limbs were then reperfused for 24 hr after ischemia. The gastrocnemius muscle was then removed, and blood was taken simultaneously. The hematologic parameters were measured, muscle neutrophil sequestration was assessed by myeloperoxidase (MPO) activity, free radical production was evaluated by the tissue lipid peroxides (LPO) levels, muscle viability was assessed by tissue levels of adenosine triphosphate (ATP) and creatine phosphate (PCr) levels, and muscle wet/dry weights were determined. Treatment with RP3 selectively and sufficiently depleted the circulating neutrophil population, markedly reduced MPO, and significantly attenuated LPO and the tissue water content after both 3- and 5-hr of ischemia. After 3 hr of ischemia, ATP and PCr levels were significantly increased by neutrophil depletion; however, after 5 hr of ischemia, the same effect was not demonstrated. These results suggest that neutrophil depletion after 3 hr of ischemia restrains free radical production and edema formation, and also attenuates skeletal muscle ischemia reperfusion injury; however, after 5 hr of ischemia, ischemic damage was so severe, that neutrophil depletion did not reduce ischemia reperfusion injury.

Tauroursodeoxycholic acid protects cholestasis in rat reperfused livers: its roles in hepatic calcium mobilization.

Tauroursodeoxycholic acid (TUDCA) is of potential benefit in cholestatic disorders. However, the effect of TUDCA on hepatic ischemia-reperfusion injury is unknown. We studied this subject with particular regard to its roles in hepatic calcium mobilization. Three doses of TUDCA were used with continuous intravenous infusion (1.0, 0.1, and 0.01 micromol/kg body weight/min). At 3 hr after 1 hr of ischemia and reperfusion in 70% rat liver, high-dose TUDCA reduced hepatic reperfused injury according to biochemical and histological findings and significantly increased bile flow after reperfusion. It significantly increased tissue calcium content and serum calcium concentration after reperfusion. Furthermore, it also enhanced biliary calcium concentration and total output during reperfusion. In conclusion, TUDCA has a salutary effect on ischemia-reperfusion injury of the liver. However, it is still unclear how the calcium mobilization induced by TUDCA is associated with the hepatoprotection against ischemia-reperfusion injury.

Phospholipase A2 secretion during intestinal graft ischemia.

The time-dependent appearance of phospholipase A2 (PLA2) activity in the preservation media of ischemic rat intestinal grafts is described. In controls, Ca2+-dependent, secretory PLA2 activity accumulated rapidly during the first 6 hr of ischemia, followed by a linear increase for up to 48 hr. LDH levels, by contrast, increased linearly throughout the 48 hr of ischemia. Addition of inhibitors of PLA2, cyclooxygenase, and lipooxygenase blocked accumulation of PLA2, but not LDH. PX-13, a novel PLA2 inhibitor, was most effective: 40 microM inhibited release by 86%, while 25 microM indomethacin (cyclooxygenase blocker) or nordihydroguiaretic acid (lipooxygenase blocker) inhibited 41 and 36%, respectively. That appearance of PLA2 activity, but not LDH, is attenuated by inhibitors of the eicosanoid cascade suggests a secretory event rather than leakage from dying cells. The secreted PLA2 is most likely the proinflammatory sPLA2 that has been implicated as a stress-induced protein and priming agent in ischemia-reperfusion injury.

A Perfluorochemical Prevents Ischemia–Reperfusion Injury of Muscle

This investigation evaluated the effect of oxygenated perfluorochemical (PFC) perfusion on the viability of ischemic skeletal muscle compared to hypothermic preservation. Twenty-five hindlimbs of 13 white rabbits were divided into five groups: a PFC group (7 hr of ischemia with 6 hr of PFC perfusion), a PFC/reperfusion group (8 hr of ischemia with 6 hr of PFC perfusion and 1 hr reperfusion), a hypothermia group (7 hr of ischemia with 6 hr of 4°C cold preservation), a hypothermia/reperfusion group (8 hr of ischemia with 6 hr of 4°C cold preservation and 1 hr of reperfusion), and a control group. The levels of adenine nucleotides, creatine phosphate, hypoxanthine, xanthine, and lipid peroxide were determined in each group. In the PFC and PFC/reperfusion groups, the ATP level remained at 90% of that in the control group. The hypoxanthine level in the hypothermia/reperfusion group was decreased to 70% of that in the hypothermia group and the xanthine level was increased to 130%. In the PFC and PFC/reperfusion groups, hypoxanthine and xanthine levels were much lower and were similar to those in the control group. Lipid peroxide levels were also lower in the PFC and PFC/reperfusion groups than in the hypothermia/reperfusion group. Electron microscopy showed that endothelial cells from the PFC/reperfusion groups were not swollen. These results suggest that PFC perfusion is superior to hypothermia in inhibiting the generation of free radicals and in preventing ischemia–reperfusion injury of skeletal muscle.

Exogenous Bradykinin Enhances Ischemia/Reperfusion Injury of Pancreas in Rats

We have investigated the effect of bradykinin on microvascular perfusion failure and enzyme release after ischemia/reperfusion of the pancreas in rats. Using intravital fluorescence microscopy in 21 anesthized Sprague-Dawley rats, quantitative analysis of the microcirculation, including functional capillary density (FCD) and leukocyte-endothelium interaction, was performed in an ischemia/reperfusion model of the pancreas. Bradykinin was dissolved in phosphate buffer and given as a bolus injection (injection group, 10 μg·kg−1body wt i.a.;n= 7) or continuously infused (infusion group, 125 μg·kg−1body wt·hr−1i.a.;n= 7) 15 min before the end of 2 hr of ischemia. Two further groups underwent sham operation (control group,n= 7) or an ischemia of 2 hr (ischemia group,n= 7) without bradykinin administration. Continuous infusion of bradykinin resulted in a significant enhancement of capillary perfusion failure after ischemia during reperfusion. In the bradykinin infusion group less than 25% of the capillaries were perfused (FCD 98 ± 9 cm−1) after 2 hr of reperfusion, whereas in the ischemia group without bradykinin 50% of capillaries were perfused (FCD 192 ± 11 cm−1). Both of these values are significantly different from the baseline value of the control group (408 ± 9 cm−1). The rise in pancreas amylase concentration was significantly more pronounced in the infusion group (basal: 1812 ± 114 U/l; 2 hr of reperfusion 3375 ± 268 U/l) when compared to the ischemia group (basal: 2386 ± 283 U/l; 2 hr of reperfusion 3486 ± 268 U/l). These findings suggest that bradykinin has an additive role in aggravation of pancreatic microcirculatory failure after ischemia/reperfusion of the pancreas.

Loss of Superficial Femoral Artery Relaxation Following Ischemia–Reperfusion

Acute ischemia followed by reperfusion in skeletal muscle is associated with tissue edema and necrosis. The purpose of this study was to demonstrate superficial femoral artery endothelial injury following complete ischemia with reperfusion. New Zealand white rabbits underwent total devascularization of one hindlimb for 3 hr followed by 0, 1, and 2 hr of reperfusion. Control rabbits underwent a sham operation. Superficial femoral artery rings were then studied for acetylcholine induced relaxationin vitro.The response to acetylcholine was measured as percentage relaxation at three incremental doses (1 × 10−7, 3 × 10−7, and 5 × 10−7M). The ischemia-only (26.30 ± 7.07, 62.63 ± 8.64, 88.08 ± 5.25%) and the 1-hr reperfusion group (19.35 ± 12.99, 39.24 ± 15.78, 62.01 ± 14.03%) showed no significant difference (P≥ 0.05, Student'sttest) in relaxation as compared to the control group (13.73 ± 2.11, 47.88 ± 7.23, 72.44 ± 9.00%). The 2-hr reperfusion group (6.10 ± 1.02, 15.33 ± 2.56, 34.67 ± 6.31%), however, had a significant loss of relaxation at all three doses of acetylcholine compared to that seen in the control group (P≤ 0.05, Student'sttest). In this model of complete ischemia, superficial femoral artery rings lose their ability to relax in response to acetylcholine following 3 hr of ischemia and 2 hr of reperfusion, demonstrating endothelial injury. However, immediately after 3 hr of ischemia or ischemia followed by only 1 hr of reperfusion, superficial femoral artery rings did not lose their ability to relax in response to acetylcholine. This study identifies a window of opportunity for therapeutic intervention after ischemia and prior to endothelial injury from reperfusion.

TNF-α Potentiates Oxidant and Reperfusion-Induced Endothelial Cell Injury

Pulmonary edema following reperfusion is a major clinical problem. Changes in endothelial cell shape induced by oxidant injury may account for immediate capillary leakage associated with reperfusion injury. In these experiments we examined the role of tumor necrosis factor-α (TNF-α) in acute endothelial cell injury following ischemia–reperfusion. Sprague–Dawley rats were treated with a neutralizing antisera directed against TNF-α prior to production of distal ischemia. These rats demonstrated a significant reduction (P< 0.05) in acute lung edema in response to 4 hr of ischemia and 30 min of reperfusion when compared to rats undergoing the same procedure without antisera treatment. Anin vitromodel was developed to determine if TNF-α had a direct effect on endothelial cell response to ischemia–reperfusion. The effects of TNF-α and oxidant stress on the integrity of cultured endothelial cell monolayers was measured. Rat pulmonary artery endothelial cell monolayers reactedin vitroto oxidant stress by an increase in permeability. The cells changed shape and an increase in diffusion of125I-albumin across cell monolayers resulted when these cells were exposed to 50 μMhydrogen peroxide (H2O2) or plasma from the ischemic hind limb of a Sprague–Dawley rat (50 μl/ml). Pretreatment of cultured cells with low levels of recombinant mouse TNF-α significantly affected both the cell shape change and the increase in permeability (P< 0.05). Increased permeability of cell monolayersin vitrowas not due to cell lysis as determined by media lactate dehydrogenase levels. The effect appeared to be due to cellular rounding and contraction seen using video time lapse microscopy. These data suggest a direct effect of TNF-α on endothelial cells, whereby the cells are rendered more susceptible to oxidant injury accompanying reperfusion.

Time Course of Leukocyte Adhesion to Endothelium in Ischemia–Reperfusion

Adhesion of leukocytes (L) to microvascular endothelium (E) is a required step in the L–E interaction leading to tissue injury in ischemia–reperfusion. To assess the optimum period for therapy aimed at ameliorating negative effects of this required step, we investigated the time course of L–E adhesion in the hamster cheek pouch using 2 hr of ischemia and 1 hr of reperfusion in our model of I–R injury (Am. J. Physiol.261: 1626, 1991). Leukocytes adhering (stationary for ≥30 sec) to postcapillary venules (15–30 μm in diameter) were counted after labeling with acridine orange. Prior to the induction of ischemia, there were no significant differences in the number of adherent leukocytes in each area chosen for study (1.9 ± 0.6 vs 2.0 ± 0.3; mean number of leukocytes/100-μm vessel length ± SD). After 10 and 20 min of reperfusion there was no significant difference in leukocyte adhesion in the ischemic area relative to the control (2.7 ± 0.5 vs 2.8 ± 0.8, and 5.3 ± 2.8 vs 2.4 ± 0.6, respectively). Leukocyte adherence increased significantly after 30 min of reperfusion and remained elevated at 1 hr of reperfusion in the postischemic area relative to the nonischemic control area (7.8 ± 1.3 vs 3.6 ± 0.6, and 8.3 ± 0.8 vs 4.1 ± 0.6, respectively;P< 0.01). Leukocyte adhesion in the postischemic area after 30 min reperfusion was not significantly different from the adhesion at the end of 1 hr reperfusion. These data suggest that (1) peak leukocyte adhesion occurs after 30 min of normal reperfusion and (2) postischemic therapeutic intervention may be most beneficial when instituted within this early time period.

Measurement of Tissue Viability Using Intravital Microscopy and Fluorescent Nuclear Dyes

Intravital microscopy used with fluorescent vital stains provides the opportunity to measure the temporal and spatial extent of tissue injury following disease processes. However, this assumes that prolonged exposure to such dyes does not alter microvascular perfusion or cellular viability. To test this hypothesis, the extensor digitorum longus (EDL) muscle in 24 male Wistar rats, anesthetized with sodium pentobarbital (Somnotal, 65 mg/kg, ip), were prepared for microscopy. The EDL was either bathed continuously (n = 6) in Krebs solution containing bisbenzimide (5 μg/ml; labels nuclei of all cells) and ethidium bromide (5 μg/ml; labels nuclei of injured cells) or had dyes topically applied 1 hr (n = 4) and 4 hr (n = 4) following dissection of the muscle. Noxious stimuli (i.e., hypoxia:FiO2 of 810% (n = 3), 95% ethanol (n = 3), and 2 hr ischemia followed by 90 min reperfusion (n = 4) were used to test the ability of ethidium bromide, when used in conjunction with intravital microscopy, to differentiate injured tissue. Video recordings at the surface of the EDL muscle were made every 30 min for 5 hr from which the number of perfused capillaries was counted (NCper). The numbers of bisbenzimide- and ethidium bromidelabeled nuclei were counted at the surface of the muscle and at two to three additional locations within the muscle (to a maximum depth of approximately 120-160 μm). The average NCper (19.05 ± 1.7) remained constant over 5 hr, while the number of nuclei stained by bisbenzimide increased linearly with time from an initial value of 1218 ± 125. The average number of ethidium bromide-labeled nuclei (380.4 ± 36.8) remained stable for at least 80-90 min, after which the number of labeled nuclei increased linearly. The number of ethidium bromide-labeled nuclei increased compared to control (P ⩽ 0.05) following all noxious stimuli. Thus, within the first 80-90 min of exposure, use of fluorescent nuclear dyes in conjunction with intravital microscopy provides a means of evaluating spatial and temporal changes in tissue viability.

Preservation of Peripheral Nerve Grafts: A Comparison of Normal Saline, Htk Organ Preservation Solution, and Dmem Schwann Cell Culture Medium

The regeneration of peripheral nerve grafts was evaluated in a rat model, after pretreating the grafts with Schwann cell culture medium, HTK organ preservation solution, and normal saline, under cold ischemic conditions for different time periods. Following orthotopic replantation of the grafts into donor animals, the quality of regeneration was assessed after 6 weeks, compared to positive controls (autologous transplantation) and negative controls (acellular grafts). The regenerative quality in the Schwann cell culture groups with ischemic periods of 32 and 72 hr was comparable to normal controls. Significantly minor regeneration was detected in specimens undergoing 14 and 120 hr of ischemia in the Schwann cell culture medium and in the HTK and normal saline groups, regardless of ischemic time. Among the conclusions was that controlled proliferation of Schwann cells seems to be a basic principle for preservation of peripheral nerve grafts.

Leukocytes express connexin 43 after activation with lipopolysaccharide and appear to form gap junctions with endothelial cells after ischemia-reperfusion.

Levels and subcellular distribution of connexin 43 (Cx43), a gap junction protein, were studied in hamster leukocytes before and after activation with endotoxin (lipopolysaccharide, LPS) both in vitro and in vivo. Untreated leukocytes did not express Cx43. However, Cx43 was clearly detectable by indirect immunofluorescence in cells treated in vitro with LPS (1 micrograms/ml, 3 hr). Cx43 was also detected in leukocytes obtained from the peritoneal cavity 5-7 days after LPS-induced inflammation. In some leukocytes that formed clusters Cx43 immunoreactivity was present at appositional membranes, suggesting formation of homotypic gap junctions. In cell homogenates of activated peritoneal macrophages, Cx43, detected by Western blot analysis, was mostly unphosphorylated. A second in vivo inflammatory condition studied was that induced by ischemia-reperfusion of the hamster cheek pouch. In this system, leukocytes that adhered to venular endothelial cells after 1 hr of ischemia, followed by 1 hr of reperfusion, expressed Cx43. Electron microscope observations revealed small close appositions, putative gap junctions, at leukocyte-endothelial cell and leukocyte-leukocyte contacts. These results indicate that the expression of Cx43 can be induced in leukocytes during an inflammatory response which might allow for heterotypic or homotypic intercellular gap junctional communication. Gap junctions may play a role in leukocyte extravasation.

A NEW CONCEPT FOR SUCCESSFUL LONG-TERM PULMONARY PRESERVATION IN A DOG MODEL

We developed a dextran-glucose-based extracellular perfusion solution (DGX) that supports limited aerobic metabolism to maintain cellular integrity of an inflated donor lung during long-term ischemia and a storage temperature of 10 degrees C. In a dog model, we compared respiratory and hemodynamic function of orthotopically transplanted left lungs preserved using this method (DGX, group I, n = 6) with function of those preserved with EuroCollins solution (EC) stored at a temperature of 4 degrees C (group II, n = 6). All lungs were inflated with room air and stored for 12 hr. Pulmonary function was monitored for 5 hr of reperfusion. Values expressed below are group means with standard deviation. Statistical significance was calculated using a two-tailed t test. For PO2 (mmHg) (FiO2 = 0.4), group I (EC): control = 193 +/- 8, 30 min p.o. = 87 +/- 20*, 300 min p.o. = 174 +/- 13*; and group II (DGX): control = 217 +/- 28, 30 min p.o. = 184 +/- 46*, 300 min p.o. = 248 +/- 5*. For pulmonary vascular resistance (dynes), group I: control = 389 +/- 22, 30 min p.o. = 1209 +/- 301, 300 min p.o. = 1025 +/- 204*; and group II: control = 401 +/- 31, 30 min p.o. = 522 +/- 129, 300 min p.o. = 458 +/- 137* (*P < 0.05 DGX vs. EC). Gas analysis performed on air samples taken from the ischemic donor lung immediately after harvest and after 12-hr storage showed (calculated as group means) a significant decrease of PO2 and a significant increase of PCO2, respectively. Histology of the lungs after 5 hr of reperfusion showed essentially normal-appearing lungs in the DGX group, whereas lungs in the EC group showed thickening of the intra-alveolar septi, marked cellular infiltration, and accumulation of protein-like material in the alveoli. In this study, preservation with DGX resulted in satisfactory respiratory and hemodynamic function of the transplanted lung even after 12 hr of ischemia. It does not cause an increase of pulmonary vascular resistance as seen after preservation with EC. Data from the intrabronchial air analysis of the donor lung suggest that aerobic metabolism continues even under preservation conditions.

N-tert-butyl-alpha-phenylnitrone improves recovery of brain energy state in rats following transient focal ischemia.

Recent results have demonstrated that the spin trapping agent N-tert-butyl-alpha-phenylnitrone (PBN) reduces infarct size due to middle cerebral artery occlusion (MCAO), even when given after ischemia. The objective of the present study was to explore whether PBN influences recovery of energy metabolism. MCAO of 2-hr duration was induced in rats by an intraluminal filament technique. Brains were frozen in situ at the end of ischemia and after 1, 2, and 4 hr of recirculation. PBN was given 1 hr after recirculation. Neocortical focal and perifocal ("penumbra") areas were sampled for analyses of phosphocreatine (PCr), creatine, ATP, ADP, AMP, glycogen, glucose, and lactate. The penumbra showed a moderate-to-marked decrease and the focus showed a marked decrease in PCr and ATP concentrations, a decline in the sum of adenine nucleotides, near-depletion of glycogen, and an increase in lactate concentration after 2 hr of ischemia. Recirculation for 1 hr led to only a partial recovery of energy state, with little further improvement after 2 hr and signs of secondary deterioration after 4 hr, particularly in the focus. After 4 hr of recirculation, PBN-treated animals showed pronounced recovery of energy state, with ATP and lactate contents in both focus and penumbra approaching normal values. Although an effect of PBN on mitochondria cannot be excluded, the results suggest that PBN acts by preventing a gradual compromise of microcirculation. The results justify a reevaluation of current views on the pathophysiology of focal ischemic damage and suggest that a therapeutic window of many hours exists in stroke.