Thesis submitted in fulfilment of the requirement for the degree Doctor of Technologiae (Biomedical Technology) in the Faculty of Health and Wellness Sciences at the Cape Peninsula University of Technology Supervisor: Prof J van Rooyen Co-supervisor: Prof JL Marnewick Bellville October 2013 / Cardiovascular disease (CVD) is without a doubt one of the most challenging health issues of our time and accounts for the highest number of deaths in both developed and developing countries. Despite the huge strides that have been achieved in the diagnosis and therapeutic intervention of CVD, the disease burden still remains enormous. Therefore, this calls for novel and innovative interventions to curb the surge of CVD. The use of plant based food with bioactive phytochemicals,has a great potential to reduce the incidence of CVD, specifically in resource-strained countries. Red palm oil (RPO) and the indigenous herbal tea, rooibos have previously been shown to exhibit potential cardioprotective effects. Their health promoting properties have largely been attributed to their antioxidant and anti-inflammatory activities and emerging evidence also showed that they have the potential to modulate cell signalling events. Substancial scientific evidence proposes oxidative stress and inflammation to play an important role in the pathogenesis of cardiovascular disease. Hence, natural plant extracts such as RPO and rooibos could be recommended as adjuvants to clinical therapy to reduce the morbidity and mortality associated with CVD. This thesis reports on three studies investigating the cardiovascular protective effects that chronic feeding of either RPO, rooibos or their combination have on 1) antioxidant enzymes and the NO-cGMP pathway in myocardial tissue of spontaneous hypertensive rats, 2) the modulation of systemic and myocardial inflammation and 3) the myocardial ischaemic/reperfusion tolerance in a rat model of lypopolysaccharide induced inflammation. The aim of the first study was to investigate the effect of RPO on cardiac function in sponteneously hypertensive rats. The role of the nitric oxide cyclic-guanosine monophosphate(NO-cGMP) pathway, (as determined by the nitric oxide (NOS) activity) and the antioxidant defence system (selected antioxidant enzymes) were also investigated. Cardiac function was monitored at stabilization and reperfusion using the Langendorff perfusion system. Antioxidant enzymes were determined from left ventricular tissue, while total NOS activity was determined in the aorta and left ventricular tissue. The results show that RPO offered cardiac protection as evidenced by improved left ventricular developed pressure (LVDevP), maximum velocity of pressure rise (+dp/dt) max and fall (-dp/dt) max during reperfusion in sponteneously hypertensive rats (SHR) compared to their control counterparts. Improved function in SHR was associated with increased myocardial superoxide dismutase 2 (SOD2) protein expression compared to the normotensive rats. There was differential modulation of the NOS activity by RPO, an increase in NOS activity was observed in the aorta while a reduction in the activity of NOS was observed in the left ventricular tissue of both RPO supplemented normotensive and hypertensive rats compared to their respective control groups. These results argue a role for elevated NO production in the aorta for endothelial function maintenance. Increased SOD2 protein might lead to reduced oxidative stress. Thus, NO-cGMP pathway and antioxidant defense systems synergistically acted to restore cardiovascular function in SHR. The aim of the second study was to investigate the effect of RPO and rooibos supplementation on the modulation of systemic and myocardial inflammation in a rat model. As RPO and rooibos contain different types of antioxidants which reside and exert their biological effects in different cellular compartments, the combination of these two natural food compounds has the potential to enhance the spectrum of available dietary antioxidants in different cellular compartments, which could result in a better protection against certain pathological conditions such as inflammation. The Langendorff system and the lypopolysaccharide (LPS)-induced inflammatory model were used to determine if RPO and rooibos could protect against the negative effect of LPS-induced inflammation on baseline cardiac function. Both inflammation and dietary supplementation did not have any effect on baseline cardiac functional parameters. Our results show that administration of LPS resulted in elevated plasma levels of IL-1β in supplemented and non-supplemented rats indicating that an inflammatory response was triggered in the LPS-treated rats. However, this increase in IL-1β was counteracted by concurrent elevation of plasma IL-10 in LPS-induced rats consuming either rooibos or RPO alone. Furthermore the combination of RPO and rooibos enhanced myocardial IL-10 levels in LPS-induced rats. This data shows a difference in response to LPS injection between the myocardium and the systemic circulation. The results indicate that the combination of these two natural food substances exhibit potential anti-inflammatory properties which could be beneficial in clinically relevant conditions where inflammation plays a role. Having shown that dietary intervention with RPO and rooibos had the potential to modulate the inflammatory response in the model of inflammation at basal conditions, we then proceeded to the third study to specifically establish if dietary RPO when supplemented alone will improve functional recovery and reduce infarct size in LPS-treated hearts. The Langendorff perfusion system was employed for determination of cardiac function and infarct size. The roles of NFkB, p38 MAPK and the myocardial antioxidant defence systems were investigated as potential mechanisms of protection. LPS-treatment caused significant increases in myocardial IL-1 β indicating that inflammation was induced. However, the levels of myocardial IL-10 was reduced in LPS-treated hearts compared to the non-treated hearts. Intervention with dietary RPO resulted in improved functional recovery and reduced infarct size, in both healthy hearts and in the LPS-treatment group. The RPO-induced cardio-protection was associated with increases in myocardial protein expression of the antioxidant enzymes, SOD1, SOD2, GPX1 as well as increased p38 phosphorylation during reperfusion. LPS treatment increased myocardial protein expression of NFkB p65 which was reversed by RPO supplementation. Reduction of myocardial NFkB protein expression, increased p38 phosphorylation and elevated mitochondrial antioxidant (SOD2 and GPX1) as well as cytosolic enzymes (SOD 1) are proposed as potential mechanisms underlying the RPO-induced cardio-protection in this model. Based on these study results, for the first time, having included vasculature aspects in the cardio-protective effects of RPO we have shown that the NO-cGMP pathway and antioxidant defense systems may act synergistically to restore cardiovascular function in spontaneously hypertensive rats. Results from the second study also provide the first scientific evidence that RPO in combination with rooibos (a flavonoid rich endemic herbal tea) could have potential anti-inflammatory activities at systemic as well as myocardial level, which may be beneficial in clinically relevant conditions where inflammation plays a role. From the third study it can be concluded that dietary RPO improved myocardial tolerance to ischaemia-reperfusion injury in a model of inflammation.

Indiana University-Purdue University Indianapolis (IUPUI) / Background: Previous studies show that prazosin, an α1-adrenergic receptor antagonist, decreases alcohol drinking in animal models of alcohol use and dependence and in alcohol-dependent men. These studies extended previous findings by using a paradigm that allows for separate assessment of prazosin on motivation to seek versus consume ethanol or sucrose in selectively bred rats given acute or chronic prazosin treatment. Methods: Alcohol-preferring P rats were trained to complete an operant response that resulted in access to either 2% (Exp. 1) or 1% (Exp.2) sucrose or 10% ethanol. In Experiment 1, a 4-week consummatory testing phase consisted of rats bar-pressing to “pay” a specified amount up front to gain access to unlimited ethanol (or sucrose) for a 20-minute period. A 4-week appetitive testing phase examined how much the rats would bar-press for ethanol in an extinction session when no reinforcer could be obtained. In Experiment 2, during testing, the response requirement was dropped to a 1 and daily session cycles of drug (3 weeks/ 14 sessions from Tues to Fri) or vehicle (2 weeks/ 9 sessions from Tues to Fri) treatment were alternated per drug dose for a total of 3 drug doses (3 cycles) per rat. After each drug cycle, a single non-reinforced extinction session was conducted with no drug ‘on board’ and no reinforcer access. On test days, rats were given IP injections of either vehicle or one of three doses of prazosin (Exp 1: 0.5, 1.0, 1.5 mg/kg; Exp 2: 0.25, 0.5, 1.0 mg/kg; balanced design; -30 min). Results: In Experiment 1, prazosin significantly decreased ethanol-seeking at all doses tested. The highest dose decreased ethanol intake and increased the latency to first lever-press and first lick. Sucrose-seeking and intake were decreased by the same doses of prazosin. In Experiment 2, prazosin significantly decreased reinforcer-seeking at the lowest and highest doses while ethanol intake was not decreased by prazosin. Conversely, sucrose-seeking was decreased at the highest dose of prazosin tested while sucrose consumption was decreased by all doses. Latency to lever-press for sucrose was increased by the lowest dose of prazosin compared to vehicle. Conclusions: These findings extend previous research and indicate that prazosin decreases motivation to seek ethanol and sucrose. The specificity of prazosin on different behaviors and over different reinforcers suggests that these findings are not due to prazosin-induced motor-impairment or malaise. These data suggest that prazosin may work by decreasing the reinforcing properties of reinforcers in general.

Indiana University-Purdue University Indianapolis (IUPUI) / Alcohol consumption produces a complex array of effects that can be divided into two types: the explicit pharmacological effects of ethanol (which can be quite separate temporally from time of intake) and the more temporally “relevant” effects (primarily olfactory and taste) that bridge the time from intake to the onset of the pharmacological effects. Dissociating these effects is essential to untangling the neurologic underpinnings of alcohol abuse and dependence. Intravenous self-administration of ethanol allows for controlled and precise dosing, bypasses first order absorption kinetics allowing for a faster onset of pharmacologic effects, and eliminates the confounding “non-pharmacological” effects associated with oral consumption. Intravenous self-administration of ethanol has been reliably demonstrated in both mouse and human experimental models; however, consistent intravenous self-administration of pharmacologically relevant levels of ethanol remains elusive in the rat. Previous work has demonstrated reliable elevated intravenous ethanol self administration using a compound reinforcer of oral sucrose and intravenous ethanol. The present study sought to elucidate the role of each component of this reinforcer complex using a multiple schedule study design. Male P rats had free access to both food and water during all intravenous self-administration sessions and all testing was performed in conjunction with the onset of the dark cycle. Once animals achieved stable operant responding on both levers for an orally delivered 1% sucrose solution (1S) on a FR4 schedule, surgery was conducted to implant an indwelling jugular catheter. Animals were habituated to the attachment of infusion apparatus and received twice daily sessions for four days to condition each lever to its associated schedule. Animals were then trained to respond on a multiple FR4-FR4 schedule composed of alternating 2.5 minute components. During one component only oral 1S was presented, while in the second component a compound reinforcer of oral 1S + IV 20% ethanol was presented (25 mg/kg/injection). Both levers were extended into the chamber during the session, with the active lever/schedule alternating as the session progressed across components. Average ethanol intake was 0.47 ± 0.04 g/kg. A significant increase in sucrose only reinforcers and sucrose lever error responding was found suggesting that sucrose not ethanol is responsible for driving overall responding. The current findings suggest that the existing intravenous ethanol self-administration methodology remains aversive in the rat.

The influence of experience during brain development was investigated on juvenile behavior, adult amphetamine sensitization, and neuronal structural plasticity in rats. Two experiential factors (i.e., tactile stimulation and stress) were studied either before or soon after birth. Early experience feminized social behavior in males; however, only stress enhanced anxiety-like behavior in males. Repeated amphetamine administration resulted in the development and persistence of behavioral sensitization. However, tactile stimulation attenuated the drug-induced behavioral sensitization whereas stress failed to influence the degree of sensitization. Neuroanatomical findings revealed that early experience altered the cortical and subcortical structures. Furthermore, drug exposure reorganized the brain structures involved in addiction but early experience prevented the drug-associated changes. Early adverse experience influences the subsequent exposure to a drug of abuse at anatomical level whereas a favorable experience has an effect both at behavioral and anatomical levels and thus may play a protective role against drug-induced sensitization and addiction. / xii, 263 leaves : ill. ; 29 cm

Indiana University-Purdue University Indianapolis (IUPUI) / Human traumatic spinal cord injuries (SCI) are primarily incomplete contusion or compression injuries at the cervical spinal level, causing immediate local tissue damage and a range of potential functional deficits. Secondary damage exacerbates initial mechanical trauma and contributes to function loss through delayed cell death mechanisms such as apoptosis and autophagy. As such, understanding the dynamics of cervical SCI and related intracellular signaling and death mechanisms is essential. Through behavior, Western blot, and histological analyses, alterations in phosphatase and tensin homolog (PTEN)/phosphatidylinositol-3-kinase (PI3K) signaling and the neuroprotective, functional, and mechanistic effects of administering the protein tyrosine phosphatase (PTP) inhibitor, potassium bisperoxo (picolinato) vanadium ([bpV[pic]) were analyzed following cervical spinal cord injury in rats. Furthermore, these studies investigated the combination of subacute Schwann cell transplantation with acute bpV(pic) treatment to identify any potential additive or synergistic benefits. Although spinal SC transplantation is well-studied, its use in combination with other therapies is necessary to complement its known protective and growth promoting characteristics. v The results showed 400 μg/kg/day bpV(pic) promoted significant tissue sparing, lesion reduction, and recovery of forelimb function post-SCI. To further clarify the mechanism of action of bpV(pic) on spinal neurons, we treated injured spinal neurons in vitro with 100 nM bpV(pic) and confirmed its neurprotection and action through inhibition of PTEN and promotion of PI3K/Akt/mammalian target of rapamycin (mTOR) signaling. Following bpV(pic) treatment and green fluorescent protein (GFP)-SC transplantation, similar results in neuroprotective benefits were observed. GFP-SCs alone exhibited less robust effects in this regard, but promoted significant ingrowth of axons, as well as vasculature, over 10 weeks post-transplantation. All treatments showed similar effects in forelimb function recovery, although the bpV and combination treatments were the only to show statistical significance over non-treated injury. In the following chapters, the research presented contributes further understanding of cellular responses following cervical hemi-contusion SCI, and the beneficial effects of bpV(pic) and SC transplantation therapies alone and in combination. In conclusion, this work provides a thorough overview of pathology and cell- and signal-specific mechanisms of survival and repair in a clinically relevant rodent SCI model.

Indiana University-Purdue University Indianapolis (IUPUI) / Tendinopathy is a common degenerative pathology that is characterized by activity related pain, focal tendon tenderness, intratendinous imaging changes, and typically results in changes in the histological, mechanical, and molecular properties of the tendon. Tendinopathy is difficult to study in humans, which has contributed to limited knowledge of the pathology, and thus a lack of appropriate treatment options. However, most believe that the pathology is degenerative as a result of a combination of both extrinsic and intrinsic factors. In order to gain understanding of this pathology, animal models are required. Because each tendon is naturally exposed to different conditions, a universal model is not feasible; therefore, an appropriate animal model must be established for each tendon susceptible to degenerative changes. While acceptable models have been developed for several tendons, a reliable model for the Achilles tendon remains elusive. The purpose of this dissertation was to develop an animal model of Achilles tendinopathy by investigating the individual and combined effects of an intrinsic and extrinsic factor on the rodent Achilles tendon. Rats selectively bred for high capacity running and Sprague Dawley rats underwent uphill treadmill running (an extrinsic factor) to mechanically overload the Achilles tendon or served as cage controls. Collagenase (intrinsic factor) was injected into one Achilles tendon in each animal to intrinsically break down the tendon. There were no interactions between uphill running and collagenase injection, indicating that the influence of the two factors was independent. Uphill treadmill running alone failed to produce any pathological changes in the histological or mechanical characteristics of the Achilles tendon, but did modify molecular activity. Intratendinous collagenase injection had negative effects on the histological, mechanical, and molecular properties of the tendon. The results of this dissertation demonstrated that the combined introduction of uphill treadmill running and collagenase injection did not lead to degenerative changes consistent with human Achilles tendinopathy. Intratendiouns collagenase injection negatively influenced the tendon; however, these changes were generally transient and not influenced by mechanical overload. Future studies should consider combinations of other intrinsic and extrinsic factors in an effort to develop an animal model that replicates human Achilles tendinopathy.

Indiana University-Purdue University Indianapolis (IUPUI) / Assessments of delay discounting in rodent lines bidirectionally selected for home cage intake and preference of alcohol have had mixed findings. The current study sought to examine if delay discounting related differentially to alcohol seeking versus and alcohol drinking, two processes underlying alcohol intake and preference. Three strains of rats were utilized to answer this question Long Evans (LE), high alcohol drinking rats (HAD2), and alcohol preferring P rats. All strains were compared in an adjusting amount delay discounting task. Operant self-administration of alcohol was then assessed in the sipper tube model, and finally home cage drinking was assessed in a 24 hour 2 bottle choice paradigm. In the delay discounting it was found that the P rats were steeper discounters than both the LE and HAD2. In the sipper tube model, P rats displayed higher levels of seeking than both the HAD2s and the LE, but both the P rats and the HAD2s had higher intakes than the LE. During 24 hour home cage access, the P rats and the HAD2s had higher intake and preference for alcohol than the LE, but were not different from each other. These results show that increased discounting of delayed rewards tracks with appetitive processes versus consummatory factors and home cage intake of alcohol. This builds on prior findings using selected line pairs by providing an explanation for discordant results, and supports the hypotheses that increased delay discounting is an intermediate phenotype that predisposes individuals to alcohol use disorders.

Indiana University-Purdue University Indianapolis (IUPUI) / Although peripheral motoneurons are phenotypically endowed with robust regenerative capacity, functional recovery is often suboptimal following peripheral nerve injury (PNI). Research to date indicates that the greatest success in achieving full functional recovery will require the use of a combinatorial approach that can simultaneously target different aspects of the post-injury response. In general, the concept of a combinatorial approach to neural repair has been established in the scientific literature but has yet to be successfully applied in the clinical situation. Emerging evidence from animal studies supports the use of electrical stimulation (ES) and testosterone as one type of combinatorial treatment after crush injury to the facial nerve (CN VII). With the facial nerve injury model, we have previously demonstrated that ES and testosterone target different stages of the regeneration process and enhance functional recovery after facial nerve crush injury. What is currently unknown, but critical to determine, is the impact of a combinatorial treatment strategy of ES and testosterone on functional recovery after crush injury to the sciatic nerve, a mixed sensory and motor spinal nerve which is one of the most serious PNI clinical problems. The results of the present study indicate that either treatment alone or in combination positively impact motor recovery. With regard to molecular effects,single and combinatorial treatments differentially alter the expression of regeneration-associated genes following sciatic nerve crush injury relative to facial nerve injury. Thus, our data indicate that not all injuries equally respond to treatment. Furthermore, the results support the importance of treatment strategy development in an injury-dependent manner and based upon the functional characteristics of spinal vs. cranial nerves.

Indiana University-Purdue University Indianapolis (IUPUI) / Aromatase inhibitors (AIs) are the current standard of care for the treatment of hormone receptor positive breast cancer in postmenopausal women. Nearly one-half of patients receiving AI therapy develop musculoskeletal toxicity that is characterized by joint and/or muscle pain and approximately one-fourth of patients discontinue their therapy as a result of musculoskeletal pain. Since there are no effective strategies for prevention or treatment, insight into the mechanisms of AI-induced pain is critical to improve treatment. However, there are few studies of AI effects in animal models of nociception. To determine whether AIs produce hypersensitivity in animal models of pain, I examined the effects of AI administration on mechanical, thermal, and chemical sensitivity in rats. The results demonstrate that (1) repeated injection of 5 mg/kg letrozole in male rats produces mechanical, but not thermal, hypersensitivity that extinguishes when drug dosing is stopped; (2) administering a single dose of 1 or 5 mg/kg letrozole in ovariectomized (OVX) rats also induces mechanical hypersensitivity, without altering thermal sensitivity and (3) a single dose of 5 mg/kg letrozole or daily dosing of letrozole or exemestane in male rats augments flinching behavior induced by intraplantar ATP injection. To determine whether the effects of AIs on nociceptive behaviors are mediated by activation or sensitization of peptidergic sensory neurons, I determined whether letrozole exposure alters release of calcitonin gene-related peptide (CGRP) from isolated rat sensory neurons and from sensory nerve endings in rat spinal cord slices. No changes in basal, capsaicin-evoked or high extracellular potassium-evoked CGRP release were observed in sensory neuronal cultures acutely or chronically exposed to letrozole. Furthermore, letrozole exposure did not alter the ability of ATP to augment CGRP release from sensory neurons in culture. Finally, chronic letrozole treatment did not augment neuropeptide release from spinal cord slices. Taken together, these results do not support altered release of this neuropeptide into the spinal cord as mediator of letrozole-induced mechanical hypersensitivity and suggest the involvement of other mechanisms. Results from this dissertation provide a new experimental model for AI-induced hypersensitivity that could be beneficial in delineating mechanisms mediating pain during AI therapy.

Indiana University-Purdue University Indianapolis (IUPUI) / Alcoholism inheritance rates have been estimated as high as 60% in a human population. Many significant features of alcohol dependence have been replicated in rodent animal models of alcoholism, however not in totality. These animal models include inbred preferring (iP) and nonpreferring (iNP) rat types. Congenic rats have been engineered from the iP and iNP strains whereby a P congenic rat has in its genome a well-chosen chromosomal portion taken from an NP rat (P.NP) and, reciprocally, an NP congenic rat has acquired the analogous DNA from a P rat (NP.P). In this case, a quantitative trait locus (QTL) from chromosome 4 is the donor genetic material for the congenic rats. It is of great interest to further study this chromosome 4 QTL because it has been found to control a significant portion of ethanol consumption behavior in iP and iNP rats. This study aimed to behaviorally profile the iP, iNP and reciprocal congenic rats. As a result of the behavioral profiling of these genetically related groups, some conclusions could be made regarding which behaviors appear to be controlled by the chromosome 4 donor DNA.This study primarily utilized the Multivariate Concentric Square Field apparatus (MCSF) to characterize behavioral profiles for the inbred and congenic rats. The Open field (OF) and Elevated plus maze (EPM) supported this effort. The MCSF is valuable in that it allows for the animals to interact within an environment that has ethological value. The 12 different zones that make up the field are characterized by some functional quality in terms of type and duration of behavior performed, etc. The behavioral data is aggregated and finally represented in terms of five functional categories, the elements of the behavioral profile: general activity, exploratory activity, risk assessment, risk taking, and shelter seeking. The study hypotheses were shaped by prior research suggesting that iPs should display lower general activity and risk taking strategy than iNPs in the MCSF. Inbred Ps should be more active in the OF and spend more time in the center of the EPM. Generally, it is expected that the iP QTL confer behavioral phenotypes to the iNP strain that deviate toward a "P" behavioral phenotype and reciprocally, the iNP QTL confer behavioral phenotypes to the iP strain that deviate toward an "NP" behavioral phenotype. The results showed that iP rats performed more risk assessment and risk taking behavior and less shelter seeking and anxiety-like behavior than iNP rats. It followed that P.NP congenic rats significantly downgraded their risk assessment and risk taking behavior when compared to iP rats. This decrease can be attributed to the chromosome 4 QTL donated from the iNP breed. All together this study concludes that risk assessment and risk taking behavior in the iP rats is controlled by the same DNA region that, in part, determines voluntary intake of ethanol consumption. Further fine mapping of the QTL region should help in discovering if the same DNA sequences that influence ethanol intake also significantly influence risk behavior.