Brain angioarchitecture and intussusceptive microvascular growth in a murine model of Krabbe disease

Abstract

Defects of the angiogenic process occur in the brain of twitcher mouse, an authentic model of human Krabbe disease caused by genetic deficiency of lysosomal b-galactosylceramidase (GALC), leading to lethal neurological dysfunctions and accumulation of neurotoxic psychosine in the central nervous system. Here, quantitative computational analysis was used to explore the alterations of brain angioarchitecture in twitcher mice. To this aim, customized ImageJ routines were used to assess calibers, amounts, lengths and spatial dispersion of CD31? vessels in 3D volumes from the postnatal frontal cortex of twitcher animals. The results showed a decrease in CD31 immunoreactivity in twitcher brain with a marked reduction in total vessel lengths coupled with increased vessel fragmentation. No significant changes were instead observed for the spatial dispersion of brain vessels throughout volumes or in vascular calibers. Notably, no CD31 ? vessel changes were detected in twitcher kidneys in which psychosine accumulates at very low levels, thus confirming the specificity of the effect. Microvascular corrosion casting followed by scanning electron microscopy morphometry confirmed the presence of significant alterations of the functional angioarchitecture of the brain cortex of twitcher mice with reduction in microvascular density, vascular branch remodeling and intussusceptive angiogenesis. Intussusceptive microvascular growth, confirmed by histological analysis, was paralleled by alterations of the expression of intussusception-related genes in twitcher brain. Our data support the hypothesis that a marked decrease in vascular development concurs to the onset of neuropathological lesions in twitcher brain and suggest that neuroinflammation-driven intussusceptive responses may represent an attempt to compensate impaired sprouting angiogenesis.

Endothelial cell dysfunction in globoid cell leukodystrophy

“…Accordingly, globotriaosylceramide induces endothelial dysfunctions in Fabry's disease (Namdar et al, ; Choi et al, ), and the Gaucher‐related metabolite β‐glucosylpsychosine (1‐β‐D‐glucosylsphingosine) exerts an inhibitory effect on endothelial cell proliferation and motility (Belleri et al, ) similar to the globoid cell leukodystrophy (GLD)‐related metabolite β‐galactosylsphingosine (psychosine, see below). Together with recent data on microvascular alterations in GLD (Belleri et al, ; Giacomini et al, ) and adrenoleukodystrophy (Musolino et al, ), these observations indicate endothelial dysfunction as a common factor in lysosomal storage disorders. In keeping with this hypothesis, morphologic endothelial alterations were observed in the CNS of acid sphingomyelinase‐deficient transgenic mice, a murine model of type A Neimann‐Pick disease (Marmiroli et al, ) as well as in knockout mice, with total deficiency of sphingolipid activator proteins required for the lysosomal degradation of sphingolipids (Oya et al, ).…”

“…Accordingly, microscopic analysis confirms the presence of intussusceptive transluminal pillars in microvessels of the brain cortex of P36 twi/twi mice (Giacomini et al, ). Intussusception occurs in twitcher brain in parallel with the modulation of genes associated with this alternative mechanism of neovascularization (Park et al, ), including a significant downregulation of the Notch target gene Hes5 and the upregulation of Fgf2 , the chemokine receptor Cxcr4 , and various proinflammatory and chemotactic mediators (Giacomini et al, ). These data suggest that structural and architectural angioadaptations may occur in twitcher mice in response to the neuroinflammatory conditions resulting from GALC deficiency (Formichi et al, ; Sakai, ).…”

“…Microvascular corrosion casting followed by scanning electron microscopy morphometry (Fig. ) confirmed the presence of significant alterations of the functional angioarchitecture of the brain cortex of twitcher mice, with reduction of microvascular density, vascular branch remodeling, vascular leakage, and intussusceptive angiogenesis (Giacomini et al, ).…”

“…The effect of GALC deficiency on postnatal CNS microvascularization has been investigated in the telencephalon of twitcher mice before the onset of neurologic signs (i.e., at P12/P17) and at P24, when pathologic alterations may occasionally occur. Brain vascularity was analyzed also at P36, when twi/twi mice show evident neurologic defects (Belleri et al, ; Giacomini et al, ). Microvascular density was measured in the cortex and periventricular areas, both characterized by a robust postnatal angiogenic response in wild‐type animals (Breier et al, ; Ogunshola et al, ).…”

“…Note the paucity of the CD31‐positive vascular network in twitcher brains. See Giacomini et al () for further details.…”