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The interrelationship of dietary cholesterol, copper and zinc on plasma lipids and tissue copper and zinc levels in the rat.Nadar, Anand. January 1992 (has links)
No abstract available. / Thesis (M.Sc.)-University of Durban-Westville, 1992.
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The interrelationship of dietary cholesterol, copper and zinc on plasma lipids and tissue copper and zinc levels in the rat.Anand, Nadar. January 1992 (has links)
No abstract available. / Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 1992.
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A quantification of heat load as assessed by indicators of tissue damage in rats.Manjoo, Mahomed. 22 October 2013 (has links)
Heatstroke is an illness that occurs when body temperature is grossly
elevated, causing widespread tissue damage. The extent of tissue
damage depends on the level of body temperature elevation and the
duration. Despite the fact that the diagnosis of heatstroke is based
on sound scientific principles, namely the elevation of serum enzyme
levels as indicators of tissue damage, the sensitivity of these
parameters of tissue damage in the prodromal period of heatstroke is
less well established, especially for sub-lethal stress conditions.
Furthermore, it is not known to what extent given elevations in serum
enzyme levels reflect the nature of various combinations of hyperthermia
and its duration as sustained during the prodromal period.
In an attempt to throw some light on the questions posed above
anaesthetized rats were exposed to three different sets of thermal
conditions. However, the amount of heat gained over and above baseline
levels was controlled to a 20% rise irrespective of the experimental
conditions. Above this increment animals did not survive thus
indicating excessive stress. Plasma enzyme levels were assayed in
each group of animals upon termination of stress, six hours post-stress
and 24 hours post-stress in order to investigate the patterns of enzyme
release as well as the sensitivity of the respective indicators of
tissue damage.
On the basis of plasma enzyme assays, the tissue damage sustained
during these particular experimental conditions was mild to moderate,
completely reversible, not indicative of heatstroke but merely of
generalized tissue damage. The results suggest that in addition to
the established positive relationship between the level and duration
of hyperthermia and tissue damage, a third component,namely the rate
of rise in body temperature, nay constitute an important factor in
the ultirrate pathology. In this regard, i.e. sub-lethal stress,
creatine kinase proved to be the most sensitive and, therefore, the
most useful parameter of tissue damage. / Thesis (M.Sc.)-University of Durban-Westville, 1984.
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Development and application of an ELISA method of analysis for fumonisinsBiden, Patricia May January 2000 (has links)
Fumonisins, mycotoxins produced by the fungus, Fusarium moniliforme, which grows on maize, are a major worldwide agricultural problem. Consumption of contaminated maize feeds causes a wide variety of toxic effects in animals depending on the species of animal. In
humans, high concentrations of fumonisins have been shown to correlate with increased incidence of oesophageal cancer (OC). Most analyses for fumonisins are done using high performance liquid chromatography (HPLC) which requires time-consuming extraction and clean-up prior to preparation of a fluorescent derivative. Enzyme-linked immunosorbent assays (ELISA), which are sensitive and specific, are a viable alternative but commercially
available antibodies and kits are extremely expensive.
Polyclonal antibodies against fumonisin B, (FB,) were raised in chickens and rabbits; all animals produced antibodies from week 2 onwards, the highest titre was at week 8 from one of the chickens. Cross-reactivities with FB, analogues were checked. A sensitive, quantitative competitive indirect ELISA (CI-ELISA) was developed and optimised; range 0.2 to 20 ng/ml
(in buffer), detection limit 0.2 nglml (in buffer), intra-assay coefficient of variation (CV) was 5.33 % and inter-assay 7.04%.
This method was adapted to analyse human plasma and urine samples. After removal of proteins by boiling, the range of recoveries of FBI were 94.7% toI12.4% at 4 ng/ml; and 94.6% to 108.7% at 8 ng/ml. Blood and urine samples from patients with OC (40 plasma, 17 urine), controls (21 plasma, 12 urine) and patients with other forms of cancer (20 plasma, 10
urine) were collected from hospitals in the Durban Metropolitan area and analysed for fumonisins. Detectable levels (>0.4 nglml) were found in 86.9% of plasma samples and 94.9% of urine samples. Statistical evaluation showed a highly significant difference between
plasma results for OC and controls (p<0.000 1) but no significant difference between the urine results. Comparison of other forms of cancer and controls showed no significant differences for either the plasma or the urine samples. However, there was a highly significant difference
between the OC and other forms of cancer results for both plasma (p<0.005) and urine (p<0.05) samples. Some samples (9 plasma, 8 urine) were checked by HPLC. For plasma samples there was correlation between the ELISA and HPLC methods (r = 0.656, p<0.005) but not for urine samples.
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Endotoxaemia in intestinal dysfunction in experimental animals : intestinal ischaemia and hyperthermia.Gathiram, Premjith. January 1988 (has links)
Endotoxins or lipopolysaccharides (LPS), highly toxic component of the outer membrane of gram-negative bacteria, are normally present in the mammalian gut lumen.In this thesis, I investigated, in laboratory animals, whether these gut-derived endotoxins play a role in pathophysiology resulting from intestinal dysfunctions caused by intestinal ischaemia and heat-stress.In primates, reperfusion of the splanchnic region after a temporary ischaemia was followed by a rapid increase in LPS concentration, first in the hepatic portal plasma and, ten minutes later, in the systemic arterial plasma. Rises in plasma LPS concentrations during or following the temporary intestinal ischaemia was prevented by prophylactic administrations of corticosteroids, anti-LPS IgG antibodies and oral, non-absorpable, antibiotics agents which appear to stabilize cellular membranes, aid the reticuloendothelial system in removal of LPS from the circulation and destroy the intestinal aerobic gramnegative bacteria respectively. In addition, administration of therapeutic anti-LPS antibodies also rapidly reduced the plasma LPS concentrations to baseline during an endotoxaemia.
In a control heat-stress model, elevations in plasma LPS concentration commenced at rectal temperatures greater than 41,SoC. Like the intestinal ischaemia model, this occurred first in the hepatic portal plasma, and 10-15 minutes later, in the systemic arterial plasma. Peak plasma LPS levels of about 0,3 ng/ml, measured in heat-stressed primates, have proved in previous studies, to be toxic. A rapid decline in mean arterial pressure was
followed by increases in plasma LPS concentrations and heart rates. Reductions in splanchnic blood flow and consequent local ischaemia coupled with thermal injury to the intestinal wall and the liver, may have permitted rises in plasma LPS concentration. Furthermore, as in the
ischaemia model, prophylactic administrations of corticosteroids, anti-LPS IgG antibodies, and oral, nonabsorbable antibiotics prevented a rise in plasma LPS concentration. Of importance, prophylaxis with intravenous corticosteroids and 'anti-LPS IgG antibodies increased the survival rates significantly in heat stroke in primates. In addition, monkeys having high titres of "natural" antiLPS IgG antibodies had lower plasma LPS concentrations and survived the induced-heat stroke. It is suggested that other pathophysiologic conditions which compromise the integrity of the gut wall would also lead to the development of an endotoxaemia, and that gutderived endotoxins contribute to the athogenesis of heat stroke and treatments with corticosteroids and anti-LPS IgG antibodies may prove beneficial in other endotoxinrelated disorders. / Thesis (Ph.D.)-University of Natal, Durban, 1988.
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An immunohistochemical evaluation of the effect of salt (NaCI) on adrenal adrenomedullin content in Dahl rats.Hariram, Arvind. January 2003 (has links)
Adrenomedullin (ADM) is a 52 amino acid vasodilator peptide isolated, in 1993, from human pheochromocytoma. It has been demonstrated in the adrenal medulla of several mammalian species, including humans and rats. There have been conflicting results of the tissue distribution in the adrenal cortex. Hypertension is a complex trait with multiple genetic and environmental influences. Furthermore, salt-sensitive hypertension is characterized by a cluster of renal, hormonal, and metabolic derangements that might favour the development of cardiovascular and renal complications. Therefore the objective of this study was to investigate the adrenal distribution of ADM as well as to semi-quantitatively assess the adrenomedullin secretory capacity of the adrenal gland in the rat model of salt sensitive hypertension. Fourty-four male weanling rats were divided into 4 experimental groups and placed on a dietary regimen for 6 weeks viz. Dahl salt sensitive (DSS) rats on a high sodium diet (8% NaCl), DSS on a normal sodium diet (1% NaCl) matched with normotensive Dahl salt resistant (DSR) rats on the same dietary treatments. Blood pressure was monitored by tail-cuff readings and by the end of the six weeks, the DSS rats developed hypertension with tachycardia irrespective of the diet they were fed. The normal sodium diet was found to delay the development of hypertension, whilst the high sodium diet exacerbated the development of hypertension. Kidney weights and heart weights were greater in DSS rats than DSR rats probably due to their renal pathology or cardiac hypertrophy. Adrenomedullin immunopositivity was found predominantly in the adrenal medulla, and to varying degrees in the zona glomerulosa and zona reticularis of the adrenal cortex. The semi-quantitative analysis indicate that there was a 6.3 fold increase in ADM content of DSS rats compared to the DSR rats, where both consumed the 1% NaCI supplemented diet (DSR : 5.98 ± 0.3 vs. DSS : 37.85 ± 0.5, P / Thesis (M.Sc.)-University of Durban-Westville, 2003.
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Immunohistochemical and ultrastructural evaluation of the pathology and aetiopathogenesis of keloid formation.Bux, Shamin. January 2013 (has links)
Introduction
Keloids are formed by the excessive production of scar tissue, which extends beyond the margins
of the original injury, often resulting in lesions of grotesque dimensions.
Keloids present a major dilemma to surgeons because of the high recurrence rate with recurrent
growth often larger than the original keloid. The high recurrence rate and the poor response of
keloids to therapy present a great challenge to surgeons. The numerous therapeutic regimens
demonstrate that to date there is no single therapy that is absolutely successful. Therefore, it is
necessary to comprehensively establish the pathology of keloids and to determine the
aetiopathogenesis of the lesion in order to eventually provide unfailing specific effective treatment
and to better understand the mechanisms regulating fibrosis in various fibroproliferative diseases.
Aim
To evaluate the pathology and aetiopathogenesis of keloid formation.
Methods
The research protocol for the study was approved by the Nelson R Mandela Faculty of Medicine
Ethics Committee. Informed consent was obtained before the biopsies were taken. Keloid and
non-lesional skin biopsies were obtained from thirty two patients who had multiple lesions in
various locations, bringing the total number of keloids and apparently normal skin biopsies
processed and examined to fifty eight. The biopsied specimens were processed for paraffin wax
embedment and routine haematoxylin and eosin, differential and immunocytochemical staining.
Sections were scrupulously examined using the Olympus BH-2 microscope; features pertinent to
the study were photographed with the Olympus DP 10 microscope digital camera system. The
stored images were studied, using the Camedia graphics processing programme.
Results
The results of the study showed that keloids comprise many distinct regions categorized as: the
zone of hyalinising collagen bundles, fine fibrous areas, areas of inflammation, zone of dense
regular connective tissue, nodular fibrous area and area of angiogenesis. Fibroblastic phenotypes
present ranged from spindle, fibrohistiocytic, epitheloid, elongated flattened condensed
fibroblastic cells to few wavy, fuzzy, polygonal and atrophic cell types. Immunocytochemically
these cells were vimentin-positive and actin- and desmin-negative. Few myofibroblastic
phenotypes were also identified and these were vimentin- and alpha smooth muscle actin-positive
and desmin-negative. The fibroblastic and myofibroblastic phenotypes were in proliferative or
degenerative stages and pathological features exhibited were the presence of vesicular, degenerate
or calcified nuclei; nuclear and plasma membrane damage; cytoplasmic and nucleoplasmic
clearing; atrophy, pyknosis and swelling.
Severe, moderate to mild paravascular inflammation was observed around the microvessels of the
sub-papillary plexus and within the keloid. There was compression and occlusion of small blood
vessels, coagulation necrosis and dissolution of mural cells of small blood vessels and small
peripheral nerves. Also present in keloids were oedematous areas, disorganised and hyalinised
connective tissue fibres and increased numbers of degranulated and degranulating mast cells.
Elastic fibres in keloids were minimal or absent whereas at the border of keloids there was an
increase.Discussion
Degenerate, occluded and compressed microvessels were a widespread pathological feature in
keloids. This resulted in impaired vascular supply to each of the keloid regions which impacted
directly on the pathology of keloids where degeneration and necrosis, manifesting the lack of
nutrients and oxygen to tissue, were found throughout the keloid. The vascular supply was
impaired because of the chronic inflammatory destruction of the microvessels and the elevated
stress within keloids. Factors contributing to increased intrinsic stress were: 1) the lack of elastic
fibres in keloids which decreased the elastic limit, leading to effects of excessive deformational
force which were compression and stiffening of tissue; 2) the high tension skin covering keloid
prone areas had low stretch and a low elastic modulus; 3). protruding hard connective tissue such
as bony prominences or cartilage into the dermis of keloid prone skin; 4) contractile forces exerted
by wound healing fibroblastic cells; and 5) external forces. Compression and occlusion of blood
vessels induced ischaemic and reperfusion tissue injury. During the reperfusion phase blood rich
in growth factors returned to tissue stimulating tissue growth. Tissue growth was also promoted
by elevated internal stress which stimulated increasing levels of gene expression, collagen
synthesis and mitotic activity. All these growth promoting effects resulted in keloid formation. / Thesis (Ph.D.)-University of KwaZulu-Natal, Durban, 2013.
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The contribution of inflammatory mediators to delayed secondary muscle damageVan de Vyver, Mari 03 1900 (has links)
Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Background: Understanding the contribution of divergent individual response patterns remains a key objective in identifying mechanisms of inflammation and potential factors limiting the resolution of inflammation. The purpose of this research project was to investigate downstream effects of inflammation following exercise-induced muscle damage in human subjects.
Methods: For three different studies, a total of 53 untrained healthy male participants were recruited and divided into a non-exercising control (n=13) and exercise-induced muscle damage groups (n=40). The study design for the three studies was the same (with few exceptions): Downhill running (DHR) (12 x 5min bouts, 10% decline, 15 km.h-1) with blood samples taken pre, post, after 2 and 4 hours post-exercise (2h, 4h) and on days 1, 2, 3, 4 and 7 (d1-d7). Serum was analysed for creatine kinase activity (CK), myoglobin (Mb), cortisol, cytokine (TNFα, IL-1ra, IL-1β, IL-4, IL-6, IL-8, IL-10, sIL-6R), chemokine (G-CSF, MIP-1β) and adhesion factor (sICAM-1, sP-selectin) concentrations. Tissue degradation was assessed by serum matrix metalloprotease (MMP-9) and myeloperoxidase (MPO) content. White blood cell differential count was determined and the surface expression of various cluster of differentiation factors (CD11b, CD163, CD68, CD88, CD34) as well as intracellular MPO were assessed in whole bood using flow cytometry. Nuclear localization of the inflammatory mediator NFĸB in isolated perhipheral blood mononuclear cells (PBMCs) was determined using immunofluorescence microscopy. Muscle biopsies (vastus lateralis) taken at baseline, 4h, d1 and d2 were analysed for fibre type, inflammatory and stress-induced pathways (STAT3, IĸBα, p38MAPK), myogenic factors (MyoD, myogenin), neutrophil activity (MPO) and satellite cell number (Pax7). Results: Participants in the DHR group were subdivided into those with a normal recovery (DHR1) and those who developed secondary damage (DHR2). CK peaked on d1 in both subgroups (DHR1: 1512 ± 413 u.L-1, DHR2: 1434 ± 202 u.L-1) and again on d4 only in the DHR2 group (1110 ± 184 u.L-1). A similar IL-6 and IL-10 response was evident immediately post DHR in all individuals. Additional IL-6 was released in the DHR2 subgroup peaking at 4h (10.3 ± 4.2 pg.mL-1) whereas IL-10 had returned to baseline. IL-1ra (23.6 ± 8.8 pg.mL-1), CD68+ (5%) and CD163+ (3%) monocytes were significantly higher in the DHR2 subgroup. Neutrophil count at 2h (DHR1: 8.6 ± 0.8 x109 cells.L-1, DHR2: 11.4 ± 1.8 x109 cells.L-1) was significantly (p<0.02) correlated to CK activity on d4. PBMC NFĸB p65 nuclear localization was slightly less at 2h in the DHR2 compared to the DHR1 and control groups. Intramuscular STAT3 signalling and MPO were significantly higher in the DHR2 compared to the DHR1 subgroup at 4h and d2 respectively. The progenitor cell response was similar for all DHR individuals with an increase in Pax7+ SC observed at 4h (0.06 ± 0.01 Pax+ SCs/fibre) and d1 (0.07 ± 0.02 Pax+ SCs/fibre).
Conclusion: Healthy young men can be divided into those with a adequate and those with a less efficient capacity to control the post damage inflammatory response. The early cytokine response, especially IL-6, seems to be a key role player in the cascade of events leading to late secondary skeletal muscle damage. / AFRIKAANSE OPSOMMING: Agtergrond: Die begrip van uiteenlopende individuele reaksie patrone, is belangrik in die identifisering van faktore asook meganismes betrokke in die ontwikkeling en resolusie van inflammasie. Die doel van hierdie navorsingsprojek was om die gevolge van oefening-geïnduseerde spierskade en inflammasie te ondersoek in menslike proefpersone.
Metodiek: ‘n Totaal van 53 gesonde mans is tydens drie verskillende studies, gegroepeer in ’n kontrole (geen oefening) (n=13) en oefening geinduseerde spier skade (DHR) groep (n=40). Die uitleg van de studies was eenders (met min uitsonderings): Afdraende hardloop (12 x 5min hardloop sessies, 10% afdraende, 15km.h-1) met bloed monsters geneem voor, na, 2 ure, 4 ure (pre, post, 2h, 4h) en op dag 1, 2, 3, 4 en 7 (d1-7). Serum is ontleed vir die volgende: kreatien kinase aktiwiteit (CK), kortisol, sitokiene (TNFα, IL-1ra, IL-1β, IL-4, IL-6, IL-8, IL-10, sIL-6R), chemokien (G-CSF, MIP-1β) en adhesie molekuul (sICAM-1, sP-selectin) konsentrasies. Weefsel degradasie is vasgestel deur die teenwoordigheid van matriks metalo-protease-9 (MMP-9) en miëloperoksidase (MPO) in serum te meet. Differensiële witbloed sel (WBC) telling asook die teenwoordigheid van sekere differensiasie faktore (CD11b, CD163, CD68, CD88, CD34) op die sel oppervlak asook intrasellulêre MPO vlakke is bepaal deur gebruik te maak van vloeisitometrie. Die lokalisering van NFĸB in die selkerne van geïsoleerde bloed mononukleêre selle (PBMC) is bepaal deur fluoriserende mikroskopie. Spierbiopsies (vastus lateralis) geneem tydens rus (basislyn), na 4h, en op d1 en d2 is ontleed vir veseltipe, inflammatoriese en stresverwante faktore (STAT3, IĸBα, p38 MAPK), miogeniese faktore (myoD, myogenin), neutrofiel aktiwiteit (MPO) en aantal satelliet selle (Pax7).
Resultate: Deelnemers in die DHR-groep is onderverdeel in twee groepe. Persone wat normaalweg herstel het is saam gegroepeer (DHR1) en diegene wat sekondêre skade ontwikkel het is saam gegroepeer (DHR2). CK aktiwiteit in serum het hoogtepunte bereik op d1 in beide subgroepe (DHR1: 1512 ± 413 u.L-1, DHR2: 1434 ± 202 u.L-1) en weer op d4 in die DHR2 groep (1110 ± 184 u.L-1). 'n Soortgelyke IL-6 en IL-10 reaksie is onmiddellik na oefening (in al die proefpersone) waargeneem. Addisionele IL-6 is vrygestel in die DHR2 subgroep en het ’n hoogtepunt bereik na 4h (10.3 ± 4.2 pg.mL-1), terwyl IL-10 reeds teruggekeer het na rustende waardes. IL-1ra (23.6 ± 8.8 pg.mL-1), CD68+ (5%) en CD163+ (3%) monosiete was aansienlik hoër in die DHR2 subgroep. Neutrofieltelling na 2h (DHR1: 8.6 ± 0.8 x109cells.L-1, DHR2: 11.4 ± 1.8 x109cells.L-1) het verband (p <0,02) gehou met CK-aktiwiteit op d4. In vergelyking met die DHR1 en kontrole groep was die lokalisering van NFĸB p65 in PBMC selkerne na 2h effens minder in die DHR2 subgroep. STAT3- en MPO-vlakke in die spiere was aansienlik hoër in die DHR2 subgroep as in die DHR1 subgroep na 4h en op d2 onderskeidelik. Die spierherstel proses was eenders vir alle individue wat aan die oefening deelgeneem het; 'n toename in Pax7+ Satelietselle (SC) is waargeneem na 4h (0.06 ± 0.01 Pax+ SC/spiervesel) en op d1 (0.07 ± 0.02 Pax+ SC/spiervesel).
Gevolgtrekking: Gesonde jong mans kan verdeel word in diegene met 'n bevoegde en diegene met 'n minder doeltreffende vermoë om oefenings-geïnduseerde spierskade en die inflammatoriese reaksie te beheer. Die sitokien-reaksie, veral IL-6, blyk om 'n belangrike rolspeler in die ontwikkeling van sekondêre skeletspierskade te wees.
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Novel therapeutic agents that blunt hyperglycemia-induced cardiac contractile dysfunctionMapanga, Rudo Fiona 03 1900 (has links)
Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Introduction
Diabetes constitutes a major health challenge. Since cardiovascular complications are common in
diabetic patients this will further increase the overall burden of disease. Furthermore, stress-induced
hyperglycemia in non-diabetic patients with acute myocardial infarction is associated with higher inhospital
mortality. Hyperglycemia-induced oxidative stress results in DNA damage and subsequent
activation of poly-ADP-ribose polymerase (PARP) as a restorative mechanism. However, PARP
attenuates glyceraldehyde–3-phosphate dehydrogenase (GAPDH) activity, thereby diverting upstream
glycolytic metabolites into damaging non-oxidative glucose pathways (NOGP). For example,
hyperglycemia-induced stimulation of four NOGP, i.e. the polyol pathway, hexosamine biosynthetic
pathway (HBP), advanced glycation end products (AGE), and PKC activation elicit cardiovascular
complications. The current thesis examined the regulation of NOGP in the setting of ischemia and
reperfusion under hyperglycemic conditions.
Here we hypothesized that administration of two unique therapeutic interventions, i.e. oleanolic acid
(OA; clove extract) and benfotiamine (BFT; vitamin B1 derivative), can blunt oxidative stress and
NOGP-induced cardiac dysfunction under hyperglycemic conditions following ischemia and
reperfusion. Our choice for these agents was based on the principle that OA possesses antioxidant
properties; and BFT stimulates transketolase (pentose phosphate pathway [PPP] enzyme) thereby
shunting flux away from the NOGP pathways. Additionally, hyperglycemia-induced oxidative stress
can also result in dysregulation of the ubiquitin-proteasome system (UPS) that removes misfolded
proteins. There are conflicting data whether increased/decreased UPS is detrimental with
hyperglycemia and/or in response to ischemia and reperfusion. In light of this, we also hypothesized
that BFT and OA act as novel cardio-protective agents by diminishing myocardial UPS activity in
response to ischemia and reperfusion under acute hyperglycemic conditions.
Materials and Methods
For the first part of the study, we employed several experimental systems: 1) H9c2 cardiac myoblasts
were exposed to 33 mM glucose for 48 hr vs. controls (5 mM glucose); and subsequently treated with
two OA doses (20 and 50 μM) for 6 and 24 hr, respectively; 2) Isolated rat hearts were perfused ex
vivo with Krebs-Henseleit buffer containing 33 mM glucose vs. controls (11 mM glucose) for 60 min,
followed by 20 min global ischemia and 60 min reperfusion ± OA treatment; 3) Infarct size was determined using Evans Blue dye and 1% 2,3,5-triphenyl tetrazolium chloride (TTC) staining with 20
min regional ischemia and 2 hr reperfusion 4) In vivo coronary ligations were performed on
streptozotocin-diabetic rats ± 0.45 mg/kg OA administration within the first two minutes of reperfusion;
and 5) Effects of long-term OA treatment (2 weeks) on heart function were assessed in streptozotocin
(STZ)-diabetic rats. Here, STZ was dissolved in citrate buffer (p.H 6.3) and diabetes was induced by
administering 60 mg/kg i.p Tissues were collected at the end of the global ischemia experiments and
analyzed for oxidative stress, apoptosis, UPS activity and HBP activation.
For the second part of the study we employed several experimental systems: 1) Isolated rat hearts
were perfused ex vivo with Krebs-Henseleit buffer containing 33 mM glucose vs. controls (11 mM
glucose) for 90 min, followed by 30 min global ischemia and 60 min reperfusion ± 25, 50 and 100 μM
BFT treatment, respectively, added during the first 20 min of reperfusion; 2) Infarct size determination
as in #3 above but with 30 min regional ischemia and 2 hr reperfusion ± 100 μM BFT treatment; and 3)
In vivo coronary ligations performed on streptozotocin-diabetic rats ± 0.50 mg/kg BFT treatment within
the first two min of reperfusion. In parallel experiments, NOGP inhibitors were added during the first 20
min of reperfusion. The following inhibitors were individually employed: AGE pathway (100 μM
aminoguanidine); PKC (5 μM chelerythrine chloride); HBP (40 μM 6-diazo-5-oxo-L-norleucine); and
polyol pathway (1 μM zopolrestat); Infarct size determination as in #2) with 30 min regional ischemia
and 120 min reperfusion ± similar treatments.
Results
Our data show decreased cardiac contractile function in response to ischemia and reperfusion under
hyperglycemic conditions. This was linked to increased PARP and attenuated GAPDH activities,
together with higher activation of the NOGP. Moreover, we found elevated myocardial oxidative stress,
UPS and cell death under these conditions. OA treatment resulted in cardio-protection, i.e. for ex vivo
and in vivo rat hearts exposed to ischemia and reperfusion under hyperglycemic conditions. In
parallel, OA decreased oxidative stress, apoptosis, HBP flux and UPS activity following ischemia and
reperfusion. Long-term OA treatment also improved heart function in streptozotocin-diabetic rats. Our
data also reveal that acute BFT treatment significantly decreased myocardial oxidative stress and
apoptosis, and provided cardio-protection in response to ischemia and reperfusion under
hyperglycemic conditions. In parallel, BFT blunted hyperglycemia-induced activation of four NOGP in
the rat heart. Acute administration of each of the NOGP inhibitors decreased PARP and enhanced GAPDH
activities, while diminishing oxidative stress and myocardial apoptosis. Moreover, each of the NOGP
inhibitors (individually) employed blunted activation of the other three pathways here examined. Hearts
treated with NOGP inhibitors also displayed improved functional recovery and smaller infarct sizes
following ischemia and reperfusion. Interestingly, NOGP inhibitors resulted in the same degree of
change (for all above-mentioned parameters evaluated) when compared to each other.
Conclusions
This study shows that acute and chronic hyperglycemia trigger myocardial oxidative stress that
eventually results in NOGP activation and contractile dysfunction following ischemia and reperfusion.
Moreover, our findings establish - for the first time as far as we are aware - that there is a convergence
of downstream NOGP effects in our model, i.e. increased myocardial oxidative stress, further NOGP
pathway activation, apoptosis, and impaired contractile function. Thus a vicious metabolic cycle is
established whereby hyperglycemia-induced NOGP further fuels its own activation by generating even
more oxidative stress, thereby exacerbating damaging effects on the heart under these conditions. We
also found that both OA and BFT treatment blunted high glucose-induced detrimental effects and
provided robust cardio-protection in response to ischemia and reperfusion under hyperglycemic
conditions (acute and chronic). These findings suggest that the UPS may be a unique therapeutic
target to treat ischemic heart disease in individuals that present with stress-induced, acute
hyperglycemia. Moreover, BFT exhibited its cardio-protective effects by NOGP inhibition after
ischemia and reperfusion under acute and chronic high glucose conditions. A similar effect was
observed at baseline although the underlying mechanisms driving this process still need to be
elucidated. In summary, the findings of this thesis are highly promising since it may eventually result in
novel, cost-effective therapeutic interventions to treat acute hyperglycemia (in non-diabetic patients)
and diabetic patients with associated cardiovascular complications. / AFRIKAANSE OPSOMMING: Inleiding
Diabetes skep ‘n groot gesondheidsuitdaging. Omrede kardiovaskulêre komplikaseis algemeen onder
diabetiese pasiënte is, sal dit oorkoepelend die las van hierdie siekte verder laat toeneem. Verder
word stresgeïnduseerde hiperglukemie in nie-diabetiese pasiënte met akute miokardiale infarksie
geassosieër met ‘n hoër binne-hospitaalmortaliteit. Hiperglukemies-geïnduseerde oksidatiewe stres
veroorsaak DNA skade, en gevolglike aktivering van poli-ADF-ribose polimerase (PARP), as ‘n
herstelmeganisme. Nietemin, PARP verminder gliseraldehied–3-fosfaatdehidrogenase (GAPDH)
aktiwiteit om sodoende die opstroom glikolitiese metaboliete te herlei na skadelike nie-oksidatiewe
glukose weë (NOGW). Byvoorbeeld, hiperglukemie-geïnduseerde stimulasie van vier NOGW, i.e. die
poliolweg, heksosamienbiosintetiese weg, (HBW), gevorderde glukasie eindprodukte (GGE), en PKC
aktivering, lei tot kardiovaskulêre komplikasies. Die huidige tesis ondersoek die regulering van NOGW
in ‘n isgemiese-reperfussie onder hiperglukemiese toestande.
Ons hipotetiseer dat die toediening van twee unieke terapeutises intervensies, i.e. oleanoliese suur
(OS, naaltjie ekstrak), en benfotiamien (BFT, vitamien B1 derivaat) oksidatiewe stress kan versag, en
NOGW geinduseerde kardiale disfunksie onder hiperglukemiese toestande na ischemie en
reperfussie. Ons keuse vir hierdie middels is gebaseer op die beginsel dat OS antioksidanteienskappe
bevat, en dat BFT transketolase (pentosefosfaat weg (PFW) ensiem) stimuleer en
sodoende die fluks weg van die NOGW weg veroorsaak. Addisioneel kan hiperglukemiegeïnduseerde
oksidatiewe stres ook tot wanregulering van die ubikwitien-proteosoomsisteem (UPS)
wat wangevoude protïene verwyder, aanleiding gee. Daar bestaan kontrasterende data oor ‘n
verhoogde/verlaagde UPS, tesame met hiperglukemie en/of in reaksie tot isgemie-reperfussie. In die
lig hiervan, hipotetiseer ons dat BFT en OS as ‘n nuwe kardiobeskermingsmiddel kan optree deur
miokardiale oksidatiewe stres en UPS aktiwiteit in reaksie op isgemie-reperfussie tydens akute
hiperglukemiese toestande kan verlaag.
Materiale en Metodes
Vir die eerste deel van die studie het ons van verskeie eksperimentele sisteme gebruik gemaak: 1)
H9c2 kardiale mioblaste is aan 33 mM glukose vir 48 uur vs. kontrole (5 mM glukose) blootgestel; en
gevolglik met twee OS dosisse (20 en 50 μM) vir 6 en 24 hr, onderskeidelik behandel; 2) geïsoleerde
rotharte is ex vivo met Krebs-Henseleit buffer, wat, 33 mM glukose vs. kontrole (11 mM glukose) bevat, vir 60 min geperfuseer, daarna is dit deur 20 min globale isgemie gevolg en 60 min reperfussie
± OS behandeling; 3) Infarkgrootte is bepaal deur Evans bou kleursel en 1% 2. 3-5 tripfeniel
tetrazoloimcholierd (TTC) kleuring met 20 minute regionale ischemie, en 2 uur reprefussie 4) In vivo
koronêre liggasies is op streptozotosien-diabetiese rotte uitgevoer ± 0.45 mg/kg OS toedienning binne
die eerste twee minute van reperfussie; en 5) effekte van langtermyn OS behandeling (2 weke) op
hartfunskie is in hierdie streptozotosien-diabetiese rotte ondersoek. Hier is STZ opgelos in ‘n
sitraatbuffer (pH 6.3), en diabetes is geinduseer deur 60mg/kg i.p. toe te dien. Weefsels is aan die
einde van die globale isgemie eksperimente versamel, en vir oksidatewe stres, apoptose, UPS
aktiwiteit en HBW aktivering, ontleed.
Vir die tweede deel van die studie het ons van verskeie eksperimentele sisteme gebruik gemaak: 1)
geïsoleerde rotharte is ex vivo met Krebs-Henseleit buffer, wat 33 mM glukose vs. kontrole (11 mM
glukose) bevat, vir 90 min geperfuseer. Daarna is dit gevolg met 30 min globale isgemie en 60 min
reperfussie ± 25, 50 en 100 μM BFT behandeling onderskeidelik, gevolg, bykomend, gedurende die
eerste 20 min reperfussie; 2) Infarkgrootte is bepaal soos in #3 hierbo, maar met 30 minute regionale
ischemie en 2 uur reperfussie ± 100 μM BFT behandeling; en 3) In vivo koronêre liggasies is op
streptozotosien-diabetiese rotte uitgevoer ± 0.50 mg/kg BFT behandeling binne die eerste twee
minute van reperfussie. Met parallele eksperimente is NOGW inhibeerders bygevoeg binne die eerste
20 min van reperfussie. Die volgende inhibeerders is individueel ontplooi: GGE weg (100 μM
aminoguanidien); PKC (5 μM chelleritrienchloried); HBW (40 μM 6-diazo-5-oxo-L-nor-leusien); en
poliolweg (1 μM zopolrestaat); 2) Infarkgrootte is bepaal soos in #2) met die uitsondering van 30 min
regionale isgemie en 120 min reperfussie ± identiese behandelings.
Resultate
Ons data toon aan dat kardiale kontraktiele funksie, in reaksie op isgemie-reperfussie onder
hiperglukemiese toestande, verlaag. Dit is verwant aan verhoogde PARP en verminderde GAPDH
aktiwiteit, tesame met ‘n hoër aktivering van die NOGW. Verder het ons bevind dat verhoogde
miokardiale oksidatiewe stres, UPS en seldood onder die toestande voorkom. OS behandeling lei tot
kardiale beskerming, i.e. vir ex vivo en in vivo rotharte wat aan isgemie-reperfussie onder
hiperglukemiese toestande blootgestel is. Parallel hiermee het OS oksidatiewe stres, apoptose, HBW
invloed, en UPS aktiwiteit na isgemie-reperfussie, verlaag. Langtermyn OS behandeling het ook
hartfunksie in streptozotosien-diabetiese rotte verbeter. Ons data vertoon verder dat akute BFT
behandeling, miokardiale oksidatiewe stres en apoptose, betekenisvol verlaag het in reaksie op isgemie-reperfussie onder hiperglukemiese toestande. Parallel hiermee het BFT hiperglukemiegeïnduseerde
aktivering van vier NOGWë in die rothart, verminder.
Akute toediening van die elk van die NOGW inhibeerders het PARP verlaag, en GAPDH aktiwiteite
verhoog, terwyl oksidatiewe stres, en miokardiale apoptose verminder. Verder het elk van die NOGW
inhibeerders wat (individueel) toegedien is, aktivering van die ander drie weë, hier ondersoek, verlaag.
Die harte wat met NOGW inhibeerders behandel is het ook ‘n verbeterde herstel en kleiner
infarkgrootte na isgemie-reperfussie getoon. Interessant is hoe die NOGW inhibeerders tot dieselfde
graad verandering (vir al die bogemelde parameters wat geevalueer is) indien dit vergelyk word teen
mekaar, gelei het.
Gevolgtrekking
Hierdie studie het bevind dat akute en chroniese hiperglukemie, miokardiale oksidatiewe stres ontlok,
en dat dit geleidelik tot NOGW aktivering en kontraktiele wanfunksionering na isgemie-reperfussie lei.
Verder het ons bevindinge vir die eerste keer, volgens ons wete, bewys dat daar ‘n ineenloping is van
afstroom NOGW effekte in ons model, i.e. verhoogde miokardiale oksidatiewe stres, verdere NOGW
weg aktivering, apoptose, en ingeperkte kontraktiele funksie. Dus, ‘n gebrekkige metaboliese siklus
word verkry waarby hiperglukemies-geïnduseerde NOGW verder sy eie aktivering aanvuur deur meer
oksidatiewe stres, en sodoende die skadelike effekte op die hart onder hierdie toestande verder
versleg. Ons het verder bevind dat beide OS en BFT behandeling, hoë glukose-geïnduseerde
skadelike effekte onderdruk, en kragtige kardiale-beskerming in reaksie op isgemie-reperfussie onder
hiperglukemiese toestande (akuut en chronies), teweeg bring. Hierdie bevindinge dui moontlik daarop
dat die UPS ‘n unieke terapeutiese teiken kan wees vir die behandeling van isgemiese hartsiekte in
individue wat presenteer met stres-geïnduseerde, akute hiperglukemie. BFT het ook sy kardiale
beskermende effekte getoon deur NOGW inhibering na isgemie-geïnduseerde reperfussie onder
aktute en chroniese hoë glukose toestande. ‘n Soorgelyke effek is tydens die basislyn waargeneem,
alhoewel die onderliggende meganisme wat hierdie proses dryf verder ondersoek moet word.
Opsommend is ons bevindinge baie belowend omrede dit daartoe kan aanleiding gee tot ‘n nuwe,
meer koste-effektiewe terapeutiese intervensie vir die behandeling van akute hiperglukemie (in niediabetiese pasiënte) en diabetiese pasiënte met geassosieërde kardiovaskulêre komplikasies. / Oppenheimer, Beit Trust and Harry Crossley
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The maladaptive effects of HIV protease inhibitors (Lopinavir/Ritonavir) on the rat heart.Reyskens, Kathleen Maria Simone Elise 12 1900 (has links)
Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Although antiretroviral treatment decreases HIV-AIDS morbidity/mortality, long-term effects include onset of insulin resistance and cardiovascular diseases. Increased oxidative stress and dysregulation of the ubiquitin-proteasome system (UPS) are implicated in protease-inhibitor (PI)-mediated cardio-metabolic pathophysiology. We hypothesized that PI treatment (Lopinavir/Ritonavir) elevates myocardial oxidative stress and concomitantly inhibits the UPS, thereby attenuating cardiac function. Lopinavir/Ritonavir was dissolved in 1% ethanol (vehicle) and injected into mini-osmotic pumps that were surgically implanted into Wistar rats for eight weeks vs. vehicle and sham controls. Subsequently, we evaluated metabolic parameters and heart function (ex vivo and in vivo methods) at baseline and following ischemia-reperfusion. PI-treated rats exhibited weight gain, increased serum LDL-cholesterol, higher tissue triglycerides (heart, liver), but no evidence of insulin resistance. It also upregulated hepatic gene expression of acetyl-CoA carboxylase β and 3-hydroxy-3-methylglutaryl-CoA-reductase, key regulators of fatty acid oxidation and cholesterol synthesis, respectively. Further, PI-treated hearts displayed impaired UPS, increased superoxide dismutase (SOD) activity and unaltered superoxide levels, and elevated peroxisome proliferator-activated receptor-γ coactivator 1-α (PGC-1α) peptide levels. Perfusion data revealed contractile dysfunction at baseline and following ischemia-reperfusion, while post-ischemic hearts exhibited decreased ATPase specific activity vs. matched controls. Early changes initiated by PI treatment resemble the metabolic syndrome and reflect a pre-atherogenic profile. Moreover, the effects of PIs on cardiac contractile function may in part be triggered by impaired UPS activity together with strain on the mitochondrial energetic system. Our study alerts to cardio-metabolic side effects of PI treatment and raises the question of the most appropriate co-therapies for patients on chronic antiretroviral treatment. / AFRIKAANSE OPSOMMING: Alhoewel anti-retrovirale behandeling MIV-VIGS morbiditeit/mortaliteit verlaag, bestaan daar langtermyn effekte soos die aanvang van insulienweerstandigheid en kardiovaskulêre siektes. Verhoogde oksidatiewe stres en wanregulering van die ubikwitien-proteosoomsisteem (UPS) word geïmpliseer met protease-inhibeerder (PI) gemediëerde kardio-metaboliese patofisiologie. Ons hipotetiseer dat PI behandeling (Lopinavir/Ritonavir) miokardiale oksidatiewe stres verhoog, en gevolglik die UPS inhibeer waardeur dit kardiale funksie verander. Lopinavir/Ritonavir is in 1% etanol (draer) opgelos en in ‘n mini-osmotiese pomp ingespuit wat chirurgies in Wistar rottes ingeplant is vir agt weke vs. draer en valskontroles. Gevolglik het ons die metabolise parameters en hartfunksie (ex vivo en in vivo metodes) op basislyn en na afloop van ischemie-reperfusie ondersoek. PI-behandelde rotte het ‘n toename in massa getoon asook verhoogde serum LDL-cholesterol, hoër weefseltrigliseriede (hart, lewer), maar geen bewys van insulienweerstandigheid nie. Dit het ook hepatiese asetielko-ensiem A karboksilase β en 3-hidrokise-3-metielglutariel KoA reduktase geenuidrukking opwaarts gereguleer, wat sleutel reguleerders van vetsuuroksidasie en cholesterolsintese onderskeidelik is. Verder, het PI-behandelde harte ingeperkte UPS, verhoogde SOD aktiwiteit en onveranderde superoksiedvlakke vertoon, asook verhoogde peroksisoomproliferator-geaktiveerde reseptor-γ ko-aktiveerder 1-α (PGC-1α) peptiedvlakke. Perfusie data toon kontraktiele wanfunskionering gedurende basislyn en na afloop van ischemie-reperfussie, terwyl post-ischemiese harte verlaagde ATPase spesifieke aktiwiteit vs gepaarde kontrole vertoon. Vroeë veranderinge wat deur PI behandeling veroorsaak word, kom ooreen met die metabolise sindroom en reflekteer op ‘n pre-aterogeniese profiel. Bowendien kan die effekte van PIs op kardiale kontraktiele funksie deels veroorsaak word deur die ingeperkte UPS aktiwiteit tesame met die las op die mitochondriale energie sisteem. Ons studie waarsku teen kardio-metaboliese newe effekte met PI behandeling en rig die vraag; wat die mees gepaste ko-behandeling vir pasiënte op chroniese anti-retrovirale behandeling is.
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