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Objectives:We have shown previously that administration of quercetin after spinal cord injury in a rat model induced significant recovery of motor function. In the same model for spinal cord compression injury, we now have correlated the treatment duration with the extent to which motor function is recovered.Methods:Seventy-four male Wistar rats were assigned to eight experimental groups. Mid-thoracic spinal cord injury was produced in the animals of seven groups. Quercetin was administered intraperitoneally in individual doses of 25 μmol kg−1. Treatment onset was 1 h after the injury. The length of treatment ranged from one single injection to 10 days, with injection frequencies of two or three times daily. BBB (Basso, Beattie and Bresnahan) scores were obtained and tissue preservation at the site of injury was analyzed.Results:None of the untreated control animals recovered motor function sufficient to walk. When quercetin was administered twice daily over a period of either 3 or 10 days, about 50% of the animals recovered sufficient motor function to walk. Stepping/walking (BBB 10) were seen in two of six animals receiving only a single injection and in one of the six animal receiving three injections. Surprisingly, none of the animals that received quercetin injections three times daily recovered the ability to walk (all BBB 9).Conclusion:Quercetin administration results in preservation of tissue bridges at the site of injury. Treatment success depends on frequency of administration and overall dose. [ABSTRACT FROM AUTHOR]

Introduction: Pelvic floor myalgia is a common cause and contributor to chronic pelvic pain [Neurourol Urodyn 4:984-1008 (2017)]. The purpose of this study was to compare in-person versus video-based teaching methods of a comprehensive assessment of the pelvic floor musculature on a pelvic model., Methods: A randomized controlled trial of 46 participants was conducted. The participants were randomized into two groups. Both groups were taught by the same pelvic floor physiotherapist using two different teaching methods on a pelvic model. Group 1 watched an instructional video, whereas group 2 had in-person training. Both groups underwent pre- and post-training assessments consisting of a written examination and an Objective Structured Clinical Examination (OSCE). Primary outcome measure was the change in participants' pre- and post-training assessment scores. Secondary outcome measures were perceived changes in both participants' comfort level in performing pelvic floor examination and applicability of the training program to clinical practice., Results: There was no statistically significant difference between the teaching methods in the degree of improvement of the participants' mean written assessment scores (p = 0.58), OSCE scores (p = 0.15), and perceived comfort level (p = 0.19). Participants' mean pre- and post-assessment scores improved significantly (p < 0.001). Participants reported the training program to be applicable towards their clinical practice., Conclusions: This study demonstrates that learners' assessment of pelvic floor musculature can be enhanced using varied teaching methods on a pelvic model.

Study Objectives: To examine the surgical indications and pathologic findings in patients undergoing a second surgery after placement of the Essure permanent birth control system to determine the role of Essure in developing new-onset pelvic pain., Design: Retrospective cohort (Canadian Task Force classification II-2)., Setting: Tertiary-level hospital., Patients: Women who have had Essure placement and subsequent second surgery., Intervention: Charts from women undergoing pelvic surgery after Essure placement from June 2002 to June 2013 were reviewed and the indication for the procedure, surgical and pathologic findings noted., Measurements and Main Results: Of 1430 patients, 62 (4.3%) had a second surgery after Essure placement, and 24 of these (1.6%) had a surgical indication not related to pain. The remaining 38 patients (2.7%) had either new-onset (n = 27) or worsening pre-existing pain (n = 11). In the new-onset pain group, 15 (1%) had surgical findings or pathology consistent with a painful gynecologic condition. In the remaining 12, 8 (0.5%) seemed to be related to some perforation or migration of the Essure device. In 4 patients (0.3%) with no other obvious cause for the new-onset pain, it was thus attributed to the Essure microinsert., Conclusion: Essure sterilization can be associated with new-onset pain or a worsening of a pre-existing painful gynecologic condition, although both are very rare. A careful and complete consent before placement and a thorough examination if pain does occur usually show some etiology for the pain other than the Essure insert., (Copyright © 2016 AAGL. Published by Elsevier Inc. All rights reserved.)

Study Objective: To compare the pain reported by patients during the Essure Micro-Insert sterilization procedure using either intravenous conscious sedation or oral analgesia., Design: Randomized, double-blind, placebo-controlled trial (Canadian Task Force classification I)., Setting: Tertiary care ambulatory women's clinic., Patients: Eighty women of reproductive age women requesting permanent sterilization., Intervention: Hysteroscopic placement of the Essure Micro-Insert permanent birth control system., Measurements and Main Results: Patients undergoing placement of the Essure Micro-Insert system for permanent contraception were randomized to receive either intravenous conscious sedation, oral analgesia, or placebo. During the procedure, pain scores were recorded using a visual analog scale. Patients in the oral analgesia group reported slightly more pain during insertion of the hysteroscope and placement of the second micro-insert; the groups were otherwise equivalent. They were also equivalent when all visual analog scale scores were combined., Conclusion: Oral analgesia is an effective method of pain control during placement of the Essure Micro-Insert permanent birth control system., (Copyright © 2011 AAGL. Published by Elsevier Inc. All rights reserved.)

Study Objective: To compare outcomes after conservative surgery for endometriosis with and without pentoxifylline and to assess the efficacy of pentoxifylline in preventing recurrence of endometriosis after conservative surgery., Design: Parallel-group, randomized, controlled trial (Canadian Task Force classification I)., Setting: Tertiary care hospital., Patients: Women undergoing conservative surgery for endometriosis., Interventions: Laparoscopic conservative surgery for endometriosis was completed by a single surgeon (J.A.T.), and all suspected endometriotic lesions were widely excised using monopolar coagulation and scissors. All specimens were submitted to pathology for confirmation of the diagnosis. Randomization to the treatment or control groups was completed preoperatively in the outpatient surgery unit by drawing colored marbles. A preoperative visual analog pain scale (VAS) was completed. After surgery, patients were discharged home with prescriptions for naproxen, hydromorphone, and pentoxifylline., Measurements and Main Results: Visual analog scale scoring was completed monthly by each patient, and each patient was seen monthly for review and pelvic examination. Analgesic use was recorded daily using an individual medication log. Ongoing treatment choice after completion of the 3-month follow-up was recorded. The relationship between the group receiving pentoxifylline and the control group as well as analysis of the VAS scores at time of surgery and 1, 2, and 3 months postoperatively was determined using a covariate mixed-model ANOVA. Forty-nine patients were enrolled in the trial. One patient became pregnant before surgery, and 1 patient's chart was not available for analysis. Of the 47 who underwent conservative surgery for endometriosis, 9 (19%) had no endometriosis noted in the pathology specimens submitted. Two patients withdrew from the trial after surgery, and 2 patients were lost to follow-up after relocating to a different city. Nineteen women completed the 3-month follow-up in the control group, 15 in the group receiving pentoxifylline. The mean age, gravidity, parity, body mass index, previous surgery for endometriosis, menstrual cycle, and preoperative analgesic use did not differ significantly between the control and treatment groups. The time to complete the conservative surgery did not vary between the 2 groups. There were no intraoperative complications: two patients were admitted postoperatively, one for nausea and vomiting, one for pain that resolved 24 hours after admission. The VAS scores did not differ at the time of surgery; and in both the control and the pentoxifylline groups, there was significant improvement at each monthly interval (p <.05). The patients receiving pentoxifylline had significantly better VAS scores at 2 and 3 months after surgery (p <.03)., Conclusions: The use of pentoxifylline after conservative surgery for endometriosis resulted in improved VAS scores at 2 and 3 months after the procedure when compared with patients having conservative surgery only. The longer-term use of pentoxifylline after conservative surgery may improve long-term outcomes after surgical treatment for endometriosis.

Objective: To assess the costs associated with the performance of outpatient total laparoscopic hysterectomy., Methods: This was a retrospective cohort study involving 224 consecutive patients undergoing total laparoscopic hysterectomy (TLH) by a single surgeon in the Regina General Hospital. Outcomes included costs associated with the initial procedure as well as those associated with any intraoperative or postoperative complications., Results: The mean age of the patients was 42.7 years. The mean uterine weight was 205 grams (range 69-1163 g), the mean operating time was 79 minutes, and the mean blood loss was 89 cc. The mean postoperative stay in the day surgery unit (DSU) was 354 minutes. Ten patients required admission from the DSU, and nine patients were admitted more than 24 hours after surgery. The total number of admission days was 24, which cost 21,900 US dollars. The total cost of all disposables was 127,373 US dollars and the cost associated with the stays in day surgery was 89,600 US dollars. The total cost for the 224 TLH procedures was 238,573 US dollars, and the average cost per TLH was 1065 US dollars., Conclusion: Outpatient TLH can be completed safely and with costs that are lower than those incurred by patients having short-stay vaginal hysterectomy in our institution. Outpatient TLH offers the opportunity to save health care costs and a procedure with excellent results.

Previously, we were able to demonstrate the neuroprotective effect of quercetin in an animal model of acute traumatic spinal cord injury. The objective of the present study was to determine whether any neuroprotective effect is seen when quercetin is administered in an animal model of traumatic brain injury. Twenty-six adult male Sprague-Dawley rats were submitted to moderate fluid percussion injury in the anterior midline position. Animals were divided into two experimental groups: one group received 25 mumol/kg quercetin starting 1 h after injury, while animals in the second group received saline vehicle (n = 13 per group). Eight animals were used as uninjured healthy controls. Eight animals in each experimental group were sacrificed at 24 h, while five animals per group were allowed to recover for 72 h following injury. Compound action potential amplitudes (CAPAs) were recorded on 400-microm vibrotome sections of the corpus callosum superfused with oxygenated artificial CSF (n = 3 per animal) in 20 experimental animals and five healthy controls. Three brains from animals in each experimental group and healthy controls were used for histological, immunocytochemical and biochemical analysis after sacrifice at 24 h. CAPAs in uninjured animals had a mean of 1.12 mV. This decreased to 0.55 mV in saline vehicle-treated injured animals by 24 h and changed little over the next 3 days. CAPAs were significantly better at 0.82 mV at 24 h and 0.76 mV at 3 days in quercetin-treated injured animals when compared to injured saline vehicle controls. Quercetin significantly prevented decrease of glutathione levels and decreased myeloperoxidase activity. We conclude that this dietary flavonoid has therapeutic potential following brain trauma.

Tumour antigen presentation by dendritic cells (DCs) to T cells in lymphoid organs is crucial for induction of anti-tumour immune responses. It has been previously reported that tumour necrosis factor-alpha (TNF-alpha) is required for DC activation and subsequent induction of optimal immune responses, and thus DCs for anti-tumour vaccination are often generated by culture in exogenous TNF-alpha. In the present study, we investigated the effect on anti-tumour immunity of vaccination with Mut1 tumour peptide-pulsed DCs engineered to express a TNF-alpha transgene. Our data shows that transfection of DCs with recombinant adenovirus AdV-TNF-alpha resulted in greater maturation of the DCs than occurred with control DCs cultured in exogenous TNF-alpha, as determined by up-regulated expression of pro-inflammatory cytokines (e.g. interleukins 1beta and 18), chemokines [e.g. interferon-gamma-inducible protein-10 and macrophage inflammatory protein-1beta (MIP-1beta)], the CC chemokine receptor CCR7, and immunologically important cell surface molecules (CD40, CD86 and intercellular adhesion molecule-1). These transgenic DCs stimulated stronger allogeneic T-cell responses in vitro and T-cell activation in vivo; displayed 2.4-fold enhanced chemotactic responses to the MIP-3betain vitro (P<0.05); and, perhaps most importantly, trafficked into the draining lymph nodes dramatically (seven-fold, P<0.01) more efficiently than the control DCs. Our data also demonstrate that vaccination of mice with Mut1 peptide-pulsed, AdV-TNF-alpha-transfected DCs stimulated more efficient in vitro Mut1-specific CD8+ cytotoxic T-cell responses and solid tumour immunity in vivo, when compared to the in vitro TNF-alpha-cultivated DCs. Thus, DCs engineered to secrete TNF-alpha may offer a new strategy in DC cancer vaccines.

The effect of hypercholesterolemia on the number, immunological phenotype and oxidative stress-dependent processes of macrophages (MPhi) and dendritic cells (DC) was studied in New Zealand White rabbits. The left ventricular myocardium was immunohistochemically analyzed in group I (control), which was on standard chow, and groups II and III, which both received a 0.5% cholesterol-enriched diet for 96 days, but thereafter, only group III was fed standard chow for 4 months. In the myocardial interstitium of group I, (1) significantly less RAM-11-immunoreactive (ir) MPhi than S-100-ir DC were found; (2) both, MPhi and DC, were similar major histocompatibility complex (MHC) class II molecules LN3-, ISCR3-, and 2.06-ir; (3) all MPhi and most DC were manganese superoxide dismutase (MnSOD)-ir and homing receptor CD44-ir. In group II, only MPhi increased about 10-fold in the myocardium in parallel to the about 40-fold increase of the serum cholesterol levels. In group III, the elevated serum cholesterol levels significantly decreased (about 90%), while the MPhi still remained significantly increased (about 8-fold). The cellular immunoreactivities of MHC class II molecules, as well as MnSOD and CD44 did not change in groups II and III in comparison to group I. We suggest that mainly the MPhi, which increase within the myocardium of rabbits after elevation of serum cholesterol levels and remain significantly increased for a long time after decrease of the blood lipid levels, might initiate or aggravate eventual complications such as coronary atherosclerosis and myocardial fibrosis., (Copyright 2003 S. Karger AG, Basel)

The multiple sclerosis (MS) lesion is characterized by an inflammatory cell mediated attack on white matter. Oxidative stress appears to play a role in the onset and progression of MS. We reasoned that decreasing oxidative stress might ameliorate MS. One way of decreasing oxidative stress is to induce phase 2 enzymes. The model chosen to test this hypothesis was experimental allergenic encephalomyelitis (EAE) induced in the Lewis rat. The 26 animals were placed into two groups: 1) those on normal rat chow, 2) those on rat chow containing 250 mumoles t-butylhydroxyanisole (BHA)/kg. After 2 weeks, animals were administered 100 micrograms guinea pig myelin basic protein and examined daily in a blinded fashion. Twenty-nine days later, animals were sacrificed, blood collected for glutathione (GSH) measurements and tissues collected for histology. Six of the 13 control chow animals developed hindlimb weakness or paralysis while 5 developed tail weakness only. Only 1 BHA fed animal exhibited symptoms--hindlimb weakness. Clinical symptoms correlated well with the extent of perivascular lymphocyte infiltration. Animals with BHA in the diet had 20% higher red cell GSH indicting induction of phase 2 enzymes. We conclude that dietary phase 2 enzyme inducers should be examined for their ability to ameliorate MS.

We systematically investigated the impact of the relative maturation levels of dendritic cells (DCs) on their cell surface phenotype, expression of cytokines and chemokines/chemokine receptors (by DNA array and RNase protection analyses), biological activities, and abilities to induce tumor immunity. Mature DCs expressed significantly heightened levels of their antigen-presenting machinery (e.g., CD54, CD80, CD86) and numerous cytokines and chemokines/chemokine receptors (i.e., Flt-3L, G-CSF, IL-1alpha and -1beta, IL-6, IL-12, CCL-2, -3, -4, -5, -17, and -22, MIP-2, and CCR7) and were significantly better at inducing effector T cell responses in vitro. Furthermore, mice vaccinated with tumor peptide-pulsed mature DCs better survived challenge with a weakly immunogenic tumor (8 of 8 survivors) than did mice vaccinated with less mature (3 of 8 survived) or immature (0 of 8 survivors) DCs. Nevertheless, intermediate-maturity DCs expressed substantial levels of Flt-3L, IGF-1, IL-1alpha and -1beta, IL-6, CCL-2, -3, -4, -9/10, -17, and -22, MIP-2, osteopontin, CCR-1, -2, -5, and -7, and CXCR-4. Taken together, our data clearly underscore the critical nature of employing DCs of full maturity for DC-based antitumor vaccination strategies.

The relation between tumour metabolism and induction of apoptosis by gene therapy was investigated in a rat Morris hepatoma (MH3924A) model expressing the HSV thymidine kinase (HSVtk) gene. In vivo the amount of glucose transporter (GLUT1 and GLUT3 isoforms) expressing cells was determined in tumours of untreated and treated animals using immunohistochemistry. In vitro uptake studies with 2-fluoro-2-deoxy-D-glucose (FDG), 3-O-methylglucose and thymidine (TdR) and a TUNEL (TdT-mediated dUTP nick end labelling) assay for the assessment of apoptosis were done immediately and 24 h after treatment of the recombinant cells with different doses of ganciclovir (GCV). Immunohistochemistry revealed a significant increase in GLUT1 in treated tumours which showed enhanced transport activity for FDG. In vitro the FDG and 3-O-methylglucose uptake increased to 186% when compared with that of the non-treated cells immediately after incubation with GCV. However, 24 h later the FDG uptake had declined to its normal level, whereas the accumulation of 3-O-methylglucose remained elevated. The uptake of TdR, which was determined simultaneously, decreased in the acid-insoluble fraction of the cells to 27% and 11%, respectively, immediately and 24 h after therapy, while in the acid-soluble fraction it increased to 229% and to 167%, respectively. Employing the TUNEL technique, 25% of cells were found to be apoptotic 24 h after the termination of GCV treatment. Inhibition of glucose transport by cytochalasin B or competition with deoxyglucose resulted in a 78% (cytochalasin B) and 88% (deoxyglucose) decrease in FDG uptake in the recombinant hepatoma cells and in an increase in the apoptotic cell fraction. It is concluded that inhibition of enhanced glucose transport in GCV-treated cells increased apoptosis. Therefore, enhanced glucose transport seems to represent a stress reaction of tumour cells dedicated for the prevention of cell death.

Apoptosis has been described as an energy-consuming process. This combined in vivo/in vitro study investigated the effects of the antineoplastic agent gemcitabine on tumour metabolism and on the induction of apoptosis. Dynamic positron emission tomography (PET) measurements of fluorine-18 fluorodeoxyglucose (FDG) uptake were done in rats bearing Morris hepatoma prior to and after therapy with 90 mg gemcitabine/kg b.w. Furthermore, thymidine (TdR) incorporation into the DNA of these tumours was determined. In vitro measurements of FDG and TdR uptake were performed immediately and 24 h after the end of gemcitabine treatment, and the amount of apoptotic cells was determined using the TUNEL reaction. In vivo an increase in FDG transport and phosphorylation occurred early after gemcitabine treatment, although TdR incorporation into the DNA of the tumours declined. In vitro, an enhanced glucose transport, an increase in TdR uptake in the cytoplasm and a decrease in TdR incorporation in the nucleic acid fraction early after treatment occurred. Inhibition of glucose transport caused an increase in the amount of apoptotic cells. The increase in glucose uptake and TdR metabolism early after therapy is interpreted as a stress reaction of the tumour cells, protecting the cells from apoptosis during this early period after exposure to cytotoxic drugs like gemcitabine.

The study aimed to 1) quantify oxidative stress in spinal cord after crush injury at T6, 2) determine whether the administration of the procysteine compound L-2-oxothiazolidine-4-carboxylate (OTC) would up-regulate glutathione (GSH) synthesis and decrease oxidative stress, and 3) determine whether decreased oxidative stress results in better tissue and function retention. We demonstrate that spinal cord compression (5 s with a 50 g aneurysm clip) at T6 in rats results in oxidative stress that is extensive (significant increases in oxidative stress seen at C3 and L4) and rapid in onset. Indices of oxidative stress used were GSH content, protein carbonyl content, and inactivation of glutathione reductase. Administration of OTC resulted in a marked decrease in oxidative stress associated with a sparing of white matter at T6 (16+/-1.9% retained in OTC-treated animals vs. less than 1% in saline-treated). Behavioral indices in control, saline-treated, and OTC-treated animals after 6 wk were respectively: angle board scores (59 degrees, 32 degrees, and 42 degrees ), modified Tarlov score (7, 2.4, and 4.1), and Basso-Beattie-Bresnahan score (21, 5.3, and 12.9). We conclude that administration of OTC after spinal cord trauma greatly decreases oxidative stress and allows tissue preservation, thereby enabling otherwise paraplegic animals to locomote.

Dietary sulfur amino acid content is a major determinant of glutathione concentration in some tissues. We examined whether brain glutathione (GSH), a key component of antioxidant defense important for minimizing ischemic injury, was also responsive to short-term sulfur amino acid deficiency. Female Long-Evans adult rats were fed a sulfur-deficient L-amino acid defined diet for five days; the control diet was supplemented with L-cystine and L-methionine (n = 6). Sulfur amino acid deficiency was confirmed by a reduction in liver cysteine and GSH concentrations, marked decreases in food intake, and weight loss. GSH concentration analyzed by reverse-phase high performance liquid chromatography was significantly depressed in the neocortex and thalamus of deficient rats. Brain cysteine was not decreased in a parallel manner. Classical glutathione peroxidase activity was increased in the liver and brain of sulfur amino acid deficient rats. This suggests an upregulation of antioxidant defense but these findings may be complicated by alterations in tissue composition. The depletion of brain GSH by a reduced supply of dietary precursors may be important during brain ischemia when the rate of GSH utilization and the need for synthesis are increased.

Glutathione (GSH) is the principal intracellular low-molecular-weight thiol and plays a critical role in the cellular defense against agents that impose oxidative stress. A common technique to measure GSH uses reversed-phase high-performance liquid chromatography (HPLC) following derivatization with 5, 5'-dithiobis(2-nitrobenzoic acid), a technique, although reliable and sensitive, that is time consuming and laborious. A common technique to measure GSH in cultured cells is to add monochlorobimane to the culture medium where it readily enters cells to form a fluorescent GSH-monochlorobimane adduct that can be measured fluorometrically. This reaction is catalyzed by glutathione S-transferase. We reasoned that adding glutathione S-transferase and monochlorobimane to tissue homogenates would allow a rapid reliable method to measure GSH. The accuracy of the new test was assessed in homogenates of rat livers. One-half of each homogenate was assayed for GSH using a HPLC approach while the other half was assayed using the monochlorobimane approach. The two methods were found to give identical results. We conclude that the monochlorobimane fluorescent method is sufficiently specific to reliably measure tissue GSH., (Copyright 2000 Academic Press.)

Low-density lipoprotein (LDL) can be transformed to an atherogenic moiety by nonoxidative, enzymatic degradation. Enzymatically degraded LDL induces macrophage foam cell formation, provokes release of cytokines, and also activates complement. To determine whether complement activation may contribute to atherogenesis, 6 pairs of homozygous C6-deficient rabbits and their non-C6-deficient heterozygous siblings were fed a cholesterol-rich diet for 14 weeks. Cholesterol levels and plasma lipoprotein profiles of the animals in the C6-competent and C6-deficient groups did not significantly differ, and the high density lipoprotein and LDL cholesterol ratios at the end of the experiment were 0.07+/-0.01 and 0.08+/-0.01 (SEM), respectively. However, differences in atherosclerotic plaque formation were discernible macroscopically, with extensive aortic lesions being visible in all C6-competent animals and absent in all C6-deficient animals. Aortas were sectioned from thorax to abdomen, and 10 sections were stained from each aorta. Quantification of atherosclerotic lesions and lumen stenosis with the use of computer-based morphometry documented a dramatic protective effect of C6 deficiency on the development of diet-induced atherosclerosis. We conclude that the terminal complement sequence is centrally involved in atherosclerotic lesion progression.

Oxidized low density lipoprotein (oxLDL) induces apoptosis in human macrophages (Mphi), a significant feature in atherogenesis. We found that induction of apoptosis in Mphi by oxLDL, C2-ceramide, tumor necrosis factor alpha (TNF-alpha), and hydrogen peroxide (H2O2) was associated with enhanced expression of manganese superoxide dismutase (MnSOD) and p53. Treatment of cells with p53 or MnSOD antisense oligonucleotides prior to stimulation with oxLDL, C2-ceramide, TNF-alpha, or H2O2 caused an inhibition of the expression of the respective protein together with a marked reduction of apoptosis. Exposure to N-acetylcysteine before treatment with oxLDL, C2-ceramide, TNF-alpha, or H2O2 reversed a decrease in cellular glutathione concentrations as well as the enhanced production of p53 and MnSOD mRNA and protein. In apoptotic macrophages of human atherosclerotic plaques, colocalization of MnSOD and p53 immunoreactivity was found. These results indicate that in oxLDL-induced apoptosis, a concomitant induction of p53 and MnSOD is critical, and suggest that it is at least in part due to an enhancement of the sphingomyelin/ceramide pathway.

We investigated the effect of alterations of blood cholesterol levels on macrophages (mphi) in the myocardium of New Zealand White (NZW) rabbits. Three groups of NZW rabbits were used: controls, rabbits fed a 0.5% cholesterol-enriched diet (CH-D) for 96 days, and rabbits fed a 0.5% CH-D for 96 days followed by normal chow for 4 months. Immunohistochemical analysis by mAbs directed against mphi (RAM-11) and Mn superoxide dismutase (MnSOD) were quantified by computer-assisted morphometry. Using cultured human and rabbit mphi, a cross-reaction of the human MnSOD mAbs was found as well as the predominant localization of MnSOD-immunoreactivity (IR) in mitochondria. In group 1, only a very few RAM-11-immunoreactive (ir) mphi occurred in the interstitial space of the myocardium. In group II blood cholesterol levels significantly increased in parallel with the numbers of mphi, which often contained lipid droplets (foam cells). Although blood cholesterol concentrations regressed about 10-fold in group III, mphi in the myocardium were found to be reduced only about 20%. Most mphi were also MnSOD-ir. In atherosclerotic coronary arteries RAM-11-IR was located in mphi and also extracellularly, whereas MnSOD-IR was found only in mphi. Drastically induced MnSOD in the mitochondria of mphi is suggested as an indicator of increased oxidative stress caused by in vitro conditions or by phagocytosis of low-density lipoprotein in vivo. Elevation of the cholesterol level leads to a long-term increase and its regression results in a delayed reduction of such mphi, which seem to play a key role in the atherogenesis of the coronary arteries as well.

The objective of the study was to analyze the intracellular antioxidative response of macrophages (Mphi) exposed to increased levels of low density lipoprotein (LDL). We studied manganese superoxide dismutase (MnSOD) and, in part, GSH in cultured human and rabbit Mphi, and in atheromatous arterial tissue of humans and heritable hyperlipidemic (HHL) rabbits. Incubation of human Mphi with oxidized-LDL (ox-LDL) resulted in an induction of MnSOD mRNA production as shown by RT-PCR. MnSOD immunoreactivity (IR) was found to be located in the mitochondria of Mphi. In HHL rabbits, MnSOD activity and GSH concentration were significantly increased in atherosclerotic intima compared to the media of the aorta, but significantly decreased (P<0.01) in larger plaques compared with smaller ones, resulting in a significant inverse correlation of MnSOD activity (r=-0.67, P<0.001) and GSH concentration (r=-0.57, P<0.01) with plaque size. Immunohistology of the atherosclerotic intima revealed MnSOD-IR in Mac-1 (CD 11b/CD 18)-immunoreactive (ir) Mphi of human arteries and, similarly, in RAM-11-ir Mphi of rabbit ones. The relation of MnSOD-ir Mphi decreased with plaque advancement, which is consistent with biochemical findings. Most MnSOD-ir Mphi in atherosclerotic plaques revealed TUNEL-positive nuclei, indicating DNA strand breaks, and p53-IR. We conclude that mitochondrial antioxidants such as MnSOD are induced in Mphi in vitro and in atherosclerotic arteries as a reply to increased mitochondrial oxidation. As normal consequences of an increased oxidative stress due to the exposure to ox-LDL nuclear DNA strand breaks occur, which are suggested to be a signal to increase p53 protein levels. Reactive oxygen species-mediated mitochondrial-dependent pathways are suggested as major contributing pathomechanisms to nuclear damage, which eventually may result in apoptosis. A common response to increased oxidative stress due to modified LDL is presumed in rabbit and human atherosclerotic plaques.