https://www.um.edu.mt/library/oar/handle/123456789/31738 Mon, 20 Apr 2026 07:11:20 GMT 2026-04-20T07:11:20Z https://www.um.edu.mt/library/oar/handle/123456789/50417 Title: Characterization of mouse model of focal ischemia and reperfusion to study the putative protective benefit of intraperitoneally administered lactate in gray and white matter regions of the brain. Abstract: Background: Stroke is a medical emergency requiring prompt intervention. In cerebral ischemia, early revascularisation is critical to salvage tissue at risk of infarction and minimize injury progression. Animal models have allowed for the characterization of several disease pathways that are amenable to therapeutic intervention. However, clinical translation remains elusive and the need for successful therapies is urgent. Future progress depends critically on animal models that allow ischemic stroke to be studied at various stages, from initial changes in blood flow, to the physiological reaction to ischemia, neuronal death, behavioural impairment and recovery. The intraluminal model of middle cerebral artery occlusion (MCAO) mimics the most common form of stroke in humans and is thus widely applicable to study putative neuroprotective strategies and test for promising compounds. Neuroprotection in cerebral ischemia, however, can only be successful if both gray and white matter components are protected through pharmacological or vascular interventions. L-lactate is a metabolite that is oxidised preferentially to glucose in conditions of high metabolic stress. lt has previously been found to be neuroprotective in vitro and in vivo when administered in a mouse model of stroke based on its sole use as an alternative energy substrate. However, in these studies, only neuronal protection in cortical gray matter regions in the brain had been assessed thus far. The aim of this study was to characterize in some histological detail a mouse model of focal ischemia to determine the structural components and the distinct cellular populations involved in this type of injury. We then tested whether L-lactate-mediated protection extends to deeper subcortical areas of white matter, apart from the already well documented gray matter protection. Recent work has suggested the involvement of lactate receptor HCA1 in mediating neuronal protection. A further extension of this study was to establish whether this receptor could be involved in L-lactate-mediated protection in white matter acting through unrecognized axon-glial interactions. Methods: The study was performed under tight control of animal physiology and important intraoperative control parameters including body temperature, intra-ischemic cerebral blood flow and vital signs. In initial studies, we selected two mouse strains (CD-1 and C57BL/6) to demonstrate a direct side-by-side comparison in histopathological and behavioural parameters between these strains that were exposed to varying degrees of ischemia to establish model reproducibility. CD-1 mice were eventually chosen to further most of these experiments because of their clear reproducibility in their depth of ischemia in correlation to the lesion size thus produced. Following in-depth histological and immunocytochemical analyses in response to 60-minute ischemia, putative protection afforded by Na L-lactate was assessed. In preliminary studies, Na L-lactate was administered intravenously (5mgkg-1). Administration was later changed to the more favourable intraperitoneal route (250mgkg-1). Single-dose administration occurred 5 minutes prior to ischemia induction or upon reperfusion onset. Putative protection was assessed via histological, immunocytochemical and functional outcome analyses. Results: In initial studies, infarct volumes and edema formation correlated well to the severity of the insult in both strains. We then demonstrated that Na L-lactate administered intraperitoneally significantly reduced infarct volumes in CD-1 and C57BL/6 strains Intraperitoneal administration of Na I –lactate resulted in the reducation of ischemia-induced neurological deficits and brain water content compared to vehicle treatment at an acute stage. Immunocytochemical characterization revealed attenuation of ischemia-induced oligodendrocyte loss and the appearance of less pyknotic nuclei in white matter at acute and subacute stages upon Na L-lactate administration before or after ischemia. Protection at both stages coincided with partially preserved phosphorylated neurofilament and axonal structure integrity. Myelin condition as observed histologically by Luxol Fast Blue (LFB) staining was ameliorated with Na L-lactate treatment in CD-1 mice. This observation extended to include the C57BL/6 strain, where improved LFB staining in WM was concomitant with partial preservation of yellow fluorescent protein {YFP) expression 24 hours following ischemia. We report for the first time the co-localization of the lactate receptor HCA1 in white matter oligodendrocytes. Increased receptor expression on oligodendrocytes was detected during ischemia, which was further elevated by Na L-lactate treatment at acute and subacute stages. Conclusion: HCA1 receptor expression on oligodendrocytes presents a role for lactate-mediated signalling in white matter. Protection in gray and white matter regions mediated by this natural and readily available substance could have important clinical implications and could provide impetus for future stroke research. Keywords: HCA1, ischemia, lactate, MCAO, white matter Description: PH.D. Sun, 01 Jan 2017 00:00:00 GMT https://www.um.edu.mt/library/oar/handle/123456789/50417 2017-01-01T00:00:00Z https://www.um.edu.mt/library/oar/handle/123456789/31839 Title: The interaction of amyloidogenic proteins with mitochondrial membranes : role of cardiolipin Abstract: BACKGROUND Neurodegenerative proteinopathies like Alzheimer's disease (AD) and Parkinson's disease (PD) are increasingly linked to direct damage to cellular membranes by aggregate complexes of amyloidogenic proteins, such as the amyloid-beta (A~) peptide, tau and a-synuclein (as). Although not related to human disease, aggregates ofthe N-terminal domain of the Eschericia coli HypF (HypF-N) protein are also membrane-active and cytotoxic. Mitochondria, abundantly located at neuronal synapses, are critically impaired in both AD and PD, leading to bioenergetics failure. OBJECTIVES In the present study, the focus of the investigation was amyloid toxicity by tau and as proteins to mitochondrial membranes. In particular, it was especially important to probe a mechanistic role for cardiolipin (CL), a non-bilayer forming acidic phospholipid unique to mitochondrial membranes, in mediating amyloid protein interaction with mitochondria. Another aspect of the present work involved validating a select group of small-molecule compounds as potential drug leads for enhancing the resilience of the mitochondrial membrane barrier against insult by amyloid aggregates. METHODOLOGY With this aim, an in vitro minimalist model system was used to eschew cellular complexity and provide a powerful means for obtaining mechanistic insights. Thus, mitochondria were freshly isolated from human neuroblastoma SH-SYSY cells and incubated with physiological (low micromolar) concentrations of tau, as, and HypF-N proteins aggregated according to established protocols. Mitochondria were subjected to assays reflecting outer or inner mitochondrial membrane integrity: organelle swelling, Cytochrome (Cyt) c release (CCR), and depolarisation of the inner mitochondrial membrane potential (LltJIm) . Further, direct binding of amyloid protein to mitochondria was assessed by immunoblotting and, in the case IV of tau, using liposomes having CL-rich membranes simulating the lipid environment of mitochondrial contact sites and the inner mitochondrial membrane. RESULTS Aggregate exposure instigated disruption of mitochondrial structural integrity as evidenced by abundant swelling, robust efflux of Cyt c from the mitochondria, and loss of the Ll'Pm (the latter not in the case of tau). An important result from a mechanistic point of view is that pharmacological inhibitors of the mitochondrial permeability transition pore (mPTP) complex could not rescue these phenotypes. Together with the mitochondrial binding studies, the data describes a scenario in which the amyloid proteins physically associate with mitochondria and possibly directly porate the mitochondrial membranes. Regarding amyloid-membrane affinity, a key finding is that the phospholipid CL appears to function as a leitmotif in targeting of amyloid proteins to mitochondrial membranes. Such a mechanism could involve either direct CL binding or sensing of, and insertion into, lipid packing and curvature defects created by the cone-shaped CL molecules. Finally, the diphenyl- pyrazole anti-aggregator anle138b, the polyphenol rosmarinic acid, and the theaflavin-rich extract from black tea, were the most effective three compounds in protecting mitochondrial organelles from perturbation by toxic amyloid entities. They may thus represent important drug leads to alleviate mitochondrial dysfunction in neurodegenerative diseases. CONCLUSION The data presented here advances a conceptual model in which CL-rich mitochondrial membranes, such as the outer membrane contact sites and the inner membrane, represent a generic target of aggregate complexes of amyloidogenic proteins. In the cytosol, toxic amyloid entities of proteins such as as and tau, would be free to target mitochondria by interacting with CL or by inserting into lipid defects created by the CL molecules, leading to compromised mitochondrial structural integrity. Hence, efforts on developing drugs that target mitochondrial lipids, and especially CL, might constitute a new strategy for pharmacological therapy of neurodegenerative proteinopathies. Description: PH.D. Sun, 01 Jan 2017 00:00:00 GMT https://www.um.edu.mt/library/oar/handle/123456789/31839 2017-01-01T00:00:00Z