Optimized heterologous transfection of viable adult organotypic brain slices using an enhanced gene gun

Abstract

Background: Organotypic brain slices (OTBS) are an excellent experimental compromise between the facility of working with cell cultures and the biological relevance of using animal models where anatomical, morphological, and cellular function of specific brain regions can be maintained. The biological characteristics of OTBS can subsequently be examined under well-defined conditions. They do, however, have a number of limitations; most brain slices are derived from neonatal animals, as it is difficult to properly prepare and maintain adult OTBS. There are ample problems with tissue integrity as OTBS are delicate and frequently become damaged during the preparative stages. Notwithstanding these obstacles, the introduced exogenous proteins into both neuronal cells, and cells imbedded within tissues, have been consistently difficult to achieve.

Results: Following the ex vivo extraction of adult mouse brains, mounted inside a medium-agarose matrix, we have exploited a precise slicing procedure using a custom built vibroslicer. To transfect these slices we used an improved biolistic transfection method using a custom made low-pressure barrel and novel DNA-coated nanoparticles (40 nm), which are drastically smaller than traditional microparticles. These nanoparticles also minimize tissue damage as seen by a significant reduction in lactate dehydrogenase activity as well as propidium iodide (PI) and dUTP labelling compared to larger traditional gold particles used on these OTBS. Furthermore, following EYFP exogene delivery by gene gun, the 40 nm treated OTBS displayed a significantly larger number of viable NeuN and EYFP positive cells. These OTBS expressed the exogenous proteins for many weeks.

Conclusions: Our described methodology of producing OTBS, which results in better reproducibility with less tissue damage, permits the exploitation of mature fully formed adult brains for advanced neurobiological studies. The novel 40 nm particles are ideal for the viable biolistic transfection of OTBS by reducing tissue stress while maintaining long term exogene expression.

Figure 1

Adult organotypic brain slice preparation and biolistic transfection. A) The adult mouse brain were embedded in DMEM-agarose with penicillin/streptomycin. This DMEM-agarose matrix, readily suitable for handling, contains phenol red, hence the red coloration. This colour gradient also helps to see the OTBS cut by the vibroslicer. Scale bar: 10 mm. B) The OTBS seen in an empty well ready for transfection by biolistics. A red arrow indicates a rod support used to maintain distance and angle. C) Scaled model showing the height of the modified barrel over the OTBS. D) Distribution pattern of the scattered micro- and nanoparticles. The affected and chosen region of interest subsequently expresses the exogene [29]. This biolistic transfection method is ideally suited for adult OTBS.

Figure 2

Organotypic brain slice viability, cellular populations, and exogene expression profile. A) Lactate dehydrogenase assay used to monitor cell viability. A significant reduction of cell viability in the 1 μm particle treated OTBS was measured using the LDH assay can be observed 5 days following treatment compared to control and 40 nm treated OTBS (n = 5, *p < 0.05). B) PI labelling at 5 days post transfection shows a significant elevation of the number of necrotic cells in the 1 μm treated OTBS compared to 40 nm treated and untreated OTBS (n = 6, *p < 0.05) as seen in representative brain regions. C) dUTP TUNEL assay at 5 days post transfection shows a slight elevation of nick end labelling in the 40 nm treated OTBS while a significant elevation in the number of dUTP positive cells compared to the untreated control can be observed in the 1 μm treated OTBS (n = 6, *p < 0.05). D) anti-NeuN and anti-GFAP immunolabelling at 5 days post transfection was used to quantify neuronal and glial populations respectively. A significant reduction in the number of NeuN positive cells was observed in comparable visual fields of the CA1 region of the hippocampus of corresponding coronal sections for the 1 μm compared to the 40 nm particle treated OTBS (n = 6, *p < 0.05). E) EYFP expression patterns seen in the 40 nm and 1 μm treated OTBS from 5 to 21 days after transfection. A significantly higher percentage of cells remained EYFP positive three weeks after transfection in the 40 nm treated OTBS compared to the 1 μm treated OTBS (n = 6, *p < 0.05) co-labelled with DAPI.

Figure 3

Microscopy images of subpopulation labelling and exogene expression profiles. A) Confocal images of the 40 nm and 1 μm treated OTBS immunolabelled with anti-NeuN, anti-GFAP, and DAPI seen at 5 and 14 days post transfection. Healthier cell morphologies can be observed in the 40 nm compared to the 1 μm treated OTBS. Scale bar: 100 μm. B) Confocal images of EYFP expression patterns seen in 40 nm and 1 μm transfected OTBS. A higher number of EYFP positive cells, maintaining neuronal morphology, can be observed in the 40 nm compared to 1 μm treated OTBS and this expression pattern remains longer (21 days). Scale bar: 100 μm.

Figure 4

Observing morphological characteristics in mature neurons of adult OTBS. A) Confocal image of neighbouring pyramidal cells, which were treated with the 40 nm particles, shows long axons oriented towards the cortex as seen by EYFP expression at 5 days post transfection. Scale bar: 25 μm. B) Another confocal image showing a population of pyramidal cells having their soma present in the CA1 region of the brain 5 days following EYFP transfection with the 40 nm particles. The radiating arborescence of neurites formed within the adult brain can clearly be seen. Cells were also stained with DAPI. Scale bar: 10 μm. C) Confocal image of another pyramidal cell 5 days following transfection with 40 nm particles showing EYFP expression. The soma also rests within the densely populated CA1 region of the hippocampus while the neurites branch out towards their respective synaptic interfaces. Cells were also stained with DAPI. Scale bar: 10 μm. This transfection procedure adequately preserves the morphological characteristics and architecture of mature neurons within these ex vivo adult OTBS.