Imaging intact mammalian brain at high resolution has always been very challenging.
Post mortem tissues can be imaged even at quite high resolution with different light based methods by making histological analysis of the sample or by labeling specific molecules. However, the sections analyzed cannot be thicker than 5-20um. Indeed, adult tissues are very opaque. The tissue contains several intracellular and extracellular structures (membranes, cytoplasm, organelles, extracellular matrix proteins,…) and when the light of a microscope hits them, it gets absorbed. Therefore, energy is lost inside the tissue resulting into poor resolution of the images acquired.
In order to avoid these problems, several approaches have been used, for instance by sectioning the sample and making serial acquisition of the different slices. However, this approach is very time consuming and prone to artifacts due to a non precise alignment of the slices. Multiphoton microscopy has partially overcome this problem by using a different technology in order to remove the “blurry effect” due to light scattering. However, by using these methods is still not possible to image sections thick enough to analyze the global complex neuronal connectivity of the brain.
Several protocols have been developed recently in order to address this problem, including Clarity, 3DISCO, Sca/e, ClearT2 and SeeDB. For instance, Clarity has been used to investigate the neurological changes in human tissue from Alzheimer’s disease, Parkinson’s disease or autism brains.
Cerebellar ataxia is a mitochondrial disease that has been associated with defects of the mitochondrial respiratory chain. The cerebellum has a very precise circuitry and several studies have shown the link between abnormalities of purkinje cells and mitochondrial diseases.
In this paper the authors used an improved Clarity protocol to analyze the effect of mitochondrial defects in the cerebellum of patients with mitochondrial diseases, in human post mortem tissues.
THE PROTOCOL:
The human tissue was either fixed in formalin for a maximum of 7 years (fresh sections were cleared faster than the ones fixed for longer times), or fresh post mortem, PFA fixed and then frozen.
Their optimized protocol consists of first incubating the sample in a hydrogel (made of acrilamide and bis acrilamide) that in few hours polymerizes around the tissue, making a matrix. After that, the cerebellum can be sliced in thick sections, up to 250/500um. This step is followed by a passive clearing of the slices with a solution containing SDS, a detergent that by removing lipids makes the sample transparent. Cleared sections are then incubated with antibodies diluted in PBS-tween at low temperatures but longer incubation times in order to increase the specificity of the antibody. The poor penetration of the antibody, that was not improved by pre-treatment with Triton-X or Proteinase-K (which instead resulted in tissue damage), forced them to use 250um slices. However, if tissue can be labeled with other techniques, with a 20X objective, after clearing, slices of up to 500um can be imaged.
THE RESULTS:
Although the human cerebellum required longer incubation times for the clearing compared to the mouse (shorter times resulted into poor quality of the staining), they were able to label myelin sheath in the granule cell layer (by MBP staining), mitochondria (by Cox1 staining, a protein of Complex-1 of the mitochondrial respiratory chain) in the cell body of purkinje cells, and axons (by neurofilament staining). The global loss of Cox1 protein expression in patients with mitochondrial diseases compared to control suggested a mitochondrial deficiency. Moreover, the co-labeling with neurofilaments provided spatial information about the distribution of this protein in axons and dendrites. These results match with previous studies showing mitochondrial abnormalities in post mortem brain tissue, but the information about neuronal projections were limited because of the thin sections used.
CONCLUSION:
The different methods used today to obtain a transparent sample to be analyzed, can use different solutions, all resulting in delipidation. All of them have different advantages and disadvantages. A major advantage of Clarity is that the native proteins are very well incorporated into the hydrogel matrix. This is a positive aspect when thinking of the possibility of stripping away the antibody and re-use the sections for a different staining, or to visualize myelin sheath with staining of the membrane protein MBP that so far has been quite challenging due to the removal of the lipid content during the clearing.
GLOSSARY BOX:
Passive clarity. While in active clarity protocols the tissue is immersed in SDS solution and then in an electric field, where SDS (negatively charged) moves into the electric field carrying lipids out of the tissue, in passive clearing the tissue is just incubated in SDS solution. SDS goes through the tissue passively, bringing lipids out of the tissue. Passive clearing is slower, but active clearing sometimes can result into tissue damage.
Multiphoton microscopy. microscopy technique where the fluorescent molecule is excited with two photons. While the energy of a single photon is not sufficient to excite the fluorophore, the sum of the energy of the two photons will be sufficient to excite the molecule. As the probability of two molecules on two different focal planes to be excited is negligible compared to the probability of exciting one molecule on the same focal plane, the signal coming from other focal planes, usually affecting the acquired image, is very small. Moreover, as in only one point of the tissue the molecule will receive enough energy to be excited, the surrounding tissue will not be affected, limiting phototoxicity.
Formalin. solution for fixation of tissues before histological analysis. It’s usually a 37% formaldehyde solution and works by cross-linking amino groups of proteins or DNA.
PFA. Paraformaldehyde. Solution for tissue fixation, resulting from polymerization of formaldehyde molecules.
PBS-Tween. phosphate buffer solution commonly used in immunohistochemistry, with addition of a small percentage of Tween detergent.
Reference:
http://www.ncbi.nlm.nih.gov/pubmed/27181107