The Neural Control of Blood Flow to Normal, Injured and Arthritic Joints

An imaging technique (laser Doppler perfusion imaging, LDI), based on measurement of backscattered Doppler-broadened laser radiation, was used to produce two-dimensional images of perfusion in animal and human joints. The advantage of this technique is that it can map the spatial distribution of tissue perfusion in a non-invasive manner. On the downside, however, LDI is unable to provide absolute measures of blood flow and it is incapable of assessing temporal changes in perfusion. The former disadvantage was addressed in this thesis by comparing LDI flux values obtained from rabbit medial collateral ligaments (MCL) with simultaneous absolute measures of blood flow determined by the coloured microsphere technique. These experiments were able to calibrate the LDI system, however, since different tissues have unique optical properties, the calibration factor produced in this study was specific to rabbit MCLs

Cytokine Dynamics, Diagnosis and Treatment of Neuroinflammation In Chronic Constriction Injury Rat Model

Chronic neuropathic pain is a serious, worldwide health problem leading to life-long treatment and the possibility of significant disability. In this study, neuropathic pain was modeled using the chronic constriction injury (CCI) in rats. The CCI rats exhibited hypersensitivity (typical neuropathic pain symptom) to mechanical stimulation of the affected paw 11 days post-surgery, at a time when sham surgery animals did not. It is known that immune cells play a role in the development of neuropathic pain and to further explore the relationship between neuropathic pain and immune cells, we hypothesized that the infiltration of immune cells into the affected sciatic nerve can be monitored in vivo by optical imaging. To test this hypothesis, an intravenous injection of a novel perfluorocarbon (PFC) nanoemulsion developed by J. M. Janjic (PharmD, PhD) and co-workers was used. Post-injection, the nanoemulsion is endocytosed by inflammatory cells (e.g. monocytes and macrophages) in a CCI rat. The nanoemulsion carried two distinct imaging agents, a near-infrared (NIR) lipophilic fluorescence reporter (DiR) and a 19F magnetic resonance imaging (MRI) tracer (PFC). This study showed that in live rats, NIR fluorescence was concentrated in the area of the affected sciatic nerve. Furthermore, the 19F MRI signal was observed in the affected sciatic nerve in the perfusion fixed rats. Histological examination of the CCI sciatic nerve sections revealed significant infiltration of CD68 positive macrophages. These results demonstrate that the infiltration of immune cells into the sciatic nerve can be visualized in live animals using these methods. Using the same strategy, a theranostic nanoemulsion containing an anti-inflammatory drug, celecoxib, along with NIR dye and 19F tracer, was used for the targeted delivery of the drug into the inflammatory cells at the site of injury while at the same time providing the ability to track inflammation. A single intravenous injection of the nanoemulsion carrying a low dose of celecoxib (0.24 mg/kg), a normally highly insoluble drug, appears to have provided a direct delivery of the drug into the inflammatory cells. Single dose intravenous injection of the theranostic nanoemulsion resulted in significant relief from the hypersensitivity behavior (mechanical allodynia) that persisted for at least four days post-injection. These findings demonstrated that celecoxib-containing theranostic nanoemulsion based therapy is an effective tool for the simultaneous tracking and treatment of the neuroinflammation in a CCI rat model. Inflammation is known to be associated with peripheral neuropathy, however the interplay among cytokines, chemokines and neurons is still unclear. We hypothesized that the neuroinflammatory interaction can be defined by using a computational modeling approach, based on the dynamics of the protein expression of various inflammatory mediators in the sciatic nerve of the CCI rats. Using Dynamic Bayesian Network inference, we identified Interleukin (IL)-18 as a central node associated with neuropathic pain in the CCI rat model. Immunofluorescence supported a role for inflammasome activation and induction of IL-18 at the site of injury. Combined in-vivo and in-silico approaches may thus highlight novel molecular targets associated with peripheral neuropathy that can be the target of future theranostic nanoemulsion-based therapy

Drug Repurposing

This book focuses on various aspects and applications of drug repurposing, the understanding of which is important for treating diseases. Due to the high costs and time associated with the new drug discovery process, the inclination toward drug repurposing is increasing for common as well as rare diseases. A major focus of this book is understanding the role of drug repurposing to develop drugs for infectious diseases, including antivirals, antibacterial and anticancer drugs, as well as immunotherapeutics

Molecular Science for Drug Development and Biomedicine

With the avalanche of biological sequences generated in the postgenomic age, molecular science is facing an unprecedented challenge, i.e., how to timely utilize the huge amount of data to benefit human beings. Stimulated by such a challenge, a rapid development has taken place in molecular science, particularly in the areas associated with drug development and biomedicine, both experimental and theoretical. The current thematic issue was launched with the focus on the topic of “Molecular Science for Drug Development and Biomedicine”, in hopes to further stimulate more useful techniques and findings from various approaches of molecular science for drug development and biomedicine

Exploring the incidence and variability of oxaliplatin-induced neuropathic pain symptoms in colorectal cancer patients, comparative in vivo / in vitro modelling of oxaliplatin/ 56MESS(IV) as an alternative cancer treatment, and minocycline administration as a prophylactic agent for chemotherapy-induced neuropathic pain

Oxaliplatin is commonly used for the treatment of advanced and recurrent colorectal cancer (CRC). However, oxaliplatin-induced neuropathic pain remains a challenge for the healthcare systems worldwide. In chapter II, the incidence and impact of acute oxaliplatin-induced neuropathic pain on chemotherapy treatment in colorectal cancer patients in the first cycle from the published research literature was explored. In chapter III, the variability of oxaliplatin-induced neuropathic pain symptoms from the Southwestern Sydney Local Health District Hospitals (SWLHDHs) database of patients who received oxaliplatin based chemotherapy treatment (2011-2015) and the implications for the management of colorectal cancer patients was assessed. In chapter IV, it was explored whether oxaliplatin can induce behavioural hypersensitivity in healthy rats using the dosing regimen that mimics the standard clinical protocols (oxaliplatin 2.5 mg/kg i.p every two weeks). In chapter V, the effects of oxaliplatin and 56MESS(IV) on the viability, PI staining (cell death) and activation (nitrite production) of RAW264.7 (macrophages) and N11 (microglia) cell lines were explored in vitro. In summary, oxaliplatin-induced neuropathic pain remains a big problem as it affects treatment compliance in quarter of CRC patients during cycle 1 and as neuropathic pain symptoms oscillate across cycles for individual patients. This warrants further detailed patient-by-patient analysis of pain symptoms in future clinical trials. Additionally, the in vivo and in vitro data showed that minocycline pre-treatment has a potential to ameliorate oxaliplatin and 56MESS(IV) induced production of pro-inflammatory chemical mediators such as nitrite in vitro cell lines, which may be relevant to in vivo models of chemotherapy-induced neuropathic pain. However, oxaliplatin-induced neuropathic pain is more likely multifactorial and research should be continued on the mechanisms of neuropathy and potential therapeutic drugs

Assessing pain and inflammation in arthritis using novel imaging methods

Enhanced bone resorption is a common pathology in destructive bone diseases. Many cytokines (e.g. IL-6 and TNF-α) and chemokines (e.g. CCL2, CCL3 and CCL5) elevated in patients exert pathological roles in leukocyte migration and inflammation, but their effect on direct bone destruction remained elusive. Published research into osteoclastogenesis was carried out in co-cultures, from which osteoclast differentiation, resorption and mediator secretion data was obtained. The main objective of this thesis was to establish a working methodology for the in vitro differentiation of CD14+ve mononuclear cells into osteoclasts and to decipher the direct effects of IL-6 and CCL3 on osteoclastogenesis and bone resorption. IL-6 trans-signalling exerted an effect in both basal and pathological osteoclastogenesis, whereas its inhibition via sgp130-Fc significantly reduced osteoclast formation and bone resorption in vitro. Although not examined in vivo, the translational use of sgp130-Fc as a therapeutic for destructive bone pathology was postulated from data showing a reduction in osteoclast differentiation and resorption after stimulation with HYPER-IL-6. Secondary to IL-6 trans-signalling, it was hypothesised that the neutralisation of CCL3 in vitro and in vivo would significantly reduce osteoclast differentiation and resorption. Osteoclast number significantly reduced in the presence of anti-CCL3, but TRAP+ve cell count was unaltered, suggesting an early role of CCL3 in osteoclast multi-nucleation/fusion. Additionally, in vivo data showed a protective effect of anti-CCL3 with significantly reduced bone erosive scores and osteoclast counts thereby presenting CCL3 as a novel biomarker of disease activity in destructive bone disease. In contrast, TNF-α, CCL2, and CCL5 were shown to have no role in direct osteoclast differentiation in our monocultures. In conclusion, for the first time this work documents novel and important roles of IL-6 trans-signalling and CCL3 in increased osteoclast differentiation and bone resorption. Our data highlights the importance of IL-6 trans-signalling and provides evidence for the use of CCL3 as a predictive biomarker in destructive bone diseases

Molecular mechanisms of nociception and pain

My thesis uses in vivo calcium imaging to investigate the cell and molecular mechanisms of two unusual pain states: congenital analgesia and cold allodynia. Genetic deletion of voltage-gated sodium channel NaV1.7 in mice and humans leads to profound pain insensitivity. Paradoxically, peripherally-targeted pharmacological antagonists of NaV1.7 fail to relieve pain in the clinic. To determine the mechanism of analgesia in NaV1.7 null mutants, I used optical, electrophysiological and behavioural methods to investigate the effect of peripheral NaV1.7 deletion on nociceptor function. Surprisingly, both calcium imaging and extracellular recording of NaV1.7-deficient sensory neurons in vivo found limited deficits in the response to noxious stimuli. Synaptic transmission from nociceptor central terminals in the spinal cord was however compromised following NaV1.7 deletion. Importantly, both synaptic deficits and behavioural analgesia were reversed by blocking central opioid receptors. Collectively, these data account for the failure of peripherally-targeted NaV1.7 blockers and point to a central mechanism of analgesia in NaV1.7 null mutants that requires opioid receptors. Chronic pain patients suffering from cold allodynia experience normally innocuous cooling as excruciating pain, but the cells and molecules driving cold allodynia remain elusive. I used in vivo calcium imaging to investigate how the activity of cold-sensing neurons was altered in three mouse models of neuropathic pain: oxaliplatin-induced neuropathy, peripheral nerve injury and ciguatera poisoning. In neuropathic mice exhibiting cold allodynia, a subset of cold-insensitive, large-diameter, peptidergic nociceptors became responsive to cooling. Diptheria toxin-mediated ablation of these silent cold-sensing neurons decreased neuropathic cold hypersensitivity. Voltage-gated potassium channels KV1.1 and KV1.2 were highly expressed in silent cold-sensing neurons and pharmacological inhibition of these channels rapidly induced cold responsiveness in cold-insensitive neurons. Taken together, I reveal that silent-cold sensing neurons contribute to cold allodynia in neuropathic pain and identify KV1 channel downregulation as a driver of de novo cold sensitivity, in vivo

Propanil (3,4-DCPA)-induced alterations of macrophage function

The pesticide 3,4-dichloropropionanilide (propanil or alternatively, DCPA) is a post-emergent herbicide predominantly used for the control of weeds on commercial rice crops worldwide. The US Environmental Protection Agency report on Pesticides Industry Sales and Usage in 2001 ranked propanil as the 17th most used herbicide in the US. It is applied on the fields via groundboom sprayers or aerial equipment suppressing photosynthesis and carbon fixation of a growing plant. The primary target of herbicide exposure is personnel working in the manufacturing and application who are required to wear personal protective equipment to minimize the exposure. The common routes of this exposure are inhalational and dermal, and to a lesser extent, oral. A number of toxic side effects are induced by propanil include hemolytic anemia, methemoglobinemia, and myelotoxicity. The majority of current knowledge about propanil\u27s toxic effects comes from animal studies. Liver and spleen were found to be the primary target organs, with many cell populations of the immune system affected, including T and B lymphocytes, NK cells and macrophages.;Macrophages play essential roles in both innate and adaptive immune responses. They recognize and destroy pathogens through phagocytosis and generation of reactive oxygen and nitrogen species (ROS and RNS). Macrophages can also function as antigen presenting cells in the initiation of an adaptive immune response and secrete a number of cytokines and inflammatory mediators thereby modulating the immune reaction at the inflamed site.;Previous studies have demonstrated that propanil was able to suppress interleukin (IL)-1beta, IL-6 and TNF-alpha cytokine production by LPS-activated murine macrophages. The analysis of nuclear factor-kappaB (NF-kappaB), responsible for TNF-alpha production, detected a decreased nuclear localization of NF-kappaB accompanied by a reduced, but not entirely abrogated binding to DNA activity.;The first study of this dissertation demonstrated the ability of propanil to inhibit TNF-alpha production by LPS-stimulated human monocytic cells line and suppress phagocytosis of fluorescent beads and Listeria monocytogenes . The ability to directly kill this bacterium and produce ROS and RNS was also assessed. The results demonstrate that DCPA has profound effects on macrophage function, and provide insight into the potential mechanisms of immunosuppression by DCPA. The second study focused on the mechanism of dramatic suppression of an inflammatory mediator secreted by activated macrophages, prostaglandin E2 (PGE2). Enzymatic activity and expression level of the proteins involved in PGE2 production were studied. However, the exact mechanism of propanil\u27s action is still unknown. The third study describes a novel behavior on NF-kappaB, called oscillations, in continuously LPS-stimulated macrophages. Binding activity of NF-kappaB was studied by two independent methods, and a mathematical model describing the oscillatory behavior was created based on experimental data. It demonstrated that propanil exposure potentiated the NF-kappaB activation process changing the oscillation behavior which could influence the transcription activity of the NF-kappaB-inducible genes.;Taken together our findings indicate that propanil exposure affects a number of important macrophage functions, such as phagocytosis, ROS and RNS production and inflammatory mediator\u27s secretion. Our studies also demonstrated a potential mechanism for the inhibition of a number of inflammatory cytokines through changes in NF-kappaB binding activity and oscillatory behavior

Antioxidant and DPPH-Scavenging Activities of Compounds and Ethanolic Extract of the Leaf and Twigs of Caesalpinia bonduc L. Roxb.

Antioxidant effects of ethanolic extract of Caesalpinia bonduc and its isolated bioactive compounds were evaluated in vitro. The compounds included two new cassanediterpenes, 1α,7α-diacetoxy-5α,6β-dihydroxyl-cass-14(15)-epoxy-16,12-olide (1)and 12α-ethoxyl-1α,14β-diacetoxy-2α,5α-dihydroxyl cass-13(15)-en-16,12-olide(2); and others, bonducellin (3), 7,4’-dihydroxy-3,11-dehydrohomoisoflavanone (4), daucosterol (5), luteolin (6), quercetin-3-methyl ether (7) and kaempferol-3-O-α-L-rhamnopyranosyl-(1Ç2)-β-D-xylopyranoside (8). The antioxidant properties of the extract and compounds were assessed by the measurement of the total phenolic content, ascorbic acid content, total antioxidant capacity and 1-1-diphenyl-2-picryl hydrazyl (DPPH) and hydrogen peroxide radicals scavenging activities.Compounds 3, 6, 7 and ethanolic extract had DPPH scavenging activities with IC50 values of 186, 75, 17 and 102 μg/ml respectively when compared to vitamin C with 15 μg/ml. On the other hand, no significant results were obtained for hydrogen peroxide radical. In addition, compound 7 has the highest phenolic content of 0.81±0.01 mg/ml of gallic acid equivalent while compound 8 showed the highest total antioxidant capacity with 254.31±3.54 and 199.82±2.78 μg/ml gallic and ascorbic acid equivalent respectively. Compound 4 and ethanolic extract showed a high ascorbic acid content of 2.26±0.01 and 6.78±0.03 mg/ml respectively.The results obtained showed the antioxidant activity of the ethanolic extract of C. bonduc and deduced that this activity was mediated by its isolated bioactive compounds