Osteogenesis Imperfecta: A Look into the Cerebellum of the Brtl Murine Model

Abstract

Osteogenesis imperfecta (OI), also known as brittle bone disease, is a rare congenital connective tissue disorder linked to collagen I defects, commonly known for its skeletal implications. However, collagen fibers play several key roles also in the central nervous system (CNS) starting from CNS development, regulating axonal growth, synaptogenesis, and terminal differentiation of Schwann cells. Collagen I plays an active role in defining brain architecture and in the developing peripheral. Furthermore, collagen is associated with astrogliosis and scar formation, processes finely regulated by molecules that are also involved in the regulation of the oxidative stress pathway. The aim of this work was to evaluate possible alterations in the cerebellum of the Brtl mouse, a well characterized model of dominant OI, focusing on cerebellar morphology using hematoxylin-eosin and Picrosirius Red staining, both in bright-field and polarized light microscopy. Additionally, ultrastructural alterations in the different cerebellar neuronal populations were evaluated using transmission electron microscopy (TEM), along with the involvement of the oxidative stress pathway by analyzing the expression levels of specific REDOX markers, such as COX4, SOD2, GPX4, and NRF2. Our results clearly demonstrate morphological and ultrastructural changes in the cerebellum of OI mice, along with increased oxidative stress detected in different regions and cell populations of this CNS area, suggesting possible CNS damage driven by the mutated form of collagen I. This work represents the first report of strong morphological and ultrastructural alterations in several cerebellar areas of Brtl mice, as well as a direct impact on the oxidative stress pathway.

Keywords: Brtl mice; Cerebellum; Collagen; In vivo; Osteogenesis imperfecta; Oxidative stress.

Fig. 1

Graphical representations of mouse cerebellar section in the sagittal plane, showing the cortical and deeper investigated areas

Fig. 2

H&E staining showing the well-preserved physiological cerebellar cytoarchitecture in male (a) and female (c) controls and Brtl mice (e, f, and h for male and female, respectively). a, c, e, and h: Low magnification micrograph shows the cerebellar cortex (subdivided into ML, PCL, and IGL) and (b, d, g, and i) the CP. f: Higher magnifications of the IGL area displaying hemorrhagic foci. Light microscopy magnification: × 40 (a–e, g–i); × 60 (f). Histograms display the quantitative valuation of ML (Panel A) and IGL (Panel B) cell density. p-values: (*) p < 0.05 and (**) p < 0.01

Fig. 3

PSR staining evaluation under bright-field (a–h) and polarized light (i–p) microscopy. Polarized light allows the discrimination between thick (red signals) and thin (green signals) collagen fibrils. Representative cerebellar specimens showing cerebellar cortex (a, c, e, g, i, k, m, and o) and CP (b, d, f, h, j, l, n, and p) from control (a, b, i, j and c, d, k, l for male and female respectively) and Brtl animals (e, f, m, n and g, h, o, p for male and female respectively). Magnification: × 40; inserts: × 60

Fig. 4

Histograms showing PSR OD measurement in bright-field (Panels A and B) and polarized light (Panels C, D, E, and F). Panel (A) and Panel (B): bright-field PSR OD measurements in meninges and CP, respectively. Panel(C) and Panel (D): polarized light PSR analyses of meninges showing the thick and thin collagen OD measurements, respectively. Panel (E) and Panel (F): polarized light PSR evaluation of CP showing the thick and thin collagen OD data respectively. p-values: (**) p < 0.01 and (***) p < 0.001

Fig. 5

Micrographs depicting immunohistochemical reaction for COX4 in the cerebellum from control (a–c and k–o for male and female respectively) and Brtl animals (f–j and p–t for male and female respectively). Non-immune control (a’). Magnification: × 40 (a’, a, d, e, f, i, j, k, n, o, p, s, and t); × 60 (b, c, g, h, l, m, q, r, and inserts)

Fig. 6

Histograms illustrating COX4-immunopositive OD (Panels A, C, D, and F) and cell density (Panels B and E) assessed in cerebellar layers and CP of control and Brtl mice. p-values: (*) p < 0.05, (**) p < 0.01, and (***) p < 0.001

Fig. 7

Micrographs depicting immunohistochemical reaction for SOD2 in the cerebellum from control (a–c and g–i for male and female respectively) and Brtl animals (d–f and j–l for male and female respectively). Non-immune control (a’). Magnification: × 40 (a’ and a–l); × 60 (inserts)

Fig. 8

Histograms illustrating SOD2-immunopositive OD (Panels A, C, and E) and cell density (Panels B and D) assessed in cerebellar layers and CP of control and Brtl mice. p-values: (**) p < 0.01 and (***) p < 0.001

Fig. 9

Micrographs depicting immunohistochemical reaction for GPX4 in the cerebellum from control (a–c and g–i for male and female respectively) and Brtl animals (d–f and j–l for male and female respectively). Non-immune control (a’). Magnification: × 40 (a’ and a-l); × 60 (inserts)

Fig. 10

Histograms illustrating GPX4-immunopositive OD (Panels A, C, and E) and cell density (Panels B and D) assessed in cerebellar layers and CP of control and Brtl mice. p-values: (**) p < 0.01 and (***) p < 0.001

Fig. 11

Micrographs depicting immunohistochemical reaction for NRF2 in the cerebellum from control (a–c and g–i for male and female respectively) and Brtl animals (d–f and j–l for male and female respectively). Non-immune control (a’). Magnification: × 40 (a’ and a–l); × 60 (inserts)

Fig. 12

Histograms illustrating NRF2-immunopositive OD (Panels A, C, and E) and cell density (Panels B and D) assessed in cerebellar layers and CP of control and Brtl mice. p-values: (*) p < 0.05, (**) p < 0.01, and (***) p < 0.001

Fig. 13

Representative cerebellar specimens investigated by transmission electron microscopy TEM from control (a–d and i–l for male and female respectively) and Brtl animals (e–h and m–p for male and female respectively). Scale bars: 2 µm

Fig. 14

Histograms illustrating ER thickness and mitochondria area (Panels A and B, respectively) measured in cerebellar cells of control and Brtl mice. p-values: (***) p < 0.001