Inhibition of nitric oxide generation and lipid peroxidation attenuates hemolysate-induced injury to cerebrovascular endothelium

Summary The mechanisms of hemolysate-induced cerebral injury following subarachnoid hemorrhage are just beginning to be clarified. This study examined the injurious effects of hemolysate on endothelial cells derived from bovine middle cerebral arteries, and evaluated the roles of lipid peroxidation and nitric oxide production in this type of damage. Cultured endothelial cells were grown to confluency on gelatin-coated plates. The cells were characterized as endothelial cells on the basis of morphology, Factor VIII-related antigen staining, and low density lipoprotein (LDL) uptake. Additional cells were grown to confluency on collagen-coated well inserts, and were treated with hemolysate for 24 hours. Prior to hemolysate exposure, cells were treated with: a) an inhibitor of iron-dependent lipid peroxidation (tirilazad mesylate 100 M), or b) an inhibitor of nitric oxide synthase (either N-nitro-L-arginine: NLA 300 M, or aminoguanidine: AG at 1.5, 7.5, 15 or 150 M). Permeability of the tracer, U-¹⁴C-sucrose, across the layer of endothelial cells was examined over a 24 hour period. Hemolysate induced a significant increase in the permeability across the endothelial cell layer. Pretreatment with tirilazad mesylate, NLA, or AG attenuated significantly hemolysate-induced changes in the endothelial cell barrier.These findings indicate that free radical generation and lipid peroxidation are critical participants in hemolysate-induced injury to the barrier function of the cerebrovascular endothelium. In addition, the results indicate that endothelial cells provide an adequate source of nitric oxide to damage their own cellular function. Finally, these findings strongly implicate free radical mechanisms in endothelial damage associated with subarachnoid hemorrhage.     

The effects of memantine on lipid peroxidation following closed-head trauma in rats

Memantine is an uncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist. Unlike other NMDA antagonists, it has been used clinically for years for the treatment of Parkinsons disease, spasticity, and dementia without serious side effects. We aimed to investigate the therapeutic efficacy of memantine on a closed head trauma model. A total of 132 adult male Sprague–Dawley rats were randomly divided into four groups: sham-operated, control (closed head trauma), sham-vehicle (closed head trauma + saline), treatment (closed head trauma + memantine, 10 mg/kg, i.p.). A cranial impact was delivered to the skull, just in front of the coronal suture, over the left hemisphere, from the height of 7 cm. Saline or memantine were applied 15 min after trauma. Rats were euthanased 0.5, 1, 2, 6, 24, 48 h after trauma. Brain tissue samples were taken 5 mm away from the left frontal pole and also from the corresponding point of the contralateral hemispheres. Malondialdehyde activity (MDA) was considered to reflect the degree of lipid peroxidation. The MDA levels continued to increase for the first 2 h after the injury, then started to decrease gradually. Memantine treatment significantly reduced lipid peroxidation levels in the treatment group compared with other groups (P<0.01). The findings of the present study indicate that memantine provides beneficial effects after closed head trauma in rats.     

Effect of hemodialysis on plasma nitric oxide levels

Nitric oxide (NO) is produced in excess in various pathological states, including sepsis and hepatic cirrhosis, and appears to be related to inflammatory status. In uremia, one would expect the levels of NO to increase. We aimed to determine whether hemodialysis (HD) would remove NO from the systemic circulation of uremic patients. Blood was collected before, after, and 1 day after HD from 12 uremic patients. Plasma nitrite and nitrate (NOx-) levels were measured by colorimetric Greiss reaction and cGMP was measured by an enzyme immunoassay kit. Our study demonstrated that uremic patients have high plasma NO levels, and HD led to a significant drop in plasma NOx- level (63 +/- 15% reduction). The level rose back to the pre-HD level on the following day. Plasma cGMP in the patients also decreased significantly after HD (27 +/- 14% reduction). In conclusion, we hypothesized that HD might be a possible approach for the removal of excess NO in pathological conditions such as sepsis and hepatic cirrhosis.     

The effect of nitric oxide in testicular ischemia-reperfusion injury

This experiment was carried out to investigate the effect of endogenous nitric oxide (NO) on the ischemia-reperfusion injury of testis.Testicular ischemia was achieved by twisting the right testis and spermatic cord 1080 counter-clockwise for 30 minutes and reperfusion was allowed for 30 minutes after detorsion of 33 rats. Animals were treated with normal saline in controls just before detorsion,NG-nitro-L-arginine methyl ester (L-NAME), and L-arginine (L-arg) in others.The tissue damage was evaluated with light microscopy, malondialdehyde (MDA) level in tissue, and the blood flow measurement using ¹³³xenon (Xe) clearence technique.MDA indicator of reperfusion injury increased 25% after detorsion when only normal saline was given, L-NAME further increased MDA, L-arginin decreased MDA to control level.Conclusion: L-arginin infusion during the detorsion reduced the reperfusion injury of testis and improved the testicular blood flow after the detorsion.     

Role of nitric oxide and lipid peroxidation in pathogenesis of acute experimental pancreatitis

In experiment, conducted on 130 Wistar line rats, there was studied up the influence of nitric oxide on the peroxidal oxidation of lipids intensity, activity of enzymes of the antioxidant defense system and morphological changes in acute pancreatitis. Modern suggestions about the influence of the nitric oxide system on pathogenetic mechanisms of an acute pancreatitis and possibilities of correcting therapy were scrutinized. It was established, that the nitrates and the nitrites content in the blood serum of experimental animals correlates positively with the pancreatic oedema index and severity of its morphological changes as well. Intraperitoneal injection of 20% water solution of L-arginine in dosage 200 mg per 100 g of an animal body mass have had aggravated an acute pancreatitis course; intravenous injection of 1% water solution of methylene blue in dosage 1 mg per 100 g of an animal body mass caused pancreatoprotective effect.     

The role of selective nitric oxide synthase inhibitor on nitric oxide and PGE2 levels in refractory hemorrhagic-shocked rats

BACKGROUND: The up-regulation of nitric oxide (NO) and cyclooxgenase-2 (COX-2) has been implicated in the pathophysiology of hemorrhagic shock. We examined the effects of aminoguanidine (AG), which is a known inducible nitric oxide synthase (iNOS) inhibitor, and NS-398, a known COX-2 inhibitor, in our rat model of refractory hemorrhagic shock (RHS). MATERIAL AND METHODS: We measured tissue iNOS and COX-2 protein expression, brain and plasma nitrate/nitrite and prostaglandin E2 (PGE2) levels, plasma creatinine and glutamic oxalacetic transaminase (GOT) levels, quantified the histological damages in kidney, liver, lung, and brain, survival rate, and mean arterial blood pressure (MABP) in RHS rats. RESULTS: Semiquantitative analysis of tissues showed iNOS protein was not detected in AG + RHS rats but was detected in normal saline and NS-398 RHS rats. Tissue COX-2 protein was not detected in AG and NS-398 RHS rats but was detected in normal saline + RHS rats. The levels of brain and plasma nitrate/nitrite and PGE2 and plasma creatinine and GOT were significantly lower in the AG + RHS rat group when compared with the normal saline RHS rat group. Histological examinations also showed a reduction in organ damage for AG + RHS rats when compared with treated RHS rats. AG + RHS rats showed significantly increased survival and MABP level when compared with treated RHS rats. CONCLUSION: Our present findings suggest that NO produced by iNOS might result in organ damages. This in turn might lead to COX-2 up-regulation, and it increases the production of reactive oxygen species and toxic prostanoids. NO-mediated organ damage might be one way in which toxic products of COX-2 might further contribute to NO's deleterious effect in the later stages of RHS. It is therefore suggested that treatment of AG via inhibition of NO might contribute to improved physiological parameters and survival rates following RHS.     

Dissociation of osmoregulation from plasma arginine vasopressin levels following thermal injury in childhood

Although the syndrome of inappropriate anti-diuretic hormone secretion has been recognised as a complication associated with burn and other trauma in adults, relatively little is known about its incidence in children. The objective of this study was to investigate whether it is a complication associated with burn injury in children. Plasma and urine levels of arginine vasopressin (anti-diuretic hormone), sodium and osmolality were measured in samples collected from 16 burn-injured children admitted to the burns unit of the regional children's hospital. No significant correlations were found between plasma vasopressin and plasma sodium or osmolality levels, but there were significant correlations between plasma vasopressin and urine osmolality, 36 (r=0.74, p=0. 009), 60 (r=0.92, p=0.000) and 84 h (r=0.84, p=0.001) after admission, respectively. There were also significant correlations between plasma sodium and plasma osmolality, 24 (r=0.7, p=0.005), 36 (r=0.57, p=0.04) and 84 h (r=0.84, p=0.004) after admission. The data suggest dissociation between the osmolar control of vasopressin secretion and vasopressin levels after burn injury in children, but do not support the incidence of inappropriate secretion of antidiuretic hormone.     

血管内放射对颈动脉内膜剥脱术后NO和ET水平的影响

目的：通过观察血管内放射对颈动脉内膜剥脱术(CEA)后NO和内皮素(ET)水平变化，探讨内放射治疗防治再狭窄的可能作用机理。方法：24只兔颈动脉内膜剥脱术后，随机分为3组，分别给予0、10、20Gy放射剂量(^32P)，术前3d、术后3、7、14、28d耳缘静脉各抽血4ml，分别测量NO、ET—1浓度，观察其水平变化。结果：术后NO水平内放射组较对照组明显升高(P＜0．01，而ET—1水平明显降低(P＜0．05)。10Gy、20Gy之间相比未见显差目。结论：内放射治疗可能是通过增加NO、降低ET—1含量来抑制平滑肌细胞增生、迁移，防治RS；血浆NO、ET—1水平的变化可作为一种观察疗效的指标。     

The effect of hemodialysis on lipid peroxidation

With regard to the variance in the levels of lipid peroxidation products (malonic dialdehyde and diene conjugates) the authors studied the impact of hemodialysis on lipid peroxidation. Transitory activation of lipid peroxidation with a subsequent decrease in the levels of lipid peroxidation products that were lower than initial ones were noted at the initial stages of hemodialysis performed with the use of a DEP-02-02 dialyzer (with a cuprophane membrane). The comparison of the input and output levels of lipid peroxidation products failed to demonstrate any generation of lipid peroxidation products inside the dialyzer. The systemic character of the activation effect in the onset of dialysis was established. It was proved that the effect did not depend on the material used for the membrane as there was no rise in the levels of lipid peroxidation products but a decrease in their concentration when the capillary dialyzers D1 and B2-100 were used. The study of the washings from the cuprophane membrane from the dialyzer DEP-02-02 demonstrated high levels of endotoxin which were several times higher than in those from the same membrane from the dialyzer D2. Endotoxin release into the circulation probably determined the transitory effect of the activation in the onset of the procedure performed with the dialyzer DEP-02-02. The authors discussed the possible origin of high initial levels of lipid peroxidation products observed in patients with terminal chronic renal failure who had undergone hemodialysis.     

The role of nitric oxide pathway in the renal ischemia-reperfusion injury in rats

INTRODUCTION: Nitric oxide (NO), synthesized from L-arginine by the enzyme nitric oxide synthase (NOS), seems to play an ambiguous role during tissue ischemia-reperfusion injury. Our objective was to evaluate the effects of L-arginine, a NO donor, and N(G)-nitro-L-arginine-methylester (L-NAME), a NOS inhibitor, on oxidative stress, renal dysfunction, histologic alterations and surgical mortality rate induced by renal ischemia-reperfusion (RIR) in uninephrectomized rats. MATERIALS AND METHODS: One-hundred and ninety-seven Wistar rats were randomized into five experimental groups. Group 1: sham operation; group 2: right uninephrectomy (UNI); group 3: UNI + RIR in the contralateral kidney; group 4: UNI + L-NAME (20 mg/kg; intraperitoneally) + RIR; and group 5: UNI + L-arginine + RIR. The effect of the drugs was evaluated by lipid peroxidation measured by the renal malondialdehyde (MD) content and chemiluminescence (CL) levels, serum creatinine (Cr) levels, urinary volume, tubular necrosis and athrophy, inflammatory infiltrate, interstitial fibrosis as histologic evaluation and surgical mortality rate after the procedures. A P value less than 0.05 was considered significant. RESULTS: Right uninephrectomy did not alter the renal parameters. RIR increased Cr levels (at 24 and 96 h of reperfusion), index of lipid peroxidation (both MD and QL levels), and worsened the histologic aspects. Pretreatment with L-arginine reduced the kidney levels of QL when compared with the non-treated group (5574 +/- 909 vs. 13 660 +/- 1104 cps/mg of protein; P < 0.05) but increased the MD levels (0.97 +/- 0.24 vs. 0.79 +/- 0.06 nmol/mg of protein; P < 0.05). Moreover, L-arginine attenuated the increment of Cr levels, inflammatory infiltrate and tubular athrophy in rats subjected to RIR (P < 0.05). On the other hand, pretreatment with L-NAME increased both CL (17 482 +/- 4397 vs. 13 660 +/- 1104 cps/mg of protein; P < 0.05) and MD levels (1.16 +/- 0.11 vs. 0.79 +/- 0.06 nmol/mg of protein; P < 0.05). Furthermore, L-NAME worsened the renal dysfunction (P < 0.05) at 192 h after the RIR, and surgical mortality rates were similar (P > 0.05). CONCLUSION: L-arginine has a tendency to exert a beneficial effect on renal damage during RIR in rats. Moreover, L-NAME seems to worsen the renal damage by increasing the kidney-levels of CL and impairment of renal function probably due to reduction of NO production.     

Early changes in plasma antioxidant and lipid peroxidation levels following coronary artery bypass surgery: a complex response

Objective: Total plasma antioxidant capacity (TPAC) quantitatively defines extracellular fluid antioxidant capacity, the mechanism of which is different from the intracellular mechanism. Patients undergoing surgery for congenital heart defects have suppressed TPAC in the early postoperative periods. Our aim was to study the early changes of TPAC following coronary artery bypass grafting (CABG), in relation to lipid peroxidation, and to identify clinical factors affecting these changes. Methods: We studied 28 consecutive patients undergoing routine uncomplicated CABG with cardiopulmonary bypass (CPB). Patients taking known antioxidants, such as captopril and allopurinol, and those receiving transfusion of blood or blood products at operation or during the first 72 postoperative hours were excluded. Serial blood samples were obtained for TPAC and lipid hydroperoxide concentration (LPX). Results: TPAC was suppressed for 72 h after the operation, while LPX exhibited a significant increase only 1 h post-operatively. TPAC time changes resulted from a simultaneous depression (50% of the baseline occurring approximately 6 h after the operation) and production (18% of the baseline occurring approximately 6 h after the operation) of plasma antioxidants. The earlier the peak of plasma antioxidant production the later and the less the plasma antioxidant depression. Plasma antioxidant depression was inversely related to LPX (r=−0.37, P=0.05 and r=−0.40, P=0.04 at 1 and 6 h respectively). Ejection fraction and operative myocardial ischaemic times significantly influenced plasma antioxidant depression. Conclusions: TPAC is suppressed for 72 h following CABG. TPAC depression may be involved in the mechanism of lipid peroxidation and is influenced by clinical factors known to be related to post CABG morbidity and mortality, like low ejection fraction and long ischaemic times.     

The effects of inhaled nitric oxide on the levels of cGMP plasma and lung tissue in a canine model of smoke inhalation injury

The effects of inhaled nitric oxide (NO) on pulmonary hypertension and their mechanisms were studied in a canine model of smoke inhalation injury. Twenty-one dogs were randomly divided into three groups: four dogs constituted the normal control group, eight dogs subjected to smoke inhalation followed by O(2) inhalation (FiO(2)=0.45) constituted the injury control group, and nine dogs inhaling a mixture of O(2) and 45ppm nitric oxide after smoke exposure served as the treatment group. The levels of cyclic guanosine monophosphate (cGMP) in arterial plasma of the treatment group were higher than that of the control group at 5, 8, and 12h after smoke exposure, while the levels of cGMP in lung tissue were also significantly higher compared with that of the control group (P<0.01). The levels of cGMP of injury control group were decreased significantly compared with normal controls (P<0.05). Pulmonary vasoconstriction following smoke inhalation was significantly attenuated by inhalation of NO (P<0.05), which exerted no apparent effect on the systemic circulation (P>0.05). Inhalation of NO may lower pulmonary hypertension induced by smoke inhalation injury in dogs. The selective effect of NO on pulmonary circulation may be attributed to an increase in level of cGMP in smooth muscle cells of the lung tissue after inhalation of NO.     

糖尿病大鼠颈动脉窦隔离灌注异丙酚对血浆内皮素及一氧化氮水平的影响

目的 探讨糖尿病大鼠颈动脉窦隔离灌注异丙酚对血浆内皮素(ET)及一氧化氮(NO)水平的影响.方法 健康清洁级成年雄性Wistar大鼠,体重180 ～ 220 g,采用腹腔注射链脲佐菌素30mg/kg的方法制备糖尿病模型.取糖尿病模型制备成功的大鼠36只,采用随机数字表法,将其随机分为3组(n=12):对照组(DC组)、低浓度异丙酚组(DP1组)和高浓度异丙酚组(DP2组).另取非糖尿病大鼠36只,采用随机数字表法,将其随机分为3组(n=12):对照组(NC组)、低浓度异丙酚组(NP组)和高浓度异丙酚组(NP2组).颈动脉窦隔离灌流K-H液(对照组)、含50 μmol/L异丙酚的K-H液(低浓度异丙酚组)和含100 μmol/L异丙酚的K-H液(高浓度异丙酚组),30 min后取股动脉血样,测定血浆ET和NO浓度.结果 与DC组比较,DP1组和DP2组血浆ET浓度降低,NO浓度升高(P＜0.05);与DP1组比较,DP2组血浆ET浓度降低,NO浓度升高,NP1组血浆ET浓度升高,NO浓度降低(P＜0.05);与DP2组比较,NP2组血浆ET浓度升高,NO浓度降低(P＜0.05).结论 异丙酚可通过对颈动脉窦压力感受器的局部作用降低糖尿病和非糖尿病大鼠血浆ET水平,升高NO水平,且对糖尿病大鼠的效应更强.     

The effect of nicotinamide on microvascular density and thermal injury in rats

The effects of nicotinamide on the microvasculature and wound healing were examined in rats subjected to thermal injury. Rats (250 g) were treated with 50 mg nicotinamide intraperitoneally twice daily for 21 days and then heart and brain biopsies were taken. Skin biopsies were removed from sites in and adjacent to the injury throughout the course of healing. Tissues were stained for alkaline phosphatase and capillary length density was determined by morphometric analysis. Significant increases were observed in the heart, brain, and dermal tissue of treated animals compared to controls. Capillary density in the injured skin was significantly greater when compared to the injured skin of saline-treated controls. The injuries of the rats that were treated systemically with nicotinamide healed significantly faster than saline-treated as determined by planimetric evaluation of the granulation bed and eschar.     

一氧化氮及其合酶在心肌缺血再灌注损伤中作用的研究

目的：用鼠的离体工作心脏研究心肌缺血再灌注损伤一氧化氮（ＮＯ）、ＮＯ合酶（ＮＯＳ）的作用。方法：ＲＴ－ＰＣＲ定量检测心肌组织结构型ＮＯＳ（ｃＮＯＳ）的ｍＲＮＡ表达,测定心肌组织的ｃＮＯＳ、诱导型ＮＯＳ（ｉＮＯＳ）及冠状动脉动脉冠脉）流出液的ＮＯ,同时检测心脏缺血再灌注前后的心功能变化。并分别于次序停跳液中加用缓激肽（ＢＫ）、Ｌ－精氨酸及ＢＫ加Ｌ－精氨酸,观察其对心肌缺血再灌注损伤的影响。结果：心肌     

Effectiveness of FK506 on lipid peroxidation in the spinal cord following experimental traumatic injury

STUDY DESIGN: An in vivo study in Wistar albino rats with injured spinal cord. SETTING: Department of Neurosurgery, Biochemistry and Pathology, Gazi University, Ankara, Turkey. OBJECTIVES: The aim of this study was to investigate and compare the effects of FK506 an immunosupressive agent with methylprednisolone (MP) on lipid peroxidation (LP) in injured spinal cord tissue. METHOD: A total of 28 adult healthy Wistar albino rats were subjected to traumatic spinal cord injuries (SCI) by using an aneurysmal clip compression technique, and they were divided into four groups. The G1 group (n=8) received FK506 (1 mg/kg); the G2 group (n=8) received FK506 (1 mg/kg) and MP (30 mg/kg); the G3 group (n=6) received only MP (30 mg/kg); and the G4 group (n=6) received no medication. The injured spinal cord tissue was studied by means of lipid peroxides, malondialdehyde (MDA), with thiobarbituric acid reaction and additionally the FK506 (G1); the MP (G3) groups were studied for histopathologic alterations 72 h after SCI with eight separate animals. RESULTS: Although LP values of G1, G2, G3 showed no statistical difference between intergroup analyses (P=0.547), a histopathological examination revealed that in the group that received MP, the oedema pattern was more significant than the group that received FK506. Another interesting finding was the presence of polymorphonuclear leucocytes in the MP group, whereas no infiltration was found in the FK506 group. CONCLUSION: Analysis of the results indicated that FK506 is a valuable pharmacological agent that could be used to decrease the LP and polymorphonuclear leucocyte infiltration and inflamatory reactions in the injured spinal cord tissue.     

The effect of duration of compression on lipid peroxidation after experimental spinal cord injury

The present study was performed to evaluate the effect of duration of acute spinal cord compression on tissue lipid peroxidation in rats. A clip compression method (1) was used to produce acute spinal cord injury. Rats were divided into 3 groups, each consisting of 10. At 1 hour after trauma all rats were sacrificed, and MDA content of the injured spinal cord segment was measured. The tissue MDA contents were 3.922 μmolMDA/gww in group 1 (control), 10.192 μmol MDA/gww in group 2 (30 seconds compression), and 12.147 μmolMDA/gww in group 3 (60 seconds compression). These results demonstrate that the length of duration of compression significantly enhances lipid peroxidation. Our study supported the view that persisting compression may cause progression of secondary mechanisms which may irreversibly eliminate any potential for recovery.     

The time course and regional variations of lipid peroxidation after diffuse brain injury in rats

Summary Free radicals are generated after head injury. These radicals rapidly react with polyunsaturated fatty acids in the cell membrane and cause membrane destruction. This process is called lipid per-oxidation. Malondialdehyde (MDA) is one of the end products of lipid peroxidation, and it is a frequently used indicator of lipid per-oxidation in biological tissues. Using a diffuse head injury animal model, we studied the time course of lipid peroxidation in different regions of injured rat brains. In the present study, the MDA levels were 36.7%, 41.8%, and 35.1% greater than sham at one hour after injury at the frontal, parietal, and brain stem, respectively (p<0.0001). The MDA levels in these regions continued to increase and peaked a 4 hours after the injury. The levels slowly decreased, and by 24 hours, they were still significantly higher than the sham control's. The elevation of MDA levels was less in the striatum and the temporal regions at one hour. They were 16.9% and 13.3%, respectively (p<0.002). The MDA levels in these two regions continued to increase even after 4 hours of injury, but the degree of elevation never exceeded 35%. The results demonstrate that there is an immediate, posttraumatic burst of MDA production, suggesting the formation of free radicals after diffuse head injury. Even though all the regions sampled show the same effect, certain regions are less affected by this diffuse head injury animal model.     

The effect of desferrioxamine on lipid peroxidation and survival of ischaemic island skin flaps in rats

To produce total necrosis, it was found necessary to subject pedicled groin flaps in rats to 16 hours of warm ischaemia (WI) (whether clamped in situ or removed and maintained at 37 degrees C ex vivo before replantation). Biochemical markers of lipid peroxidation (Schiff bases and thiobarbituric acid (TBA)-reactive material) in homogenates of full thickness skin or of subcutaneous fat rose significantly (p less than 0.001) after 14 hours of WI and reperfusion in vivo. Desferrioxamine (15 mg/kg i.v.) administered systemically either before 14 h WI only, before reperfusion only after 14 h WI or in both circumstances inhibited these rises to near-control (fresh tissue) levels. In survival experiments, this treatment also protected these flaps from necrosis (p less than 0.01).