Laser capture microdissection protocol for gene expression analysis in the brain.

Laser capture microdissection (LCM) allows the isolation of specific cell populations from complex tissues that can be then used for gene expression studies. However, there are no reproducible protocols to study RNA in the brain and, particularly, in the substantia nigra. RNA is a very labile biomolecule that is easily degraded during manipulation. LCM studies use low amounts of material and special precautions must be taken to preserve RNA yield and integrity, which are decisive for PCR analysis. The RNA yield and/or integrity can be affected negatively by tissue manipulation, LCM process and RNA extraction. We have optimized these three critical steps using nigral tissue sections, and developed a LCM protocol to obtain high-quality RNA for gene expression analysis. The optimal LCM protocol requires the use of 20 µm-thick tissue sections mounted on glass slides and processed for rapid tyrosine hydroxylase immunofluorescence. Additionally, a total microdissected tissue area of 1 mm2 and a column-based RNA extraction method were used to obtain a high RNA yield and integrity. In the rat substantia nigra, we demonstrated the expression of RNA for the angiotensin type 1 and type 2 receptors using this optimized LCM protocol. In conclusion, the LCM protocol reported here can be used to study the expression of both scarcely or abundantly expressed genes in the different brain regions of mammals under both physiological and pathological conditions.

Modification of the number and phenotype of striatal dopaminergic cells by carotid body graft.

In non-human primates, striatal tyrosine hydroxylase-immunoreactive (TH-ir) cells are increased in number after dopamine depletion and in response to trophic factor delivery. As carotid body cells contain the dopaminotrophic glial cell line-derived neurotrophic factor (GDNF), we evaluated the number, morphology and neurochemistry of these TH-ir cells, in the anterior and posterior striatum of five monkeys treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) which received a graft of carotid body cell aggregates (CBCA) (n = 3) or sham surgery (n = 2), and six MPTP-monkeys that were sacrificed 6 months and 3 years after the last MPTP dose [MPTP I (n = 3) and MPTP II (n = 3), respectively]. Three intact monkeys served as controls. A disability rating scale was used for the assessment of parkinsonism in all lesioned animals, both before and after surgery. For the neurochemical examination, tissue sections were double-labelled with antibodies to TH, dopamine transporter, dopa decarboxylase-67, vesicular monoamine transporter 2, glutamic acid decarboxylase -67, calbindin, parvalbumin, calretinin, neuronal nitric oxide synthase and GDNF. Only animals receiving CBCA graft showed a moderate but significant recovery of parkinsonism that persisted 12 months after the graft. The grafted striatum contained the greatest TH-ir cell density (120.4 +/- 10.3 cells/100 mm2), while the control striatum displayed the lowest (15.4 +/- 6.8 cells/100 mm2), and MPTP I, MPTP II and sham-operated monkeys showed a similar intermediate value (66.1 +/- 6.2, 58.3 +/- 17.2 and 57.7 +/- 7.0 cells/100 mm2, respectively). In addition, in the post-commissural striatum, only CBCA graft induced a significant increase in the TH-ir cell density compared to control animals (47.9 +/- 15.9 and 7.9 +/- 3.2, respectively). Phenotypically, TH-ir cells were striatal dopaminergic interneurons. However, in the grafted animals, the phenotype was different from that in control, MPTP and sham-operated monkeys, with the appearance of TH/GDNF-ir cells and the emergence of two TH-ir subpopulations of different size as the two main differentiating features. Our data confirm and extend previous studies demonstrating that striatal CBCA grafts produce a long-lasting motor recovery of MPTP-monkeys along with an increase in the number and phenotype changes of the striatal TH-ir interneurons, probably by the action of the trophic factors contained in carotid body cells. The increased number of striatal TH-ir cells observed in the grafted striatum may contribute to the improvement of parkinsonism observed after the graft.