(563a) Probing Brain Structure-Function Relationships in Neurodegeneration Using Organotypic Whole-Hemisphere Slice Models and Multiple Particle Tracking Technology

Background: Aging is a common denominator in neurodegenerative pathogenesis, however the mechanisms by which healthy aging deviates towards a pathological state remain unclear. We hypothesize that the structural integrity of brain becomes compromised due to metabolic stress on neurons, rendering the brain less capable of maintaining healthy states and becoming more susceptible to pathologies such as plaque buildup or Lewy body aggregation. The use of nanoparticles with real-time tracking technology has increased access to structural information on live brain tissue – prior work using nanoparticle tracking techniques has identified design parameters that enable penetration in the brain microenvironment and detection of microstructural changes associated with different brain ages (1,2).

In this work, develop a neurodegeneration model in organotypic whole hemisphere (OWH) brain slice tissue. OWH brain slices are robust, stimuli-responsive platforms for recapitulating in vivo functional states with high throughput and spatiotemporal resolution (3,4). To model pathological processes in neurodegeneration in OWH slices, we induce chronic energy depletion in brain cells. Metabolic decline, driven by mitochondrial abnormality, is a classical hallmark of pathological aging and onset of neurodegeneration (5,6). Continuous, low-grade exposure to the neurotoxin rotenone (ROT) inhibits Complex I of the mitochondrial respiratory chain at intercellular concentrations above 20 nM (7). 50 nM ROT exposure has been shown to result in sufficient damage to dendritic spines and reduce neuronal connectivity (8). The upper limit of ROT exposure, without compromising viability, is reported to be 20 μM (9). We use the ROT-exposed OWH brain slice model in combination with multiple particle tracking, confocal microscopy, and molecular biology techniques to probe micro and macroscopic changes to native extracellular architecture and cellular function.

Methods: We develop the OWH platform to: 1) model the development of pathological processes that contribute to neurodegeneration, 2) characterize regional hallmarks of neurodegeneration at the inter-, intra-, and extra-cellular level, and 3) correlate these data with microstructural information. Based on the previously reported limits of ROT exposure, ROT doses in this study range from 50 nM to 10 μΜ. ROT is diluted in DMSO at 10 mM and further diluted into aqueous culture media at 10 μΜ (DMSO < 0.01% v/v). Slices are cultured 4 days in normal media – this is based on evidence that viability loss from the slicing procedure recovers after 4 days in vitro (4DIV) for slices from postnatal day 10 (P10) rats (10). This postnatal age is well-suited to study chronic effects of ROT exposure as P10 slices maintain viability for up to three weeks post extraction. At 4DIV, media is replaced with ROT media and changed every day for 6 days of ROT exposure. We assume that continuous ROT exposure establishes a uniform concentration profile of ROT in cells. Healthy control slices (NC) continue to receive normal media and are provided fresh media at the same time points as ROT-exposed slices.

Structure-function relationships are studied by correlating changes in tissue microstructure with effects of ROT exposure. Fluorescent bioinert nanoparticles are incubated in OWH slices and tracked dynamically using live-video epifluorescence microscopy. Nanoparticle trajectories are segmented using ImageJ TrackMate (11) and fed into a lab-developed Python package diff_classifier to compute trajectory mean-squared displacements (MSD) and geometric features (12). Particle trajectories can be classified as Brownian and non-Brownian, for which Brownian MSD values directly interface with governing transport equations to yield values for local effective diffusivity and viscosity. We perform multiple particle tracking (MPT) analysis in multiple brain regions to study the regional effect of ROT-exposure on the extracellular space.

We further assess the effects of ROT-exposure at the organelle, cellular, and extracellular levels. We isolate synaptosomic mitochondria from NC and ROT slices and assayed for activity (Sigma-Aldrich, MAK259). Slices are homogenized, decanted, and centrifuged in a mitochondrial isolation buffer to collect crude mitochondrial pellet, further separated into synaptosomic and non-synaptosomic mitochondria by discontinuous Percoll gradient in MIB (13). At the cellular level, we examine whole-slice viability and the functional health of neurons by region. Whole-slice viability is assessed by a lactate dehydrogenase (LDH) assay, for which slice culture supernatants are assayed at each time point from NC and ROT-exposed slices. The cumulative percent of damaged cells is computed relative to a maximum death control corresponding to the highest dose (10 μM) and longest exposure (6d) OWH slices treated with Triton-X detergent. We evaluate neuronal health in the following regions of interest (ROIs): cortex (CTX), striatum (STR), hippocampus (HP), and thalamus (TH). Neuronal death by ROI is quantified by co-localization of propidium iodide signal (a marker of dying or dead cells) with neurons, imaged using confocal microscopy. Slices are also stained with the neurodegenerative marker FluoroJade-C (FJC) and microtubule marker MAP-2, both of which assess structural integrity of neurons in response to ROT (8, 14). At the extracellular level, we quantify matrix degradation by extracting proteoglycans and using liquid-chromatography with mass-spectrometry (LC/MS) to survey reductions in average molecular weight of components such as hyaluronic acid and chondroitin sulfate. Collectively, this set of studies interfaces structural information gathered by nanoparticle probes with multi-level biological response to metabolic decline.

Results: We observe tunable responses to ROT exposure in our OWH model. Assaying for whole-slice viability demonstrated dose- and time- dependent responses to ROT exposure. The increase in cumulative cytotoxicity, relative to NC slices, was proportional to the dosing level and this relationship was maintained over 6 days of continuous ROT exposure for each dose. At 6 days of ROT exposure, slices exposed to 50 nM ROT experience a 25% increase in cumulative cytotoxicity. The most severe exposure (highest dose, longest time) reached a cumulative cytotoxicity of 86%. We observed a proportional increase in the expression of matrix-metalloprotease 9 (MMP-9) in response to ROT exposure, providing preliminary evidence that ROT exposure drives structural modifications while simultaneously inflicting cellular damage. Regionally, MMP-9 expression (mean fluorescence signal, n=10 images per group) increased 5-fold in the cortex and 3.5-fold in the striatum following 6 days of 1 μM ROT exposure, compared to 1.7- and 1.8-fold increases in NC slices. Diffusion data from MPT shows a change in nanoparticle diffusion ability dependent on both ROT exposure time, dose, and region. Diffusivity profiles for acute exposure (24h) contrast with long exposure (6-day) results. Nanoparticles were tracked at 2h, 6h, and 24h for 50 nM and 10 μΜ exposure. 50 nM ROT showed similar acute decay profiles for effective diffusivity (D_eff, μm²/s) in both the cortex and striatum, decreasing from 0.55 to 0.14 (CTX) and 0.46 to 0.14 (STR) over 24h. For 10 μΜ exposure, both regions experienced a sharp drop in diffusivity within 2h (CTX, 0.55 to 0.13; STR, 0.46 to 0.03) followed by an increase to 71% (CTX) and 43% (STR) of the initial NC D_eff within 24h. Interestingly, for longer exposure, response diverged by region and dose. Following 6 days of ROT exposure, NC effective diffusivity in the cortex increased from 0.55 (NC) to 0.70 (50 nM), 0.60 (5 μM), and 0.59 (10 μM), relative to a 6d NC diffusivity of 0.14. In the striatum, effective diffusivity responded differently, increasing from 0.46 (NC) to 0.67 (50 nM), and decreasing to 0.08 (5 μM) and 0.42 (10 μM), relative to a 6d NC diffusivity of 0.09. The extent to which ROT-driven mitochondrial malfunction contributes to neuronal abnormality and tissue structural modifications will continue to be revealed by the intercellular and cellular analyses described above.

Conclusions: The ROT-exposure OWH slice model provides a tunable platform to survey the effects of brain cell metabolic stress on short- and long-term neuronal health and tissue structural integrity. We precisely tune and conserve whole-slice cellular response by dose and exposure time, represented by LDH assay data. Observable region-dependent changes in surrogate features of tissue microstructure enable further investigation into the time- and severity- dependent impacts of metabolic stress on neuronal health and brain microstructure. Our ROT-OWH model can equally screen recovery from exposure to explore the questions of irreversibility and how permutable disease states mediate tissue resiliency.

References:

1. Nance et. al. (2012). 1126/scitranslmed.3003594

2. McKenna et. al. (2021). 10.1021/acsnano.1c00394

3. Joseph et. al. (2020). 1002/btm2.10175

4. Wood et. al. (2020). 10.1002/btm2.10265

5. Lau et. al. (2013). 10.1038/nrn3550

6. Grimm & Eckert. (2017). 1111/jnc.14037

7. Higgins et. al. (1996). 10.1523/jneurosci.16-12-03807.1996

8. Testa et. al. (2005). 1016/j.molbrainres.2004.11.007

9. Pakrashi et. al. (2020). 10.1007/s11064-020-03061-8

10. McKenna et. al. (2022). In review

11. Schneider et. al. (2012). 1038/nmeth.2089

12. Curtis et. al. (2019). 10.21105/joss.00989

13. Lampl et. al. (2015). 3791/52076

14. Lapointe et. al. (2004). 10.1096/fj.03-0677fje