PRISM :: Browsing by Author "Trang, Tuan"

Browsing by Author "Trang, Tuan"

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Open Access
Behavioural and microglial effect of cannabinoids in neuropathic pain
(2022-09) Davidson, Chloé E. D.; Trang, Tuan; Borgland, Stephanie; Hill, Matthew
Chronic pain is a disease affecting 1 in 5 Canadians and the leading cause of disability worldwide. Among the most debilitating forms of chronic pain is neuropathic pain, which is caused by injury or disease of the nervous system. Neuropathic pain is difficult to manage clinically because it is often resistant to strong analgesics including opioids. Since therapeutic options are limited, some patients resort to medical cannabis to alleviate pain symptoms. Pain is the most common medical reason to use cannabis and cannabinoids. However, the efficacy of cannabis for pain, as well as its potential health impacts, are not well defined. My project addresses this knowledge gap by characterizing the behavioural and cellular effects of cannabis in the rat spared nerve injury (SNI) model. This neuropathic pain model replicates the mechanical pain sensitivity reported in neuropathic pain patients, including mechanical allodynia, which is an abnormal sensitivity to tactile stimuli in which even light touch can elicit pain. Prior research shows that microglia, the resident immune cells of the central nervous system, play a central role in the development of chronic neuropathic pain after peripheral nerve injury in males. In this study, we assessed the effects of vaporized whole cannabis extract and a combination of THC and CBD on mechanical hypersensitivity in spared nerve injury (SNI) male and female Sprague-Dawley rats. I also investigated the effect of cannabinoid exposure on spinal microglia. I find that cannabis and cannabinoid extracts transiently reversed mechanical allodynia in the spared nerve injury model of neuropathic pain in a sex- and compound-dependent manner. Additionally, I demonstrate a compound-dependent effect of cannabinoids on microglial reactivity.
Open Access
Characterization of Different Isoforms of the K+-dependent Na+- Ca2+ Exchangers: A Look into Ca2+ Coordination and Functional Consequences of Mutations Associated with Different Genetic Diseases
(2016) Jalloul, Ali; Schnetkamp, Paul; Braun, Andrew; Altier, Christophe; Trang, Tuan; Chen, S. R. Wayne
K+-dependent Na+-Ca2+ exchangers (NCKXs) belong to the Solute Carrier 24 gene family of membrane transporters. Five different exchangers have been identified in humans and contribute to many biological processes including vision, enamel maturation, Melanocortin-4-receptor-dependent satiety, olfaction and skin pigmentation. Here, I examined the differences in cation coordination of NCKX1-4 and tested the effect of single residue substitutions in the α-repeats on Ca2+ affinity in NCKX2. In addition, I investigated the functional consequences of mutations in different NCKX genes associated with genetic diseases. I measured NCKX-mediated Ca2+ transport activity of WT and mutant NCKX proteins while manipulating external or internal ion concentrations. I concluded that the differences observed among these exchangers do not explain the variation in their tissue distribution. Also, 13 single residue substitutions significantly lowered Ca2+ affinity in NCKX2. Moreover, the functional data presented about the mutations associated with genetic diseases support that the genetic analysis describing these mutations.
Open Access
Characterization of Embryonic Microglia in the Developing Hypothalamus of Germ-Free Mice
(2022-08) Malik, Faizan; Kurrasch, Deborah; Trang, Tuan; Reimer, Raylene; McCoy, Kathy
Emerging evidence suggests that the maternal microbiome may influence the colonization, development, and function of microglia during embryonic brain development. Our objective was to conduct an unbiased and comprehensive morphometric evaluation of microglia morphology over several embryonic timepoints in the germ-free (GF) and specific pathogen free (SPF) tuberal hypothalamus. We hypothesized that hypothalamic microglia exhibit subtle differences in colonization, morphology, and/or dynamics in the embryonic brain of germ-free mice. This investigation was performed using two primary imaging modalities including histological, and acute organotypic slice time-series, imaging. Few morphological disturbances were observed between GF and SPF microglia across all embryonic timepoints analyzed. However, the changes we did observe, were robust and striking. GF microglia along the ventricular zone of the E14.5 tuberal hypothalamus exhibited several significant differences indicative of excess ramification. This finding was corroborated by the marked absence of an ameboid and highly phagocytic population of microglia in GF acute organotypic brain slices, otherwise present in the SPF condition. Furthermore, we observed a global decrease in CD68 expression among microglia in the tuberal hypothalamus. Interestingly, microglia in the parenchyma appeared unaffected by the absence of the microbiome. Together this data suggests that the maternal microbiome may discriminately influence discrete subpopulations of microglia in the tuberal hypothalamus at developmentally relevant time-points.
Open Access
Chronic morphine regulates TRPM8 channels via MOR-PKCβ signaling
(2020-04-14) Iftinca, Mircea; Basso, Lilian; Flynn, Robyn; Kwok, Charlie; Roland, Corinne; Hassan, Ahmed; Defaye, Manon; Ramachandran, Rithwik; Trang, Tuan; Altier, Christophe
Abstract Postoperative shivering and cold hypersensitivity are major side effects of acute and chronic opioid treatments respectively. TRPM8 is a cold and menthol-sensitive channel found in a subset of dorsal root ganglion (DRG) nociceptors. Deletion or inhibition of the TRPM8 channel was found to prevent the cold hyperalgesia induced by chronic administration of morphine. Here, we examined the mechanisms by which morphine was able to promote cold hypersensitivity in DRG neurons and transfected HEK cells. Mice daily injected with morphine for 5 days developed cold hyperalgesia. Treatment with morphine did not alter the expressions of cold sensitive TREK-1, TRAAK and TRPM8 in DRGs. However, TRPM8-expressing DRG neurons isolated from morphine-treated mice exhibited hyperexcitability. Sustained morphine treatment in vitro sensitized TRPM8 responsiveness to cold or menthol and reduced activation-evoked desensitization of the channel. Blocking phospholipase C (PLC) as well as protein kinase C beta (PKCβ), but not protein kinase A (PKA) or Rho-associated protein kinase (ROCK), restored channel desensitization. Identification of two PKC phosphorylation consensus sites, S1040 and S1041, in the TRPM8 and their site-directed mutation were able to prevent the MOR-induced reduction in TRPM8 desensitization. Our results show that activation of MOR by morphine 1) promotes hyperexcitability of TRPM8-expressing neurons and 2) induces a PKCβ-mediated reduction of TRPM8 desensitization. This MOR-PKCβ dependent modulation of TRPM8 may underlie the onset of cold hyperalgesia caused by repeated administration of morphine. Our findings point to TRPM8 channel and PKCβ as important targets for opioid-induced cold hypersensitivity.
Open Access
Early Life Regulation of TRPV1+ Nociceptors by the Microbiome: Implications for Pathological Pain?
(2024-04-30) Abdullah, Nasser Salem; Altier, Christophe; Trang, Tuan; Hirota, Simon
Pain is essential for the survival and wellbeing of organisms. Dysregulation in the pain pathway leads to pathological pain in part due to poor pain management stemming from a lack of understanding of the underlying mechanisms that lead to pathological pain. Pain is initiated by specialised primary afferent neurons called nociceptors. TRPV1+ nociceptors play a central role in multiple pathological pain conditions, including inflammatory pain, where their sensitization can lead to chronic pain. In this thesis, we shed light on how TRPV1+ nociceptors participate in chronic pain and the factors that contribute to their regulation. This thesis provides two studies divided into 3 chapters. The first study (Chapter 3) is focused on the role of TRPV1+ nociceptors in initiating chronic visceral pain. The second study (Chapter 4 and 5) is focused on understanding how the early life microbiome regulates the sensitivity of TRPV1+ nociceptors. In Chapter 3, we present findings indicating that in a murine model of colitis, TRPV1+ nociceptors activate spinal microglia leading to visceral hypersensitivity, demonstrating their essential role for the transition from acute to chronic pain in the context of colitis. In Chapter 4, we investigated the role of the early life microbiome on TRPV1+ nociceptor specification and pain sensitivity using germ-free mice and then germ-free mice colonized before or after weaning. We found that a lack of microbiome in early life leads to hyposensitivity to heat and capsaicin, The hyposensitive phenotype was not due to changes in nociceptor specification, innervation, or TRPV1 expression, but it correlated with a reduction in TRPV1 trafficking to the cell membrane. In Chapter 5, we investigated the underlying mechanisms for early life microbiome induced hyposensitivity and identified that the early life microbiome regulates the sensitivity of nociceptors and the trafficking of TRPV1 through regulating mast cell derived NGF. Altogether, this study demonstrates the central role of TRPV1+ nociceptors in inducing the transition to chronic pain and the important role of the early life microbiome in regulating the sensitivity of these nociceptors through mast cell derived NGF.
Open Access
Effect of Cafeteria Diet Induced Obesity on Neuroinflammation in the Orbitofrontal Cortex
(2016) Kaur, Manpreet; Borgland, Stephanie; Pittman, Quentin J.; Trang, Tuan
Palatable food consumption is associated with obesity and induce inflammation in brain. The orbitofrontal cortex is implicated in compulsive behavior. Glial changes lead to synaptic remodeling and altered behavior. However it is not clear if metabolic changes associated with cafeteria diet consumption lead to glial changes in OFC. My study addressed this important question whether intake of cafeteria diet leads to glial changes in OFC. I observed that rats with extended access to cafeteria diet become obese, exhibited inflexible behaviour and increased GFAP and IBA1 expression in the hippocampus and the hypothalamus. Further, there was an increase observed in several proinflammatory cytokines in the hypothalamus. My work has shown that extended intake of cafeteria diet is associated with increased GFAP expression in OFC without any changes in IBA1 expression, proliferation and the levels of proinflammatory cytokines. This is the first study reporting astrocytic activation in OFC associated with compulsive food intake.
Open Access
Fundamental Mechanisms of Astrocyte and Cerebrovascular Regulation by Potassium in the Neocortex
(2018-05-10) Shin, Steven Seungjae; Gordon, Grant Robert J.; Trang, Tuan; Bains, Jaideep Singh
Astrocytes can control local cerebral arteriole tone through Ca2+ dependent mechanisms. Modest elevations of the external K+ concentration in the brain are thought to have little effect on astrocyte free Ca2+, yet K+ dilates parenchymal arterioles. It remains unclear whether fluctuations in external K+, within the low to moderate range, controls arteriole diameter through an astrocyte Ca2+ dependent mechanism. Using relative and quantitative two-photon fluorescence Ca2+ imaging in acute brain slices of the somatosensory cortex from Sprague Dawley rats, we found that elevations in external K+ from 2.5 mM to 3.5 or 5.0 mM decreased astrocyte free Ca2+ and caused arteriole dilation. The phenomenon relied on external Ca2+, Cl-, and bicarbonate, as well as, unexpectedly, the potassium chloride cotransporter (KCC). These data highlight novel aspects of K+-astrocyte dynamics that involve the subtle regulation of free astrocyte Ca2+ via Cl- and bicarbonate fluxes with corresponding changes to arteriole tone.
Open Access
Hippocampal Oxygen Dynamics During and Following Febrile Seizures
(2021-01-22) Harris, Sydney; Teskey, Gordon Campbell; Scantlebury, Morris H.; Kurrasch, Deborah M.; Trang, Tuan
Objective: Febrile seizures are the most common convulsive event, with anywhere from 2-14% of the population having at least one between 6 months and 5 years of age. Febrile seizures are linked to memory impairments and increased seizure susceptibility. In adults, prolonged seizures resulting from a brain damaging event such as status epilepticus result in hyperoxia. Conversely, brief self-terminating seizures lead to postictal hypoxia. It is currently unknown the dynamic changes in oxygen levels and the mechanisms involved in febrile seizures. Methods: Eight-day-old rat pups were implanted with a hippocampal electrode and oxygen sensing optode. Following surgery rat pups received an immune challenge through 4, once daily injections of lipopolysaccharide. Febrile seizures were then induced using a modified heated dry air model. Before, during and after seizure induction hippocampal local field potentials and oxygen levels were recorded. Separate groups of pups received injections of drugs targeting COX-1, COX-2, L-type calcium channels, CB1 receptors, TRPV1 receptors and adenosine receptors prior to febrile seizure induction. Following febrile seizures, a subset of pups were raised to young adulthood then tested in a novel object recognition paradigm to test learning deficits Seizure thresholds following pentylenetetrazol administration were also tested in adulthood. Results: Febrile seizures result in oxygen dynamics that are related to epileptiform activity. Oxygen following seizures are the same in both male and female rat pups. Oxygen dynamics appear to be linked to seizure length and severity. When a seizure starts there is an initial increase in oxygen, followed by a local oxygen minimum. While oxygen is at the lowest rat pups behaviourally recovery from the seizure, but epileptiform activity in the brain persists. Oxygen then climbs for a second time and does not begin to fall until the epileptiform activity terminates. Calcium channels are involved in seizure behavioural termination with inhibition prolonging recovery while activation decreases time to behavioural recovery without altering epileptiform activity termination. Additionally, inhibiting calcium channels decreases hyperoxia during and following the seizure. Inhibiting COX-1, CB1 and TRPV1 receptors all lead to less hyperoxia postictally. Caffeine had a proconvulsant effect and resulted in less oxygen fluctuations in the hippocampus. A single febrile seizure leads to decreased memory formation but did not alter seizure thresholds in adulthood. Significance: Febrile seizure postictal brain oxygenation does not follow the same pattern or have the same mechanisms as a brief focal seizure or status epilepticus in adults. A single febrile seizure can lead to lasting consequences in memory formation. Brain oxygen dynamics may be an important consideration in the development of treatments for febrile seizures.
Open Access
Increased Microglial Reactivity Alters Morphine Analgesia
(2017) Leduc-Pessah, Heather; Trang, Tuan; Pittman, Quentin; Altier, Christophe; Gilch, Sabine; Gendron, Louis
Opioids are a potent class of analgesics in the management of both moderate to severe acute pain and chronic pain. Although neuronal response to opioids is well described, we are lacking a complete characterization of the diverse interplay of neurons with other cells types in response to opioids. Microglia, the immune cells of the CNS, are key targets of opioids and their response to repeated opioid exposure is implicated in the severe side effects associated with prolonged opioid use: opioid analgesic tolerance, opioid-induced hyperalgesia and opioid use disorder. In addition, increased microglial reactivity can alter analgesic response to opioids. Thus, understanding the role of microglia in response to opioids is both critical for improving the analgesic efficacy of opioids and for interfering with the negative side effects associated with prolonged opioid use. This thesis explores the contributions of the microglial P2X7 receptor and the microglial transcription factor Runx1 in morphine analgesia and in the development of adverse effects. My over-arching hypothesis is that increased microglial reactivity diminishes the analgesic potential of opioids, such as morphine. Here I show that repeated morphine causes a potentiation in microglial P2X7R function mediated by µ-receptor activation of Src kinase. Specifically, I identified tyrosine residues 382-384 on the P2X7R C-terminal domain as a critical site of phosphorylation and found that interfering with this site attenuated the development of tolerance in rats. I also show that the microglial transcription factor Runx1 regulates microglial reactivity in vitro and in vivo and that inhibition of Runx1 causes a decline in morphine analgesia. I characterized a novel strain of microglial-specific Runx1 knock-out mice and show that Runx1 deficiency causes a reduction in acute morphine analgesia and an exacerbation of opioid tolerance, hyperalgesia, and naloxone-precipitated withdrawal. Collectively, in this thesis I found that increased microglial reactivity, through variable mechanisms causes a reduction in the antinociceptive response to morphine in rodents. In conclusion, the work of this thesis has identified novel targets (P2X7R and Runx1) for interfering with the side effects associated with prolonged opioid use and in the acute analgesic response.
Open Access
Investigating the Heat Shock Protein 110 as a Modifier Of Prion Infection In Vitro and In Vivo
(2021-11-22) Marrero Winkens, Cristóbal; Schätzl, Hermann M; Trang, Tuan; Braun, Janice E. A.; Ousman, Shalina S.; Nussbaum-Krammer, Carmen
Prion diseases are fatal transmissible neurodegenerative disorders affecting humans and wild or domesticated animals. Molecularly, they are caused by a misfolding of the cellular prion protein (PrPC) into a highly pathogenic isoform (PrPSc). Over time, PrPSc aggregates into fibrils which are fragmented to recruit and convert further PrPC. Even though this process is essential to the replication of PrPSc, the molecular players involved remain unknown. Based on reports showing that the molecular chaperone Hsp110 forms part of a mammalian disaggregation machinery, we hypothesize that Hsp110 is critical for the fragmentation of PrPSc in prion propagation. To test this, Hsp110 levels were manipulated in prion-infected cultured cells: Transient knock-down was found to reduce PrPSc levels in neuronal and non-neuronal cells, while transient overexpression increased PrPSc in a dose-dependent manner in neuronal cells. Knockout of Hsp110 by CRISPR/Cas9 reduced the susceptibility of neuronal cells to prion infection. Overall, the effects of Hsp110 manipulation in cultured cells are consistent with an involvement of Hsp110 in prion propagation. Next, mice overexpressing Hsp110 (TgApg-1 mice) were inoculated with the 22L and Me7 prion strains. Compared to WT mice, TgApg-1 mice showed significantly prolonged survival after Me7- but not 22L-inoculation. To test whether this prolongation of survival occurred due to altered PrPSc propagation, the biochemical features of PrPSc were examined in terminally-diseased animals. No difference was found between PrPSc of WT or TgApg-1 mice, suggesting that Hsp110 overexpression may have been protective independently of PrPSc. Finally, the effect of compounds broadly inducing the expression of heat shock proteins was examined. WT FVB mice inoculated with RML or Me7 prions were treated with either celastrol, geranylgeranylacetone or vehicle. No difference in survival time was found between the groups, despite evidence of heat-shock response induction in drug-treated animals. These results suggest that broad heat-shock induction is not protective in prion infection. Overall, our in vitro studies are consistent with a role of Hsp110 in PrPSc fragmentation, but our in vivo work is inconclusive in this regard. We, therefore, propose further research aimed at exploring prion fragmentation and the role of Hsp110 in prion infection.
Open Access
Investigating the Role of Central Endocannabinoids and Inflammation in Comorbid Anxiety-Like Behaviour and Colitis
(2020-01-08) Vecchiarelli, Haley Alleson; Hill, Matthew Nicholas; Sharkey, Keith A.; Trang, Tuan
There is a well-established connection between inflammatory disorders and stress-associated neuropsychiatric disorders. For example, in patients with chronic inflammatory diseases, there are increased indices of anxiety and depression. However, despite this clinically significant relationship, there is not a comprehensive insight of the mechanisms linking inflammatory diseases with co-morbid mood and anxiety disorders. The endocannabinoid system, which is the system in the body that the psychoactive constituents of cannabis act on, regulates both anxiety and inflammation—indicating that it has the potential to underly these comorbidities. We hypothesize that in a rodent model of peripheral (gastrointestinal) inflammation, there would be alterations in endocannabinoid signaling that drive anxiety-like behaviours; that by boosting endocannabinoid signaling, these behaviours can be reversed; that neuroinflammation contributes to changes in central endocannabinoids.; and that endocannabinoids have the potential to regulate inflammatory processes. We find, using a rat model of colitis (intracolonic administration of 2,4,6-trinitrobenzenesulfonic acid (TNBS)), that there are reductions of an endocannabinoid, anandamide, in brain regions that regulate anxiety, which contributes to the generation of anxiety-like behaviour. We further show that these reductions are driven through corticotropin releasing factor receptor 1 (CRF-R1) and neuroinflammation mechanisms. Using a transgenic mouse model which presents elevated anandamide levels, we find a role for anandamide in regulating peripheral and central inflammatory changes induced by colitis. These studies link endocannabinoids and neuroinflammation to anxiety-like behaviour and show a protective role for endocannabinoid signaling in colitis-induced anxiety in rats and in peripheral and central inflammation in mice. This work contributes to our understanding of the mechanisms underlying inflammation induced anxiety.
Open Access
Mechanistic Exploration of Exercise and Neuropathic Pain
(2023-12-08) Vogel, Holly; Trang, Tuan; Kurrasch, Deborah; Whelan, Patrick
Peripheral nerve injury caused by disease or damage of the peripheral nerves is often accompanied by neuropathic pain, a debilitating chronic pain condition plagued by abnormal pain sensitivity. Current treatment options are limited and provide sub-optimal pain relief. Recent pre- clinical and clinical studies suggest that exercise reduces pain and improves motor recovery following nerve injury, but how this occurs is not clear. In this study, I characterized the development of neuropathic pain using the spared nerve injury (SNI) model which produces robust mechanical allodynia, a painful response to an innocuous stimulus. Utilizing a voluntary running wheel exercise paradigm, I identified key timepoints after nerve injury where exercise improves recovery following the development of mechanical allodynia. Focusing on the spinal cord as it is a hub for sensory nerve input and contains important motor circuitry that is altered following nerve injury, I assessed the impact of SNI and exercise on sensory peripheral nerve endings and spinal motor neurons as nerve injury induces persistent motor deficits. I also test the hypothesis that exercise modulates microglia, immune cells residing in the central nervous system that become reactive following SNI, contributing to the sequelae of neuropathic pain. Using a genetic mouse strain to visually distinguish peripheral immune cells from resident microglia, I demonstrate that SNI increases, while exercise decreases, the infiltration of peripheral immune cells into the spinal cord at a key timepoint where nerve injured animals given access to a running wheel no longer display mechanical allodynia. Further analysis of the spinal cord using gene profiling arrays uncover dysregulated gene expression due to SNI and exercise unique to the different regions of the spinal cord related to sensory and motor processing. Specifically, I identify genes Grin2b, Ptges3, and Faah that are over expressed two weeks after SNI in animals given access to a running wheel. At four weeks after SNI, expression of these genes is comparable to sham control. Ultimately these findings uncover specific dorsal and ventral horn temporal disease related transcriptional changes engaged by exercise that future studies may target to improve the treatment of neuropathic pain by mimicking the beneficial effects of exercise.
Open Access
Microglial pannexin-1 is a cellular determinant of opioid withdrawal
(2018-06-28) Burma, Nicole Elizabeth; Trang, Tuan; Teskey, G. Campbell; Borgland, Stephanie Laureen; Finn, David P.; Dyck, Richard Henry
Opioid analgesics are indispensable for treating acute post-operative pain, and a variety of chronic pain conditions. However, an over-reliance on opioids can put individuals at risk of developing severe side effects. For chronic opioid users, stopping or decreasing opioid use is difficult as many individuals experience a severe withdrawal syndrome. Opioid withdrawal is characterized by a host of debilitating signs and symptoms, including somatic and autonomic physical symptoms and an aversive affective component. The adverse effects associated with opioid use have become increasingly linked to the activity of microglia, which are immune cells in the central nervous system. Yet, the cellular mechanisms mediating this microglial response remain poorly understood. This thesis investigates the core cellular mechanisms by which microglial pannexin-1 (Panx1) channels underlie opioid withdrawal and other adverse effects associated with chronic opioid use. My over-arching hypothesis is that microglial Panx1 critically contributes to opioid withdrawal. Here, I show that morphine produces a preferential increase in Panx1 expression and function on microglia, and that genetic ablation of microglial Panx1 is sufficient to attenuate the physical signs and aversive component of morphine withdrawal. I provide novel evidence for direct microglia-neuron signaling in opioid withdrawal, and identify that Panx1-mediated ATP release is a key spinal substrate of physical withdrawal signs. I also demonstrate that morphine analgesia, opioid-induced hyperalgesia, analgesic tolerance, and reward behaviours are notably intact in microglial Panx1-deficient mice. This suggests that the side effects of repeated opioid use may be mechanistically separable, and that microglial Panx1 preferentially underlies the expression of opioid withdrawal. Finally, I show the potent amelioration of opioid withdrawal using the clinically utilized broad-spectrum Panx1 blocker, probenecid, indicating that Panx1 may represent a feasible therapeutic target for combating withdrawal in the clinic. In conclusion, this thesis identifies microglial Panx1 as a novel cellular determinant and unexpected target for combating opioid withdrawal, and as a result, represents a paradigm shift in understanding how opioid withdrawal occurs.
Open Access
Microglial Panx1 as a therapeutic target for opioid withdrawal
(2020-03-31) Komarek, Kristina; Trang, Tuan; Teskey, Gordon Campbell; Borgland, Stephanie Laureen
Open Access
Microglial Panx1 is a Spinal Determinant of Arthritis Joint Pain
(2018-09-27) Mousseau, Michael James; Trang, Tuan; Matyas, John Robert; Altier, Christophe; Salo, Paul T.
Pain is a debilitating feature of arthritis. Despite recent advances in treating joint inflammation and the widespread use of pain medications by arthritis patients, adequate pain control is an ongoing medical problem. A major challenge is that joint pain is poorly understood: some individuals with severe joint damage report little pain, whereas paradoxically others with seemingly minor joint damage complain of debilitating pain. This thesis examines how microglia in the spinal cord signal through pannexin-1 (Panx1) channels to mediate joint pain. My overarching hypothesis is that microglial Panx1 channels critically modulate arthritis joint pain and that silencing peripheral afferents suppresses microglial reactivity following joint injury. In rats, joint pain caused by intra-articular injection of monosodium iodoacetate (MIA) was associated with spinal adenosine triphosphate (ATP) release and an increase in spinal microglial reactivity. Here, I provide evidence that both C- and A-afferents modulate the development of mechanical allodynia in a model of MIA-induced joint pain. I also demonstrate that specific ablation of either peptidergic or non-peptidergic afferents can attenuate mechanical allodynia, decrease spinal microglial reactivity and suppress extracellular ATP concentration in the CSF of animals with MIA-induced joint damage. Following joint injury, I show a microglial-specific upregulation of P2X7-receptors (P2X7R). P2X7Rs drive pannexin-1 (Panx1) channel activation and, in rats with mechanical allodynia, Panx1 function is increased in spinal microglia. Specifically, I demonstrate that microglial Panx1-mediated release of the pro-inflammatory cytokine interleukin-1 (IL-1) induces mechanical allodynia in the MIA-injected hindlimb. Furthermore, I provide evidence that mice with a microglial-specific genetic deletion of Panx1 are protected from developing mechanical allodynia. Finally, I show that the clinically utilized broad-spectrum Panx1 blocker, probenecid, attenuates MIA-induced mechanical allodynia in two experimental models of arthritis and normalizes responses in the dynamic weight-bearing test, without impacting acute nociception. In conclusion, this thesis identifies that spinal microglial Panx1 channels are critically involved in the development of arthralgia and that Panx1-targeted therapy is a new mechanistic approach for alleviating joint pain.
Open Access
Minocycline treatment timing and its influence on serotonin expression following spinal cord injury
(2019-11) Flood, Jennifer Margaret; Whelan, Patrick J.; Trang, Tuan; Casha, Steven; Ousman, Shalina S.
The effects of incomplete traumatic spinal cord injury (SCI) can be partly reversed by the plasticity of local and spared descending projections. A promising window of plasticity occurs for a number of weeks following injury and involves the control of neuroinflammatory processes. The FDA-approved drug, minocycline, is a promising drug for treating SCI since it decreases microglia activity, reduces macrophage activity, and generally provides neuroprotective properties. In this thesis I established a timeline of injury, looking at both serotonin (5-HT) and microglia/macrophage (Iba-1) immunoreactivity (ir), and I targeted a time point before a significant reduction of descending serotonergic fibers, in the form of 5-HTir, took place (i.e. 1-week). I found that the administration of minocycline increased 5-HTir caudal and ipsilateral to the lesion, compared to shams and controls. Using the selected time point, 1-week post-SCI, I administered minocycline and found a decrease in lesion size and an increase of 5-HTir both caudal and ipsilateral to the injury as well as rostral and contralateral to the injury. In this thesis, I provide evidence that minocycline impacts 5-HT expression when administered acutely and one week following SCI. These data suggest that the timing of minocycline treatment influences the neuroprotective properties previously reported and also influences descending 5-HT expression post-SCI.
Open Access
Non-Psychotropic cannabinoids attenuate visceral pain in colitis
(2023-05-25) Svendsen, Kristofer; Altier, Christophe; Sharkey, Keith; Nasser, Yasmin; Trang, Tuan; Ma, Chris
The inflammatory bowel diseases (IBD), Crohn’s disease and ulcerative colitis, are complex chronic diseases that affect an increasing proportion of the population. Abdominal pain is a major clinical symptom, but current treatments are limited and a source of frustration for patients, many of whom seek alternatives such as cannabis. Cannabis contains many compounds with therapeutic potential that do not have the prohibitive psychotropic effects of tetrahydrocannabinol (THC). These non-psychotropic cannabinoids (npCBs) have a variety of effects including analgesia and anti-inflammatory actions and show potentiating effects when administered in combination. This project explored the analgesic effects of cannabichromene (CBC), cannabidiol (CBD), cannabidivarin (CBDV), and cannabigerol (CBG), individually and in combination, in the treatment of colitis-evoked visceral hypersensitivity use the acute dextran sulfate sodium model. Abdominal pain was quantified by electromyographic recordings of the reflexive contraction of the external oblique muscle in response to colorectal distension using an animal of experimental colitis. Activation of the spinal cord was assessed using immunohistochemistry to the neuronal activity marker c-Fos in neurons of the lumbosacral dorsal horn. A single injection intraperitoneal injection of 10 mg/kg of either CBD or CBG was found to reduce both nocifensive behaviors in the functional assay and c-Fos activity in spinal cord in animals with colitis. Similarly, a combination of npCBs consisting of 5 m/kg CBD with 1 m/kg each CBC, CBDV, and CBG—all sub-therapeutic dosages—reduced both measures to the level of untreated control animals. Investigations of mechanism of actions via whole-cell patchclamp electrophysiology of primary dorsal root ganglia neurons revealed CBD to act via a voltage-gated calcium channel with preliminary evidence indicating a high-voltage activated isoform. Preliminary data also suggest that the mixture of npCBs may act through a similar mechanism. These results suggest CBD, CBG, and a mixture of npCBs may be beneficial in managing pain associated with IBD.
Open Access
Novel Approaches to Fight Prion Diseases
(2020-04-29) Thapa, Simrika; Schaetzl, Hermann M.; Gilch, Sabine; Trang, Tuan; van Marle, Guido; Coffin, Carla S.; Telling, Glenn C.
Prion diseases are fatal neurodegenerative disorders caused by PrPSc, the misfolded and infectious isoform of the cellular prion protein (PrPC). Currently, no preventive or therapeutic measures are available. In this work, we focused on therapeutic and prophylactic strategies against prion infections. In the therapeutic approach, we targeted cellular pathways and investigated the role of the quality control (QC) proteins, ERp57 and VIP36, on prion propagation. We found that the overexpression of ERp57 or VIP36 significantly reduced PrPSc levels in persistently prion-infected cells and decreased the susceptibility of uninfected cells to de novo prion infection. Moreover, lentiviral-mediated overexpression of ERp57 prolonged the survival of prion-infected mice. Mechanistically, we found that ERp57 overexpression reduced endoplasmic reticulum (ER) stress. To translate this proof-of-concept into potential drug therapy, we investigated the anti-prion effect of Sephin1, shown to prolong the phosphorylation of eIF2α and lower ER stress in the cells. In persistently prion-infected neuronal cells, we found that treatment with Sephin1 markedly reduced PrPSc levels. Moreover, Sephin1 reduced ER stress-induced PrP aggregates in cells and significantly extended the survival of prion-infected mice. These data provide the basis for targeting these cellular pathways as novel anti-prion therapy. In our prophylactic approach, we hypothesized that active vaccination is useful to contain chronic wasting disease (CWD), a contagious and expanding prion disease of cervids. Here, we vaccinated transgenic mice expressing elk prion protein with adjuvant CpG alone, or one of four recombinant PrP (rPrP) immunogens: deer dimer (Ddi), deer monomer (Dmo), mouse dimer (Mdi), and mouse monomer (Mmo). After challenging the animals with CWD prions intraperitoneally, we found that all vaccinated groups had longer survival times than the CpG control group. Interestingly, the Mmo-immunized group revealed that survival was extended by 60%. We also observed 28.4% and 24.1% prolongation in Dmo and Ddi groups, respectively. Our preliminary study in reindeer showed substantial humoral immune response induced by Mdi and Ddi, and the sera from the Ddi-vaccinated reindeer significantly reduced CWD prions in a cell culture model. Taken together, this study describes potential vaccine candidates against CWD. However, their protective effect in the natural cervid host needs further investigation.
Open Access
Orexin- and Drug-induced Potentiation of Dopamine Neurotransmission and Reward-related Behaviours
(2021-01-14) Thomas, Catherine S.; Borgland, Stephanie, L.; Trang, Tuan; McGrath, Daniel
Mesolimbic dopamine (DA) plays a pivotal role in orchestrating numerous motivated behaviours, including seeking and consumption of rewards and the attribution of motivational (incentive) value to reward-related stimuli, such as food and drug cues. Repeated exposure to drugs of abuse produces enduring changes in mesolimbic DA neurotransmission, including potentiated responses to future drug experiences, known as sensitization. A prominent theory of addiction proposes that sensitization of mesolimbic DA by drugs of abuse renders individuals hypersensitive to the motivational properties of drug stimuli (i.e., incentive sensitization), allowing them to gain powerful control over behaviour and ultimately driving addiction. However, it remains unknown how rapidly incentive value can be attributed to drug cues, or if previous drug exposure potentiates cue-evoked mesolimbic DA and attribution of incentive value to drug cues. Furthermore, the hypothalamic neuropeptide orexin (Ox), is hypothesized to sculpt mesolimbic DA and guide reward-related behaviours, including the drug-induced sensitization. However, the effects of endogenous Ox release in the ventral tegmental area (VTA) on mesolimbic DA and reward - related behaviour remains to be characterized. Chapter 1 of this thesis critically evaluates literature regarding motivation and related neurobiology, the effects of drugs of abuse, current theories of addiction, and the influence of Ox. Chapters 2 and 3 of this thesis demonstrate that repeated exposure to drugs of abuse and orexin release in the VTA increase mesolimbic DA and reward-related behaviour. Firstly, pre-treatment with cocaine or the short-acting opioid remifentanil increases rapid attribution of incentive value to a cocaine or remifentanil cue. Secondly, remifentanil pre-treatment potentiates remifentanil-evoked and cue-evoked mesolimbic DA across a single Pavlovian conditioning session. Thirdly, endogenous Ox release in the VTA leads to place preference and increases motivated food-cue directed behaviour. Lastly, Ox release in the VTA potentiates electrically evoked mesolimbic DA. The significance, caveats, and wider implications of this work is discussed chapter 4.
Open Access
Pannexin 1 Channels as a Therapeutic Target: Structure, Inhibition, and Outlook
(2020-01) Navis, Kathleen E; Fan, Churmy Y; Trang, Tuan; Thompson, Roger J; Derksen, Darren J
Pannexin 1 (Panx1) channels are transmembrane proteins that release adenosine triphosphate and play an important role in intercellular communication. They are widely expressed in somatic and nervous system tissues, and their activity has been associated with many pathologies such as stroke, epilepsy, inflammation, and chronic pain. While there are a variety of small molecules known to inhibit Panx1, currently little is known about the mechanism of channel inhibition, and there is a dearth of sufficiently potent and selective drugs targeting Panx1. Herein we provide a review of the current literature on Panx1 structural biology and known pharmacological agents that will help provide a basis for rational development of Panx1 chemical modulators.