Smaller Absolute Quantities but Greater Relative Densities of Microvessels Are Associated with Cerebellar Degeneration in Lurcher Mice

Abstract

Degenerative affections of nerve tissues are often accompanied by changes of vascularization. In this regard, not much is known about hereditary cerebellar degeneration. In this study, we compared the vascularity of the individual cerebellar components and the mesencephalon of 3-month-old wild type mice (n = 5) and Lurcher mutant mice, which represent a model of hereditary olivocerebellar degeneration (n = 5). Paraformaldehyde-fixed brains were processed into 18-μm thick serial sections with random orientation. Microvessels were visualized using polyclonal rabbit anti-laminin antibodies. Then, the stacks comprised of three 5-μm thick optical sections were recorded using systematic uniform random sampling. Stereological assessment was conducted based on photo-documentation. We found that each of the cerebellar components has its own features of vascularity. The greatest number and length of vessels were found in the granular layer; the number of vessels was lower in the molecular layer, and the lowest number of vessels was observed in the cerebellar nuclei corresponding with their low volume. Nevertheless, the nuclei had the greatest density of blood vessels. The reduction of cerebellum volume in the Lurcher mice was accompanied by a reduction in vascularization in the individual cerebellar components, mainly in the cortex. Moreover, despite the lower density of microvessels in the Lurcher mice compared with the wild type mice, the relative density of microvessels in the cerebellar cortex and nuclei was greater in Lurcher mice. The complete primary morphometric data, in the form of continuous variables, is included as a supplement. Mapping of the cerebellar and midbrain microvessels has explanatory potential for studies using mouse models of neurodegeneration.

Keywords: Lurcher; blood microvessels; cerebellum; cerebral degeneration; laminin; mice; quantitative histology; stereology.

Figure 1

Quantification of cerebellar and midbrain microvessels. (A) —The dorsal view of the wild type (WT) and Lurcher (Lc) fixed brain contained the following parts: cerebral cortex (1), midbrain (2), cerebellum (3), pons and medulla (4); (B)—The preparations were processed into serial of equidistant randomly orientated sections (a1, a2, …ai …an) with constant step k; (C)—Sagittal section through the midbrain and cerebellum: nuclei (N), white matter (WM), granular (GL), and molecular layers (ML) of the cerebellum. The microscopic regions of interest (ROI) in the x–y plane were selected at a constant interval. This was performed for the cerebellum and midbrain separately. Only fields marked with the green dots were taken into account; (D–F)—One of the microscopic fields is shown as a stack with three 5 μm-thick optical sections in the z-axis that illustrates a disector volume probe. Vessel profiles (marked by dots) and valence of nodes (n) are marked with respective dots. Scale bars: (C) 500 μm; (D–F) 10 μm.

Figure 2

Comparing quantitative parameters of the microvascular bed of midbrain and cerebellar anatomical compartments in wild type mice (left) and Lurcher mice (right). (A)—Volume of the cerebellum, its individual parts and the volume of the midbrain; (B)—Diffusion distance of vessels; (C)—Total number of vessels; (D)—Numerical density of vessels; (E)—Total length of vessels; (F)—Length density of vessels. Significant results of the Wilcoxon matched pairs test within group are connected with lines (p < 0.05). Corresponding anatomical compartments between groups were compared using the Mann-Whitney U test (significant results are presented within the diagrams: ^*p < 0.05, ^**p < 0.01).

Figure 3

Comparing quantitative parameters of microvessels between wild type (WT mice) and Lurcher mice. (A)—Relative proportions of the individual components of the cerebellum; (B)—Mean length of vessels in the cerebellum, its individual components and in the midbrain. Corresponding anatomical compartments were compared using the Mann-Whitney U test (significant results are presented within the diagrams: ^*p < 0.05, ^**p < 0.01).

Figure 4

Histological comparison of microcirculation in the separated cerebellar layers and the midbrain in the wild type (A–C) and Lurcher (D–F) mice. (A,D)—The complete absence of Purkinje cells (red arrow) with intensive degeneration of the molecular (ML) and granular (GL) layers in Lurcher mice; (B,E)—Morphological features of vascularity in the white matter (WM) and nuclei (N). The boundary between the molecular and granular layers marked by the purple dotted lines; between the granular layer and white matter by the green and between the white matter and the nuclei by the blue dotted lines. (C,D)—No significant difference in the midbrain vascularity. Immunohistochemical detection of the microvessel laminin outlines, visualization with horseradish peroxidase/diaminobenzidine (brown), counterstaining with haematoxylin. Scale bar: 50 μm, uniform magnification in (A–F).