Browsing by Author "Raimondo, Joseph Valentino"

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    Establishing in vitro models of neuroinflammation to investigate neuroimmune responses in neurocysticercosis
    (2024) Awala, Amalia Naita; Raimondo, Joseph Valentino
    Neurocysticercosis (NCC), a parasitic infection of the central nervous system (CNS) caused by the larvae of the cestode Taenia solium, is the leading cause of adult-acquired epilepsy in the world. A surprising clinical manifestation of NCC is that viable larvae can exist in the brain for extended periods with no symptomatology, but when they die, clinical symptoms develop. Clinical evidence suggests that the hallmark of symptomatic NCC is neuroinflammation; however, the neuroinflammatory mechanisms underlying this disease remain grossly understudied, and how innate immune cells respond to this infection is still debated. One of the reasons for this is that there is a lack of reliable experimental models of inflammation at the level of the brain that allow for cell-type specific tracking of inflammation in innate immune cells. Thus, this thesis's first aim was to establish the rodent-derived organotypic brain slice culture (OBSC) system as an in vitro model for investigating neuroinflammatory signalling and activation of microglia and astrocytes in the brain. To validate this model of neuroinflammation, OBSCs from neonatal mice were treated with the pro-inflammatory stimulant lipopolysaccharide (LPS) for 24 hours and compared to untreated control slices. Inflammatory activation of microglia and astrocytes was measured by tracking the activation of the inflammatory transcription factor nuclear factor for interleukin 6 (NF-IL6), a robust biomarker for tracking neuroinflammation in glial cells. Inflammation was confirmed by measuring the concentrations of pro-inflammatory cytokines IL-6 and TNF-α released by OBSCs in the culture medium. Lastly, we used single-nucleus RNA sequencing to investigate these inflammatory changes at a transcriptomic level. My results show that LPS significantly increased NF-IL6 activation in microglia and astrocytes and increased the release of both IL6 and TNF-α. At the transcriptomic level, I observed an upregulation of major inflammatory genes such as CCL5 and Timp1 in both microglia and astrocytes in response to LPS treatment, further confirming inflammation. Having demonstrated that OBSCs present a robust platform for investigating neuroinflammatory mechanisms in brain infections, the second aim of this thesis sought to use this established model to investigate how viable Taenia larvae modulate neuroinflammation in NCC. The potential immunomodulatory effects of the Taenia larvae on glial activation and inflammation was assessed by concurrently treating OBSCs with both LPS and Taenia larvae homogenate. I found that the co-application of LPS and Taenia larvae homogenate suppressed the LPS-induced microglial and astrocytic activation, proinflammatory cytokine release, and prevented the upregulation of key inflammatory genes. Together, this observed anti-inflammatory effect could explain how Taenia larvae can exist in the human brain without eliciting symptomatology from the host. This thesis's final aim was to set up a comparable translational in vitro human model of neuroinflammation using human II acute and organotypic brain slice cultures (hOBSCs). Using the inflammatory transcription factor NF-IL6 as a marker for glial activation, I found that at baseline, hOBSCs displayed higher levels of microglial and astrocytic activation than human acute slices. Additionally, untreated control and LPS-treated hOBSCs both displayed high levels of NF-IL6 activation. However, cytokine data revealed low concentrations of pro-inflammatory cytokines IL-6 and TNF-α in culture medium in untreated control hOBSCs that increased when slices were exposed to LPS, highlighting an inflammatory reaction. Taken together, these findings provide novel insights into understanding the neuroinflammatory mechanisms underlying NCC and highlight the utility of organotypic brain slice cultures in studying neuroimmune responses in diseases of an inflammatory nature such as NCC.
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    Exploring molecular and cellular mechanisms underlying seizures in neurocysticercosis
    (2021) de Lange, Anja; Raimondo, Joseph Valentino
    Neurocysticercosis is a disease in which larvae of the tapeworm, Taenia solium, infect the central nervous system of humans. Seizures are the most common symptom of NCC, occurring in between 70 % and 90 % of all symptomatic NCC cases. Neurocysticercosis impacts heavily on the quality of life of patients, and further presents a significant drain on the economic resources of endemic countries. Despite its considerable global impact, the molecular and cellular mechanisms underlying seizures in neurocysticercosis remain largely unknown. In this thesis I have explored novel models for neurocysticercosis by combining mouse hippocampal organotypic brain slice cultures with various preparations of a model parasite, Taenia crassiceps. Utilising these models, I first explored, using patch clamp and local field potential electrophysiology, how Taenia larval extracts directly affect neuronal excitability. I report that extracts of Taenia crassiceps resulted in a significant acute excitation of neurons and triggered seizure-like events in brain slices. Further investigation revealed that this excitation was mediated by the activation of glutamate receptors and that, indeed, the larvae of both Taenia crassiceps and Taenia solium contain and produce levels of glutamate sufficient to explain this effect. Chronic exposure of brain slices to intact, living, larvae did not, however, result in any changes in network excitability. Next, I investigate whether Taenia larvae produce acetylcholinesterases, as these enzymes have the potential to affect neuronal signaling by digesting the neurotransmitter acetylcholine. Ellman's assays, in situ acetylcholinesterase activity assays, and patch clamp electrophysiology reveal that both Taenia crassiceps and Taenia solium larvae produce acetylcholinesterases and that the activity of Taenia acetylcholinesterases is sufficient to digest acetylcholine at a concentration that alters neuronal signaling. Finally, I explore the effect that Taenia larval extracts have on the innate immune cells of the brain, as the responses of these cells can also alter neuronal excitability. Through the measurement of brain slice cytokine release using enzyme-linked immunosorbent assays, I discover that Taenia crassiceps extracts have robust antiinflammatory effects, which involve lipid, protein, and glycan elements. This thesis presents novel findings that reveal ways in which Taenia larvae interact with both neuronal and nonneuronal resident brain cells. It further delves into how these interactions could contribute to seizure generation in neurocysticercosis and proposes some potential new therapeutic approaches to treat seizures in neurocysticercosis.
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    Investigating neural responses in models of neurocysticercosis
    (2020) Tomes, Hayley Sarah; Raimondo, Joseph Valentino; Kellaway, Lauriston
    Epilepsy is more frequent in sub-Saharan Africa than the rest of the world due to high levels of brain infections by larvae of the pig cestode Taenia solium, a condition termed neurocysticercosis. Despite the large nature of the problem, little is known about how neurocysticercosis modulates neuronal responses to result in the development of seizures. In this thesis I have used the cestode Taenia crassiceps to develop multiple in vitro and in vivo models of neurocysticercosis in rodents. Utilising patch-clamp electrophysiology in organotypic hippocampal brain slices and chronic, wireless electrocorticographic recordings in freely moving animals I have explored how cestode larvae affect neuronal excitability in the brain across a range of time scales. First I demonstrate that homogenate of Taenia crassiceps larvae has a strong, acute excitatory effect on neurons, which is sufficient to trigger seizurelike events. The excitatory component of the homogenate was found to strongly activate glutamate receptors and not acetylcholine receptors nor acid-sensing ion channels. An enzymatic assay showed that the larval homogenate contains high levels of glutamate, explaining its acute excitatory effects on neurons. In the second part of my thesis I demonstrate that longer-term incubation of Taenia crassiceps homogenate with organotypic brain slices over the course of a day does not affect the intrinsic properties of pyramidal neurons nor the excitability of the neuronal network. In the final part of my thesis I established an in vivo model of neurocysticercosis. I found that intradermal inoculation together with multiple intracerebral injections of Taenia crassiceps homogenate did not result in the development of seizures over 3 months of chronic electrocorticography recordings. In addition, the seizure-threshold to picrotoxin, an excitotoxin, was not altered by Taenia crassiceps homogenate injection. Immunohistological analysis of the tissue below the injection site revealed no difference in astrocytes nor the number of microglia. However, microglial processes were observed to be retracted in the Taenia crassiceps group reflecting a moderate neuroinflammatory response. Together the data in my thesis provides novel insight into the acute and chronic effects of Taenia crassiceps homogenate on the excitability of neuronal networks with relevance to our understanding of neurocysticercosis.