A novel ex vivo Huntington's disease model for studying GABAergic neurons and cell grafts by laser microdissection

Abstract

Organotypic brain slice cultures have been recently used to study neurodegenerative disorders such as Parkinson's disease and Huntington's disease (HD). They preserve brain three-dimensional architecture, synaptic connectivity and brain cells microenvironment. Here, we developed an innovative model of Huntington's disease from coronal rat brain slices, that include all the areas involved in the pathology. HD-like neurodegeneration was obtained in only one week, in a single step, during organotypic slice preparation, without the use of neurotoxins. HD-like histopathology was analysed and after one week, a reduction of 40% of medium spiny neurons was observed. To analyse new therapeutic approaches in this innovative HD model, we developed a novel protocol of laser microdissection to isolate and analyse by RT-qPCR, grafted cells as well as surrounding tissue of fresh organotypic slices. We determined that laser microdissection could be performed on a 400μm organotypic slice after alcohol dehydration protocol, allowing the analysis of mRNA expression in the rat tissue as well as in grafted cells. In conclusion, we developed a new approach for modeling Huntington's disease ex vivo, and provided a useful innovative method for screening new potential therapies for neurodegenerative diseases especially when associated with laser microdissection.

Fig 1. Protocol to obtain coronal HD organotypic slices.

(A) The head must be dissected quickly and carefully to preserve brain structure. (B) The brain was glued onto the chuck of an aqueous solution-cooled vibratome, and 400μm thick coronal brain sections were cut and collected. (C) Brain slices were disposed on a 0.4 μm membrane insert. Coronal slices display different brain areas such as cortex (Cx), striatum (St), globus pallidus (GP) and corpus callosum (CC) associated with HD. (N = 25).

Fig 2. Slice plane selection to model HD and choice of pup’s age.

(A) Rat's brain was cut to obtain sagittal, coronal or horizontal brain slices. In each condition, the major areas involved in HD could be observed. (B) DARPP32 was detected in brain slices by immunofluorescence at day 0 (just after sectioning) and 7 days after transversal, coronal and sagittal sections. (C) NeuN, GAD67 and DARPP32 expression was determined in coronal brain slices by immunochemistry from P5-6 and P9-11 rats immediately after sacrifice, at day 0. The maturity of brain structure in slices from P9-11 rats is closer to adult brain than slices from P5-6 rats (N = 3).

Fig 3. Morphology and viability of organotypic slices.

Immunohistochemistry against NeuN in two brain regions: (A) cortex and (B) the striatum at day 0, 11, 19 and at day 30. (C) Quantification of NeuN-positive cells in cortex, striatum and lateral septum shows no important loss of staining in the cortex after 30 days in culture while a significant loss is observed in the striatum. (N = 4).

Fig 4. Expression of specific MSN markers.

Immunohistochemistry against striatal (A) DARPP32 -positive neurons and (B) GAD67 -positive neurons at day 0, 11, 19 and 30. (C) Striatal DARPP32 -positive neuron number decreased progressively until day 19 when depopulation became total in comparison to day 0 set as 100%. (D) Striatal GAD67 -positive neuron number also decreased progressively with a 30% decrease at day 4, 45% at day 7 and 100% at day 30, compared to day 0 representing 100%. (N = 4) (SD in dotted lines).

Fig 5. GABAergic degeneration depending on the slice section.

After vibrosection, plan sections are different, and we do not consider them similarly. (A) We distinguish caudal and rostral sections of brain slices obtained after vibrosection. (B) Immunohistochemistry against striatal DARPP32 positive neurons at day 0 and day 11. (C) Immunohistochemistry against striatal GAD67 positive neurons at day 0 and day 21. The expression of those markers remained over time in rostral sections. (D) Quantifications of DARP32-positive cells over time in rostral and caudal sections. (E) Quantification of GAD67-positive cells among time in rostral and caudal sections. (F) Schematic representations of neuronal pathways observed on a sagittal view and in a 3D projection. Yellow arrows represent dopaminergic pathways. Red arrows represent Glutamatergic pathways. Green arrows represent GABAergic pathways. The top schemas represent the morphology of the brain slices obtained in this model (taken from http://atlas.brain-map.org).

Fig 6. Slice dehydration protocol development to allow LMC.

Entire organotypic slice with main brain areas labelled (A). Scale bar: 2mm. Striatum of organotypic slices observed by brightfield microscopy. GFP-positive hNSCs were implanted in the striatum. Striatum delimitations can be easily determined by some landmarks as the anterior commissure-posterior (ACP), the corpus callosum (CC) and the lateral ventricule (LV). Scale bar is 300μM. (B) The 1^st protocol was based on water sublimation after 2h incubation at -20°C followed by 1h at 30°C. The 2^nd protocol was based on ethanol dehydration. Four 4 min successive baths of ethanol were performed, to induce a complete desiccation of the slice. The slices were then put on a foil bottom dish to perform LMD. (C) After water sublimation, the slice was still too hydrated blocking laser dissection. (D) After ethanol dehydration, a clean laser dissection could be performed. Scale bar: 300μm (E) Organotypic slices after LMD observed by brightfield microscopy. A clear-cut of the whole striatum was performed. Collected samples observed by brightfield microscopy and by fluorescent microscope. GFP-positive NSCs can easily be observed in the collected striatum. Scale bar: 300 μm. (N = 3).

Fig 7. mRNA quality analysis.

(A) Experion gel electrophoresis analysis. The only electrophoresis close to the control one is the alcohol treatment + RNA Later buffer conditions. (B) Electropherogram generated by Experion system of the different samples. The only high RNA quality indicator (RQI) was obtained with the alcohol treatment + RNA Later buffer condition. Conditions: L: Ladder, C: Positive control made of in vitro NSCs, 1: alcohol treatment + RNA later buffer, 2: alcohol treatment + lysis Buffer, 3: water sublimation + RNA Later buffer and 4: water sublimation + lysis Buffer. (N = 3).