A rotenone organotypic whole hemisphere slice model of mitochondrial abnormalities in the neonatal brain

HIGHLIGHTS

What: The authors develop an organotypic whole-hemisphere (OWH) brain slice model of mitochondrial dysfunction in the neonatal brain. The authors demonstrate the ability of OWH slice cultures exposed to ROT to capture time-, region-, and severity-dependent responses across relevant scales.
Who: Brendan Butler from the Department of Chemical, University of Washington, Seattle, WA, USA have published the research work: A rotenone organotypic whole hemisphere slice model of mitochondrial abnormalities in the neonatal brain, in the Journal: (JOURNAL)
How: For confocal imaging a 10 × tilescan of each slice was obtained to provide a representative region map followed by additional images at 240x (60 × lens with 4 × zoom) for representative mitochondrial morphology and complex I association. Most ROT models study the effects on neurons and how microglial interactions modulate neuronal survival therefore the authors chose to focus the studies to these cell types.
Future: Future work could probe causational relationships that contribute to diffusive differences by quantifying hyaluronan fractionation and ECM protein expression nanoparticle uptake and clearance osmotic_pressure and ionic strength and cell-specific proliferation . The results highlight severity- time- and region-dependent responses and establish a complimentary model system of mitochondrial abnormalities for high-throughput or live-tissue experimental needs. Future work can incorporate male rats and different postnatal ages into the study design to consider sex-based and age-based differences if it is desired to implement ROT exposure to study therapeutic efficacy of drugs that target mitochondrial regulation in neonates or pediatric populations. Future work should consider other cell types such as astrocytes and oligodendrocytes when studying neonatal mitochondrial abnormalities or developing celltargeted treatments.

SUMMARY

The authors have demonstrated that OWH slices obtained from postnatal (P) day 10 rats, term equivalent to the human neonate, retain viability and metabolic activity up to two weeks in_vitro. The authors use ROT to inflict mitochondrial damage and examine the tissue-level, extracellular, cell-type, organelle, and RNA-level responses to repeat ROT exposure and single ROT exposure in two distinct brain regions over 6 days in culture. The results highlight severity-, time-, and region-dependent responses and establish ROT exposure in term-equivalent OWH cultures as a model system complimentary to traditional in_vivo or in_vitro ROT models. At 4 days in_vitro (4DIV), SCM is replaced with RSCM at 50 nM (Fig 1). This study establishes the utility of ROT in OWH slice cultures as a compliment to in_vitro and in_vivo ROT models, and also demonstrates the exposure regime and time dependence of multi-scalar responses to the mitochondrial poison (Fig 9). Prior to this study, most in_vitro ROT organotypic models focus on individual subcortical structures. The authors hypothesized that the tradeoffs been cell numbers and % cell damage may be attributed to the microglial response to neuronal damage, an effect which has previously been shown in_vitro. This study established a complimentary model system to common in_vitro and in_vivo ROT models to study mitochondrial dysfunction in the neonatal brain with high experimental throughput. Quantifying neuronal damage, microglial localization with damaged cells, and microglial density suggested differences in microglial responses and microglia-neuron interactions that contribute to regional differences in cell damage and density in response to exposure regime and culture time. @@

LAY DEFINITIONS

rotenone: A botanical insecticide that is an inhibitor of mitochondrial electron transport.
brain: A brain is an organ that serves as the center of the nervous system in all vertebrate and most invertebrate animals. It is located in the head, usually close to the sensory organs for senses such as vision
Carboxylate: A carboxylate is the conjugate base of a carboxylic acid.
Dimethylsulfoxide: A highly polar organic liquid, that is used widely as a chemical solvent. Because of its ability to penetrate biological membranes, it is used as a vehicle for topical application of pharmaceuticals
Lactate dehydrogenase: A tetrameric enzyme that, along with the coenzyme NAD+, catalyzes the interconversion of LACTATE and PYRUVATE. In vertebrates, genes for three different subunits (LDH-A, LDH-B and LDH-C) exist
slice: A wafer (slice or substrate) is a thin slice of semiconductor material, such as a crystalline silicon, used in electronics for the fabrication of integrated circuits and in photovoltaics for conventional, wafer-based solar cells. The wafer serves as the substrate for microelectronic devices built in and over the wafer and undergoes many microfabrication process steps such as doping or ion implantation, etching, deposition of various materials, and photolithographic patterning
Electron transport chain: An electron transport chain is a series of compounds that transfer electrons from electron donors to electron acceptors via redox reactions, and couples this electron transfer with the transfer of protons across a membrane. This creates an electrochemical proton gradient that drives the synthesis of adenosine triphosphate, a molecule that stores energy chemically in the form of highly strained bonds
Confocal microscopy: Confocal microscopy, most frequently confocal laser scanning microscopy, is an optical imaging technique for increasing optical resolution and contrast of a micrograph by means of adding a spatial pinhole placed at the confocal plane of the lens to eliminate out-of-focus light. It enables the reconstruction of three-dimensional structures from the obtained images by collecting sets of images at different depths within a thick object
mitochondrial: Dihydrolipoamide dehydrogenase (DLD), also known as dihydrolipoyl dehydrogenase, mitochondrial, is an enzyme that in humans is encoded by the DLD gene. DLD is a flavoprotein enzyme that oxidizes dihydrolipoamide to lipoamide
Dynamic light scattering: Dynamic light scattering (DLS) is a technique in physics that can be used to determine the size distribution profile of small particles in suspension or polymers in solution. In the scope of DLS, temporal fluctuations are usually analyzed by means of the intensity or photon auto-correlation function (also known as photon correlation spectroscopy or quasi-elastic light scattering)
Extracellular matrix: In biology, the extracellular matrix (ECM) is a collection of extracellular molecules secreted by cells that provides structural and biochemical support to the surrounding cells. Because multicellularity evolved independently in different multicellular lineages, the composition of ECM varies between multicellular structures; however, cell adhesion, cell-to-cell communication and differentiation are common functions of the ECM
Institutional animal care and use committee: Institutional committees established to protect the welfare of animals used in research and education. The 1971 NIH Guide for the Care and Use of Laboratory Animals introduced the policy that institutions using warm-blooded animals in projects supported by NIH grants either be accredited by a recognized professional laboratory animal accrediting body or establish its own committee to evaluate animal care; the Public Health Service adopted a policy in 1979 requiring such committees; and the 1985 amendments to the Animal Welfare Act mandate review and approval of federally funded research with animals by a formally designated Institutional Animal Care and Use Committee (IACUC)
Cellular metabolism: Metabolism is the set of life-sustaining chemical reactions in organisms. The three main purposes of metabolism are: the conversion of food to energy to run cellular processes; the conversion of food/fuel to building blocks for proteins, lipids, nucleic acids, and some carbohydrates; and the elimination of metabolic wastes
Oxidative phosphorylation: Oxidative phosphorylation (or electron transport-linked phosphorylation or terminal oxidation) is the metabolic pathway in which cells use enzymes to oxidize nutrients, thereby releasing energy which is used to reform adenosine triphosphate (ATP). In most eukaryotes, this takes place inside mitochondria
Polystyrene latex: Polymerized forms of styrene used as a biocompatible material, especially in dentistry. They are thermoplastic and are used as insulators, for injection molding and casting, as sheets, plates, rods, rigid forms and beads
Diethylpyrocarbonate: Preservative for wines, soft drinks, and fruit juices and a gentle esterifying agent.
Propidium iodide: Propidium iodide (or PI) is a fluorescent intercalating agent that can be used to stain cells and nucleic acids. PI binds to DNA by intercalating between the bases with little or no sequence preference
Reactive oxygen species: Reactive oxygen species are chemically reactive chemical species containing oxygen. Examples include peroxides, superoxide, hydroxyl radical, and singlet oxygen

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Site reference: https://jbioleng.biomedcentral.com/counter/pdf/10.1186/s13036-024-00465-w

DOI reference: https://www.doi.org/10.1186/s13036-024-00465-w

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